CN106011141B - Ming River lily inducible promoter and its application - Google Patents

Abstract

The invention discloses a kind of Ming River lily inducible promoter LrP1 and its application, the nucleotide sequence such as SEQ ID NO of LrP1：Shown in 1, the present invention confirms that Ming River lily promoter L rP1 responds several plant hormone, biology and abiotic stress by molecular biology and genetic engineering relation technological researching；It Ming River lily promoter L rP1 of the present invention is connect the expression cassette being built into beta-glucosiduronatase gene is transferred in tobacco and express, the glucuronidase activity of transgene tobacco is detected by fluorescence standard measure, the results showed that transgene tobacco glucuronidase activity after gibberellin, ethylene, abscisic acid, NaCl, injury, Fusarium oxysporum, sclerotinite, Botrytis cinerea processing is remarkably reinforced；It can be seen that Ming River lily promoter L rP1 is induced by several hormones, biology and the abiotic stress factor, Genes For Plant Tolerance adverse circumstance genetic engineering can be used for.

Description

Ming River lily inducible promoter and its application

Technical field

The present invention relates to molecular biology and genetic engineering Related Research Domains, and in particular to a kind of inducible promoter LrP1 and its application.

Background technology

Promoter is to be located at structural gene 5'The DNA sequence dna for holding upstream, can activate RNA polymerase, be allowed to and template DNA standard True combination and the specificity with transcription initiation.Common promoter has constitutive promoter, organizing specific type to open in plant Mover and inducible promoter.Constitutive promoter (constitutive promoter) refer to such promoter control under, The expression somewhat constant of structural gene does not have notable difference on certain level, in different tissues, position expression.Tissue is special Different promoter (tissue-specific promoter) is also known as organ specific promoters, and it is special to be divided into root-specific promoter, stem Different promoter etc..Under this kind of promoter regulation, gene is often only expressed at certain specific organ or tissue positions, and is showed Go out the characteristic of growth adjustment.Using genetic engineering method by rice (Oryza sativa) sucrose synthase gene promoterRSP1 WithRSP2It is transferred to rice, the results showed thatRSP1WithRSP2Can drive beta-glucuronidase (β-glucuronidase, GUS) gene efficient specifically expressing in root, stem, leaf and glume, but do not expressed in embryo and endosperm（Li Yongchun, Zhang Xianyin, Xue In celebrating sucrose synthase genes promoter clone and its transgenic rice plant in specific expressed Acta Agronomica Sinicas, 2002, 28(5):586-590）.

Inducible promoter (inducible promoter) refers to the stimulation in certain specific physically or chemically signals Under, the transcriptional level of gene can be significantly increased in the promoter of this type.Inducible promoter can plant by The expression quantity that gene is improved when extraneous physics or chemical stress stops the regulation and control to gene after removal is coerced.Induction type starts Son this characteristic can guarantee played when plant is by stress from outside protection plant, resist environmental stimuli effect, and Ensure that plant energy does not waste in suitable environment.In genetic engineering, it is transferred to the foreign gene of plant if not being controlled, meeting The great expression in plant causes albumen largely to accumulate and wastes energy.Inducible promoter is added on carrier can be The expression for regulating and controlling target gene when environmental stimuli, solves the problems, such as foreign gene without limiting expression well.

Inducible promoter includes abiotic inducible promoter and biological factor inducible promoter.Abiotic factor lures Conductivity type promoter includes the inducible promoters such as arid, salt stimulation, temperature；Biological factor inducible promoter refers to pest and disease damage and lures The promoter led.Using genome ends random amplification technology, from spot thatch (Erianthus arundinaceus) PR10 bases It because terminal amplification goes out the promoter of 592bp, and connects and is transferred in tobacco, rice and sugarcane with gus reporter gene, the results showed that spot Thatch PR10 promoters can respond the processing of injury, methyl jasmonate and abscisic acid quickly（Chakravarthi M, Syamaladevi DP, Harunipriya P, Augustine SM, Subramonian N. A novel PR10 promoter from Erianthus arundinaceus directs high constitutive transgene expression and is enhanced upon wounding in heterologous plant systems. Mol Biol Rep, 2016, 43(1):17-30）.From western kahikatea（Pinus monticola）Middle clone obtainsPmPR10-1.13 Promoter and three 5 ' missing ends, connect Reporter gene GUS after be transferred to arabidopsis.As a result show that GUS appears in 2-3 days earliest The plumular axis and cotyledon of seedling, after plant top great expression.But in adult plants,PmPR10-1.13Promoter responds Pathogen infection and wound stress, showPmPR10-1.13Promoter response biology or abiotic stress（Liu JJ, Ekramoddoullah AK, Piggott N, Zamani A. Molecular cloning of a pathogen/wound- inducible PR10 promoter from Pinus monticola and characterization in transgenic Arabidopsis plants. Planta, 2005, 221(2):159-169）.Asparagus (Asparagus officinalis) in clone obtain a PR10 gene (AoPR10) promoter, willAoPR10Promoter connects reporter geneGUSIt is transferred in arabidopsis and gives adverse circumstance processing appropriate,GUSGene can be in injured, pathogen infection and H₂O₂Treatment site It expresses and accumulates, showAoPR10Promoter is induced by above-mentioned biology and the abiotic stress factor（Mur LA, Sturgess FJ, Farrell GG, Draper J. The AoPR10 promoter and certain endogenous PR10 genes respond to oxidative signals in Arabidopsis. Mol Plant Pathol, 2004, 5(5): 435-451）.

Invention content

It is an object of the present invention to provide a kind of inducible promoter LrP1, derive from Ming River lily, nucleotide sequence such as SEQ ID NO：Shown in 1.

The present invention is another object is that the promoter is applied in genetic engineering, i.e., as induced expression under environment stress Specific high-efficiency expression of the promoter regulation foreign gene in transgene receptor plant.

The present invention relates to separant induction type promoter fragment and identify its expression activity, the present invention is cloned from Ming River lily Inducible promoter is obtained, which is 1489bp；Bioinformatic analysis shows to include one in inducible promoter The different cis-acting elements of series, such as tissue-specific element, a variety of hormone (abscisic acid, gibberellin, ethylene, salicylic acid, jasmines Jasmine acid methyl esters) response element, abiotic stress (moisture, with high salt, injury) response element, Analysis of Defence Genes Involved responds pathogenic bacterium inducing The element etc. of transcription.WRKY transcription factors play important regulating and controlling effect in plant disease-resistant defense response, the inducible promoter There are three the cis-acting elements w box of WRKY for sequence tool（C/TTGACC/T）.

The inducible promoter segment of present invention separation clone is replaced to the 35s promoters of the CaMV on pBI121 carriers, Reporter gene is driven by inducible promoterGUSExpression cassette, by Agrobacterium tumefaciems (Agrobacterium tumefaciens) mediate will be transferred to model plant tobacco (Nicotiana tabacum) in expression, and pass through further experiment The expression characterization for disclosing inducible promoter, for later-stage utilization, the promoter regulation foreign gene is efficient in transfer-gen plant Specifically expressing lays the foundation.This promoter is named as LrP1 by inventor.

LrP1 promoters in the present invention are drivenGUSExpression cassette be transferred in tobacco, using several plant hormone, biology and Abiotic stress handles transgenic tobacco plant, and carries out the active quantitative fluorescence analysis of GUS, and testing result shows that LrP1 is opened Mover responds the processing of several abiotic, biotics and several plant hormone, gibberellin, ethylene, abscisic acid, NaCl, injury, Fusarium oxysporum, Botrytis cinerea (Botrytis cinerea), sclerotinite (Sclerotinia sclerotiorum) can be apparent The activity of evoked promoter LrP1.

Above-mentioned promoter L rP1 can be applied to the induced expression of foreign gene in genetic engineering, and concrete operations are as follows：

(1) using the special primer of amplification LrP1, genomic DNA is extracted from Ming River lily tender tissue, passes through polymerization The full length sequence that enzyme chain reaction (polymerase chain reaction, PCR) amplifies LrP1, is subsequently attached to On pGEM-T carriers, being obtained through sequencing, there is sequence correctly to clone；

(2) digestion with restriction enzyme pGEM-T-LrP1 carriers are used, promoter fragment is recycled；Simultaneously using suitable limit Composition type expression promoter on property endonuclease digestion removal plant expression vector processed, recycles to obtain carrier large fragment by glue； Obtained LrP1 segments are connect with pCAMBIA2300S carrier segments again, build plant inducible expression carrier；Later by institute's structure The plant inducible expression carrier built is recombinated with target gene, and is transferred in recipient plant by Agrobacterium tumefaciens mediated, and expression turns When being infected by NaCl, injury stress or Fusarium oxysporum, sclerotinite, Botrytis cinerea, promoter L rP1 drives the plant of gene Dynamic target gene can induce and up-regulated expression is horizontal, and internal external gibberellin, ethylene, abscisic acid can also induce purpose in addition High level gene expression.

The present invention is that the promoter of a new induced expression is provided in plant genetic engineering application.It is planted in genetic engineering Object overexpression vector commonly uses the 35S promoter from cauliflower mosaic virus, which is composition type expression promoter, purpose The expression somewhat constant of gene does not have notable difference on certain level, in different tissues, position expression, so being transferred to plant Its expression of the foreign gene of object is uncontrolled, causes albumen largely to accumulate and wastes energy.And inducible promoter can plant The expression quantity that gene is improved when object is influenced by stress from outside or chemical factor lowers mesh after removal stress or chemical treatment Gene expression, it is ensured that played when plant is by environment stress protection plant, resist environmental stimuli effect, otherwise The energy of plant is not wasted in suitable environment.In genetic engineering application, addition inducible promoter can overcome on carrier The problem of target gene is without limiting expression.Several hormones (gibberellin, ethylene, abscisic acid), abiotic stress (NaCl, injury), Biotic (Fusarium oxysporum, sclerotinite, Botrytis cinerea) obviously induces the expression of inducible promoter LrP1 in the present invention to live Property, therefore the present invention has broad application prospects in the genetic engineering of antibiont or abiotic stress.

Description of the drawings

Fig. 1 is the glue recovery product testing result of promoter L rP1 in the present invention (A figures) and pBI-121 carriers (B figures)；

Fig. 2 is pBI121- LrP1- in the present inventionGUSThe positive colony testing result of Escherichia coli is converted, wherein positive Control is reacted by the PCR of template of pGEM-T-LrP1 plasmids, and blank control is reacted by the PCR of template of sterile water；

Fig. 3 is part pBI121-LrP1- in the present inventionGUSThe PCR the selection results of transgene tobacco, wherein positive control It is with plasmid pBI121-LrP1-GUSIt is reacted for the PCR of template；WT：Non-transgenic tobacco (wild type) total DNA carries out for template PCR；

Fig. 4 is the standard curve of GUS enzyme activity in the present invention；

Fig. 5 is pBI121-LrP1- in the present inventionGUSTransgene tobacco is in gibberellin, ethylene, abscisic acid treated GUS Activity, wherein CK are the pBI121-LrP1- of normal growthGUSThe GUS activity of transgene tobacco；

Fig. 6 is pBI121-LrP1- in the present inventionGUS transgene tobaccos in NaCl, aggrieved treated GUS activity, wherein CK is the pBI121-LrP1- of normal growthGUSThe GUS activity of transgene tobacco；

Fig. 7 is pBI121-LrP1- in the present inventionGUSTransgene tobacco connects in Fusarium oxysporum, Botrytis cinerea, sclerotinite GUS activity after kind, wherein CK are the pBI121-LrP1- of normal growthGUSThe GUS activity of transgene tobacco.

Specific implementation mode

Below by drawings and examples, the present invention is further described, but the scope of the present invention is not limited in described Hold, method operating according to a conventional method unless otherwise specified in the present embodiment, the use of agents useful for same unless otherwise specified is normal Rule reagent or the reagent configured according to a conventional method.

Specific implementation mode

Below by drawings and examples, the present invention is further described, but the scope of the present invention is not limited in described Hold, method operating according to a conventional method unless otherwise specified in the present embodiment, the use of agents useful for same unless otherwise specified is normal Rule reagent or the reagent configured according to a conventional method.

Embodiment 1：The clone of Ming River lily inducible promoter LrP1 and sequence analysis

Using the Ming River lily kan gene group DNA of extraction as template, with the special primer (sense primer of amplification promoter L rP1 For：5 ' TCGAGTTTGGTGTTATTGTTATTAG3 ' ', downstream primer are：5 ' GATGTGTTTGAGAAGGAGGTGT3 ') it is upper Downstream primer passes through the sequence of PCR cloning promoters LrP1.Reaction system (20 μ L) is 0.5 μ of Ming River lily genomic DNA G, 2 10 × Advantage of μ L, 2 PCR Buffer, 1.8 μ L dNTP Mix (10mM each), 0.2 μ L sense primers (10 μM), 0.2 μ L downstream primers (10 μM), 0.2 μ L Advantage, 2 PCR Polymerase Mix, 14.6 μ L PCR-Grade water.PCR reaction conditions：94℃ 5 min；94 DEG C of 30 s, 65 DEG C of 30 s, 72 DEG C of 2 min, 32 cycles； 72℃ 5 min.After PCR, take 8 μ L into row agarose gel electrophoresis, to detect the specificity of amplified production and big It is small.

Acquired PCR product only has a DNA band, therefore directly carries out TA clones to PCR product, and the kit used is PGEM-T vector system (Promega), reaction system and operating process are：1.5 μ L PCR products are taken, are sequentially added 1 μ L pGEM-T vector (50 ng/ μ L) and 2.5 μ 2 × Ligation of L solution I, mixing is placed on 16 DEG C of mistakes Night reacts.Connection product is transferred in bacillus coli DH 5 alpha competence by heat-shock transformed method.With containing ampicillin The LB solid medium screening positive clones of (ampicillin, Amp).Several single bacterium colonies are selected, amplification LrP1 is used after shaking bacterium Special primer detection multiple cloning sites be inserted into LrP1 clone.Obtained positive colony is sequenced, what is finally obtained opens 1489 bp of mover LrP1 long.The cis-acting elements of promoter is predicted using PLANTCARE, predicts promoter sequence In with the relevant cis-acting elements of environment stress (table 1).Include a series of different cis-acting elements, such as group in promoter Specific element is knitted, the response element of a variety of hormones (abscisic acid, gibberellin, ethylene, salicylic acid, methyl jasmonate) is abiotic Stress (moisture, with high salt, injury) response element, Analysis of Defence Genes Involved respond the element etc. of pathogenic bacterium inducing transcription；WRKY transcription factors Important regulating and controlling effect is played in plant disease-resistant defense response, there are three the cis-acting elements w of WRKY for promoter sequence tool box（C/TTGACC/T）.

Table 1：Cis-acting elements prediction result in promoter sequence

。

Embodiment 2：LrP1-GUSExpression vector establishment

PBI121 multiple cloning sites haveHinIII Hes of dBamI restriction enzyme sites of H, therefore in the special primer point of amplification promoter It does not addHinIII Hes of dBamThe recognition site of H I.Using a small amount of extraction agent boxes of SanPrep pillar Plasmid DNA (Shanghai life work) The escherichia coli plasmid pGEM-T-LrP1 and plant expression vector pBI121 plasmids of LrP1 is inserted into extraction, takes 1 μ L for fine jade Sepharose electrophoresis is to detect the integrality and concentration level of extracted plasmid.Use restriction enzymeBamI Hes of HHinD III divides Other to carry out double digestion (100 μ L systems) to plasmid pGEM-T-LrP1 and pBI121, reaction system and operating process are：It takes respectively 20 μ L pGEM-T-LrP1 and pBI121 plasmids sequentially add 10 μ L 10 × H buffer, 5 μ LBamHⅠ、5 μL Hind Ⅲ、60 μL ddH₂O centrifuges after mixing, is placed in 37 DEG C of reaction overnights in short-term.All digestion products are subjected to Ago-Gel electricity Then swimming uses SanPrep pillar DNA plastic recovery kits (Shanghai life work) large stretch of to promoter fragment and pBI121 carriers Section carries out glue recycling respectively, and 1 μ L recovery products is taken to detect the size and concentration of recycling segment by agarose gel electrophoresis, The results are shown in Figure 1.

Using T4 DNA Ligase (TaKaRa), the promoter dna fragment of recycling is connected with pBI121 carrier segments Get up, reaction system (20 μ L) and operating process are：10 μ L LrP1 DNA fragmentations are taken to sequentially add 2 μ L pBI121 carriers DNA、2 μL 10×T4 DNA Ligase Buffer、1 μL T4 DNA Ligase、5μL ddH₂O, after mixing in short-term from The heart, then 16 DEG C of water-bath reaction overnights.Then connection product is transferred in bacillus coli DH 5 alpha using heat-shock transformed method, with containing The solid medium screening positive clone of 50 mg/L kanamycins (kanamycin, Km).Picking individual colonies shake bacterium, are with bacterium solution Template carries out PCR with the special primer of amplification promoter L rP1, picks out the clone that LrP1 and pBI121 is successfully connected, is obtaining Positive strain in glycerine is added is placed in -80 DEG C and saves backup.

It is extracted using SanPrep pillar plasmid extraction kits and purifies the pBI121- in above-mentioned bacillus coli DH 5 alpha LrP1-GUSPlasmid.Then use frozen-thawed method by the plant expression vector pBI121-LrP1 of above-mentioned structure-GUSIt is transferred to made In standby agrobacterium tumefaciens lba4404 competent cell.Operating procedure is：Take 0.2 μ g pBI121-LrP1-GUSPlasmid is added In centrifuge tube containing 200 μ L competent cells, gently 5 min of ice bath after mixing, then continues in liquid nitrogen and freezes 1 min, so After be immediately placed in 37 DEG C of 5 min of water-bath, then 2 min of ice bath, 500 μ L LB liquid mediums are added later in 28 DEG C of shaken cultivations 4 h.Agrobacterium after activation is applied on the LB solid mediums containing 50 mg/L Km, 28 DEG C are inverted culture.Select single bacterium It falls and shakes bacterium, then PCR reactions are carried out with the specific primer of amplification LrP1, detect pBI121-LrP1-GUSWhether Agrobacterium is transferred to In.For positive colony shown in Fig. 2, addition glycerine is placed on -80 DEG C and saves backup.

Embodiment 3：Agriculture bacillus mediated Genetic Transformation in Higher Plants and genetically modified plants screening

The transgene receptor of this experiment is tobacco, and 75% alcohol of tobacco seed is impregnated 30 s, is used after sterile water washing 0.1% HgCl₂Impregnate 8 min, then again with sterile water washing several times, be seeded on 1/2 MS culture mediums, 28 DEG C of light cultures 5-8 d go to illumination box (25 DEG C, 16h/d illumination) after germination, monthly use MS culture medium subcultures primary later.

Contain pBI121-LrP1 by what is preserved in -80 DEG C of refrigerators-GUSThe Agrobacterium LBA4404 bacterium solution of plasmid is taken out, and takes 10 μ L bacterium solutions are inoculated in the LB liquid medium that 1 mL contains 20 mg/L rifampins and 50 mg/L Km, 28 DEG C 200 Rpm shaken cultivations are to muddiness.Drawing 500 μ L bacterium solutions, to be spread evenly across the LB containing 20 mg/L rifampins and 50 mg/L Km solid On body culture medium, 28 DEG C are inverted culture to growing lawn.It is inoculated in 40 mL with oese scraping 3-5 ring lawns and contains 25 mg/mL In the MGL culture mediums of acetosyringone, 28 DEG C of 220 rpm shaken cultivation is until OD₆₀₀About 0.6.By sterile tobacco tissue-cultured seedling Blade is cut into about 1 cm²The leaf dish of size is soaked in the MGL culture mediums containing suspension Agrobacterium, 25 DEG C of shake cultures 15 min.It is transferred to tobacco after being blotted the bacterium solution on leaf dish surface with aseptic filter paper and co-cultures culture medium（MS+0.02 mg/L 6-BA+ + 6 g/L agar of 2.1 mg/L NAA+30 g/L sucrose）In, 22 DEG C of light cultures 2 days.Leaf dish after co-cultivation is transferred to cigarette Careless screening and culturing medium（+ 50 mg/L Km+ of+6 g/L agar of MS+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L sucrose 200 mg/L cephalosporins）On, it is incubated at illumination box (25 DEG C, 16 h/d illumination).Culture about 3 weeks will differentiate and Tobacco seedling cut and subculture in the root media containing 50 mg/L Km and 300 mg/L cephalosporins（MS+30 g/L + 50 mg/L Km+300 mg/L cephalosporins of+6 g/L agar of sucrose）Upper carry out culture of rootage.

The genomic DNA that transgenic tobacco plant blade is extracted using CTAB methods takes 1 μ L genomic DNAs to carry out agarose Its integrality of detected through gel electrophoresis and concentration.The spy of promoter L rP1 is expanded using the genomic DNA of transfer-gen plant as template Different primer carries out PCR reactions.After PCR, take 8 μ L products for agarose gel electrophoresis to detect positive transgenic plant. The amplification of partial transgenic tobacco plant is as shown in figure 3, Ming River lily inducible promoter LrP1 transgene tobaccos sieve altogether Choose 22 plants of positive transgenic plant.

Embodiment 4：The GUS fluorogenic quantitative detections of transgene tobacco

To the active quantitative fluorescence analysis of transgene tobacco root GUS with reference to (Jefferson R. such as Jefferson Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep. 1987,5(4):387-405) method, reaction mechanism are：GUS can be reacted with substrate 4-MUG, and catalysis generates 4- MU, 4-MU generate fluorescence under the conditions of excitation wavelength is 365 nm, launch wavelength is 455 nm, and the fluorescent value of generation can pass through Sepectrophotofluorometer is quantitative determined.

The Tobacco Root anticipated is placed in grind into powder in the mortar equipped with liquid nitrogen, 400 μ L GUS extractions are added Homogenate is transferred in 1.5 mL centrifuge tubes by buffer solution, and 10 min are centrifuged in 4 DEG C, 12000 g.After centrifugation, supernatant is collected In new centrifuge tube.The 4-MUG solution (1 mmol/L) of 1 mL is taken to preheat 10 min for 37 DEG C in 2.0 mL centrifuge tubes in advance. It takes 50 μ L supernatants to be added into the GUS reaction buffers of preheating, shakes up rapidly, and 200 μ L reaction mixtures is taken to set immediately In the stop buffer of 1.8 mL (operating time is less than 30 s), as 0 point sample of enzymatic reaction (as sky when fluoremetry White control), remaining liq continues 37 DEG C and reacts and start timing.200 are taken respectively when reacting 15 min, 30 min, 45 min μ L reaction mixtures are added into 1.8 mL stop buffers, are used for fluoremetry.Using sepectrophotofluorometer in excitation wave A length of 365 nm, launch wavelength measure the fluorescent value of each sample under conditions of being 455 nm.Make 4-MU standard curves：By 1 mM 4-MU mother liquors are diluted to 50 nM, 100nM, 200 nM, 400 nM, the difference of 500 nM and 1000 nM with reaction terminating liquid respectively Gradient liquid is 365 nm in excitation wavelength, and launch wavelength measures the fluorescent value of each gradient liquid under conditions of being 455 nm, with reaction Terminate liquid is blank control, and standard curve (as shown in Figure 4) is drawn with the concentration of the fluorescent value and 4-MU measured.It takes on 10 μ L Clear liquid, using the protein content of the Coomassie Brilliant Blue determination sample of improvement.1 nmol 4- are generated with one minute catalysis 4-MUG The enzyme amount of MU is a unit of activity, and GUS enzyme activity is calculated with the enzyme activity of every mg total proteins, that is, is expressed as 4-MU nmol/min/ Mg (albumen).By standard curve, the GUS activity of transgene tobacco is calculated.

In order to detect response of the Ming River lily promoter to plant hormone, biotic and abiotic stress, respectively with several plantations The root of object hormone, biotic and abiotic stress factor treatment transgene tobacco, and by above method measurement handle before GUS activity afterwards, as a contrast (CK) with the untreated transgene tobacco root GUS activity of normal growth.Such as Fig. 5, red mould After element, abscisic acid and ethylene processing, the apparent up-regulation of GUS activity in Ming River lily promoter L rP1 transgene tobacco roots, to opening From the point of view of the induction degree of promoter activity, Chi Meisu >Tuo Luosuan >Ethylene.The GUS activity of transgene tobacco after NaCl, aggrieved processing As shown in fig. 6, injury and NaCl both abiotic stress factors notable activity of up-regulation promoter L rP1, are coerced with NaCl It compares, promoter L rP1 is stronger to the responsiveness of injury stress, and after injury processing, the GUS activity of LrP1 drivings is far above NaCl Stress and control.The root that transgene tobacco is handled with three kinds of pathogen Fusarium oxysporums, sclerotinite and Botrytis cinerea, also obviously lures The activity for having led promoter, from induction degree, Botrytis cinerea;Jian Baoliandaojun >Sclerotinite (Fig. 7).Above-mentioned experiment knot Fruit shows the processing of Ming River lily promoter L rP1 response several plant hormone, abiotic stress and biotic, gibberellin, Abscisic acid, ethylene, NaCl, injury, Botrytis cinerea, Fusarium oxysporum, sclerotinite can obviously raise the GUS activity of LrP1 drivings. Obviously, Ming River lily promoter L rP1 is a Plant Hormone, biotic and abiotic stress factor inducible promoter, can Applied to plant stress-resistance genetic engineering.

Sequence table

<110>Kunming University of Science and Technology

<120>Ming River lily inducible promoter and its application

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acacccacca tttactctaa taatattatc ttaagagatc ttcattataa atatctcttc 180

ttcgaatgaa tttgcttgat ttttttttat attcttcttc tgatgatgac aatcacgatc 240

aaatgattgg gctttccata atggcagtcg gttccttttg gcttcttctt taggccacta 300

ttcttcttaa aagaagtgat attcttagcc tttagtttct tgacatcatt actctcattt 360

gtattggcct tatgggactt aaagttgtca tctttcttct tttgttttct tgaatcctct 420

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ttatctacaa ggtcaatccc gacatgcttt atatcatcaa caatcaattg caggtcataa 600

acctgatcga gggtttactt atcattcacc attttataat taacataatt ggaaattaga 660

aatttctaat taatgccttt caaagctcct ttgcacttgc tgcttatcga taatggtggt 720

acaaagtctt tgaaagctcg ttggagggtg tgtctcttgc acatgaattc attttccttt 780

tgcttgtgac gtttctacct cagaacttca atattgtttg atttggattg aaaatggttt 840

caaaattctt atccagaatg taacccaact tcatcgtcgt gaggaaaaat ataacattat 900

gttacgacga gtgtagtttg tcatgctaaa atgcttgagt caaaccagat cttgattcat 960

atctcacaga gacttgaaaa tggtggagtt catactgtag ccaaaaaaat tgtctaattc 1020

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gtcctaaaaa gattgcagcg tacaagtcaa caagatgatg actgttgttt gaatagcgtg 1320

ttgggcatat taaactggga ggtgtgatta ttaaaacctc atcataagtc ggcggaactc 1380

ttttatatgg cggaagacgt ttagatatag cttggcttgc agtgggacta agcacaccaa 1440

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Claims (2)

1. a kind of inducible promoter LrP1 derives from Ming River lily, nucleotide sequence such as SEQ ID NO：Shown in 1.

2. inducible promoter LrP1 described in claim 1 is in environment stress down regulation foreign gene in transgene receptor plant In specific high-efficiency expression；

Described refers in gibberellin, ethylene, abscisic acid, NaCl, injury, Fusarium oxysporum, sclerotinite or ash under environment stress In the presence of grape spore stress factors.