CN1232645C

CN1232645C - Method for protecting plants

Info

Publication number: CN1232645C
Application number: CNB971816107A
Authority: CN
Inventors: J·A·赖亚尔斯; S·J·尤肯斯; A·莫里那弗南德咨; L·B·弗里德里克
Original assignee: Syngenta Participations AG
Current assignee: Syngenta Participations AG
Priority date: 1996-12-27
Filing date: 1997-12-23
Publication date: 2005-12-21
Anticipated expiration: 2017-12-23
Also published as: HUP0000654A2; CA2275854A1; JP2001508288A; IL130452A0; HU224480B1; TR199901491T2; AU5859798A; KR100500751B1; WO1998029537A2; EP1009838A2; CN1245537A; KR20000069743A; AU725767B2; WO1998029537A3; AR010855A1

Abstract

The present invention concerns a method of protecting plants from pathogen attack through synergistic disease resistance attained by applying a conventional microbicide to immunomodulated plants. Immunomodulated plants are those in which SAR is activated and are therefore referred to as ''SAR-on'' plants. Immunomodulated plants may be provided in at least three different ways: by applying to plants a chemical inducer of SAR such as BTH, INA, or SA; through a selective breeding program based on constitutive expression of SAR genes and/or a disease-resistant phenotype; or by transforming plants with one or more SAR genes such as a functional form of the N/M1 gene. By concurrently applying a microbicide to an immunomodulated plant, disease resistance is unexpectedly synergistically enhanced; i.e., the level of disease resistance is greater than the expected additive levels of disease resistance.

Description

The method of protective plant

The present invention relates to come protective plant to avoid the method for pathogenic agent invasion and attack by microorganism agent being applied to the collaborative disease resistance that the immunomodulatory plant obtains.

Plant often is subjected to the attack that multiple causal organism comprises virus, bacterium, fungi and nematode.Crop plants is especially fragile because they are usually as the consistent monoculture cultivation of heredity; When disease was attacked, loss can be very serious.Yet most of plants have the natural immunology defense of resisting causal organism.Plant breeder and pathologist have identified to be had the natural mutation of resistance and has cultivated into the multiple kinds of crops plant phytopathogen.These natural disease resistence genes produce high-caliber resistance or the immunity of resisting pathogenic agent usually.

Systemic acquired resistance (SAR) be a plant moiety being used to protect the compound system of self avoiding pathogenic agent injury (Hunt and Ryals, Crit.Rev.in plant Sci.15,583-606 (1996) is incorporated herein by reference; Ryals etc., Plant is thin Born of the same parents8,1809-1819 (1996) is incorporated herein by reference).Also, be incorporated herein by reference referring to United States Patent (USP) 5,614,395.SAR be plant-pathogenic agent send out the aspect that should be even more important because it be pathogen-inducible comprise virus to extensively infecting the factor, system's resistance of bacterium and fungi.When the SRA signal transduction pathway was closed, plant was responsive more to morbific pathogenic agent under normal circumstances, they to normal circumstances non-pathogenic infect the factor also become responsive (Gaffney etc., Science261,754-756 (1993) is incorporated herein by reference; Delaney etc., Science266,1247-1250 (1994) is incorporated herein by reference; Delaney etc., institute of NAS newspaper, 92,6602-6602 (1995) is incorporated herein by reference; Delaney, Plant physiology113,5-12 (1997) is incorporated herein by reference; Bi etc., The plant magazine8,235-245 (1995) is incorporated herein by reference; Mauch-Mani and Slusarenko, Vegetable cell8,203-212 (1996) is incorporated herein by reference).These observations show that the SAR signal transduction pathway is vital for keeping plant health.

Conceptive, the SAR reaction can be divided into two stages.At initial period, pathogenic agent infects and is identified and releasor, and signal phoresys to remote organization by phloem.Target cell recognition system signal, and the expression response by SAR gene and disease resistance.The SAR maintenance stage is meant for some time of the whole vital process of a few Zhou Zhizhi plants, this in stage plant be in quasi-stationary state, disease resistance is maintained (Ryals etc., 1996).

It seems that Whitfield's ointment (SA) accumulation be that the SAR signal transduction is essential.Because with the back transgene expression of giving birth to inhibition or salicylate hydroxylase of special inhibitor processing, phenylalanine ammonia-lyase, the plant that can not accumulate SA can not be induced SAR genetic expression or disease resistance (Gaffney etc., 1993; Delaney etc., 1994; Mauch-Mani and Slusarenko 1996; Maher etc., Institute of NAS newspaper91,7802-7806 (1994) is incorporated herein by reference; Pallas etc., The plant magazine10,281-293 (1996) is incorporated herein by reference).The possessor advises that SA may be as system signal to the greatest extent, but still disputable at present, what determine at present a bit is if SA can not accumulate, the blocking-up of SAR signal transduction [Pallas etc., 1996; Shulaev etc., vegetable cell 7,1691-1701 (1995) is incorporated herein by reference; Vernooij etc., Vegetable cell6,959-965 (1994) is incorporated herein by reference].

Recently, Arabidopis thaliana begins mode system (Uknes etc., vegetable cell 4, the 645-656 (1992) as research SAR; Be incorporated herein by reference; Uknes etc., Mol.plant-Microbe. Interact.6,692-698 (1993) is incorporated herein by reference; Cameron etc., Plant The thing magazine5:715-725 (1994) is incorporated herein by reference; Mauch-Mani and Slusarenko; Mol.plant-Microbe.Interact.7,378-383 (1994) is incorporated herein by reference; Dempsey and Klessig, Bulletin de L ' Institut.Pasteur 93,167-186 (1995) is incorporated herein by reference).Verified, SAR can be activated as SA, 2 6-dichloro-isonicotinic acid (INA) and benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters (BTH) (Uknes etc., 1992 by pathogenic agent and chemical in Arabidopis thaliana; Vernooij etc. Mol.Plant-Microbe Interact., 8,228-234 (1995) is incorporated herein by reference; Lawton etc., The plant magazine10,71-82 (1996) is incorporated herein by reference).After INA or BTH handle or pathogenic agent infects, have three kinds of pathology (PR) protein gene of be correlated with at least, i.e. the appearance of PR-1, PR-2 and PR-5 and resistance is worked in coordination with simultaneously and is induced (Uknes etc., 1992,1993).In feature is understood the most clearly the species tobacco, with pathogenic agent or immunocomplex handle induce at least nine cover expression of gene (Ward etc., Plant Cell3,1085-1094 (1991) is incorporated herein by reference).By prepared disease resistance transgenic plant (United States Patent (USP) 5,614,395) with multiple SAR gene-transformed plant.

Be separated to the multiple arabidopsis mutant body (Delaney, 1997) of SAR signal transduction with modification.First batch of mutant in these mutant be so-called lsd (damage stimulate sick) mutant and acd2 (necrocytosis of acceleration) (Dietrich etc., Cell77,565-577 (1994) is incorporated herein by reference, Greenberg etc., Cell77,551-563 (1994) is incorporated herein by reference).These mutant have the formation of the spontaneous downright bad damage of some degree on its leaf, the SA level of rising, the accumulation of SAR mRNA and the disease resistance that obviously improves.At least isolation identification different lsd mutant (Dietrich etc., 1994 of seven strains; Weymann etc., Vegetable cell7,2013-2022 (1995) is incorporated herein by reference).Another kind of interesting mutant be cim (composing type immunity) mutant (Lawton etc., " molecular biology of systemic acquired resistance ", in " Defence is anti-in the plant Should mechanism" in, B.Fritig and M.Legrand compile (Dordrecht, The Netherlands:Kluwer Academic Publishers), and 422-432 page or leaf (1993) is incorporated herein by reference).Also see international pct application WO94/16077, be incorporated herein by reference.Identical with the acd2 mutant with lsd, the cim mutant has SA and the SAR genetic expression and the resistance of rising, and different with lsd or acd2, does not occur observable damage on their leaf.Cpr1 ( PRThe constitutive expression person of gene) may be one type cim mutant; Yet, because also get rid of atomic little damage is not arranged on the leaf, Cpr1 may be one type the lsd mutant (Bowling etc., Vegetable cell6,1845-1857 (1994) is incorporated herein by reference).

Also be separated to the mutant that the SAR signal path is blocked.Ndr1 (non-specific specificity disease resistance) be the normal avirulent strains growth of the pseudomonas syringae (Pseudomonas syringae) that allows to comprise various nontoxic genes and parasitic downy mildew (Peronospora parasitica) mutant (Century etc., Institute of NAS newspaper92,6597-6601 (1995) is incorporated herein by reference).Clearly, this mutant is blocked in early days at the SAR signal path.Npr1 (the non-expresser of PR gene) be the mutant that after INA handles, can not induce the SAR signal pathway to express (Cao etc., Vegetable cell6,1583-1592 (1994) is incorporated herein by reference).Eds (enhanced disease susceptibility) according to its behind the low bacterial concentration of inoculation, can support the ability of bacterial invasion obtain separating (Glazebrook etc., Genetics143,973-982 (1996) is incorporated herein by reference; Parker etc., Vegetable cell8,2033-2046 (1996) is incorporated herein by reference).Very similar on some eds mutation type surface to npr1, and show that recently eds5 and eds53 and npr1 are homotopic (Glazebrook etc., 1996).Nim1 (non-induction type immunity) handles mutant (Delaney etc., 1995 that parasitic downy mildew (being the virulence factor of oidium) growth is supported in the back at INA; WO94/16077).Nim1 can accumulate SA although pathogenic agent infects the back, and it can not induce SAR genetic expression or disease resistance, and showing suddenlys change has blocked the SA downstream pathway.Nim1 also sustains damage to the ability of INA or BTH reaction, shows that blocking-up is present in downstream (Delaney etc., 1995 of these chemical effects; Lawton etc., 1996).

Recently, separate and identified two Arabidopis thaliana allelotrope, they be the mutant of being responsible for nim1 and npr phenotype respectively (Ryals etc., Vegetable cell9,425-439 (1997) is incorporated herein by reference; Cao etc., Cell88,57-63 (1997) is incorporated herein by reference).Wild-type NIM1 gene product has participated in causing in the Arabidopis thaliana signal transduction cascade reaction (Ryals etc., 1997) of SAR and gene pairs gene (gene-for-gene) disease resistance.Ryals etc., 1997 have also reported other 5 allelic separation of nim1, they show a series of phenotypes that are damaged to very strong blocking-up from the PR-1 genetic expression of chemical induction and fungus resistant a little less than.The not only complementary sudden change of npr1 mutant, recovery SAR inductive reactivity and the disease resistance relevant with PR genetic expression are gone in wild-type NPR1 gene transformation, and the render transgenic plant has stronger resistance (Cao etc., 1997) not having under the SAR inductive situation pseudomonas syringae infected.

NF-к B/ к B signal transduction pathway has participated in the disease resistance reaction of multiple biology from the fruit bat to the Mammals.In Mammals, NF-к B/ к B signal transduction can be induced by multiple different stimulated, comprise cells contacting lipopolysaccharides, tumour necrosis factor, interleukin 1 (IL-1) or virus infection (Baeuerle and Baltimore, Cell87,13-20 (1996); Baldwin, Immunity is commented academic year14,649-681 (1996)).The activatory approach causes participating in the synthetic of the inflammatory reaction and the immunoreactive multiple factor, as interleukin II, interleukin 6, interleukin 8 and granulocyte/macrophage colony stimulating factor (deMartin etc., Gene152,253-255 (1995)).In transgenic mice research, the knocking out of NF-к B/I к B signal transduction cause the defective type immune response comprise enhanced to the susceptibility of bacterium and viral pathogen (Beg and Baltimore, Science274,782-784 (1996); Van Antwerp etc., Science274,787-789 (1996); Wang etc., science 274,784-787 (1996); Baeuerle and Baltimore (1996)).In Arabidopis thaliana, SAR is similar on function to inflammatory reaction, because the normal resistance process in SAR activation back strengthens, causes enhanced disease resistance (Bi etc., 1995; Cao etc., 1994; Delaney etc., 1995; Delaney etc., 1994; Gaffney etc., 1993; Mauch-Mani and Slusarenko 1996; Delaney; 1997).And the inactivation of this approach causes the increase to bacterium, virus and fungal pathogens susceptibility.Be enjoyably, it is reported, the activation of NF-к B in the SA blocking-up mammalian cell (Kopp and Ghosh, Science265,956-959 (1994)), and the signal transduction in the SA activation Arabidopis thaliana.Thereby the bacterial invasion fruit bat activated the signal transduction cascade reaction cause a large amount of antifungal protein such as cercropinB, defensin, diptericin and drosomycin synthetic (Ip etc., Carefully Born of the same parents75,753-763 (1993); Lemaitre etc., Cell86,973-983 (1996)).In fly, thisly induce the gene product of two homologue dorsal depending on NF-к B and dif and suppressed by the homologue cactus of I к B.Antimycotic and the proteic synthetic reduction of antibacterium greatly reduces the resistance to infecting.

Although big quantity research and application have been done in skilled and meticulous crop protection measure, comprise the genetic transformation of plant, because the loss that disease causes still is tens dollars every year.So, still constantly to seek development based on the new crop protection measure of ever-increasing understanding to the disease resistance of plant hereditary basis.

Based on above-mentioned, the present invention preferably relates in one aspect to by the insecticidal microorganism agent being applied to collaborative disease resistance protective plant that the immunomodulatory plant obtains and avoids the novel method of pathogenic agent invasion and attack.The immunomodulatory plant is meant that SAR is activated, and shows than the stronger SAR genetic expression of wild-type usually thereby is called as " SAR-opens " plant.The immunomodulatory plant that is applied in the inventive method can obtain by three kinds of diverse ways at least: with SAR pharmaceutical chemicals inductor such as BTH, INA or SA are applied to plant; By selection breeding system based on moulding expression of SAR genome and/or disease resistance phenotypic screen plant; Transform the genetically engineered plants that plant obtains by functional form with one or more SAR genes such as NIM1 gene.The microbicide that is applied to the immunomodulatory plant can be conventional microbicide such as mycocide metalaxyl (matalaxyl), if perhaps be applied to the immunomodulatory plant that obtains by selection breeding or genetically engineered, microbicide can be chemical inducer such as BTH, INA or the SA of SAR.

Immunomodulatory provides the disease resistance of certain level to plant.Similarly, microbicide is applied to the disease resistance that plant provides certain level.The expected results of immunomodulatory and microbicide being used the associating use is that controlled levels has reflected by single adduction controlled levels that the disease resistance method is provided of planting.Yet by simultaneously microbicide being applied to the immunomodulatory plant, disease resistance is worked in coordination with reinforcement beyond expectationly, and promptly the disease resistance level is than the disease resistance adduction level height of expecting.

Correspondingly, the present invention relates to immunomodulatory cultivation of plants and an amount of conventional microbicide is applied to this plant.Particularly preferred embodiment of the present invention relates to gene engineering method to be comprised plant and expresses functional form or its homologue or its variant of NIM1 gene.

Method of the present invention obtains than passing through only with immunomodulatory or the higher pathogenic agent control of microbicide.Immunomodulatory offers the disease resistance of plant certain level.Similarly, microbicide is applied to the disease resistance that plant provides certain level.The desired result of immunomodulatory and microbicide being used the associating use is that controlled levels has reflected by single adduction controlled levels that the disease resistance method is provided of planting.Yet by simultaneously microbicide being applied to the immunomodulatory plant, disease resistance is worked in coordination with reinforcement beyond expectationly, and promptly the disease resistance level is than the disease resistance adduction level height of expecting.

Except disease resistance increased, another advantage of the present invention was that the inventive method reaches the required microbicide of disease resistance level than lacking with the amount of common wild-type plant needs.Consequent result is that the microbicide economic drain is low, causes the probability of adverse environment problem to reduce by some microbicide toxicity.And the method for protective plant of the present invention is regulated by combined immunization and the effect of microbicide prolongs and the crop yield raising antipathogen action time.Another advantage of present method is that the threat that forms resistance is avoided effectively because the combined action pattern of these two kinds of pathogenic agent controls is different fully each other.

Therefore the present invention relates to avoid the method for pathogenic agent invasion and attack by collaborative disease resistance protective plant; May further comprise the steps:

(a) provide immunomodulatory plant with one-level disease resistance; And

(b) use at least a microbicide to give the secondary disease resistance to described immunomodulatory plant;

(c) described thus microbicide is given collaborative enhanced three grade disease resistances bigger than firsts and seconds disease resistance summation to using of described immunomodulatory plant.

Preferably according to method of the present invention, wherein said immunomodulatory plant is composing type immunity (cim) mutant plant.

Particularly preferably be according to method of the present invention, wherein said cim mutant plant is elected from plant population according to the following step:

(a) expression of assessment SAR gene in the plant that phenotype is infected normally, the plant of wherein being infected lacks the damage model phenotype; And

(b) screen when not having virus, bacterium or fungal infection the plant of being infected of constitutive expression SAR gene.

Preferably according to method of the present invention, wherein said immunomodulatory plant is a damage simulation mutant plant.

Particularly preferably be according to method of the present invention, wherein said damage simulation mutant plant is selected from plant population according to the following step:

(a) expression of assessment SAR gene in having the plant of being infected of damage model phenotype; And

(b) screening is not having virus, when bacterium or fungal infection, and the plant of being infected of constitutive expression SAR gene.

Preferably according to method of the present invention, wherein said immunomodulatory plant is by recombinant expressed obtain of SAR gene in plant.

Particularly preferably be according to method of the present invention, wherein said SAR gene is the functional form of NIM1 gene.

More preferably according to method of the present invention, the NIM1 albumen of wherein said NIM1 genes encoding has participated in causing in the plant signal transduction cascade reaction of systemic acquired resistance.

Particularly preferably be according to method of the present invention, wherein said NIM1 albumen comprises the aminoacid sequence shown in the SEQ ID NO:2.

Particularly preferably be according to method of the present invention, wherein said NIM1 gene is hybridized with the encoding sequence shown in the SEQ ID NO:1 under following condition: at 1%BSA; 520mM NaPO ₄, pH7.2; 7% sodium lauryl sulphate salt; 1mM EDTA; In 55 ℃ of hybridization 18-24 hour, in 6 * SSC, washed 3 times 15 minutes in the 250mM sodium-chlor in 55 ℃; In 3 * SSC, washed 1 time 15 minutes.

Particularly preferably be according to method of the present invention, wherein said NIM1 gene comprise the encoding sequence shown in the SEQ ID NO:1 and under medium stringent condition with all dna moleculars of its hybridization.

Particularly preferably be according to method of the present invention, wherein said SAR genes encoding is as proteic the changing form of NIM1 of the dominant regulator of SAR signal transduction pathway.

More preferably according to method of the present invention, wherein said NIM1 is proteic, and to change form in 55 and 59 the amino acid position that is equivalent to SEQ ID NO:2 be L-Ala rather than Serine.

Particularly preferably be according to method of the present invention, proteic the changing form of wherein said NIM1 comprises the aminoacid sequence shown in the SEQ ID NO:8.

Particularly preferably be according to method of the present invention, wherein said dna molecular comprise the nucleotide sequence shown in the SEQID NO:7 and under medium stringent condition with all dna moleculars of its hybridization.

Particularly preferably be according to method of the present invention, wherein said dna molecular is at the nucleotide sequence hybridization shown in following condition and the SEQ ID NO:7: at 1%BSA, and 520mM NaPO ₄, pH7.2; 7% sodium lauryl sulphate salt; 1mM EDTA in 55 ℃ of hybridization 18-24 hour, washed in 6 * SSC 15 minutes in 55 ℃ in the 250mM sodium-chlor, and 3 times, 15 washed 1 time 15 minutes in 3 * SSC.

More preferably according to method of the present invention; Wherein said NIM1 is proteic the change form amino acid of the terminal brachymemma of N-with the 1-125 amino acid position that is about as much as SEQ ID NO:2.

Particularly preferably be according to method of the present invention, proteic the changing form of wherein said NIM1 comprises the aminoacid sequence shown in the SEQ ID NO:10.

Particularly preferably be according to method of the present invention, wherein said dna molecular comprise the nucleotide sequence shown in the SEQID NO:9 and under medium stringent condition with all dna moleculars of its hybridization.

Particularly preferably be according to method of the present invention, wherein said dna molecular under following condition with the nucleotide sequence hybridization shown in the SEQ ID NO:9; At 1%BSA; 520mMNaPO ₄, pH7.2; 7% sodium lauryl sulphate; 1mM EDTA; In 55 ℃ of hybridization 18-24 hour, in 6 * SSC, washed 3 times 15 minutes in the 250mM sodium-chlor in 55 ℃; In 3 * SSC, washed 1 time 15 minutes.

Particularly preferably be according to method of the present invention wherein said NIM1 is proteic the change form amino acid of the terminal brachymemma of C-with the 522-593 amino acid position that is about as much as SEQ ID NO:2.

Particularly preferably be according to method of the present invention, proteic the changing form of wherein said NIM1 comprises the aminoacid sequence shown in the SEQ ID NO:12.

Particularly preferably be according to method of the present invention, wherein said dna molecular comprise the nucleotide sequence shown in the SEQID NO:11 and under medium stringent condition with all dna moleculars of its hybridization.

Particularly preferably be according to method of the present invention, wherein said dna molecular under following condition with the nucleotide sequence hybridization shown in the SEQ ID NO:11: at 1%BSA; 520mMNaPO ₄, pH7.2; 7% sodium lauryl sulphate salt; 1mM EDTA; In 55 ℃ of hybridization 18-24 hour, in 6 * SSC, washed 15 minutes in 55 ℃ in the 250mM sodium-chlor, 3 times, in 3 * SSC, washed 1 time 15 minutes.

More preferably according to method of the present invention, wherein said NIM1 is proteic change form the terminal brachymemma of N-with the 1-125 amino acid position that is about as much as SEQ ID NO:2 amino acid and be about as much as the amino acid of the terminal brachymemma of C-of the 522-593 amino acids of SEQ ID NO:2.

Particularly preferably be according to method of the present invention, proteic the changing form of wherein said NIM1 comprises the aminoacid sequence shown in the SEQ ID NO:14.

Particularly preferably be according to method of the present invention, wherein said dna molecular comprise the nucleotide sequence shown in the SEQID NO:13 and under medium stringent condition with all dna moleculars of its hybridization.

Particularly preferably be method according to the present invention, wherein said dna molecular under following condition with the nucleotide sequence hybridization shown in the SEQ ID NO:13; At 1%BSA; 520mMNaPO ₄PH7.2; 7% sodium lauryl sulphate salt; 1mM EDTA; In 55 ℃ of hybridization 18-24 hour, in 6 * SSC, washed 15 minutes in 55 ℃ in the 250mM sodium-chlor, 3 times, in 3 * SSC, washed 1 time 15 minutes.

More preferably according to method of the present invention, proteic the changing form of wherein said NIM1 is made up of the ankyrin motif of the 103-362 amino acid position that is about as much as SEQ ID NO:2 basically.

Particularly preferably be according to method of the present invention, proteic the changing form of wherein said NIM1 comprises the aminoacid sequence shown in the SEQ ID NO:16.

Particularly preferably be according to method of the present invention, wherein said dna molecular comprise the nucleotide sequence shown in the SEQID NO:15 and under medium stringent condition with all dna moleculars of its hybridization.

Particularly preferably be according to method of the present invention, wherein said dna molecular under following condition with the nucleotide sequence hybridization shown in the SEQ ID NO:15: at 1%BSA; 520mMNaPO ₄, pH7.2; 7% sodium lauryl sulphate salt; 1mM EDTA; In 55 ℃ of hybridization 18-24 hour, in 6 * SSC, washed 3 times 15 minutes in the 250mM sodium-chlor in 55 ℃; In 3 * SSC, washed 1 time 15 minutes.

The example of target crop disclosed herein is including, but not limited to the following plants kind: cereal (corn, wheat, barley, rye, oat, paddy rice, Chinese sorghum and relevant crop); Beet (beet and fodder beet); The operatic circle, drupe and mushy fruit (apple, pears, Lee, peach, apricot, cherry, strawberry, rasp berry and blackberry, blueberry); Leguminous plants (Kidney bean, root of Szemao crotalaria, pea, soybean); Oilseed plant (rape, mustard seed, opium poppy platymiscium, olive, Sunflower Receptacle, coconut, castor-oil plant, cocoa beans, Semen arachidis hypogaeae); Mellon plant (pumpkin, cucumber, muskmelon); Textile plant (cotton, flax, hemp, jute); Citrus plant (orange, lemon, natsudaidai, oranges and tangerines); Vegetables (spinach, lettuce, asparagus, Caulis et Folium Brassicae capitatae, Radix Dauci Sativae, onion, tomato, potato, capsicum); Bay (avocado, cinnamon, camphor tree); Or such as tobacco, nut, coffee, sugarcane, tea, the plant of hop banana and natural rubber and ornamental plant (flowering plant, shrub, deciduous tree and evergreen tree such as softwood tree).This catalogue is not represented any restriction.

Method of the present invention can be used for giving to the resistance of phytopathogen widely, and wherein pathogenic agent includes but not limited to: viruses such as virus or viroid such as tobacco or cucumber mosaic virus, ring spot virus or necrosis virus, pelargonium leaf curl virus, red clover mottle virus, tomato bushy stunt virus; Ascomycetes fungi such as Venturia, Caulococcus, Erysiphe, chain sclerotinia sclerotiorum genus, mycosphaerella and snag shell are mould; Basidiomycetes such as hunchbacked spore Rust, Rhizoctonia and Puccinia; Deuteromycotina such as Staphlosporonites, Helminthosporium, Rhynchosporium spp, Fusarium (promptly inferior sticking group series connection sickle spore, Septoria, Cercospora, Alternaria, Pyricularia and pseudo-cercospora (being P.herpotrichoides); Oomycete fungi such as phytophthora (being phytophthora parasitica), Peronospora (being the tobacco downy mildew), Bremia, pythium and Plasmopara; And other fungi such as big spore refer to that epidemic disease is mould, Sclerophthora rayissiae, standing grain life refer to obstruct mould, Peronosclerosporasorghi, Peronosclerospora philippinensis, Peronosclerosporasacchari and Peronosclerospora maydis, Physopella zeae, Cercospora zeae-maydis, standing grain are given birth to thorn dish spore, red mould, the Exserohilumturcicum of corn chinese sorghum, corn chinese sorghum ball stalk spore and Bipolaris maydis; Bacterium such as pseudomonas syringae, tobacco pseudomonas and the general bacterium of Si Shi; Insect such as aphid are as black peach aphid; Belong to kind with lepidopteran such as Heliothus; And nematode such as Meloidogyne incognita.

Obtain the immunomodulatory plant

Below three kinds of common approaches that obtain the immunomodulatory plants be correlated with because they all are fit to the SAR signal transduction pathway model described in the Ryals etc. (1996).In case the activation of SAR signal transduction pathway just obtains disease resistance, no matter take any of three kinds of following approach, thereby series of identical SAR gene is opened the generation disease resistance.The difference of these three kinds of approach only is which site unlatching of SAR approach; The net result of three kinds of approach is consistent.So, can extrapolate and be applicable to the immunomodulatory plant that obtains with different approaches to immunomodulatory plant analysis and observations by what a kind of approach obtained.

The application of A systemic acquired resistance inductor

Article one approach that obtains the immunomodulatory plant relates to the chemical that can induce SAR to the plant use.Strong especially SAR chemical inducer is diazosulfide such as benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters (BTH).The diazosulfide derivative that can be used as conditioning agent is described in United States Patent (USP) 5,523, and 311 and 5,614,395, both are incorporated herein by reference.Can in the various plants of field, provide BTH inductive SAR at the provide protection of wide spectrum disease at Freidrich etc., The plant magazine10 (1), 61-70 (1996); Lawton etc., Plant is assorted Will10 (1), 71-82 (1996) and Gorlach etc., Vegetable cell8,629-643 has detailed description in (1996), and these two pieces of articles are incorporated herein by reference.Other the SAR chemical inducer that can be used for obtaining the used immunomodulatory plant of the inventive method comprises iso-nicotinic acid compound as 2,6-dichloro-isonicotinic acid (INA) and lower alkyl esters thereof and salicylic acid compound (SA).The example of suitable INA and SA compound is described in United States Patent (USP) 5,614,395.

B cultivates composing type immunity (CIM) mutant plant

Second approach that obtains the immunomodulatory plant is constitutive expression and/or the disease resistance phenotypic screen scheme according to the SAR gene.Mass data shows the property of being closely related (Ward etc. (1991) between SAR genetic expression and the systemic acquired resistance itself; Uknes etc. (1992); Uknes etc. (1993); Lawton etc. (1993); With (1993) such as Alexander, NAS Institute's newspaper90,7327-7331 is incorporated herein by reference).In Arabidopis thaliana, the SAR gene of determining is PR-1, PR-2 and PR-5, and the highest background of PR-1 expression level is minimum.

In order to identify and screen the plant of constitutive expression SAR gene, carry out Northern and analyze to detect the SAR expression of gene.Carry out cross experiment with the method that (1992) such as Uknes are described with known SARDNA sequence.The method of hybridization and cloning nucleic acid sequences is well known.(see, for example, molecular cloning, laboratory manual, second edition, 1-3 volume, Sambrook etc. (editor), press of cold spring harbor laboratory (1989) and the reference of quoting thereof).Be separated to the SAR signal transduction mutant of at least two group constitutive expression SAR genes.One group is called as " lsd " mutant (the lsd=damage stimulates sick), is also referred to as " cim I group " mutant.See WO94/16077.Lsd (cim I group) mutant is spontaneous formation damage on leaf, accumulation high density SA, and the mRNA of high-caliber PR-1, PR-2 and PR-5 has resistance (Dietrich etc., 1994 to fungi and bacterial pathogens; Weymann etc., 1995).Second group is called as " cim " (immunity of cim=composing type) mutant, is also referred to as " cim II group " mutant.See WO94/16077.The cim mutant has all features of the lsd mutant except that spontaneous damage, and promptly the visible phenotypic of cim mutant is normal.

In case screen the plant of constitutive expression SAR gene, they can be used for breeding system to mix in the plant strain system with the constitutive expression of SAR gene with to the resistance of pathogenic agent.The method of knowing according to the field of plant breeding technician is screened the offspring who is used for further mating according to SAR expression of gene and disease resistance and other for the feature of producing and quality is important.For example, because the lsd mutant shows that damage forms and be downright bad, although the cim mutant with normal phenotype if desired, also can be selected the lsd mutant for use for being used for such breeding system and method for the present invention is preferential.

C SAR gene-transformed plant

Article three, the approach that obtains the immunomodulatory plant is by use the SAR gene, preferably the functional form of NIM1 gene conversion plant.

1. wild-type NIM1 gene is recombinant expressed

The reorganization overexpression of wild-type NIM1 (SEQ ID NO:1) has produced the transgenic plant with disease resistance phenotype.See that common unsettled u.s. patent application serial number 08/880,179 is incorporated herein by reference.The disease resistance effect that active NIM1 protein level rising produces is with identical with the SA chemical induction with pharmaceutical chemicals such as BTH, INA.Preferably, NIM1 expression of gene level is more than the twice of NIM1 gene expression dose in the wild-type plant at least, more preferably is that the wild-type expression level is more than ten times at least.The part that is called " recombinant DNA technology " is below described the method be used in the transgenic plant with the level recombinant expressed wild-type NIM1 gene higher than wild-type plant.Alternatively, the method described of available Cao etc. (1997) with wild-type NPR1 gene-transformed plant to produce the disease resistance plant.

2.NIM1 gene change form recombinant expressed

The immunomodulatory plant that is used for the inventive method by the recombinant expressed acquisition of changing form of NIM1 gene, the change of NIM1 gene has utilized two kinds of understanding thus, and promptly parallel the and NIM1 gene product of the function of the NF-к B/I к B regulation mechanism in the performance of SAR approach and Mammals and the fly is the structure homologue of mammalian signal transduced element I к B α subclass in the plant.See common unsettled PCT application " with the method for NIM1 gene conferring disease resistance in plants "; Be incorporated herein by reference.

(SEQ ID NO:1) is used for blast search with the NIM1 gene order, homology according to suitable conservative region in the NIM1 gene and ankyrin zone, in multiple protein such as spectrin, ankyrin, NF-к B and I к B, coupling (Michaely and Bennett have been identified Trends Cell Biol.2,127-129 (1992)).Between the known albumen that contains ankyrin of NIM1 albumen (SEQ ID NO:2) and 70, carry out visual inspection in twos, find surprising similarity (Baeuerle and Baltimore 1996 with the sub-I к of transcriptional control B α class members; Baldwin, 1996).As shown in Figure 1, NIM1 albumen (SEQ ID NO:2) has tangible homology with the I к B α albumen of mouse, rat and pig (being respectively SEQ ID NO:3,4 and 5).In whole albumen, NIM1 comprises several important I к B αJie Gouyus, comprise ankyrin district (in Fig. 1, marking), two aminoterminal Serines (55 and 59 amino acids of NIM1), a pair of Methionin (99 among NIM1 and 100 amino acids) and acid (end by discontinuous underscore.Generally, it is identical that NIM1 and I к B α have 30% residue, and 50% residue is that conservative property is replaced.Therefore, I к B α and NIM1 have homology in whole albumen, and total similarity of 80% is arranged.

I к B α albumen is brought into play function in signal transduction a kind of method is by combining with kytoplasm transcription factor NF-к B, stop it to enter nucleus, thereby changes (Baeuerle and Baltimore, 1996 of transcribing of target gene; Baldwin, 1996).The target gene of NF-к B is regulated (activate or prevent) several cell processes, comprises antiviral, antimicrobial and necrocytosis reaction (Baeuerle and Baltimore, 1996).When signal transduction pathway activates, two serine residue phosphorylations of I к B (the 32nd and 36 amino acid of mouse I к B α).This starts the ubiquitination at two Methionin places (21 and 22 amino acids of mouse I к B α).Behind the ubiquitination, NF-к B/I к B mixture is carried by proteoplast, and I к B α is degraded on proteoplast, and NF-к B is released in the nucleus.

The successive conserved sequence from 35 to 84 amino acids (Fig. 1) is conservative for the serine residue of the phosphorylation that I к B α function is played an important role.Form contrast with I к B α, two Methionins are positioned at about 15 the amino acid places from this protein N terminal; In NIM1, two Methionins are positioned at from 40 amino acid places of C-terminal.And there is high homology in the carboxyl terminal zone abundant at NIM1 and I к B α serine/threonine, this zone for basal metabolic rate(BMR) be important (Sun etc., Molecular cytobiology16,1058-1065 (1996)).According to the present invention, based on structural homology analysis and known existence to the important element of I к B α function, NIM1 is as I к B α effect in expection, and promptly regulation and control have similar effect to plant gene.

When containing the plant of wild-type NIM1 gene, estimate that plant has the phosphorylation of more NIM1 gene product (I к B homologue) or NIM1 gene product still less (I к B homologue) with the chemical inducer processing.According to this model, the result is that plant NF-к B homologue is retained in outside the nucleus and generation SAR genetic expression and resistance reaction.In nim1 mutant plant, the NIM1 gene product of non-functional has appearred.So according to this model, NF-к B homologue freely enters nucleus, suppress resistance and SAR genetic expression.

Identical of views therewith, improve with I к B stability in the acid-treated zooblast of bigcatkin willow/abundance, activated NF-к B reduces (Kopp and Ghosh, 1994) in the nucleus.The mutation effect of known I к B for super suppress son or dominant (Britta-Mareen Traenckner etc., EMBO14:2876-2883 (1995); Brown etc., Science267:1485-1488 (1996); Brockman etc., Molecule and cytobiology15:2809-2818 (1995); Wang etc., Science274; 784-787 (1996)).The mutant form of these I к B combines with NF-к B but also ubiquitination not of phosphorylation not, so be not degraded.NF-к B still combines with I к B and does not enter in the nucleus.

With regard to above-mentioned, can produce the changing form of NIM1 of the dominant regulator that act as the SAR signal transduction pathway.The plant that has transformed the dominant form of NIM1 has opposite phenotype with nim1 mutant plant, shows composing type SAR genetic expression so has the CIM phenotype because transformed the plant that changes form of NIM1, and promptly transgenic plant are immunoregulatory.Because NIM1 is residing position in the SAR signal transduction pathway, can predict except this is concrete disclosed, causing the constitutive expression of SAR gene, so produce the CIM phenotype for many changes of this gene.Following title is used in changing form of expression ratio wild-type level is high in the transgenic plant NIM1 gene for the described method of " recombinant DNA technology " part.Changing form of several NIM1 genes of can be used as SAR signal transduction pathway dominant regulator is described below.

A becomes alanine residue with 55 and 59 serine residues.

Among the people I к B α serine phosphorylation for thorn activating signal activation I к B α degraded to activate NF-к B thus be essential.Serine residue (S among the people I к B α ₃₂And S ₃₆) sport alanine residue and suppressed stimulation inductive phosphorylation, therefore sealed degraded (Traenckner etc., 1995 of I к B α proteoplast mediation; Brown etc., 1996; Brockman etc., 1995; Wang etc., 1996).The I к B α that changes form seals downstream events as dominant form functionating thus by NF-к B is remained in the tenuigenin.According to aminoacid sequence between NIM1 shown in Figure 1 and the I к B relatively, the 55th (S among the NIM1 (SEQ ID NO:2) ₅₅) and 59 (59) Serines and people I к B α in S32 and S ₃₆Homology.In order to make up is the NIM1 of property inactivation form, and 55 and 59 Serine is mutated into alanine residue.Therefore, in a preferred embodiment, the NIM1 gene is changed and makes that its coded product corresponding 55 and 39 amino acids in Arabidopis thaliana NIM1 aminoacid sequence (SEQ IDNO:2) are L-Ala rather than Serine.

The terminal deletion of b N-

People I к B α 1-36 amino acids (Brockman etc., 1995; Sun etc., 1996) or the deletion of 1-72 amino acids (Sun etc., 1996), comprised ubiquitination lysine residue K ₂₁And K ₂₂And phosphorylation site S ₃₂And S ₃₆, caused in people's cell culture of transfection, having occurred dominant I к B α phenotype.Terminal the 125th the amino acid whose deletion of NIM1 gene product N-will be removed as 8 lysine residues in ubiquitination site and the S discussed above of supposition ₅₅And S ₅₉Phosphorylation site.Therefore, in a preferred embodiment, the NIM1 gene is changed and makes its coded product compare with natural Arabidopis thaliana NIM1 aminoacid sequence (SEQ IDNO:2), has lacked 125 amino acid of precontract.

The terminal deletion of c C-

The deletion of people I к B α 261-317 amino acids has strengthened intrinsic stability by the composing type phosphorylation of terminal Serine of sealing C-and threonine residues.The I к B α that changes form estimates with dominant form performance function.The abundant district of Serine and Threonine is positioned at the 522-593 amino acids of NIM1 C-end.Therefore, in a preferred embodiment, the NIM1 gene is changed and makes its coded product compare with natural Arabidopis thaliana NIM1 aminoacid sequence (SEQ IDNO:2), has lacked the C-terminal portions that comprises the 522-593 amino acids approximately.

Terminal mosaic of d N-end/C-and ankyrin zone

The product that changes form of NIM1 gene can be used as C-end and the deletion of N-terminal fragment or mosaic and is prepared.In another embodiment of the invention, provide the construct that comprises NIM1 gene ankyrin zone.

3 other SAR genes recombinant expressed

By recombinant expressed multiple SAR gene, described in (1991) such as Ward, preparation is used for the immunomodulatory plant of the inventive method.For example see, United States Patent (USP) 5,614,395, it has been described throughput and has expressed one or more PR protein genes and prepare the disease resistance plant.Although it has referred to the recombinant expressed of NIM1 gene form especially, below title be used in the transgenic plant with horizontal expression other SAR gene higher, as the PR protein gene for the described method of part of " recombinant DNA technology " than wild-type.

Recombinant DNA technology

Wild-type or changing form of NIM1 gene by enhanced SAR genetic expression conferring disease resistance in plants can be incorporated in the vegetable cell with conventional recombinant DNA technology.Usually, with the cloning process of this area standard, the NIM1 dna molecular of the selected above-mentioned form of coding is inserted in the expression system, wherein dna molecular is allogenic (being not exist under the normal circumstances).This carrying agent comprises the albumen coded sequence of insertion and transcribes and translate essential element.Variety carrier known in the art can be used, for example plasmid, phage virus and other modified virus.Suitable carriers includes but not limited to, virus vector is such as λ carrier system λ gtl1, λ gtl0 and Charon4; Plasmid vector is such as pBI121, pBR322, PACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII; The system similar with other.The composition of expression system can be modified to improve and be expressed.For example, can utilize the sequence of brachymemma, nucleotide substitution or other modification.In fact expression system described here can transform any crop cell under appropriate condition.Cell transformed can be reentered whole plant so that the NIM1 gene of selected form activates SAR in transgenic plant.

The structure of A expression of plants box

Be used for gene order that transgenic plant express and will be assemblied at first that be arranged in can be at the expression cassette after the suitable promotor that plant is expressed.Can comprise the essential or optional sequence of any transgene expression in the expression cassette.Such sequence includes but not limited to transcription terminator, strengthens outside sequence such as intron, great-hearted sequence and the sequence that gene product is positioned to specific cells device and cell cell expressed.Hereinafter describe these expression cassettes and be transferred to the Plant Transformation carrying agent easily.The various compositions of typical expression cassette have hereinafter been described.

1 promotor

The selection that is used for the promotor of expression cassette will determine transgenosis between transgenic plant are prominent and the time phraseology.Selected promotor will be in particular cell types (as leaf epidermal cell, mesophyll cell, root cortex cell) or in particular organization or organ (root, leaf or flower, for example) express transgenic, and select to reflect the accumulation position that gene product is wished.Alternatively, selected promotor can drive genetic expression under multiple inductive condition.Promotor intensity promptly starts the ability difference of transcribing.According to used host cell systems, any one in available many suitable promotors.Be the example that can be used for the countless promotor of expression cassette below.

The a constitutive expression, the CaMV35S promotor:

The structure of plasmid pCGN1761 is described in disclosed patent application EP0392225 (embodiment 23), is incorporated herein by reference.PCGN1761 comprises " two " CaMV35S promotor and tm1 transcription terminator, single EcoRI site is arranged between promotor and terminator and have pUC type skeleton.Make up the derivative of pCGN1761, it has modified polylinker, except already present EcoRI site, also comprises NotI and XhoI site.This derivative is named as pCGN1761ENX.PCGN1761ENX can be used for cDNA sequence or gene order (comprising microorganism ORF sequence) are cloned in its polylinker, so that they are expressed under the control of 35S promoter in transgenic plant.Whole 35S promoter-gene order in the construct-tm1 terminator expression cassette can be by the Hind III near promotor 5 ' end like this, Sphl, Sal I and Xba I site and near the Xba I of 3 of terminator ' end, BamH I and the cutting of Bgl II site are transferred to such as conversion carrier described below being used for.And with Hind III, Sph I, SalI, Xba I or PstI carry out 5 ' cutting, carry out with any restriction site (EcoRI, Not I or Xho I) of polylinker that 3 ' cutting is replaceable to be another promotor.If desired, carry out cloning site modification on every side by introducing the sequence that strengthens translation.If carry out overexpression, this point is particularly useful.For example, according to United States Patent (USP) 5,639,949 embodiment 37 are described to be modified pCGN1761ENX by optimizing translation initiation site, and this patent is incorporated herein by reference.

B expresses under the adjustable promotor of chemistry/pathogenic agent

Two 35S promoters among the pCGN1761ENX can be replaced by any other alternative promotor of suitable high expression level that causes.For example, United States Patent (USP) 5,614, the chemically inducible promoter of describing in 395 can replace this 35S promoter.Alternative promotor preferably being limited property enzyme is downcut from its source, and a kind of alternative method is to obtain with the primer PCR amplification with suitable end limit site.If the employing pcr amplification behind the promotor insertion targeting vector with amplification, should check order to detect the amplification mistake again to promotor.The tobacco PR-la promotor of chemistry/pathogen-inducible is downcut from plasmid pCIB1004 (building process is seen to be incorporated herein by reference the embodiment 21 of EP0332104) is transferred to plasmid pCGN1761ENX (Uknes etc., 1992).With Nco I cutting pCIB1004,3 ' overhang of the linear fragment of generation is handled with the T4 archaeal dna polymerase and is made it become flush end.Cut this fragment with Hind III then, the fragment that contains the PR-la promotor that gel-purified obtains, and it is cloned among the pCGN1761ENX that removes 35S promoter.This is by with Xho I cutting, and the T4 polysaccharase is mended flat, then with the HindIII cutting, separates that the fragment of the carrier that contains big insertion pCIB1004 promotor finishes.This has produced a pCGN1761ENX derivative, and it has the PR-la promotor, tm1 terminator and the insertion polylinker with single EcoR I and Not I site.Selected encoding sequence can be inserted in this carrier, and fusion product (being promotor-gene-terminator) can be transferred to any selected conversion carrier, comprises hereinafter described carrier.The number of chemical conditioning agent can be used for comprising United States Patent (USP) 5,523 to induce the expression of selected encoding sequence in conversion of the present invention plant, disclosed diazosulfide, Yi Yansuan and salicylic acid compound in 311 and 5,614,395.

The c constitutive expression, actin promoter:

Known several Actin muscle isomer is expressed in most of cell types, so actin promoter is a good constitutive promoter.Particularly, the promotor of paddy rice Actl gene has obtained the clone, has identified (McElroy etc., vegetable cell 2: 163-171 (1990)).1.3kb promoter fragment be included in and express all required regulatory elements in the rice protoplast.And multiple is that the expression vector of fundamental construction is specifically designed to monocotyledons (McElroy etc., Mol Gen Genet. with the ActI promotor 231: 150-160 (1991)).They are the ActI-intron, AdhI 5 ' flanking sequence and AdhI-intron (from the maize alcohol dehydrogenase gene) and combine from the sequence of CaMV35S promotor.35S and Act I intron or Act I 5 ' lateral order row are the highest with the vector expression that Act I intron merges.The optimization of sequence (sequence of gus reporter gene) also strengthens expression around the initiator codon ATG.By (Mol.Gen.Genet. such as McElory 231: 150-160 (1991)) promoter expression cassettes of Miao Shuing is easy to be used for genetic expression and be specially adapted to the monocotyledons host by modification.For example, downcut two 35S promoters that the fragment that comprises promotor is used for replacing pCGN1761ENX from the McElroy construct, it can be in order to insert the special genes sequence.The fusion gene that is built into like this can be transferred to suitable conversion carrier.In another was reported separately, paddy rice Act I promotor and its intron can instruct high expression level (Chibbar etc., Plant Cell Rep. at the barley cell of cultivating 12: 506-509 (1993)).

The d constitutive expression, the ubiquitin promotor

Ubiquitin is another known its gene product in the accumulation of broad variety cell, its promotor from several plant the clone and be used for transgenic plant (as, Sunflower Receptacle-Bine etc., plant science 79: 87-94 (1991) and corn-Christensen etc., molecular biology of plants 12: 619-632 (1989)).Corn ubiquitin promotor is developed in transgenosis monocotyledons system, and its sequence and vector construction that is used for the monocotyledons conversion is disclosed in patent disclosure EP0 342 926 (to Lubrizol); Be incorporated herein by reference.(Plant cellRep. such as Taylor 12491-495 (1993)) describes a carrier (pAHC25) that comprises corn ubiquitin promotor and first intron thereof, when it imports by microparticle bombardment, in multiple monocot plant cell suspension, very high activity is arranged.The ubiquitin promotor is suitable for genetic expression in the transgenic plant, particularly monocotyledons.Any conversion carrier that suitable carriers has the derivative of pAHC25 or describes in this application, they obtain modifying by introducing suitable ubiquitin promotor and/or intron sequences.

The different expression of e Gent

The mode of another genetic expression is that root is expressed.De Framond (FEBS 290103-106 (1991)) and described a suitable root promotor among the disclosed patent application EP 0 452 269, be incorporated herein by reference.Change this promotor over to suitable carriers such as pCGN1761ENX to insert selected gene, change whole promotor-genes-terminator box over to the purpose conversion carrier subsequently.

The promotor of f wound-induced

The promotor of wound-induced also is applicable to genetic expression.Many such promotors have been described (as Xu etc., molecular biology of plants 22573-588 (1993), Logemann etc., vegetable cell 1: 151-158 (1989), Rohrmeier ﹠amp; Lehle, molecular biology of plants 22: 783-792 (1993), Firek etc., molecular biology of plants 22: 129-142 (1993), Warner etc., the plant magazine: 3: 191-201 (1993)) and be applicable to the present invention.Logemann etc. have described 5 ' upstream sequence of dicotyledons potato Wunl gene.Xu etc. show that dicotyledons potato wound-induced promotor (pin2) has activity in monocotyledon rice.Further, Rohrmeier ﹠amp; Lehle has described the clone of the corn Wipl cDNA of wound-induced, and it can be used for separating homologous promoter with standard technique.Similar, Firek etc. and Warner etc. have described the wound-induced gene from the monocotyledons officinalis, and it can be expressed at local wound and pathogenic agent invasion and attack site.Use clone technology well known in the art, these promotors can change in the suitable carriers, and the gene fusion relevant with the present invention is used for expressing these genes in the plant wound site.

The preferential expression of g pith:

Patent application WO93/07278 is incorporated herein by reference, and has described the separation at the preferential corn trpA gene of expressing of myelocyte.Between the transcription initiation site of gene order and the promotor 1726bp is arranged.With the Protocols in Molecular Biology of standard, this promotor or its part can change in the carrier such as pCGN1761, and it can replace 35S promoter and start expression of exogenous gene in the preferential mode of marrow.In fact, comprise the preferential promotor of marrow or its a part of fragment can change any carrier over to, be used for transgenic plant after modified.

H leaf specifically expressing:

Hudspeth ﹠amp; Grula (molecular biology of plants 12579-589 (1989)) corn gene of coding phosphoric acid enol carboxylase (PEPC) has been described.Use standard molecular biological technique, the promotor of this gene is used in transgenic plant and starts any expression of gene in the special mode of leaf.

2 transcription terminators

Multiple transcription terminator can be used for expression cassette.They are responsible for Transcription Termination after the transgenosis and correct polyadenylation thereof.Suitable transcription terminator is that those knownly work in plant, comprises the CaMV35S terminator, tm1 terminator, nopaline synthase terminator and pea rbcS E9 terminator.They both can be used for monocotyledons and also can be used for dicotyledons.

3 strengthen or regulate the sequence of expression

But the multiple sequence reinforcing gene expression in the transcription unit, these sequences can improve their expression in transgenic plant with gene combined utilization of the present invention.

Multiple intron sequences can strengthen expression, particularly in monocot plant cell.For example, when importing maize cell, the intron of corn AdhI gene obviously improves the expression that is positioned at the wild type gene under its homologous promoter.Introne 1 is effective especially, can strengthen expression (Callis etc., gene development in the fusion constructs that contains chloramphenicol acetyl transferasegene 11183-1200 (1987)).In same experimental system, the intron of corn bronzel gene is having similar effect aspect the enhancing expression.Intron sequences is incorporated in the plant conversion carrier routinely, and intron typically is positioned at the untranslated leader.

Many untranslated leaders from virus can strengthen expression, and they are effective especially in the dicotyledons cell.Tobacco mosaic virus (TMV) (TMV, " W-sequence ") particularly, the leader sequence of corn chlorotic mottle poison (MCMV) and alfalfa mosaic virus (AMV) strengthen effectively expression (as, Gallie etc., nucleic acids research 158693-8711 (1987); Skuzeski etc., molecular biology of plants 1565-79 (1990)).

4 gene products are at intracellular target

The mechanism that has multiple target gene product in plant has been done comparatively careful evaluation to the sequence of controlling these mechanism performance functions.For example, gene product target chloroplast(id) is the control of the aminoterminal signal sequence of multiple protein by recommending, when be transported to chloroplast(id) this signal sequence when producing maturation protein cut (as Comal etc., journal of biological chemistry 263: 15104-15109 (1988)).These signal sequences can with heterologous gene products merge with influence the allos product to the transportation of chloroplast(id) (an den Broeck, etc., naturally 313: 358-363 (1985)).Can be from coding RUBISCO albumen, CAB albumen, EPSP synthase, the terminal DNA that separates coding appropriate signals sequence of the proteic cDNA 5 ' of GS2 albumen and other multiple known guiding chloroplast(id).Referring to United States Patent (USP) 5,639,949 embodiment, 37 titles are " the chloroplast(id) guiding is expressed " part.

Other gene product be positioned other organoid such as plastosome and peroxysome (as, Unger etc., molecular biology of plants 13: 411-418 (1989)).May command is encoded the cDNA of these products to influence the guiding of heterologous gene products to these organoids.The example of sequence is the sequence of the special aspartate aminotransferase isomer of nuclear ATP enzyme and plastosome of encoding like this.Rogers etc. have described target (institute of the NAS newspaper of cell protein body 82: 6512-6516 (1985)).

In addition, identified the sequence that causes other cell cell of gene product target.Amino terminal sequence is responsible for endoplasmic reticulum, apoplast and from target (the Koehler ﹠amp of aleurone cell to cell exocrine; Ho, vegetable cell 2: 769-783 (1990)).In addition, amino terminal sequence and carboxyl terminal sequence are responsible for vacuole location (Shinshi etc., molecular biology of plants jointly 14357-368 (1990)).

By above-mentioned suitable target sequence and purpose transgenic sequence are merged, any organoid or cell cell may lead transgene product.For the chloroplast(id) guiding, for example, RUBISCO gene, CAB gene, the chloroplast(id) signal sequence of epsp synthase gene or GS2 gene press framework and transgenosis aminoterminal ATG merges.Selected signal sequence should comprise known cleavage site, the aminoacid sequence behind the cleavage site that the structure syzygy should be considered to need to cut.In some cases, this requirement can be passed through to increase a small amount of several amino acid between cleavage site and genetically modified ATG, or the several amino acid of replacing in the transgenic sequence satisfies.With Bartlett etc., at (editor) chloroplast(id) molecular biology methods such as Edelmann, Elsevier 1081-1091 page or leaf (1982) and Wasmann etc., Mol, Gen.Genet 205: the technology that 446-453 (1986) describes, thus the fusion constructs that the detection of chloroplast(id) absorption efficient is used for the chloroplast(id) transportation measured by the external translation of detection in-vitro transcription construct and the absorption of chloroplast(id) subsequently.These constructing technologies are well known and are equally applicable to plastosome and peroxysome.

Above-mentioned cell guiding mechanism not only can be used jointly with its homologous promoter, and can use jointly with allogeneic promoter, with the specific cell-targeting of influence under the promoter transcription regulation and control, the phraseology of promotor is different with the phraseology of the promotor in target signal source.

The structure of B plant conversion carrier

The many conversion carriers that are used for Plant Transformation are known by the those of ordinary skill in Plant Transformation field, and the gene relevant with the present invention can use jointly with any one such carrier.The target species of preferred transformation technology and conversion are depended in the selection of carrier.For some target species, microbiotic or weedicide selective marker that can be preferably different.The selective marker that routine is used to transform comprises npt II gene, produces resistance (the Messing ﹠amp to kantlex and associated antibiotic; The Vierra gene 19: 259-268 (1982); Bevan etc., nature 304: 184-187 (1983)), the bar gene produces resistance (White etc., nucleic acids research to the weedicide phosphinothricin 18: 1062 (1990); Spencer etc., Theor.Appl.Genet 79: 625-631 (1990)), the hph gene creates antagonism and gives birth to resistance (the Blochinger ﹠amp of plain Totomycin; The Diggelmann molecular cytobiology 4: 2929-2931) with the dhfr gene, produce resistance (Bourouis etc., EMBO's magazine to methotrexate 2 (7): 1099-1104 (1983)), and the EPSPS gene, produce glyphosate resistance (United States Patent (USP) 4,940,935 and 5,186,642).

1 is applicable to the carrier that edaphic bacillus transforms

A lot of carriers are applicable to Agrobacterium tumefaciems and transform.They contain at least one T-DNA border sequence usually and comprise carrier such as pBIN19 (Bevan, nucleic acids research (1984) and pXYZ.Hereinafter describe two and typically be suitable for the carrier that edaphic bacillus transforms.

A pCIB200 and pCIB2001.

Binary vector pCIB200 and pCIB2001 are used to use the structure of the recombinant vectors of edaphic bacillus, make up in the following manner.With Nar I digestion pTJS75 (Schrnidhauser ﹠amp; Helinski, the bacteriology magazine 164: excision tetracycline resistance gene 446-455 (1985)), insert the Acc I fragment of carrying NPT II from pUC4K then and obtain pTJS75Kan (Messing﹠amp; Vierra, gene 19: 259-268 (1982); Bevan etc., nature 304: 184-187 (1983); McBride etc., molecular biology of plants 14: 266-276 (1990)).Xho I joint is connected with the EcoRV fragment of PCIB7, it comprises T-DNA border, the left and right sides, plant selectable nos/npt II mosaic gene and pUC polylinker (Rothstein etc., gene 53:153-161 (1987)), Xho I digestion fragment is cloned into the pTJS75 Kan of Sal digestion to produce pCIB200 (see EP0332104, embodiment 19).PCIB200 comprises following single polylinker restriction site: EcoR I, Sst I, Kpn I, Bgl II, Xba I and Sal I.PCIB2001 inserts the derivative that other restriction site produces in the pCIB200 polylinker.Single restricted point of contact in the pCIB2001 polylinker is EcoR I, Sst I, Kpn I, Bgl II, Xba I, Sal I, Mln I, Bc II, Avr II, Apa I, Hpa I and StuI.PCIB2001 is except comprising these restriction sites, also comprise the screening of plant and bacterium kantlex, left and right T-DNA border can be used for agrobacterium-mediated conversion, from the trfA function of moving between intestinal bacteria and other host of RK2 and from OriT and the OriV function of RK2.The pCIB2001 polylinker is suitable for cloning the expression of plants box that contains self conditioning signal.

The derivative of b pCIB10 and hygromycin selection thereof

Binary vector pCIB10 comprise the gene that is coded in the kalamycin resistance that screens in the plant and left and right sides T-DNA border sequence and from wide spectrum host range plasmid pRK252, integrate make it can be in intestinal bacteria and all reproducible sequence of edaphic bacillus.It makes up by (genes such as Rothstein 53: 153-161 (1987)) describe.Made up the multiple pCIB10 derivative that is integrated with the hygromycin B phosphotransferase gene, by (genes such as Gritz 25: 179-188 (1983)) describe.These derivative render transgenic vegetable cells can only on (pCIB743) or Totomycin and kantlex on the Totomycin, screen (pCIB715, pCIB717).

2 are suitable for the carrier that non-agrobacterium transforms

Make in selected conversion carrier without the conversions of Agrobacterium tumefaciems and needn't require the T-DNA sequence; So lack the carrier of these sequences also can use except the carrier that uses the above-mentioned T-DNA of containing sequence.The transformation technology that does not rely on edaphic bacillus comprises that by particle bombardment protoplastis is taken in (as PEG and electroporation) and microinjection.The selection of carrier depends primarily on the preferential selection that transforms species.The structure of the typical carriers that is suitable for the non-agrobacterium conversion has hereinafter been described.

a pCIB3064

PCIB3064 is the carrier that derives from pUC, is suitable for the combined utilization of direct gene transfer techniques and weedicide basta (or phosphinothricin) screening.Plasmid pCIB246 comprises CaMV35S promotor and the CaMV35S transcription terminator that merges with the regulation and control of intestinal bacteria gus gene, is described among the disclosed application of the PCT WO93/07278.The 35S promoter of this carrier comprise two initiation sites 5 ' the ATG sequence.Suddenly change these sites to remove ATG and to produce restriction site Ssp I and Pvu II with the standard round pcr.New restriction site distance single Sal I site 96 and 37bp, actual initiation site 101 of distance and 42bp.The pCIB246 derivative that obtains is named as pCIB3025.Gus gene with among Sal I and the Sac I digestion excision pCIB3025 reconnects end-filling to produce plasmid pCIB3060.Plasmid pJIT82 is from JohnInnes Centre, and Norwich contains Hpa I site (Thompson etc., EMBO's magazine from the cut pCIB3060 of being inserted into of bar gene 400bp fragment of streptomyces viridochromogenes 6: 2519-2523 (1987)).Produced pCIB3064 thus, it is included in the bar gene that is used for herbicide screening under CaMV 35S promoter and the terminator control, ammonia benzyl resistant gene (being used for the intestinal bacteria screening) and have single site Sph I, PstI, the polylinker of Hind III and BamH I.This carrier is applicable to that the clone comprises the expression of plants box of self conditioning signal.

B pSOG19 and pSOG35

PSOG35 is a conversion carrier of giving the methotrexate resistance with intestinal bacteria Tetrahydrofolate dehydrogenase (DFR) as selection markers.Utilize PCR from the increase GUS untranslated leader sequence of intron 6 (about 550bp) and 18bp of 35S promoter (about 800bp), corn Adhl gene of pSOG10.Also the increased 250bp fragment of coding intestinal bacteria Tetrahydrofolate dehydrogenase II type gene, these two fragments assemble up by the Sac I-Pst I fragment that comprises pUC19 carrier framework and nopaline synthase terminator from pB1221 (Clontech).These segmental assemblings produce pSOG19, and it comprises 35S promoter, GUS leader, DHFR gene and the nopaline synthase terminator that merges with intron 6 sequences.The GUS leader that replaces among the pSOG19 with corn chlorotic mottle poison (MCMV) leader sequence produces carrier pSOG35.PSOG19 and pSOG35 have the pUC gene of ammonia benzyl resistance and are used to clone the Hind III of allogenic material, SphI, Pst I and EcoR I site.

C transforms

In case the purpose encoding sequence is cloned into expression system, just it is converted in the vegetable cell.The method of conversion and plant regeneration is known in the art.For example, the Ti-plasmids carrier has been used to transport foreign DNA, and directly DNA takes in, liposome, electroporation, microinjection and microparticle bombardment.In addition, the bacterium of Agrobacterium also can be used for transformed plant cells.Hereinafter described and be used to transform dicotyledons and monocotyledonous typical technology.

The conversion of 1 dicotyledons

The dicotyledons transformation technology is well known, comprises based on the technology of edaphic bacillus and the technology that do not need edaphic bacillus.The non-agrobacterium technology relates to protoplastis or cell is directly taken in exogenous genetic material.This can be by the absorption of PEG or electroporation mediation, and the transhipment or the microinjection of particle bombardment mediation realize.Paszkowski etc. is seen in the description of the example of these technology, EMBO's magazine 3: 2717-2722 (1984), Potrykus etc., Mol.Gen.Genet. 199: 169-177 (1985); Reich etc., biotechnology 41001-1004 (1986) and Klein etc., nature 327: 70-73 (1987).Under each situation, make cell regeneration become whole strain plant with standard technique well known in the art.

Agrobacterium-mediated conversion is to transform the preferred technology of dicotyledons, because its transformation efficiency is higher and be suitable for using various plants.Edaphic bacillus transforms the binary vector (as pCIB200 or pCIB2001) that relates generally to carry the external source target DNA and is transferred to suitable edaphic bacillus bacterial strain, depend on vir gene that the strain of host's edaphic bacillus carries additional be (as to be used for CIB542 strain (Uknes etc., the vegetable cell of pCIB200 and pCIB2001 on the Ti-plasmids or on karyomit(e) 5: 159-169 (1993)).The reorganization binary vector is transferred to edaphic bacillus and realizes by the triparental mating method, and its uses the intestinal bacteria of carrying the reorganization binary vector, carries plasmid such as pRK2013 and the reorganization binary vector can be moved to the auxiliary e. coli strains of target edaphic bacillus strain.Alternatively, the reorganization binary vector can be transferred to edaphic bacillus (Hofgen ﹠amp by DNA; Willmitzer, nucleic acids research 16: 9877 (1988)).

Transform the explant that the target plant is usually directed to cultivate altogether with method well known in the art edaphic bacillus and plant with the reorganization edaphic bacillus.Regenerate on the microbiotic that being organized in of transforming contained between the double source plasmid T-DNA border or the selection substratum of Herbicid resistant mark.

Another method with gene-transformed plant relates to non-activity or has bioactive particle to be advanced on plant tissue and the cell.This technology is published in the United States Patent (USP) 4,945,050,5,036,006 and 5,100 of authorizing Sanford etc., in 792.Usually, this method relates to and is infiltrating cell outer surface effectively and be incorporated under its inner condition non-activity or have bioactive particle to be advanced on the cell.When using the non-activity particle, involvedly wish that the particle of gene is with the carrier transfered cell by wrapping.Alternatively, surround target cell, activate by particulate and bring carrier into cell with carrier.The particle of biologically active (as the yeast cell of doing, dried bacterium or phage, each comprises the DNA that needs importing) can be used for being advanced in the plant cell tissue.

2 monocotyledonous conversions

Most of monocotyledonous conversions have become routine.Preferred technology comprises with PEG or electroporation technology directly transgenosis in protoplastis; Transfer in the callus with a son bombardment.Available single DNA kind or multiple DNA (being cotransformation) transform, and these two kinds of technology all are applicable to the present invention.The advantage of cotransformation is to avoid complete vector construction and preparation goal gene and the not chain transgenic plant of selection markers, does not need selection markers in the feasible filial generation subsequently, and is optional if this point is considered to.Yet the shortcoming of cotransformation is that single DNA is incorporated into probability in the genome less than 100% (Schocher etc., biotechnology 41093-1096 (1986)).

Patent application EPO 292 435, the technology that EPO 392 225 and WO93/07278 describe has from corn original seed inbred lines and prepares callus and protoplastis, transforms protoplastis with PEG or electroporation, from the protoplast regeneration maize plant that transforms.(vegetable cell such as Gordonkamm 2603-618 (1990)) and (biotechnology such as Fromm 8: 833-839 (1990)) announced the technology that transforms the corn system in A188 source with a son bombardment.And, (biotechnologys such as WO93/07278 and Koziel 11: 194-200 (1993)) described by a technology of son bombardment maize transformation original seed inbred lines.This technology has utilized long prematurity embryo and the PDS-1000He Biolistics instrument of 1.5-2.5mm that downcuts from 14-15 days the mealie in back of pollinating to bombard.

Also can be by the direct gene transportation techniques with protoplastis or particle bombardment rice transformation.Conversion (Zhany etc., the vegetable cell breeding of the protoplastis mediation that Japonica type and Indica type are carried out have been described 7: 379-384 (1988); Shimamoto etc., natural 338:274-277 (1989); Datta etc., biotechnology 8: 736-740 (1990)).Alpha bombardment can carry out routine to two types and transform (Christou etc., biotechnology 9: 957-962 (1991)).

Patent application EP 0332581 has described preparation, the technology of conversion and regeneration Pooideae protoplastis.These technology can be used for transforming Dactylis and wheat.And, (biotechnology such as Vasil 10: 667-674 (1992)) describe regenerate for a long time wheat of callus cell of C type that alpha bombardment carries out and transformed (biotechnology such as Vasil 11: (plant physiology such as 1553-1558 (1993) and Week 102: 1077-1084 (1993)) describe alpha bombardment and carried out the wheat conversion of the callus in prematurity embryo and prematurity embryo source.Yet preferential wheat transformation technology relates to by particle bombardment conversion prematurity embryo and comprises that gene transports preceding high-sucrose or high malt sugar step.Before bombardment, the embryo of arbitrary number (0.75-1mm is long) is seeded in and contains 3% sucrose (Murashiga ﹠amp; Skoog, plant physiology 15: with the inductor cell stage, this process lucifuge is carried out 473-497 (1962)) and on the MS substratum of 3mg/l 2.4-D.In that day of selected bombardment, the embryo taken out to be placed in the permeate agent from inducing culture (be to contain sucrose or the maltose that is hopeful concentration in the inducing culture, be generally 15%.Embryo's plasmolysis was bombarded after 2-3 hour.Usually every target flat board has 24 embryos, but this is not crucial.The plasmid (as pCIB3064 or pSG35) that will carry suitable gene with standard method is deposited on the gold grain of millimeter size.80 orders screen with standard passes through DuPont Biolistics with the percussive pressure of about 1000psi ^The helium instrument bombards the embryo on each flat board.After the bombardment, the embryo is returned to about 24 hours (still in permeate agent) of recovery in the dark.After 24 hours, the embryo taken out from permeate agent be put back into one month that before regeneration, stops in the inducing culture.After about 1 month, embryo's explant with embryo's generation callus of growth is transferred to (MS+1mg/l NAA in the regeneration culture medium, 5mg/l GA), further comprise suitable selective agent (for pCIB3064 is 10mg/l basta, is 2mg/lmethotrexate for pSOG35).After about 1 month, the bud of growing is transferred in the big sterile chamber " GA7S ", it comprises half strength MS, the selective agent of 2% sucrose and same concentrations.

Recently, described and used the edaphic bacillus transforming monocots.See WO94/00977 and United States Patent (USP) 5,591,616, both are referred to herein as a reference.

Breeding

The immunomodulatory plant that obtains by the NIM1 gene transformation with SAR gene such as a kind of form can be any in the various plants, comprises monocotyledons and dicotyledons; Yet, be used for the inventive method the immunomodulatory plant optimization be selected from the hereinafter listed target crop of agriculture importance.The NIM1 expression of gene of selected form and other can be incorporated a plant lines to the feature of producing and quality is significant by breeding.Breeding method and technology are well known.As see Welsh J.R. plant genetics and breeding basis John Wiley﹠amp; Sons, NY (1981); Crop breeding, Wood D.R. (editor) American Society of AgronomyMadison, Wigconsin (1983); Mayo O. plant breeding theory, second edition, Clarendon Press Oxford (1987); Singh, D.P.; Disease and pest resistance breeding, Springer-Verlag; NY (1986); And Wricke and Weber, quantitative genetics and selection plant breeding, Walter cle Gruyter and Co., Berlin (1986).

Above-mentioned pass through the genetics feature that gene engineering method is incorporated in transgenic seed and the plant and can in progeny plants, keep and breed like this by the sexual propagation and the transmission of nourishing and growing.Usually said keep and breed used known for the method that is fit to the specific purpose development as ploughing sowing or results.Special method such as water planting or greenhouse technology also can be used.Because growing crop is subjected to by insect easily or infects attack or the damage that causes and will compete with ruderal plant, thus the weeds of will taking measures to control, plant disease, insect, nematode and other unfavourable condition are to improve output.This comprises mechanical measure such as soil tillage, weeds or infected the removal of plant, and use agrochemicals such as weedicide, mycocide, gametocide, nematocides, growth regulator, ripener and sterilant.

The superiority inheritance feature of transgenic plant of the present invention and seed can be utilized by plant breeding, its objective is that the plant that makes cultivation has the feature such as the insect of improvement, weedicide or stress resistance, the nutritive value that improves, the output of raising or the structure of improvement make because the loss that lodging or breaking-up cause reduces.Multiple breeding step all has clearly people's the participation of regulation as selecting the hybridization strain, instructs the pollination that the parent is or selects suitable progeny plants.According to the feature of hope, adopt different breeding measures.Correlation technique is known in the art including, but not limited to hybridization, inbreeding, and back cross breeding, multi-thread breeding, kind is mixed, species hybridization, aneuploid technology etc.Hybridization technique comprises that also plant is sterile, by machinery, chemistry or the biological chemistry means produce male or female sterile plants.Pollination has guaranteed that male sterile, female performance educate plant and obtain feature from both sides parent system between the pollen of one strain male sterile plants and different strains.Like this, transgenic seed of the present invention and plant can be used for improveing the breeding of plant lines, promptly for example, improve the validity of traditional method such as weedicide or sterilant or make it because the hereditary feature of tool improvement can be propagated by this method.Alternatively, can obtain having the new crop of the stress resistance of improvement, because the heredity " equipment " of its optimization, the product of generation is better than the product of the crop that can not resist identical unfavorable developmental condition.

In seed was produced, the homogeneity of germination quality and seed was basic product feature, and gathered in the crops the germination quality and the homogeneity of seed of sale by the farmer inessential.Because be difficult to make and do not have other crop and weed seed in the crop, control produces the seed with good germination quality from the disease of seed, and seed producers has developed very extensive and clear and definite seed production practice, they are in growth, and it is experienced to nurse one's health and sell purebred sub-aspect.Therefore, for the farmer, purchase meets the attested seed of extra fine quality standard and do not use the seed of gathering in the crops from own crops is very common way.Reproductive material as seed is processed with the protective material bag usually, and it comprises weedicide, sterilant, mycocide, bactericide, nematocides, invertebrate poison or its mixture.Normally used protective material bag is comprised compound such as actidione, carboxin, thiram (TMTD ^) metalaxyl (metalaxyl) (Apron) and pririmiphos_methyl (Actellic ^).If desired, these compounds can with formulation art further carrier commonly used, surfactant or promote the adjuvant of using to make preparation not to be subjected to the damage that causes by bacterium, fungi or pest with protection.The protective material bag can be carried out application by the preparation combined packet of soaking with liquid preparation or wet or do.Other application method also is possible, as direct processing bud or fruit.

The further method of the present invention provides new Agricultural methods, enumerates as mentioned, is characterized in using transgenic plant of the present invention, transgenic plant material or transgenic seed.

These seed can comprise that suitable wrapping material at bag, provide in container or the pipe, and this bag or container can be sealed to comprise seed.This bag, container, pipe can be used for for a long time, but short-term or not only can be for a long time but short-term preserve seed.The example of suitable wrapping material comprises paper, such as kraft paper, and hard or plastelast or other polymeric material, glass or metal.Preferred bag, container or pipe are made up of the wrapping material of the identical or different type of multilayer.In one embodiment, the bag that provides, container or pipe can prevent or limit water and contact seed with moisture.In one embodiment, seal bag, the mouth of container or pipe, for example the heat-sealing mouth enters to prevent water or moisture.In another embodiment, be placed on water-absorbing material between the wrapping material or near the wrapping material.In another embodiment, form bag by processing, the material of container or pipe suppresses disease with restriction, the disadvantageous effect in pollution or other storage or transportation kind of the subprocess.An example of Chu Liing is sterilization like this, as by chemical means or pass through radiation exposure.Being contained in of the present invention is a commodity bag, and it contains NIM1 gene or the proteic transgenic plant seed of a kind of NIM that comprises a kind of form; The NIM1 gene transforms expression level in plant than the height in wild plant at this, with seed, also have suitable carriers, also have its application of label explanation, can make plant produce disease resistance widely.

Microbicide is applied to the immunomodulatory plant

As described herein, the inventive method of protective plant relates to two steps: at first, activation SAR approach to be providing the immunomodulatory plant, and the second, insecticidal microorganism is applied to this immunomodulatory plant to reach collaborative enhanced disease resistance.

The microbicide that A is traditional

The method according to this invention, any commercial that buy or microbicide can be applicable to by any immunomodulatory plant that obtains of above-mentioned three kinds of approach.The example of suitable microbicide includes but not limited to following mycocide: 4-[3-(4-chloro-phenyl-)-3-(3, the 4-Dimethoxyphenyl) acryloyl] morpholine (" dimethomorph ") (reference: C.Tomlin (editor): sterilant handbook, the 10th edition, Farnhan, Britain, 1994 351-352 pages or leaves); The 5-methyl isophthalic acid, 2,4-triazole [3,4-b] [1,3] benzothiazole (" tricyclazole ") (reference: C.Tomlin (editor): sterilant handbook, the 10th edition, Farnham, Britain, 1994, the 1017-1018 pages or leaves); 3-allyloxy-1,2-[4-morpholinodithio-1,1-dioxide (" allyl group isothiazole ") (reference: C.Tomlin (editor): sterilant handbook, the 10th edition, Farnham, Britain, 1994, the 831-832 pages or leaves); α-[2-(4-chloro-phenyl-) ethyl]-α-(1, the 1-dimethyl ethyl)-1H-1,2,4-triazole-1-ethanol (" tebuconazole ") (reference: EP-A-4 345); 1-[[3-(2-chloro-phenyl-)-2-(4-fluorobenzene) oxyethane-2-yl] methyl]-1H-1,2,4-triazole (" epoxy azoles alcohol "), (reference: EP-A-196038); α-(4-chloro-phenyl-)-α-(1-ring sec.-propyl ethyl)-1H-1,2,4-triazole-1-ethanol (" cyproconazole ") (reference: the U.S. 4 664 696); 5-(4-oxygen phenyl)-2,2-dimethyl-1-(1H-1,2,4-triazol-1-yl methyl)-cyclopentanol (" metconazole ") (reference: EP-A-267 778); 2-(2,4 dichloro benzene base)-3-(1H-1,2,4-triazol-1-yl)-propyl group-1,1,2,2-tetrafluoro ethyl ether, (" fluorine ether azoles ") (reference: EP-A-234.242); Methyl-(E)-2{2-[6-(2-cyanogen phenoxy group) pyrimidine-4-base oxygen base] phenyl }-3-methoxy acrylate (" ICI A5504 ", " azoxystrobin ") (reference: EP-A-382 375); Methyl-(E)-2-methoxyimino-2-(α-(O-tolyloxy)-O-phenyl] acetic ester (" BAS 490F ", " kresoxime methyl ") (reference: EP-A-400 417); 2-(2-phenoxy phenyl)-(E)-2-methoxyimino-N-methylacetamide (reference: EP-A-398 692), [2-(2,5-dimethyl Phenoxymethyl)-phenyl]-(E)-2-methoxyimino-N-methylacetamide (reference: EP-A-398 692); (1R, 3S/1S, 3R)-2,2-two chloro-N-[(R)-1-(4-chlorine (phenyl) ethyl]-1-ethyl-3-methyl cyclopropane methane amide (" KTU3616 ") (reference: EP-A-341 475); Two (dithiocarbamic acid) manganese polymer-zinc complexes of ethylene; (" zinc manganese ethylenebisdithiocarbamate ") (reference: the U.S. 2 974 156); 1-[2-(2,4 dichloro benzene base)-4-propyl group-1,3-dioxolane-2-methylene radical] 1H-1,2, the 4-triazole; (" Wocosin 50TK ") (reference: GB-1522657); 1-{2-[2-chloro-4-(4-chlorophenoxy) phenyl]-the 4-methyl isophthalic acid, 3-dioxolane-2-ylmethyl)-H-1,2, the 4-triazole (Difenoconazole ") (reference: GB-209860); 1-[2-(2,4 dichloro benzene base) amyl group-1H-1,2,4-triazole (" Topaze ") (reference: (GB-1589852); Suitable-4-[3-(4-tert-butyl-phenyl)-2-methyl-propyl]-2, the 6-thebaine; (" fenpropimorph ") (reference: DE2752135); 1-[3-(4-tert-butyl-phenyl)-2-methyl-propyl] piperidines (" fenpropidin ") (reference: DE2752135); 4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidine ammonia (" cyprodinil ") (reference: EP-A-310550); (RS)-and N-(2,6-3,5-dimethylphenyl-N-(methoxy ethanoyl)-alanine methyl ester (" metalaxyl ") (reference: GB-1500581); (R)-and N-(2,6-3,5-dimethylphenyl-N-(methoxy ethanoyl)-alanine methyl ester (" R-metalaxyl ") (reference: GB-1500581); 1,2,5,6-tetrahydrochysene-4H-pyrrolo-[3,2,1-ij] quinoline-4-ketone (" pyroquilon ") (reference: GB-1394373); Phosphoric acid hydrogen ethyl ester (" phosethyl Al ") (reference: C.Tomlin (editor): sterilant handbook, l0 version, Farnhan, Britain, 1994, the 530-532 pages or leaves); And copper hydroxide (reference: C.Tomlin (editor): sterilant handbook, the 10th edition, Farnhan, Britain, 1994, the 229-230 pages or leaves).

Selected microbicide preferably with preparation technique in further carrier commonly used, tensio-active agent or other application of stimulus adjuvant are applied to protected immunomodulatory plant with the form of composition.Suitable carriers and adjuvant can be that solid can be a liquid, and they are materials commonly used in the preparation technique, as natural or regenerated mineral substance, solvent, dispersion agent, wetting agent, tackiness agent, intensifier, tackiness agent and fertilizer.

Using the microbicide composition preferable methods is the over-ground part that it is applied to plant, especially is applied to (foliage applying) on the leaf.Frequency of using and speed depend on biology and living condition weather of pathogenic agent.Yet, if (cultivating as paddy rice) or microbicide are soaked into solid form in the site of plant by liquid preparation, as be introduced into soil (soil application) with particle form, microbicide can infiltrate into whole strain plant by root by soil or by water (systemic effect).In order to handle seed, microbicide also can be applied to seed (bag by), can by soak with microbicide or with the preparation bag wet or that do that has merged by stem tuber or grain, microbicide is applied to seed (bag quilt).In addition, under special circumstances, other possible application process be can take, bud or fruit bunches for example handled.

Microbicide can be not modified form use or preferably, unite use with adjuvant commonly used in the preparation technique, so will make with known method,, can wrap the paste of quilt as emulsifiable concentrate, the solution that can directly spray or dilute, the emulsion of dilution, wettable powder, soluble powder, pulvis, granule or encapsulated in as the polymer material.According to the character of composition, consistent with intended purposes and overall situation, select application process such as spray, spraying, dusting, scattering, bag by or pour into.The potence ratio that microbicide is used is generally 50g-2kg activeconstituents/hectare, 100g-1000g activeconstituents/hectare preferably, 150g-700g activeconstituents/hectare particularly, handle kind of a period of the day from 11 p.m. to 1 a.m, using ratio is every 100kg seed 0.5g-1000g, preferably 0.5g-100g activeconstituents.

Prepare preparation in a known way, as mixing by homology and/or with extension agent such as solvent, solid carrier and if use, surface active cpd (tensio-active agent) grinding microbicide.

Suitable solvent is: aromatic hydrocarbons, preferably include the part of 8-12 carbon atom, as dimethylbenzene cyanogen mixture or alternate naphthalene, phthalamic acid, as dioctyl phthalate (DOP), the aliphatic carbons hydrate is as hexanaphthene or paraffin, pure and mild glycerine and ether thereof and ester, as ethanol, ethylene glycol, glycol monomethyl methyl or single ethyl glycol ether, ketone, as pimelinketone, intensive polar solvent is as the N-N-methyl-2-2-pyrrolidone N-, dimethyl sulfoxide (DMSO) or dimethyl formamide and vegetables oil or epoxidized vegetable oil are as epoxidation Oleum Cocois or soybean oil; Or water.

Solid carrier, as be used for pulvis and dispersible powder, normally natural mineral filler, such as calcite, talcum, kaolin, montmorillonite or attapulgite.In order to improve physical property, might add highly dispersed silicic acid or highly dispersed absorption agent polymer.Suitable particle adsorptive support is a multi-hole type, for example, pumice, brickbat, sepiolite or bentonite and suitable non-adsorptive type carrier are, as calcite or sand.In addition, can use multiple pre-granular inorganic and organic substance, for example particularly rhombspar or ground plant residue.

According to the character of microbicide, suitable surfactivity mixture is to have good emulsification, nonionic, positively charged ion and/or the anion surfactant of dispersion and wetting property.Term " tensio-active agent " can be understood that to comprise surfactant mixtures.

Using of having superiority especially promotes that adjuvant is natural or the phosphatide of synthetic kephalin and lecithin series, as phosphatidylethanolamine, and phosphatidylserine, phosphatidyl glycerol and lysolecithin.

The agrochemicals composite medicine generally includes 0.1-99%, the active microbicidel composition of 0.1-95% preferably, and 99.9-1% is 99.9-5% solid or liquid adjuvant and 0-25% preferably, preferably the 0.1-2.5% tensio-active agent.

Although commerical prod is preferably made concentrated solution, the end user uses the dilution preparation usually.

B plant activatory microbicide

If microbicide is applied to the immunomodulatory plant (selecting breeding or genetic engineering) that obtains by second or the 3rd above-mentioned approach, it can be that the chemical inducer (plant activatory microbicide) of SAR is as the diazosulfide compound, iso-nicotinic acid compound or salicylic acid compound, they are described in United States Patent (USP) 5,523, in 311 and 5,614,395.Therefore, two kinds of immunoregulatory methods have been used simultaneously.By plant activatory microbicide being applied to immunomodulatory plant by selecting breeding approach or genetic engineering approach to obtain, produced " outer-immunomodulatory ", obtained collaborative enhanced disease resistance.

As mentioned below, the transgenosis immunomodulatory plant of overexpression NIM1 is faster and the BTH of much lower dosage reacted to the reaction of BTH than wild-type plant, and this point can be found out from PR-1 genetic expression with to the resistance of parasitic downy mildew.See the Northern trace of embodiment 35 and Fig. 3.Only use 10uM BTH and just can in NIM1 overexpression body, reach collaborative enhanced disease resistance, be not enough to bring into play any effectiveness under this concentration normal circumstances.By utilizing synergy, can avoid using tool phytotoxicity under the normal circumstances of inducing SAR or not wish the pharmaceutical chemicals of concentration.In addition, object has been the immunomodulatory plant if SAR induces the pharmaceutical chemicals utilization, and as NIM1 overexpression body, people can utilize the change of SAR activatory time course.And, reach the given needed SAR of protective plant level and induce the minimizing of amount of chemicals used that certain economic benefits is also arranged.

Conventional microbicide of C and plant activatory microbicide combined utilization

In order to obtain bigger disease resistance, the microbicide and the plant activatory microbicide of routine can be applied to simultaneously by selecting the immunomodulatory plant of breeding approach or genetic engineering approach acquisition.With separately by immunomodulatory, microbicide by immunomodulatory and one type, or compare by the disease resistance level that the microbicide (conventional with plant activatory) of using two types simultaneously obtains, produce higher levels of collaborative disease resistance like this.See the table 35 among the embodiment 19.

The disease resistance assessment

Carry out the disease resistance assessment with method well known in the art.See Uknes etc. (1993) molecule phytomicroorganism interaction 6:680-685; Gorlach etc. (1996) vegetable cell 8: 629-643; Alexander etc., institute of NAS newspaper 907327-7331 (1993).For example, hereinafter having described several typical disease resistances measures

A phytophthora parasitica (phytophthora parasitica) (balck shank) resistant determination

Being determined at of the microorganism phytophthora parasitica that causes balck shank reported on the plant in six ages in week of the method growth that 90:7327-7331 (1993) describes by institutes of NAS such as Alexander carry out.Plant is got wet, make it parch, then inoculation 10ml sporocyst suspension (300 sporocysts/ml) in soil.The plant of inoculation is placed the greenhouse, keep 23-25 ℃ of day temperature, temperature 20-22 ℃ of night.The standard of measuring wilt disease is: 0=is asymptomatic; 1=is asymptomatic; 1=has the symptom of some wilting, and turgidity reduces; The 2=symptom of significantly wilting; Do not rot or dwarfing; The 3=symptom of significantly wilting is downgraded, and it is mashed not have tangible stem rot; 4=seriously wilts, some damage that visible stem rot is mashed and root system is united; 5=is with 4, but plant is gone down near dead or dead, serious root system system.All mensuration is to carry out on the plant that design is arranged at random.

B pseudomonas syringae resistant determination

With concentration is that every ml contains 10 ⁶Or 3 * 10 ⁶The aqueous solution of pseudomonas syringae tobacco disease mutation #551 strain be expelled to two of a few strain 6-7 plants in age in week than on the low blades.Each point in time measurement 6 strain plant.With the plant grading that 5 some disease seriousness infect the wildfire pseudomonas, 5=100% extremely organizes, and 0=is asymptomatic.T check (LSD) is carried out in the assessment of every day, after average disease rating value, indicated group.Behind the assessment numerical value on the same day, there are the data of same letter not have notable difference statistically.

C tobacco tail spore (Cercospora nicotianae) resistant determination

With the spore suspension of tobacco tail spore (ATCC#18366) (100,000-150, kept plant 5 days until outflow on the surface of 000 spore/ml) be sprayed onto leaf under 100% humidity.Use water mist spray plant 5-10 time then every day.Each time point is estimated 6 strain plants.Blade area percentage ratio according to the performance illness is graded to tobacco tail spore.T-check (LSD) is carried out in the assessment of every day, after average disease rating value, indicated group.On the statistics certificate, there is not notable difference in the data that same letter is arranged behind the assessment numerical value on the same day.

The parasitic downy mildew resistant determination of D

On plant, carry out parasitic downy mildew resistant determination by (1993) such as Uknes are described.By spraying conidium suspension (about 5 * 10 ⁴Individual spore/ml) inoculate competitive parasitic downy mildew isolate to plant.The plant of inoculation grows under 17 ℃ of certain humidity conditions in the growth room with 14 hours sunshine/10 hour dark cycles.After inoculation 3-14 days, preferably detected the existence of conidiophore on the plant in 7-12 days.In addition, select a few strain plants of every processing to carry out lactophenol trypan blue dyeing carrying out micrography (Keogh etc., Trans Br.Mycol Soc.74:329-333 (1980)) at random.

The accompanying drawing summary

Fig. 1 is the sequence contrast of the I к B α of NIM1 protein sequence and mouse, rat and pig.It is identical (matrix value=1.5) that sequence top vertical line is represented the amino acid between NIM1 and the I к B α sequence; The two point (:) of sequence top is represented similar value＞0.5; The single-point () of sequence top is represented similar value＜0.5 but greater than＞0.0; Numerical value＜0.0 is represented no similarity sequence top and is not had mark (seeing embodiment).According to de Martin etc., gene 152,253-255 (1995) have been determined the position in Mammals I к B α ankyrin district.Point in the sequence has been represented the breach between NIM1 and the I к B α albumen.Broken string has indicated five ankyrins among the I к B α to repeat below sequence.Marked amino acid whose number according to NIM1 albumen, introduced breach in suitable place.Plus sige (+) is placed in outside per 10 amino acid in sequence top.

Fig. 2 is that the aminoacid sequence of NIM1 albumen zone (numeral is equivalent to amino acid position among the SEQ ID NO:2) and paddy rice EST protein product (SEQ ID NO:17-24) compares.

The result that on behalf of Northern, Fig. 3 analyze, show that water or BTH handle after, the time course that the PR-1 gene is expressed in wild-type and NIM1 overexpression strain system.Prepare and analysis RNA from treated plant by the method for describing among the embodiment." Ws " is the wild-type Arabidopis thaliana Ws ecotype." 3A ", " 5B " " 6E " and " 7C " are the independently NIM1 overexpression plant strain systems according to embodiment 21 preparations." 0 BTH " is with water treatment; " 10 BTH " handles with 10 μ M BTH; " 100 BTH " handles with 100 μ M BTH." 0 " is the 0th day control sample; " 1 ", " 3 " and " 5 " are the 1st, 3 and 5 day samples.

Sequence summary in the sequence table

SEQ ID NO:1 is the 5655bp gene order that comprises wild-type Arabidopis thaliana NIM1 gene coding region.

SEQ ID NO:2 is the proteic aminoacid sequence of wild-type Arabidopis thaliana NIM1 by the encoding sequence coding of SEQ ID NO:1.

SEQ ID NO:3 is the mouse I к B alpha amino acid sequence among Fig. 1.

SEQ ID NO:4 is a rat I к B alpha amino acid sequence among Fig. 1.

SEQ ID NO:5 is a pig I к B alpha amino acid sequence among Fig. 1.

SEQ ID NO:6 is the cDNA sequence of Arabidopis thaliana NIM1 gene.

SEQ ID NO:7 and 8 is respectively the dna encoding sequence and the amino acid sequence coded of the proteic dominant form of NIM1, and this proteic the 55th and 59 amino acids is L-Ala rather than Serine.

SEQ ID NO:9 and 10 is respectively the dna encoding sequence and the amino acid sequence coded of the proteic dominant form of NIM1 of N-terminal deletion.

SEQ ID NO:11 and 12 is respectively the dna encoding sequence and the amino acid sequence coded of the proteic dominant form of NIM1 of C-terminal deletion.

SEQ ID NO:13 and 14 is respectively the dna encoding sequence and the amino acid sequence coded of the NIM1 gene of N-terminal and the C-terminal dominant form of deleting.

SEQ ID NO:15 and 16 is respectively the dna encoding sequence and the amino acid sequence coded in NIM1 ankyrin zone.

SEQ ID NO:17 is paddy rice among Fig. 2-1 aminoacid sequence 33-155.

SEQ ID NO:18 is paddy rice among Fig. 2-1 aminoacid sequence 215-328.

SEQ ID NO:19 is paddy rice among Fig. 2-2 aminoacid sequence 33-155.

SEQ ID NO:20 is paddy rice among Fig. 2-2 aminoacid sequence 208-288.

SEQ ID NO:21 is paddy rice among Fig. 2-3 aminoacid sequence 33-155.

SEQ ID NO:22 is paddy rice among Fig. 2-3 aminoacid sequence 208-288.

SEQ ID NO:23 is paddy rice among Fig. 2-4 aminoacid sequence 33-155.

SEQ ID NO:24 is paddy rice among Fig. 2-4 aminoacid sequence 215-271.

SEQ ID NO:25-32 is an Oligonucleolide primers.

Definition

Following definition will help to understand the present invention:

Vegetable cell: structure of plant and physiological structure, form by protoplastis and cell walls.Term " vegetable cell " refers to a part of cell of plant or from the cell of plant.The example of some cell comprises it being the noble cells of a viable cell part; The noble cells of cultivating; The undifferentiated cell of cultivating; The cell of indifferent tissue such as callus or tumour; Seed, embryo, the noble cells of propagulum and pollen.

Plant tissue: a group vegetable cell that is organized into 26S Proteasome Structure and Function unit.Any tissue that comprises plant or cultivation.This term includes but not limited to whole plant, plant organ, plant seed, tissue culture or be organized into structure and/or any vegetable cell group of functional unit.The plant tissue of this term and above-mentioned any specific type or thus the plant tissue of any specific type of comprising of definition unite that to use or use separately be not the plant tissue that will get rid of any other type.

Protoplastis: the vegetable cell that does not have cell walls.

Progeny plants: the following plant from generation to generation so that sexual mode or asexual mode produce includes but not limited to progeny plant.

Transgenic plant: in genome stable integration the plant of recombinant DNA.

Recombinant DNA: with any dna molecular different sources or that be connected to form with the dna fragmentation of recombinant DNA technology preparation.

Recombinant DNA technology: external preparation recombinant DNA and the technology that recombinant DNA changes the cell that it can express or breed over to (seen, biomedicine and molecular biosciences concise dictionary, Juo edits, the Boca Raton of CRC press (1996)), for example, in a variety of forms DNA is transferred to protoplastis or cell, DNA comprises (1) annular, the naked DNA of linear or superhelix shape, (2) be contained in the DNA of nucleosome or karyomit(e) or nucleus or its plant, the DNA that (3) are compound or relevant with other molecule, (4) are contained in liposome, spheroplast, the DNA (as Agrobacterium tumefaciems) of the biology beyond the host is changeed in the DNA in cell or the protoplastis or (5).These and other multiple method that changes recombinant DNA over to cell is known in the art; Can be used to prepare transgenic cell of the present invention or transgenic plant.

Recombinant DNA technology also comprises Treco that can be used for improving peroxidase activity in the monocotyledons etc., WO94/12650 and Treco etc., the homologous recombination method of describing among the WO95/31560.Particularly, regulatory region (as promotor) can be changed in the Plant Genome to improve the expression of external source peroxidase.

Recombinant DNA technology also comprises selects the peroxidase coding sequence insertion monocotyledons of expression signal also to analyze owing to the external source control sequence in the monocotyledons shortage, and the expression of peroxidase improves in the transgenosis monocotyledons.This will cause the rising of plant endoperoxide enzyme encoding sequence copy number.

According to traditional plant breeding method rather than technological method described herein, at first recombinant DNA is inserted R ⁰The genome of plant is not defined as finishing.After initial insertion, can the breeding transgene offspring with basic traditional breeding method.

Mosaic gene: comprise the dna molecular of at least two allos part, promptly to be pre-existing in state not relevant with it from the part of the dna sequence dna that is pre-existing in, and preferably prepares these sequences by recombinant DNA technology.

Expression cassette: be included in the dna molecular that can insert encoding sequence between promotor and the terminator.

Encoding sequence: dna molecular causes polypeptide or albumen to form when transcribing and translating.

Gene: comprise that being responsible for control expresses the regulating DNA sequence promptly transcribing and translate and the discontinuous chromosomal region of encoding sequence, when transcribing and translate, this encoding sequence has produced unique polypeptide or albumen.

Acd: the mutant plant that quickens necrocytosis

AFLP: amplified fragment length polymorphism

AvrRpt2: virulent gene Rpt2, separate from pseudomonas syringae

BAC: bacterial artificial chromosome

BTH: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

CIM: composing type immunophenotype (SAR composing type activatable)

Cim: composing type immunity mutant plant

CM: centimorgan

Cprl: PRThe constitutive expression body of gene mutation body plant

Col-O: the environmental Columbia of Arabidopis thaliana

ECs: enzyme combination

Emwa: with the environmental compatible parasitic downy mildew isolate of Arabidopis thaliana Ws-O

EMS: ethylmethane sulfonate

INA:2, the 6-dichloro-isonicotinic acid

Ler: the environmental Landsberg erecta of Arabidopis thaliana

Lsd: damage stimulates the disease mutant plant

NahG: the salicylate hydroxylase pseudomonas putida that Whitfield's ointment is transformed into catechol

NahG: with the Arabidopis thaliana strain system of nahG gene transformation

Ndr: non-species specific disease resistance mutant plant

Nim: non-induction of immunity mutant plant

NIM1: wild type gene participates in SAR signal transduction cascade reaction

NIM1: the albumen of wild-type NIM1 genes encoding

The mutation allele of nim1:NIM1 makes plant to disease-susceptible humans; Also refer to have NIM1

The arabidopsis mutant plant of nim1 mutation allele

Noco: with the environmental compatible parasitic downy mildew isolate of Arabidopis thaliana Col-O

ORF: open reading frame

PCs: combination of primers

PR: pathology is correlated with

SA: Whitfield's ointment

SAR: systemic acquired resistance

SAR-on:SAR activatory immunomodulatory plant is usually than wild-type SAR genetic expression

Height also has the disease resistance phenotype

SSLP: SSLP

UDS: general disease-susceptible humans phenotype

Wela: with the environmental compatible parasitic downy mildew chorista of Arabidopis thaliana Weiningen

Ws-O: the environmental Issilewskija of Arabidopis thaliana

WT: wild-type

YAC: yeast artificial chromosome

Embodiment

Further describe the present invention by hereinafter detailed method, prescription and embodiment.Embodiment is just in order to illustrate rather than to limit the scope of the invention.

This place accepted standard recombinant DNA and molecule clone technology are known in the art; Sambrook etc. Molecular cloning, editor, press of cold spring harbor laboratory, cold spring port, New York (1989), T.J.Silhavy, M.L.Berman and L.W.Enquist. Gene fusion is real Test, press of cold spring harbor laboratory, cold spring port; New York (1984) and Ausubel F.M. etc., Modern molecular biology techniqueGreene Publishing Assoc and Wiley-Interscience have also described these technology in (1987).

I. with systemic acquired resistance chemical inducer and the co-administered collaborative disease resistance effect that produces in plant of conventional microbicide

In this group embodiment, by the SAR of utilization SAR chemical inducer such as diazosulfide inducing plant.In addition, Chang Gui microbicide also is applied to these plants.Make the pathogenic pressure of plant acceptance then from various pathogenic agent.The method combined utilization (inducing chemical+microbicide) of two kinds of opposing pathogenic agent produces the promptly collaborative disease resistance of the effect bigger than adduction.This decides by synergy multiple (SF), the ratio of promptly observed effect (O) and desired effects (E).

The symphyogenetic desired effects of given activeconstituents (E) can be described by so-called Colby formula, calculates (Colby, S.R. " calculating symphyogenetic the working in coordination with antagonism of weedicide reacts " weeds, the 15th volume, 20-22 page or leaf (1967)) as follows:

Activeconstituents in every liter of spraying mixture of ppm=(=a.i.) milligram number

The effect percentage ratio that produces when X=is used with p ppm activeconstituents by activeconstituents I,

The effect percentage ratio that produces when Y=is used with q ppm activeconstituents by activeconstituents II,

The desired result that produces when E=is used with p+q ppm activeconstituents by activeconstituents I+II

(additivity effect).

The Colby formula is

E = X + Y - \frac{X \times Y}{100}

Embodiment 1 on barley to the effect of standing grain powdery mildew

The residue provide protection: use spraying mixture (being up to 0.02% activeconstituents) sprays the high barley plants of about 8cm and reaches the degree that blade tip drips, and the conidium with fungi after 3-4 days is sprayed plant.The plant-growth of being infected is in 22 ℃ of greenhouses.Usually infect infecting of 10 days postevaluation fungies.

Systemic effect: the high barley plants of use spraying mixture (be up to 0.002% activity, depend on the volume of soil) pouring 8cm.Careful spraying mixture makes it to contact the over-ground part of plant.Conidium with fungi after 3-4 days is sprayed plant.The plant-growth of being infected is in 22 ℃ of greenhouses.Usually infecting infecting of back 10 days assessment fungies.

The effect of the white standing grain powder bacterium of opposing on table 1 barley

Composition I: diazosulfide-7-carboxylic acid

Composition II: metconazole

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1 2 3	0.6 2 6					Composition I	Composition II	O (observed value)	E (predictor)	0 40 89
4 5 6 7	1 2 3	0.6 2 6				0.6 2 6 20		10 40 51 65			0 40 89
4 5 6 7	8 9 10 11	0.6 0.6 0.6 0.6		0.6 2 6 20	1∶1 1∶3 1∶10 1∶30	0.6 2 6 20		10 40 51 65			37 59 81 78	10 40 51 65	3.7 1.5 1.6 1.2
12 13	8 9 10 11	0.6 0.6 0.6 0.6	2 2	0.6 2 6 20	1∶1 1∶3 1∶10 1∶30	6 20	1∶3 1∶10	78 98	71 79	1.1 1.2	37 59 81 78	10 40 51 65	3.7 1.5 1.6 1.2

The effect of the white standing grain powder bacterium of opposing on table 2 barley

Composition I: diazosulfide-7-carboxylic acid

Composition II: fluorine ether azoles

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1 2	0.6 2					Composition I	Composition II	O (observed value)	E (predictor)	14 27
3 4	1 2	0.6 2				0.6 2		45 63			14 27
3 4	5 6	0.6 0.6		0.6 2	1∶1 1∶3	0.6 2		45 63			70 82	53 68	1.3 1.2
7	5 6	0.6 0.6	2	0.6 2	1∶1 1∶3	0.6	3∶1	79	60	1.3	70 82	53 68	1.3 1.2

The effect of table 3 dialogue standing grain powder bacterium on barley

Composition I: benzo 1,2,3} thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: metconazole

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1 2	0.6 2					Composition I	Composition II	O (observed value)	E (predictor)	0 33
3 4 5	1 2	0.6 2				6 20 60		17 33 50			0 33
3 4 5	6 7 8	0.6 0.6 0.6		6 20 60	1∶10 1∶30 1∶100	6 20 60		17 33 50			33 50 83	17 33 50	1.9 1.5 1.7

Embodiment 2: the effect of opposing Hu Lu section's hair disc spore (Colletotrichum lagenarium) on the cucumber

Cultivate after 10-14 days, use from the spraying mixture of wettable detection compound pulvis preparation and spray cucumber plant.After 3-4 days, with the spore suspension (1.0 * 10 of fungi ⁵Spore/ml) infect plant was cultivated 30 hours in 23 ℃ under high humidity.Under conventional humidity, continue to cultivate then in 22-23 ℃.According to fungal infection, infect after 7-10 days and assess provide protection.

Cultivate after 10-14 days,, use from the spraying mixture of wettable detection compound pulvis preparation and spray cucumber plant by the method for soil application.After 3-4 days, with the spore suspension (1.5 * 10 of fungi ⁵Spore/ml) infect plant was cultivated 30 hours in 23 ℃ under high humidity.Under conventional humidity, continue to cultivate then in 22 ℃.According to infecting of fungi, infect after 7-10 days and assess provide protection.

The effect of table 4 cucumber/foliage applying opposing Hu Lu section hair disc spore

Composition I: diazosulfide-7-carboxylic acid

Composition II:azoxystrobin

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1 2 3	0.06 0.2 2					Composition I	Composition II	O (observed value)	E (predictor)	0 5 22
4 5 6 7	1 2 3	0.06 0.2 2				0.06 0.2 0.6 6		5 9 12 17			0 5 22
4 5 6 7	8	0.06		0.06	1∶1	0.06 0.2 0.6 6		5 9 12 17			16	5	3.2
9 10 11	8	0.06	2 2 2	0.06	1∶1	0.2 0.6 6	10∶1 3∶1 1∶3	65 49 44	29 31 35	2.2 1.6 1.3	16	5	3.2

The effect of table 5 cucumber/soil application opposing Hu Lu section hair disc spore

Composition I: diazosulfide-7-carboxylic acid

Composition II:azoxystrobin

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1 2 3 4	0.006 0.02 0.06 0.2					Composition I	Composition II	O (observed value)	E (predictor)	0 40 49 91
5 6 7 8	1 2 3 4	0.006 0.02 0.06 0.2				0.2 0.6 2 6		0 9 28 66			0 40 49 91
5 6 7 8	9 10 11	0.006		0.2 0.6 2	1∶30 1∶100 1∶300	0.2 0.6 2 6		0 9 28 66			11 30 83	0 9 28	3.3 3.0
12 13	9 10 11	0.006	0.02 0.06	0.2 0.6 2	1∶30 1∶100 1∶300	6 6	1∶300 1∶100	97 100	80 82	1.2 1.2	11 30 83	0 9 28	3.3 3.0

^*The synergy multiple can not calculate

The effect of table 6 cucumber/foliage applying opposing Curcurbitaceae hair disc spore

Composition I: diazosulfide-7-carboxylic acid

Composition II:kresoxime methyl

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1 2	0.2 0.6					Composition I	Composition II	O (observed value)	E (predictor)	3 51
3 4	1 2	0.2 0.6				2 20		0 41			3 51
3 4	5 6	0.2 0.2		2 20	1∶10 1∶100	2 20		0 41			15 61	3 43	5 1.4

The effect of table 7 cucumber/foliage applying opposing Curcurbitaceae hair disc spore

Composition I: benzo 1,2,3} thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II:azoxystrobin

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1 2 3	0.06 0.2 6					Composition I	Composition II	O (observed value)	E (predictor)	16 22 60
4 5	1 2 3	0.06 0.2 6				2 6		18 75			16 22 60
4 5	6 7	0.06 0.2		2 2	1∶30 1∶10	2 6		18 75			43 57	31 36	1.4 1.6

The effect of table 8 cucumber/soil application opposing Curcurbitaceae hair disc spore

Composition I: benzo 1,2,3} thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II:azoxystrobin

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition .II	O (observed value)	E (predictor)
	1 2 3 4	0.006 0.02 0.06 0.2					Composition I	Composition .II	O (observed value)	E (predictor)	0 6 23 36
5 6 7 8 9	1 2 3 4	0.006 0.02 0.06 0.2				0.02 0.06 0.6 2 6		1 5 27 61 93			0 6 23 36
5 6 7 8 9	10 11 12	0.006 0.006 0.006		0.02 0.6 2	1∶3 1∶100 1∶300	0.02 0.06 0.6 2 6		1 5 27 61 93			26 44 84	1 27 61	26 1.6 1.4
13 14	10 11 12	0.006 0.006 0.006	0.02 0.02	0.02 0.6 2	1∶3 1∶100 1∶300	0.02 2	1∶1 1∶100	23 77	7 64	3.3 1.2	26 44 84	1 27 61	26 1.6 1.4
13 14	15 16	0.06 0.06	0.02 0.02	0.02 2	3∶1 1∶30	0.02 2	1∶1 1∶100	23 77	7 64	3.3 1.2	42 92	24 70	1.8 1.3
17	15 16	0.06 0.06	0.2	0.02 2	3∶1 1∶30	2	1∶10	93	75	1.2	42 92	24 70	1.8 1.3

Embodiment 3: the effect of opposing tobacco tail spore on the tobacco plant

Formulation soln with detection compound (concentration: maximum value is 0.02% activeconstituents) sprays tobacco plant (6 age in week).Handled back 4 days, (the sporocyst suspension inoculation plant of 150,000 spores/ml) is placed after 4-5 days under high humidity and further cultivates at the illumination/dark rhythm and pace of moving things of routine with tobacco tail spore.According to the symptom in the leaf surface assessment experiment of having infected fungi.

Table 9 on tobacco plant to the effect of tobacco tail spore

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: tebuconazole

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor
	1 2 3 4	0.2 2 6 20					Composition I	Composition II	O (observed value)	E (predictor	0 17 55 78
5 6	1 2 3 4	0.2 2 6 20				2 6		0 0			0 17 55 78
5 6	7 8	0.2 0.2		2 6	1∶10 1∶30	2 6		0 0			87 97	0 0
9 10	7 8	0.2 0.2	2 2	2 6	1∶10 1∶30	2 6	1∶1 1∶3	87 94	17 17	5.1 5.5	87 97	0 0
9 10	11 12	6 6	2 2	2 6	3∶1 1∶1	2 6	1∶1 1∶3	87 94	17 17	5.1 5.5	87 90	55 55	1.6 1.6
13 14	11 12	6 6	20 20	2 6	3∶1 1∶1	2 6	10∶1 3∶1	97 97	78 78	1.2 1.2	87 90	55 55	1.6 1.6

The effect of opposing tobacco tail spore on table 10 tobacco plant

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: cyproconazole

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1 2 3 4	0.2 2 6 20					Composition I	Composition II	O (observed value)	E (predictor)	0 17 55 78
5 6	1 2 3 4	0.2 2 6 20				2 6		0 0			0 17 55 78
5 6	7 8	0.2 0.2		2 6	1∶10 1∶30	2 6		0 0			78 84	0 0
9 10	7 8	0.2 0.2	2 2	2 6	1∶10 1∶30	2 6	1∶1 1∶3	90 94	17 17	5.3 5.5	78 84	0 0
9 10	11 12	6 6	2 2	2 6	3∶1 1∶1	2 6	1∶1 1∶3	90 94	17 17	5.3 5.5	87 93	55 55	1.6 1.7
13 14	11 12	6 6	20 20	2 6	3∶1 1∶1	2 6	10∶1 3∶1	100 100	78 78	1.3 1.3	87 93	55 55	1.6 1.7

The effect of opposing tobacco tail spore on table 11 tobacco plant

Composition I: diazosulfide-7-carboxylic acid

Composition II: fenpropimorph

Test No.	Kg activeconstituents/hectare		I∶II	The % effect		SF O/E
	Kg activeconstituents/hectare			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	0 1 2 3 4	0.2 0.6 2 6					Composition I	Composition II	O (observed value)	E (predictor)	0 (contrast) 03 69 79
5 6 7	0 1 2 3 4	0.2 0.6 2 6				2 6 10		13 23 42			0 (contrast) 03 69 79
5 6 7	8 9 10 11	0.2 0.2 0.6 6		2 6 2 6	1∶1 0 1∶30 1∶3 1∶1	2 6 10		13 23 42			52 61 71 100	13 23 16 83	4 2.7 4.4 1.2

The effect of tobacco tail spore on table 12 tobacco plant

Composition I: diazosulfide-7-carboxylic acid

Composition II: Difenoconazole

Test No.	Kg activeconstituents/hectare		I∶II	The % effect		SF O/E
	Kg activeconstituents/hectare			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	0 1 2 3	-- 2 6 20		--			Composition I	Composition II	O (observed value)	E (predictor)	0 (contrast) 69 79 100
4 5 6	0 1 2 3	-- 2 6 20		--		0.6 2 6		3 23 32			0 (contrast) 69 79 100
4 5 6	7 8	2 6		0.6 0.6	3∶1 10∶1	0.6 2 6		3 23 32			90 100	70 80	1.3 1.3

Embodiment 4: on rice plants to the effect of Pyricularia oryzae

The rice plants in two ages in week is placed in the container that spraying mixture (maximum value 0.006% activeconstituents) is housed together with the soil around its root.After 96 hours, infect rice plants with the conidium suspension of this fungi.Assess infecting of fungi after 5 days being infected plant in 24 ℃ of cultivations under the 95-100% relative humidity.

The effect of opposing Pyricularia oryzae on table 13 rice plant

Composition I: benzo [1,2,3] thiadiazoles-7-carboxylic acid-S-methyl esters

Composition II:KTU 3616

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1	6					Composition I	Composition II	O (observed value)	E (predictor)	15
2 3 4 5 6 7	1	6				0.02 0.06 0.2 0.6 2 6		0 28 47 79 83 91			15
2 3 4 5 6 7	8 9 10 11 12 13	6 6 6 6 6 6		0.02 0.06 0.2 0.6 2 6	300∶1 100∶1 30∶1 10∶1 3∶1 1∶1	0.02 0.06 0.2 0.6 2 6		0 28 47 79 83 91			42 76 98 98 100 98	15 39 55 82 86 92	2.8 1.9 1.8 1.2 1.2 1.1

At a 12m ²The soil on, use the spraying mixture spray water rice plant of making, natural infection from wettable activeconstituents powder.Infect to measure in back 44 days and assessed by the area of the leaf of fungal infection.The result is as follows:

The effect of opposing Pyricularia oryzae on the table 14 field rice plant

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: pyroquilon

Test No.	Kg activeconstituents/hectare		1	The % effect	SF O/E
	Kg activeconstituents/hectare			The % effect		Composition I	Composition II	O (observed value)	E (predictor)
	-	--		--		Composition I	Composition II	O (observed value)	E (predictor)	0 (contrast)
1 2	-	--	0.25 0.5	--			22 50			0 (contrast)
1 2	3 4		0.25 0.5	0.75 1.5			22 50			46 82
5 6	3 4		0.25 0.5	0.75 1.5	0.75 0.75	1∶3 1∶1.5	80 85	58 73	1.4 1.2	46 82

The rice plants in two ages in week is placed in the container that spraying mixture is housed together with the soil around its root.The assessment fungi is invaded and harassed after 36 days.The dissemination percentage ratio of undressed plant is 0.

The effect of opposing Pyricularia oryzae on table 15 rice plant

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: tricyclazole

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1 2 3 4	0.5 0.25 0.1 0.05					Composition I	Composition II	O (observed value)	E (predictor)	65 39 18 5
5 6 7 8	1 2 3 4	0.5 0.25 0.1 0.05				1 0.5 0.25 0.1		74 71 48 32			65 39 18 5
5 6 7 8	9 10 11 12 13	0.25 0.1 0.1 0.05 0.05		0.25 0.25 0.1 1 0.25	1∶1 1∶2.5 1∶1 1∶20 1∶5	1 0.5 0.25 0.1		74 71 48 32			75 69 61 80 58	68 57 44 75 50	1.1 1.2 1.4 1.1 1.2

Embodiment 5 belongs to the effect of kind (anthraenose) and Cercospora genus kind (leaf spot) to Colletotrichum on capsicum

Effect to crop yield: at about 10m ²The soil on (test position: Cikampek, Java Indonesia), spray spraying mixture 7 times altogether with the amount that per hectare 500-700 rises to plant, about 7 days at interval.After spraying 3 days first, manually infect plant with fungi.

The effect that table 16 opposing hair disc spore belongs to: infecting of pepper fruit assessed by analyzing the 5th sprinkling back

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: zinc manganese ethylenebisdithiocarbamate

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect	SF O/E
	Mg activeconstituents/liter (ppm)			The % effect		Composition I	comp.II	O (observed value)	E (predictor)
	1 2	5		100		Composition I	comp.II	O (observed value)	E (predictor)	55 12
3	1 2	5	5	100	100	1∶20	77	59	1.3	55 12

The effect of table 17 opposing Cercospora: infecting of leaf assessed by analyzing the 6th sprinkling back

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: zinc manganese ethylenebisdithiocarbamate

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect	SF O/E
	Mg activeconstituents/liter (ppm)			The % effect		Composition I	Composition II	O (observed value)	E (predictor)
	1 2	5		100		Composition I	Composition II	O (observed value)	E (predictor)	76 8
3	1 2	5	5	100	100	1∶20	87	78	1.1	76 8

The effect of table 18 pair crop yield: the 6th sprinkling back results capsicum.

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: zinc manganese ethylenebisdithiocarbamate

Test No.	Mg activeconstituents/liter (ppm)		I∶II	Per hectare crop yield (kg)	SF O/E
	Mg activeconstituents/liter (ppm)			Per hectare crop yield (kg)		Composition I	Composition II	O (observed value)	E (predictor)
	1 2	5		100		Composition I	Composition II	O (observed value)	E (predictor)	459 8
3	1 2	5	5	100	100	1∶20	1400	About 460	About 3	459 8

Embodiment 6: the effect of opposing Puccinia recondita in the wheat

Spray the wheat plant of 7 ages in days to dripping point with the spraying mixture for preparing from the activeconstituents of making or the combination of activeconstituents.After 4 days, infect processed plant with this fungus conidium suspension, processed subsequently plant was cultivated two days at 20 ℃ under relative humidity 90-100%.Infected the assessment fungal infection back 10 days.

The effect of opposing Puccinia recondita in table 19 wheat

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: Wocosin 50TK

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	- 1	-- 100		--			Composition I	Composition II	O (observed value)	E (predictor)	0 (contrast) 51
2	- 1	-- 100		--		5		10			0 (contrast) 51
2	3	100		5	20∶1	5		10			79	56	1.4

The effect of opposing Puccinia recondita in table 20 wheat

Composition I: diazosulfide-7-carboxylic acid

Composition II: fenpropidin

Test No.	Kg activeconstituents/hectare		I∶II	The % effect		SF O/E
	Kg activeconstituents/hectare			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	- 1 2	-- 6 20		--			Composition I	Composition II	O (observed value)	E (predictor)	0 (contrast) 20 40
3 4	- 1 2	-- 6 20		--		20 60		40 60			0 (contrast) 20 40
3 4	5 6	6 6		20 20	1∶3 1∶10	20 60		40 60			73 75	52 68	1.4 1.1

Embodiment 7: in wheat to the effect of standing grain powdery mildew

(10m in field test ²), use from the spraying mixture of wettable activeconstituents powdered preparation and spray the winter wheat that is in vegetative period.Natural infection infects back 10 days assessment fungal infections.Obtained following result:

The effect of opposing standing grain powdery mildew in table 21 wheat

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: Wocosin 50TK

Test No.	G activeconstituents/hectare		I∶II	The % effect		SF O/E
	G activeconstituents/hectare			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	- 1	-- 5		--			Composition I	Composition II	O (observed value)	E (predictor)	0 (contrast) 29
2 3	- 1	-- 5		--		50 100		2 31			0 (contrast) 29
2 3	4 5	5 5		50 100	1∶10 1∶20	50 100		2 31			49 59	32 51	1.5 1.2

The effect of opposing standing grain powdery mildew in table 22 wheat

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II:cyprodinil

Embodiment 8 in banana to the effect of Mycosphaerella fijiensis

Spray 300m with the spraying mixture of making from wettable activeconstituents pulvis with 17-19 days interval ²40 banana plants on the soil, 6 times altogether.Natural infection.Measuring the leaf of fungal infection assesses.Obtained following result:

The effect of opposing Mycosphaerella fijiensis in table 23 banana

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: Wocosin 50TK

Test No.	G activeconstituents/hectare		I∶II	The % effect		SF O/E
	G activeconstituents/hectare			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	- 1	-- 50		--			Composition I	Composition II	O (observed value)	E (predictor)	0 (contrast) 19
2	- 1	-- 50		--		50		26			0 (contrast) 19
2	3	50		50	1∶1	50		26			46	40	1.15

Embodiment 9: in tomato to the effect of target chain lattice spore

Spray 7m with spraying mixture with 7 days intervals from wettable activeconstituents powdered preparation ²Tomato plant on the soil, 9 times altogether.Natural infection.Measuring the leaf of fungal infection assesses.Obtained following result:

The effect of opposing target chain lattice spore in the big Tanaka's of table 24 the tomato

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II:cyprodinil

Test No.	G activeconstituents/hectare		I∶II	The % effect		SF O/E
	G activeconstituents/hectare			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	- 1	-- 2.5		--			Composition I	Composition II	O (observed value)	E (predictor)	0 (contrast) 32
2 3	- 1	-- 2.5		--		12.5 25		30 51			0 (contrast) 32
2 3	4 5	2.5 2.5		12.5 25	1∶5 1∶10	12.5 25		30 51			79 80	53 67	1.5 1.2

Embodiment 10 in tomato to the effect of phytophthora infestans

Drip with spraying mixture or activeconstituents combination sprinkling tomato plant " Roter Gnom " to blade tip from wettable activeconstituents powdered preparation.Spray the plant of handling with allergenic ascus suspension after 4 days, in cell, cultivated two days in 18-20 ℃ at relative air humidity 90-100% subsequently.Infect after 5 days and assess fungal infection.Obtained following result:

The effect of opposing phytophthora infestans on table 25 tomato

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: metalaxyl

Test No.	Mg is alive, and generation divides/liter		I∶II	The % effect		SF O/E
	Mg is alive, and generation divides/liter			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	- 1 2 3 4	-- 5 25 100 500		--			Composition I	Composition II	O (observed value)	E (predictor)	0 (contrast) 14 36 61 72
5 6 7 8	- 1 2 3 4	-- 5 25 100 500		--		0.1 1 10 50		13 23 35 68			0 (contrast) 14 36 61 72
5 6 7 8	9 10 11 12 13 14 15 16	5 5 5 5 25 100 100 500		0.1 1 10 50 50 10 50 10	50∶1 5∶1 1∶2 1∶10 1∶2 10∶1 2∶1 50∶1	0.1 1 10 50		13 23 35 68			50 62 87 84 92 85 95 97	25 34 44 73 80 75 88 82	2.0 1.8 2.0 1.2 1.2 1.1 1.1 1.2

The effect of opposing phytophthora infestans in table 26 tomato

Composition I: diazosulfide-7-carboxylic acid

Composition II: metalaxyl

Test No.	The mg activeconstituents/liter		I∶II	The % effect	SF O/E
	The mg activeconstituents/liter			The % effect		Composition I	Composition II	O (observed value)	E (predictor.
	-	--		--		Composition I	Composition II	O (observed value)	E (predictor.	0 (contrast)
1 2 3 4	-	--	0.1 0.5 1 5	--			0 9 22 45			0 (contrast)
1 2 3 4	5 6 7 8		0.1 0.5 1 5	1 10 50 100			0 9 22 45			13 33 63 83
9 10 11 12 13	5 6 7 8		0.1 0.5 1 1 5	1 10 50 100	1 1 1 10 1	1∶10 1∶2 1∶1 1∶10 5∶1	36 29 57 79 61	13 21 32 48 52	2.8 1.4 1.8 1.6 1.2	13 33 63 83

The effect of the false downy mildew of opposing Cuba in embodiment 11 cucumber

Drip with spraying cucumber plant (" Wisconsin ") to the blade tip in 16-19 days ages from the spraying mixture of wettable activeconstituents powdered preparation or activeconstituents combination.Use Cuba false downy mildew (365 strains, Ciba after 4 days; 5000/milliliter of maximum values) sporocyst infects the plant of processing, cultivates 1-2 days in 18-20 ℃ under relative air humidity 70-90% subsequently.Infect back 10 days assessment fungal infections and compare with infecting of the plant of being untreated.Obtain following result:

The effect of the false downy mildew of opposing Cuba in table 27 cucumber

Composition I: diazosulfide-7-carboxylic acid

Composition II: metalaxyl

Test No.	The mg activeconstituents/liter		I∶II	The % effect	SF O/E
	The mg activeconstituents/liter			The % effect		Composition I	Composition II	O (observed value)	E (predictor)
	- 1	-- 0.05		--		Composition I	Composition II	O (observed value)	E (predictor)	0 (contrast) 0
2 3	- 1	-- 0.05	0.5 5	--			6 66			0 (contrast) 0
2 3	4 5 6		0.5 5	0.5 5 50			6 66			31 66 91
7 8 9 10	4 5 6		0.05 0.05 0.5 0.5	0.5 5 50	0.5 5 0.5 5	1∶10 1∶100 1∶1 1∶10	66 83 83 83	31 66 35 68	2.1 1.3 2.4 1.2	31 66 91

The effect of opposing tobacco downy mildew on embodiment 12 tobacco plants

The spray solution tobacco plant made from detection composition (6 age in week).Handle the sporocyst suspension inoculation plant of using fungi after 4 days, under high humidity, cultivate the natural back of 4-5 and continue to continue in proper order to cultivate with normal illumination/dark rhythm and pace of moving things.According to by the symptom in the blade face evaluation test of fungal infection.The dissemination percentage ratio of plant of being untreated is 0.

The effect of opposing tobacco downy mildew on table 28 tobacco plant

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: dimethomorph

Test No.	Mg activeconstituents/liter (ppm)		I∶II	The % effect		SF O/E
	Mg activeconstituents/liter (ppm)			The % effect			Composition I	Composition II	O (observed value)	E (predictor)
	1 2 3	0.03 0.1 0.3					Composition I	Composition II	O (observed value)	E (predictor)	14 34 88
4 5	1 2 3	0.03 0.1 0.3				0.3 1		52 52			14 34 88
4 5	6 7 8	0.03 0.1 0.1		1 0.3 1	1∶33 1∶3 1∶10	0.3 1		52 52			74 92 95	59 68 68	1.3 1.4 1.4

The effect of the parasitic downy mildew of opposing in embodiment 13 Arabidopis thalianas

With the mycocide metalaxyl, phosethyl Al and copper hydroxide and SAR activator benzo (1,2,3)-thiadiazoles-7 hydroxy acid S-methyl esters (BTH) makes 25% respectively with wettable powder carrier, 80%, 70% and 25% activeconstituents, they are applied on the plant in three ages in week with the form of meticulous dirt powder.Use wettable powder separately in contrast.After three days, the method for pressing (1995) descriptions such as Delaney is with parasitic downy mildew conidium suspension inoculation plant.With compatible parasitic downy mildew isolate Emwa (1-2 * 10 ⁵The inoculation of spore/m) Ws plant; With compatible parasitic downy mildew isolate Noco2 (0.5-1 * 10 ⁵The inoculation of spore/ml) Col plant.After the inoculation, cover plant to be keeping high humidity, and it is placed in 17 ℃ the growth room with 14 hours illumination/10 hour dark cycle (Uknes etc., 1993).Results tissue after 8 weeks of inoculation.

By under dissecting microscope, conidial growth being graded, follow the tracks of 12 days (Delaney etc. (1994) of fungal infection process; Dietrich etc. (1994)).Independent blade is carried out the dyeing of lactophenol trypan blue to observe fungi growth in the leaf texture.Make primer with NS1 and NS2, parasitic downy mildew EmWa DNA carries out the rRNA fungi probe that PCR obtains as template according to the method for (1990, PCR method, methods and applications guide, 315-322 page or leaf) such as White fungal growth is carried out quantitatively.With phenol/sedimentary method of chloroform extracting post chlorization lithium from frozen tissue purifying RNA (Lagrimini etc., 1987 institutes of NAS newspapers, 84:7542-7546).According to Ausbel etc. 1987) describe, by formaldehyde agarose gel electrophoresis sample separation (7.5 μ g) and with isolating sample transfer (Hybond-N to nylon membrane ⁺, Amersham).According to Church and Gilbert (1984, institute of NAS newspaper, method 81:1991-1995) is hybridized and is washed.According to the explanation Phosphor Imager of manufacturer (Molecular Dynamics, Sunnyvale, CA) relative quantity of mensuration transcript.By with constitutive expression, b-microtubule Arabidopis thaliana cDNA surveys strip filter paper and carries out sample stdn on the sample.It is zero that the fungal growth that untreated plant is infected suppresses.Obtained following result:

The effect of the parasitic downy mildew NoCo2 of opposing in table 29 Arabidopis thaliana (Col-O)

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: metalaxyl

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			BTH	Metalaxyl	O (observed value)	E (predictor)
	Contrast	--	--	0		BTH	Metalaxyl	O (observed value)	E (predictor)
1	Contrast	--	--	0	0.01mM	--	0
1	2	--	0.1mg/l	0	0.01mM	--	0
3	2	--	0.1mg/l	0	0.01mM	0.1mg/l	40.7	0	∞

The effect of the parasitic downy mildew Emwa of opposing in table 30 Arabidopis thaliana (Ws)

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: metalaxyl

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			BTH	Metalaxyl	O (observed value)	E (predictor)
	Contrast	--	--	0		BTH	Metalaxyl	O (observed value)	E (predictor)
1 2	Contrast	--	--	0	0.01mM 0.003mM		20 0
1 2	3 4 5		2.5mg/l 0.5mg/l 0.1mg/l	75 50 50	0.01mM 0.003mM		20 0
6 7 8 9	3 4 5		2.5mg/l 0.5mg/l 0.1mg/l	75 50 50	0.01mM 0.01mM 0.01mM 0.003mM	2.5mg/l 0.5mg/l 0.1mg/l 2.5mg/l	100 95 88 100	90 70 70 75	1.1 1.4 1.3 1.3

The effect of the parasitic downy mildew Emwa of opposing in table 31 Arabidopis thaliana (Ws)

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: phosethyl Al

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			BTH	Phosethyl Al	O (observed value)	E (predictor)
	Contrast	--	--	0		BTH	Phosethyl Al	O (observed value)	E (predictor)
1	Contrast	--	--	0	0.01mM		30
1	2 3 4		1.0g/l 0.2g/l 0.04g/l	40 10 0	0.01mM		30
5 6 7	2 3 4		1.0g/l 0.2g/l 0.04g/l	40 10 0	0.01mM 0.01mM 0.01mM	1.0g/l 0.2g/l 0.04g/l	100 100 95	70 40 30	1.4 2.5 3.2

The effect of the parasitic downy mildew Emwa of opposing in table 32 Arabidopis thaliana (Ws)

Composition I: benzo [1,2,3] thiadiazoles-7-thiocarboxylic acid-S-methyl esters

Composition II: copper hydroxide

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			BTH	Cu(OH) ₂	O (observed value)	E (predictor)
	Contrast	--	--	0		BTH	Cu(OH) ₂	O (observed value)	E (predictor)
1	Contrast	--	--	0	0.01mM	--	30
1	2		0.01g/l	0	0.01mM	--	30
3	2		0.01g/l	0	0.01mM	0.01g/l	85	30	2.8

As can be seen from Table 29, shown collaborative disease resistance effect in the wild-type Arabidopis thaliana Col-O plant.Do not observe fungal growth and suppress by 0.01mM BTH or 0.0001g/L metalaxyl being applied to plant separately, because these concentration under normal circumstances are not enough to effective force.Yet, use simultaneously with two kinds of compounds of the not enough concentration of normal circumstances, observe 40.7% fungal growth inhibition, clearly be synergistic effect.Table 30-32 has represented the collaborative disease resistance effect in the wild-type Arabidopis thaliana Ws plant.0.01mM BTH is applied to the Ws plant, and the fungal growth that can only observe 20-30% suppresses.Yet simultaneously with BTH and metalaxyl, phosethyl Al or copper hydroxide are applied to plant simultaneously, can be observed collaborative disease resistance.The associating antifungal effect has reduced control pathogenic agent required mycocide and 13TH effective concentration, stops the required chemical dose of fungal growth thereby reduced, so can relax because the leaf that the chemistry tolerance has caused destroys.

II is by being applied to the collaborative disease resistance effect that composing type immunity (CIM) mutant plant obtains with conventional microbicide and/or systemic acquired resistance chemical inducer

In this group embodiment, the Northern trace Screening and Identification that has developed high throughput has the mutant plant of high density PR-1 mRNA in normal growth, think also representation system acquired resistance of these mutant plants.Obtained the mass mutation body with this screening; They not only show the accumulation of PR-1 but also show PR-2 and the accumulation of PR-5 mRNA (Lawton etc. (1993); (1995) such as Dietrich etc. (1994) and Weymann.The SA level raises in these mutant, and the opposing pathogenic agent infects, and confirms that this method can be used for separating SAR signal transduction mutant.

With this screening and separating to two groups of SAR signal transduction mutant.One group is named as lsd mutant (the lsd=damage stimulates disease).This group mutant is disclosed as " cim organizes I " in WO94/16077, this openly is incorporated herein by reference.Lsd group (aka cim I group) is spontaneous formation damage on leaf, the SA of accumulation high density, and high-caliber PR-1, PR-2 and PR-5 mRNA have resistance (Dietrich etc., 1994 to fungi and bacterial pathogens; Weymann etc., 1995).

Second group, be called as cim (immunity of cim=composing type) and describe to some extent hereinafter and have the feature of lsd mutant all except that spontaneous damage.Second group (cim) is equivalent to WO94/16077 disclosed " cim organizes II " mutant.It is dominant mutation that cim3 mutant plant described below system belongs to cim group (cim organize II), and its wild-type phenotype is the stable high-caliber SA of expression, and the SAR gene mRNA also has disease resistance widely.

Embodiment 14 has the separation and the evaluation of the cim mutant of composing type SAR genetic expression

With 1100 independent M ₂Mutagenesis (EMS) arabidopsis thaliana grows in (lehle seeds, Round Rock, TX) growth, every group about 100 in the Aracon dish.Press above-mentioned Uknes etc., the 1993 method culturing plants of describing are paid special attention to avoid excessively to water and pathogenic agent infects.In brief, be upgraded to one group of autoclaving 3 times 70 minutes with water saturation Metro Mix360 and with 10.Fully stir between each autoclaving and carry kind of a mixture.Seed is placed 20%, and (lorox carried out disinfection in 5 minutes, prior to seeding with sterilization washing 7 times.With the sowing seed vernalization 3-4 days after in the room, cultivating in week with 15 hour night of 9 hour daytime aspire to 22 ℃.When plant grow to three to around during age, gather in the crops the leaf of 1 to 2 50-100mg weight, separate total RNA (Verwoerd etc. (1989) nucleic acids research 17,2362) with fast micro-RNA preparation method.Analyze PR-1 genetic expression (Lagrimini etc. (1987), institute of NAS newspaper 84,7542-7546 by the Norther engram analysis; Ward etc., 1991).Each group plant comprises undressed and handles (0.25mg/m) Arabidopis thaliana of two days in contrast with INA.Keeping according to (1995) such as Weymann are described of all plants carried out.

Identified that 80 strains may accumulate the mutant of high-level PR-1 mRNA.Its offspring is tested, selected 5 strains and further identified.Do not having pathogenic agent or inducing under the situation of processing, the cim mutant SAR genetic expression of supposing raises.Supposition cim mutant offspring is tested the PR-1 expression that confirms composing type can heredity.In the cim mutant, cim2 and cim3 level are the highest, have identified that further the most stable PR-1 expresses.

There is not mao proterties as identifying F with recessiveness ₁The mark and the Columbia of filial generation backcross.Before scattering, pollen removes the Col-gll bud, immediately with the pollen F that uses from mutant ₁Plant grows in soil, identifies the outcross plant by the appearance of fragrant flowers.

After cim2 and cim3 and environmental Col-O or the La-er hybridization, identify the F of most SAR gene high expression ₁Plant shows that these proterties are dominance.For cim2, some rather than whole F ₁Plant constitutive expression SAR gene.If the cim2 mutant be dominance and also exist among the parent with the form of heterozygote, just can expect to obtain such result.The further genetic test of cim2 shows F ₂Have continuous variable to separate in generation, this is consistent with Incomplete dominance.

Cim3 is at F ₁Show separation in 1: 1 in generation, two F that express high-level PR-1mRNA ₁The plant selfing produces F ₂Colony.By PR-1 mRNA is accumulated divided rank, F ₂Be separated into 93 strain F ₂Plant high expression level PR-1 mRNA, 52 strain F ₂Plant does not have tangible PR-1 mRNA accumulation, and ratio is 3: 7: 1 (C ²=1.77; 0.5＞P＞0.1), this is that the hypothesis of dominance single gene mutation is consistent with cim3.Outcross subsequently confirms that cim3 is as dominant mutation heredity.

For cim3, the initial M2 plant and the M3 colony that identify in screening look normal.Yet because the cim plant is selfing, it is lower that some expresses best strain fertility.Obtain having normal performance and fertility and PR-1 after backcrossing with Col-gll and expressed strong plant

During initial the evaluation, some is short a little for cim3; And have thin and leaf distortion.Yet, with the F of wild-type Col-gl hybridization generation ₂Plant has kept the SAR gene high expression, and can not distinguish with wild-type plant.This shows that the phenotype of short sex change is by causing with the irrelevant independent mutation of composing type SAR genetic expression.When plant grows under aseptic condition, also can be observed the cim3 mutant phenotype, confirmed that PR-1 mRNA accumulation is not to be caused by pathogenic agent.

Embodiment 15:SARA genetic expression

Except that PR-1, cim3 is the other two kinds of SAR genes of high expression level also, PR-2 and PR-5.SAR gene expression dose difference among the offspring, but always high 10 times than not processed contrast, similar to the level that obtains through the INA of induction of resistance (0.25mg/ml) processing wild-type plant.

Embodiment 16: the Whitfield's ointment analysis

After the necrosis of pathogen-inducible Arabidopis thaliana, the rising of endogenous SA concentration (Uknes etc., 1993, above-mentioned).According to Uknes etc.; 1993 is described, analyzed Whitfield's ointment and glucose conjugate thereof.Gather in the crops leaf texture the plant 4 ages in week from 10 strain cim3 and 10 strains contrast.The leaf of results individual plants is also analyzed its PR-1 genetic expression.Measure the SA level of expressing the PR-1 plant.High 3.4 times (being respectively 233 ± 35VS, 69 ± 8ng/g fresh weight) of the wild-type Arabidopis thaliana that the concentration ratio of free SA is infected among the cim3.SA glucose conjugate (SAG) is than the wild-type Arabidopis thaliana that is infected high 13.1 times (being respectively 4519 ± 473VS, 344 ± 58ng/g fresh weight) among the cim3.The tissue that the level that SA and SAG raise and the pathogenic agent of report infect or the level of epr mutant can be compared.

Embodiment 17: disease resistance

Assessed the resistance of cim3 to the parasitic downy mildew of Arabidopis thaliana oidium virulence factor (NoCo2).According to Uknes etc. 1991, above-mentioned, described parasitic downy mildew is inoculated cim (expressed confirm by PR-1RNA) 30 strains in about 4 ages in week, control plant (wild-type Columbia) 30 strains.After 7 days, according to (1980) Trans Br Mycol.Soc 74 such as keogh, 329-333 and koch and Slusarenko (1990) Vegetable cellThe described analysis plant spore of 2 437-445 forms and observes the fungi structure with expecting that platform indigo plant dyes.Wild-type (Col-O) plant is supported mycelia, conidium and oospore growth, and can not support fungal growth with wild-type plant and cim plant that INA (0.25mg/ml) handles.The resistance of typical cim3 mediation can infect the position in pathogenic agent and see a small set of dead cell.Resistance in this resistance and lsd mutant (Dietrich etc., 1994, above-mentioned, Weymann etc., 1995, above-mentioned) or the derivative wild-type plant of SAR (Uknes etc., 1992, above-mentioned) is similar.Usually, resistant phenotype in the middle of can be observed comprises the necrosis that spreads of trailing the mycelia point in the cim3 plant.Spread find in the downright bad wild-type plant with the SA of low dosage or INA processing similar (Uknes etc., 1992 is above-mentioned, Uknes etc. are 1993, above-mentioned).Yet, never see oospore growth on the cim3 plant and all control plants all show the oospore growth.Expect that the blue dyeing of platform do not see on the nonvaccinated cim3 leaf that spontaneous damage is arranged.

Except the parasitic downy mildew of fungal pathogens is had the resistance, cim3 also has resistance to infecting of bacterial pathogens pseudomonas syringae DC3000.With pseudomonas syringae DC3000 the suspension inoculation wild-type (± INA processing) and the cim3 plant in six ages in week, the progress of disease is followed the tracks of in the growth of the bacterium of extracting from infect blade by monitoring in time course.At the 0th day and the 2nd day Col-O, the difference between Col-O+INA and the cim3 bacterial titer was not obvious statistically.Yet in the time of the 4th day, the bacterial growth of wild-type plant and cim3 plant has 31 times reduction (P＜0.003, Sokal and Rohlf, 1981).The visual inspection plant is to detect illness.The leaf of wild-type plant seriously moves back green, and illness is well beyond initial inoculation zone.On the contrary, almost not have illness in pretreated wild-type plant of INA or the cim3 plant.

In this embodiment, (agar plate or liquid) is gone up and is cultivated pseudomonas syringae tomato strain DC3000 (Walen etc. (1991) on 28 ℃ of King B substratum that containing Rifampin (50 μ g/ml) Vegetable cell3,45-59).Dilution overnight culture and with 2-5 * 10 ⁵Cell/ml is suspended in 10mM MgCl again ₂In and be expelled in the Arabidopis thaliana leaf.Prick a hole with No. 28 syringe needles in the middle and upper part of leaf, the bacterial solution that will about 250ul dilutes with the 1cc syringe is expelled in the hole then.At different time points, get 3 blade perforated sheet at random with No. 1 cork punch tool from 10 strain plants of each processing, always have 10 chance samples.3 perforated sheet are placed on contain 300ul 10mM MgCl ₂Eppendorf pipe in and with grinding the bar grinding.The bacterial suspension that obtains of dilution and it is seeded on the King B substratum that contains Rifampin (50 μ g/ml) was cultivated 4 days in 28 ℃ suitably.The counting bacterial clone carries out Student data analysis (Sokal and Rohlf (1981), Biometry, second edition, New York: W.H.Freemanand Company) to data.

In this embodiment, with 2,6-dichloro-isonicotinic acid (INA) is suspended in and forms 25% activeconstituents (0.25mg/ml, the 325 μ M that make in the wettable pulvis in the aseptic deionized water; Kessmann etc. (1994) plant pathology academic year comments 32,439-59).All plants with water or I spray solution to being about to effusive degree.

The effect of embodiment 18:SA in SAR genetic expression and disease resistance

In order to study SA among the cim3, the relation between SAR genetic expression and the resistance is hybridized (Delaney etc., 1994) to the arabidopsis thaliana of expressing salicylate hydroxylase (nahG) gene.By utilizing agrobacterium-mediated conversion that nahG gene transformation to the Arabidopis thaliana that 35S drives has been prepared " NahG plant ".See Huang H, Ma, H. (1992) Plant Mol.Biol. Rep.10,372-383 is incorporated herein by reference; Gaffney etc. (1993) Science261,754-756 is incorporated herein by reference; And Delaney etc. (1994) Science266,1247-1250 is incorporated herein by reference.The Col-nahG Arabidopis thaliana also carries the dominance kalamycin resistance gene except tool dominance nahG gene, so Col-nahG is as the pollen donor.With F ₁Seed aquation 30 minutes in water, (lorox 0.05% polysorbas20 carried out surface sterilization 5 minutes and used the sterilized water cleaning down with 10% then.Seed is inoculated in the cultivation that contains the 25mg/ml kantlex (GM, Murashige and Skoog substratum contain 10g/L sucrose, 0.5g/L 2-(morpholine) ethyl sulfonic acid, regulating pH with KOH is 5.7) with screening F ₁Plant.See institute of (1988) NAS newspapers 85 such as Valvekens, 5536-5540.The F that will have kalamycin resistance after 18 days ₁Plant is transferred in the soil.Confirm in all experiments, the existence of nahG gene and the expression of PR-1 are arranged all by the Northern engram analysis.

Because cim mutant and nahG phenotype all are dominance, can be at F ₁Analyze the epistasis between these two genes in the plant.70 strain F of cim3 * nahG hybridization have been analyzed ₁PR-1 in the plant and nahG genetic expression.In the Norther engram analysis that mRNA expresses, the existence of nahG gene is relevant with the inhibition of SAR genetic expression.The F that obtains by analysis ₂PR-1 mRNA in the chorista confirms each strain F ₁The existence of cim is all arranged.

In order to determine that whether for disease resistance cim3 sudden change be upper for nahG, 5 strain F of cim3 * nahG hybridization ₁20-30 grain F has been planted in the plant selfing ₂Seed, wherein 5 strain F ₁Plant has been proved nahG and has lacked PR-1mRNA.Analyzed F ₂NahG in the colony in the individual plants and the expression of PR-1mRNA are then with parasitic downy mildew (NoCo ₂) attack these plants to assess its disease susceptibility.The disease resistance of being given by cim3 has been eliminated in the existence of nahG gene, shows that nahG is upper for cim3 in SAR genetic expression and disease resistance phenotype.

Embodiment 19: microbicide and/or BTH are applied to the collaborative disease resistance that the cim mutant obtains

Inoculate preceding 3 days in pathogenic agent, to make the chemical inducer systemic acquired resistance BTH (benzo [1 of 25% activeconstituents (ai) with wettable powder carrier, 2,3] (Metraux etc. thiadiazoles-7-carboxylic acid-S-methyl esters), 1991) and/or make the microbicide metulaxyl (cGA48988) of 25% activeconstituents, or separately with wettable powder with the form of meticulous dust be applied to 4 age in week plant leaf.With the parasitic downy mildew NoCo of compatible pathogenic agent ₂Conidium suspension (1.8 * 10 ⁵The inoculation of spore/ml) plant.After the inoculation, cover plant is to keep high humidity and it is placed in 17 ℃ the Percival growth room in 14 hours daytime/10 hour cycle at night (Uknes etc., 1993).Inoculate back 8 days results plants.

Make the parasitic downy mildew EmWa of primer DNA as template in order to NS1 and NS2, according to White etc. (1990; PCR method: methods and applications guide, 315-322 page or leaf) the rRNA fungi probe assay fungal growth that the method for PCR obtains.By phenol/sedimentary method of chloroform extracting post chlorization lithium purifying RNA (Lagrimini etc., 1987: institute of NAS reports 84:7542-7546) from the refrigerated tissue.Pass through formaldehyde agarose gel electrophoresis sample separation (7.5ng) and trace (Hybond-N to nylon membrane according to what (1987) such as Ausbel were described ⁺, Amersham).According to Church and Gilbert (1984, institute of NAS newspaper 81; Method 1991-1995) is hybridized and is washed.According to the explanation PhosphorImager of manufacturer (Molecular Dynamics, Sunnyvale, CA) relative quantity of mensuration transcript.Carry out sample stdn on the sample by surveying strip filter paper with constitutive expression b-microtubule Arabidopis thaliana cDNA.It is zero that the fungal growth that not processed plant is infected suppresses.

Separately with metalaxyl, " plant activator " BTH or with metalaxyl and BTH be applied to simultaneously above-mentioned cim plant produced bigger than additivity, promptly collaborative disease resistance effect.Ratio measurement effect with (E) effect of promptly observed (O) effect of synergy multiple (SF) and expectation.Obtained following result.

The effect of the parasitic downy mildew of opposing in table 33 Arabidopis thaliana

Composition I:cim3 sudden change

Composition II: metalaxyl

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			cim3	Metalaxyl	O (observed value)	E (predictor)
	Contrast	wt	--	0		cim3	Metalaxyl	O (observed value)	E (predictor)
1	Contrast	wt	--	0	cim3	--	12.5
1	2 3 4 5	wt wt wt wt	12.5mg/l 2.5mg/l 0.1mg/l 0.02mg/l	52.7 0 0 ND	cim3	--	12.5
6 7 8	2 3 4 5	wt wt wt wt	12.5mg/l 2.5mg/l 0.1mg/l 0.02mg/l	52.7 0 0 ND	cim3 cim3 cim3	12.5mg/l 2.5mg/l 0.1mg/l	ND 82.2 57.8	ND 12.5 12.5	ND 6.6 4.6
6 7 8	9	cim3	0.02mg/l	55.6	cim3 cim3 cim3	12.5mg/l 2.5mg/l 0.1mg/l	ND 82.2 57.8	ND 12.5 12.5	ND 6.6 4.6	ND	ND

WT=wild-type Col-0

The ND=undetermined

The effect of the parasitic downy mildew of opposing in table 34 Arabidopis thaliana

Composition I:cim3 sudden change

Composition II:BTH

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			cim3	BTH	O (observed value)	E (predictor)
	Contrast	wt	--	0		cim3	BTH	O (observed value)	E (predictor)
1	Contrast	wt	--	0	cim3	--	12.5
1	2 3 4	wt wt wt	0.1mM 0.03mM 0.01mM	85.7 20.8 0	cim3	--	12.5
5 6 7	2 3 4	wt wt wt	0.1mM 0.03mM 0.01mM	85.7 20.8 0	cim3 cim3 cim3	0.1mM 0.03mM 0.01mM	ND 73.1 16.6	98.2 33.3 12.5	ND 2.2 1.3

WT=wild-type Col-0

The ND=undetermined

The effect of the parasitic downy mildew of opposing in table 35 Arabidopis thaliana

Composition I:cim3 sudden change

Composition II:BTH and metalaxyl (M)

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			cim3	BTH+M	O (observed value)	E (predictor)
	Contrast	wt	--	0		cim3	BTH+M	O (observed value)	E (predictor)
1	Contrast	wt	--	0	cim3	--	12.5
1	2	wt	BTH 0.01mM +M 0.5mg/l	100	cim3	--	12.5
3 4	2	wt	BTH 0.01mM +M 0.5mg/l	100	wt wt	BTH 0.01mM +M 0.1mg/l BTH 0.01mM +M 0.02mg/l	40.7 ND
3 4	5 6 7	cim3 cim3 cim3	BTH 0.01mM +M 0.5mg/l BTH 0.01mM +M 0.1mg/l BTH 0.01mM +M 0.02mg/l	ND	wt wt	BTH 0.01mM +M 0.1mg/l BTH 0.01mM +M 0.02mg/l	40.7 ND			100 77.7	100 53.2 ND	ND 1.9 ND

WI=wild-type Col-0

The ND=undetermined

By in the last tabulation as can be seen, by using metalaxyl separately, use BTH separately and unite and use metalaxyl and BTH, cim3 plant can show collaborative disease resistance effect.For example, compare with not processed wild plant, visible not processed cim3 plant has 12.5% fungal growth to suppress; This shows that the constitutive expression of SAR gene in the cim3 mutant is relevant with disease resistance.Yet as shown in Table 30, by metalaxyl is applied to immunoregulatory (SAR-opens) cim3 plant with 0.0001g/l (being not enough to effective concentration under the normal circumstances), observed fungal growth inhibition level is increased to 57.8%.From these data computation to synergy multiple 4.6 clearly illustrate that by microbicide being applied to the synergistic effect that the immunomodulatory plant obtains.

The data of listing in the table 31 show and collaborative obtain by systemic acquired resistance chemical inducer such as BTH being applied to immunoregulatory (SAR-opens) cim3 plant.For example, in wild-type plant, be not enough to give effective disease resistance under the BTH normal circumstances of 0.03mM, only provide 20.8% fungal growth to suppress.Yet in the cim3 plant, the BTH of not enough concentration can provide 73.1% fungal growth to suppress under the normal circumstances, and almost the inhibition level with the effective concentration 0.1mM of the BTH that recommends is the same high.The synergy multiple 2.2 that calculates in the data from table 31 clearly illustrates that by BTH being applied to the synergistic effect that the immunomodulatory plant obtains and obtains by other approach.

When BTH and metalaxyl were applied to the cim3 plant simultaneously, the disease resistance effect was more obvious.Embodiment 13 (table 29) is described as mentioned, in wild-type plant, uses 0.01mM BTH or 0.0001g/l metalaxyl separately and can not obtain the fungal growth inhibition, because be not enough to produce effect under these concentration normal circumstancess.Yet, by simultaneously these compositions in the not enough concentration of normal circumstances being applied to plant, can be observed 40.7% fungal growth and suppress, be a kind of synergistic effect for wild-type plant.In the cim3 plant, to use 0.01mM BTH and 0.001g/l metalaxyl simultaneously and can produce the inhibition of 100% fungal growth, this shows further synergistic activity.

Therefore, lower concentration invalid under normal circumstances chemistry medicine is united be used for disease resistance that immunomodulatory cim plant obtains clearly advantage is provided for the agriculture field technician.By the synergetic property advantage of utilizing this paper to show, can avoid deleterious or do not wish the pharmaceutical chemicals of concentration under the normal circumstances.In addition, reduce the income that can realize economically owing to reach the amount of chemicals used of given plant protection level needs.

The collaborative disease resistance effect that III is applied to the transgenic plant acquisition that comprises NIM1 gene form by microbicide and/or systemic acquired resistance chemical inducer with routine

The NIM1 gene is the key component (Ryals etc., 1996) of systemic acquired resistance (SAR) approach of plant.Be associated with the activation of SAR by chemistry and biotic induce agent NIM1 gene, linking to each other with these inductors is that SAR and SAR genetic expression are essential.Determine the position of NIM1 gene by the known genome that carries sudden change nim1 gene plant of molecular biological analysis, thereby this gene makes host plant to multiple pathogenic agent is extremely responsive they can not be replied to pathogenic agent and SAR chemical inducer.Bent to the wild-type NI of Arabidopis thaliana because of having carried out mapping and order-checking (SEQ ID NO:1).Wild-type NIM1 gene product (SEQ ID NO:2) has participated in causing the signal transduction cascade reaction (Ryals etc., 1997) of SAR and gene-right-gene disease resistance in the Arabidopis thaliana.The reorganization overexpression of the NIM1 of wild-type form makes the immunomodulatory plant have composing type immunity (CIM) phenotype, therefore can give the transgenic plant disease resistance.The disease resistance effect that the rising of active NIM1 protein level produces with induce pharmaceutical chemicals such as BTH, the chemical induction disease resistance effect that INA and SA etc. produce is identical.See that common pendent U. S. application 08/880,179 is incorporated herein by reference.

And, shown that it is the structure homologue (Ryals etc., 1997) of mammalian signal transduced element I к B α subclass that the NIM1 gene produces.The sudden change of I к B can be used as super repressor or the dominant thing that NF-к B/I к B regulates program.Therefore, the dominance-inactivation instrumentality that changes form and can be used as the SAR signal transduction pathway of some NIM1.The phenotype that the changing form of these NIM1 given the plant that has transformed them is opposite with the nim1 mutant, that is, the immunomodulatory plant that changes form that has transformed NIM1 shows composing type SAR genetic expression and CIM phenotype.See that common pendent PCT application " with the method for NIM1 gene conferring disease resistance in plants " is incorporated herein by reference.

Embodiment 20: transform plant with the clay clone who comprises wild-type NIM1 gene

Clay D7 (being stored in American type culture collection with ATCC97736 on September 25th, 1996) produces and the next wild-type NIM1 gene (SEQ ID NO:1) that therefore comprises from comprising the DIM1 gene regions.Clay E1 produces and the next wild-type NIM1 gene (SEQ ID NO:1) that therefore also comprises from comprising the NIM1 gene region.Describe according to U.S. Patent application 08/880,179, by conjugal transfer clay D7 and E1 are moved into Agrobacterium tumefaciems AGL-1 with triparental mating by auxiliary strain AB101 (pRK2013).With vacuum filtration with these clays transform the kantlex sensitivities nim1 sudden change Arabidopis thaliana system (Mindrinos etc., 1994, cell 78,1089-1099).The seed of results suction filtration plant also makes it germinate containing on the GM agar plate of 50mg/ml kantlex as selective agent.Drawing the seedling that will stand to screen after dull and stereotyped about two weeks is transferred in the soil.

The plant that is transferred in the soil is approximately growing a week in artificial climate chamber after the transfer.300mM INA is used with meticulous dust and complete cover plant with Chromister.Make an appointment with two days later, the results leaf is with extracting RNA and carry out the PR-1 expression analysis.Spray plant with parasitic downy mildew (isolate EmWa), in the growth room of 19 ℃ of daytime/17 temperature at ℃ night and 8 hours illumination/16 hour dark cycles, growing under the high humidity.After fungal infection 8-10 days, the assessment plant is divided positive or negative to fungal growth.In each group experiment, handle Ws and nim1 plant in the same way in contrast.

From the tissue of collecting, extract total RNA (Verwoerd etc., 1989, nucleic acids research, 2362) with LiCl/ phenol extraction buffer.Be transferred at isolation of RNA sample on the formaldehyde agarose gel and with it on GeneScreen Plue (DuPont) film.With ³²The PR-1 cDNA probe of P mark is hybridized trace.The trace that obtains is exposed to determine to induce the transformant of PR-1 expression after INA handles to film.

Whether D7 and E1 transformant overexpression NIM1 are that the elementary transformant that comprises D7 or E1 clay carries out selfing and collects the T2 seed owing to insert site (position) effect in order to observe.To in soil and by above-mentioned method, grow from the planting seed of strain E1 system and 95 strain D7 system.When the T2 plant has 4 true leaves at least, collect a slice leaf of every strain plant respectively.From tissue, extract RNA, analyze PR-1 and NIM1 and express.Analyzed fungal growth in back 10 days with parasitic downy mildew (EmWa) inoculation plant and in infecting.Most of transformant fungal growths are lower than normally, and wherein 4 is D7-2, D7-74, and D7-89 and E1-1 system do not have the visible fungal growth at all.Plant is higher and have fungus resistant and show that the NIM1 overexpression gives disease resistance than normal NIM1 and PR-1 expression level.

Embodiment 21:NIM1 is overexpression under its natural promoter

Transform the plant that the Ws wild-type plant produces constitutive expression NIM1 gene with the BamHI-HindIII NIM1 genomic fragment that comprises the 1.4kb promoter sequence (SEQID NO:1-base 1249-5655).This fragment cloning is gone into pSGCG01 and be transformed into edaphic bacillus bacterial strain GV3101 (pMP90, Koncz and Schell (1986), Mol Gen Genet 204:383-396).Press preceding method suction filtration Ws plant.The seed that obtains of results also is inoculated in it on GM agar that contains the 50ug/ml kantlex.The plant of survival is transferred in the soil and detects resistance parasitic downy mildew isolate Emwa by preceding method.Selecteed plant selfing also continues two generations of screening and isozygotys with generation and be.The planting seed of several strains wherein in soil, is cultivated three weeks of 15-18 strain plant of every kind of strain, under the prerequisite that need not induce pharmaceutical chemicals to handle, detect the Emwa resistance once more.Handled the back about 24 hours fungi, 48 hours sky results merge the tissue of freezing each strain.Plant continues to grow to back 10 days of inoculation in the growth room, says then and estimates its resistance to Emwa.

From all samples of collecting, prepare RNA and analyze (Delaney etc., 1995) by preceding method.Trace and arabidopsis gene probe PR-1 are hybridized (Uknes etc., 1992).Five strains in the 13 analyzed strain transgenic plant show PR ₁The early evoking of genetic expression.In these strains, it is apparent in view that fungi is handled the 24 or 48 hours PR-1 mRNA in back.These five strains do not have the visible fungal growth yet.Dye blade to confirm not having hypha,hyphae in the blade according to (Dietrich etc., 1994) are described with lactophenol indigo plant.Do not have the abduction delivering of PR-1 gene in the time of 48 hours in other 8 strains, these plants are to the Emwa non-resistant.

The raising to bacterial pathogens pseudomonas syringae DC3000 resistance that has also detected subgroup resistant strain system experimentizes according to the method that (1996) such as Lawton are described basically with the resistance scope of assessment according to description such as Uknes (1993).Those bacterial growths strain slowly also show composing type resistance to Emwa.This plant that shows NIM1 gene overexpression under its natural promoter has the composing type immunity to pathogenic agent.

In order to assess the further feature of CIM phenotype in these strains, in the plant that assessment is infected freely and with glucose link coupled Whitfield's ointment, dye blade to assess the appearance of micro-damage with lactophenol indigo plant.Resistance plant is hybridized to set up the superordination of resistant phenotype and other mutant with SAR mutant such as NahG and ndrl in sexual mode, and how the dominant negative mutant of assessment NIM1 influences the feedback loop that Whitfield's ointment relies on.

The overexpression of the NIM1 that embodiment 22:35S starts

With total length NIM1 cDNA (SEQ ID NO:6) be cloned into pCGN1761 ENXr EcoRI site (Comai etc. (1990) molecular biology of plants 15,373-381).From the plasmid that produces, obtain containing enhanced CaMV 35S promoter, transcribed the Xba I fragment of the NIM1 cDNA and the tm1 3 ' terminator of correct direction.This fragment is cloned into binary vector pCIB200 and is changed over to GV3101.Press preceding method suction filtration Ws plant.The seed that obtains of results also is seeded in it on GM agar that contains the 50ug/ml kantlex.The young plant of survival is transferred in the soil, detects by preceding method.Selected plant carries out selfing and continued to select two generations to isozygoty with generation.When handling with Emwa and pharmaceutical chemicals of no use has 9 to show resistance in tested 58 strains when handling.Therefore, the overexpression of NIM1 cDNA also can produce disease resistance.

Embodiment 23:NIM1 is the homologue of I к B α

Between the protein gene product of NIM1 and I к B, carry out the multisequencing contrast, show that the NIM1 gene product is the homologue (Fig. 1) of I к B α.Carry out the search of sequence homologue with BLAST (Al+schul etc., molecular biology magazine 215,403-410 (1990)).(M adison, WI) part of the Lasergene Biocomputing Software of company bag has made up the multisequencing contrast as DNASTAR with ClustV (Higgin etc., CABIO 5,151-153 (1989)).Being used for correlated sequence is NIM1 (SEQ ID NO:2), mouse I к B α (SEQ IDNO:3, gene pool typing number 1022734), rat I к B α (SEQ ID NO:4, gene pool typing numbers 57674 and X63594; Tewari etc., nucleic acids research 20,607 (1992) and pig I к B α (SEQ ID NO:5, gene pool typing Z21968; De Martin etc., Europe molecular biology is organized magazine 12,2773-2779 (1993); Gene typing numbers 517193, deMartin etc., gene 152,253-255 (1995)).The parameter that is used for the Clustal analysis is gap penalty 10 and gap length penalty 10.Calculate evolutionary divergence distance (Dayhoff etc., " a kind of albumen evolution variation model with PAM250 weight table.Detect relational matrix far away ", protein sequence and structure atlas, the 5th volume augments 3, M.O.Dayhoff edits (national biomedical research foundation, Washiagton D.C.) 345-358 page or leaf (1978)).Calculate residue similarity (Schwartz and Dayhoff with improved Dayhoff table, " a kind of albumen evolution variation model ", protein sequence and structure atlas, M.O.Dayhoff edits (national biomedical research foundation, Washington, D.C.) 353-358 page or leaf (1979); Gribskov and Burgess nucleic acids research 14,6745-6763 (1986)).

The homology search shows NIM1 and several albumen, comprises ankyrin, and there is homology in the ankyrin zone of NF-к B and I к B.Total homology is best is homology (Fig. 1) with I к B and associated molecule.NIM1 has two Serines the 55th and the 59th amino acids; The 59th Serine is in (D/ExxxxS) in the proximity effect sequence, and this position (N-end) relies on phosphorylation, but the induced degradation effect of ubiquitin mediation is consistent.All I к B α contain the terminal Serine of these N-, and are that I к B inactivation and NF-к B subsequently discharge necessary.NIM1 has ankyrin zone (amino acid 260-290 and 323-371).It is believed that the ankyrin zone relates to protein and interacts; Be the universals of I к B and NF-к B molecule.The C-terminal of I к B can be dissimilar.There is some similarity in the abundant district of the QL-of NIM1 and some I к B C-end (amino acid 491-499).

24: the 55 and 59 serine residues of embodiment change the preparation that changes form of the NIM1 of alanine residue into

The phosphorylation of serine residue is that stimulation activatory I к B α degraded and activation NF-к B thereof are necessary among the people I к B α.Become L-Ala to suppress to stimulate the inductive phosphorylation to seal degraded (E.Britta-Mareen Traenckuer etc., the magazine 14:2876-2883 of EMBO (1995) of I к B α proteoplast-mediation thus serine residue (S32-S36) mutagenesis among the people I к B α; Broun etc., science 267:1485-1488 (1996); Brockman etc., molecule and cytobiology 15:2809-2818 (1995); Wang etc., science 274:784-787 (1996)).

The I к B α that this changes form is arrested in NF-к B in the cytoplasm as a kind of dominant form, seals downstream signal transduction incident thus.According to the sequence between NIM1 and the I к B relatively, S32 and the S36 homology among the Serine 55 (S55) of NIM1 and 59 (S59) and the people I к B α.In order to make up the NIM1 of dominant form, the 55th and 59 Serine is mutated into alanine residue.This can be undertaken by method well known to those skilled in the art, such as, with QuikChange site-directed mutagenesis test kit (#200518:Strategene).

With following primer (SEQ ID NO:6, position 192-226), use the total length NIM1 cDNA (SEQID NO:6) of the 3 ' UTR that comprises 42bp5 ' non-translated sequence (UTR) and 187bp can be prepared into the mutagenesis construct according to manufacturer's explanation: 5 '-CAA CAG CTT CGA A GC CGT CTTTGA C GC GCC GGA TG-3 ' (SEQ ID NO:25) and 5 ' CAT CCG GCG CGT CAAAGA CGG CTT CGA AGC TGT TG-3 ' (SEQ ID NO:26), wherein the base of underscore is the sudden change position.Technological line is: will be cloned into the NIM1 cDNA sex change of carrier pSE936 (Elledeg etc., institute of NAS report 88:1731-1735 (1991)) and include the primer annealing that changes base.Archaeal dna polymerase (pfu) thus extending primer by non-strand displacement produces the endless chain of incising is arranged.With restriction endonuclease DpnI dna digestion, it only cuts the site (template DNA of non-mutagenesis) that methylates.Remaining circular double-stranded DNA is in order to transformed into escherichia coli strain XL-1-BLue.Extracting plasmid and carry out the sequence order-checking from the clone who obtains does not have other sudden change to occur to confirm having mutating alkali yl to exist simultaneously.

Be cloned into pCGN1761 with the NIM1 cDNA of restriction endonuclease EcoRI digestion mutagenesis and with it, it is under the transcriptional control of the two 35S promoters of cauliflower mosaic virus.Partly digest by XbaI and will comprise 35S promoter, the conversion box of NIM1 cDNA and tm1 terminator also is connected to the XbaI site of dephosphorylized pCIB200 with it.SEQ ID NO:7 and 8 has represented the dna encoding sequence and the amino acid sequence coded thereof that change form of NIM1 gene respectively.

The preparation that changes form of the NIM1 of the terminal deletion of embodiment 25:N

With people I к B α 1-36 amino acids (Brockman etc.; Sun etc.) or the 1-72 amino acids comprise K21, K22, S32 and S32 deletion has caused transforming the dominant I к B α phenotype in people's cell culture.The terminal deletion of the N-of NIM1 cDNA coded product about 125 amino acid have been removed may be as 8 lysine residues and the possible phosphorylation site (seeing embodiment 2) of S55 and S59 position in ubiquitination site.By any technology well known to those skilled in the art, can prepare the gene construct of change.For example, with the method for gene 77:51-59 (1989) such as Ho, can prepare the deleted NIM1 form of preceding approximately 125 the amino acid whose DNA of coding.Following primer can produce the PCR product (SEQ ID NO:6:418-2011) of a 1612bp: 5 '-ggaattca- ATGGAT TCG GTT GTG ACT GTT TTG-3 ' (SEQ ID NO:27) and 5 '-gga att cTA CAA ATC TGT ATA CCA TTG G-3 ' (SEQ ID NO:28), wherein the synthetic initiator codon is by underscore (ATG), and the EcoRI joint sequence is a small letter.

With comprising the 0.1-100ng template DNA, 10mM Tris pH8.3/50mM KCl/2mMMgCl ₂The final volume of/0.00% gelatin/every kind of primer of every kind of dNTP/0.2mM of 0.25mM and 1 rTth of unit archaeal dna polymerase is reaction mixture amplified fragments on Perkin Elmer Cetus 9600PCR machine of 50 μ l.The PCR condition is as follows: 94 ℃ 3 minutes: 35 * ℃ (94 ℃ 30 seconds: 52 ℃ 1 minute: 72 ℃ 2 minutes): 72 ℃ 10 minutes.The PCR product directly is cloned into pCR2.1 carrier (Invitrogen).The insertion fragment that PCR is produced with restriction enzyme EcoRI digestion discharges from the PCR carrier and with it EcoRI site that is connected to dephosphorylized pCGN1761, makes it to be under the transcriptional control of two 35S promoters.Construct is carried out sequencing to confirm existing and confirming not take place in the PCR process other sudden change of synthetic initial ATG.To comprise 35S promoter by Xba I part restrictive diges-tion, the conversion box of the NIM1cDNA of modification and tm1 terminator discharges the Xba I site that is connected to pCIB200 from pCGN1761.SEQ ID 9 and 10 has represented to have the dna encoding sequence and the aminoacid sequence thereof that change form of the NIM1 gene of-terminal amino acid deletion respectively.

The preparation that changes form of the NIM of embodiment 26 C-terminal deletion

The 261-317 amino acids that it is believed that deletion people I к B α can cause enhanced intrinsic stability by the Serine of blocking-up C-terminal and the composing type phosphorylation of threonine residues.The 522-593 amino acids of NIM1C end contains one section Serine and the abundant district of Threonine.Nucleotide sequence by deletion coding 522-593 amino acids can be modified the C-terminal coding region of NIM1 gene.Method with (1989) such as Ho, the terminal coding region of C-and the 3 ' UTR of NIM1cDNA (SEQ ID NO:6:1606-2011) have been deleted by PCR, produced the fragment of a 1623bp, used primer is: 5 '-cggaattcGATCTCTTTAATTTGTGAATTTC-3 ' (SEQ ID NO:29) and 5 '-ggaattc TCAACAGTTCATAATCTGGTCG-3 ' (SEQ ID NO:30), wherein, a synthetic terminator codon is by underscore (being TGA on the complementary strand), the EcoRI joint sequence is a lowercase.The PCR reacted constituent is with previously described identical, and loop parameter is as follows: 94 ℃ 3 minutes: 35 * (94 ℃ 30 minutes: 52 ℃ 30 seconds: 72 ℃ 2 minutes); 72 ℃ 10 minutes].The PCR product directly is cloned into pCR2.1 carrier (Invitrogen).Carry out the EcoRI site that restriction endonuclease digestion dissociates out with the insertion fragment of PCR generation in the PCR carrier and is connected to the dephosphorylized pCGN1761 that comprises two 35S promoters with EcoRI.This construct is carried out sequencing to confirm existing of the in-frame terminator codon of synthetic, confirm not occur in the PCR process other sudden change simultaneously.To comprise promotor by carrying out the part restrictive diges-tion with Xba I, the NIM1 cDNA of decorations and the conversion box of tm1 terminator discharge the Xba I site that is connected to dephosphorylized pCIB200 from pCGN1761.SEQ ID NO11 and 12 has represented a kind of dna encoding sequence and amino acid sequence coded of NIM1 gene of the C-terminal amino group acid deletion that changes form respectively.

The preparation that changes form of the chimeric NIM1 of the terminal deletion of embodiment 27:N-end/C-

The NIM1 (SEQ ID NO:6) that can prepare N-end and C-terminal deletion form with a single KpnI restriction site of the 819th (SEQ ID NO:6).The terminal delete form of N-(embodiment 25) is to produce with the digestion of EcoRI/KpnI restriction endonuclease, reclaims the fragment of the 415bp consistent with the N-terminal of modifying by gel electrophoresis.Equally, the C-terminal delete form produces with the digestion of EcoRI/KpnI restriction endonuclease, reclaims the fragment of the 790bp consistent with the C-end of modifying by gel electrophoresis.Fragment in 15 ℃ of connections, is cloned into the EcoRI site of dephosphorylized pCGN1761 with EcoRI digestion with elimination EcoRI concatermer and with it.The NIM1 of the terminal delete form of Nlc is under the transcriptional control of two 35S promoters.Similarly, the phosphorylation site S55/S59 (embodiment 24) that can prepare by the supposition of mutagenesis deletes the NIM1 that (embodiment 26) merge the chimeric form that forms with the C-end.This construct passes through method for preparing.This construct is carried out sequencing with the fidelity that confirms the initial sum terminator codon and confirm to occur in clone's process sudden change.By with Xba I part restrictive diges-tion separately comprise 35S promoter, the conversion box of NIM1 mosaic and tm1 terminator discharges the Xba I site that is connected to dephosphorylized pCIB200.SEQ ID NO13 and 14 represents the dna encoding sequence and the amino acid sequence coded thereof of the terminal and NIM1 gene that C-terminal amino acid all lacks of the N-that changes form respectively.

Embodiment 28: the preparation that changes form of the NIM1 in ankyrin zone

NIM1 has homology at about 103-362 amino acid place and ankyrin motif.Method with (1989) such as Ho, by the PCR[condition: 94 ℃ 3 minutes: 35 * (94 ℃ 30 seconds: 62 ℃ 30 seconds: 72 ℃ 2 minutes): 72 ℃ 10 minutes] from the dna sequence dna (SEQ ID NO:1:3093-3951) in the ankyrin zone that NIM1 cDNA (SEQ ID NO:6:349-1128) amplification coding is inferred, the primer is: 5 '-ggaattca ATGGACTCCAACAACACCGCCGC-3 ' (SEQ ID NO:31) and 5 '-ggaattc TCAACCTTCCAAAGTTGCTTCTGATG-3 ' (SEQ ID NO:32).Digest the EcoRI site that the generation that obtains is connected to dephosphorylized pCGN1761 then with the EcoRI restriction endonuclease, it is under the transcriptional control of two 35S promoters.Construct is carried out sequencing to confirm synthetic initiator codon (ATG), and the existence of in-frame terminator codon (TGA) confirms that simultaneously other sudden change does not appear in the PCR process.By comprising 35S promoter with the digestion of Xba I part restriction endonuclease, the conversion box of ankyrin zone and tm1 terminator discharges and is connected to the Xba I site of dephosphorylized pCIB200 from pCGN1761.SEQ ID NO15 and 16 has represented the dna encoding sequence and the amino acid sequence coded thereof in NIM1 ankyrin zone respectively.

Embodiment 29: the structure of mosaic gene

Base 1249-5655) and/or the EcoRV/BamHI fragment of a 5655bp (SEQ IDNO:1: base 1-5655) be used to prepare the changing form of NIM1 of the foregoing description 24-28 the possibility of expressing for the suitable room and time that changes form that improves NIM1,4407bp comprises HindIII/BamHI fragment (the SEQ ID NO:1: of NIM1 promotor and gene.Although construction step may be different, it is similar that thinking is stated above-described embodiment therewith.The strong overexpression that changes form might be a lethality.So changing form of the NIM1 gene of describing among the embodiment 24-28 can be placed under the control of endogenic NIM1 promotor promotor in addition, including, but not limited to the little subunit of no promotor or Rubisco promotor.Equally, the NIM1 that changes form can express (United States Patent (USP) 5,614,395) under the control of pathogenic agent-reaction promotor PR-1.Such expression only allows the strongly expressed that changes form of NIM1 under pathogenic agent attack or other SAR activation condition.And, when handling with the SAR activator compound (being BTH or INA) that does not under normal circumstances activate the concentration of SAR, under the PR-1 promoter regulation, express in the transformant of the NIM that changes form, disease resistance can be obvious, activate feedback loop (Weymann etc., (1995) vegetable cell 7:2013-2022) thus.

Embodiment 30: changing form of NIM1 is transformed into Arabidopis thaliana

The construct (embodiment 24-29) of preparation enters the GV3101 strain by electroporation and moves into Agrobacterium tumefaciems.These constructs by vacuum filtration (Mindrinos etc., Cell78,1089-1099 (1994)) or pass through the conversion of standard root in order to environmental Col-O of arabidopsis thaliana transformation and Ws-O.Gather in the crops the seed of these plants and they are germinateed on the agar plate of kantlex (or other suitable microbiotic) as selective agent.Have only the young plant of conversion can remove the toxicity of selective agent and survive.The seedling of standing to screen is transferred to and detects its CIM (composing type immunity) phenotype in the soil.By comparing, assess observable phenotypic difference with wild-type plant.

Embodiment 31: assessment has transformed the CIM phenotype of the plant that changes form of wild-type NIM1 gene or NIM1 gene

Gather in the crops a slice leaf of the elementary transformant of each strain, isolation of RNA (Verwoerd etc., 1989, nucleic acids research, 2362) is expressed (Uknes etc., 1992) by rna blot analysis test set moulding PR-1.Assess the enhanced disease resistance reaction (Uknes etc., 1992) of the expression composing type SAR expression analysis of each strain plant.Prepared two kinds of compatible parasitic downy mildew isolates, Emwa and Noco, concentration be 5-10 * 10 ⁴Spore/ml conidium suspension (that is, these fungi strains cause the disease of wild-type Ws-O and Col-O plant respectively) according to the wild-type of transformant, sprays transformant with suitable isolate.At the 7th day plant is carried out the disease classification, results a slice leaf carries out rna blot analysis with probe, and a kind of method of measuring fungal infection is provided.

Produce the T of the transformant of performance CIM phenotype ₁In generation, also identified the homozygote plant.According to following transformant is carried out a series of disease resistance test.Describe according to (1994) such as Dietrich, repeat fungal infection, dye leaf existing with the evaluation hypha,hyphae with lactophenol indigo plant with Noco and Emwa.Describe according to (1993) such as Uknes, infect transformant to assess the resistance scope with bacterial pathogens pseudomonas syringae DC3000.The freedom and the glucose link coupled SA of leaf infected in assessment, dyes the appearance of leaf with the assessment microlesion with lactophenol indigo plant.To set up the superordination of resistant phenotype and other mutant, how the dominant negative mutant of assessment NIM1 influences the feedback loop of SA dependence to resistance plant by sexual mode and SAR mutant such as NahG (United States Patent (USP) 5,614,395) and hdr1 hybridization.

The separation of embodiment 32:NIM1 homologue

By can obtain the NIM1 homologue with Arabidopis thaliana or preferred and oligonucleotide probe hybridization from Arabidopis thaliana NIM1 gene under medium stringent condition, this probe comprises its encoding sequence sequential portion of at least 10 Nucleotide.The factor that influences heterozygote stability has determined the preciseness of hybridization.Wherein one of factor is melting temperature(Tm) Tm, according to dna probe, and George HKeller and Mark M.Manak; Macmillan Publisher Ltd, 1993 first parts; Molecular hybridization; The 8th page of formula that provides is easy to calculate the Tm value.Preferred hybridization temperature is than the low about 25 ℃ temperature of the melting temperature(Tm) Tm that calculates, if oligonucleotide is the temperature than low about 5-10 ℃ of melting temperature(Tm) Tm.

Make probe with NIM1 cDNA (SEQ ID NO:6), by screening gene library of different crops or the homologue that Arabidopis thaliana NIM1 is determined in the cDNA library, these farm crop are listed in the embodiment 33.The standard technique of finishing this work comprises screening by hybridization (plaque or the clone in dull and stereotyped DNA library; See as Sambrook etc., Molecular cloning, editor, press of cold spring harbor laboratory (1989)) and with Oligonucleolide primers by pcr amplification (see, as Innis etc., PCR method, the methods and applications guideEditor, Academic Press (1990)).The homologue of identifying is incorporated into herein in the expression vector by genetic engineering and is transformed in the above-mentioned farm crop.Assess the enhanced disease resistance of transformant with the related diseases substance of tested crop plants.

By the southern blotting technique analyzing and testing cucumber, tomato, tobacco, corn, the NIM1 homologue in wheat and the oat gene group.From cucumber, tomato, tobacco, corn, isolation of genomic DNA in wheat and the barley is used BamHI, HindIII, the restricted cutting said gene of XbaI or SalI group, the genome after electrophoresis separation cuts on 0.8% sepharose is also transferred to them on the nylon membrane by the capillary trace.Behind UV-cross fixation DNA, use ³²P-radiolabeled Arabidopis thaliana NIM1 cDNA and film hybridize under low stringency condition [(I%BSA; 520mM NaPO ₄, pH7.2; 7% sodium lauryl sulphate; 1mM EDTA; 250mM sodium-chlor) in 55 ℃ of hybridization 18-24 hour].After the hybridization, under low stringency condition, wash trace [6 * SSC 15 minutes (* 3), 3 * SSC 15 minutes (* 1) is in 55 ℃; 1 * SSC is 0.15M NaCl, 15mM Trisodium Citrate (pH7.0)], x-ray film is exposed to observe the band consistent with NIM1.

In addition, by can be used for separating homologue with the expressed sequence tag (EST) of the similar evaluation of NIM1 gene.For example, by having identified several paddy rice expressed sequence tag (ESTs) with the similarity of NIM1 gene.With ClustalV (Higgins, Desmond G. and Panl M.Sharp (1989), the multisequencing contrast of the quick sensitivity of minicomputer; CABIOS5:151-153) as DNA ^*The part of (1228 South Park Street, Madison Wisconsin, 53715) Lasergen Biocomputing Software package for theillacintosh (1994) is built into the multisequencing contrast.The amino acid sequence homologous of the rice cDNA protein product that proteic some zone of NIM1 is different with 4.Determined homology in the GenBankBLAST search with the NIM1 sequence.The homology zone of NIM1 and rice cDNA product relatively list in Fig. 2 (seeing SEQ ID NO:2 and SEQ ID NO17-24).This NIM1 protein fragments and 4 paddy rice products have 36-48% common aminoacid sequence.These paddy rice EST are particularly useful for separate the NIM1 homologue from other monocotyledons.

Also can obtain homologue by PCR.In this method, between known homologue (as paddy rice and Arabidopis thaliana), compare.Amino acid can be used as the PCR primer with the highly similar or identical zone of DNA.The regional back that amino acid M and W enrich is amino-acid residue F preferably, Y, and C, H, Q, the zone that K and E are abundant is because these amino acid are by the codon coding of limited quantity.In case determined suitable zone, can prepare that regional primer by substituting of the 3rd different bit codons.The target species limit the 3rd alternate diversity.For example abundant because corn is GC, if possible, during the design primer at the 3rd with G or C.Under the multiple standards condition, cDNA or genomic dna are carried out the PCR reaction.If a band, then clone it also/or to its order-checking to determine that it is the NIM1 homologue.

Embodiment 33: a kind of NIM1 expresses in crop plants

The construct of giving Col-O or Ws-O CIM phenotype is transformed into crop plants and assesses.Alternatively, isolating altered natural NIM1 gene is put back in separately the plant from previous embodiment.Although the NIM1 gene can be inserted into any vegetable cell of broad range, it is particularly useful for crop plant cells, such as paddy rice, and wheat, oat, rye, corn, potato, Radix Dauci Sativae, sweet potato, beet, Kidney bean, pea, witloof, lettuce, Caulis et Folium Brassicae capitatae, Cauliflower, asparagus broccoli, turnip, Radix Raphani, spinach, asparagus, onion, garlic, eggplant, pepper, celery, Radix Dauci Sativae, summer squash, pumpkin, zucchini, cucumber, apple, pears Quinces Quince, muskmelon, Lee, cherry, peach, nectarine, apricot, strawberry, grape, rasp berry, blackberry, blueberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, jowar and sugarcane.The enhanced disease resistance of assessment transformant.In a preferred embodiment of the present invention, NIM1 expression of gene level is the twice of natural NIM1 expression level in the wild-type plant very less, and is preferably, high 10 times than wild-type expression level.

Embodiment 34: the collaborative disease resistance that obtains by the transgenic plant that conventional microbicide are applied to overexpression NIM1

The plant lines that is used for present embodiment (6E and 7C) is described according to embodiment 21, obtains with BamHI-HindIII NIM1 gene fragment (SEQ ID NO:1-base 1249-5655) conversion wild-type arabidopsis thaliana (environmental Ws).Mycocide metalaxyl phosethyl Al and copper hydroxide are made on 25%, 80% and 70% activeconstituents (ai) was applied to for three ages in week with the form of meticulous dust the leaf of transgenic plant Ws of constitutive expression NIM1 gene with wettable powder carrier respectively.Use wettable powder separately in contrast.After three days, according to (195) such as Delaney, Zhou Jisheng downy mildew isolate Emwa conidium suspension (1-2 * 10 ⁵The inoculation of spore/ml) plant.After the inoculation, cover plant to be keeping high humidity, and it is placed in the Pervical growth room of 17 ℃ of 14 hours illumination/10 hour dark cycles (Uknes etc., 1993).Inoculate back 8 days results tissues.

By under dissecting microscope, observing to conidial growth divided rank (Delaney etc. (1994); Dietrich, etc., 1994), followed the tracks of the fungal infection process 12 days.Individual blade is carried out the dyeing of lactophenol trypan blue to observe fungal growth in the blade.In order to NS1 and NS2 is primer, is template with parasitic downy mildew EmWa DNA, according to White etc. (1990; PCR method, methods and applications guide, 315-322 page or leaf) method by the rRNA fungi probe that PCR obtains fungal growth is carried out quantitatively.By phenol/sedimentary method of chloroform extracting post chlorization lithium (Lagrimini etc., 1987, institute of NAS reports 84:7542-7546), purifying RNA from freezing tissue.Described according to (1987) such as Ausbel by formaldehyde agarose gel electrophoresis sample separation (7.5ug) and with its mark (Hybond N to nylon membrane ⁺, Amersham).Hybridize and wash according to Church and Gilbert (1984, institute of NAS reports 81:1991-1995).(Molecular DynamicsSunnyvale CA) determines the relative quantity of transcript with Phosphor Image according to manufacturer explanation.Carry out sample stdn on the sample by surveying strip filter paper with constitutive expression b-microtubule Arabidopis thaliana cDNA.It is zero that the fungal growth that not processed plant is infected suppresses.

With metalaxyl, the plant lines that phosethyl Al or copper hydroxide are applied to overexpression NIM1 produces a promptly collaborative disease resistance effect bigger than additivity.Effect is measured according to synergy multiple (SF), and SF is the ratio of (E) effect of (O) effect of observing and expectation.Obtain following result:

The effect of the parasitic downy mildew of opposing in table 36 Arabidopis thaliana

Composition I:NIM1 overexpression (6E strain system)

Composition II: metalaxyl

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			NIM1	Metalaxyl	O (observed value)	E (predictor)
	Contrast	wt	--	0		NIM1	Metalaxyl	O (observed value)	E (predictor)
1	Contrast	wt	--	0	NIM1	--	10
1	2 3	wt wt	0.0125g/l 0.0012g/l	59 27	NIM1	--	10
4	2 3	wt wt	0.0125g/l 0.0012g/l	59 27	NIM1	0.0125g/l	76	69	1.1
4	5	NIM1	0.0012g/l	56	NIM1	0.0125g/l	76	69	1.1	37	1.5

WT=wild-type Ws

The effect of the parasitic downy mildew of opposing in table 37 Arabidopis thaliana

Composition I:NIM1 overexpression (6E strain system)

Composition II: phosethyl Al

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			NIM1	Phosethyl Al	O (observed value)	E (predictor)
	Contrast	wt	--	0		NIM1	Phosethyl Al	O (observed value)	E (predictor)
1	Contrast	wt	--	0	NIM1	--	10
1	2 3 4	wt wt wt	5.0g/l 0.5/l 0.05g/l	7 2 0	NIM1	--	10
5 6 7	2 3 4	wt wt wt	5.0g/l 0.5/l 0.05g/l	7 2 0	NIM1 NIM1 NIM1	5.0g/l 0.5g/l 0.05g/l	93 83 42	17 12 10	5.5 6.9 4.2

WT=wild-type Ws

The effect of the parasitic downy mildew of opposing in table 38 Arabidopis thaliana

Composition I:NIM1 overexpression (7C system)

Composition II: phosethyl Al

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			NIM1	Phosethyl Al	O (observed value)	E (predictor)
	Contrast	wt	--	0		NIM1	Phosethyl Al	O (observed value)	E (predictor)
1	Contrast	wt	--	0	NIM1	--	14
1	2 3	wt wt	5.0g/l 0.5g/l	7 2	NIM1	--	14
4 5	2 3	wt wt	5.0g/l 0.5g/l	7 2	NIM1	5.0g/l 0.5g/l	80 56	21 16	3.8 3.5
4 5		NIM1			NIM1	5.0g/l 0.5g/l	80 56	21 16	3.8 3.5

WT=wild-type Ws

The effect of the parasitic downy mildew of opposing in table 39 Arabidopis thaliana

Composition I:NIM1 overexpression (6E strain system)

Composition II: copper hydroxide

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			NIM1	Cu(OH) ₂	O (observed value)	E (predictor)
	Contrast	wt	--	0		NIM1	Cu(OH) ₂	O (observed value)	E (predictor)
1	Contrast	wt	--	0	NIM1	--	10
1	2 3 4	wt wt wt	2.0g/l 0.2g/l 0.02g/l	0 0 0	NIM1	--	10
5 6 7	2 3 4	wt wt wt	2.0g/l 0.2g/l 0.02g/l	0 0 0	NIM1 NIM1 NIM1	2.0g/l 0.2g/l 0.02g/l	66 14 20	10 10 10	6.6 1.4 2.0

WT=wild-type Ws

The effect of the parasitic downy mildew of opposing in table 40 Arabidopis thaliana

Composition I:NIM1 overexpression (7C strain system)

Composition II: copper hydroxide

Test No.	Composition		Fungal growth suppresses %		Synergy multiple O/E
	Composition		Fungal growth suppresses %			NIM1	Cu(OH) ₂	O (observed value)	E (predictor)
	Contrast	wt	--	0		NIM1	Cu(OH) ₂	O (observed value)	E (predictor)
1	Contrast	wt	--	0	NIM1	--	14
1	2 3 4	wt wt wt	2.0g/l 0.2g/l 0.02g/l	0 0 0	NIM1	--	14
5 6 7	2 3 4	wt wt wt	2.0g/l 0.2g/l 0.02g/l	0 0 0	NIM1 NIM1 NIM1	2.0g/l 0.2g/l 0.02g/l	77 51 55	14 14 14	5.5 3.6 3.9

WT=wild-type Ws

From above-mentioned table as can be seen, by using metalaxyl, phosethyl Al and copper hydroxide, the plant of overexpression NIM1 shows collaborative disease resistance effect.For example, (6E system) has 10% fungal growth to suppress with respect to untreated wild-type plant in the untreated NIMI plant; This SAR genetic expression that shows composing type among this NIM1 overexpression person is relevant with disease resistance.Yet, can find out from table 37 that by phosethyl Al is applied to immunoregulatory (SAR-opens) NIM1 overexpression plant with 5.0g/l (being not enough to effective concentration under the normal circumstances), observed fungal growth suppresses to be increased to 93%.5.5 the synergy multiple that calculates from these data has clearly illustrated that microbicide has been applied to the synergistic effect that immunomodulatory plant (SAR-opens) obtains.In another embodiment, not processed NIM1 plant (7C system) has 14% fungal growth inhibition with respect to untreated wild-type plant, shows that the SAR genetic expression of composing type among this NIM1 overexpression person is relevant with disease resistance.Yet, can find out from table 40, by copper hydroxide is applied to immunoregulatory (SAR-opens) NIM1 overexpression plant with 2.0g/l (being not enough to effective concentration under the normal circumstances), observed fungal growth suppresses to be increased to 77%.5.5 the synergy multiple that calculates from these data has clearly illustrated microbicide has been applied to the synergistic effect that immunomodulatory plant (SAR-opens) obtains.

Therefore, with the immunomodulatory plant of overexpression NIM1 and low, the advantage that the microbicide combined utilization that under normal circumstances is not enough to effective concentration obtains disease resistance for the technician of agriculture field clearly.Utilize the synergetic property advantage that shows herein, can avoid undesirable concentrations of microbiocibles poisonous or other under the normal circumstances.In addition, reduce, can realize economic benefit owing to reach the amount of the needed microbicide of given protective plant level.

Embodiment 35 is applied to the collaborative disease resistance that the transgenic plant of overexpression NIM1 obtain with the SAR chemical inducer

Also analyzed include the NIM1 genomic DNA fragment (embodiment 21) under the control of self promotor plant with respect to of the reaction of wild-type Ws strain to the BTH of different concns.Cultivate the seed of each strain according to the preceding method sowing.Plantation about 3 weeks of back, fasten results leaf sample (comparing in the 0th day) from each product, remaining plant is with H ₂O, 10uM BTH or 100uM

BTH handle.Handle

1,3 and 5 day other sample of results.After having gathered in the crops the 3rd day sample, a subgroup plant of each strain is removed and handles with parasitic downy mildew isolate Emwa according to aforesaid method.From the tissue of results, prepare RNA, carry out Northern with Arabidopis thaliana PR-1 gene probe and analyze.Infect and the plant epiphyte resistance carried out divided rank in back 8 days.

Northern analytical results to Ws and 4 strain NIM overexpression strains (3A, 5B, 6E and 7E) is shown in Fig. 3.After low-level 10uM BTH handles (effective concentration of BTH is 100-300uM under the normal circumstances), almost detect genetic expression in the wild-type Ws strain less than PR-1.Through the Ws of this processing plant still to parasitic downy mildew (Emwa) sensitivity of fungal pathogens.Yet, in all NIM-1 overexpression strains, strong many PR-1 genetic expression reaction is arranged after low-level BTH handles.In addition, all NIM1 overexpression strains of handling with 10uM BTH show all or part of resistance of parasitic downy mildew.Dye leaf to identify exist (Dietrich etc., 1994) of hypha,hyphae with lactophenol indigo plant) confirm do not have fungal growth in the NIM1 overexpression strain.For wild-type, after 100uM BTH handles, strong many and faster of the PR-1 genetic expression in the leaf texture of NIM1 overexpression strain.Therefore, from PR-1 genetic expression and to the resistance of parasitic downy mildew as can be seen, the immunomodulatory plant can be made fast many reactions to low many BTH.These data show by will being that acquired resistance chemical inducer such as BTH is applied to immunomodulatory (SAR-opens) plant such as NIM1 overexpression plant and can obtains collaborative disease resistance.

Therefore, the advantage that the immunomodulatory plant of overexpression NIM1 and the low microbicide combined utilization that under normal circumstances is not enough to effective concentration is obtained disease resistance for the technician of agriculture field clearly.Utilize the synergetic property advantage that shows herein, can avoid undesirable concentrations of microbiocibles poisonous or other under the normal circumstances.In addition, reduce, can realize economic benefit owing to reach the amount of the required microbicide of given protective plant level.

Sequence table

(1) essential information

(i) applicant:

(A) title: NOVARTIS AG

(B) street: Schwarzwaldallee 215

(C) city: Basel

(D) country: Switzerland

(F) postcode: 4002

(G) phone :+41 61 696 11 11

(H) fax :+41 61 696 79 76

(H) fax: 962 991

(ii) denomination of invention: the method for protective plant

(iii) sequence number: 32

(iv) computer-reader form

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, version #1.30

(2) about the information of SEQ ID NO:1:

(i) sequence signature

(A) length: 5655 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: DNA (genome)

(iii) suppose: do not have

(iii) antisense: do not have

(ix) feature:

(A) title/key: exon

(B) position: 2787..3347

(D) out of Memory :/product=" first exon of NIM1 "

(ix) feature:

(A) title/key: exon

(B) position: 3427..4162

(D) out of Memory :/product=" second exon of NIM1 "

(ix) feature:

(A) title/key: exon

(B) position: 4271..4474

(D) out of Memory :/product=" the 3rd exon of NIM1 "

(ix) feature:

(A) title/key: exon

(B) position: 4586..4866

(D) out of Memory :/product=" the 4th exon of NIM1 "

(ix) feature:

(A) title/key: CDS

(B) position: connect (2787..3347,3427..4162,4271..4474,4586..4866).

(xi) sequence description: SEQ ID NO:1:

TGTGATGCAA GTCATGGGAT ATTGCTTTGT GTTAAGTATA CAAAACCATC ACGTGGATAC 60

ATAGTCTTCA AACCAACCAC TAAACAGTAT CAGGTCATAC CAAAGCCAGA AGTGAAGGGT 120

TGGGATATGT CATTGGGTTT AGCGGTAATC GGATTGAACC CTTTCCGGTA TAAAATACAA 180

AGGCTTTCGC AGTCTCGGCG TATGTGTATG TCTCGGGGTA TCTACCATTT GAATCACAGA 240

ACTTTTATGT GCGAAGTTTT CGATTCTGAT TCGTTTACCT GGAAGAGATT AGAAAATTTG 300

CGTCTACCAA AAACAGACAG ATTAATTTTT TCCAACCCGA TACAAGTTTC GGGGTTCTTG 360

CATTGGATAT CACGGAACAA CAATGTGATC CGGTTTTGTC TCAAAACCGA AACTTGGTCC 420

TTCTTCCATA CTCCGAACTC TGATGTTTTC TCAGGATTAG TCAGATACGA AGGGAAGCTA 480

GGTGCTATTC GTCAGTGGAC AAACAAAGAT CAAGAAGATG TTCACGAGTT ATGGGTTTTA 540

AAGAGCAGTT TTGAAAAGTC GTGGGTTAAA GTGAAAGATA TTAAAAGCAT TGGAGTAGAT 600

TTGATTACGT GGACTCCAAG CAACGACGTT GTATTGTTTC GTAGTAGTGA TCGTGGTTGC 660

CTCTACAACA TAAACGCAGA GAAGTTGAAT TTAGTTTATG CAAAAAAAGA GGGATCTGAT 720

TGTTCTTTCG TTTGTTTTCC GTTTTGTTCT GATTACGAGA GGGTTGATCT GAACGGAAGA 780

AGCAACGGGC CGACACTTTA AAAAAAAAAT AAAAAAAATG GGCCGACAAA TGCAAACGTA 840

GTTGACAAGG ATCTCAAGTC TCAAGTCTCA ATTGGCTCGC TCATTGTGGG GCATAAATAT 900

ATCTAGTGAT GTTTAATTGT TTTTTATAAG GTAAAAAGGA ATATTGAATT TTGTTTCTTA 960

GGTTTATGTA ATAATACCAA ACATTGTTTT ATGAATATTT AATCTGATTT TTTGGCTAGT 1020

TATTTTATTA TATCAAGGGT TCCTGTTTAT AGTTGAAAAC AGTTACTGTA TAGAAAATAG 1080

TGTCCCAATT TTCTCTCTTA AATAATATAT TAGTTAATAA AAGATATTTT AATATATTAG 1140

ATATACATAA TATCTAAAGC AACACATATT TAGACACAAC ACGTAATATC TTACTATTGT 1200

TTACATATAT TTATAGCTTA CCAATATAAC CCGTATCTAT GTTTTATAAG CTTTTATACA 1260

ATATATGTAC GGTATGCTGT CCACGTATAT ATATTCTCCA AAAAAAACGC ATGGTACACA 1320

AAATTTATTA AATATTTGGC AATTGGGTGT TTATCTAAAG TTTATCACAA TATTTATCAA 1380

CTATAATAGA TGGTAGAAGA TAAAAAAATT ATATCAGATT GATTCAATTA AATTTTATAA 1440

TATATCATTT TAAAAAATTA ATTAAAAGAA AACTATTTCA TAAAATTGTT CAAAAGATAA 1500

TTAGTAAAAT TAATTAAATA TGTGATGCTA TTGAGTTATA GAGAGTTATT GTAAATTTAC 1560

TTAAAATCAT ACAAATCTTA TCCTAATTTA ACTTATCATT TAAGAAATAC AAAAGTAAAA 1620

AACGCGGAAA GCAATAATTT ATTTACCTTA TTATAACTCC TATATAAAGT ACTCTGTTTA 1680

TTCAACATAA TCTTACGTTG TTGTATTCAT AGGCATCTTT AACCTATCTT TTCATTTTCT 1740

GATCTCGATC GTTTTCGATC CAACAAAATG AGTCTACCGG TGAGGAACCA AGAGGTGATT 1800

ATGCAGATTC CTTCTTCTTC TCAGTTTCCA GCAACATCGA GTCCGGAAAA CACCAATCAA 1860

GTGAAGGATG AGCCAAATTT GTTTAGACGT GTTATGAATT TGCTTTTACG TCGTAGTTAT 1920

TGAAAAAGCT GATTTATCGC ATGATTCAGA ACGAGAAGTT GAAGGCAAAT AACTAAAGAA 1980

GTCTTTTATA TGTATACAAT AATTGTTTTT AAATCAAATC CTAATTAAAA AAATATATTC 2040

ATTATGACTT TCATGTTTTT AATGTAATTT ATTCCTATAT CTATAATGAT TTTGTTGTGA 2100

AGAGCGTTTT CATTTGCTAT AGAACAAGGA GAATAGTTCC AGGAAATATT CGACTTGATT 2160

TAATTATAGT GTAAACATGC TGAACACTGA AAATTACTTT TTCAATAAAC GAAAAATATA 2220

ATATACATTA CAAAACTTAT GTGAATAAAG CATGAAACTT AATATACGTT CCCTTTATCA 2280

TTTTACTTCA AAGAAAATAA ACAGAAATGT AACTTTCACA TGTAAATCTA ATTCTTAAAT 2340

TTAAAAAATA ATATTTATAT ATTTATATGA AAATAACGAA CCGGATGAAA AATAAATTTT 2400

ATATATTTAT ATCATCTCCA AATCTAGTTT GGTTCAGGGG CTTACCGAAC CGGATTGAAC 2460

TTCTCATATA CAAAAATTAG CAACACAAAA TGTCTCCGGT ATAAATACTA ACATTTATAA 2520

CCCGAACCGG TTTAGCTTCC TGTTATATCT TTTTAAAAAA GATCTCTGAC AAAGATTCCT 2580

TTCCTGGAAA TTTACCGGTT TTGGTGAAAT GTAAACCGTG GGACGAGGAT GCTTCTTCAT 2640

ATCTCACCAC CACTCTCGTT GACTTGACTT GGCTCTGCTC GTCAATGGTT ATCTTCGATC 2700

TTTAACCAAA TCCAGTTGAT AAGGTCTCTT CGTTGATTAG CAGAGATCTC TTTAATTTGT 2760

GAATTTCAAT TCATCGGAAC CTGTTG ATG GAC ACC ACC ATT GAT GGA TTC GCC 2813

Met Asp Thr Thr Ile Asp Gly Phe Ala

1 5

GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC GCT ACC GAT AAC ACC 2861

Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val Ala Thr Asp Asn Thr

10 15 20 25

GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA GTA CTC ACC GGA CCT 2909

Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln Val Leu Thr Gly Pro

30 35 40

GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC TTC GAA TCC GTC TTT 2957

Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser Phe Glu Ser Val Phe

45 50 55

GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG CTT GTT CTC TCC GAC 3005

Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys Leu Val Leu Ser Asp

60 65 70

GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG TCA GCG AGA AGC TCT 3053

Gly Arg Glu Val Ser Phe His Arg Cys Val Leu Ser Ala Arg Ser Ser

75 80 85

TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG GAG AAA GAC TCC AAC 3101

Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys Glu Lys Asp Ser Asn

90 95 100 105

AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG ATT GCC AAG GAT TAC 3149

Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile Ala Lys Asp Tyr

110 115 120

GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC 3197

Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser

125 130 135

AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG 3245

Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu

140 145 150

AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG GAT TTC ATG TTG GAG 3293

Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu

155 160 165

GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC 3341

Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu

170 175 180 185

TAT CAG GTAAAACACC ATCTGCATTA AGCTATGGTT ACACATTCAT GAATATGTTC 3397

Tyr Gln

TTACTTGAGT ACTTGTATTT GTATTTCAG AGG CAC TTA TTG GAC GTT GTA GAC 3450

Arg His Leu Leu Asp Val Val Asp

190 195

AAA GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA 3498

Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile

200 205 210

TGT GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT 3546

Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile

215 220 225

GTC AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA 3594

Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu

230 235 240

GAG CTT GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG 3642

Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu

245 250 255

GTA CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC 3690

Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp

260 265 270 275

TCG GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC 3738

Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr

280 285 290

AAT CTA GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT 3786

Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn

295 300 305

GTG AAG ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC 3834

Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn

310 315 320

CAT AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG 3882

His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg

325 330 335

AAG GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA 3930

Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala

340 345 350 355

TCA GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA 3978

Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln

360 365 370

GCC ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT 4026

Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His

375 380 385

TCT CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA 4074

Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys

390 395 400

CGA GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC 4122

Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala

405 410 415

GAT GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA G 4162

Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg

420 425 430

GTATCTATCA AGTCTTATTT CTTATATGTT TGAATTAAAT TTATGTCCTC TCTATTAGGA 4222

AACTGAGTGA ACTAATGATA ACTATTCTTT GTGTCGTCCA CTGTTTAG TT GCA CTT 4278

Val Ala Leu

435

GCT CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC 4326

Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala

440 445 450

GAA ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC 4374

Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp

455 460 465

CGT CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT 4422

Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro

470 475 480

TTC AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA 4470

Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys

485 490 495

ACC G GTATGGATTC TCACCCACTT CATCGGACTC CTTATCACAA AAAACAAAAC 4524

Thr

500

TAAATGATCT TTAAACATGG TTTTGTTACT TGCTGTCTGA CCTTGTTTTT TTTATCATCA 4584

G TG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC 4629

Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu

505 510 515

GAC CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA 4677

Asp Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu

520 525 530

GAC GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA 4725

Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu

535 540 545

ATA CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA 4773

Ile Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu

550 555 560

GGA AAT TCG TCC CTG ACA GAT TCG ACT TCT TCC ACA TCG AAA TCA ACC 4821

Gly Asn Ser Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr

565 570 575

GGT GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 4866

Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg *

580 585 590

GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 4926

TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 4986

ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 5046

ATTTGTAATA TATATTTATG TACATCAACA ATAACCCATG ATGGTGTTAC AGAGTTGCTA 5106

GAATCAAAGT GTGAAATAAT GTCAAATTGT TCATCTGTTG GATATTTTCC ACCAAGAACC 5166

AAAAGAATAT TCAAGTTCCC TGAACTTCTG GCAACATTCA TGTTATATGT ATCTTCCTAA 5226

TTCTTCCTTT AACCTTTTGT AACTCGAATT ACACAGCAAG TTAGTTTCAG GTCTAGAGAT 5286

AAGAGAACAC TGAGTGGGCG TGTAAGGTGC ATTCTCCTAG TCAGCTCCAT TGCATCCAAC 5346

ATTTGTGAAT GACACAAGTT AACAATCCTT TGCACCATTT CTGGGTGCAT ACATGGAAAC 5406

TTCTTCGATT GAAACTTCCC ACATGTGCAG GTGCGTTCGC TGTCACTGAT AGACCAAGAG 5466

ACTGAAAGCT TTCACAAATT GCCCTCAAAT CTTCTGTTTC TATCGTCATG ACTCCATATC 5526

TCCGACCACT GGTCATGAGC CAGAGCCCAC TGATTTTGAG GGAATTGGGC TAACCATTTC 5586

CGAGCTTCTG AGTCCTTCTT TTTGATGTCC TTTATGTAGG AATCAAATTC TTCCTTCTGA 5646

CTTGTGGAT 5655

(2) about the information of SEQ ID NO:2:

(i) sequence signature

(A) length: 594 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:2:

Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser

1 5 10 15

Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu

20 25 30

Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu

35 40 45

Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr

50 55 60

Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His

65 70 75 80

Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala

85 90 95

Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu

100 105 110

Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val

115 120 125

Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro

130 135 140

Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys

145 150 155 160

Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile

165 170 175

Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp

180 185 190

Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu

195 200 205

Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys

210 215 220

Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser

225 230 235 240

Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu

245 250 255

Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys

260 265 270

Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu

275 280 285

Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala

290 295 300

Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala

305 310 315 320

Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala

325 330 335

Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly

340 345 350

Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile

355 360 365

Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln

370 375 380

Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln

385 390 395 400

Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala

405 410 415

Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg

420 425 430

Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met

435 440 445

Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu

450 455 460

Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys

465 470 475 480

Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala

485 490 495

Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser

500 505 510

Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala

515 520 525

Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg

530 535 540

Tyr Met Glu Ile Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn

545 550 555 560

Leu Glu Leu Gly Asn Ser Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser

565 570 575

Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg

580 585 590

Arg *

(2) about the information of SEQ ID NO:3:

(i) sequence signature

(A) length: 314 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:3:

Met Phe Gln Pro Ala Gly His Gly Gln Asp Trp Ala Met Glu Gly Pro

1 5 10 15

Arg Asp Gly Leu Lys Lys Glu Arg Leu Val Asp Asp Arg His Asp Ser

20 25 30

Gly Leu Asp Ser Met Lys Asp Glu Glu Tyr Glu Gln Met Val Lys Glu

35 40 45

Leu Arg Glu Ile Arg Leu Gln Pro Gln Glu Ala Pro Leu Ala Ala Glu

50 55 60

Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu

65 70 75 80

Ala Ile Ile His Glu Glu Lys Pro Leu Thr Met Glu Val Ile Gly Gln

85 90 95

Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln

100 105 110

Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Gly Ile Ala Glu

115 120 125

Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly

130 135 140

Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val

145 150 155 160

Ala Val Leu Thr Gln Thr Cys Thr Pro Gln His Leu His Ser Val Leu

165 170 175

Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Thr

180 185 190

His Gly Tyr Leu Ala Ile Val Glu His Leu Val Thr Leu Gly Ala Asp

195 200 205

Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala

210 215 220

Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly

225 230 235 240

Ala Asp Val Asr Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu

245 250 255

Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu

260 265 270

Thr Leu Glu Asn Leu Gln Met Leu Pro Glu Ser Glu Asp Glu Glu Ser

275 280 285

Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp

290 295 300

Cys Val Phe Gly Gly Gln Arg Leu Thr Leu

305 310

(2) about the information of SEQ ID NO:4:

(i) sequence signature

(A) length: 314 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:4:

Met Phe Gln Pro Ala Gly His Gly Gln Asp Trp Ala Met Glu Gly Pro

1 5 10 15

Arg Asp Gly Leu Lys Lys Glu Arg Leu Val Asp Asp Arg His Asp Ser

20 25 30

Gly Leu Asp Ser Met Lys Asp Glu Asp Tyr Glu Gln Met Val Lys Glu

35 40 45

Leu Arg Glu Ile Arg Leu Gln Pro Gln Glu Ala Pro Leu Ala Ala Glu

50 55 60

Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu

65 70 75 80

Ala Ile Ile His Glu Glu Lys Thr Leu Thr Met Glu Val Ile Gly Gln

85 90 95

Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln

100 105 110

Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Gly Ile Ala Glu

115 120 125

Ala Leu Leu Lys Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly

130 135 140

Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val

145 150 155 160

Ala Val Leu Thr Gln Thr Cys Thr Pro Gln His Leu His Ser Val Leu

165 170 175

Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile

180 185 190

His Gly Tyr Leu Gly Ile Val Glu His Leu Val Thr Leu Gly Ala Asp

195 200 205

Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala

210 215 220

Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly

225 230 235 240

Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu

245 250 255

Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu

260 265 270

Thr Leu Glu Asn Leu Gln Thr Leu Pro Glu Ser Glu Asp Glu Glu Ser

275 280 285

Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp

290 295 300

Cys Val Phe Gly Gly Gln Arg Leu Thr Leu

305 310

(2) about the information of SEQ ID NO:5:

(i) sequence signature

(A) length: 314 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:5:

Met Phe Gln Pro Ala Glu Pro Gly Gln Glu Trp Ala Met Glu Gly Pro

1 5 10 15

Arg Asp Ala Leu Lys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Ser

20 25 30

Gly Leu Asp Ser Met Lys Asp Glu Glu Tyr Glu Gln Met Val Lys Glu

35 40 45

Leu Arg Glu Ile Arg Leu Glu Pro Gln Glu Ala Pro Arg Gly Ala Glu

50 55 60

Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu

65 70 75 80

Ala Ile Ile His Glu Glu Lys Ala Leu Thr Met Glu Val Val Arg Gln

85 90 95

Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln

100 105 110

Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Glu Ile Ala Glu

115 120 125

Ala Leu Leu Glu Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly

130 135 140

Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val

145 150 155 160

Gly Val Leu Thr Gln Pro Arg Gly Thr Gln His Leu His Ser Ile Leu

165 170 175

Gln Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile

180 185 190

His Gly Tyr Leu Gly Ile Val Glu Leu Leu Val Ser Leu Gly Ala Asp

195 200 205

Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala

210 215 220

Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly

225 230 235 240

Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu

245 250 255

Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu

260 265 270

Thr Leu Glu Asn Leu Gln Met Leu Pro Glu Ser Glu Asp GLu Glu Ser

275 280 285

Tyr Asp Thr Glu Ser Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp

290 295 300

Cys Val Leu Gly Gly Gln Arg Leu Thr Leu

305 310

(2) about the information of SEQ ID NO:6:

(i) sequence signature

(A) length: 2011 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: cDNA

(vi) primary source:

(A) biology: Arabidopis thaliana

(ix) feature:

(A) title/key: misc_feature

(B) position: 1..2011

(D) out of Memory :/remarks=" the cDNA sequence of NIM1 "

(ix) feature:

(A) title/key: CDS

(B) position: 43..1824

(D) out of Memory :/product=" NIM1 albumen "

(xi) sequence description: SEQ ID NO:6:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr

1

ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102

Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val

5 10 15 20

GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150

Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln

25 30 35

GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198

Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser

40 45 50

TTC GAA TCC GTC TTT GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246

Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys

55 60 65

CTT GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294

Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu

70 75 80

TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342

Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys

85 90 95 100

GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390

Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu

105 110 115

ATT GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG 438

Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu

120 125 130

GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT 486

Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser

135 140 145

GAA TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG 534

Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val

150 155 160

GAT TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT 582

Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro

165 170 175 180

GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630

Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys

185 190 195

GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678

Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys

200 205 210

GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC 726

Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val

215 220 225

AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774

Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu

230 235 240

CTT GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG GTA 822

Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val

245 250 255 260

CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG 870

Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser

265 270 275

GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT 918

Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn

280 285 290

CTA GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG 966

Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val

295 300 305

AAG ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC CAT 1014

Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His

310 315 320

AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG 1062

Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys

325 330 335 340

GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110

Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser

345 350 355

GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC 1158

Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala

360 365 370

ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206

Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser

375 380 385

CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254

Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln GLu Asp Lys Arg

390 395 400

GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302

Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp

405 410 415 420

GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA GTT GCA CTT GCT 1350

Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala

425 430 435

CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398

Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu

440 445 450

ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446

Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg

455 460 465

CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT TTC 1494

Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe

470 475 480

AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542

Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr

485 490 495 500

GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590

Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp

505 510 515

CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA GAC 1638

Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp

520 525 530

GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA ATA 1686

Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu Ile

535 540 545

CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GGA 1734

Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly

550 555 560

AAT TTG TCC CTG ACA GAT TCG ACT TCT TCC ACA TCG AAA TCA ACC GGT 1782

Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr G1y

565 570 575 580

GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 1824

Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg *

585 590

GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 1884

TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 1944

ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 2004

ATTTGTA 2011

(2) about the information of SEQ ID NO:7:

(i) sequence signature

(A) length: 2011 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: cDNA

(ix) feature:

(A) title/key: CDS

(B) position: 43..1824

(D) out of Memory :/product=" NIM1 changes form "

/ remarks=" wild-type NIM1 gene product is changed into alanine residue at the serine residue of 55 and 59 amino acids "

(ix) feature:

(A) title/key: misc_feature

(B) position: 205..217

(D) out of Memory :/remarks=" compare with wild-type sequence, 205 and 217 Nucleotide become G from T "

(xi) sequence description: SEQ ID NO:7:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr

1

ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102

Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val

5 10 15 20

GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150

Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln

25 30 35

GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198

Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser

40 45 50

TTC GAA GCC GTC TTT GAC GCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246

Phe Glu Ala Val Phe Asp Ala Pro Asp Asp Phe Tyr Ser Asp Ala Lys

55 60 65

CTT GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294

Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu

70 75 80

TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342

Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys

85 90 95 100

GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390

Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu

105 110 115

ATT GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG 438

Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu

120 125 130

GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT 486

Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser

135 140 145

GAA TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG 534

Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val

150 155 160

GAT TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT 582

Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro

165 170 175 180

GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630

Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys

185 190 195

GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678

Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys

200 205 210

GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC 726

Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val

215 220 225

AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774

Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu

230 235 240

CTT GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG GTA 822

Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val

245 250 255 260

CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG 870

Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser

265 270 275

GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT 918

Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn

280 285 290

CTA GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG 966

Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val

295 300 305

AAG ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC CAT 1014

Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His

310 315 320

AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG 1062

Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys

325 330 335 340

GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110

Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser

345 350 355

GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC 1158

Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala

360 365 370

ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206

Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser

375 380 385

CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254

Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg

390 395 400

GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302

Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp

405 410 415 420

GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA GTT GCA CTT GCT 1350

Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala

425 430 435

CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398

Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu

440 445 450

ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446

Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg

455 460 465

CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT TTC 1494

Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe

470 475 480

AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542

Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr

485 490 495 500

GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590

Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp

505 510 515

CAG ATT ATG AAC TGT GAG GAC TTG ACT CAA CTG GCT TGC GGA GAA GAC 1638

Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala Cys Gly Glu Asp

520 525 530

GAC ACT GCT GAG AAA CGA CTA CAA AAG AAG CAA AGG TAC ATG GAA ATA 1686

Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg Tyr Met Glu Ile

535 540 545

CAA GAG ACA CTA AAG AAG GCC TTT AGT GAG GAC AAT TTG GAA TTA GGA 1734

Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn Leu Glu Leu Gly

550 555 560

AAT TTG TCC CTG ACA GAT TCG ACT TCT TCC ACA TCG AAA TCA ACC GGT 1782

Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser Lys Ser Thr Gly

565 570 575 580

GGA AAG AGG TCT AAC CGT AAA CTC TCT CAT CGT CGT CGG TGA 1824

Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg Arg *

585 590

GACTCTTGCC TCTTAGTGTA ATTTTTGCTG TACCATATAA TTCTGTTTTC ATGATGACTG 1884

TAACTGTTTA TGTCTATCGT TGGCGTCATA TAGTTTCGCT CTTCGTTTTG CATCCTGTGT 1944

ATTATTGCTG CAGGTGTGCT TCAAACAAAT GTTGTAACAA TTTGAACCAA TGGTATACAG 2004

ATTTGTA 2011

(2) about the information of SEQ ID NO:8:

(i) sequence signature

(A) length: 594 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:8:

Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser

1 5 10 15

Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu

20 25 30

Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu

35 40 45

Leu Ser Asn Ser Phe Glu Ala Val Phe Asp Ala Pro Asp Asp Phe Tyr

50 55 60

Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His

65 70 75 80

Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala

85 90 95

Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu

100 105 110

Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val

115 120 125

Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro

130 135 140

Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys

145 150 155 160

Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile

165 170 175

Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp

180 185 190

Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu

195 200 205

Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys

210 215 220

Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser

225 230 235 240

Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu

245 250 255

Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys

260 265 270

Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu

275 280 285

Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala

290 295 300

Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala

305 310 315 320

Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala

325 330 335

Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly

340 345 350

Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile

355 360 365

Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln

370 375 380

Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln

385 390 395 400

Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala

405 410 415

Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg

420 425 430

Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met

435 440 445

Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu

450 455 460

Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys

465 470 475 480

Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala

485 490 495

Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser

500 505 510

Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala

515 520 525

Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg

530 535 540

Tyr Met Glu Ile Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn

545 550 555 560

Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser

565 570 575

Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg

580 585 590

Arg *

(2) about the information of SEQ ID NO:9:

(i) sequence signature

(A) length: 1597 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: cDNA

(ix) feature:

(A) title/key: CDS

(B) position: 1..1410

(D) out of Memory :/product=" NIM1 changes form "

/ remarks=" compare deleted the N end with wild-type sequence "

(xi) sequence description: SEQ ID NO:9:

ATG GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC AGC AGA GTG 48

Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val

1 5 10 15

AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG AAT TGC TGC 96

Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys

20 25 30

CAC GTG GCT TGC CGG CCG GCG GTG GAT TTC ATG TTG GAG GTT CTC TAT 144

His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr

35 40 45

TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC TAT CAG AGG 192

Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg

50 55 60

CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT ATA GAG GAC ACA TTG GTT 240

His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val

65 70 75 80

ATA CTC AAG CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAG CTA TTG 288

Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu

85 90 95

GAT AGA TGT AAA GAG ATT ATT GTC AAG TCT AAT GTA GAT ATG GTT AGT 336

Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser

100 105 110

CTT GAA AAG TCA TTG CCG GAA GAG CTT GTT AAA GAG ATA ATT GAT AGA 384

Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg

115 120 125

CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG 432

Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser

130 135 140

AAT GTA CAT AAG GCA CTT GAC TCG GAT GAT ATT GAG TTA GTC AAG TTG 480

Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu

145 150 155 160

CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT GCG TGT GCT CTT CAT 528

Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His

165 170 175

TTC GCT GTT GCA TAT TGC AAT GTG AAG ACC GCA ACA GAT CTT TTA AAA 576

Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys

180 185 190

CTT GAT CTT GCC GAT GTC AAC CAT AGG AAT CCG AGG GGA TAT ACG GTG 624

Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val

195 200 205

CTT CAT GTT GCT GCG ATG CGG AAG GAG CCA CAA TTG ATA CTA TCT CTA 672

Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu

210 215 220

TTG GAA AAA GGT GCA AGT GCA TCA GAA GCA ACT TTG GAA GGT AGA ACC 720

Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr

225 230 235 240

GCA CTC ATG ATC GCA AAA CAA GCC ACT ATG GCG GTT GAA TGT AAT AAT 768

Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn

245 250 255

ATC CCG GAG CAA TGC AAG CAT TCT CTC AAA GGC CGA CTA TGT GTA GAA 816

Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu

260 265 270

ATA CTA GAG CAA GAA GAC AAA CGA GAA CAA ATT CCT AGA GAT GTT CCT 864

Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro

275 280 285

CCC TCT TTT GCA GTG GCG GCC GAT GAA TTG AAG ATG ACG CTG CTC GAT 912

Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp

290 295 300

CTT GAA AAT AGA GTT GCA CTT GCT CAA CGT CTT TTT CCA ACG GAA GCA 960

Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala

305 310 315 320

CAA GCT GCA ATG GAG ATC GCC GAA ATG AAG GGA ACA TGT GAG TTC ATA 1008

Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile

325 330 335

GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AAG AGA ACA TCA 1056

Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser

340 345 350

CCG GGT GTA AAG ATA GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT 1104

Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser

355 360 365

AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC 1152

Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe

370 375 380

CCG CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT GAG GAC TTG l200

Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu

385 390 395 400

ACT CAA CTG GCT TGC GGA GAA GAC GAC ACT GCT GAG AAA CGA CTA CAA 1248

Thr Gln Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln

405 410 415

AAG AAG CAA AGG TAC ATG GAA ATA CAA GAG ACA CTA AAG AAG GCC TTT 1296

Lys Lys Gln Arg Tyr Met Glu Ile Gln Glu Thr Leu Lys Lys Ala Phe

420 425 430

AGT GAG GAC AAT TTG GAA TTA GGA AAT TTG TCC CTG ACA GAT TCG ACT 1344

Ser Glu Asp Asn Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr

435 440 445

TCT TCC ACA TCG AAA TCA ACC GGT GGA AAG AGG TCT AAC CGT AAA CTC 1392

Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu

450 455 460

TCT CAT CGT CGT CGG TGA GACTCTTGCC TCTTAGTGTA ATTTTTGCTG 1440

Ser His Arg Arg Arg *

465 470

TACCATATAA TTCTGTTTTC ATGATGACTG TAACTGTTTA TGTCTATCGT TGGCGTCATA 1500

TAGTTTCGCT CTTCGTTTTG CATCCTGTGT ATTATTGCTG CAGGTGTGCT TCAAACAAAT 1560

GTTGTAACAA TTTGAACCAA TGGTATACAG ATTTGTA 1597

(2) about the information of SEQ ID NO:10:

(i) sequence signature

(A) length: 470 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:10:

Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val

1 5 10 15

Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys

20 25 30

His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr

35 40 45

Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg

50 55 60

His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val

65 70 75 80

Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu

85 90 95

Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser

100 105 110

Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg

115 120 125

Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser

130 135 140

Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu

145 150 155 160

Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His

165 170 175

Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys

180 185 190

Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val

195 200 205

Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu

210 215 220

Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr

225 230 235 240

Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn

245 250 255

Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu

260 265 270

Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro

275 280 285

Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp

290 295 300

Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala

305 310 315 320

Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile

325 330 335

Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser

340 345 350

Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser

355 360 365

Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe

370 375 380

Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu

385 390 395 400

Thr Gln Leu Ala Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln

405 410 415

Lys Lys Gln Arg Tyr Met Glu Ile Gln Glu Thr Leu Lys Lys Ala Phe

420 425 430

Ser GLu Asp Asn Leu Glu Leu Gly Asn Leu Ser Leu Thr Asp Ser Thr

435 440 445

Ser Ser Thr Ser Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu

450 455 460

Ser His Arg Arg Arg *

465 470

(2) about the information of SEQ ID NO:11:

(i) sequence signature

(A) length: 1608 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: cDNA

(ix) feature:

(A) title/key: CDS

(B) position: 43..1608

(D) out of Memory :/product=" NIM1 changes form "/remarks=" compare deleted the N end with wild-type sequence "

(xi) sequence description: SEQ ID NO:11:

GATCTCTTTA ATTTGTGAAT TTCAATTCAT CGGAACCTGT TG ATG GAC ACC ACC 54

Met Asp Thr Thr

1

ATT GAT GGA TTC GCC GAT TCT TAT GAA ATC AGC AGC ACT AGT TTC GTC 102

Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser Thr Ser Phe Val

5 10 15 20

GCT ACC GAT AAC ACC GAC TCC TCT ATT GTT TAT CTG GCC GCC GAA CAA 150

Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu Ala Ala Glu Gln

25 30 35

GTA CTC ACC GGA CCT GAT GTA TCT GCT CTG CAA TTG CTC TCC AAC AGC 198

Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu Leu Ser Asn Ser

40 45 50

TTC GAA TCC GTC TTT GAC TCG CCG GAT GAT TTC TAC AGC GAC GCT AAG 246

Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr Ser Asp Ala Lys

55 60 65

CTT GTT CTC TCC GAC GGC CGG GAA GTT TCT TTC CAC CGG TGC GTT TTG 294

Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His Arg Cys Val Leu

70 75 80

TCA GCG AGA AGC TCT TTC TTC AAG AGC GCT TTA GCC GCC GCT AAG AAG 342

Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala Ala Ala Lys Lys

85 90 95 100

GAG AAA GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG 390

Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu

105 110 115

ATT GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG 438

Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu

120 125 130

GCT TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT 486

Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser

135 140 145

GAA TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG 534

Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val

150 155 160

GAT TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT 582

Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro

165 170 175 180

GAA TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA 630

Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys

185 190 195

GTT GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT 678

Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys

200 205 210

GGT AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC 726

Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val

215 220 225

AAG TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG 774

Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu

230 235 240

CTT GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG GTA 822

Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val

245 250 255 260

CCT AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG 870

Pro Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser

265 270 275

GAT GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT 918

Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn

280 285 290

CTA GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG 966

Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val

295 300 305

AAG ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC CAT 1014

Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His

310 315 320

AGG AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG 1062

Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys

325 330 335 340

GAG CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA 1110

Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser

345 350 355

GAA GCA ACT TTG GAA GGT AGA ACC GCA CTC ATG ATC GCA AAA CAA GCC 1158

Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile Ala Lys Gln Ala

360 365 370

ACT ATG GCG GTT GAA TGT AAT AAT ATC CCG GAG CAA TGC AAG CAT TCT 1206

Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln Cys Lys His Ser

375 380 385

CTC AAA GGC CGA CTA TGT GTA GAA ATA CTA GAG CAA GAA GAC AAA CGA 1254

Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln Glu Asp Lys Arg

390 395 400

GAA CAA ATT CCT AGA GAT GTT CCT CCC TCT TTT GCA GTG GCG GCC GAT 1302

Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala Val Ala Ala Asp

405 410 415 420

GAA TTG AAG ATG ACG CTG CTC GAT CTT GAA AAT AGA GTT GCA CTT GCT 1350

Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg Val Ala Leu Ala

425 430 435

CAA CGT CTT TTT CCA ACG GAA GCA CAA GCT GCA ATG GAG ATC GCC GAA 1398

Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met Glu Ile Ala Glu

440 445 450

ATG AAG GGA ACA TGT GAG TTC ATA GTG ACT AGC CTC GAG CCT GAC CGT 1446

Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu Glu Pro Asp Arg

455 460 465

CTC ACT GGT ACG AAG AGA ACA TCA CCG GGT GTA AAG ATA GCA CCT TTC 1494

Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys Ile Ala Pro Phe

470 475 480

AGA ATC CTA GAA GAG CAT CAA AGT AGA CTA AAA GCG CTT TCT AAA ACC 1542

Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala Leu Ser Lys Thr

485 490 495 500

GTG GAA CTC GGG AAA CGA TTC TTC CCG CGC TGT TCG GCA GTG CTC GAC 1590

Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser Ala Val Leu Asp

505 510 515

CAG ATT ATG AAC TGT TGA 1608

Gln Ile Met Asn Cys *

520

(2) about the information of SEQ ID NO:12:

(i) sequence signature

(A) length: 522 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:12:

Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser

1 5 10 15

Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu

20 25 30

Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu

35 40 45

Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr

50 55 60

Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His

65 70 75 80

Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala

85 90 95

Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu

100 105 110

Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val

115 120 125

Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro

130 135 140

Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys

145 150 155 160

Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile

165 170 175

Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp

180 185 190

Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu

195 200 205

Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys

210 215 220

Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser

225 230 235 240

Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu

245 250 255

Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys

260 265 270

Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu

275 280 285

Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala

290 295 300

Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala

305 310 315 320

Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala

325 330 335

Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly

340 345 350

Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile

355 360 365

Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln

370 375 380

Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln

385 390 395 400

Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala

405 410 415

Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg

420 425 430

Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met

435 440 445

Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu

450 455 460

Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys

465 470 475 480

Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala

485 490 495

Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser

500 505 510

Ala Val Leu Asp Gln Ile Met Asn Cys *

515 520

(2) about the information of SEQ ID NO:13:

(i) sequence signature

(A) length: 1194 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: cDNA

(ix) feature:

(A) title/key: CDS

(B) position: 1..1194

(D) out of Memory :/product=" NIM1 changes form "/remarks=" N end/C holds mosaic "

(xi) sequence description: SEQ ID NO:13:

ATG GAT TCG GTT GTG ACT GTT TTG GCT TAT GTT TAC AGC AGC AGA GTG 48

Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val

1 5 10 15

AGA CCG CCG CCT AAA GGA GTT TCT GAA TGC GCA GAC GAG AAT TGC TGC 96

Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys

20 25 30

CAC GTG GCT TGC CGG CCG GCG GTG GAT TTC ATG TTG GAG GTT CTC TAT 144

His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr

35 40 45

TTG GCT TTC ATC TTC AAG ATC CCT GAA TTA ATT ACT CTC TAT CAG AGG 192

Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg

50 55 60

CAC TTA TTG GAC GTT GTA GAC AAA GTT GTT ATA GAG GAC ACA TTG GTT 240

His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val

65 70 75 80

ATA CTC AAG CTT GCT AAT ATA TGT GGT AAA GCT TGT ATG AAG CTA TTG 288

Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu

85 90 95

GAT AGA TGT AAA GAG ATT ATT GTC AAG TCT AAT GTA GAT ATG GTT AGT 336

Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser

100 105 110

CTT GAA AAG TCA TTG CCG GAA GAG CTT GTT AAA GAG ATA ATT GAT AGA 384

Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg

115 120 125

CGT AAA GAG CTT GGT TTG GAG GTA CCT AAA GTA AAG AAA CAT GTC TCG 432

Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser

130 135 140

AAT GTA CAT AAG GCA CTT GAC TCG GAT GAT ATT GAG TTA GTC AAG TTG 480

Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu

145 150 155 160

CTT TTG AAA GAG GAT CAC ACC AAT CTA GAT GAT GCG TGT GCT CTT CAT 528

Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His

165 170 175

TTC GCT GTT GCA TAT TGC AAT GTG AAG ACC GCA ACA GAT CTT TTA AAA 576

Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys

180 185 190

CTT GAT CTT GCC GAT GTC AAC CAT AGG AAT CCG AGG GGA TAT ACG GTG 624

Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val

195 200 205

CTT CAT GTT GCT GCG ATG CGG AAG GAG CCA CAA TTG ATA CTA TCT CTA 672

Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu

210 215 220

TTG GAA AAA GGT GCA AGT GCA TCA GAA GCA ACT TTG GAA GGT AGA ACC 720

Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr

225 230 235 240

GCA CTC ATG ATC GCA AAA CAA GCC ACT ATG GCG GTT GAA TGT AAT AAT 768

Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn

245 250 255

ATC CCG GAG CAA TGC AAG CAT TCT CTC AAA GGC CGA CTA TGT GTA GAA 816

Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu

260 265 270

ATA CTA GAG CAA GAA GAC AAA CGA GAA CAA ATT CCT AGA GAT GTT CCT 864

Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro

275 280 285

CCC TCT TTT GCA GTG GCG GCC GAT GAA TTG AAG ATG ACG CTG CTC GAT 912

Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp

290 295 300

CTT GAA AAT AGA GTT GCA CTT GCT CAA CGT CTT TTT CCA ACG GAA GCA 960

Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala

305 310 315 320

CAA GCT GCA ATG GAG ATC GCC GAA ATG AAG GGA ACA TGT GAG TTC ATA 1008

Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile

325 330 335

GTG ACT AGC CTC GAG CCT GAC CGT CTC ACT GGT ACG AAG AGA ACA TCA 1056

Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser

340 345 350

CCG GGT GTA AAG ATA GCA CCT TTC AGA ATC CTA GAA GAG CAT CAA AGT 1104

Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser

355 360 365

AGA CTA AAA GCG CTT TCT AAA ACC GTG GAA CTC GGG AAA CGA TTC TTC 1152

Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe

370 375 380

CCG CGC TGT TCG GCA GTG CTC GAC CAG ATT ATG AAC TGT TGA 1194

Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys *

385 390 395

(2) about the information of SEQ ID NO:14:

(i) sequence signature

(A) length: 398 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:14:

Met Asp Ser Val Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val

1 5 10 15

Arg Pro Pro Pro Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys

20 25 30

His Val Ala Cys Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr

35 40 45

Leu Ala Phe Ile Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg

50 55 60

His Leu Leu Asp Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val

65 70 75 80

Ile Leu Lys Leu Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu

85 90 95

Asp Arg Cys Lys Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser

100 105 110

Leu Glu Lys Ser Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg

115 120 125

Arg Lys Glu Leu Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser

130 135 140

Asn Val His Lys Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu

145 150 155 160

Leu Leu Lys Glu Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His

165 170 175

Phe Ala Val Ala Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys

180 185 190

Leu Asp Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val

195 200 205

Leu His Val Ala Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu

210 215 220

Leu Glu Lys Gly Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr

225 230 235 240

Ala Leu Met Ile Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn

245 250 255

Ile Pro Glu Gln Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu

260 265 270

Ile Leu Glu Gln Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro

275 280 285

Pro Ser Phe Ala Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp

290 295 300

Leu Glu Asn Arg Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala

305 310 315 320

Gln Ala Ala Met Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile

325 330 335

Val Thr Ser Leu Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser

340 345 350

Pro Gly Val Lys Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser

355 360 365

Arg Leu Lys Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe

370 375 380

Pro Arg Cys Ser Ala Val Leu Asp Gln Ile Met Asn Cys *

385 390 395

(2) about the information of SEQ ID NO:15:

(i) sequence signature

(A) length: 786 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: cDNA

(ix) feature:

(A) title/key: CDS

(B) position: 1..786

(D) out of Memory :/product=" NIM1 changes form "/remarks=" the ankyrin structural domain of NIM1 "

(xi) sequence description: SEQ ID NO:15:

ATG GAC TCC AAC AAC ACC GCC GCC GTG AAG CTC GAG CTT AAG GAG ATT 48

Met Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile

1 5 10 15

GCC AAG GAT TAC GAA GTC GGT TTC GAT TCG GTT GTG ACT GTT TTG GCT 96

Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala

20 25 30

TAT GTT TAC AGC AGC AGA GTG AGA CCG CCG CCT AAA GGA GTT TCT GAA 144

Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu

35 40 45

TGC GCA GAC GAG AAT TGC TGC CAC GTG GCT TGC CGG CCG GCG GTG GAT 192

Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp

50 55 60

TTC ATG TTG GAG GTT CTC TAT TTG GCT TTC ATC TTC AAG ATC CCT GAA 240

Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu

65 70 75 80

TTA ATT ACT CTC TAT CAG AGG CAC TTA TTG GAC GTT GTA GAC AAA GTT 288

Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val

85 90 95

GTT ATA GAG GAC ACA TTG GTT ATA CTC AAG CTT GCT AAT ATA TGT GGT 336

Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly

100 105 110

AAA GCT TGT ATG AAG CTA TTG GAT AGA TGT AAA GAG ATT ATT GTC AAG 384

Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys

115 120 125

TCT AAT GTA GAT ATG GTT AGT CTT GAA AAG TCA TTG CCG GAA GAG CTT 432

Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu

130 135 140

GTT AAA GAG ATA ATT GAT AGA CGT AAA GAG CTT GGT TTG GAG GTA CCT 480

Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro

145 150 155 160

AAA GTA AAG AAA CAT GTC TCG AAT GTA CAT AAG GCA CTT GAC TCG GAT 528

Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp

165 170 175

GAT ATT GAG TTA GTC AAG TTG CTT TTG AAA GAG GAT CAC ACC AAT CTA 576

Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu

180 185 190

GAT GAT GCG TGT GCT CTT CAT TTC GCT GTT GCA TAT TGC AAT GTG AAG 624

Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys

195 200 205

ACC GCA ACA GAT CTT TTA AAA CTT GAT CTT GCC GAT GTC AAC CAT AGG 672

Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg

210 215 220

AAT CCG AGG GGA TAT ACG GTG CTT CAT GTT GCT GCG ATG CGG AAG GAG 720

Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu

225 230 235 240

CCA CAA TTG ATA CTA TCT CTA TTG GAA AAA GGT GCA AGT GCA TCA GAA 768

Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu

245 250 255

GCA ACT TTG GAA GGT TGA 786

Ala Thr Leu Glu Gly *

260

(2) about the information of SEQ ID NO:16:

(i) sequence signature

(A) length: 262 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:16:

Met Asp Ser Asn Asn Thr Ala Ala Val Lys Leu Glu Leu Lys Glu Ile

1 5 10 15

Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val Val Thr Val Leu Ala

20 25 30

Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro Lys Gly Val Ser Glu

35 40 45

Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys Arg Pro Ala Val Asp

50 55 60

Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile Phe Lys Ile Pro Glu

65 70 75 80

Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp Val Val Asp Lys Val

85 90 95

Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu Ala Asn Ile Cys Gly

100 105 110

Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys Glu Ile Ile Val Lys

115 120 125

Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser Leu Pro Glu Glu Leu

130 135 140

Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu Gly Leu Glu Val Pro

145 150 155 160

Lys Val Lys Lys His Val Ser Asn Val His Lys Ala Leu Asp Ser Asp

165 170 175

Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu Asp His Thr Asn Leu

180 185 190

Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala Tyr Cys Asn Val Lys

195 200 205

Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala Asp Val Asn His Arg

210 215 220

Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala Ala Met Arg Lys Glu

225 230 235 240

Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly Ala Ser Ala Ser Glu

245 250 255

Ala Thr Leu Glu Gly *

260

(2) about the information of SEQ ID NO:17:

(i) sequence signature

(A) length: 41 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:17:

Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val

1 5 10 15

Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala

20 25 30

Val His Tyr Ala Val Gln His Cys Asn

35 40

(2) about the information of SEQ ID NO:18:

(i) sequence signature

(A) length: 38 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:18:

Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro

1 5 10 15

Asp Met Val Ser Val Leu Leu Asp His His Ala Asp Xaa Asn Phe Arg

20 25 30

Thr Xaa Asp Gly Val Thr

35

(2) about the information of SEQ ID NO:19:

(i) sequence signature

(A) length: 41 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:18:

Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp lle Glu Leu Val

1 5 10 15

Lys Leu Met Val Met Gly Glu Giy Leu Asp Leu Asp Asp Ala Leu Ala

20 25 30

Val His Tyr Ala Val Gln His Cys Asn

35 40

(2) about the information of SEQ ID NO:20:

(i) sequence signature

(A) length: 27 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:20:

Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val

1 5 10 15

Ser Pro Asp Met Val Ser Val Leu Leu Asp Gln

20 25

(2) about the information of SEQ ID NO:21:

(i) sequence signature

(A) length: 41 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:21:

Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val

1 5 10 15

Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala

20 25 30

Val His Tyr Ala Val Gln His Cys Asn

35 40

(2) about the information of SEQ ID NO:22:

(i) sequence signature

(A) length: 27 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:22:

Arg Arg Pro Asp Ser Lys Thr Ala Leu His Leu Ala Ala Glu Met Val

1 5 10 15

Ser Pro Asp Met Val Ser Val Leu Leu Asp Gln

20 25

(2) about the information of SEQ ID NO:23:

(i) sequence signature

(A) length: 41 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:23:

Ile Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val

1 5 10 15

Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Asp Ala Leu Ala

20 25 30

Val His Tyr Ala Val Gln His Cys Asn

35 40

(2) about the information of SEQ ID NO:24:

(i) sequence signature

(A) length: 19 amino acid

(B) type: amino acid

(C) chain: irrelevant

(D) topological framework: irrelevant

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:24:

Pro Thr Gly Lys Thr Ala Leu His Leu Ala Ala Glu Met Val Ser Pro

1 5 10 15

Asp Met Val

(2) about the information of SEQ ID NO:25:

(i) sequence signature

(A) length: 35 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: other nucleic acid

(A) describe :/desc=" oligonucleotide "

(xi) sequence description: SEQ ID NO:25:

CAACAGCTTC GAAGCCGTCT TTGACGCGCC GGATG 35

(2) about the information of SEQ ID NO:26:

(i) sequence signature

(A) length: 35 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: other nucleic acid

(A) describe :/desc=" oligonucleotide "

(xi) sequence description: SEQ ID NO:26:

CATCCGGCGC GTCAAAGACG GCTTCGAAGC TGTTG 35

(2) about the information of SEQ ID NO:27:

(i) sequence signature

(A) length: 32 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: other nucleic acid

(A) describe :/desc=" oligonucleotide "

(xi) sequence description: SEQ ID NO:27:

GGAATTCAAT GGATTCGGTT GTGACTGTTT TG 32

(2) about the information of SEQ ID NO:28:

(i) sequence signature

(A) length: 28 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: other nucleic acid

(A) describe :/desc=" oligonucleotide "

(xi) sequence description: SEQ ID NO:28:

GGAATTCTAC AAATCTGTAT ACCATTGG 28

(2) about the information of SEQ ID NO:29:

(i) sequence signature

(A) length: 31 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: other nucleic acid

(A) describe :/desc=" oligonucleotide "

(xi) sequence description: SEQ ID NO:29:

CGGAATTCGA TCTCTTTAAT TTGTGAATTT C 31

(2) about the information of SEQ ID NO:30:

(i) sequence signature

(A) length: 29 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: other nucleic acid

(A) describe :/desc=" oligonucleotide "

(xi) sequence description: SEQ ID NO:30:

GGAATTCTCA ACAGTTCATA ATCTGGTCG 29

(2) about the information of SEQ ID NO:31:

(i) sequence signature

(A) length: 31 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: other nucleic acid

(A) describe :/desc=" oligonucleotide "

(xi) sequence description: SEQ ID NO:31:

GGAATTCAAT GGACTCCAAC AACACCGCCG C 31

(2) about the information of SEQ ID NO:32:

(i) sequence signature

(A) length: 33 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: other nucleic acid

(A) describe :/desc=" oligonucleotide "

(xi) sequence description: SEQ ID NO:32:

GGAATTCTCA ACCTTCCAAA GTTGCTTCTG ATG 33

Claims

1. avoid the method for pathogenic agent invasion and attack by collaborative disease resistance protective plant, may further comprise the steps:

(a) provide the immunomodulatory plant with one-level disease resistance, wherein said immunomodulatory plant is

(i) composing type immunity mutant plant; Or

(ii) obtain by recombinant expressed SAR gene in plant, wherein said SAR gene is the NIM1 gene of the proteic functional form of coding NIM1, causes the signal transduction cascade reaction of systemic acquired resistance in this NIM1 albumen involved in plant;

And

Described thus microbicide is given collaborative enhanced three grade disease resistances bigger than the horizontal summation of firsts and seconds disease resistance to using of described immunomodulatory plant.

2. method according to claim 1, wherein said composing type immunity mutant plant is selected from plant population according to the following step:

(a) assessment SAR gene is infected expression in the plant normally in phenotype, and wherein said plant of being infected lacks damage simulation phenotype, and

3. method according to claim 1, wherein said NIM1 albumen comprises the aminoacid sequence shown in the SEQ ID NO:2.

4. method according to claim 1, wherein said NIM1 gene comprises the encoding sequence shown in the SEQ ID NO:1.

5. according to any described method among the claim 1-4, wherein said microbicide is the mycocide that is selected from following colony:

The 4-3[(4-chloro-phenyl-)-and 3-(3, the 4-Dimethoxyphenyl) acryloyl] morpholine;

The 5-methyl isophthalic acid, 2,4-triazole [3,4-b] [1,3] benzothiazole;

3-allyloxy-1,2-[4-morpholinodithio-1,1-dioxide;

α-[2-(4-chloro-phenyl-) ethyl]-α-(1, the 1-dimethyl ethyl)-1H-1,2,4-triazole-1-ethanol;

1-[3-(2-chloro-phenyl-)-2-(4-fluorobenzene) oxyethane-2-yl] methyl]-1H-1,2, the 4-triazole;

α-(4-chloro-phenyl-)-α-(1-ring sec.-propyl ethyl)-1H-1,2,4-triazole-1-ethanol;

5-(4-chloro-phenyl-)-2,2-dimethyl-1-(1H-1,2,4-triazol-1-yl methyl)-cyclopentanol;

2-(2,4 dichloro benzene base)-3-(1H-1,2,4-triazol-1-yl)-propyl group-1,1,2,2-tetrafluoro ether;

Methyl-(E)-2-{2-[6-(2-cyanogen phenoxy group) pyrimidine-4-base oxygen base] phenyl }-3-methoxy acrylate;

Methyl-(E)-2-methoxyimino-2-[α-(O-tolyloxy)-O-tolyl] acetic ester;

2-(2-phenoxy phenyl)-(E)-2-methoxyimino-N-methylacetamide;

[2-(2,5-dimethyl Phenoxymethyl)-phenyl)]-(E)-2-methoxyimino-N-methylacetamide;

(1R, 3S/1S, 3R)-2,2-two chloro-N-[(R)-1-(4-chlorine (phenyl) ethyl]-1-ethyl-3-methyl cyclopropane methane amide;

Two (dithiocarbamic acid) manganese polymer-zinc complexes of ethylene;

1-[2-(2,4 dichloro benzene base)-4-propyl group-1,3-dioxolane-2-ylmethyl]-1H-1,2, the 4-triazole;

1-{2-[2-chloro-4-(4-chlorophenoxy) phenyl]-the 4-methyl isophthalic acid, 3-dioxolane-2-ylmethyl)-1H-1,2, the 4-triazole;

1-[2-(2,4 dichloro benzene base) amyl group-1H-1,2, the 4-triazole;

Suitable-4-[3-(4-tert-butyl-phenyl)-2-methyl-propyl]-2, the 6-thebaine;

1-[3-(4-tert-butyl-phenyl)-2-methyl-propyl] piperidines;

4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidine ammonia;

(RS)-and N-(2,6-3,5-dimethylphenyl-N-(methoxy ethanoyl)-alanine methyl ester;

(R)-and N-(2,6-3,5-dimethylphenyl-N-(methoxy ethanoyl)-alanine methyl ester;

1,2,5,6-tetrahydrochysene-4H-pyrrolo-[3,2,1-ij] quinoline-4-ketone; With phosphoric acid hydrogen ethyl ester.

6. method according to claim 5, wherein said mycocide be (RS)-N-(2,6-3,5-dimethylphenyl-N-(methoxy ethanoyl)-alanine methyl ester.

7. according to any described method among the claim 1-6, wherein said microbicide is diazosulfide compound, iso-nicotinic acid compound or salicylic acid compound.

8. according to any described method among the claim 1-7, wherein two kinds of microbicides are applied to described immunomodulatory plant simultaneously.

9. method according to claim 8, wherein said microbicide are the mycocide that is selected from following colony:

The 5-methyl isophthalic acid, 2,4-triazole [3,4-b] [1,3] benzothiazole;

3-allyloxy-1,2-[4-morpholinodithio-1,1-dioxide;

Methyl-(E)-2-methoxyimino-2-[α-(O-tolyloxy)-O-tolyl] acetic ester;

2-(2-phenoxy phenyl)-(E)-2-methoxyimino-N-methylacetamide;

[2-(2,5-dimethyl Phenoxymethyl)-phenyl)]-(E)-2-methoxyimino-N-methylacetamide;

(1R, 3S/1S, 3R)-2,2-two chloro-N-[(R)-1-(4-chlorine (phenyl) ethyl] 1-ethyl-3-methyl cyclopropane methane amide;

Two (dithiocarbamic acid) manganese polymer-zinc complexes of ethylene;

1-[2-(2,4 dichloro benzene base) amyl group-1H-1,2, the 4-triazole;

Suitable-4-[3-(4-tert-butyl-phenyl)-2-methyl-propyl]-2, the 6-thebaine;

1-[3-(4-tert-butyl-phenyl)-2-methyl-propyl] piperidines;

4-cyclopropyl-6-methyl-N-phenyl-2-pyrimidine ammonia;

(RS)-and N-(2,6-3,5-dimethylphenyl-N-(methoxy ethanoyl)-alanine methyl ester;

(R)-and N-(2,6-3,5-dimethylphenyl-N-(methoxy ethanoyl)-alanine methyl ester;

And another kind of microbicide is diazosulfide compound, iso-nicotinic acid compound or salicylic acid compound.

10. method according to claim 9, wherein mycocide be (RS)-N-(2,6-3,5-dimethylphenyl-N-(methoxy ethanoyl)-alanine methyl ester, another kind of microbicide is the diazosulfide compound.