CN101056981A - Method for down-regulating gene expression in fungi - Google Patents

Abstract

The present invention concerns methods for controlling fungus infestation via dsRNA mediated gene silencing, whereby the intact fungus cell(s) are contacted with a double-stranded RNA from outside the fungal cell(s) and whereby the double-stranded RNA is taken up by the intact fungal cell(s). In one particular embodiment, the methods of the invention are used to alleviate plants from fungus pests. Alternatively, the methods are used for treating and/or preventing fungal infestation on a substrate or a subject in need of such treatment and/or prevention. Suitable fungal target genes and fragments thereof, dsRNA constructs, recombinant constructs and compositions are disclosed.

Description

Negative method of regulating genetic expression in the fungi

Technical field

The present invention relates to control the method that fungi grows on cell or organism, method of prevention fungal infection cell or organism and the negative method of regulating genetic expression in the fungi of use double-stranded RNA.The present invention also relates to the resistance of transgenic plant to fungal infection.

Background technology

RNA disturbs or " RNAi " is by expressing the method (Fire of (being also referred to as " gene silencing " or " gene silencing of RNA mediation ") with treating a kind of sequence-specific negative regulator gene that the negative target gene region sequence complementary double-stranded RNA of regulating (dsRNA) starts, A.TrendsGenet.Vol.15,358-363,1999; Sharp, P.A.Genes Dev.Vol.15,485-490,2001).

In the past several years rely on RNA to disturb (RNAi) negative adjusting to target gene in multicellular organisms to become a kind of very perfect technology.Generally speaking, RNAi comprises organism is contacted with having corresponding to the double-stranded RNA fragment for the treatment of negative regulator gene (" target gene ") sequence to small part (perhaps as the complementary single stranded RNAs of two annealed or as the hair clip construct).General introduction for the RNAi technology can be with reference to International Application No. WO 99/32619 (Carnegie Institute of Washington), International Application No. WO 99/53050 (Benitec) and Fire et al., Nature, Vol.391, pp.806-811, February 1998.

In nematode, can be by with the RNAi fragment or comprise the segmental bacterial isolates of RNAi or just can the segmental bacterial isolates of expressed rna i feed nematode once nematode picked-up and implement RNAi.For this so-called " RNAi through feeding " is described, can (see with reference to International Application No. WO 00/01846, the 1998 East Coast Worm Meetingabstract 180-Timmons and Fire that submit to by the applicant Www.elegans.swmed.edu/wli/[ecwm98p180]/) and can be with reference to WO99/32619.

The means as protective plant opposing plant nematode with RNAi have also been proposed; promptly by in plant (for example in whole plants or in part, tissue or the cell of plant) express one or more form corresponding to plant nematode in the segmental nucleotide sequence of dsRNA of target gene, wherein said target gene is essential for growth, breeding and/or the survival of this plant nematode.Explanation for this technology can be with reference to United States Patent (USP) 6,506,559 (based on WO 99/32619), the 11st is listed as the 55th row is listed as the 9th row and the 13rd to the 12nd and is listed as the 61st row and is listed as the 11st row to the 14th), by the 7th page of 11-8 capable and International Application No. WO 01/96584, WO 01/37654 and WO 03/052110 in the International Application No. WO 00/01846 of the applicant submission.

Elbashir et al. (Nature, 411,494-498,2001) is verified to use the long dsRNA fragment (being also referred to as siRNA s or siRNAs) of 21 Nucleotide to carry out the gene silencing of RNAi mediation effectively in mammalian cell.These short siRNAs have proved effective and specific gene silencing, avoided the non-specific reduction of genetic expression of Interferon, rabbit mediation simultaneously, it is observed (Stark G.R.et al. in the situation of application greater than the long dsRNAs of 30bp in mammalian cell, Ann RevBiochem.1998,67:227-264; Manche, L et al., MoI Cell Biol., 1992,12:5238-5248).Therefore, in order in mammalian cell, to carry out the negative adjusting or the gene silencing of target gene specific, advised the work-around solution that RNAi is used as antisense technology.

Although the RNAi technology has been understood many years widely in the field of plant, nematode and mammalian cell, come negative regulator gene to express to be still up to today for application RNAi in fungi and know little about it.

Kadotani et al., (2003) Mol Plant Microbe Interac.16:769-776 has described at ascomycetous fungus rice blast fungus (Magnaporthe oryzae) (pathogenic bacterium of rice blast) and (had been called as Magnaporthe grisea in the past; Anamorph Pyricularia oryzae Cav. and Pyricularia grisae) in the gene silencing of the RNA mediation carried out according to mechanism with characterization of molecules consistent with RNAi.Expression by dsRNA in the fungal cell realizes gene silencing: use the DNA construct that can express double-stranded RNA to transform the fungi protoplastis in the laboratory, so double-stranded RNA is transcribed in the fungal cell.

Cogoni ﹠amp; Macino, (1999) Nature.399:166-169 have described in filamentous fungus Neuraspora crassa (Neurospora crassa) gene silencing by RNAi.Thereby realize gene silencing by in the fungal cell, transcribing double-stranded RNA with the transgenosis transformed eukaryotic mycetocyte that can express double-stranded RNA, permission.

The RNA that Liu et al., (2002) Genetics.160:463-470 have described in people pathomycete Cryptococcus neoformans (Cryptococcus neoformans) disturbs.Equally, by in culture, using the DNA construct transformed eukaryotic mycetocyte of coding double-stranded RNA, realize RNAi thereby make double-stranded RNA original position in the fungal cell transcribe.

These studies confirm that RNA interference path works in many different fungi kinds.Yet, up to now only by with DNA construct or the transgenosis transformed eukaryotic mycetocyte that can transcribe suitable dsRNA thus, in the fungal cell, transcribe dsRNA subsequently and realize RNAi.

It is useful need being used in the experimental research of RNAi technology in the laboratory of instructing the DNA construct transformed eukaryotic mycetocyte that dsRNA produces in the fungal cell; but be clear that very much it and be not suitable for the many potential application in practice of RNAi; for example need dsRNA is introduced among many fungal cells on a large scale or in the field; so that for example protective plant is resisted phytopathogenic fungi, or handle matrix (substrates) on a large scale and avoid fungal infection or be used for human or animal's treatment or the purposes medicinal or for animals of prevention fungal infection to protect it.

Summary of the invention

Nowadays the inventor has been found that, in fungi by making complete fungal cell (promptly, have complete cell walls) and double-stranded RNA is in the extracellular (promptly, outside at cell walls) contacts negative regulator gene expression specifically, wherein double-stranded RNA comprises the annealed complementary strand, and a chain wherein has the nucleotide sequence with the target nucleotide sequences corresponding (promptly with its at least a portion complementation) for the treatment of the negative fungi target gene of regulating.

It has surprisingly been found that when complete fungal cell contacted in the cell walls outside with double-stranded RNA, the fungal cell caused growth inhibiting amount absorption double-stranded RNA with enough specificitys.Thisly in fungi, avoided in order to introduce the transgenosis that can instruct double stranded rna expression in the fungal cell and to the needs of complex transformations step through the method for RNAi.Therefore, for fungi self, do not need, and particularly need not use non-natural operation transformed eukaryotic mycetocyte in order to be introduced in the DNA construct that instructs dsRNA to express in the fungal cell by genetic manipulation.Therefore, this technology is simple and has great application in practice, started a diverse field of using in the original in the prior art unpractical fungi of RNAi.

Method of the present invention can obtain application in practice in expecting the pathogenic or communicable any technical field that suppresses viability, growth, growth or the breeding of fungi or reduce fungi.Method of the present invention also in expectation specificity downward modulation fungi one or more expression of target gene branches showed off application in practice.Useful especially application in practice includes, but not limited to (1) protective plant opposing phytopathogenic fungi; (2) medicinal or for animals (for example the controlling, treat or prevent people's fungi infestation) in humans and animals; (3) opposing of protection material is by fungus-caused infringement; (4) protection perishables (such as food, seed etc.) opposing is by fungus-caused infringement; (5) gene function and needs control fungi and/or needs prevention the common any application by fungus-caused infringement aspect of the functional genomics in fungi to illustrate the fungi target gene.

According to one embodiment of the invention relate to be used to control among cell or the organism or on fungal growth or the be used to method of preventing to be subject to the fungal infection of the cell of fungal infection or organism, this method comprises makes the fungal cell contact with double-stranded RNA from fungal cell's outside, wherein double-stranded RNA comprises the annealed complementary strand, a chain wherein has the nucleotide sequence complementary nucleotide sequence to small part with the fungi target gene, and double-stranded RNA is ingested and enters the fungal cell and control growing or prevention are infected thus whereby.

According to other embodiments, the present invention relates to be used for negative method of regulating the fungi expression of target gene, this method comprises makes the fungal cell contact with the outside of double-stranded RNA the fungal cell, wherein double-stranded RNA comprises the annealed complementary strand, a chain wherein has and treats the negative fungi target gene of regulating partial nucleotide sequence complementary nucleotide sequence at least, and double-stranded RNA is ingested and enters fungal cell and the negative thus fungi target gene expression of regulating whereby.

According to an embodiment, method of the present invention depends on the double-stranded RNA picked-up that will be present in fungi outside (that is the outside of cell walls) and enters the fungal cell and do not need the expression of double-stranded RNA in the fungal cell.In addition, the present invention also comprises the aforesaid method that fungal cell wherein contacts with the composition that comprises this double-stranded RNA.

Described double-stranded RNA can be by protokaryon (such as but not limited to bacterium) or eucaryon (such as but not limited to yeast) host cell or host organisms expression.

According to another embodiment, method of the present invention depends on wherein the GMO method by cell fungal infection or that be subject to fungal infection or organism expressing double-stranded RNA.Preferably, described cell is that vegetable cell or described organism are plants.

Therefore the present invention also relates to and is used to produce the method that phytopathogenic fungi is had the plant of resistance, and it may further comprise the steps:

(a) with comprising the recombinant precursor transformed plant cells of at least a adjusting sequence, described adjusting sequence operably be connected to and be selected from target fungal gene nucleotide sequence in the group of forming by following nucleotide sequence to small part complementary sequence:

(i) at least 17, preferably at least 18,19,20 or 21, more preferably at least 22, the coding of 23 or 24 Nucleotide length and the nucleotide sequence of fungi target gene complementary RNA, described fungi target gene comprises with the sequence that is selected from following any sequence and has at least 75%, preferably at least 80%, 85%, 90%, or more preferably at least 95%, 98% or 99% conforming sequence: SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15,48 and 110, or their complementary sequence, perhaps wherein said fungi target gene is to comprise SEQ ID Nos 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, in 48 and 110 the fungi of the gene of arbitrary sequence directly to homologue and

(ii) comprise the sense strand that contains (i) described nucleotide sequence and contain the nucleotide sequence of antisense strand of the complementary sequence of (i) described nucleotide sequence, wherein can form double-stranded RNA by described nucleotide sequence coded transcript,

(b) from plant transformed cell regeneration plant; With

(c) make the plant transformed growth under the condition that is fit to the express recombinant construct, the conversion plant of described growth and unconverted plant compare fungi and have resistance, produce fungus resistant plants thus.

Still according to other embodiments, in the method for the invention, express double-stranded RNA from the construct of reorganization, wherein said construct comprises at least a adjusting sequence that operably is connected to described nucleotide sequence, the described nucleotide sequence of described nucleotide sequence and the described fungi target gene for the treatment of negative adjusting to the small part complementation.

The fungal cell can be any fungal cell, and it means and is present in or derives from any cell that belongs to the mycota organism.Method of the present invention be applicable to through RNA interferential gene silencing sensitivity and can be from them all fungies and the fungal cell of internalization double-stranded RNA the direct environment.

In one embodiment of the invention, fungi can be a mould, or filamentous fungus more particularly.In another embodiment of the invention, fungi can be a yeast.

In one embodiment, fungi can be an ascus class fungi, promptly belongs to the fungi of Ascomycota.

Preferred but do not have in the restricted embodiment in the present invention, the fungal cell is selected from the group of being made up of following fungi:

(1) fungal cell of phytopathogenic fungi, or come from the cell of phytopathogenic fungi, be such as but not limited to little Acremonium (Acremoniella spp.), Alternaria (Alternariaspp.) (for example wild cabbage blackspot chain lattice spore (Alternaria brassicola) or alternaria solani sorauer (Alternaria solani)), shell two born of the same parents Pseudomonas (Ascochyta spp.) (for example pea shell two born of the same parents bacterium (Ascochyta pisi)), Staphlosporonites (Botrytis spp.) (for example Botrytis cinerea (Botrytis cinerea) or Fu Shi grape spore cup fungi (Botryotinia fuckeliana)), cladosporium belongs to (Cladosporium spp.), cercospora (Cercospora spp.) is Kikuchi tail spore (Cercospora kikuchii) or corn tail spore (Cercospora zaea-maydis) for example), cladosporium belongs to (Cladosporium spp.) (for example yellow branch spore (Cladosporium fulvum)), Colletotrichum (Colletotrichum spp.) (for example beans thorn dish spore (Colletothchumlindemuthianum)), Curvularia (Curvularia spp.), shell look list is every spore (Diplodiaspp.) (for example corn husk look list is every spore (Diplodia maydis)), Erysiphe (Erysiphespp.) (wheat powdery mildew (Erysiphe graminis f.sp.graminis) for example, big wheat powdery mildew (Erysiphe graminis f.sp.hordei) or pea powdery mildew (Erysiphe pisi)), Erwinia armylovora, fusarium (Fusarium spp.) (is for example avenged rotten sickle spore (Fusariumnivale), intend branch spore sickle spore (Fusarium sporotrichioides), Fusarium oxysporum (sharp sickle spore), F.graminearum schw (Fusarium graminearum), Fusariumgerminearum, yellow sickle spore (Fusarium culmorum), eggplant sickle spore (Fusariumsolani), beading sickle spore (Fusarium moniliforme) or pink sickle spore (Fusariumroseum)), the top softgel shell belongs to (Gaeumanomyces spp.) (for example wheat top softgel shell (Gaeumanomyces graminis f.sp.tritici)), Gibberella (Gibberella spp.) (for example Gibberella zeae (Gibberella zeae)), Helminthosporium (Helminthosporiumspp.) (the leaf blight length spore (Helminthosporium turcicum) of wriggling for example, bipolaris carbonum wilson (Helminthosporium carbonum), long spore (Helminthosporiummavdis) or the bent long spore (Helminthosporium sigmoideum) of wriggling of fork of wriggling of Zea mays), rice ball cavity bacteria (Leptosphaeria salvinii), shell ball spore belongs to (Macrophomina spp.) (for example Kidney bean shell ball spore (Macrophomina phaseolina)), Magnaportha spp. (for example rice blast fungus (Magnaporthe oryzae)), mycosphaerella (Mycosphaerella spp.), Nectria (Nectria spp.) (for example Nectria heamatococca), Peronospora (Peronosporaspp.) (for example northeast downy mildew (Peronospora manshurica) or tobacco downy mildew (Peronospora tabacina)), Phoma (Phoma spp.) (Li such as Chard dish stem point mould (Phoma betae)), Phakopsora (Phakopsora spp.) (for example Macroptilium layer rest fungus (Phakopsora pachyrhizi)), knurl stalk spore belongs to (Phymatotrichum spp.) (for example how main knurl stalk spore (Phymatotrichum omnivorum)), phytophthora (Phytophthoraspp.) (camphor tree epidemic disease mould (Phytophthora cinnamomi) for example, Phytophthora cactorum (Phytophthoracactorum), Kidney bean epidemic disease mould (Phytophthora phaseoli), phytophthora parasitica (Phytophthoraparasitica), oranges and tangerines brown rot epidemic disease mould (Phytophthora citrophthora), big male epidemic disease mould (Phytophthora megasperma f.sp.soiae) or phytophthora infestans (Phytophthorainfestans)), Plasmopara (Plasmopara spp.) (for example grape is given birth to single shaft mould (Plasmopara viticola)), Podosphaera (Podosphaera spp.) (for example white cross hair list softgel shell (Podosphaera leucotricha)), Puccinia (Puccinia spp.) (corn handle rest fungus (Puccinia sorghi) for example, bar shaped handle rest fungus (Pucciniastriiformis), wheat handle rest fungus (Puccinia graminis f.sp.tritici), Radix Asparagi handle rest fungus (Puccinia asparagi), Puccinia recondita (Puccinia recondita) or Semen arachidis hypogaeae handle rest fungus (Puccinia arachidis)), pythium (Pythium spp.) (for example melon and fruit corruption mould (Pythium aphanidermatum)), nuclear cavity Pseudomonas (Pyrenophora spp.) (for example couchgrass nuclear cavity bacteria (Pyrenophora tritici-repentens) or circle nuclear cavity bacteria (Pyrenophora teres)), Coniosporium (Pyricularia spp.) (for example piricularia oryzae (Pyricularia oryzae)), pythium (Pythium spp.) (for example ultimate corruption mould (Pythiumultimum)), Rhincosporium secalis, Rhizoctonia (Rhizoctonia spp.) (dry thread Pyrenomycetes (Rhizoctonia solani) for example, withered spot rhizoctonia of rice (Rhizoctonia oryzae) or cereal rhizoctonia (Rhizoctonia cerealis)), Rhizopus (Rhizopus spp.) (for example zhizopchin (Rhizopus chinensid)), sclerotium (Scerotium spp.) (for example Sclerotium rolfsii (Scerotium rolfsii)), Sclerotinia (Sclerotinia spp.) (for example sclerotinite (Sclerotinia sclerotiorum)), Septoria (Septoria spp.) (tomato septoria musiva (Septoria lycopersici) for example, soybean septoria musiva (Septoria glycines), grain husk withered septoria musiva (Septoria nodorum) or wheat septoria (Septoria tritici)), Thiclaviopsis (Thielaviopsis spp.) (for example thielaviopsis sp (Thielaviopsis basicola)), Tilletia (Tilletia spp.), Trichoderma (Trichoderma spp.) (for example viride (Trichoderma virde)), Uncinula (Uncinula spp.) (for example grape snag shell (Uncinula necator)), Ustilago maydis (Ustilago maydis) (for example Ustilago maydis (D C.) Corola.), Venturia (Venturia spp.) (for example black star bacterium (Venturia pirina) of venturia inaequalis (Venturiainaequalis) or pears) or Verticillium (Verticilliumspp.) (for example Garden Dahlia wheel branch spore (Verticillium dahliae) or black and white wheel branch spore (Verticillium albo-atrum));

(2) can infect the people fungi the fungal cell or derive from the cell of the fungi that can infect the people, such as, but be not limited to mycocandida (Candida spp.), especially Candida albicans (Candida albicans); Dermatophytes (Dermatophytes) comprises Epidermophyton (Epidermophyton spp.), Trichophyton (Trichophyton spp.) and Microsporon (Microsporum spp.); Aspergillus (Aspergillus spp.) (especially flavus (Aspergillus flavus), Aspergillus fumigatus (Aspergillus fumigatus), Aspergillus nidulans (Aspergillus nidulans), aspergillus niger (Aspergillus niger) or terreus (Aspergillus terreus)); Dermatitis budding yeast (Blastomyces dermatitidis); Paracoccidioides brasiliensis (Paracoccidioides brasiliensis); Posadasis spheriforme (Coccidioides immitis); Novel Cryptococcus (Cryptococcus neoformans); Capsule tissue's spore slurry bacterium mutation (Histoplasma capsulatum Var.capsulatum) or capsule tissue's spore slurry bacterium Du Shi mutation (Var.duboisii); Sporothrix schenckii (Sporothrixschenckii); Fusarium (Fusarium spp.); Short handle broom mould (Scopulariopsisbrevicaulis); Fonsecaea (Fonsecaea spp.); Penicillium (Penicilliumspp.); Or zygomycota fungi (especially absidia corymbifera (Absidia corymbifera), Rhizomucor pusillus (Rhizomucor pusillus) or rhizopus arrhizus (Rhizopus arrhizus));

(3) can infect animal fungi the fungal cell or derive from the cell of the fungi that can infect animal, such as, but be not limited to mycocandida (Candida spp.), Microsporon (Microsporum spp.) (especially little spore of dog mould (Microsporum canis) or the little spore of gypsum shape mould (Microsporum gypseum)), trichophyton mentagrophytes (Trichophytonmentagrophytes), Aspergillus (Aspergillus spp.) or novel Cryptococcus (Cryptococcus neoforman);

With

(4) to matrix or material cause unfavorable infringement fungi the fungal cell or come from this cell, such as the fungi of invasion and attack food, seed, wooden thing, coating, plastics, dress material etc.The example of this fungi is a mould, includes but not limited to grape ear mould (Stachybotrys spp.), Aspergillus (Aspergillus spp.), Alternaria (Alternaria spp.), Cladosporium (Cladosporium spp.), Penicillium (Penicillium spp.) or Phanerochaete chrysosporium (Phanerochaete chrysosporium).

Preferred phytopathogenic fungi is Cercospora (Cercospora spp.) (for example Kikuchi tail spore (Cercospora kikuchii) or a corn tail spore (Cercosporazaea-maydis)) according to the present invention, and it causes blackspot type and macula lutea type leaf spot in the banana for example; Colletotrichum (Colletotrichum spp.) (for example beans thorn dish spore (Colletotrichumlindemuthianum)), it causes the anthrax in the corn for example; Curvularia (Curvularia spp.), it causes seedling blight; Diplodia (Diplodia spp.) (for example corn husk look list is every spore (Diplodia maydis)), it causes for example rotting of mealie, grain and stalk; Fusarium (Fusarium spp.) (for example avenging rotten sickle spore (Fusarium nivale), sharp sickle spore (Fusarium oxysporum), F.graminearum schw (Fusarium graminearum), Fusarium germinearum, yellow sickle spore (Fusarium culmorum), eggplant sickle spore (Fusarium solani), beading sickle spore (Fusarium moniliforme) or pink sickle spore (Fusarium roseum)), it causes the rotting of for example mealie, grain and stalk, cotton sickle spore bacterium blight and banana Panama disease; Gibberella (Gibberella spp.), it causes for example rotting of mealie, grain and stalk; Magnaportha spp. (for example rice blast fungus (Magnaportheoryzae)), it causes rice blast; Mycosphaerella (Mycosphaerella spp.) causes for example black yellow leaf spot of banana; Phakopsora (Phakopsora spp.) (for example Macroptilium layer rest fungus (Phakopsora pachyrhizi)), it causes for example soybean rust; Phytophthora (Phytophthora spp.) (for example camphor tree epidemic disease mould (Phytophthora cinnamomi), Phytophthora cactorum (Phytophthora cactorum), Kidney bean epidemic disease mould (Phytophthora phaseoli), phytophthora parasitica (Phytophthora parasitica), oranges and tangerines brown rot epidemic disease mould (Phytophthoracitrophthora), big male epidemic disease mould (Phytophthora megasperma f.sp.soiae) or phytophthora infestans (Phytophthora infestans)), it causes for example potato and tomato late blight; Puccinia (Puccinia spp.) (for example corn handle rest fungus (Puccinia sorghi), bar shaped handle rest fungus (Puccinia striiformis) (yellow rust)), wheat handle rest fungus (Pucciniagraminis f.sp.tritici), Radix Asparagi handle rest fungus (Puccinia asparagi), Puccinia recondita (Puccinia recondita) or Semen arachidis hypogaeae handle rest fungus (Puccinia arachidis)), it causes for example corn coventional type rust; Rhizoctonia (Rhizoctonia spp.) (for example dry thread Pyrenomycetes (Rhizoctonia solani), the withered spot rhizoctonia of rice (Rhizoctonia oryzae) or cereal rhizoctonia (Rhizoctonia cerealis)), it causes for example rice sheath blight disease or target; Rhizopus (Rhizopus spp.) (for example Rhizopus chinensid (zhizopchin)) causes seedling blight); Trichoderma (Trichoderma spp.) (for example viride (Trichodermavirde)), it causes seedling blight; Or Verticillium (Verticillium spp.) (for example Garden Dahlia wheel branch spore (Verticillium dahliae) or black and white wheel branch spore (Verticillium albo-atrum)), it causes for example cotton verticillium wilt.

The especially preferred phytopathogenic fungi of the present invention is rice blast fungus (Magnaporthe oryzae), and it causes for example rice blast; Rhizoctonia (Rhizoctonia spp.) (for example dry thread Pyrenomycetes (Rhizoctonia solani), the withered spot rhizoctonia of rice (Rhizoctonia oryzae) or cereal rhizoctonia (Rhizoctonia cerealis)), it causes for example rice sheath blight disease; Curved spore mould (Curvularia spp.), Rhizopus (Rhizopus spp.) (for example zhizopchin (Rhizopuschinensid)), Trichoderma (Trichoderma spp.) (for example viride (Trichodermavirde)), it causes the paddy rice seedling blight; Phakopsora (Phakopsora spp.), it causes for example soybean rust; Phytophthora (Phytophthora spp.) (for example camphor tree epidemic disease mould (Phytophthora cinnamomi), Phytophthora cactorum (Phytophthora cactorum), Kidney bean epidemic disease mould (Phytophthora phaseoli), phytophthora parasitica (Phytophthora parasitica), oranges and tangerines brown rot epidemic disease mould (Phytophthora citrophthora), big male epidemic disease mould (Phytophthoramegasperma f.sp.soiae) or phytophthora infestans (Phytophthora infestans)), it causes for example tomato and the late blight of potato; Cercospora (Cercospora spp.) (for example Kikuchi tail spore (Cercospora kikuchii) or corn tail spore (Cercospora zaea-maydis)) or mycosphaerella (Mycosphaerella spp.), it causes for example black yellow leaf spot of banana; Or fusarium (Fusarium spp.) (for example avenging rotten sickle spore (Fusarium nivale), sharp sickle spore (Fusarium oxysporum), F.graminearum schw (Fusarium graminearum), Fusarium germinearum, yellow sickle spore (Fusarium culmorum), eggplant sickle spore (Fusarium solani), beading sickle spore (Fusarium moniliforme) or pink sickle spore (Fusarium roseum)), it causes for example banana Panama disease.

Especially preferred phytopathogenic fungi is the rice blast fungus (Magnaportheoryzae) that causes rice blast.

The fungal cell can be complete fungal cell, this means that the fungal cell has cell walls.In this nonrestrictive embodiment, thereby the fungal cell is contacted with double-stranded RNA by complete fungal cell is contacted with double-stranded RNA.With the cell walls that does not need to remove the fungal cell before double-stranded RNA contacts.Therefore, when the fungal cell has cell walls, method of the present invention comprises makes the fungal cell contact with at least a double-stranded RNA, it is outside or contact at this that wherein dsRNA is added into fungal cell's the outside, fungal cell's cell walls, wherein double-stranded RNA comprises the annealed complementary strand, and a chain wherein has and the target nucleotide sequences corresponding nucleotide sequences for the treatment of the negative target fungal gene of regulating.DsRNA is ingested through cell walls by the fungal cell.

Term " fungal cell " comprises all types and the fungal cells of all etap, comprises that special catabolic propagated cell is such as sexual and asexual spore.As what use in this article, the fungal cell comprises fungi as referred, also comprises the fungi of other life form, such as haustorium, conidium, mycelium, invasion silk, spore, zoospore etc.

Fungi not only by sexual but also by vegetative situation under, these fungies have two names: when fungi carries out sexual propagation with the name WD fungi of teleomorph; Name with anamorph when fungi carries out vegetative propagation is represented fungi.The name of teleomorph is represented " whole fungi (whole fungus) ", comprises two kinds of propagation methods.When mentioning one of these names in the present invention, be expression " whole " fungi.

According to one embodiment of the invention, the fungal cell who contacts with dsRNA is the phytopathogenic fungi cell that is in the outside life stage of vegetable cell, for example is the form of mycelia, germ tube, appressorium, conidium (asexual spore), ascoma, cleistothecium or thecaspore (at exophytic sexual spore).According to another embodiment of the invention, the fungal cell who contacts with dsRNA is the phytopathogenic fungi cell that is in the inner life stage of vegetable cell, for example such as the pathogenic form of invading silk, mycelia, spore or haustorium.

The present invention relates to can be by the negative any interested gene of regulating (it can be called as " target gene " in this article) in the fungi.

Term " the negative adjusting of genetic expression " and " inhibition of genetic expression " are used interchangeably in this article, and be illustrated on the level from the protein of target gene and/or mRNA product the measuring or elimination fully that observable reduction or detectable gene are expressed of genetic expression.When the negative adjusting of target gene or when suppressing to take place in the mode that the other gene of fungal cell not have obviously influence, the negative adjusting or the inhibition of genetic expression is " specific ".

Depend on the person's character of target gene, the negative adjusting of genetic expression or suppress analysis that can be by pair cell or whole fungi phenotype or connect immunosorbent assay (ELISA), Western trace, radioimmunoassay (RIA), other immunoassay or fluorescence activated cell analysis (FACS) such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, the determination of gene expression of application microarray, antibodies, enzyme the measurement of mRNA or protein expression is confirmed among the fungal cell by using molecular engineering.

" target gene " can be in fact any gene that expectation suppresses, because it disturbs fungi growth or pathogenic or infectivity.For example, if method of the present invention will be used to prevent fungi growth and/or infect, so preferential the selection is necessary target gene or relates to epiphyte pathogenic or infectious any gene viability, growth, growth or the breeding of fungi, thereby the specificity of target gene is suppressed to cause the phenotype or the reduction of lethality or stop fungal infection.

According to a non-limiting embodiments, target gene is such, and promptly method of the present invention is negative regulates or when suppressing it and expressing when using, and the fungal cell is killed or fungal cell's breeding or growth are stopped or stop.Such target gene is considered to be essential and be called as indispensable gene for fungal cell's viability.Therefore, the present invention includes the method for describing herein, wherein target gene is an indispensable gene.

The specific gene that must target gene relates to keep the cell basic function of cell survival, cell g and D and breeding that is suitable for the inventive method.Other suitable target gene examples that relate to different cell processes have been described at table 1 in 2.

According to other non-limiting embodiments, target gene is such, promptly when using method of the present invention to its negative adjusting, the infecting or infect of fungi, by fungus-caused infringement and/or fungal infection or infection host organism and/or cause that the ability of such infringement is lowered.Term " infects " and " infection " or " infectivity " and " infectivity " can exchange use usually in this article.Such target gene is considered to participate in the pathogenic or infectious of fungi.Therefore, the present invention prolongs and described herein method, and wherein target gene participates in the pathogenic or infectious of fungi, the preferred fungi target gene relate to germ tube formation, conidially adhere to, the formation of appressorium, formation or conidial formation of invasion silk.The advantage of selecting the target gene of latter type is the formation of blocking-up fungi to plant or further infection of plant part or offspring.Use to participate in other advantage pathogenic or infective target gene and be when fungi when inside plants is grown, dsRNA can be absorbed by fungi, so that formed spore can not infect other plant part.

According to an embodiment, target gene is conservative gene or fungi specific gene.

Some are preferred but do not have the example of restricted suitable target gene to be listed in table 1 and 2.

Therefore, the present invention relates to one or more rice blast fungus (Magnaporthe grisea) target gene of listing in above-mentioned table 1 is carried out the negative adjusting or the inhibition of RNAi mediation, and also relate to homology in the different fungi kinds of listing in table 2/straight negative adjusting to the homology target gene.Therefore, the method that the present invention prolongs and describes herein, wherein the fungi target gene participates in as defined any cell function in the table 1 (right column).A non-limiting instance is a method for example described herein, and wherein the fungi target gene is the gene that participates in rrna, proteasome, spliceosome, APC complex body function; Perhaps participate in nuclear translocation (nuclear transport), translation startup, the transportation of transcribing (activation of for example transcribing), cell inner membrance, dna replication dna, mitotic spindle formation, the transhipment of vesicle or the gene of cytoskeleton.

In addition, can instruct the RNAi of fungi target gene or the gene silencing of RNA mediation or any suitable double-stranded RNA fragment of inhibition can be used for method of the present invention.

In the method for the invention, dsRNA is used to suppress to grow or disturb the pathogenic or infectious of fungi.

Therefore the present invention relates to comprise the isolating double-stranded RNA of complementary strand of annealing, a chain wherein have with the fungi target gene to small part complementary target nucleotide sequences.Target gene can be any target gene of describing herein, perhaps their the performance part of sample function.

According to one embodiment of the invention, the isolating double-stranded RNA that comprises the complementary strand of annealing is provided, a chain wherein have with fungi target gene nucleotide sequence to small part complementary nucleotide sequence, wherein said fungi target gene is essential for viability, growth, growth or the breeding of fungi, and preferred described fungi target gene participates in any cell function as defined in Table 1; Perhaps wherein said fungi target gene participates in the pathogenic or infectious of fungi, preferred described fungi target gene participate in germ tube formation, conidially adhere to, the formation of appressorium, formation or conidial formation of invasion silk, described nucleotide sequence can suppress target gene expression.In one embodiment, described target gene comprise be selected from following in the sequence of any sequence representative sequence set have at least 75%, 80% or 85% consistence, preferably at least 90%, 95%, 96%, or more preferably at least 97%, 98% or 99% conforming sequence: SEQ IDNOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15,48 and 110, or its complementary sequence, perhaps wherein said fungi target gene is to comprise SEQ ID Nos 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, the fungi of the gene of any sequence is directly to homologue in 48 and 110.

Dependence is used for measuring the detection of gene silencing, compare with the target cell of handling by contrast dsRNA, growth-inhibiting can be quantified as greater than about 5%, 10%, and more preferably from about 20%, 25%, 33%, 50%, 60%, 75%, 80%, most preferably from about 90%, 95% or about 99%.

According to another embodiment of the invention, isolating double-stranded RNA is provided, wherein at least one described annealing complementary strand comprises by SEQ ID NOs 99,100,111,112,113,114,115,116,101,102,97,98,124,106,109,126,119,127,103,104,105,118,108,121,125,123,107,122, the RNA equivalent of at least one nucleotide sequence of any sequence representative in 120 and 110, perhaps their at least 17, preferably at least 18,19,20 or 21, more preferably at least 22,23 or 24 double-stranded fragments that base pair is long.Preferred described isolating double-stranded RNA comprises the RNA equivalent by at least one nucleotide sequence of any sequence representative among the SEQ ID NOs192,201,202,193,190,191,196,199,200,194,195,198 and 197, perhaps their 17, the long double-stranded fragments of preferred at least 18,19,20 or 21, more preferably at least 22,23 or 24 base pairs.

If method of the present invention be used to control fungi among host cell or the host organisms or on growth or infect, preferably double-stranded RNA is not with any host gene or do not enjoy any significant homology with any indispensable gene of host at least.In this case, preferably double-stranded RNA demonstrates any gene with host cell and has less than 30%, is more preferably less than 20%, is more preferably less than 10% even be more preferably less than 5% consensus nucleic acid sequence.The per-cent of sequence identity should calculate with the total length in double-stranded RNA district.If the genomic sequence data of host organisms is available, people can use the information biology instrument crosscheck of standard and the sequence identity of double-stranded RNA.In one embodiment, the Nucleotide for 21 adjacency between dsRNA and host sequences does not have sequence identity, this means under this background, and preferably 21 of dsRNA do not appear in the genome of host organisms in abutting connection with base pair.In another embodiment, have less than about 10% or less than about 12.5% sequence identity in abutting connection with Nucleotide and any nucleotide sequence of planting for 24 of dsRNA from the host.

Double-stranded RNA comprises the annealed complementary strand, and wherein one has and the target nucleotide sequences corresponding nucleotide sequences for the treatment of negative adjusting target gene.Another chain of double-stranded RNA can carry out base pairing with first chain.

" target region " of target fungal gene and " target nucleotide sequences " this statement can be any appropriate area of gene or nucleotide sequence.Target region should comprise at least 17, at least 18 or at least 19 continuous nucleotides of target gene, more preferably at least 20 or at least 21 Nucleotide, and more preferably at least 22 of target gene, 23 or 24 Nucleotide still.

Preferably double-stranded RNA (to small part) will be enjoyed 100% sequence identity with the target region of fungi target gene.It should be understood, however, that 100% sequence identity on the double-stranded region total length suppresses optional for effective RNA.Also have been found that for target sequence, have insertion, to suppress for RNA be effective to the RNA sequence of disappearance and single point sudden change.Term " corresponding to/with ... correspondence " or " be complementary to/with ... complementation " can exchange use in this article, and when these terms are used to represent sequence correspondence between double-stranded RNA and the target gene target region, they should so be explained, promptly not be the sequence identities of absolute demand 100%.Yet sequence identity per-cent between double-stranded RNA and target region will be normally at least 80% or 85%, preferably at least 90%, 95%, 96% or more preferably at least 97%, 98%, still more preferably at least 99%.

The term of Shi Yonging " complementary " had not only related to the complementarity of DNA-DNA but also had related to the complementarity of DNA-RNA in this article.By parity of reasoning, and " RNA equivalent " is illustrated in fact in the dna sequence dna, and base " T " can be replaced by the corresponding base " U " of normal presence in Yeast Nucleic Acid.

Although dsRNA contains the sequence corresponding with the target region of target gene, corresponding with the sequence of target region for dsRNA integral body is not the sin qua non's.For example, dsRNA can comprise the short non-target region that is positioned at target-specific sequence flank, as long as such sequence is not influencing the performance of dsRNA in RNA suppresses on the substantial extent.

In order to optimize the performance in RNAi, dsRNA can comprise one or more replacement bases.Even the replacement of a mononucleotide can exert an influence to the activity of dsRNA in RNAi.In order to optimize the performance of specifying dsRNA, the activity (for example, in suitable vitro detection system) that how one after the other to change each base of dsRNA and detect gained siRNAs will be very tangible for the technician.

For example in order to strengthen the stability between preservation period or to strengthen resistance to nuclease degradation, dsRNA can also comprise the modifier of DNA base, non-natural base or non-natural main chain connexon or sugar-phosphate backbone.

Before reported, approximately the formation of the short interfering rna s (siRNAs) of 21bp is desired for efficient gene silencing.Yet, in applicant's application, show, in order to be absorbed the preferably about at least 80-100bp of the minimum length of dsRNA effectively by some harmful organism.There are indications, invertebrates such as free living nematodes Caenorhabditis elegans (C.elegans) or plant parasitic nematodes Meloidogyne incognita (Meloidogyneincognita) in, these long fragments are more effective in gene silencing, and this may be because these long dsRNA are more effectively absorbed by invertebrates.

Also proposed by the 27-mer flush end recently or had the stem of 29bp and synthetic RNA duplex that the bob of 2 Nucleotide 3 ' overhangs folder (sh) RNAs forms is than the more effective RNA interference of the 21-mer siRNAs of routine inductor.Therefore, based on top definite target and 27-mer flush end or have the stem of 29bp and bob folder (sh) RNAs of 2 Nucleotide 3 ' overhangs is included in the scope of the present invention equally.

Therefore, in one embodiment, double-stranded RNA fragment (or zone) self will be preferably at least 17bp long, preferred 18 or 19bp is long, more preferably 20bp, more preferably 21bp or 22bp or 23bp or 24bp, 25bp, 26bp or 27bp is long at least at least at least at least at least at least.Word " double-stranded RNA fragment " or " double-stranded RNA zone " expression little double-stranded RNA entity corresponding with (part) target gene.

Usually, double-stranded RNA is preferably approximately 17-1500bp, even more preferably about 80-1000bp, most preferably approximately 17-27bp or approximately 80-250bp; Double-stranded RNA zone such as about 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, 25bp, 27bp, 50bp, 80bp, 100bp, 150bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 550bp, 600bp, 650bp, 700bp, 900bp, 100bp, 1100bp, 1200bp, 1300bp, 1400bp or 1500bp.

The upper limit of double-stranded RNA length may depend on i) fungal cell absorbs the needs of dsRNA and ii) dsRNA is treated to the segmental needs of guide RNA i in cell.Selected length also can be subjected to RNA synthetic method and RNA is delivered to the influence of the mode of cell.Preferably, the double-stranded RNA that is used for the inventive method will be less than 10, and 000bp is long, more preferably 1000bp or less, more preferably 500bp or less, more preferably 300bp or less or more preferably 100bp or less.For any specified target gene and fungi, can be definite for the dsRNA optimum length of effective inhibition by testing.

Double-stranded RNA can be completely or partial two strands.Partial double-stranded RNA s can comprise short strand overhang at one or two end of two strands part, as long as RNA still can be and guided RNAi by fungal cell's picked-up.

Method of the present invention can comprise simultaneously or provide two or more different double-stranded RNA s or RNA constructs to same fungal cell continuously, thereby realizes the negative adjusting or the inhibition of a plurality of target genes or realize the more effective inhibition of single target gene.

Selectively, hit a plurality of targets, and can more effectively suppress single target corresponding to the segmental existence of the double-stranded RNA of target gene above a copy by a kind of double-stranded RNA that hits a plurality of target sequences is provided.Therefore, in one embodiment of the invention, the double-stranded RNA construct comprises a plurality of dsRNA zone, at least one chain in each dsRNA zone comprise with the target nucleotide sequences of fungi target gene to small part complementary nucleotide sequence.According to the present invention, the dsRNA zone in the RNA construct can be complementary to identical or different target genes and/or dsRNA zone and can be complementary to from identical or from the target of different fungi kinds.Therefore this dsRNA construct being applied in plant host cell set up the more effective resistance to single or multiple fungi kinds in the plant.

In one embodiment, the double-stranded RNA zone comprise a plurality of copies with target gene complementary nucleotide sequence.Selectively, dsRNA hits the other target sequence in the identical target gene.

At least two, at least three, at least four, at least five, at least six of term " a plurality of " expressions etc. in the context of the present invention.

Word " other target gene " or " at least a other target gene " expression be second, the 3rd or the 4th 's etc. target gene for example.

The combination of hitting the dsRNA that surpasses a kind of above-mentioned target or resisting the different dsRNA of above-mentioned different targets can be developed or be used for method of the present invention.

Therefore the present invention relates to isolating double-stranded RNA construct, its comprise two copies at least by SEQ ID NOs 99,100,111,112,113,114,115,116,101,102,97,98,124,106,109,126,119,127,103,104,105,118,108,121,125,123,107,122, the RNA equivalent of at least one nucleotide sequence of any sequence representative perhaps comprises their at least 17 in 120 and 110, preferably at least 18,19,20 or 21, more preferably at least 22,23 or 24 at least one double-stranded fragments that base pair is long.Preferably, described double-stranded RNA comprises the RNA equivalent by the nucleotide sequence of SEQ ID NO 117 representatives, perhaps its at least 17, the long double-stranded fragments of preferred at least 18,19,20 or 21, more preferably at least 22,23 or 24 base pairs.

Thus, the present invention prolongs and described herein method, wherein dsRNA comprises the annealed complementary strand, a chain wherein have with the target nucleotide sequences of fungi target gene to small part complementary nucleotide sequence, and it comprises at least one extra dsRNA zone, at least one chain wherein comprise with the nucleotide sequence of at least one other fungi target gene to small part complementary nucleotide sequence.This other target gene can be described herein any target gene.According to a preferred embodiment, dsRNA hits at least one for fungi viability, growth, growth or breed necessary target gene and hit the above-mentioned pathogenic or infective gene of at least a this paper of relating to.Selectively be, dsRNA hits a plurality of genes of identical category, and for example, dsRNA hits at least 2 indispensable genes or at least 2 and relates to pathogenic gene or at least 2 genes that relate to any cell function described in the table 1.According to other embodiments, dsRNA hits at least 2 target genes, and wherein said target gene relates to the different cell functions that are selected from those functions described in the table 1.For example dsRNA hits two or more genes that relate to the formation (for example 'beta '-tubulin gene) (such as the gene that is shown in Fig. 3) of albumen synthetic (for example ribosomal subunit), protein degradation (for example proteasome subunit), microtubule cytoskeleton.

Preferably, the present invention prolongs and described herein method, wherein said fungi target gene comprises with the sequence that is selected from the sequence set of following any sequence representative and has at least 75%, preferably at least 80%, 85%, 90%, more preferably at least 95%, 98% or 99% conforming sequence: SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15,48 and 110, or its complementary sequence, perhaps wherein said fungi target gene is to comprise SEQ ID Nos 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, the fungi of the gene of any sequence is directly to homologue in 48 and 110.

DsRNA zone (or fragment) in double-stranded RNA can be as follow combined:

A) when a plurality of dsRNA zone of the single target gene of target is combined, they can be combined with initial order (being the order that described zone occurs in target gene) in the RNA construct,

B) selectively be, can ignore segmental initial order, thereby they are mixed with any order randomly or intentionally and are combined into the double-stranded RNA construct,

C) selectively be, a single fragment can be repeated several times in the dsRNA construct, and for example from 1 to 10 time, for example 1,2,3,4,5,6,7,8,9 or 10 time, or

D) dsRNA zone (target genes that target is single or different) can be there to be justice or antisense orientation combined.

In addition, target gene to be made up can be selected from one or more in the following gene type:

E) aforesaid " essential " gene or " pathogenic gene " comprise most important and cause the gene of lethal or serious (for example ingest, breed, grow) phenotype during by silence when it for one or more target fungies.The strong selection that causes death target gene causes effective RNAi effect.In RNA construct of the present invention, a plurality of dsRNA zone that can make up target identical or different (very effective) lethal gene acts on effectiveness, effect or speed in the fungi control with further increase RNAi.

F) " weak " gene comprises having the target gene that interested especially function is arranged in some cellular pathways described herein, but can cause weak phenotype effect when this target gene quilt is reticent independently.In RNA construct of the present invention, can make up a plurality of dsRNA zone of the single or different weak genes of target, thereby obtain stronger RNAi effect.

G) " fungi specificity " gene comprises as searching for (for example passing through blast search) determined gene that does not have substantive homologous corresponding part in non-fungal organism by the information biology homology.The selection of fungi specific target gene causes the RNAi effect of species specificity, and does not influence or do not have the influence of essence (unfavorable) in non-target organism.

H) " conservative gene " to be included between target organism and the non-target organism be the gene of conservative (on amino acid levels).In order to reduce to the possible influence of non-target species, analyze this effectively but conservative gene and selecting from the target sequence of the Variable Area of these conservative genes target as dsRNA zone in the RNA construct.At this, on the level of nucleotide sequence, assess conservative property.Therefore such Variable Area comprises the part that has minimum conservative property on the nucleotide sequence level of conservative target gene.

I) " conservative approach " gene comprises the gene that relates to identical biological approach or cell processes or is included in the gene that has the same functionality that causes specificity and effective RNAi effect and the control of more effective fungi in the different fungi kinds;

J) selectively, according to a plurality of genes from different biopathways of RNA construct target of the present invention, it causes cell RNA i effect widely and the control of more effective fungi.

Preferably, all double-stranded RNA zones comprise at least one with the nucleotide sequence of any target gene described herein to small part or local complementary chain.Yet, if one of double-stranded RNA zone comprise at least one with the part complementary chain of the nucleotide sequence of arbitrary target gene described herein, another double-stranded RNA zone can comprise at least one with the part complementary chain of any other fungi target gene (comprising known target gene).

Still according to another embodiment of the invention, provide isolating double-stranded RNA construct, it also comprises sequence that at least one is extra and connexon randomly.In one embodiment, described extra sequence is selected from the group that comprises following sequence: the sequence that (i) helps to produce on a large scale the dsRNA construct; (ii) realize the sequence that dsRNA stability increases or reduces; Thereby (iii) allow protein or other molecule in conjunction with the sequence that promotes the fungal cell to the picked-up of RNA construct; Thereby (iv) on the binding to fungal cell surface or tenuigenin in acceptor or the sequence of the adaptive son of the molecule picked-up, endocytosis and/or the transcytosis that promote the fungal cell; Or (the v) additional sequences of the processing in catalysis dsRNA zone.In one embodiment, described connexon is the RNA sequence that the conditionality oneself shears, preferred pH susceptibility connexon or hydrophobic susceptibility connexon.In one embodiment, described connexon is an intron.

In one embodiment, a plurality of dsRNA zone that connects the double-stranded RNA construct by one or more connexons.In another embodiment, described connexon is present in the site of RNA construct, and the dsRNA zone is separated with another interesting areas.Different connexon types at the dsRNA construct are provided in the present invention.

In another embodiment, a plurality of dsRNA zone of double-stranded RNA construct connects without connexon.

In specific embodiments of the present invention, connexon can be used to separate less dsRNA zone in harmful organisms.Advantageously, in this case, the connexon sequence can promote long dsRNA to be divided into less dsRNA zone under specific situation, thereby causes the release in the dsRNA zone that separates under these situations, and causes these more effective gene silencings in less dsRNA zone.The example that the appropriate condition oneself shears connexon is the RNA sequence that the oneself shears under high pH condition.Borda et al. (NucleicAcids Res.2003 May 15; 31 (10): 2595-600) described the suitable example of this RNA sequence, the document is by with reference to being incorporated herein.This sequence derives from the catalytic core of hammerhead ribozyme HH16.

In another aspect of this invention, the position that connexon is arranged in the RNA construct can make the dsRNA zone with other, for example extra, interested sequence separately, described interested sequence preference provides some additional function for the RNA construct.

In specific embodiment of the present invention, dsRNA construct of the present invention is equipped with the adaptive son that promotes fungi picked-up dsRNA.The material that this adaptive son is designed to be absorbed with fungi combines.Such material can be fungi or plant origin.A specific examples of described adaptive son is and transmembrane protein, for example the adaptive son of transmembrane protein bonded of fungi.Selectively be that described adaptive son can be in conjunction with (plant) metabolite or the nutrition by fungi absorbed.

Selectively, connexon oneself in endosome shears.This may be favourable fungi when endocytosis or transcytosis absorb construct of the present invention and therefore it separated in the endosome at fungal species.Described endosome can have low pH environment, causes the shearing of connexon.

When dsRNA construct of the present invention is used to when another cell is transferred in the transportation of cell through cell walls, for example when passing through the cell walls of fungi harmful organisms, the above-mentioned connexon that carries out oneself's shearing under hydrophobic conditions is useful especially in dsRNA construct of the present invention.

Intron also can be used as connexon." intron " used herein can be any non-coding RNA sequence of messenger RNA(mRNA).The intron sequences especially suitable for construct of the present invention is that (1) is rich in U (35-45%); (2) average 100bp long (about 50 and about 500bp between change), its base pair can be selected at random or can be based on known intron sequences; (3) originate in have-AG:GT-or-CG:GT-5 ' end and/or (4) they 3 ' end have-AG:GC-or-AG:AA.

Noncomplementation RNA sequence from about 1 base pair to about 10,000 base pair scopes also can be used as connexon.

Do not wish to be subject to any specific theory or machine-processed, supposition is added long dsrna s to the fungal cell and is ingested by natural mechanism and enters cell from the outside, the fungal cell absorbs material such as endocytosis approach for example by described natural mechanism from their next-door neighbours' environment.The double-stranded RNA s that cell is advanced in picked-up subsequently in cell the effect by the endogenous endonuclease be processed to short dsrna s (being called as siRNA s (siRNAs)).The siRNAs that is obtained is subsequently by the polycomponent RNase complex body formation mediate rna i of (be named as RISC or RNA and disturb silencing complex).

In order in the fungal cell, to realize negative adjusting to target gene, the double-stranded RNA that is added on the cell walls outside can be any dsRNA or dsRNA construct, they can be ingested into cell and be processed to the siRNAs of mediate rna i subsequently subsequently in cell, and the RNA self that perhaps adds outside to can be ingested into cell and the siRNA of guide RNA i thus.

SiRNAs normally has the short dsrna s that length is 19-25 base pair or 20-24 base pair.In preferred embodiments, can use corresponding to treat negative regulate target gene have 19,20,21,22,23,24 or 25 base pairs and especially a siRNAs of 21 or 22 base pairs.Yet the present invention there is no and is limited to the intention of using this siRNAs.

SiRNAs can comprise the strand overhang at one or two end, and it is positioned at the flank of double-stranded part.In particularly preferred embodiments, siRNA can contain 3 ' outstanding Nucleotide, preferred two 3 ' outstanding Thymine deoxyriboside (dTdT) or uridines (UU).If being right after the target-gene sequence that is contained in the double-stranded partial sequence of dsRNA upstream is AA, can in siRNA, comprise 3 ' TT or UU overhang so.This allows TT or UU overhang and target gene hybridization in siRNA.Although the other end at siRNA also can comprise 3 ' TT or UU overhang, the target sequence that is contained in the sequence downstream in the double-stranded part of siRNA needn't have AA.Under this background, in also being encompassed in as the chimeric siRNAs of RNA/DNA.These mosaics comprise, for example, comprise the double-stranded RNA that has as above DNA base (for example dTdT) 3 ' overhang of discussing and also comprise as wherein one or more RNA bases or ribonucleotide or even the whole piece chain on all ribonucleotides by the siRNAs of the double-stranded RNA s of DNA base or the polynucleotide that deoxyribonucleotide replaced.

Can be by two (justice and antisense are arranged) RNA chain formation dsRNA that separate by annealing through (non-covalent) base pairing together.Alternatively, dsRNA can have folding stem ring or hairpin structure, and wherein two of dsRNA annealing chains connect with covalent manner.What in this embodiment, the different zones of the single polynucleotide molecule of self-complementary formed dsRNA from the part has justice and an antisense strand.If treat to synthesize dsRNA, for example in the host cell of following argumentation or organism or through in-vitro transcription, the RNAs that has this structure so is easily by expression in vivo.The accurate person's character and the sequence that connect " ring " of two RNA chains are not substantial to the present invention usually, except it should not weaken the ability of double-stranded part mediate rna i of this molecule.(for example see WO 99/53050, its content by with reference to being incorporated herein) that is used for that the feature of " hair clip " or " stem-ring " RNA of RNAi is known in the art with the CSIRO name.In another embodiment of the invention, ring structure can comprise aforesaid connexon sequence or other sequence.

Double-stranded RNA or construct can be prepared in self known mode.For example, can be at external use chemistry well known in the art or the synthetic double-stranded RNA s of enzymatic RNA synthetic technology.In a method, can synthesize two RNA chains that separate separately and with after annealing forms double-stranded.In other embodiments, can synthesize double-stranded RNA s or construct by in host cell or organism, carrying out cell inner expression from suitable expression vector.This method is at length discussed below.

The amount of the double-stranded RNA that contacts with the fungal cell is to realize the negative amount of regulating described one or more target genes of specificity.Can be to allow sending the amount of at least one copy to introduce RNA to each cell delivery.Yet in certain embodiments, the higher dosage of double-stranded RNA (for example, each cell at least 5,10,100,500 or 1000 copies) can produce more effective inhibition.For any fungal gene target, the optimum quantity that is suitable for effective dsRNA that suppresses can be determined by the laboratory method of routine.

The fungal cell is contacted with double-stranded RNA, thereby allow the direct picked-up of fungi double-stranded RNA.For example, the fungal cell can for example be contained the aqueous solution of dsRNA with pure or pure substantially form, contacts with double-stranded RNA.In this embodiment, fungi simply " immersion " in containing the aqueous solution of double-stranded RNA.In other embodiments, can it be contacted with double-stranded RNA by spraying the fungal cell with the liquid composition that contains double-stranded RNA.

Alternatively, in order to increase the picked-up of fungi, double-stranded RNA can be connected to the food component of fungi, on the food component such as the Mammals pathomycete to dsRNA.

In other embodiments, the fungal cell can contact with the composition that comprises double-stranded RNA.Except dsRNA, described composition can also comprise other vehicle, diluent or carrier.The preferred feature of such composition is discussed in greater detail below.

Double-stranded RNA also can be incorporated in the substratum of fungal growth be incorporated into the material that is subjected to fungal infection or matrix among or on or inject matrix or the material that is subject to fungal infection.

Another aspect of the present invention is the target nucleotide sequences of disclosed in this article fungi target gene.Such target nucleotide sequences is even more important for design consideration dsRNA of the present invention.Such target nucleotide sequences preferably at least 17, preferably at least 18,19,20 or 21, more preferably at least 22,23 or 24 Nucleotide is long.The limiting examples of preferred target nucleotide sequences provides in an embodiment.The present invention includes by SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, at least one sequence of any sequence representative in 48 and 110, or their complementary sequence, comprise and contain SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, the fragment of at least 17 Nucleotide of any sequence in 48 and 110, or the isolating nucleotide sequence of their complementary sequence composition.

According to an embodiment, the invention provides the isolating nucleotide sequence of encode double-stranded RNA as described herein or double-stranded RNA construct.

According to another embodiment, the invention provides the fungi target gene, its be included in herein by SEQ ID NO 192,117,201,202,193,190,191,196,199,200,194,195,198 and 197 or they at least 17, the sequence of the fragment representative of preferred at least 18,19,20 or 21, more preferably at least 22,23 or 24 Nucleotide, and described target gene is contained in the method for the invention.

According to more particular embodiment, the present invention relates to by SEQ ID NOs 3,99,100,192,39,111,112,113,114,115,116,117,201,202,5,101,102,193,184,97,98,190,191,37,124,9,188,106,196,13,109,199,200,33,126,23,119,35,127,7,186,103,104,105,194,195,29,118,17,108,198,25,121,19,125,31,123,11,107,197,27,122,21,120, the sequence of any sequence representative in 15 and 110, or they at least 17, preferably at least 18,19,20 or 21, more preferably at least 22, the isolated nucleic acid sequences that the fragment of 23 or 24 Nucleotide is formed.

Person of skill in the art will appreciate that the homologue that can find these target genes, and these homologues also are useful in the method for the invention.

If protein or nucleotide sequence demonstrate the sequence similarity of " significantly " level, they may be exactly homologue so.Real homologous sequence is by the divergence generation contact from common ancestor's gene.The sequence homology thing can be two types: (i) be present in the situation of different plant species at homologue, known they be directly to homologue, for example the α-Zhu Danbai gene in mouse and people is directly to homologue.(ii) in single species, the parallelism homologue is a homologous gene, for example in mouse α-and the beta-globin gene be the parallelism homologue.

Preferred homologue is to comprise and be selected from by SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15,48 and 110, or the sequence in the sequence set of their complementary sequence representative has at least about 85% or 87.5%, still more preferably from about 90%, still more preferably at least about 95%, most preferably at least about the gene of 99% conforming sequence.The method of determining sequence identity in the art is a routine techniques, comprises that Blast software and GAP analyze the application of (GCG program).

Other homologue is as comprising by SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, the allelic gene of the gene of any sequence representative sequence in 48 and 110.Preferred homologue in addition is and comprises by SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, the gene of any sequence representative sequence is compared in 48 and 110, comprises the gene of at least one single nucleotide polymorphism (SNP).

According to another embodiment, the present invention includes as the fungi that contains the gene of any sequence among the SEQ ID Nos 206-458 directly to the target gene of homologue.Preferably directly to homologue by SEQ ID Nos 206-353 in any sequence representative.Preferred directly to homologue by SEQID NOs 206-337 in any sequence representative.

In a preferred embodiment of the invention, dsRNA can be by host cell or host organisms, as the organic host cell or the organism expressing (for example transcribing within it) that are subject to the fungal infection influence or attack.In this embodiment, in fungi the gene silencing of one or more target genes of RNAi mediation can be used as among the host organisms or on control fungal growth and/or prevention or reduce the mechanism of host organisms fungal infection.Therefore, double-stranded RNA can be given to specific fungi or to the resistance of certain class fungi in the intracellular expression of host organisms.Hit at dsRNA and to surpass under a kind of situation of fungi target gene, double-stranded RNA can be given surpassing a kind of fungi or surpassing the resistance of a class fungi in the intracellular expression of host organisms.

In preferred embodiments, host organisms is a plant, and fungi is a phytopathogenic fungi.In this embodiment, by infecting or be subject in plant that phytopathogenic fungi infects or the vegetable cell and express double-stranded RNA being subjected to phytopathogenic fungi, the fungal cell is contacted with double-stranded RNA.

In the present context, term " plant " comprises any hope treatment, prevention or reduces fungal growth or and/or the vegetable material of fungal infection.This comprises, especially whole plants, seedling, breeding or reproductive material be such as seed, cutting, grafting, explant etc., and also comprise vegetable cell and tissue culture.Described vegetable material will be expressed corresponding to the dsRNA of one or more fungi target genes or be had the ability of expressing described dsRNA.

Therefore, the present invention provides breeding or the reproductive material of plant, preferred transgenic plant or (transgenosis) plant aspect other or expresses and maybe can express the plant cell cultures of at least a double-stranded RNA, wherein double-stranded RNA comprises the annealed complementary strand, a chain wherein has the nucleotide sequence to the small part complementary with the target nucleotide sequences of fungi target gene, rely on the interaction of plant-fungi thus, fungi picked-up double-stranded RNA, described double-stranded RNA can disturb by RNA and suppress target gene or the negative target gene expression of regulating.Target gene can be any target gene as herein described, and for example for viability, growth, the growth of fungi or breed necessary target gene, preferred described fungi target gene participates in defined any cell function in the table 1; Or for example participate in epiphyte pathogenic or infective fungi target gene, preferred described fungi target gene relate to germ tube formation, conidially adhere to, the formation of appressorium, formation or conidial formation of invasion silk.

In this embodiment, the fungal cell can be any fungal cell, but the fungal cell of preferred plant pathogenic fungus.Preferred plant pathogenic fungi includes, but are not limited to those fungies listed above.

The plant or the transgenic plant according to the present invention that are used for the inventive method comprise any plant, but preferably be subject to the plant that plant pathogenic fungi infects, include but not limited to following plant: paddy rice, corn, soybean, cotton, potato, banana or tomato, cereal grass comprise wheat, oat, barley, rye, grape, apple, pears, Chinese sorghum, grain, beans (beans), Semen arachidis hypogaeae, Semen Brassicae campestris (rapeseed), Sunflower Receptacle, sugarcane.Most preferably described plant is paddy rice, corn, soybean, cotton, potato, banana or tomato.

Therefore, the present invention prolongs and described herein method, and wherein said plant is wheat, Chinese sorghum, grain, beans (beans), Semen arachidis hypogaeae, Semen Brassicae campestris, Sunflower Receptacle, sugarcane, paddy rice, corn, soybean, cotton, potato, banana or tomato.In preferred embodiments, described plant is paddy rice, corn, soybean, cotton, potato, banana or tomato.。

In one embodiment, the present invention prolongs and described herein method, wherein said plant is a paddy rice, and this target gene is from the gene that is selected from the fungi in the group of being made up of following fungi: Magnaporthe spp. (for example rice blast fungus (Magnaporthe oryzae) or Magnaporthe grisae), Rhizoctonia (Rhizoctonia spp.) (dry thread Pyrenomycetes (Rhizoctonia solani) for example, withered spot rhizoctonia of rice (Rhizoctonia oryzae) or cereal rhizoctonia (Rhizoctonia cerealis)), fusarium (Fusarium spp.) (for example pink sickle spore (Fusarium roseum)), little Acremonium (Acremoniella spp.) (for example black little single head spore mould (Acremoniella atra)), pythium (Pythium spp.) (for example many male rotten mould (Pythium arrhenomanes), group's knot rotten mould (P.myriotylum), or wide male rotten mould (P.dissotocum), (for example rice is bent spore (Curvulariaoryzae) to Curvularia (Curvularia spp.), curved spore (Curvularia lunatas)), Trichoderma (Trichoderma spp.) (for example viride (Trichoderma virde)) and Rhizopus (Rhizopus spp.) (for example zhizopchin (Rhizopus chinensis)); In another embodiment, the present invention prolongs and method as described herein, wherein plant is a corn, and this target gene is the gene that is selected from the fungi in the group of being made up of following fungi: Colletotrichum (Colletotrichum spp.) (for example beans thorn dish spore (Colletotrichum lindemuthianum)), Gibberella belongs to (Gibberella spp.), fusarium (Fusarium spp.) (is for example avenged rotten sickle spore (Fusarium nivale), point sickle spore (Fusarium oxysporum), F.graminearum schw (Fusarium graminearum), Fusarium germinearum, yellow sickle spore (Fusarium culmorum), eggplant sickle spore (Fusarium solani), beading sickle spore (Fusarium moniliforme) or pink sickle spore (Fusarium roseum)), shell look list is every spore (Diplodia spp.) (for example corn husk look list is every spore (Diplodia maydis)) or Puccinia (Puccina spp.) (corn handle rest fungus (Puccinia sorgh) for example, bar shaped handle rest fungus (Puccinia triiformis) (causing yellow rust), wheat handle rest fungus (Puccinia graminis f.sp.tritici), Radix Asparagi handle rest fungus (Puccinia asparag), Puccinia recondita (Puccinia recondita) or Semen arachidis hypogaeae handle rest fungus (Puccinia arachidis)); In another embodiment, the present invention has expanded method as described herein, wherein plant is a soybean, and target gene is the gene that is selected from the fungi Phakopsora (Phakopsora spp.) (for example yam bean layer rest fungus (Phakopsora pachyrhizi)); In another embodiment, the present invention has expanded method as described herein, wherein plant is a cotton, and target gene is to be selected from by fusarium (Fusarium spp.) (for example to avenge rotten sickle spore (Fusarium nivale), point sickle spore (Fusarium oxysporum), F.graminearum schw (Fusarium graminearum), Fusarium germinearum, yellow sickle spore (Fusarium culmorum), eggplant sickle spore (Fusarium solani), beading sickle spore (Fusarium moniliforme) or pink sickle spore (Fusarium roseum)) or the group formed of Verticillium (Verticillium spp.) (for example Garden Dahlia wheel branch spore (Verticillium dahliae) or black and white wheel branch spore (Verticillium albo-atrum)) in the gene of fungi; In another embodiment, the present invention prolongs and described herein method, wherein plant is a potato, and target gene is to be selected from by phytophthora (Phytophthora spp.) (camphor tree epidemic disease mould (Phytophthora cinnamomi) for example, Phytophthora cactorum (Phytophthora cactorum), Kidney bean epidemic disease mould (Phytophthora phaseoli), phytophthora parasitica (Phytophthora parasitica), oranges and tangerines brown rot epidemic disease mould (Phytophthora citrophthora), big male epidemic disease mould (Phytophthoramegasperma f.sp.soiae) or phytophthora infestans (Phytophthora infestans)), Rhizoctonia (Rhizoctonia spp.) (dry thread Pyrenomycetes (Rhizoctonia solani) for example, withered spot rhizoctonia of rice (Rhizoctonia oryzae) or cereal rhizoctonia (Rhizoctonia cerealis)) or cause wilt disease, the gene of fungi in the group that the fungi kind of rot or scurf is formed; In another embodiment, the present invention prolongs and described herein method, wherein plant is a banana, and target gene is to be selected from by mycosphaerella (Mycosphaerella spp.), cercospora (Cercospora spp.) (for example Kikuchi tail spore (Cercospora kikuchii) or corn tail spore (Cercospora zaea-maydis)) or fusarium (Fusarium spp.) (are for example avenged rotten sickle spore (Fusarium nivale), point sickle spore (Fusarium oxysporum), F.graminearum schw (Fusarium graminearum), Fusarium germinearum, yellow sickle spore (Fusarium culmorum), eggplant sickle spore (Fusarium solani), beading sickle spore (Fusarium moniliforme) or pink sickle spore (Fusarium roseum)) the gene of fungi in the group formed; In another embodiment, the present invention prolongs and method as described herein, wherein plant is a tomato, and target gene is to be selected from by phytophthora (Phytophthoraspp.) (camphor tree epidemic disease mould (Phytophthora cinnamom) for example, Phytophthora cactorum (Phytophthoracactorum), Kidney bean epidemic disease mould (Phytophthora phaseoli), phytophthora parasitica (Phytophthoraparasitica), oranges and tangerines brown rot epidemic disease mould (Phytophthora citrophthora), big male epidemic disease mould (Phytophthora megasperma f.sp.soiae) or phytophthora infestans (Phytophthorainfestans)) or cause leaf disease, the gene of fungi in the group that the fungi kind of wilt disease or fruit rot is formed.

In specific embodiment, described plant is a paddy rice, and fungi is to cause for example rice blast fungus of rice blast.In another embodiment, described plant is a paddy rice, fungi is Rhizoctonia (Rhizoctonia spp.) (for example dry thread Pyrenomycetes (Rhizoctoniasolani), the withered spot rhizoctonia of rice (Rhizoctonia oryzae) or a cereal rhizoctonia (Rhizoctoniacerealis)), and it causes for example banded sclerotial blight.Still in another embodiment, described plant is a paddy rice, and described fungi is Rhizoctonia (Rhizoctonia spp.) (dry thread Pyrenomycetes (Rhizoctonia solani) for example, withered spot rhizoctonia of rice (Rhizoctonia oryzae) or cereal rhizoctonia (Rhizoctonia cerealis)), fusarium (Fusarium spp.) (for example pink sickle spore (Fusarium roseum)), little Acremonium (Acremoniella spp.) (for example black little single head spore mould (Acremoniella atra)), pythium (Pythium spp.) (for example many male rotten mould (Pythium arrhenomanes), group's knot rotten mould (P.myriotylum), or wide male rotten mould (P.dissotocum)), (for example rice is bent spore (Curvularia oryzae) to Curvularia (Curvularia spp.), curved spore (Curvularia lunatas)), Trichoderma (Trichoderma spp.) (for example viride (Trichoderma virde)) or Rhizopus (Rhizopus spp.) (for example zhizopchin (Rhizopus chinensis)) cause seedling blight; Described in another embodiment plant is that soybean and described fungi are Phakopsora (Phakopsora spp.) (for example yam bean layer rest fungus (Phaopsora pachyrhizi)), and it causes for example soybean rust; In another embodiment, described plant is a potato, and described fungi is phytophthora (Phytophthora spp.) (camphor tree epidemic disease mould (Phytophthoracinnamomi) for example, Phytophthora cactorum (Phytophthora cactorum), Kidney bean epidemic disease mould (Phytophthoraphaseoli), phytophthora parasitica (Phytophthora parasitica), oranges and tangerines brown rot epidemic disease mould (Phytophthora citrophthora), big male epidemic disease mould (Phytophthora megaspermaf.sp.soiae) or phytophthora infestans (Phytophthora infestans)), it causes for example late blight; In another embodiment, described plant is a banana, and described fungi is cercospora (Cercospora spp.) (for example Kikuchi tail spore (Cercospora kikuchii) or corn tail spore (Cercospora zaea-maydis)) or mycosphaerella (Mycosphaerella spp.), and it causes for example black yellow leaf spot.In another embodiment, described plant is a banana, and described fungi is fusarium (Fusarium spp.) (for example avenging rotten sickle spore, sharp sickle spore (Fusarium oxysporum), F.graminearum schw (Fusarium graminearum), Fusarium germinearum, yellow sickle spore (Fusarium culmorum), eggplant sickle spore (Fusarium solani), beading sickle spore (Fusarium moniliforme) or pink sickle spore (Fusarium roseum)), and it causes for example banana Panama disease; In another embodiment, described plant is a tomato, and described fungi is phytophthora (Phytophthora spp.) (camphor tree epidemic disease mould (Phytophthora cinnamomi) for example, Phytophthora cactorum (Phytophthoracactorum), Kidney bean epidemic disease mould (Phytophthora phaseoli), phytophthora parasitica (Phytophthoraparasitica), oranges and tangerines brown rot epidemic disease mould (Phytophthora citrophthora), big male epidemic disease mould (Phytophthora megasperma f.sp.soiae) or phytophthora infestans (Phytophthorainfestans)), it causes for example late blight; In another embodiment, described plant is a corn, and described fungi be Colletotrichum (Colletotrichum spp.) (for example beans thorn dish spore (

Lindemuthianum)), it causes for example anthrax.In another embodiment, described plant is a corn, and described fungi is that shell look list is every spore (Diplodia spp.) (for example corn husk look list is every spore (Diplodia maydis)), fusarium (Fusarium spp.) (is for example avenged rotten sickle spore (Fusarium nivale), point sickle spore (Fusariumoxysporum), F.graminearum schw (Fusarium graminearum), Fusariumgerminearum, yellow sickle spore (Fusarium culmorum), eggplant sickle spore (Fusariumsolani), beading sickle spore (Fusarium moniliforme) or pink sickle spore (Fusariumroseum)) or Gibberella (Gibberella spp.), it causes for example fringe, rotting of grain and stalk.In another embodiment, described plant is a corn, and described fungi is Puccinia (Puccinia spp.) (for example corn handle rest fungus (Puccinia sorghi), bar shaped handle rest fungus (Puccinia striiformis), wheat handle rest fungus (Puccinia graminis f.sp.tritici), Radix Asparagi handle rest fungus (Puccinia asparagi), Puccinia recondita (Pucciniarecondita) or a Semen arachidis hypogaeae handle rest fungus (Puccinia arachidis)), and it causes for example coventional type rust; In another embodiment, described plant is a cotton, and described fungi is fusarium (Fusarium spp.) (for example avenging rotten sickle spore (Fusarium nivale), sharp sickle spore (Fusarium oxysporum), F.graminearum schw (Fusarium graminearum), Fusarium germinearum, yellow sickle spore (Fusarium culmorum), eggplant sickle spore (Fusarium solani), beading sickle spore (Fusarium moniliforme) or pink sickle spore (Fusarium roseum)), and it causes for example sickle spore bacterium blight.In another embodiment, described plant is a cotton, and described fungi is Verticillium (Verticillium spp.) (for example Garden Dahlia wheel branch spore (Verticillium dahliae) or a black and white wheel branch spore (Verticilliumalbo-atrum)), and it causes for example verticillium; In another embodiment, described plant is a potato, and described fungi is for example dry thread Pyrenomycetes (Rhizoctonia solani), the withered spot rhizoctonia of rice (Rhizoctonia oryzae) or a cereal rhizoctonia (Rhizoctonia cerealis) of Rhizoctonia (Rhizoctonia spp.)), it causes for example early blight.In another embodiment, described plant is a potato, and described fungi is to cause for example fungi kind of wilt disease, rot or scurf; In another embodiment, described plant tomato, and described fungi is the fungi kind that causes for example leaf disease, wilt disease or fruit rot.

The top specifically described plant species that transgenic plant according to the present invention prolong and all have resistance to top specifically described corresponding fungi kind.Preferred transgenic plant (perhaps reproduction of transgenic plant or reproductive material, or the transgenic plant cells of cultivating) is plant (perhaps reproduction of transgenic plant or reproductive material, or the transgenic plant cells of cultivating), wherein said fungi target gene comprises and is selected from by SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, sequence in 48 and 110 in the sequence set of any sequence or its complementary sequence representative has at least 75%, preferably at least 80%, 85%, 90%, more preferably at least 95%, 98% or 99% conforming sequence, perhaps wherein said fungi target gene are to comprise SEQ ID Nos 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, the fungi of the gene of any sequence is directly to homologue in 48 and 110.

Can provide described plant with the form of in one or more (kind) cell, cell type or tissue, expressing (transcribing) double-stranded RNA actively.Alternatively, this plant can " can be expressed ", this means that its be encoded transgenosis of dsRNA of expectation transforms, but when described plant (and be in when this plant is provided form) was provided, described transgenosis was not in active state in plant.

Therefore, according to another embodiment, provide the recombinant DNA construction body of the nucleotide sequence that comprises the coding dsRNA or the dsRNA construct that are connected at least one adjusting sequence according to the present invention with handling.Preferably, described adjusting sequence is selected from and comprises constitutive promoter or tissue-specific promoter as described in the present invention.

Target gene can be any target gene of describing herein.Preferably, described regulatory element is to have active regulatory element in vegetable cell.More preferably be that regulatory element is to come from plant.

Term " adjusting sequence " should understood its meaning in the scope widely, and expression can realize can handling with it modulability nucleic acid of the sequence expression that couples together.

Aforementioned term is contained promotor and activation or is strengthened the nucleic acid or the synthetic fusion molecule or derivatives thereof of expression of nucleic acid, promptly so-called activator or enhanser.Term " operably connection " is illustrated in the functional connection between promoter sequence and the gene of interest as used in this article, makes promoter sequence can start transcribing of gene of interest.

For example, the transgenosis nucleotide sequence of coding double-stranded RNA can be placed under the control of derivable or growth or etap specificity promoter, by adding at the inductor of inducible promoters maybe when reaching particular growth or etap, described promotor allows to start transcribing of this dsRNA.

Selectively be, the transgenosis of coding double-stranded RNA is placed under the strong constitutive promoter, described promotor for example is to be selected to comprise CaMV35S promotor, two CaMV35S promotor (doubled CaMV35S promoter), ubiquitin promoter, actin promoter, ribulose-1,5-bisphosphate, 5-bisphosphate carboxylase promotor (rubisco promotor), GOS2 promotor, figwort mosaic virus (Figwort mosaic virus, FMV) any promotor in the group of 34S promotor.

Selectively be, the transgenosis of coding double-stranded RNA is placed under the tissue-specific promoter, and described promotor for example is to be selected from the root-specific promoter (root specific promoters of genes encoding PsMTA ClassIII Chitinase) that comprises coding PsMTA III shell polysaccharase gene, photosynthetic tissue's specificity promoter such as cab1 and the proteinic promotor of cab2, rbcS, gapA, gapB and ST-LS1, JAS promotor, chalcone synthetase promoters with from any promotor in the group of the RJ39 promotor of strawberry.

In addition, when using method of the present invention to be used to develop the antifungal property transgenic plant, will place according to the nucleic acid of coding double-stranded RNA of the present invention under tissue-specific promoter's control and may benefit.In order to improve dsRNA, can be preferably at first enter or cause in the plant part of infringement and express dsRNA the plant harmful organism from the transfer of vegetable cell to harmful organism.Under the situation of phytopathogenic fungi, the preferred tissue of expressing dsRNA is root, leaf and stem.Therefore, in the method for the invention, can use the preferred promotor of plant tissue, such as root-specific promoter, leaf specificity promoter or root-specific promoter.The suitable example of root-specific promoter is PsMTA (Fordam-Skelton, A.P., et al., 1997 PlantMolecular Biology 34:659-668.) and III shell polysaccharidase promotor.Leaf-and the example of root-specificity or photosynthetic tissue's specificity promoter (also being photoactivated) be two kinds of chlorophyll-binding protein matter (cab1 and cab2) (Stahl D.J. from beet, et al., 2004 BMCBiotechnology 20044:31), ribulose-bisphosphate carboxylase (Rubisco) (Nomura M.et al., 2000 Plant MoI.Biol.44:99-106) by the rbcS coding, A of chloroplast(id) glyceraldehyde 3-phosphate dehydro-genase (gapA) and (the ConleyT.R.et al.1994 MoI.Cell Biol.19:2525-33 of B (gapB) subunit; Kwon H.B.et al.1994Plant Physiol.105:357-67) promotor, coding leaf and the proteinic potato of root-specific (ST-LS1) (Solanum tuberosum) gene promoter (Zaidi M.A.et al., 2005 Transgenic Res.14:289-98), the stem modulability, the defence induced gene promoter is such as JAS promotor (patent publication number 20050034192/US-A1), flower specific promoter such as chalcone synthetase promoters (Faktor O.et al.1996 Plant.MoI.Biol.32:849) and fruit-specific promoter such as RJ39 promotor (WO 9831812) from strawberry.

In other embodiment of the present invention, use to can be used for expressing other promotor of dsRNA, include but not limited to RNA Poll, RNA Polll, RNA Pollll, the promotor of T7 RNA polymerase or SP6 RNA polymerase.These promotors are normally used for produced in vitro dsRNA, subsequently this dsRNA are included in the anti-mycotic agent, for example antimycotic liquid, spraying or powder.

Therefore, the present invention also comprises the method that produces any double-stranded RNA of the present invention or RNA construct.This method may further comprise the steps

Allowing nucleic acid or recombinant DNA construction body to produce under the condition of dsRNA or RNA construct,

Isolating nucleic acid of the present invention or recombinant DNA construction body are contacted with acellular component; Or

B. in cell, introduce (for example by conversion, transfection or injection) isolating nucleic acid of the present invention or recombinant DNA construction body.

Therefore, the present invention also comprises the cell that comprises any described nucleotide sequence or recombinant DNA construction body herein.The present invention also comprise prokaryotic cell prokaryocyte (such as, but be not limited to Gram-positive and gram negative bacterium cell) and eukaryotic cell (such as, but be not limited to yeast cell or vegetable cell).Preferred described cell is bacterial cell or vegetable cell.

Randomly, one or more transcription termination sequences also can be incorporated in the recombinant precursor of the present invention.Term " transcription termination sequence " is included in the control sequence of transcriptional units end, and it sends about 3 ' end processing and the polyadenylic acidization of primary transcript and the signal of Transcription Termination.The regulatory element that other adds such as enhanser that transcribe or translation, can be incorporated in the expression construct.

Recombinant precursor of the present invention can also comprise replication orgin, and it is essential for keeping and/or duplicating in concrete cellular type.Example is that the requirement expression construct is as the free genetic elements in the cell (for example plasmid or clay molecule) when maintaining in the bacterial cell.Preferred replication orgin includes, but not limited to f1-ori and colE1 ori.

Recombinant precursor can randomly comprise selectable marker gene.As what use in this article, term " selectable marker gene " comprises that any cell of expressing therein to it gives phenotype, to promote through the evaluation of the cell with expression construct of the present invention of transfection or conversion and/or the gene of selection.But the example of suitable selective marker comprises the resistant gene of anti-penbritin (Ampr), tsiklomitsin (Tcr), kantlex (Kanr), phosphinothricin and paraxin (CAT) gene.Suitable marker gene in addition provides metabolism proterties, for example manA.Also visual marker gene be can use, for example β-glucuronidase (GUS), luciferase and green fluorescent protein (GFP) comprised.

Be encoded the plant of transgenosis stable conversion of dsRNA can be inactive expression dsRNA, but have this ability the form of seed, reproductive material, reproductive material or cell culture provide.

Therefore, the present invention includes plant (for example paddy rice class plant) or the seed (for example rice paddy seed) or the cell (for example bacterium or vegetable cell) of any nucleotide sequence that contains coding dsRNA described herein or dsRNA construct.The present invention also comprises plant (for example paddy rice, barley, rye, wheat, grain, Herba Eragrostidis pilosae or lady's-grass plant) or seed (seed of paddy rice, barley, rye, wheat, grain, Herba Eragrostidis pilosae (lovegrass) or lady's-grass (crabgrass)) or the cell (for example bacterium or vegetable cell) that contains any dsRNA described herein or dsRNA construct.Preferably, these plants or seed or cell comprise recombinant precursor, and the nucleotide sequence of wherein encoding according to dsRNA of the present invention or dsRNA construct operably is connected at least one aforesaid regulatory element.Preferably, described plant or seed or cell are paddy rice or rice paddy seed or rice cell.

In plant, express the routine techniques that exogenous double-stranded RNA is used for the RNAi purpose and be well known in the art (see Baulcombe D, 2004, Nature.431 (7006): the RNA silence in the 356-63. plant, its content is incorporated herein by reference).More specifically, in plant, express double-stranded RNA and be used for that negative to regulate the plant harmful organism also be known such as the method for the genetic expression of nematode or insect in the art.In order in plant, to express double-stranded RNA to be used for negative method of regulating the applications similar in a similar fashion of phytopathogenic fungi expression of target gene purpose.In order to realize such effect, only need plant in the plant part that will directly contact fungi, to express (transcribing) double-stranded RNA, thereby double-stranded RNA can be absorbed by fungi.Depend on fungi person's character and with the relation of host plant, the expression of dsRNA can take place in the cell or tissue of plant, fungi also is present in its life cycle in the cell or tissue of described plant, perhaps RNA can be entered space between the cell by secretion, such as apoplast, it is that fungi is occupied in life cycle.In addition, dsRNA can be in the vegetable cell, for example in cytosol, or the cell organelle of vegetable cell such as chloroplast(id), plastosome, vacuole or endoplasmic reticulum in.

Selectively, dsRNA can be secreted by vegetable cell, and by plant secretion to the plant outside.Like this, dsRNA can form protective layer on plant surface.

In another embodiment, the present invention relates to be used to control the composition of fungal growth and/or prevention or reduction fungal infection, it comprises at least a double-stranded RNA, wherein said double-stranded RNA comprises the annealed complementary strand, a chain wherein have in the nucleotide sequence of fungi target gene to small part complementary nucleotide sequence, and randomly also comprise at least a suitable carriers, vehicle or thinner.Target gene can be described herein any target gene.Preferably, this fungi target gene is viability, growth, the growth for fungi or breeds necessaryly that for example the fungi target gene relates to any as cell function represented in the table 1; Perhaps the fungi target gene relates to the pathogenic or infectious of fungi, for example the fungi target gene relate to germ tube formation, conidially adhere to, the formation of appressorium, formation or conidial formation of invasion silk.

Another aspect of the present invention relates to aforesaid composition, wherein the fungi target gene comprises and is selected from by SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, sequence in 48 and 110 in the sequence set of any sequence or its complementary sequence representative has at least 75%, preferably at least 80%, 85%, 90%, more preferably at least 95%, 98% or 99% conforming sequence, perhaps wherein said fungi target gene are to comprise SEQ ID Nos 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, the fungi of the gene of any sequence is directly to homologue in 48 and 110.

The invention still further relates to the composition that comprises at least a double-stranded RNA as described herein, at least a double-stranded RNA construct, at least a nucleotide sequence and/or at least a recombinant DNA construction body, it randomly also comprises at least a suitable carriers, vehicle or thinner.

Described composition can comprise the other component that plays stabilizing dsrna and/or prevent the dsRNA Degradation during the composition prolonged preservation.

Described composition still can also comprise enhancing or promote the fungal cell to absorb the component of dsRNA.These components can comprise, for example, mainly promote for example lipofectamin etc. of chemical agent that the RNA picked-up enters cell, and the enzyme or the chemical agent that can digest fungal cell wall, for example chitinase.

In order to be applied to fungal cell, matrix, cell (for example vegetable cell) or to be subjected to organism fungi infestation or that be subject to fungi infestation, described composition can be any nonlimiting examples of suitable physical.

Expect that " composition " of the present invention can provide with the form of " box parts ", it comprises the diluent or carrier that is in a double-stranded RNA in the container and is in the suitable RNA in the container separately.The present invention also relates to double-stranded RNA is provided separately and without any other component.In these embodiments, dsRNA can provide with spissated form, such as the spissated aqueous solution.Even can provide with the refrigerated form or with lyophilize or freeze dried form.The latter is more stable for prolonged preservation, and can be thawed before facing application and/or restore with suitable diluent.

The invention still further relates to the medical usage of described any double-stranded RNA s, double-stranded RNA construct, nucleotide sequence, recombinant DNA construction body or composition herein.

In a particular, described composition is to be respectively applied for treatment or prevention human or animal's the fungal disease or the medicinal or veterinary composition of infection.Such composition will comprise at least a double-stranded RNA or RNA construct or the nucleotide sequence or the recombinant DNA construction body of encode this double-stranded RNA or RNA construct, wherein double-stranded RNA comprises the annealed complementary strand, a chain wherein has the nucleotide sequence corresponding to the target nucleotide sequences of the fungi target gene that causes disease or infection, and comprises at least a medicinal carrier, vehicle or the thinner of being suitable for.

Described composition can be the composition that is suitable for topical application, such as the composition of on animal or human's skin, using, for example be applied to the fluid composition of skin or be used for the sprays, ointment, ointment etc. of topical application or as the patch of transdermal as drops, gel, aerosol form or by brushing.

Alternatively, described fungi dsRNA is by being included in the food and producing as the bacterium (for example Bacterium lacticum) that the oral vaccine of anti-fungal infection works.

Also other conventional pharmaceutical dosage forms be can produce, tablet, capsule, vaginal suppository, transdermal patch, suppository etc. comprised.The disease characteristic that selected formulation will depend on the characteristic of target fungi and wish thus to be treated.

Preferably go into the pathogenic and pathogenic target fungi of animal and include, but are not limited to following fungi:

In the people: mycocandida (Candida spp.), especially Candida albicans (Candidaalbicans); Dermatophytes (Dermatophytes) comprises Epidermophyton (Epidermophytonspp.), Trichophyton (Trichophyton spp.) and Microsporon (Microsporumspp.); The set of Aspergillus (Aspergillus spp.), especially flavus (Aspergillus flavus), Aspergillus fumigatus (Aspergillus fumigatus), Aspergillus nidulans (Aspergillus nidulans), aspergillus niger (Aspergillus niger) or terreus (Aspergillus terreus); Dermatitis budding yeast (Blastomyces dermatitidis); Paracoccidioides brasiliensis (Paracoccidioides brasiliensis); Posadasis spheriforme (Coccidioides immitis); Novel Cryptococcus (Cryptococcus neoformans); Capsule tissue's spore slurry bacterium mutation (Histoplasma capsulatum Var.capsulatum) or capsule tissue's spore slurry bacterium Du Shi mutation (Var.duboisii); Sporothrix schenckii (Sporothrix schenckii); Fusarium (Fusarium spp.); Short handle broom mould (Scopulariopsis brevicaulis); And Fonsecaea (Fonsecaea spp.); Penicillium (Penicillium); Zygomycota fungi, especially absidia corymbifera (Absidia corymbifera), Rhizomucor pusillus (Rhizomucorpusillus) or rhizopus arrhizus (Rhizopus arrhizus);

In animal: mycocandida (Candida spp.); The little spore of Microsporon (Microsporumspp.), especially dog mould (Microsporum canis), the little spore of gypsum shape mould (Microsporum gypseum); Trichophyton mentagrophytes (Trichophyton mentagrophytes); Aspergillus (Aspergillus spp.); Or Cryptococcus neoformans (Cryptococcus neoformans).

In a specific embodiments, for protect matrix avoid infecting of fungi and/or prevent, stop or reduce fungi on matrix growth and prevent thus by fungus-caused infringement, described composition can be the coating that can be applied to matrix.In this embodiment, described composition can be used to protect and anyly be subject to fungal infection or cause the matrix or the material of infringement by fungi, for example food and other perishable material and such as the matrix of timber one class.For the preferred target fungi kind of this embodiment including, but not limited to following fungi: grape ear mould (Stachybotrys spp.), Aspergillus (Aspergillus spp.), Alternaria (Alternariaspp.) or cladosporium belong to (Cladosporium spp.).

The characteristic of vehicle and the physical form of composition can depend on desired processing matrix characteristic and change.For example, described composition can be brushed or be sprayed on pending material or the matrix or seal liquid within it.

The present invention also comprises the method that treats and/or prevents fungal infection on the matrix, comprises any described composition of using significant quantity to described matrix herein.

The present invention also comprises the method that treats and/or prevents fungal disease or illness, comprise to this object that treats and/or prevents of needs and use described any composition herein, described composition comprises at least a double-stranded RNA or the double-stranded RNA construct that comprises the complementary strand of annealing, a chain wherein have with cause fungal disease or illness the fungi target gene nucleotide sequence to small part complementary nucleotide sequence.

In another embodiment of the invention, described composition is used as plant or plant propagation or reproductive material, such as the mycocide on the seed.As an example, described composition can be used as by spraying or being administered on the plant tissue or spraying on soil before or after plantlet grows or mycocide that hybrid mode is used.

In another embodiment, the invention provides the breeding or the fungal growth of reproductive material and/or the method for fungal infection that treat and/or prevent plant or plant, comprise described any composition of using significant quantity to breeding or the reproductive material of plant or plant herein.

In another embodiment, the present invention relates to double-stranded RNA or the nucleotide sequence of RNA construct or the purposes of recombinant DNA construction body described in described any double-stranded RNA or RNA construct or coding this paper herein, perhaps relate to any composition that comprises described material, be used to control fungi growth; Be used to prevent be subject to the fungal infection of the plant of fungal infection; Or be used for the treatment of the fungal infection of plant.The concrete plant that is ready to use in the fungi infestation that treatment causes by concrete fungi kind and is included in the described purposes as previously mentioned.

The invention still further relates to and comprise as at least a double-stranded RNA that is used for the treatment of fungi infestation in the plant of description earlier or the test kit of double-stranded RNA construct or nucleotide sequence or recombinant DNA construction body or cell or composition.

According to embodiment further, the present invention prolongs and is used to increase the method for plant biomass, comprises with effable form introducing any nucleotide sequence as described herein or recombinant DNA construction body in plant.Be included in plant and first describe the same in this method.Preferably, described plant is a paddy rice.

In a specific embodiments, method of the present invention also can be used as the instrument of experimental study, especially in the field of functioning gene group.In order to study the function of gene, by RNAi to the target of fungal gene downward modulation can in external or body, use in the detection method, its similar method is at nematode Caenorhabditis elegans (C.elegans) and in drosophila melanogaster (Drosophila melanogaster) description was arranged equally.Regulate based on the orientation of specific fungal gene is negative, thereby the detection method that causes measuring phenotype also can constitute the screening compound basis of novel anti Fungicide.

The explanation of accompanying drawing and form

Fig. 1: dsRNA is to the influence of rice blast fungus (Magnaporthe grisea) mycelial growth.That show is relevant 'beta '-tubulin target gene (MG00604.4 (nt 1151-1344, see Table 3) (a), MG00884.4 (nt 845-1044, see Table 3) (b), MG07031.4 (nt 251-500, see Table 3) (c) and the data of MG04484.4 (nt 211-409 sees Table 3) dsRNA (d).Each detects by 4 in 96 orifice plates and repeats to form, and with contrast dsRNA and compare such as detection from the dsRNA of GST.Each dsRNA is added in 1250 fungal spores with the amount of 0.5mg/ml, and absorbancy is read in after adding dsRNA 0,24 and 30 hour.The inhibition per-cent that is caused by the dsRNA of 'beta '-tubulin had significance (P＜0.05) in the time of 24 and 30 hours.Dotted line indicates the background absorbancy at the O.D.595nm place.

Fig. 2: the plasmid map of plant expression vector pMH115

Fig. 3: the target gene catalogue that comprises encoding sequence.Rule below initial sum ending place of sequence of initial sum terminator codon.

Fig. 4: hairpin tabulation.The sequence of runic is represented Sl intron (SEQ ID NO204).

Table 1: the example of suitable fungi target gene.DNA that provides in table and protein sequence come from Pyricularia oryzae Cav fungi rice blast fungus (Magnaporthe grisea) (being also referred to as rice blast fungus (Magnaporthe oryzae)).Identifier is corresponding to the genetic identifier of rice blast fungus (M.grisea) gene order-checking.Also provided the homologous gene of budding yeast-yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), identifier is corresponding to yeast belong genome database (SGD ^TM) genetic identifier, and provided specified gene function on yeast data basis.

Table 2: the example of suitable fungi target gene.The DNA that provides in table is directly consistent to homologue with the fungi of the Pyricularia oryzae Cav fungi rice blast fungus (Magnaporthe grisea) of table 1 with protein sequence.Identifier is corresponding to typing numbering and version number.

Table 3: clone's details summary of rice blast fungus (Magnaporthe grisea) target gene cDNA.The PCR condition is as follows: A: 5 ' 94 ℃ of high-fidelity pcr amplification systems (Roche), 10 ' 72 ℃ of 30 circulations (1 ' 94 ℃, 1 ' 58 ℃, 1 ' 72 ℃); B: 5 ' 94 ℃ of high-fidelity pcr amplification systems (Roche), 30 circulations (58 ℃, 45 of 45 " 94 ℃, 45 " " 72 ℃), 10 ' 72 ℃; C: 5 ' 94 ℃ of high-fidelity pcr amplification systems (Roche), 30 circulations (58 ℃, 45 of 45 " 94 ℃, 45 " " 72 ℃), 10 ' 72 ℃; D: 5 ' 94 ℃ 30 circulations of high-fidelity pcr amplification system (Roche) (1 ' 94 ℃, 1 ' 60 ℃, 3 ' 72 ℃), 5 ' 72 ℃; 3 ' 95 ℃ of E:Taq polysaccharases (Bangalore Genei), 10 circulations (55 ℃ of 30 " 95 ℃, 30 ", 3 ' 72 ℃), 25 circulations (55 ℃ of 30 " 95 ℃, 30 ", 3 ' (+5 "/circulation) 72 ℃), 7 ' 72 ℃; With 5 ' 94 ℃ of F:GC polysaccharases (BD Biosciences), 10 ' 68 ℃ of 30 circulations (1 ' 94 ℃, 1 ' 58 ℃, 3 ' 68 ℃).

Table 4: the details of using gDNA to clone rice blast fungus (Magnaporthe grisea) target gene exon as template is summarized.The PCR condition is as follows: 5 ' 94 ℃ of A:Pwo polysaccharases (Roche), 5 ' 72 ℃ of 30 circulations (60 ℃ of 30 " 94 ℃, 30 ", 1 ' 72 ℃); 5 ' 94 ℃ of B:Pwo polysaccharases (Roche), 5 ' 72 ℃ of 30 circulations (58 ℃, 30 of 30 " 94 ℃, 30 " " 72 ℃); 5 ' 94 ℃ of C:Pwo polysaccharases (Roche), 5 ' 72 ℃ of 30 circulations (1 ' 94 ℃, 1 ' 61 ℃, 1 ' 30 " 72 ℃); 5 ' 94 ℃ of D:Pwo polysaccharases (Roche); 5 ' 72 ℃ of 30 circulations (60 ℃, 30 of 30 " 94 ℃, 30 " " 72 ℃); 5 ' 94 ℃ of E:Pwo polysaccharases (Roche), 5 ' 72 ℃ of 30 circulations (60 ℃, 45 of 45 " 94 ℃, 45 " " 72 ℃); 10 ' 94 ℃ of F:Pwo polysaccharases (Roche), 10 ' 72 ℃ of 30 circulations (1 ' 94 ℃, 1 ' 60 ℃, 3 ' 72 ℃).

Table 5: the selected target nucleotide sequences of fungi target gene.(*: the test of operation)

Table 6: in pMH115, be used for the primer that hair clip makes up

By will further understanding the present invention with reference to following non-limiting example.

Embodiment

Embodiment 1: the clone of target gene

The method that is used for the gene silencing of fungi dsRNA mediation is as described herein resisted plant pathogenic fungi applicable to the dsRNA that uses corresponding to the phytopathogen target gene.From fungi, identify and separate suitable phytopathogen target gene by following.

Use is used for the RNeasy Mini test kit (QIAGEN Cat.No.74904) of plant from the total RNA of fungi rice blast fungus (Magnaporthe grisea) preparation, uses Superscript ^TMDouble-stranded cDNA synthetic agent box (Invitrogen) prepares cDNA from total RNA.In order to separate the encoding sequence of rice blast fungus (Magnaporthe grisea) target gene MG00884.4, MG04484.4, MG07472.4, MG06292.4, MG03668.4, MG05169.4 and MG03872.4, implement PCRs as template with the cDNA or the genomic dna of rice blast fungus (Magnaporthe grisea).PCR condition and primer in form, have been summarized for target gene.Each PCR carries out in duplicate.On sepharose, analyze from resulting two the PCR products independently of each target gene, separate and be cloned into pTZ57R/T carrier (MBI Fermentas).For each PCR product, at least to three cloning and sequencings.To compare from the sequence that obtained of clone and the sequence of public database, and select one or more clones further to test each target gene.Provide in clone's details form in this article of the encoding sequence of these genes.

Optional clone's scheme: the clone of cDNA exon:

For can not be from the cDNA of total RNA amplification, from genomic dna (gDNA) thus amplification exon and exon linked together form cDNA.Application from Naqvi etal. (Molecular Breeding, 1995,1:341-348) improved plan prepares the genomic dna of rice blast fungus (Magnaporthe grisea).With the dried mycelia of about 150mg in liquid nitrogen grind into powder and extracting damping fluid (0.25M NaCl carries out extracting in 30 minutes genomic dna in 1%SDS) under 65 ℃ for 10mM Tris pH 8.0,1mM EDTA.Potassium ethanoate by adding 5M pH4.8 is also deposited in-20 ℃ of refrigerators and was come sedimentation cell fragment and protein in 15 minutes.By removing precipitation in centrifugal 10 minutes with 14000rpm.Supernatant liquor is transferred in the new pipe and uses phenol/CH ₃The Cl/ iso pentane alcohol mixture extracts.The water layer isopropanol precipitating.Clean the DNA precipitation with 70% ethanol, air-dry and be resuspended among the TE again.This prepared product is used as template and is used for exon through pcr amplification fungi target gene.

In order to separate the coding region of rice blast fungus (Magnaporthe grisea) target gene MG00170.4, MG07031.4, MG02946.4 and MG10192.4, each exon of each target gene is implemented two independently PCRs.Use from the full-length gene group gene order of gDNA amplification as template each exon that increases, thereby editor comprises the cDNA of coding region.In table 4, summarized PCR condition for each exon of target gene.Subsequently,, on sepharose, analyze the exon that the increases/PCR product that comes from identical target, it is separated, and connect together with correct order each other in order to obtain the encoding sequence of total length.Such connection is as follow finished:

Step 1: use check and correction enzyme Pwo polysaccharase (Roche) to carry out the pcr amplification of each exon, produce flush end.

Step 2: the polynucleotide kinase (PNK, Bangalore Genei) of using 1 unit in having the 15 μ l reaction systems of 1mM ATP makes the second exon phosphorylation of 2.5pmols.

Step 3: the flush end of two exons connects.

Step 4: the gene of applying gene specificity forward and reverse primer pcr amplification total length from be connected mixture.This step guarantees only to select the connection product of those exons correctly to be linked in sequence.

In having target gene MG07031.4 (3) situation of 5 exons, two exons in front (exons 1 and 2 is respectively 70bp and 21bp) prepare with synthesis mode.Also as follow connect as 3 exons in general introduction amplification back more than this paper:

1-connects and pcr amplification exon 4+5, thereby obtains exon 4,5;

2-connects and pcr amplification exon 3+ exon 4,5, thereby obtains exon 3,4,5;

3-is connected to exon 3,4,5 with synthetic exons 1+2, obtains exons 1,2,3,4,5 (through the gene of montage) thereby then increase.

Connect product and be cloned into pTZ57R/T carrier (MBI Fermentas).Connect product for each, at least 3 cloning and sequencings.To compare from the sequence of clone's acquisition and the sequence of public database, and select one or more clones to be used for further test each target gene.Provide clone's details of the exon that is joined together to form these target gene encoding sequences in the table 4 in this article.CDNAs according to other target gene of one of above method clone.

The template of starfish AFP (autofluorescent protein) is inner clone pGR37, from pGEX4T-1 amplification paddy Guang sweet phthalein S-transferring enzyme (GST), clones pDW2821 amplification GFP internally.Directly from gDNA amplification 'beta '-tubulin gene extron and be used as dsRNA synthetic template.

Embodiment 2: select the target nucleotide sequences of the target gene of rice blast fungus (Magnaporthe grisea) to be used for the gene silencing that RNAi mediates

The intravital further RNAi external or as embodiment 4,5 and 6 described in that select the fragment of described target gene herein to be used for as described in example 3 above test.These fragments in table 5, have been listed.Person of skill in the art will appreciate that, can in rice blast fungus (Magnaporthe grisea) sequence, determine other fragment of all lengths, and the present invention prolongs equally and these fragments and their purposes in the silence of the RNAi of fungal gene mediation.Preferably, such fragment does not influence such as paddy rice or people non-target organism.

Use the fragment of these fungi target genes or target nucleotide sequences as described in example 3 above at external generation dsRNA, perhaps cloned and in the hair clip construct so that in vegetable cell, produce dsRNA (seeing Table 4).

Embodiment 3: the effect of reticent target gene in external rice blast fungus (Magnaporthegrisea) in vitro detection

The conidium that has demonstrated sprouting absorbs material by endocytosis actively from substratum.In following detection, use the germination conidia of rice blast fungus (Magnaporthe grisea) to prove that fungi is in external picked-up to dsRNA.Make conidia germination under hydrophilic conditions, this has simulated the sprouting situation in leaf after fungi is invaded leaf.On hydrophilic surface, conidium grows up to mycelium in vegetative mode.

As follow from the dsRNA of genomic dna or cDNA preparation corresponding to the target gene target region.Set two PCR reaction: one is used for amplification adopted RNA chain, another sense-rna chain that is used for increasing is arranged.The forward primer of each reaction comprises the T7 promoter sequence, and the back is corresponding to by the sequence of target sequence, and reverse primer only comprises the sequence that is complementary to target sequence.Use QIAquick PCR purification kit (Qiagen) purified pcr product, and be used as the in-vitro transcription template subsequently to produce double-stranded RNA (T7 RiboMAXExpress RNAi System Promega).Precipitation dsRNA, to it quantitatively and be dissolved in the water of no RNase.

1. appressorium detection method on water repellent surface:

By being exposed to light, radicula byssoidea produces conidium (asexual spore) over 7-10 days.With 10 ⁴The aquation conidium that the density of individual conidium/ml will just have been collected is resuspended in the water, and it is seeded in artificial rust, and (GelBond film is on water repellent surface Cambrex).In water, in the final volume of 20 μ l, check dsRNA individually corresponding to each fungi target gene (seeing Table 5) with the concentration of 0.1-1mg/ml.Use contrasts as negative corresponding to the dsRNA of a GST part.After 28 ℃ are cultivated 16-24 hour, with C.I. 42685 the spore of sprouting is dyeed, so that more clearly illustrate the structure of cell.

Examine under a microscope the formation of appressorium on artificial rust.The inhibition that germ tube and appressorium form is because the direct indication of passing through RNAi inhibition expression of target gene that complete fungi picked-up dsRNA causes.Carry out soak test in triplicate.Number by appressorium in 3 in the microscope appointment visuals field calculates the per-cent of appressorium formation divided by the number of spore, and each at least 200 spores of double counting.

2. the detection of mycelial growth on hydrophilic surface

Collect the conidium of rice blast fungus (Magnaporthe grisea), and be resuspended in the potato glucose nutrient solution, and in each hole of wetting ability 96 orifice plates (Falcon 3072) 1250 conidiums of five equilibrium (in about 90 μ l volumes).28 ℃ 0-2 hours pre-sprout handle after, add dsRNA in the final volume of 100 μ l to 0.1 or the final concentration of 0.5mg/ml.Detection is corresponding to the long dsRNA fragment of about 200bp of the different target regions of target gene.Use contrasts as negative corresponding to the dsRNA of the part of GST, starfish AFP or GFP.After 28 ℃ were cultivated 16-24 hour, (GENiosTecan Austria) upward quantized mycelial growth in the hole by reading the optical density value of 96 orifice plates at 595am wavelength place in plate reader.Compared with the control, fungi demonstrates its growth-inhibiting when having target dsRNA fragment (see Table 5 and Fig. 1).The growth-inhibiting phenotype is because the direct indication of passing through RNAi inhibition expression of target gene that complete fungi picked-up dsRNA causes.Carry out soak test in quadruplicate, and calculate the inhibition per-cent that soaks sample with respect to contrast.Comprise at least in each the detection that thereby two different negative contrasts guarantee to suppress result's consistence.Use Analyse-it software and implement statistical analysis.

Embodiment 4: clone's hair clip construct and plant expression vector are used for producing dsRNA at vegetable cell

Because RNA interferential mechanism works by the dsRNA fragment, the target nucleotide sequences of the as above selected and target gene that provides in table 5 is with antisense and have the justice orientation to be cloned, and by from Arabidopis thaliana (Arabidopsis thaliana) gene X (SI, SEQ ID NO:204) the synthetic intron (SI) of 189bp separates, thereby forms dsRNA hair clip construct.These hair clip constructs are cloned a plurality of cloning sites that enter plant expression vector pMH115 (SEQ ID NO:205), wherein comprise two CaMV35S promotors and CaMV35S3 ' element.Use the template of aforesaid cDNA clone as PCRs.Produced hair clip construct by these clonogenic assay for each target gene, its have promotor-have adopted fragment-segmental structure of SI-antisense or more preferably promotor-antisense fragment-SI-adopted segmental structure is arranged, wherein there is adopted fragment in table 5, to provide, and wherein said promotor is the steerable promotor of any plant, preferred strong constitutive promoter is such as the CaMV35S promotor.The complete sequence of several hair clip constructs (antisense fragment-SI-has adopted fragment) provides in Fig. 4.

Aforesaid hair clip construct is inserted among the binary vector pMH115 with two CaMV35S promotors, and described carrier is suitable for transforming and enters agrobacterium tumefaciens (A.tumefaciens), transform and enter paddy rice, and expresses hairpin structure in paddy rice.

Selectively, use other carrier to transform, and use other promotor to express for the hair clip dsRNA of described target gene herein.For example, such promotor is selected from strong constitutive promoter, it comprises strong constitutive promoter such as CaMV35S promotor, two CaMV35S promotor, ubiquitin promoter, actin promoter, ribulose-1,5-bisphosphate, 5-bisphosphate carboxylase promotor (rubisco promotor), GOS2 promotor and FMV promotor.

The plant expression vector that comprises rice blast fungus (Magnaporthe grisea) hairpin structure is entered in the agrobacterium tumefaciens (Agrobacterium tumefaciens) (seeing embodiment 5) by conversion subsequently.

Embodiment 5: the rice plants of anti-rice blast fungus (Magnaporthe grisea)

Rice transformation callus and be regenerated as bud and complete plant described in document.Plant is transferred in the greenhouse and cultivates until maturation and produce seed.Leaf texture to the T1 plant implements genome PCR and/or Southern trace, with homozygosity/heterozygosity and the genetically modified copy amount of insertion of determining integrator locus.Further analyze the transgenic positive plant by Northern trace and/or RT-PCR, to detect dsRNA and siRNA.Foundation demonstrates to be expressed isozygotying of dsRNA and/or siRNA and is and is used for fungi infestation research.

Application carries out infecting at Pyricularia oryzae Cav the initial analysis of resistance from the explant (each has 15-20 repetition) of T1 plant (comprising heterozygosis and the intasome that isozygotys).Downcut the plant leaf of 20 days sizes and insert in the kinetin agar plate leaf is terminal.The small droplets (containing 200-1000 spore in 20 μ l water) of rice blast fungus (Magnaporthe grisea) spore is inoculated on the leaf.Relatively test and negative infection rate and the lesion size that contrasts between the leaf.

With 10 ⁵The fungal spore of/ml density sprays the dsRNA that (in planta) infection has been set up in the plant and expresses plant, covers with the plastics bag of having beaten the hole subsequently.These plants are maintained in the environmental cabinet (Convirons) until demonstrating disease symptoms.The time that manifests, the size of damage and amount and plant wilt rate are the indications (Valent is 80:57-67 B.1990.Phytopathology) to the susceptibility of rice blast fungus (Magnaporthe grisea) infection.In each test, each is transformed system and use 50-100 repetition.Further test the resistant rice plant in Convirons and in the field condition.

It will be understood by those skilled in the art that as shown in specific embodiments, can make various variations and/or improvement to the present invention, and not break away from the broadly described spirit or scope of the present invention.Therefore, these embodiments all are considered illustrative in all respects, and unrestriction.

In addition, be to be understood that, run through that word that whole specification sheets uses " comprises " or its version means such as " comprising " or " containing " and comprises key element, integer or the step stated or the set of key element, integer or step, but do not get rid of any other key element, integer or step or the set of key element, integer or step.

Those skilled in the art can recognize or only use the routine test method can determine many equivalents for each specific embodiments of invention described herein.These equivalents are intended to be included in the following claim.

All disclosed in this article reference comprise patent documentation, all are incorporated herein in full by the reference mode.

Trl1p has the activity of phosphodiesterase, polynucleotide kinase and ligase enzyme; Every kind of activity is positioned at different structural domains.In tRNA processing, from the tRNA transcript, remove intron by many subunits tRNA montage endonuclease (by SEN2, SEN15, SEN34 and SEN54 coding); Half point that connects gained by Trl1p.The disappearance of TRL1 is fatal; TRNA montage intermediate is accumulated in the cell that eliminates Trl1p or is limiting</entry></row></tbody></tgroup></table></tables>

				Be accumulated under the temperature condition in the temperature-sensitive mutation body.
					23 and 52	24	MG04056.4	YDR407C	The component of target complex body (TRAPP) relates to the transportation of ER to the Golgi film.Invalid body is nonviable.
21 and 51	22	MG08911.4	YJL039C	The essential structure subunit of nuclear pore complex (NPC) is positioned around the nuclear of nucleopore, with people p205 be homologous.Invalid body is nonviable.
					25 and 53	26	MG06314.4	YGR047C	Transcription factor tau (TFIIIC) subunit 13 1.
27 and 54	28	MG08863.4	YKL033W	Really, the non-marked protein positioning is in plastosome.
					31 and 56	32	MG07222.4	YPR175W	The second largest subunit of dna polymerase i I (archaeal dna polymerase ε), it is essential to be that normal yeast chromosomal duplicates; The border that is expressed in the G1/S phase peaks; Potential Cdc28p substrate.Null mutant can't be survived; Conditional mutant demonstrates the defective of dna replication dna aspect.
37 and 59	38	MG01760.4	YJL085W	The essential 70kDa subunit of exocytosis capsule complex body (Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p and Exo84), it has the essential function to the polarized target of exocytosis avtive spot of mediation secretory vesicle.Invalid body also is nonviable.
					19 and 50	20	MG07116.4	YIL129C	In seeking to activate the mutant process that COH1 transcribes, identify; The transcriptional activator of OCH1
33 and 57	34	MG03872.4	YJL010C	The rna binding protein that relates to RNA processing.The necessary protein of cell survival.
					35 and 58	36	MG04185.4	YBL034C	The component of mitotic spindle, it plays an important role in conjunction with the interpolar microtubule and in their ability that outward force on the spindle pole is provided.Null mutant shows the defective of spindle assembling aspect

Table 2

The rice blast fungus identifier	SEQ ID NO	Directly to homologue identifier (" registration number " " version number ")	Directly to the homology kind
				MG00170.4	210	XM_387062.1	Gibberella zeae PH1
214	XM_745627.1	Aspergillus fumigatus
			229		XM_446439.1	Candida glabrata
230	DR709833.1	Black-koji mould
			233		XM_709731.1	Candida albicans
236	CA582255.1	Paracoccidioides brasiliensis
			243		AJ636229.1	Standing grain green-ball chamber bacterium
245	XM_408069.1	Aspergillus nidulans FGSC A4
			254		DN795961.1	Sclerotinite
271	XM_571562.1	The Cryptococcus neoformans mutation
			272		XM_402498.1	Ustilago maydis
296	AW790794.1	Standing grain powdery mildew (Blumeria graminis f.sp.)
			301		CO136158.1	Aspergillus flavus
321	CV968020.1	Phytophthora infestans
			324		BI189607.1	Intend branch spore sickle spore
406	BW644197.1	Point sickle spore (Fusarium oxysporum f.sp.)
			409		XM_369074.1	Rice blast fungus 70-15
MG00604.4	208	XM_713903.1					Candida albicans
			215	AL117033.1	Fu Shi grape spore cup fungi
	216	CN239178.1				Paracoccidioides brasiliensis
			217	DR046029.1	Phaeosphaeria nodorum
	219	DR709051.1				Black-koji mould
			222	DR439037.1	Phytophthora parasitica
	223	CV966689.1				Phytophthora infestans
			224	DR439037.1	Phytophthora parasitica
	231	CO138830.1				Aspergillus flavus
			234	BQ110893.1	Garden Dahlia wheel branch spore
	241	BI191775.1				Intend branch spore sickle spore
			244	BI191775.1	Intend branch spore sickle spore
	249	DN477093.1				Wild cabbage blackspot chain lattice spore
			325	BQ621948.1	Conidiobolus coronatus
	329	AW789096.1				Standing grain powdery mildew (Blumeria graminis f.sp.)
			338	AY337716.1	Point sickle spore (Fusarium oxysporum f.sp.)
	410	AF218256.1				Ultimate corruption is mould
			411	AF257329.1	The Cruciferae ball cavity bacteria
	412	AY763789.1				Trichophyton purpureatum
			413	X81961.1	The pea powdery mildew
	414	XM_368640.1				Rice blast fungus 70-15
			415	XM_389706.1	Gibberella zeae PH-1
	416	XM_403443.1				Ustilago maydis
			417	XM_405319.1	Aspergillus nidulans FGSC A4
	418	XM_448766.1				Candida glabrata

	419	XM_569650.1	The Cryptococcus neoformans mutation
				420	XM_747363.1	Aspergillus fumigatus
	MG00884.4	220	AL116400.1				Fu Shi grape spore cup fungi
239				CO145866.1	Aspergillus flavus
		250	XM_716188.1			Candida albicans
251				XM_716459.1	Candida albicans
		255	XM_566480.1			The Cryptococcus neoformans mutation
256				XM_400867.1	Ustilago maydis
		289	AJ639036.1			Standing grain green-ball chamber bacterium
294				DR706305.1	Black-koji mould
		312	CN251795.1			Paracoccidioides brasiliensis
330				CF844359.1	Soybean phytophthora
		366	CV959275.1			Phytophthora infestans
421				XM_368360.1	Rice blast fungus 70-15
		422	XM_386221.1			Gibberella zeae PH-1
423				XM_406904.1	Aspergillus nidulans FGSC A4
		424	XM_749902.1			Aspergillus fumigatus
MG01760.4				263	XM_750434.1		Aspergillus fumigatus
	267	XM_750434.1	Aspergillus fumigatus
				275	XM_410347.1	Aspergillus nidulans FGSC A4
	335	DR707382.1	Black-koji mould
				355	CO152071.1	Aspergillus flavus
	367	AW180181.1	Standing grain green-ball chamber bacterium
				374	BQ493216.1	Paracoccidioides brasiliensis
	403	XM_399460.1	Ustilago maydis
				404	XM_449482.1	Candida glabrata
	425	XM_363834.1	Rice blast fungus 70-15
				426	XM_382095.1	Gibberella zeae PH-1
	MG02946.4	207	XM_389109.1				Gibberella zeae PH-1
225				XM_408897.1	Aspergillus nidulans FGSC A4
		276	DR710305.1			Black-koji mould
278				DR710305.1	Black-koji mould
		287	XM_749869.1			Aspergillus fumigatus
336				XM_397920.1	Ustilago maydis
		352	CA582353.1			Paracoccidioides brasiliensis
357				XM_566533.1	The Cryptococcus neoformans mutation
		389	XM_717783.1			Candida albicans
405				CV949205.1	Phytophthora infestans
		427	XM_366870.1			Rice blast fungus 70-15
MG03668.4				209	XM_570418.1		The Cryptococcus neoformans mutation
	237	XM_446411.1	Candida glabrata
				252	XM_708682.1	Candida albicans
	268	CN239360.1	Paracoccidioides brasiliensis
				283	BQ110666.1	Garden Dahlia wheel branch spore
	290	DR703485.1	Black-koji mould
				304	AJ635696.1	Standing grain green-ball chamber bacterium
	361	DN478339.1	Wild cabbage blackspot chain lattice spore
				384	CF843394.1	Soybean phytophthora
	393	CV916642.1	Phytophthora infestans

	428	XM_361125.1	Rice blast fungus 70-15
				429	XM_389630.1	Gibberella zeae PH-1
	430	XM_399990.1	Ustilago maydis
				431	XM_405043.1	Aspergillus nidulans FGSC A4
	432	XM_747845.1	Aspergillus fumigatus
				MG03872.4	319	XM_387214.1	Gibberella zeae PH-1
328	XM_749415.1	Aspergillus fumigatus
			377		CO147563.1	Aspergillus flavus
381	XM_706881.1	Candida albicans
			402		CF645358.1	Ustilago maydis
433	XM_361398.1	Rice blast fungus 70-15
			MG04056.4		206	XM_410670.1	Aspergillus nidulans FGSC A4
212	XM_742501.1	Aspergillus fumigatus
				376	CO151047.1	Aspergillus flavus
378	DR164852.1	Paracoccidioides brasiliensis
				386	XM_714273.1	Candida albicans
394	DN477226.1	Wild cabbage blackspot chain lattice spore
				408	XM_570072.1	The Cryptococcus neoformans mutation
434	XM_361582.1	Rice blast fungus 70-15
				435	XM_386415.1	Gibberella zeae PH-1
MG04185.4	280	XM_747547.1					Aspergillus fumigatus
			343	XM_405132.1	Aspergillus nidulans FGSC A4
	345	XM_402160.1				Ustilago maydis
			346	XM_708987.1	Candida albicans
	354	AL111352.1				Fu Shi grape spore cup fungi
			370	BI187889.1	Intend branch spore sickle spore
	372	BI187889.1				Intend branch spore sickle spore
			391	XM_569869.1	The Cryptococcus neoformans mutation
	436	XM_361711.1				Rice blast fungus 70-15
			437	XM_386367.1	Gibberella zeae PH-1
	MG04484.4	221				XM_362039.1	Rice blast fungus 70-15
257			BU064817.1	Gibberella zeae
		258			XM_390042.1	Gibberella zeae PH-1
260			CO134330.1	Aspergillus flavus
		261			XM_409937.1	Aspergillus nidulans FGSC A4
264			CO140779.1	Black-koji mould
		265			DR706673.1	Aspergillus flavus
266			XM_749981.1	Aspergillus fumigatus
		269			CD488085.1	Ustilago maydis
273			CN241271.1	Paracoccidioides brasiliensis
		284			DT932969.1	Leptosphaeria maculans
288			AL113061.1	Fu Shi grape spore cup fungi
		291			AJ638244.1	Standing grain green-ball chamber bacterium
305			BQ110718.1	Garden Dahlia wheel branch spore
		317			CF845455.1	Soybean phytophthora
326			CV949997.1	Phytophthora infestans
		333			XM_718100.1	Candida albicans
337			DN478895.1	Wild cabbage blackspot chain lattice spore
		341			XM_570672.1	The Cryptococcus neoformans mutation

	342	DR438661.1	Phytophthora parasitica
				358	BQ283973.1	Standing grain powdery mildew (Blumeria graminis f.sp.)
	MG05169.4	240	XM_449481.1				Candida glabrata
297				XM_716256.1	Candida albicans
		308	XM_398486.1			Ustilago maydis
331				CN239627.1	Paracoccidioides brasiliensis
		349	XM_570638.1			The Cryptococcus neoformans mutation
368				AL116927.1	Fu Shi grape spore cup fungi
		438	XM_359608.1			Rice blast fungus 70-15
439				XM_391770.1	Gibberella zeae PH-1
		440	XM_405433.1			Aspergillus nidulans FGSC A4
441				XM_747243.1	Aspergillus fumigatus
		MG06292.4	226			X59269.1	The naked shell spore of structure nest bacterium
227	XM_750103.1			Aspergillus fumigatus
			282		XM_566728.1	The Cryptococcus neoformans mutation
285	XM_712829.1			Candida albicans
			286		XM_712763.1	Candida albicans
353	XM_399704.1			Ustilago maydis
			373		CO142798.1	Aspergillus flavus
399	CN240192.1			Paracoccidioides brasiliensis
			442		XM_369777.1	Rice blast fungus 70-15
443	XM_385668.1			Gibberella zeae PH-1
			MG06314.4		293	XM_385552.1	Gibberella zeae PH-1
295	XM_385552.1	Gibberella zeae PH-1
				320	XM_404544.1	Aspergillus nidulans FGSC A4
369	XM_745154.1	Aspergillus fumigatus
				383	XM_444925.1	Candida glabrata
390	CA581516.1	Paracoccidioides brasiliensis
				392	XM_397947.1	Ustilago maydis
395	XM_567461.1	The Cryptococcus neoformans mutation
				407	DR439107.1	Phytophthora parasitica
444	XM_369799.1	Rice blast fungus 70-15
				MG07031.4	211	XM_367106.1	Rice blast fungus 70-15
238	DR668205.1	Gibberella moniliformis
			242		CN811486.1	Gibberella zeae
247	DR710271.1	Black-koji mould
			248		XM_746981.1	Aspergillus fumigatus
253	AL111548.1	Fu Shi grape spore cup fungi
			259		CN248132.1	Paracoccidioides brasiliensis
270	CO151671.1	Aspergillus flavus
			298		XM_447214.1	Candida glabrata
299	XM_408594.1	Aspergillus nidulans FGSC A4
			302		XM_716840.1	Candida albicans
306	BI201970.1	Intend branch spore sickle spore
			316		XM_401794.1	Ustilago maydis
332	XM_569789.1	The Cryptococcus neoformans mutation
			350		CV222840.1	Phanerochaete chrysosporium
356	DR440327.1	Phytophthora parasitica
			359		BE775707.1	Phytophthora infestans

	360	DR440327.1	Phytophthora parasitica
				379	DN479774.1	Wild cabbage blackspot chain lattice spore
	MG07116.4	303	XM_716554.1				Candida albicans
363				CA582713.1	Paracoccidioides brasiliensis
		364	XM_397938.1			Ustilago maydis
445				XM_367191.1	Rice blast fungus 70-15
		446	XM_389963.1			Gibberella zeae PH-1
447				XM_404731.1	Aspergillus nidulans FGSC A4
		448	XM_745894.1			Aspergillus fumigatus
MG07222.4				277	XM_744655.1		Aspergillus fumigatus
	300	XM_411576.1	Aspergillus nidulans FGSC A4
				351	CN247150.1	Paracoccidioides brasiliensis
	385	XM_446786.1	Candida glabrata
				387	XM_400375.1	Ustilago maydis
	396	XM_571676.1	The Cryptococcus neoformans mutation
				397	XM_714233.1	Candida albicans
	449	XM_367297.1	Rice blast fungus 70-15
				450	XM_390075.1	Gibberella zeae PH-1
	MG07472.4	218	XM_386292.1				Gibberella zeae PH-1
279				XM_742945.1	Aspergillus fumigatus
		292	XM_412317.1			Aspergillus nidulans FGSC A4
307				AL115094.1	Fu Shi grape spore cup fungi
		314	XM_572276.1			The Cryptococcus neoformans mutation
318				DR708959.1	Black-koji mould
		322	CO143369.1			Aspergillus flavus
334				XM_400233.1	Ustilago maydis
		347	CV957053.1			Phytophthora infestans
380				CA581803.1	Paracoccidioides brasiliensis
		388	XM_714102.1			Candida albicans
451				XM_367561.1	Rice blast fungus 70-15
		MG08863.4	281			XM_385749.1	Gibberella zeae PH-1
311	XM_750141.1			Aspergillus fumigatus
			339		XM_410205.1	Aspergillus nidulans FGSC A4
362	AL111561.1			Fu Shi grape spore cup fungi
			375		XM_569100.1	The Cryptococcus neoformans mutation
400	BQ499912.1			Paracoccidioides brasiliensis
			401		XM_448856.1	Candida glabrata
452	XM_364018.1			Rice blast fungus 70-15
			MG08911.4		213	XM_748293.1	Aspergillus fumigatus
313	BI191623.1	Intend branch spore sickle spore
				348	CO143726.1	Aspergillus flavus
382	DR706257.1	Black-koji mould
				398	XM_447745.1	Candida glabrata
453	XM_364066.1	Rice blast fungus 70-15
				454	XM_385694.1	Gibberella zeae PH-1
MG10192.4	228	XM_403070.1					Ustilago maydis
			232	XM_706539.1	Candida albicans
	235	XM_706539.1				Candida albicans
			246	DR701435.1	Black-koji mould

	262	BQ110649.1	Garden Dahlia wheel branch spore
				274	AL114181.1	Fu Shi grape spore cup fungi
	309	BI201406.1	Intend branch spore sickle spore
				310	XM_570890.1	The Cryptococcus neoformans mutation
	315	BW644576.1	Point sickle spore (Fusarium oxysporum f.sp.)
				323	DR045681.1	Phaeosphaeria nodorum
	327	BQ622080.1	Conidiobolus coronatus
				340	BE187972.1	Yellow branch spore
	344	CN242658.1	Paracoccidioides brasiliensis
				365	AJ637851.1	Standing grain green-ball chamber bacterium
	371	BE776281.1	Phytophthora infestans
				455	XM_365972.1	Rice blast fungus 70-15
	456	XM_387413.1	Gibberella zeae PH-1
				457	XM_406880.1	Aspergillus nidulans FGSC A4
	458	XM_745176.1	Aspergillus fumigatus

Table 3

Target gene ID	PCR	Auele Specific Primer FW	Auele Specific Primer RV	SEQ ID NO cDNA clone
					MG00884.4	A	(SEQ ID NO：61) atgaaggacgagacatcacg	(SEQ ID NO：62) ttacatgccctcgaattctcc	(SEQ ID NO：3)MG00884.4TA/02A#01
MG04484.4	B	(SEQ ID NO：63) atgggtatcgatcttaagaagcacc	(SEQ ID NO：64) ttagaccttgaaaccacgcg	(SEQ ID NO：186)MG04484.4TA/04A#07
					MG07472.4	B	(SEQ ID NO：65) atgcactcgccgccgcggaagttg	(SEQ ID NO：66) tcaatcgtcgtcctccaccc	(SEQ ID NO：11)MG07472.4TA/07A#01
MG06292.4	C	(SEQ ID NO：67) atggcacctggtagcagtg	(SEQ ID NO：68) tcagccatcaccgttgggtag	(SEQ ID NO：17)MG06292.4TA/08A#03
					MG03668.4	D	(SEQ ID NO：69) atggcgacgtcagacatc	(SEQ ID NO：70) tcaagtctgctgcatgac	(SEQ ID NO:13) MG03668.4TA/09A#01 and MG03668.4TA/09B#05
D	(SEQ ID NO：71) atggcgacgtcagacatc	(SEQ ID NO：72) tcaagtctgctgcatgac
			MG05169.4	E		(SEQ ID NO：73) taaaatggccaccgatgcccaagggcct	(SEQ ID NO：74) ccctcagaattttgacaggaccgcc	(SEQ ID NO:29) MG05169.4TA/11A#12 and MG05169.4TA/11B#15
E	(SEQ ID NO：75) taaaatggccaccgatgcccaagggcct	(SEQ ID NO：76) ccctcagaattttgacaggaccgcc
				MG03872.4	F	(SEQ ID NO：77) atgggtaaacgaaagagcgaacag	(SEQ ID NO：78) tcactcatctgcctcctgcttttttc		(SEQ ID NO：33)MG03872.4TA/30B#01

Table 4

Target gene ID	Exon	PCR	Auele Specific Primer FW	Auele Specific Primer RV	SEQ ID NO of cDNA clone
						MG00170.4	1	A	(SEQ ID NO：79)1 FP： atggacaccctggtagcccgctac	(SEQ ID NO：80)1 Ex1 RP： atgtgctactggcggaacc	(SEQ ID NO：184)MGD0170.4TA/01A#04
2	A	(SEQ ID NO：81)Ex2 FP： ccctcatcttggctccgca	(SEQ ID NO：82)1RP： ctagtccagcgcactcgtaac
				MG07031.4	1		Synthetic			(SEQ ID NO:5) MG07031.4TA/03A#05 and MG07031.4TA/03B#02
2	Synthetic

	3	B	(SEQ ID NO：83)3 Ex3 FP： gttcttttggggatcacgggcaa	(SEQ ID NO：84)3 Ex3 RP： tggtgtctccaggctcgc
						4	B	(SEQ ID NO：85)3 Ex4 FP： tccagtttgcggagtaca	(SEQ ID NO：86)3 Ex4 RP： tgagcatatccatgcgca
	5	B	(SEQ ID NO：87)3 Ex5 FP： gtactactgcctctccat	(SEQ ID NO：88)3 Ex5 RP： tcaagcactcttgacaatcttg
						MG02946.4	1	C	(SEQ ID NO：89)6 FP： tttatggcgaccaacggcgat	(SEQ ID NO：90)6 Ex1 RP： tcctcatgtgctcctcccactc	(SEQ ID NO：188)MG02946.4TA/06A#1
2	D	(SEQ ID NO：91)6 Ex2 FP： tcgaactgctggatccca	(SEQ ID NO：92)6 RP： ccgttattcctttttgtctcc
				MG10192.4	2		E	(SEQ ID NO：93)10 FP： atgcctcctccaccgcatc	(SEQ ID NO：94)10 Ex2 RP：ctctatcgtgccgacatttgtg	(SEQ ID NO:15) MG10192.4TA/10A-exon 2 #H1 and MG10192.4TA/10B-exon 3 #09
3	F	(SEQ ID NO：95)10 Ex3 FP： cttgttttgaaaaaattcatcgagctcgccgc	(SEQ ID NO：96)10 RP： ctagttcccggcattctcacg

Table 5

Target gene (ID)	Target fragment	Growth in vitro suppresses
			MG00170.4 (Nucleotide 11-306)	(SEQ ID NO：97) tggtagcccgctacagccgcccggcttaccagcagaacgagacattcacagaagatgatcagcaagacctttgcgattccgtcccaagtctttcactcaagttt gcggttccgccagtagcacatccctcatcttggctccgcacagcaacggacgaccacgcaaacccaaactgccccatcaagatcgcacacggaacgacg acgctcgctttcaggtttcagggaggcatcatcgttgcgaccgactctcgtgccaccgccggcaactggattgcttcgcagacggtcaagaag(296bp)	*
MG00170.4 (Nucleotide 1-835)	(SEQ ID NO：98) atggacaccctggtagcccgctacagccgcccggcttaccagcagaacgagacattcacagaagatgatcagcaagacctttgcgattccgtcccaagtctt tcactcaagtttgcggttccgccagtagcacatccctcatcttggctccgcacagcaacggacgaccacgcaaacccaaactgccccatcaagatcgcaca cggaacgacgacgctcgctttcaggtttcagggaggcatcatcgttgcgaccgactctcgtgccaccgccggcaactggattgcttcgcagacggtcaagaa ggtcatcgagatcaactcttgcctgctcggcaccatggccggcggtgccgcagactgccagtactggctcgcctggctgggcatgcagtgccgtctgcacga gctccgccacaagcgccgcatctcggtcgccgccgcctccaagattctcgccaacctcgtctaccagtacaagggcatgggcctcagcatgggtaccatgtg cgccggcgtcaccaaggaggagggtcccgccctgtactacatcgacagcgacggcaccaggcttgccggcaatctgttctgtgtgggatccggtcagacctt tgcctatggtgtgctggatgccgagtacaagtacgacctgtcggatgaggatgcgctcgagctcggccgcaggagcattctcgccgccacccacagggatg cctactccggtggttttatcaacttgtaccacgtcaaggaggacggttgggtcaagcacggattcaacgacacgaaccctatcttctggaagactaagctggag aagggcgagtttaccaacg(835bp)	*

MG00884.4 (Nucleotide 852-1139)	(SEQ ID NO：99) cgacctgctcgtctcacatacacagttccccgagaatgctgcaaacaaccaagttgcgcggtatctctactaccttggtcggattcgcgccatacagctgcgct acacggaggcacacgagcacctgacggctgcgacgcggaaggcaccgagctctaactgcgcggttggtttctcgcagacggccaccaagcttttgctggtg gttgagcttcttatgggcgatattcctgatcgcgcgaccttccgccaaccgaccctcgaggaggcactgcacccttacttcct(288bp)	*
			MG00884.4 (Nucleotide 845-1044)	(SEQ ID NO：100) accaggccgacctgctcgtctcacatacacagttccccgagaatgctgcaaacaaccaagttgcgcggtatctctactaccttggtcggattcgcgccataca gctgcgctacacggaggcacacgagcacctgacggctgcgacgcggaaggcaccgagctctaactgcgcggttggtttctcgcagacggccaccaag (200bp)	33% (seeing Fig. 1 b)
MG07031.4 (Nucleotide 261-543)	(SEQ ID NO：101) ggagtacatcatggcaaacgcgaagctctattcgatgcgtaacgagaccgatctttcaccgtccgcattggcgcattacgtccgtggcgagcttgcctcgagct tgaggtcgcggaagccatacacagtcaacctcctcttgggcggtgtcgaccccatcacacacaagccgagcctctactggctggactaccttgcggctctggc gccggtcccatatgctgcgcatggatatgctcagtactactgcctctccatcctcgacaaacatcaccatcccgat(283bp)	*
			MG07031.4 (Nucleotide 251-500)	(SEQ ID NO：102) tccagtttgcggagtacatcatggcaaacgcgaagctctattcgatgcgtaacgagaccgatctttcaccgtccgcattggcgcattacgtccgtggcgagcttg cctcgagcttgaggtcgcggaagccatacacagtcaacctcctcttgggcggtgtcgaccccatcacacacaagccgagcctctactggctggactaccttgc ggctctggcgccggtcccatatgctgcgcatggatatgctca(250bp)	43% (seeing Fig. 1 c)
MG04484.4 (Nucleotide 7-276)	(SEQ ID NO：103) atcgatcttaagaagcaccacgtgcgcagcacgcaccgcaaggcccccaagagcgacaatgtctacctcaagctcttggtgaagctctaccgcttcctggcc cgcaggaccgactccagcttcaacaaggttgtccttcgccgcctctttatgtcgcgcaccaaccgccctcccgtctccctgtcgcgcatcgcaggcaacctgaa gaacggcaacgagaagaagaccgttgtcgttgtcggcaccgtcaccgacgacaaccgcctggtt(270bp)	*
			MG04484.4 (Nucleotide 1-555)	(SEQ ID NO：104) atgggtatcgatcttaagaagcaccacgtgcgcagcacgcaccgcaaggcccccaagagcgacaatgtctacctcaagctcttggtgaagctctaccgcttc ctggcccgcaggaccgactccagcttcaacaaggttgtccttcgccgcctctttatgtcgcgcaccaaccgccctcccgtctccctgtcgcgcatcgcaggcaa cctgaagaacggcaacgagaagaagaccgttgtcgttgtcggcaccgtcaccgacgacaaccgcctggttgagtgccccaaggcccaggtcgctgccctt cgcttcaccgccaccgcccgtgcccgcatcgttgccgccggcggccaggccatcacccttgaccagctggctcttgagaagcccactggtgccaacaccctc ctcctccccaagaacgcccgtgaggctgtcaagcacttcggcttcggtccccacaagcacaagaagccttacgtccagtccaagggccgcaagttcgagaa ggcccgtggtcgcagacgatcgcgtggtttcaaggtctaa(555bp)	*
MG04484.4 (Nucleotide 211-409)	(SEQ ID NO：105) aagaacggcaacgagaagaagaccgttgtcgttgtcggcaccgtcaccgacgacaaccgcctggttgagtgccccaaggcccaggtcgctgcccttcgctt caccgccaccgcccgtgcccgcatcgttgccgccggcggccaggccatcacccttgaccagctggctcttgagaagcccactggtgccaacaccctcc (199bp)	30% (seeing Fig. 1 d)
			MG02946.4 (Nucleotide 58-333)	(SEQ ID NO：106) actgcggccgcggcacagctcgacggcctcaggcctcccgagggcgtcatcattccgccacccggtgagatccgcgaggtgattgaaaagacggccggg tacgtggcgcgtggtggactgggcatcgagcagcgcctacgcgagaaccacagcgggaacccaaagttcagcttcgtcacgagccagagcgacgcgta caatccgtactacgagtggcgcaaggccgagtacaaggccgggcgtggcacggcgctggcggctggtcgggcggatgcg(276bp)	*

MG07472.4 (Nucleotide 603-886)	(SEQ ID NO：107) ccaatcatctggtttcgagggcggcatcaacgaggacaacccttatgacaagccgctttttgctgttcaggacgccatcagtagtatctaccggccccgtgcga acaacgatgatgaggacgaggcggctggcgatgcggagatggccaagatacaaaaggcgagcagatatggtgaggtgctaggccgaggtacattctcc ggtgccggggatgtggaagcccgagaaggtccagtgcagttcgaaaaggatgctgcaggcgcggaccctttcaatgtggaca(284bp)	*
			MG06292.4 (Nucleotide 1684-1978)	(SEQ ID NO：108) tatgctgaagcagaggcctccttcaagcgcttaagaacattggcaccgaatcggctggaggacatggaggtctattccacagtactttggcacctgaagaag gaaacagaggcatcgtttctggcacacgagcttgttgacatagcgtggcattcaccgcacgcctggtgcgccttgggcaatgcatggtcgctggcatcagatc gcgaacaagcgctgcggtgcttcaagcgtgctacgcagctcgacgcaaaatttgcatatgcctacacacttcaagggcatgagcactttg(295bp)	*
MG03668.4 (Nucleotide 1325-1614)	(SEQ ID NO：109) cctgctccgaggctattgaccccagccagcacctcgagcctctgatccgctcgttgtttgctggcctgctaaacactcccaagatggccgcctcgtgctgttggg ctctgatgaacctggcggaacgttttgccggtgagcccggagcgccccagaatgccatcactgcctacttcaacgacagtgtgcgcagcctcctggacgtca cccgccaagaacgattgcgactcggctgtcaggacggccgcgtatgaggttctcaacgctttcatcgtcaatgcggcaaacgatt(290bp)	*
			MG10192.4 (Nucleotide 2825-3103)	(SEQ ID NO：110) gtgacagggtcccatcttcaaacgtcggagccgctccctcggagcgttctgagcgtccctcggagcgccctgctcctgcccgaactggtacccccatggagc gcactgattcaaacgatcgtgctggtggtccgccccggcttaaccttgctcgtgctgacggtgccaagcccagctggcgtgaacgtgagcaggccaaggcta gcggtggccctgagcgggatcttcctcccagcagggcggcttctggaaggggcccaccgatgcacaggaccgatt(279bp)	*
MG00604.4 (Nucleotide 160-759)	(SEQ ID NO：111) gcctccggcaacaagcatgttccccgtgctgtcctcgtcgatctcgagcccggcaccatggacgccgtccgtgccggtccttttggccagctcttccgccccga caacttcgtcttcggtcagtctggtgctggaaacaactgggccaagggtcactacactgagggtgccgagcttgtcgaccaggtccttgacgtcgtccgtcgtg aggctgagggctgtgactgcctccagggtttccagatcacccactccctgggtggtggtaccggtgccggtatgggtactctgctgatctccaagatccgcgag gagttccccgaccgtatgatggccaccttctcggtcgttccctcgcccaaggtttccgacaccgtcgttgagccctacaacgctaccctctcggtccaccagctg gtcgagaactctgacgagaccttctgcattgacaacgaggctctgtacgacatctgcatgcgcaccctgaagctgtcgaacccctcatacggtgacctgaact acctggtttcggccgtcatgtctggcgtcaccacctgcttgcgtttccccggccagctcaactctgatctccgcaagctt(600bp)	*
			MG00604.4 (Nucleotide 361-950)	(SEQ ID NO：112) cgtcgtgaggctgagggctgtgactgcctccagggtttccagatcacccactccctgggtggtggtaccggtgccggtatgggtactctgctgatctccaagatc cgcgaggagttccccgaccgtatgatggccaccttctcggtcgttccctcgcccaaggtttccgacaccgtcgttgagccctacaacgctaccctctcggtccac cagctggtcgagaactctgacgagaccttctgcattgacaacgaggctctgtacgacatctgcatgcgcaccctgaagctgtcgaacccctcatacggtgacc tgaactacctggtttcggccgtcatgtctggcgtcaccacctgcttgcgtttccccggccagctcaactctgatctccgcaagcttgccgtcaacatggttcccttcc ctcgtctgcacttcttcatggttggcttcgctcctttgaccagccgtggtgcccactctttccgcgctgtcaccgttcccgagttgacccagcagatgttcgacccca agaacatgatggctgcttctgacttcaggaatggtcgttacctgacctgctctgccatctt(590bp)	*
MG00604.4 (Nucleotide 952-1344)	(SEQ ID NO：113) cgtggaaaggtttccatgaaggaggtcgaggaccagatgcgcaacgtccagaacaagaactcgtcgtacttcgtcgagtggatccccaacaacatccaga ccgctctctgctctatcccgccccgcggcctcaagatgtcgtcgactttcatcggaaactcgaccgccatccaggagctgttcaagcgtgtcggtgagcagttca ctgccatgttcaggcgcaaggctttcttgcattggtacactggtgagggtatggacgagatggagttcactgaggccgagtccaacatgaacgatcttgtttccg agtaccagcagtaccaggatgctggtgttgacgaggaggaagaggagtacgaggaggaggcccctcttgagggcgaggagtag(393bp)	*

MG00604.4 (Nucleotide 160-359)	(SEQ ID NO：114) gcctccggcaacaagcatgttccccgtgctgtcctcgtcgatctcgagcccggcaccatggacgccgtccgtgccggtccttttggccagctcttccgccccga caacttcgtcttcggtcagtctggtgctggaaacaactgggccaagggtcactacactgagggtgccgagcttgtcgaccaggtccttgacgtcgt(200bp)	10-50％
			MG00604.4 (Nucleotide 760-950)	(SEQ ID NO：115) gccgtcaacatggttcccttccctcgtctgcacttcttcatggttggcttcgctcctttgaccagccgtggtgcccactctttccgcgctgtcaccgttcccgagttgac ccagcagatgttcgaccccaagaacatgatggctgcttctgacttcaggaatggtcgttacctgacctgctctgccatctt(191bp)	20-50％
MG00604.4 (Nucleotide 1151-1344)	(SEQ ID NO：116) agttcactgccatgttcaggcgcaaggctttcttgcattggtacactggtgagggtatggacgagatggagttcactgaggccgagtccaacatgaacgatcttg tttccgagtaccagcagtaccaggatgctggtgttgacgaggaggaagaggagtacgaggaggaggcccctcttgagggcgaggagtag(194bp)	50-90% sees Fig. 1 a
			MG00604.4 (3X Nucleotide 1151-1344)	(SEQ ID NO：117) agttcactgccatgttcaggcgcaaggctttcttgcattggtacactggtgagggtatggacgagatggagttcactgaggccgagtccaacatgaacgatcttg tttccgagtaccagcagtaccaggatgctggtgttgacgaggaggaagaggagtacgaggaggaggcccctcttgagggcgaggagtagagttcactgcc atgttcaggcgcaaggctttcttgcattggtacactggtgagggtatggacgagatggagttcactgaggccgagtccaacatgaacgatcttgtttccgagtac cagcagtaccaggatgctggtgttgacgaggaggaagaggagtacgaggag9aggcccctcttgagggcgaggagtagagttcactgccatgttcaggc gcaaggctttcttgcattggtacactggtgagggtatggacgagatggagttcactgaggccgagtccaacatgaacgatcttgtttccgagtaccagcagtac caggatgctggtgttgacgaggaggaagaggagtacgaggaggaggcccctcttgagggcgaggagtag	*
MG05169.4 (Nucleotide 31-330)	(SEQ ID NO：118) ctcgggaacaaggcagccgcctcagatggcatgccaaatatggtgatgccatatgaacctcaggaccccgctgtcatcgcagagatggtccgcgtgctcga tgagcatacaaagggtggcgccaagggtcgtttccgtatcaagaagaccaagtttgccgttactggttctccttccaaggtcactgtagattcctggaagctaca ggactgggactacaagaaacctgggctgcctacatatgcccgtggcctcttcaccacgcgcctcccaaataatgtaccagaaattgccgtcaga(300bp)	*
			MG04056.4 (Nucleotide 2377-2676)	(SEQ ID NO：119) gcaggtgcatcgacggcgcgcgaatcccaggacgttgatcctctaattcacaacccgttcctgaagccgcccgacaaggcagcagtagaaagtaccctggt tgcgttcgagcccgcgactttcaaattgactttacaaaatccgtatgaaatcgaggttgaaattgaaagcgtgcggttagacacggaaggggtcgagtttgaat cctctgtcgacagcgccctcattggcccatatcgcacgcagatacttcgtctagtcggcataccgaaggctgcggggtctctaaagattaccggc(300bp)	*
MG08911.4 (Nucleotide 2448-2747)	(SEQ ID NO：120) ggtgatggaatggatgttcaacggccaggttgtagatgccatatttgaaacgatacaccaacagtccagcgatatcggaagcgtttcgcccgattcgcccttga tattgggcattatacgagctgttgaggtggtgtcgaaagttttggagcttcaggacacatatgttgatcttgtgcggcaagtcataaaacagcacacaggacaac gccatagacatgtgcctcacgcttcctacgcctccttcgaagaagggtttgctcatcacctcgaggtcgtcgcagatctcggccgatattg(300bp)	*
			MG06314.4 (Nucleotide 551-850)	(SEQ ID NO：121) ggcggcacgcgctcgaggcaaaacgtatccaggcctcactcaccaagggcgacacacagctttggcttggtactgcagacttggctttgcacatggtagatg gcgcctatcaggattcacccgaggaaatcgacaagactttgaaagtagctatggactgctacaggagcgtattacagatcgacaagacaaaccccgttgctc gcctgggcaaagccgacatcctcgcggacttgggacagtccagcaaggccgttgctgcttatctcgactacctcaaacagaaaccttataatcttc(300 bp)	*

MG08863.4 (Nucleotide 1419-1718)	(SEQ ID NO：122) cgtggccgacgctgaatttgaaggggagatgagtgtcaaccccacagacttggttacacggccgggtgaaacgcgaacattcaagcccatcatgctggcgc atgggagtcaagcaggtacacgtgatgccttcctcaggttggtttccggtattggctcgccatctcagcaggcagatctggctaccgacatgatcagtctggcaa gggatttgtcaggaatagatcaggtcacagccttatggctagcgtttgagttgctgaaagtagcactagccaagaacgagcaggacgagcagtt(300bp)	*
			MG07222.4 (Nucleotide 161-460)	(SEQ ID NO：123) ccgctcacacactgcggcccttcaacccgcaaaagcccgccggagcagccatcctcccgatcctcttaccgccagcaacactccgcccattggccttccgg acctttaccaaaaaacacagcctgaccctcacatcgtctgctctacaagagctggcgtcctttgtcggccgacactgcggctcaggatggcgtgaggaaggg ctcgccgagcgtgtgctagaggaggtggcaaaatcgtggaaggctggcaatggcggggtgatagtcgatggggccagccccatcctgaaggacatcc (300bp)	*
MG01760.4 (Nucleotide 1241-1540)	(SEQ ID NO：124) gtggcggtggcgatgccttaccaagcctggcctcctttgacgttggagcgaatggccaggagatctttgcggattactgcagtgatactatcgacattcttttgctct cgctggatggcaaagcacggatgatgaatggaaagaagccggttgtcggtgtcttcatagcaaacagtattgccatcaccgagcggtcgattcgcgagtctg atctggcgcctttgatggagacacgcctgggaatattggaaacataccgaaagaaggcgaagctttattacacagagccttgcaaggatg(300bp)	*
			MG07116.4 (Nucleotide 959-1258)	(SEQ ID NO：125) tgatgttgtggaggaagagcaaaagcgatgccgcgagtgacgcgcgcaagcagctgcaaatgtccaaatccgtaccgcccagcggtcctctcctgaggag aaacacagagccactgcagttgatgtcaggccctaatcctcaagaccagaacggttccatatcgccatcgcctctcgcagcgaagcaggagtatgttgtgca aacagaaagacggtcaagcgtctccacctatctgctatgccgtgtcctcctcgaggtcatcagccaaagcagtttgagtctgatcactcctgaaatgg(300 bp)	*
MG03872.4 (Nucleotide 1125-1424)	(SEQ ID NO：126) gagcgaagagacctcggaagcggcaaggtttgtcagtggcacgatctacgactcgataggatcacgcctgctcgaaacactcgtaagccattgcccaggc aagatattcaaaggcctgtggagtcacatcattgggcctcggtttgagacactcctccgtaacgagacggcaacatatgtagcaatgaaggcattggtcaggc tgagtcgagaggacttggcggacgccgtgcttaagacgataccgaaagtggaaatgctagtgtcgaaaggccgcttcaatatcttgacgctgctgtt(300 bp)	*
			MG04185.4 (Nucleotide 2637-2g36)	(SEQ ID NO：127) actggtgactataggtgacccttcggaaatggtcgtagttctgacacaggccctgcagggtgtcgagatggacgcaaggggttgtcggtcgctcaacatgggc ttgcacgttctgagggaagttgtcgatgcgcgtccggattttcatccgaccgaatctgaacttgccgccctgagcgggttg9ccgcgcgctgccttgaaagccat gagtctggcgtgcggatggatgcagtccaattgtgtgttgccctgcatgcccgtgtgggcgatacacgcttctgggacaacatcaagggtgt(300bp)	*
Seastar#2 AFP (Nucleotide 162-453)	(SEQ ID NO：128) cgatatcttgtcaaacgcgttcgcgtacggaaacagagcattgaccaaatacccagacgatatagcagactatttcaagcagtcgtttcccgagggatattcct gggaaagaagcatgacttttgaagacaaaggcattgtcaccgtggaaactgacgtcagcatggaagacgactgctttatctataaaattcgttttgatggggtg aactttcctcccaatggtccagtaatgcagaagaaaactttgaggtgggaaccatccactgagattatgtacgttcgtgatggc(292bp)	*
			Seastar#2 AFP (Nucleotide 161-360)	(SEQ ID NO：129) acgatatcttgtcaaacgcgttcgcgtacggaaacagagcattgaccaaatacccagacgatatagcagactatttcaagcagtcgtttcccgagggatattcc tgggaaagaagcatgacttttgaagacaaaggcattgtcaccgtggaaactgacgtcagcatggaagacgactgctttatctataaaattcgtttt(200bp)	As 100%, negative contrast

GST (Nucleotide 1-200)	(SEQ ID NO：130) atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcga tgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacaca(200bp)	As 100%, negative contrast
			GFP	(SEQ ID NO：131) ttatggtgttcaatgcttttcaagatacccagatcatatgaaacggcatgacttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaa agatgacgggaactacaagacacgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcaggtgctgaagtcaagtttgaaggtgataccctt gttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattg(279bp)	As 100%, negative contrast

Table 6

Target gene rice blast fungus identifier	The primer name	Primer sequence	Justice (S)/antisense (AS) is arranged	Restriction enzyme
					MG00170.4	oBG109	(SEQ ID NO：132)GAATTCATGGACACCCTGGTAGCC	S	EcoRI
oBG110	(SEQ ID NO：133)CTGCAGCGTTGGTAAACTCGCCCT	S	PstI
				oBG107		(SEQ ID NO：134)CCCGGGATGGACACCCTGGTAGCC	AS	XmaI
oBG108	(SEQ ID NO：135)AGATCTCGTTGGTAAACTCGCCCT	AS	BglII
				MG00884.4		oBG33	(SEQ ID NO：136)GAATTCCGACCTGCTCGTCTCACATA	S	EcoRI
oBG34	(SEQ ID NO：137)CTGCAGAGGAAGTAAGGGTGCAGTGC	S	PstI
					oBG35	(SEQ ID NO：138)CCCGGGCGACCTGCTCGTCTCACATA	AS	XmaI
oBG36	(SEQ ID NO：139)CCATGGAGGAAGTAAGGGTGCAGTGC	AS	NcoI
					MG07031.4	oBG37	(SEQ ID NO：140)GAATTCGGAGTACATCATGGCAAACG	S	EcoRI
oBG38	(SEQ ID NO：141)CTGCAGATCGGGATGGTGATGTTTG	S	PstI
				oBG39		(SEQ ID NO：142)CCCGGGGGAGTACATCATGGCAAACG	AS	XmaI
oBG40	(SEQ ID NO：143)CCATGGATCGGGATGGTGATGTTTG	AS	NcoI
				MG04484.4		oBG087	(SEQ ID NO：144)GAATTCATGGGTATCGATCTTAAGAAGCACCACG	S	EcoRI
oBG088	(SEQ ID NO：145)CTGCAGTTAGACCTTGAAACCACGCGATCGTCT	S	PstI
					oBG089	(SEQ ID NO：146)CCCGGGATGGGTATCGATCTTAAGAAGCACCACG	AS	XmaI
oBG090	(SEQ ID NO：147)CCATGGTTAGACCTTGAAACCACGCGATCGTCT	AS	NcoI
					MG02946.4	oBG49	(SEQ ID NO：148)GAATTCACTGCGGCCGCGGCACAG	S	EcoRI
oBG50	(SEQ ID NO：149)CTGCAGCGCATCCGCCCGACCAGC	S	PstI
				oBG51		(SEQ ID NO：150)CCCGGGACTGCGGCCGCGGCACAG	AS	XmaI
oBG52	(SEQ ID NO：151)CCATGGCGCATCCGCCCGACCAGC	AS	NcoI

MG07472.4	oBG53	(SEQ ID NO：152)GAATTCCCAATCATCTGGTTTCGAGG	S	EcoRI
					oBG54	(SEQ ID NO：153)ACGCGTTGTCCACATTGAAAGGGTCC	S	MluI
	oBG55	(SEQ ID NO：154)CCCGGGCCAATCATCTGGTTTCGAGG	AS	XmaI
					oBG56	(SEQ ID NO：155)CCATGGTGTCCACATTGAAAGGGTCC	AS	NcoI
	MG06292.4	oBG57	(SEQ ID NO：156)GAATTCTATGCTGAAGCAGAGGCCTC	S					EcoRI
oBG58					(SEQ ID NO：157)CTGCAGCAAAGTGCTCATGCCCTTG	S	PstI
		oBG59	(SEQ ID NO：158)CCCGGGTATGCTGAAGCAGAGGCCTC	AS				XmaI
oBG60					(SEQ ID NO：159)CCATGGCAAAGTGCTCATGCCCTTG	AS	NcoI
		MG03668.4	oBG61	(SEQ ID NO：160)GAATTCCCTGCTCCGAGGCTATTG				S	EcoRI
oBG62	(SEQ ID NO：161)CTGCAGATCGTTTGCCGCATTGAC				S	PstI
			oBG63	(SEQ ID NO：162)CCCGGGCCTGCTCCGAGGCTATTG			AS	XmaI
oBG64	(SEQ ID NO：163)CCATGGATCGTTTGCCGCATTGAC				AS	NcoI
			MG10192.4	oBG65			(SEQ ID NO：164)GAATTCGTGACAGGGTCCCATCTTCA	S	EcoRI
oBG66	(SEQ ID NO：165)CTGCAGAATCGGTCCTGTGCATCG	S			PstI
				oBG67		(SEQ ID NO：166)CCCGGGGTGACAGGGTCCCATCTTCA	AS	XmaI
oBG68	(SEQ ID NO：167)AGATCTAATCGGTCCTGTGCATCG	AS			BglII
				MG00604.4 (11.1)		oBG093	(SEQ ID NO：168)GAATTCGCCTCCGGCAACAAGCATGTTC	S	EcoRI
oBG094	(SEQ ID NO：169)CTGCAGAAGCTTGCGGAGATCAGAGTTGAGCTGG	S	PstI
					oBG091	(SEQ ID NO：170)CCCGGGGCCTCCGGCAACAAGCATGTTC	AS	XmaI
oBG092	(SEQ ID NO：171)AGATCTAAGCTTGCGGAGATCAGAGTTGAGCTGG	AS	BglII
					MG00604.4 (11.2)	oBG097	(SEQ ID NO：172)GAATTCCCGTCGTGAGGCTGAGGGCTGTG	S	EcoRI
oBG098	(SEQ ID NO：173)CTGCAGAAGATGGCAGAGCAGGTCAGGTAACGA	S	PstI
				oBG095		(SEQ ID NO：174)CCCGGGCCGTCGTGAGGCTGAGGGCTGTG	AS	XmaI
oBG096	(SEQ ID NO：175)CCATGGAAGATGGCAGAGCAGGTCAGGTAACGA	AS	NcoI
				MG00604.4 (11.3)		oBG101	(SEQ ID NO：176)GAATTCCCGTGGAAAGGTTTCCATGAAGGAGG	S	EcoRI
oBG102	(SEQ ID NO：177)CTGCAGCTACTCCTCGCCCTCAAGAGGG	S	PstI
					oBG099	(SEQ ID NO：178)CCCGGGCCGTGGAAAGGTTTCCATGAAGGAGG	AS	XmaI
oBG100	(SEQ ID NO：179)CCATGGCTACTCCTCGCCCTCAAGAGGG	AS	NcoI
					MG00604.4 (11.4)	oBG105	(SEQ ID NO：180)GAATTCAGTTCACTGCCATGTTCAG	S	EcoRI
oBG106	(SEQ ID NO：181)CTGCAGCTACTCCTCGCCCTCAAG	S	PstI
				oBG103		(SEQ ID NO：182)CCCGGGAGTTCACTGCCATGTTCAG	AS	XmaI
oBG104	(SEQ ID NO：183)CCATGGCTACTCCTCGCCCTCAAG	AS	NcoI

Claims (76)

1. being used to control cell or organism goes up fungal growth or is used to prevent be subject to the cell of fungi infestation or the method for the fungal infection of organism, comprise the fungal cell is contacted with double-stranded RNA from the fungal cell is outside, wherein double-stranded RNA comprises the annealed complementary strand, a chain wherein have with the nucleotide sequence of fungi target gene to small part complementary nucleotide sequence, double-stranded RNA is ingested and enters the fungal cell and control growing or prevention are infected thus whereby.

2. negative method of regulating expression of target gene in the fungi, comprise the fungal cell is contacted with the outside of double-stranded RNA the fungal cell, wherein double-stranded RNA comprises the annealed complementary strand, a chain wherein have with the nucleotide sequence for the treatment of the negative fungi target gene of regulating to small part complementary nucleotide sequence, double-stranded RNA is ingested and enters fungal cell and the negative thus fungi target gene expression of regulating whereby.

3. according to the method for claim 1 or 2, wherein said fungal cell contacts with the composition that comprises described double-stranded RNA.

4. according to the method for claim 1 or 2, wherein said fungal cell contacts with the double-stranded RNA of being expressed by protokaryon (such as bacterial cell) or eucaryon (such as yeast cell) host cell or host organisms.

5. according to the process of claim 1 wherein by by described fungal infection or be subject to the described cell or the described double-stranded RNA of organism expressing of described fungal infection.

6. according to the method for claim 5, wherein said cell is that vegetable cell or wherein said organism are plants.

7. according to each described method of claim 1-6, wherein express described double-stranded RNA from recombinant precursor, this construct comprises at least a adjusting sequence that is connected to described nucleotide sequence of handling, described nucleotide sequence and the described nucleotide sequence for the treatment of the negative described fungi target gene of regulating to the small part complementation.

8. according to each described method of claim 1-7, wherein said fungi target gene comprises with the sequence that is selected from the following sequence set and has at least 75% conforming sequence: SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15,48 and 110, or its complementary sequence, perhaps wherein said fungi target gene is to comprise SEQ ID Nos 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, the fungi of the gene of arbitrary sequence is directly to homologue in 48 and 110.

9. produce the method that plant pathogenic fungi is had the plant of resistance, may further comprise the steps:

A. with comprising the recombinant precursor transformed plant cells of at least a adjusting sequence, described adjusting sequence operably be connected to and be selected from the target fungal gene in the group of forming by following nucleotide sequence nucleotide sequence to small part complementary sequence:

(i) coding of at least 17 Nucleotide length and the nucleotide sequence of fungi target gene complementary RNA, described fungi target gene comprises with the sequence that is selected from following any sequence representative sequence set and has at least 75% conforming sequence: SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15,48 and 110, or its complementary sequence, perhaps wherein said fungi target gene is to comprise SEQ ID Nos 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, in 48 and 110 the fungi of the gene of any sequence directly to homologue and

(ii) comprise the sense strand that contains (i) described nucleotide sequence and contain the nucleotide sequence of antisense strand of the complementary sequence of (i) described nucleotide sequence, wherein can form double-stranded RNA by described nucleotide sequence coded transcript,

B. from plant transformed cell regeneration plant; With

C. allow plant transformed grow under the condition that is fit to the express recombinant construct, the conversion plant of described growth and unconverted plant compare fungi and have resistance.

10. according to each described method of claim 1-9, wherein said fungal cell is the cell that is selected from down the plant pathogenic fungi of group: little Acremonium (Acremoniellaspp.), Alternaria (Alternaria spp.) (for example wild cabbage blackspot chain lattice spore (Alternariabrassicola) or alternaria solani sorauer (Alternaria solani)), shell two born of the same parents Pseudomonas (Ascochytaspp.) (for example pea shell two born of the same parents bacterium (Ascochyta pisi)), Staphlosporonites (Botrytis spp.) (for example Botrytis cinerea (Botrytis cinerea) or Fu Shi grape spore cup fungi (Botryotiniafuckeliana)), cladosporium belongs to (Cladosporium spp.), cercospora (Cercosporaspp.) is Kikuchi tail spore (Cercospora kikuchii) or corn tail spore (Cercosporazaea-maydis) for example), cladosporium belongs to (Ciadosporium spp.) (for example yellow branch spore (Cladosporium fulvum)), Colletotrichum (Colletotrichum spp.) (for example beans thorn dish spore (Colletothchum lindemuthianum)), Curvularia (Curvulariaspp.), shell look list is every spore (Diplodia spp.) (for example corn husk look list is every spore (Diplodiamaydis)), Erysiphe (Erysiphe spp.) (wheat powdery mildew (Erysiphegraminis f.sp.graminis) for example, big wheat powdery mildew (Erysiphe graminis f.sp.hordei) or pea powdery mildew (Erysiphe pisi)), Erwinia armylovora, fusarium (Fusariumspp.) (is for example avenged rotten sickle spore (Fusarium nivale), intend branch spore sickle spore (Fusariumsporotrichioides), point sickle spore (Fusarium oxysporum), F.graminearum schw (Fusariumgraminearum), Fusarium germinearum, yellow sickle spore (Fusariumculmorum), eggplant sickle spore (Fusarium solani), beading sickle spore (Fusariummoniliforme) or pink sickle spore (Fusarium roseum)), the top softgel shell belongs to (Gaeumanomyces spp.) (for example wheat top softgel shell (Gaeumanomyces graminisf.sp.tritici)), Gibberella (Gibberella spp.) (for example Gibberella zeae (Gibberellazeae)), Helminthosporium (Helminthosporium spp.) (the leaf blight length spore (Helminthosporium turcicum) of wriggling for example, bipolaris carbonum wilson (Helminthosporiumcarbonum), long spore (Helminthosporium mavdis) or the bent long spore (Helminthosporium sigmoideum) of wriggling of fork of wriggling of Zea mays), rice ball cavity bacteria (Leptosphaeriasalvinii), shell ball spore belongs to (Macrophomina spp.) (for example Kidney bean shell ball spore (Macrophomina phaseolina)), Magnaportha spp. (for example rice blast fungus (Magnaporthe oryzae)), mycosphaerella (Mycosphaerella spp.), Nectria (Nectria spp.) (for example Nectria heamatococca), Peronospora (Peronosporaspp.) (for example northeast downy mildew (Peronospora manshurica) or tobacco downy mildew (Peronospora tabacina)), Phoma (Phoma spp.) (Li such as Chard dish stem point mould (Phoma betae)), Phakopsora (Phakopsora spp.) (for example Macroptilium layer rest fungus (Phakopsora pachyrhizi)), knurl stalk spore belongs to (Phymatotrichum spp.) (for example how main knurl stalk spore (Phymatotrichum omnivorum)), phytophthora (Phytophthoraspp.) (camphor tree epidemic disease mould (Phytophthora cinnamomi) for example, Phytophthora cactorum (Phytophthoracactorum), Kidney bean epidemic disease mould (Phytophthora phaseoli), phytophthora parasitica (Phytophthoraparasitica), oranges and tangerines brown rot epidemic disease mould (Phytophthora citrophthora), big male epidemic disease mould (Phytophthora megasperma f.sp.soiae) or phytophthora infestans (Phytophthorainfestans)), Plasmopara (Plasmopara spp.) (for example grape is given birth to single shaft mould (Plasmopara viticola)), Podosphaera (Podosphaera spp.) (for example white cross hair list softgel shell (Podosphaera leucotricha)), Puccinia (Puccinia spp.) (corn handle rest fungus (Puccinia sorghi) for example, bar shaped handle rest fungus (Pucciniastriiformis), wheat handle rest fungus (Puccinia graminis f.sp.tritici), Radix Asparagi handle rest fungus (Puccinia asparagi), Puccinia recondita (Puccinia recondita) or Semen arachidis hypogaeae handle rest fungus (Puccinia arachidis)), pythium (Pythium spp.) (for example melon and fruit corruption mould (Pythium aphanidermatum)), nuclear cavity Pseudomonas (Pyrenophora spp.) (for example couchgrass nuclear cavity bacteria (Pyrenophora tritici-repentens) or circle nuclear cavity bacteria (Pyrenophora teres)), Coniosporium (Pyricularia spp.) (for example piricularia oryzae (Pyricularia oryzae)), pythium (Pythium spp.) (for example ultimate corruption mould (Pythiumultimum)), Rhincosporium secalis, Rhizoctonia (Rhizoctonia spp.) (dry thread Pyrenomycetes (Rhizoctonia solani) for example, withered spot rhizoctonia of rice (Rhizoctonia oryzae) or cereal rhizoctonia (Rhizoctonia cerealis)), Rhizopus (Rhizopus spp.) (for example zhizopchin (Rhizopus chinensid)), sclerotium (Scerotium spp.) (for example Sclerotium rolfsii (Scerotium rolfsii)), Sclerotinia (Sclerotinia spp.) (for example sclerotinite (Sclerotinia sclerotiorum)), Septoria (Septoria spp.) (tomato septoria musiva (Septoria lycopersici) for example, soybean septoria musiva (Septoria glycines), grain husk withered septoria musiva (Septoria nodorum) or wheat septoria (Septoria tritici)), Thiclaviopsis (Thielaviopsis spp.) (for example thielaviopsis sp (Thielaviopsis basicola)), Tilletia (Tilletia spp.), Trichoderma (Trichoderma spp.) (for example viride (Trichoderma virde)), Uncinula (Uncinula spp.) (for example grape snag shell (Uncinula necator)), Ustilago maydis (Ustilago maydis) (for example Ustilago maydis (D C.) Corola.), Venturia (Venturia spp.) (for example black star bacterium (Venturia pirina) of venturia inaequalis (Venturiainaequalis) or pears) or Verticillium (Verticilliumspp.) (for example Garden Dahlia wheel branch spore (Verticillium dahliae) or black and white wheel branch spore (Verticillium albo-atrum)).

11. according to the method for claim 10, wherein said plant pathogenic fungi is selected from down in the group: Cercospora (Cercospora spp.) (for example Kikuchi tail spore (Cercospora kikuchii) or corn tail spore (Cercospora zaea-maydis)), Colletotrichum (Colletotrichumspp.) (for example beans thorn dish spore (Colletotrichum lindemuthianum)), Curvularia (Curvularia spp.), Diplodia (Diplodia spp.) (for example corn husk look list is every spore (Diplodia maydis)), fusarium (Fusarium spp.) (is for example avenged rotten sickle spore (Fusarium nivale), point sickle spore (Fusarium oxysporum), F.graminearum schw (Fusarium graminearum), Fusarium germinearum, yellow sickle spore (Fusarium culmorum), eggplant sickle spore (Fusarium solani), beading sickle spore (Fusarium moniliforme) or pink sickle spore (Fusarium roseum)), Gibberella (Gibberella spp.), Magnaportha spp. (for example rice blast fungus (Magnaportheoryzae)), mycosphaerella (Mycosphaerella spp.), Phakopsora (Phakopsora spp.) (for example Macroptilium layer rest fungus (Phakopsora pachyrhizi)), phytophthora (Phytophthoraspp.) (camphor tree epidemic disease mould (Phytophthora cinnamomi) for example, Phytophthora cactorum (Phytophthoracactorum), Kidney bean epidemic disease mould (Phytophthora phaseoli), phytophthora parasitica (Phytophthoraparasitica), oranges and tangerines brown rot epidemic disease mould (Phytophthora citrophthora), big male epidemic disease mould (Phytophthora megasperma f.sp.soiae) or phytophthora infestans (Phytophthorainfestans)), Puccinia (Puccinia spp.) (corn handle rest fungus (Pucciniasorghi) for example, bar shaped handle rest fungus (Puccinia striiformis) (yellow rust)), wheat handle rest fungus (Puccinia graminis f.sp.tritici), Radix Asparagi handle rest fungus (Puccinia asparagi), Puccinia recondita (Puccinia recondita) or Semen arachidis hypogaeae handle rest fungus (Pucciniaarachidis)), Rhizoctonia (Rhizoctonia spp.) (dry thread Pyrenomycetes (Rhizoctonia solani) for example, withered spot rhizoctonia of rice (Rhizoctonia oryzae) or cereal rhizoctonia (Rhizoctonia cerealis)), Rhizopus (Rhizopus spp.) (for example Rhizopuschinensid (zhizopchin)), Trichoderma (Trichoderma spp.) (for example viride (Trichoderma virde)) and Verticillium (Verticillium spp.) (for example Garden Dahlia wheel branch spore (Verticillium dahliae) or black and white wheel branch spore (Verticillium albo-atrum)).

12. according to each described method of claim 6-11, wherein said plant is selected from paddy rice, corn, soybean, cotton, potato, banana, tomato, wheat, Chinese sorghum, grain, beans (beans), Semen arachidis hypogaeae, Semen Brassicae campestris, Sunflower Receptacle and sugarcane; Be preferably selected from paddy rice, corn, soybean, cotton, potato, banana and tomato.

13. according to each described method of claim 6-12, wherein said plant is a paddy rice, and wherein said target gene is from the gene that is selected from down the fungi in the group: Magnaporthe spp., Rhizoctonia (Rhizoctonia spp.), little Acremonium (Acremoniella spp.), pythium (Pythium spp.), Curvularia (Curvulariaspp.), Trichoderma (Trichoderma spp.), fusarium (Fusarium spp.) and Rhizopus (Rhizopus spp.).

14. according to each described method of claim 6-12, wherein said plant is a corn, and wherein said target gene is from the gene of fungi that is selected from down in the group: Colletotrichum (Colletotrichum spp.), Gibberella (Gibberella spp.), fusarium (Fusariumspp.), shell look list are every spore (Diplodia spp.) and Puccinia (Puccina spp.).

15. according to each described method of claim 6-12, wherein said plant is a soybean, and wherein said target gene is the gene from Phakopsora (Phakopsora spp.).

16. according to each described method of claim 6-12, wherein said plant is a cotton, and wherein said target gene is from the gene that is selected from the fungi in fusarium (Fusarium spp.) and the Verticillium (Verticillium spp.).

17. according to each described method of claim 6-12, wherein said plant is a potato, and wherein said target gene is from the gene of fungi that is selected from down in the group: phytophthora (Phytophthora spp.), Rhizoctonia (Rhizoctonia spp.) and cause the fungal species of wilt disease, canker or scurf.

18. according to each described method of claim 6-12, wherein said plant is a banana, and wherein said target gene is the gene from the fungi that is selected from mycosphaerella (Mycosphaerellaspp.), cercospora (Cercospora spp.) and fusarium (Fusarium spp.).

19. according to each described method of claim 6-12, wherein said plant is a tomato, and wherein said target gene is from being selected from phytophthora (Phytophthora spp.) and causing the gene of the fungi among the fungi kind of leaf disease, wilt disease or fruit rot.

20. according to each described method of claim 1-19, wherein said fungi target gene is viability, growth, the growth for this fungi or breeds necessaryly that preferred described fungi target gene relates to any cell function as defined in Table 1; Or wherein said fungi target gene relates to the pathogenic or infectious of fungi, and formation, the conidium that preferred described fungi target gene relates to germ tube adheres to, formation or conidial formation of the formation of appressorium, invasion silk.

21. transgenic plant or be used for the breedings of transgenic plant or reproductive material or the transgenic plant cells cultivated, its expression maybe can be expressed at least a double-stranded RNA, wherein said double-stranded RNA comprises the annealed complementary strand, a chain wherein have with the nucleotide sequence of fungi target gene to small part complementary nucleotide sequence, and wherein said double-stranded RNA interacts through plant-fungi and is absorbed by the fungal cell.

22. according to the transgenic plant of claim 21 or be used for the breedings of transgenic plant or reproductive material or the transgenic plant cells cultivated, wherein said fungi target gene is viability, growth, the growth for fungi or breeds necessaryly that preferred described fungi target gene relates to any cell function as defined in Table 1; Or wherein said fungi target gene relates to the pathogenic or infectious of fungi, and formation, the conidium that preferred described fungi target gene relates to germ tube adheres to, formation or conidial formation of the formation of appressorium, invasion silk.

23. transgenic plant according to claim 21 or 22, the breeding or the reproductive material that perhaps are used for transgenic plant, the perhaps transgenic plant cells of Pei Yanging, wherein said fungi target gene comprises the sequence identical with the sequence at least 75% that is selected from down group: SEQ ID NOs3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15,48 and 110, perhaps its complementary sequence, perhaps wherein said fungi target gene is to comprise SEQ ID Nos 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, the fungi of the gene of any sequence is directly to homologue in 48 and 110.

24. according to each transgenic plant of claim 21-23, the breeding or the reproductive material that perhaps are used for transgenic plant, the perhaps transgenic plant cells of Pei Yanging, it is a rice plants, the perhaps breeding of rice plants or reproductive material, the perhaps rice plants cell of Pei Yanging, wherein target gene is the gene from the fungi that is selected from down group: Magnaporthe spp., Rhizoctonia (Rhizoctonia spp.), little Acremonium (Acremoniella spp.), pythium (Pythium spp.), Curvularia (Curvularia spp.), Trichoderma (Trichodermaspp.), fusarium (Fusarium spp.) and Rhizopus (Rhizopus spp.).

25. according to claim 21-23 each transgenic plant or be used for the breedings of transgenic plant or reproductive material or the transgenic plant cells cultivated, it is the corn plant cell of breeding or the reproductive material or the cultivation of maize plant or maize plant, and wherein target gene is the gene from the fungi that is selected from down group: Colletotrichum (Colletotrichumspp.), Gibberella (Gibberella spp.), fusarium (Fusarium spp.), shell look list are every spore (Diplodia spp.) and Puccinia (Puccina spp.).

26. according to claim 21-23 each transgenic plant or be used for the breedings of transgenic plant or reproductive material or the transgenic plant cells cultivated, it is the soybean plant cell of breeding or the reproductive material or the cultivation of soybean plants or soybean plants, and wherein target gene is the gene from Phakopsora (Phakopsora spp.).

27. according to claim 21-23 each transgenic plant or be used for the breedings of transgenic plant or reproductive material or the transgenic plant cells cultivated, it is the cotton plant cell of breeding or reproductive material or the cultivation of vegetable lamb or vegetable lamb, and wherein target gene is from the gene that is selected from the fungi in fusarium (Fusarium spp.) and the Verticillium (Verticillium spp.).

28. according to claim 21-23 each transgenic plant or be used for the breedings of transgenic plant or reproductive material or the transgenic plant cells cultivated, it is the potato plants cell of breeding or the reproductive material or the cultivation of potato plants or potato plants, and wherein target gene is from being selected from phytophthora (Phytophthora spp.), Rhizoctonia (Rhizoctonia spp.) and causing the gene of the fungi among the fungi kind of wilt disease, canker or scurf.

29. according to claim 21-23 each transgenic plant or be used for the breedings of transgenic plant or reproductive material or the transgenic plant cells cultivated, it is the banana plant cell of breeding or the reproductive material or the cultivation of banana plant or banana plant, and wherein target gene is the gene from the fungi that is selected from down group: mycosphaerella (Mycosphaerellaspp.), cercospora (Cercospora spp.) and fusarium (Fusarium spp.).

30. according to claim 21-23 each transgenic plant or be used for the breedings of transgenic plant or reproductive material or the transgenic plant cells cultivated, it is the breeding of tomato plants or tomato plants or reproductive material or the tomato plants cell cultivated, and wherein target gene is from being selected from phytophthora (Phytophthora spp.) and causing the gene of the fungi among the fungi kind of leaf disease, wilt disease or fruit rot.

31. isolating double-stranded RNA, wherein comprise the annealed complementary strand, a chain wherein have with the nucleotide sequence of fungi target to small part complementary nucleotide sequence, wherein said fungi target gene is viability, growth, the growth for fungi or breeds necessaryly that preferred described fungi target gene relates to any cell function as defined in Table 1; Or wherein said fungi target gene relates to the pathogenic or infectious of fungi, and formation, the conidium that preferred described fungi target gene relates to germ tube adheres to, formation or conidial formation of the formation of appressorium, invasion silk.

32. isolating double-stranded RNA according to claim 31, wherein comprise the annealed complementary strand, a chain wherein have with the nucleotide sequence of fungi target gene to small part complementary nucleotide sequence, wherein said target gene comprises the sequence identical with being selected from following sequence at least 75%: SEQ ID NOs 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15,48 and 110, perhaps its complementary sequence, perhaps wherein said fungi target gene is to comprise SEQID Nos 3,42,99,100,39,60,111,112,113,114,115,116,117,5,43,101,102,1,41,184,185,97,98,37,59,124,9,45,188,189,106,13,47,109,33,57,126,23,52,119,35,58,127,7,44,186,187,103,104,105,29,55,118,17,49,108,25,53,121,19,50,125,31,56,123,11,46,107,27,54,122,21,51,120,15, the fungi of the gene of any sequence is directly to homologue in 48 and 110.

33. isolating double-stranded RNA according to claim 31 or 32, at least one of wherein said annealing complementary strand comprises RNA equivalent at least a in the nucleotide sequence of following any sequence representative: SEQ ID NOs 99,100,111,112,113,114,115,116,101,102,97,98,124,106,109,126,119,127,103,104,105,118,108,121,125,123,107,122,120 and 110, the perhaps long double-stranded fragment of its at least 17 base pairs.

34. isolating double-stranded RNA according to claim 33, wherein said nucleotide sequence comprises at least a by in any sequence representative sequence in SEQ ID NOs 192,201,202,193,190,191,196,199,200,194,195,198 and 197, the perhaps long double-stranded fragment of its at least 17 base pairs.

35. isolating double-stranded RNA is as the purposes of medicine, wherein said double-stranded RNA comprises the annealed complementary strand, a chain wherein have with the nucleotide sequence of fungi target to small part complementary nucleotide sequence.

36. according to each isolating double-stranded RNA of claim 31-34 as the purposes of medicine.

37. according to each isolating double-stranded RNA construct of claim 31-33, its comprise two copies at least and the nucleotide sequence fungi target to the described nucleotide sequence of small part complementary.

38. isolating double-stranded RNA construct according to claim 37, it comprises at least a RNA equivalent: SEQ ID NOs 99,100,111,112,113,114,115,116,101,102,97,98,124,106,109,126,119,127,103,104,105,118,108,121,125,123,107,122,120 and 110 in following any sequence representative nucleotide sequence of two copies at least, or comprises their at least one long double-stranded fragment of at least 17 base pairs.

39. according to the isolating double-stranded RNA or the RNA construct of claim 38, it comprises the RNA equivalent as the nucleotide sequence of representative among the SEQ ID NO 117.

40. according to each isolating double-stranded RNA or RNA construct of claim 31-39, it comprises the dsRNA zone that at least one is extra, its at least one chain comprise with the nucleotide sequence of at least one other fungi target gene to small part complementary nucleotide sequence.

41., also comprise at least one extra sequence and connexon randomly according to each isolating double-stranded RNA construct of claim 31-40.

42. according to the isolating double-stranded RNA construct of claim 41, wherein said extra sequence is selected from the group that comprises following sequence: (i) promote the extensive sequence that produces this dsRNA construct; (ii) realize the sequence that this dsRNA stability increases or reduces; Thereby (iii) allow protein or other molecule in conjunction with promoting the fungal cell to absorb the sequence of RNA construct; Thereby (iv) as on the binding to fungal cell surface or tenuigenin in acceptor or the sequence of the adaptive son of the molecule picked-up, endocytosis and/or the transcytosis that promote the fungal cell.

43. according to each isolating double-stranded RNA construct of claim 37-42 as the purposes of medicine.

44. coding according to claim 31-35 each double-stranded RNA or according to each the isolating nucleotide sequence of double-stranded RNA construct of claim 37-42.

45. isolating nucleotide sequence according to claim 44, it comprises the sequence by any sequence representative in SEQ IDNOs 192,117,201,202,193,190,191,196,199,200,194,195,198 and 197, or the fragment of their at least 17 Nucleotide.

46. an isolating nucleotide sequence, it is by SEQ ID NOs 3,99,100,192,39,111,112,113,114,115,116,117,201,202,5,101,102,193,184,97,98,190,191,37,124,9,188,106,196,13,109,199,200,33,126,23,119,35,127,7,186,103,104,105,194,195,29,118,17,108,198,25,121,19,125,31,123,11,107,197,27,122,21,120, the sequence of any sequence representative in 15 and 110, or the fragment of their at least 17 Nucleotide is formed.

47. the recombinant DNA construction body wherein comprises and operably connects each described nucleotide sequence of claim 44-46 that at least one regulates sequence.

48. recombinant DNA construction body according to claim 47, wherein said adjusting sequence is selected from and comprises constitutive promoter and comprise the CaMV35S promotor such as any being selected from, two CaMV35S promotors, ubiquitin promoter, actin promoter, ribulose-1,5-bisphosphate, 5-bisphosphate carboxylase promotor (rubisco promotor), the GOS2 promotor, promotor in the promotor group of figwort mosaic virus (FMV) 34S, or tissue-specific promoter is such as any root-specific promoter that is selected from the gene that comprises coding PsMTA III shell polysaccharidase, photosynthetic tissue's specificity promoter is such as cab1 and cab2, rbcS, gapA, the proteinic promotor of gapB and ST-LS1, the JAS promotor, chalcone synthetase promoters and from the promotor in the group of the RJ39 promotor of strawberry.

49. make the purposes of medicine according to the recombinant DNA construction body and function of claim 47 or 48.

50. comprise the composition of at least a double-stranded RNA, wherein said double-stranded RNA comprises the annealed complementary strand, a chain wherein have with the nucleotide sequence of fungi target gene to small part complementary nucleotide sequence, and comprise at least a suitable carriers, vehicle or thinner.

51. a composition wherein comprises at least aly according to each double-stranded RNA or at least a according to each double-stranded RNA construct of claim 37-42 of claim 31-34, and also comprises at least a suitable carrier, vehicle or thinner.

52. a composition wherein comprises at least a according to each nucleotide sequence and/or at least a recombinant DNA construction body according to claim 47 or 48 of claim 44-46; And also comprise at least a suitable carrier, vehicle or thinner.

53. according to each composition of claim 50-52 as the purposes of medicine.

54. the composition according to claim 50 or 51 is used for purposes medicinal or for animals.

55. be used as the purposes of the mycocide of plant or plant propagation or reproductive material according to the composition of claim 50 or 51.

56. be used to control the purposes of fungal growth according to the composition of claim 50 or 51.

57. the composition according to claim 50 or 51 is used to prevent to be subject to the plant of fungi infestation influence by the purposes of fungal infection.

58. comprise claim 44-46 each nucleotide sequence or the cell of the recombinant DNA construction body of claim 47 or 48.

59. the cell of claim 58, wherein said cell are prokaryotic cell prokaryocyte or eukaryotic cell.

60. the cell of claim 59, wherein said cell is a bacterial cell.

61. the cell of claim 59, wherein said cell is a vegetable cell.

62. a kind of plant wherein comprises at least a according to each double-stranded RNA or at least a according to each double-stranded RNA construct, at least a according to each nucleotide sequence, at least a according to the recombinant DNA construction body of claim 47 or 48 or the vegetable cell of at least a claim 61 of claim 44-46 of claim 37-42 of claim 31-34.

63. the plant of claim 60, wherein said plant is selected from the group that comprises paddy rice, barley, rye, wheat, grain, Herba Eragrostidis pilosae or lady's-grass.

64. the plant of claim 62 or 63, wherein said plant is a paddy rice.

65. comprise at least a according to claim 44-46 each nucleotide sequence or the seed of at least a recombinant DNA construction body according to claim 47 or 48.

66. the seed of claim 65, wherein said seed are the seeds as the defined plant of claim 63.

67. the seed of claim 65 or 66, wherein said seed is a rice paddy seed.

68. treat and/or prevent the method for fungal infection on the matrix, it comprises the composition according to claim 50 or 51 of using significant quantity to described matrix.

69. treat and/or prevent breeding or the fungal growth of reproductive material and/or the method for fungal infection of plant or plant, it comprises the composition according to claim 50 or 51 of using significant quantity to the breeding of plant or plant or reproductive material.

70. treat and/or prevent the method for fungal disease or illness, it comprises to this object that treats and/or prevents of needs uses composition according to claim 50 or 51, described composition comprises at least a double-stranded RNA or the double-stranded RNA construct that contains the complementary strand of annealing, a chain in the wherein said annealing complementary strand have with cause this fungal disease or illness the fungi target gene nucleotide sequence to small part complementary nucleotide sequence.

71. according to claim 31-34 each double-stranded RNA, according to claim 37-42 each the double-stranded RNA construct or according to claim 44-46 each nucleotide sequence according to the recombinant DNA construction body of claim 47 or 48 or according to claim 59-61 each cell or be used for the treatment of the purposes of the fungi infestation of plant according to the composition of claim 50 or 51.

72., be used for the treatment of the fungi infestation in each defined plant of claim 12-19 according to the purposes of claim 71.

73. test kit, wherein comprise according to claim 31-34 each double-stranded RNA, according to claim 37-42 each the double-stranded RNA construct or according to claim 44-46 each nucleotide sequence according to the recombinant DNA construction body of claim 47 or 48 or according to claim 59-61 each cell or according to the composition of claim 50 or 51, be used for the treatment of the fungi infestation of plant.

74. increase the method for plant biomass, its be included in the plant with effable form introduce according to claim 44-46 each nucleotide sequence or according to the recombinant DNA construction body of claim 47 or 48.

75. according to the method for claim 74, wherein said plant is selected from the group that comprises paddy rice, barley, rye, wheat, grain, Herba Eragrostidis pilosae or lady's-grass.

76. according to the method for claim 74 or 75, wherein said plant is a paddy rice.

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