CN1831128A - Inducible promoter separated from leaf mustard - Google Patents

Abstract

The invention discloses a MeJA inducement promoter separated from mustard that could construct recombination expression carrier using the connecting of the external source gene. When the external gene is anti-insect gene, the transformed plant would be gained.

Description

The inducible promoter of separated from leaf mustard

Technical field

The invention belongs to the genetically engineered field, it relates to a kind of plant promoter.Specifically, relate to from being hurt of being separated to of leaf mustard, methyl jasmonate (MeJA) inductive promotor.

Background technology

Plant materials can only conform passively because can not independently move.Through evolution of long period of time, plant materials has been set up a cover protection mechanism, to resist the infringement of disadvantage factor such as disease and pest, as forming very thick physical barriers such as cell walls; And start some expression of gene fast, and in order to resist the attack of disease, insect pest, as chitinase gene, protease inhibitor gene, and course of disease protein gene etc.It is found that the intravital hormone of plant when disease and pest takes place, the significant variation takes place in the content as methyl jasmonate (MeJA), dormin (ABA), Whitfield's ointment (SA) etc., infer that these hormones have brought into play vital role in plant disease-resistant insect pest process, therefore these hormones are used to disease-resistant worm, and the expression of gene study on regulation.Different kinds, the ability of individual disease-resistant worm differ, and people cultivate disease-resistant worm kind by traditional methods such as selection, hybridization, for high yield, the stable yields of farm crop have been made huge contribution.Along with development of molecular biology, plant disease-resistant worm mechanism is able to be described on molecular level, and molecular breeding is achieved and paid more and more attention.

Utilize engineered method, people import special genes in the plant, improve the insect resistance capacity of plant.As the Bt gene importing cotton of Monsanto Company, realized the effective control of cotton to bollworm with the CaMV35S promoters driven.In recent years transgenic plant industrialization paces are constantly accelerated.1,700,000 hectares of the areas of global genetically modified crops in 1996,2003 6770 ten thousand hectares, increased nearly 40 times in 7 years.By 2005, the genetically modified crops area reached 1,500,000,000 mu, accounted for 10% of the total cultivated area in the whole world.The U.S. is in absolute predominance in this field, and cultivated area accounts for more than 60% of the global total area.But national genetically modified crops cultivated area amplification is very fast in the developed recently, and for example India's transgene cotton area in 2005 increased by 260% than 2004, reached more than 130 ten thousand hectares; The genetically engineered soybean area of Brazil increased by 88% than 2004, reached more than 930 ten thousand hectares.2,800,000 hectares of China's plantation transgene cottons account for 4% of the global total area, account for 66% of the Cotton in China total area.Genetically modified crops are planted by existing 21 countries in the whole world at present.From these development trends, it is matter of time that the genetically modified crops kind " routinizes ".Over more than 20 year, comparatively widely used promotor has the CaMV35S promotor from cauliflower mosaic virus in the plant genetic engineering, from the CsVMV35S promotor of cassava vein mosaic virus, from promotors such as Adh1, the Act1 of plant itself, Ubi1.These all are constitutive promoters.

Because the gene that constitutive promoter drives all has expression in various degree in plant different tissues organ, expose some problems in the application gradually.As a rule, people do not wish foreign gene wide expression in the whole plant of transgenic plant, whole growing, because can increase the metabolism burden of plant on the one hand, on the other hand, some foreign protein is unfavorable for plant normal growth to plant and nonessential even poisonous; In addition, in some plant organs, the expression activity of constitutive promoter strong (Chinese science C collects life science for Ren Maozhi etc., the promoter function analysis of cotton propagating organ's predominant expressed gene, 2005,35 (1): 22-28).

The research of plant inducible promoter is a lot, is a research focus of plant biotechnology field.Research contents mainly concentrates on two aspects, and the one, about the research of natural inducible promoter, comprise the promotor that light, temperature, water, salt, wound, pathogenic bacteria invasion and attack etc. are replied, be partial to study the Regulation Mechanism of genetic expression more.The 2nd, about the research of artificial induction's type promotor, promptly utilize achievement in research to natural promoter, but make up foreign gene abduction delivering (the Xu Bet al of system, Methyl jasmonate induced expression of the tobacco putrescine N-methyltransferase genes requires both G-box and GCC-motif elements, PlantMolecular Biology, 2004,55 (5): 743-61; Wait quietly on the road, higher plant promotor and applied research progress thereof, natural science progress, 2004,14 (8): 856-862).Particularly, for example Guevara-Garcia etc. has analyzed (the Ni of the wound-induced element of mannopine synthase gene promoter on the Ti-plasmids with Ni etc., M.et al., Sequence-specific interaction of wound-inducible nuclear factors with mannopinesynthase 2 ' promoter wound-responsive elements, Plant Molecular Biology, 1996,30 (1): 77-96); Siebertz etc. (1989), Zhu etc. (1993), Rouster etc. (1997), Devoto etc. (1998), Xu etc. (2004) have studied potato wunl gene respectively, paddy rice chitinase gene RCH10, the barley fat oxygenase gene, the soybean poly closes galacturonic acid enzyme inhibitory enzyme gene and tobacco putrescine N-methyltransferase gene promoter sequence and wound or JA inductive relation (Devoto, A.et al., The promoter of a geneencoding a polygalacturonase-inhibiting protein of Phaseolus vulgaris L.Isactivated by wounding but not by elicitors or pathogen infection.Planta, 1998,205 (2): 165-174; Rouster, J.et al., Identification of a methyl jasmonate-responsiveregion in the promoter of a lipoxygenase l gene expression in barley grain, Plant Journal, 1997,11 (3): 513-523; Xu B et al, Methyl jasmonate induced expressionof the tobacco putrescine N-methyltransferase genes requires both G-box andGCC-motif elements, Plant Molecular Biology, 2004,55 (5): 743-61).The promotor block (motif) relevant with wound-induced that has identified at present has 6 kinds at least: AG-motif (AGATCCAA), 13-bp (TGGTAGGTGAGAT), G-box (CACGTG), L-box (ATTCTCACCTACCA), P-box (CTCCAACAAACCCCTTC) and A-box (CCGTCC); Induce relevant promotor block to have 4 kinds at least with jasmonic (JA): G-box, 13-bp, Hexamer-motif (TGACGT) and GCC-box.Above-mentioned block helps the researchist that the promoter fragment that obtains is predicted, tentatively to determine whether to belong to inducible promoter.

(Zhao and Chye in the research that some of the staff of the present invention participate in about the leaf mustard chitinase, Methyljasmonate induces expression of a novel Brassica juncea chitinase with twochitin-binding domains, Plant Molecular Biology, 1999, Vol 40, p1009-1018), relate to clone's chitinase gene (BjCHI1) in leaf mustard.The important difference of BjCHI1 and previous plant chitinase gene is to have two chitin binding domain coding regions, and the protein that shows this genes encoding in research subsequently has chitinase activity and activity of lectin concurrently, can suppress fungi growth (Fung etc., Tobacco-expressedBrassica juncea chitinase BjCHI1 shows anti-fungal activity in vitro, PlantMolecular Biology, 2002, Vol 50, p283-294; Ou Yangshiwen etc., the research of the structure-function relationship of the novel chitinase BjCHI1 of leaf mustard,, Ph D dissertation in 2002; Tang etc., Functional analyses of thechitin-binding domains and the catalytic domain of Brassica juncea chitinaseBjCHI1, Plant Molecular Biology, 2004, Vol 56, thereby may have multiple function and purposes p285-298).When the BjCHI1 gene is studied, the researchist has been noted that: 1. keep silent under the BjCHI1 normal circumstances, but run into JA (jasmonate), wound, worm (Pieris rapae) food, fungi (Aspergillus niger) invasion and attack, will start rapidly, strong and express (Fung KL, Zhao, KJ, Deng, Tobacco-expressed Brassicajuncea chitinase BjCHI1 shows anti-fungal activity in vitro, Plant MolecularBiology, 2002,50 (2): 283-294; Zhao, K.J etc., Methyl jasmonate induces expressionof a novel Brassica juncea chitinase with two chitin-binding domains, PlantMolecular Biology, 1999,40 (6): 1009-1018); 2. its double base CBD constitutional features determines it to have the dual nature of chitinase and lectin concurrently, tool unique effect (Chye ML in phytopathy worm system of defense, Zhao KJ etc., An agglutinating chitinase with two chitin-binding domains confers fungalprotection in transgenic potato, Planta, 2005,220 (5): 717-730); Tang CM, ChyeML, Ramalingam S, Ouyang SW, Zhao KJ etc., Functional analyses of the chitin-bindingdomains and the catalytic domain of Brassica juncea chitinase BjCHl1, PlantMolecular Biology, 2004,56 (2): 285-98; Ou Yangshiwen, Zhao Kaijun etc. have the plant chitinase BjCHI1 of activity of lectin, biotechnology journal, 2002,18 (5): 51-56) concurrently.The promotor that the inventor feels regulation and control BjCHI1 gene deeply has researching value.By inventor's effort, successfully be separated to the promotor (being called BjC-p) of control BjCHI1 gene in leaf mustard finally, and prove that by experiment this promotor is subjected to the induced strong of wound.

Summary of the invention

The purpose of this invention is to provide and a kind ofly for example can bring into play the inducible promoter of useful effect in the tobacco plant.This is the present inventor through making great efforts with great concentration, a kind of strong inducible promoter that from the leaf mustard genome, is separated to, thus finish the present invention.

The present invention relates to derive from the inducible promoter of leaf mustard (Brassica juncea), it controls the expression of chitinase gene BjCHIl, and it has the nucleotide sequence shown in the SEQ ID NO:1.Inducing herein is meant that injury induces (as insect pest) and methyl jasmonate to induce.

Studies show that promotor of the present invention also comprises by the promotor that nucleotide sequence is formed shown in the 92nd to 1378 among the SEQ ID NO:1, by nucleotide sequence is formed shown in the 302nd to 1378 among the SEQ ID NO:1 promotor, by the 556th to 1378 promotor that Nucleotide is formed shown in the 456th to 1378 promotor that Nucleotide is formed shown in the SEQ ID NO:1 or the SEQ ID NO:1.They still possess is hurt or JA inductive promoter activity.

The present invention also provides the complementary sequence of above-mentioned each promotor.

Promotor of the present invention also be included under the stringent condition and SEQ ID NO:1 shown in the 556th to 1378 nucleotide sequence shown in the 456th to 1378 nucleotide sequence shown in the 302nd to 1378 nucleotide sequence, the SEQ IDNO:1 or the SEQ ID NO:1 among the 92nd to 1378 nucleotide sequence, the SEQ ID NO:1 in the nucleotide sequence, SEQ ID NO:1, and the DNA isolation of their complementary sequence hybridization, and still possess and be hurt or JA inductive promoter activity.Wherein, described stringent hybridization condition, comprise: be typically at about 2X and (release rare obtaining in by 20X SSC mother liquor to about 10X SSC, this mother liquor comprises the distilled water solution of 3 mol sodium-chlor and 0.3 mol Trisodium Citrate, pH 7.0), about 2.5X extremely about 5X Denhardt ' s solution (releases rare obtaining by the 50X mother liquor, this mother liquor comprises 1% (w/v) bovine serum albumin, and 1% (w/v) ficoll and 1% (w/v) polyvinylpyrrolidone are in distilled water), about 10 mg/ml to about 100 mg/ml fish sperm DNAs and about 0.02% (w/v) to about 0.1% (w/v) SDS in about 50 ℃ to about 70 ℃ of incubation a few hours to spending the night to finish nucleic acid hybridization.High rigorous condition optimization is in 55 ℃ of incubation a few hours among 6X SSC, 5X Denhardt ' s solution, 100 mg/ml fish sperm DNAs and 0.1% (w/v) SDS.

The invention provides a kind of recombinant expression vector that comprises promotor of the present invention, under the control of described promotor, this carrier can contain other structure gene, as an example, preferred as anti insect gene such as Bt protein gene, disease-resistant gene such as chitinase gene etc.Above-mentioned recombinant vectors can import in the proper host cell.Choosing proper host cell is well known to a person skilled in the art.For example, described host cell comprises eucaryon or prokaryotic cell prokaryocyte.For prokaryotic cell prokaryocyte, be preferably intestinal bacteria; For eukaryotic cell, be preferably yeast cell, vegetable cell etc.Wherein, make up recombinant expression vector and import to host cell and can utilize the conventional method that adopts in this area to finish.

The present invention further provides a kind of method that transforms plant that produces, comprising:

(a) provide above-mentioned recombinant expression vector;

(b) transform plant with described recombinant expression vector, obtain transformed plant, and the conversion plant that obtains can the described foreign gene of abduction delivering when being subjected to insect pest.

Thereby with recombinant expressed importing host plant body of the present invention, preferred plant materials is a dicotyledons, can obtain useful conversion plant by aforesaid method.Because promotor of the present invention belongs to inducible promoter, when foreign gene is anti insect gene, when being subjected to insect bite and hindering, plant can express described anti insect gene to obtain resistance to insect pest when transforming.

The present invention also provide a kind of in plant the method for expression alien gene, comprising:

(a) provide above-mentioned recombinant expression vector;

(b) transform plant with described recombinant expression vector, obtain transformed plant;

(c) cultivate described conversion plant;

(d) with injury or the described conversion of methyl jasmonate treatment plant, with the foreign gene of abduction delivering importing.

For the technology that recombinant expression vector is imported Plant Genome well-known in the art.For example, use and to comprise agriculture bacillus mediated conversion, will import the genome of target vegetable cell according to recombinant expression vector of the present invention effectively with the whole bag of tricks of protoplastis DNA conversion, vacuum infiltration and other mechanical DNA transfer techniques of the tungsten of DNA parcel or gold grain biolistic bombardment, electroporation or polyoxyethylene glycol (PEG) mediation.The transgenic plant cells that contains promotor of the present invention can be bred in suitable condition.And can use currently known methods and condition to be prepared into the reproductive material of complete plant or plant from original transgenic cell.

Brief description of drawings

Figure 1B jC-p promotor total length (the 92nd to 1378 nucleotide sequence among the SEQ ID NO:1, carrier pBICHP0) drives gus gene and slanders the GUS histochemical stain result who expresses before and after the harm reason at tobacco leaf.

Illustrate: A: as the wild-type tobacco K326 blade of negative control, a left side is injury 0 hour, and right is the sample that injury was got in 3 hours; B: transform the tobacco leaf of pBICHP0, a left side is injury 0 hour, the right sample C that got in 3 hours for injury: as the tobacco leaf of the conversion pBI121 of positive control, a left side is injury 0 hour, the right sample of getting in 3 hours for injury.

The truncated segment of Fig. 2 BjC-p promotor different lengths drives gus gene before and after tobacco leaf is slandered the harm reason, the tissue staining of GUS expression activity and fluorometric analysis result.

Illustrate: top is divided into GUS tissue staining result, A, and the tobacco leaf of conversion pBICHP1, a left side is injury 0 hour, right is the sample that injury was got in 3 hours; B, the tobacco leaf of conversion pBICHP2, a left side is injury 0 hour, right is the sample that injury was got in 3 hours; C, the tobacco leaf of conversion pBICHP3, a left side is injury 0 hour, right is the sample that injury was got in 3 hours.The bottom is divided into GUS active fluoro analytical results, and the longitudinal axis is the GUS expression amount, and transverse axis is the carrier name, and Wt is the wild-type tobacco K326 as negative control.

Embodiment

Conventional experimental methods of molecular biology can be referring to molecular cloning: experiment guide, and J.Sambrook waits and writes, second edition, cold spring harbor laboratory publishes, cold spring port, N.Y., 1989.Reagent and other material are from commercial source or as particularly pointing out.

Embodiment 1 by joint PCR from the leaf mustard gene element from BjCHI1 gene promoter (BjC-p)

1.1 the preparation of leaf mustard genomic dna

Plant in indoor one month leaf mustard (Brassica juncea), get about 3 grams of tender leaf, method (McCouch S.P. with reference to McCouch etc., et al., Molecular mapping of rice chromosome, Theor Appl Genet, 1988,76:815-829) adopt the SDS method to extract genomic dna.The leaf mustard genomic dna that is extracted detects through agarose gel electrophoresis, and ultraviolet spectrophotometer is measured after the concentration in-20 ℃ of preservations.

1.2 the enzyme of leaf mustard genomic dna is cut and is connected with joint

Method (Siebert P.D. with reference to people such as Siebert, et al., An improved PCR method for walking inuncloned genomic DNA, Nucleic Acids Research, 1995,23 (6): 1087-1088), get 2 microgram leaf mustard genomic dnas, cut fully with restriction enzyme EcoRV, Dra I and Sma I respectively, 20 microlitre enzymes are cut and are added each 60U of above-mentioned enzyme in the system respectively, and 37 ℃ are incubated 9 hours.Enzyme is cut product through the extracting of isopyknic phenol chloroform once, 70% ethanol sedimentation, and 20 microlitre TE damping fluids redissolve.Enzyme behind the purifying is cut product and is connected with joint, joint long-chain sequence: 5 '-CTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CCG CCC GGG CAG GT-3 ' (SEQID NO:2), joint short chain sequence: 5 '-ACC TGC CC-NH ₂-3 ' (SEQ ID NO:3).Linked system: contain joint (25 micromoles per liter) 4 microlitres in the 20 microlitre systems, the enzyme of purifying is cut product 8 microlitres, and 10 times connect damping fluid 2 microlitres, T4 ligase enzyme (3U/ μ L) 2 microlitres, ultrapure water 2 microlitres.16 ℃ connect 12 hours.

1.3PCR process

According to joint sequence design upstream primer (joint primer) AP1 and AP2, sequence is respectively: AP1:5 '-GGA TCCTAA TAC GAC TCA CTA TAG GGC-3 ' (SEQ ID NO:4), AP2:5 '-AAT AGG GCT CGAGCG GC-3 ' (SEQ ID NO:5); According to BjCHI1 gene cDNA sequence design downstream primer (gene specific primer) GSP1 and GSP2, sequence is respectively: GSP1:5 '-ACA CTG CGG GGT TGG AGG A-3 ' (SEQ IDNO:6), GSP2:5 '-GCA TAG ACC GTT GGG GCA G-3 ' (SEQ ID NO:7).Cutting the connection product with leaf mustard genomic dna enzyme is template, is that primer carries out first round PCR with AP1 and GSP1, and the PCR program is: 94 ℃, and 4 minutes; 94 ℃, 45 seconds; 62 ℃, 2 minutes; 72 ℃, 3 minutes; 35 circulations; 72 ℃, 15 minutes.With first round PCR product is template, is primer with AP2 and GSP2, carry out second and take turns PCR, and the same first round of PCR program, but only carry out 30 circulations.

1.4 clone and sequential analysis

Sepharose reclaims second and takes turns the PCR product, is connected into cloning vector pGEM-T Easy carrier, imports e. coli jm109, order-checking.Institute's calling sequence carries out the homology comparison with the B1ast instrument on the NCBI website, and compares in twos with the cDNA sequence of BjCHI1 gene, and the new sequence of conclusive evidence gained is the upstream sequence of BjCHI1 gene.After removing 5 ' terminal sequence of BjCHI1 gene cDNA, the upstream sequence length of gained BjCHI1 gene is 1378bp, and its sequence is referring to SEQID NO:1.

The checking of embodiment 2 promoter functions

By promotor is connected with reporter gene, change plant materials afterwards over to, the examining report expression of gene is carried out.

2.1 the structure of recombinant expression vector pBICHP0

PBICHP0 forms with the 35S promoter sequence construct that BjC-p promotor (by the 92nd to 1378 Nucleotide shown in the SEQ ID NO:1) sequence replaces gus gene upstream among the expression vector pBI121.Contain neomycin phosphotransferase gene (Npt II) and as beta-Glucuronidase (GUS) gene of reporter gene as the plant binary expression vector pBI121 of expression vector.Method by PCR obtains BjC-p promoter sequence (by the 92nd to 1378 Nucleotide shown in the SEQ ID NO:1) from the leaf mustard genome, and used PCR primer is right:

KjXeB11：5′-TAA AGC TTT CTC AAA TAT GAG GTT TTA CTA TTC AT-3′(SEQID NO：8)

KjWXF7：5′-TAG GAT CCG TTT CTC TGA GCT GTA TGG TTG-3′(SEQ ID NO：9)

Owing to increased HindIII and BamH I restriction enzyme site at 5 of primer ' end respectively, so pcr amplified fragment is cut with HindIII and BamH I enzyme, with its be connected with the big fragment of pBI121 that BamH I enzyme is cut through HindIII equally, electric shocking method changes bacillus coli DH 5 alpha over to, extract plasmid from DH5 α again, through enzyme cut, the sequence verification sequence errorless after, recombinant plasmid called after pBICHP0, electric shock changes agrobacterium tumefaciens EHA105 over to again.

2.2 agriculture bacillus mediated Plant Transformation

The plant expression vector pBICHP0 that builds changes among the tobacco K326 by agrobacterium-mediated transformation.Tobacco seed steeped 40 seconds with 70% ethanol, and 0.1% mercuric chloride bubble 2 minutes is seeded in the 1/2MS substratum, and 25 ℃, 16 hours illumination/skies.Get the leaf dish that tender leaf is cut into 0.5 * 0.5 centimetre of size after 40 days,, stir with the bacterium liquid immersion of the EHA105 that contains pBICHP0, take out after 5 minutes, aseptic filter paper blots, and places on the common substratum (additional 1 mg/litre 6-BA of MS substratum and 0.1 mg/litre NAA), 25 ℃, secretly cultivated 60 hours.Contain the preparation of the EHA105 bacterium liquid of pBICHP0: (every liter contains 10 gram Tryptoness at the LB substratum that adds 50 mg/litre Kan and 25 mg/litre Rif, 5 gram yeast extracts, 5 gram sodium-chlor) go up a stroke single bacterium colony, the fresh single bacterium colony of picking shakes bacterium in the LB substratum of 2 milliliters of additional 50 mg/litre Kan and 25 mg/litre Rif, 28 ℃, 200 rev/mins were shaken 12 hours, take out 100 microlitres again and in the LB substratum of 100 milliliters of additional 50 mg/litre Kan and 25 mg/litre Rif, shake bacterium, 28 ℃, 200 rev/mins are shaken to OD ₆₀₀Value is 0.8,3000g, and 15 minutes centrifugal collection thalline are diluted to OD with the MS liquid nutrient medium ₆₀₀Value is 0.8.After dark cultivation finishes, the leaf dish is earlier with aseptic washing 15 minutes, again with the aseptic washing that contains 500 mg/litre Car 15 minutes, washed 20 minutes with the liquid MS that contains 500 mg/litre Car at last, afterwards the leaf dish is moved into (the additional 100 mg/litre Kan of MS substratum and 500 mg/litre Car and 1 mg/litre 6-BA and 0.1 mg/litre NAA) on the bud inducing culture, 25 ℃, 16 hours illumination/skies, every fortnight change fresh culture one time.Deng bud length is about 1 centimetre, and bud is downcut, and moves into root media (the additional 100 mg/litre Kan of MS substratum and 250 mg/litre Car and 0.1 mg/litre IAA), and 25 ℃, 16 hours illumination/skies, every fortnight change fresh culture one time.When being about two centimetres, seedling being moved in the native medium temperature chamber of sterilization and cultivate, until the results seed Deng the length of root.

2.3pBICHP0 the GUS expression activity of the transfer-gen plant that transforms is analyzed

The GUS histochemical stain

Get transgene tobacco T1 and carry out the GUS histochemical stain for blade.Method, when treating that the cigarette seedling grows 5 true leaves, injure processing on the 3rd true leaf: with middle vein is boundary, cuts half as 0 hour sample of injury reason, and simultaneously, second half uses the pinprick hole, and sampling after 3 hours is as 3 hours sample of injury reason.Blade is cut into about 3 millimeters * 3 millimeters fragment, places on ice and soaked 30 minutes, use the phosphoric acid salt of 1 mol (to contain NaH in 200 milliliters of ultrapure waters afterwards with 90% acetone ₂PO ₄7.78 gram, Na ₂HPO ₄51.624 gram, pH 7.2) wash twice, each 15 minutes; (containing final concentration is Na to add the X-Gluc reaction solution afterwards ₂HPO ₄, 50 mmoles/liter; NaH ₂PO ₄, 50 mmoles/liter; EDTA, 1 mmole/liter; Triton-X100,0.4%; K ₄Fe (CN) ₆, 1 mmole/liter; K ₃Fe (CN) ₆, 1 mmole/liter; X-Gluc, 0.5 mg/litre) 37 ℃ of reactions are 24 hours; With the reaction solution sucking-off, use 70% ethanol decolorization, visual inspection and photomicrography.

The result as shown in Figure 1, the active strong very dark blueness that demonstrates of GUS, no blueness then represents not have the GUS activity or the GUS activity is very low.Transform pBICHP0 and can dye very blue color, then do not have blueness as the wild-type tobacco blade of negative control as the tobacco leaf that positive control transforms pBI121.Explanation has very strong injury inductive promoter activity by sequence shown in the 92nd to 1378 Nucleotide among the SEQ ID NO:1.

The promoter activity analysis of embodiment 3 brachymemmas

The investigator thinks and has the active region in the above-mentioned total length promotor, therefore to having carried out brachymemma by the total length promotor shown in the SEQ ID NO:1 (the 92nd to 1378 Nucleotide shown in the SEQ ID NO:1), obtain corresponding brachymemma promotor, to analyze their activity.

3.1 the structure of recombinant expression vector

With the pBICHP0 plasmid is template, uses corresponding primer to obtain the promoter sequence of three brachymemmas to carrying out pcr amplification, makes up following expression vector:

1) the 302nd to 1378 Nucleotide shown in the SEQ ID NO:1 is obtained by primer KjXeB1 and KjWXF7 amplification as promotor, and the carrier of structure claims pBICHP1;

2) the 456th to 1378 Nucleotide shown in the SEQ ID NO:1 is obtained by primer KjXeB2 and KjWXF7 amplification as promotor, and the carrier of structure claims pBICHP2;

3) the 556th to 1378 Nucleotide shown in the SEQ ID NO:1 is obtained by primer KjXeB3 and KjWXF7 amplification as promotor, and the carrier of structure claims pBICHP3;

KjXeB1：5′-TAA AGC TTC CCA TCC AAA AGAGTC CAA A-3′(SEQ ID NO：10)；

KjXeB2：5′-TAA AGC TTT TTT CGC CAA AAC CAT AAA ATA-3′(SEQ ID NO：11)；

KjXeB3：5′-TAA AGC TTC AAG CGA ACC CAT CCT ATT G-3′(SEQ ID NO：12)；

KjWXF7：5′-TAG GAT CCG TTT CTC TGA GCT GTA TGG TTG-3′(SEQ ID NO：9)

Specific operation process can be with reference to embodiment 2.

3.2 agriculture bacillus mediated Plant Transformation

Specific operation process can be with reference to embodiment 2.

3.3GUS activation analysis

The GUS histochemical stain

Sampling: the T1 that gets transgene tobacco plants in soil for seed, cultivates in the greenhouse, and 28 ℃, 16 hours illumination/skies.When treating that the cigarette seedling grows 5 true leaves, injure processing on the 3rd true leaf: with middle vein is boundary, cuts half as 0 hour sample of injury reason, simultaneously, second half uses the pinprick hole, sampling after 3 hours, as 3 hours sample of injury reason, carry out the GUS histochemical stain.Concrete dyeing process is with embodiment 2.

The result is shown in Fig. 2 top, for the tobacco plant that above-mentioned three recombinant expression vectors transform, after injury was handled 3 hours, blue obviously eager to excel in whatever one does than 0 hour illustrated that the promoter fragment that comprises among pBICHP1, pBICHP2, the pBICHP3 all has the injury inducibility.

The GUS active fluoro is analyzed

The T1 that gets transgene tobacco plants in soil for seed, cultivates in the greenhouse, and 28 ℃, 16 hours illumination/skies.When treating that the cigarette seedling grows 5 true leaves, injure processing on the 3rd true leaf: with middle vein is boundary, cuts half as 0 hour sample of injury reason, and second half uses the pinprick hole, and sampling after 3 hours is as 3 hours sample of injury reason.The sample retention of taking off is in-70 ℃ of refrigerators.Tobacco sample is grind into powder under liquid nitrogen, changes 1.5 milliliters of centrifuge tubes over to and places on ice, and (extraction buffer is formed: 50 mmoles/rise NaH to add the per 100 milligrams of samples of 400 microlitre extraction buffers ₂PO ₄, 10 mmoles/rise EDTA, 0.1%TritonX-100,0.1% sarcosyl, 10 mmoles/rise beta-mercaptoethanol), be ground into slurry with pestle, afterwards 4000 rpms centrifugal 10 minutes, get supernatant ,-70 ℃ of preservations.Get 10 microlitre supernatant liquors and be used to measure the GUS activity.The reaction system of 200 microlitres, 190 microlitre reaction solutions (contain final concentration in the extraction buffer and be 1 mmole/liter 4-MUG) in add 10 microlitre supernatant liquors, mixing is placed in 37 ℃ the water-bath, takes out 20 microlitres in therefrom per 10 minutes and with the yellow soda ash termination reaction of 180 microlitres, 0.2 mol.On 96 microwell plates, measure, use the multi-functional microplate reader of GENios, excitation wavelength 340 nanometers, absorb light wavelength 465 nanometers.Make standard substance production standard curve with 4-MUP.The sample protein Determination on content is used the Bradford method, with BSA production standard curve, surveys the OD value of its 595 nanometers behind the adding Xylene Brilliant Cyanine G R-250 mixing.The GUS activity is represented with picomole 4-MU/ branch/milligram protein.

The result is shown in table 1 or Fig. 2 lower section:

Table 1 transgene tobacco GUS activation analysis

Carrier	GUS activity (unit: picomole 4-MU/ minute/milligram protein)
			0 hour	3 hours
	pBICHP1	7192			23001
pBICHP2			11333	22115
	pBICHP3	6631			11525
Wt			6999	7513

Annotate: pBICHP1, pBICHP2, pBICHP3 represent it is with carrier pBICHP1, pBICHP2, pBICHP3, transformation of tobacco gained; The transfer-gen plant of being analyzed all is its T1 generation, and data are the mean values of three different T1 for plant; Wt is the wild-type tobacco K326 as negative control.

The result of table 1 and Fig. 2 shows, recombinant expression vector pBICHP1, pBICHP2 and pBICHP3 transformation of tobacco plant all show injury abduction delivering activity in the fluorometric analysis.

Art technology people will understand under the situation that does not deviate from wide limits of the present invention and scope, can carry out many variations to above-mentioned illustrational promotor and the construct that contains promotor.

Sequence table

＜110〉Chinese Academy of Agricultural Sciences's crop investigations institute

＜120〉inducible promoter of separated from leaf mustard

<130>

<140>

<141>

<160>

<170>PatentIn Ver.2.1

<210>1

<211>1387

<212>DNA

＜213〉leaf mustard (Brassica juncea)

<400>1

ATTAATATAA TTATATGTAA GTTACTAAAT CATAAAAGCT AAAATAAAAA ATAAATAGCA 60

TTTTAAAATT TTGAATAACA GAATAATAGA ATCTCAAATA TGAGGTTTTA CTATTCATAC 120

CTTTAAAATT AGAAGGTTTA AAAATCTGTT AAAAAGTTAA AATTCTCAAA ATTTAAATGT 180

TTAAAAATCT ATTGAGAATG CTCTAAAAAC AAATATGTTT ATCGTTAACA TAGGAATCAT 240

CCAAATGATC CATCCAGATA AATTTTAAGA TTTTAATTTT AAATAAAATT CATCAAAATA 300

ACCCATCCAA AAGAGTCCAA AATAACAAAT GACAATTTCA CCTTTATTTT AACAAAAATT 360

ATATTCAAAC TAGACAAACA TGATAATTGT GATATAGTTT CTCAACAAGT TTCAAAAACA 420

ATTATAGTTT TCCACCATAA TTGTAAACAC ACATATTTTC GCCAAAACCA TAAAATATAT 480

CTTCTCGCTA AAACAACAAG AAAAACATTT GTCCGTGAAT TCCATCTCTT CTCATAAACT 540

TCATCTTCCA TCGTATCAAG CGAACCCATC CTATTGGGGG GTATGTGATT CATGAGCCTT 600

AATTAGGGTG GTACAGTCAG CGACTCCTGA GCTTCAGAGA GGGTAGTCCA GCAGCTAGTG 660

ACTCTGCTAG AGATAGTGTG GTCCAGCTAG TGAGTAGTGA CTCATGAGGT AGAGAGAGGG 720

TGGTCCATCA TATATCGTGT TAGACAAATT CTATATAGAC AATGAGGTAA ATGTGGGATA 780

TGAAGGTGGT GGCAAATTTT AGAACAATTA GTATTTAAGT ATAGTAAACA TAAAAACAAA 840

CCAGCACTGC TTATCTTCGA TTCTATCAAG CGAACCCACC TTATATTGAA TTAATTTATG 900

GTAGCAGTGA TTCGTGAACG TCAGAGAGGG TGGTCCCTCT AGTGACTCAT GAGCTAGAGA 960

GAGGGTGGTC CAGCTATCGT GTAGAAATAT TCTATAGACC ATTAGATAAA CGTGCCAAAT 1020

GAACTTGGTG GTGACAAAAT AGAAGTTTAG AATGTTTGGG TTCCCGGTAA AAAGGTGAAA 1080

CCACCTTTTT TTTTTTTTTT TTTTACCAAC GTTGAATTGG CCTAATGATA AAAGGGTTAC 1140

GCTTGTAACT ACCGCCACCC AGGTTTAATT CACGGTGGGA GAGACTATTT ATAATGCTTA 1200

GGTTTCTGGC AAAGAAGTGA AACCACTTTT TTTCTTTCCA GAAAAAATAG AAGTCTAGAA 1260

AACATAAAAA CAAGTATATA AAATTCGATG TGATTTAACA CTAACTGATT TTGTACATTG 1320

CATGTTGCTA TATATACCCT TACTTGCCTC TTAAAACAAC CATACAGCTC AGAGAAAC 1378

<210>2

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>2

CTAATACGAC TCACTATAGG GCTCGAGCGG CCGCCCGGGC AGGT 44

<210>3

<211>8

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>3

ACCTGCCC-NH ₂ 8

<210>4

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>4

GGATCCTAAT ACGACTCACT ATAGGGC 27

<210>5

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>5

AATAGGGCTC GAGCGGC 17

<210>6

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>6

ACACTGCGGG GTTGGAGGA 19

<210>7

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>7

GCATAGACCG TTGGGGCAG 19

<210>8

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>8

TAAAGCTTTC TCAAATATGA GGTTTTACTA TTCAT 35

<210>9

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>9

TAGGATCCGT TTCTCTGAGC TGTATGGTTG 30

<210>10

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>10

TAAAGCTTCC CATCCAAAAG AGTCCAAA 28

<210>11

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>11

TAAAGCTTTT TTCGCCAAAA CCATAAAATA 30

<210>12

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer

<400>12

TAAAGCTTCA AGCGAACCCA TCCTATTG 28

Claims (9)

1, a kind of isolating promotor comprises:

1) DNA of sequence or its complementary sequence shown in the SEQ ID NO:1;

2) DNA of sequence shown in the 92nd to 1378 Nucleotide or its complementary sequence among the SEQ ID NO:1;

3) DNA of sequence shown in the 302nd to 1378 Nucleotide or its complementary sequence among the SEQ ID NO:1;

4) DNA of sequence shown in the 456th to 1378 Nucleotide or its complementary sequence among the SEQ ID NO:1;

5) DNA of sequence shown in the 556th to 1378 Nucleotide or its complementary sequence among the SEQ ID NO:1; Or

6) under stringent condition with 1) or 2) or 3) or 4) or 5) the DNA isolation of DNA hybridization, and still possess promoter activity.

2, promotor according to claim 1 is characterized in that its separated from leaf mustard.

3, a kind of recombinant expression vector is characterized in that: contain the described promotor of claim 1.

4, recombinant expression vector according to claim 3 is characterized in that: also comprise the foreign gene that can be operatively connected with described promotor, described expression of exogenous gene is subjected to the control of described promotor.

5, recombinant expression vector according to claim 4 is characterized in that: described foreign gene is anti insect gene or disease-resistant gene.

6, the host cell that contains each described recombinant expression vector of claim 3-5.

7, the described host cell of claim 6, it is intestinal bacteria.

8, a kind of method that transforms plant that produces comprises:

(a) provide recombinant expression vector according to claim 4;

(b) transform plant with described recombinant expression vector, obtain transformed plant, and the transformed plant that obtains is when being subjected to insect pest, described promotor is induced activation, starts described expression of exogenous gene.

9, a kind of in plant the method for expression alien gene, comprising:

(a) provide recombinant expression vector according to claim 4;

(b) transform plant with described recombinant expression vector, obtain transformed plant;

(c) cultivate described conversion plant;

(d) with injury or the described conversion of methyl jasmonate treatment plant, with the foreign gene of abduction delivering importing.