CN100415887C - Cotton tissue specific and pathogenic bacterium inducing promoter and its use - Google Patents

Abstract

The present invention relates to cotton tissue specific and pathogenic bacterium inducing promoter and their application, and belongs to the field of biotechnology. The promoter contains CAAT-box, TATA-box, ethylene, methyl jasmonate, abscisic acid response element, pathogenic bacterium inducer response elements W boxes, GT-1, MYB, MYBST1, MYB1LEPR, etc. There are also specific root expressing elements to enhance the expression of GUS gene, which expresses specifically in the phloem of root, bud and stem, the vein of foliage and the guard cell of air pore. Paraffin section observation shows that GHNBS promoter expresses in phloem companion cell. After treatment with SA, MeJA, Ethylene, pathogenic bacteria of blight and pseudomonas syringae DC3000, GUS will have obviously raised activity, so that it is one organ specific and pathogenic bacterium relevant promoter.

Description

Cotton tissue specific and pathogenic bacterium inducing promotor and application thereof

One, technical field

The present invention relates to plant gene promoter and application thereof, a cotton tissue specific and pathogenic bacterium inducing promotor are provided.Belong to biological technical field.Be used for improving disease resistance of plant and other useful production traitss by plant gene engineering technology.

Two, background technology

The plant disease that is caused by fungi, bacterium, virus and nematode is that crop production has been brought tremendous influence.Plant has the different resistance mechanism of two covers: the one, and Passive Defence mechanism, be meant the morphological structure that some resistances are relevant, as epidermis and hard cell walls (Brady J.D., Fry S.Formation of di-isodityrosine and loss of isodityrosinein the cell walls of tomato cell-suspension cultures treated with fungal elicitors or H2O2.PlantPhysiol 1997,115:87-92.).Another one is initiatively defence, and initiatively defence is that resistant gene is discerned with pathogenic bacteria and done caused mutually.The exciton of pathogen nontoxic gene (avr) coding acts on the generation disease resistance response mutually with the acceptor molecule of the corresponding disease-resistant gene of host plant (R) coding exciton.The plant disease-resistant defense response generally experiences signal identification, signal conduction, three links of defence genetic expression.Initiatively all types of disease-resistant mechanism all will rely on defence genetic expression in the defence, and the validity of opposing depends on the space-time characterisation of genetic expression.Just because of the spatial and temporal expression characteristic of defence gene, its promotor often has some special cis-acting elements, and people can utilize defence gene, especially its cis element to provide favourable instrument for the genetically engineered improvement.Most disease-resistant genes are all encoded and are had nucleotide binding site and leucine multiple albumen (NBS-LRR), (McHale L, Tan X in the plant, Koehl P, MichelmoreRW.Plant NBS-LRR proteins:adaptable guards, Genome Biol.2006,26; 7 (4): 212).But the promotor research for this genoid is also fewer.

Plant gene promoter is important cis-acting elements, is the dna sequence dna that is positioned at structure gene 5 ' end upstream, is the center of transcriptional control.Since the strain transgenic plant were come out from nineteen eighty-three first, promotor was engineered research focus always, selects the promotor of suitable and effective expression to establish solid basis for engineered research.The organizing specific promotor can make expression of exogenous gene only occur in some specific organ or tissue position, and often shows the characteristic of growing adjusting; Inducible promoter can make foreign gene that some signal is produced response, only expresses under distinctive signal stimulates.The great advantage of these promotors is: it has overcome, and foreign gene that constitutive promoter starts is nonspecific in recipient plant to continue, efficiently expresses the waste that is caused, and satisfies the demand of some needs foreign gene specifically expressing.Tissue specificity or abduction delivering promotor all are the emphasis and the difficult points of plant genetic engineering research all the time.

At present, find some tissue specificities or abduction delivering promotor, and found the corresponding cis factor of existence on it.For promotor research is afterwards laid a good foundation.The Sar8.2b gene of tobacco is induced by Whitfield's ointment (SA), has found several cis acting factors on its promotor, comprises the as-1 factor, GT21 and Dof binding sequence.SA induces Sar812b genetic expression may be present in (Song F.Goodman R M.Cloning and identification of the promoter of the tobacco Sar8.2bgene.a gene involved in systemic acquired resistance.Gene in the cis factor in-728 to-927bp and-197 to-351bp zone, 2002,290 (122): 115～124.).1 of corn sucrose synthase is expressed (Yang N S in the phloem cell, Russell D.Maize sucrosesynthase-1 promoter directs phloem cell specific expression of Gus gene in transgenic tobaccoplants.Proc Natl Acad Sci 1990,87 (11): 4144～4148.).The PsGNS2 promotor is specifically expressing (Buchner P in seed only, Rochat C, Wuilleme S, Boutin J P.Characterization of a tissue-specific anddevelopmentally regulated beta-1,3-glucanase gene in pea (Pisum sativum) .Plant Mol Biol2002,49 (2): 171～186.).

Cotton is the important cash crop in the world, and the fungal disease of cotton, Micobial Disease and nematode all can cause output to reduce and quality descends.The clone of cotton disease resistance gene and the separation of promotor can make us understand the mutual work of host and pathogenic bacteria better and lay the foundation for genetically engineered improves.So far, the separation of the promotor of cotton disease resistance gene and functional study yet there are no report.The present inventor had before passed through according to the proteic conserved domain of NBS-LRR, by the design degenerated primer, (the GENBANK number of landing is: DQ785169), and then obtained the promoter sequence of this gene to have obtained the NBS-LRR gene order of cotton from normal anti-cotton.Because the difficulty of cotton regenerated system, some research groups adopt model plant tobacco (Hsu CY, Creech RG, Jenkins JN, Ma DP.Analysis of promoteractivity of cotton lipid transfer protein gene LTP6 in transgenic tobacco plants.Plant Sci1999; 143:63-70.) or Arabidopis thaliana (Wang S, Wang J W, Yu N, Li Ch H, Luo B, Gou J Y, Wang L J, Chen X Y.Control of Plant Trichome Development by a Cotton Fiber MYB Gene.The PlantCell 2004 16:2323-2334) comes the cotton promotor is analyzed, its result of study show gus reporter gene fully can be in these model plants normal expression.

Three, summary of the invention

Technical problem

The objective of the invention is: a cotton tissue specific and pathogenic bacterium inducing promotor are provided, this promotor can be located to express the phloem companion cell cell of root, flower bud, stem, the vein of blade and the guard cell of pore, can also be activated by cotton wilt pathogenic bacteria Fusarium oxysporum Schl.f.sp.vasinfectum, pseudomonas syringae DC3000, simultaneously, also can be activated by Whitfield's ointment, ethene, methyl jasmonate treatment.Can utilize promotor of the present invention to be built into various plant expression vectors, be applied to the Agricultural biotechnologies breeding to improve crop disease-resistant, pest-resistant proterties and resisting abiotic adverse circumstance.

Technical scheme

A cotton tissue specific provided by the present invention and pathogenic bacterium inducing promotor pGHNBS derive from upland cotton (Gossypium hirsutum), are one of following nucleotide sequence:

1) dna sequence dna shown in the SEQ ID NO.1 or part dna sequence dna in the sequence table; The nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with SEQ ID NO.1 in the sequence table.

The rigorous condition of described height is at 0.1 * SSPE (15mM NaCl, 1mM NaH ₂PO ₄, 0.1mM EDTA), in the solution of 0.1 * SSC (15mM NaCl, 1.5mM Trisodium Citrate), 0.1%SDS (sodium laurylsulfonate), wash film under 65 ℃ of conditions.

SEQ ID NO.1 in the sequence table is by 1600 based compositions.From 5 ' end the 1532nd bit base is transcription initiation site, be designated as+1.

Wherein the TATA frame is positioned at transcription initiation site upstream-39 to-34 bit bases, and CAAT box is positioned at-136 to-139 bit bases, these elements are basic promoter element in transcribing.

From the-1460 to-1455 at 5 ' end ,-1360 to-1355 bit bases are Induced by Salicylic Acid or photoinduction element ASF1; From the-1424 to-1419 at 5 ' end ,-566 to-561 ,-550 to-545 bit bases are the different Expression element OSE1ROOTNODULE of Gent; From the-1052 at 5 ' end ,-767 ,-441 ,-420 ,-186 ,-121 ,-1149 ,-875 ,-349 ,-1200 ,-881 ,-469 ,-333 ,-100 bit bases are Whitfield's ointment or photoinduction element GT1CONSENSUS; From the-1168 at 5 ' end ,-920 ,-860 ,-531 bit bases are enhancer element EECCRCAH1; From the-1052 at 5 ' end ,-767 ,-100 bit bases are pathogenic bacterium inducing Expression element GT-1; From 5 ' end, the-1065 bit bases is disease-resistant gene Expression element MYB1LEPR; From the-1503 at 5 ' end ,-1160 ,-1144 ,-926 ,-905 ,-672 ,-238 is ABA abduction delivering element; From the-1248 at 5 ' end ,-181 ,-919 ,-530 bit bases are sugared response element WBOXHVISO1; From the-612 at 5 ' end ,-182 ,-1465 ,-1359 ,-918 ,-234 bit bases are Induced by Salicylic Acid element WBOXATNPR1.The The sequencing results of GHNBS promotor pGHNBS shows that the GHNBS gene is subjected to the regulation and control of complicated factor in cotton resistance and stress-inducing process.

The invention provides the expression vector and host bacterium and the arbitrary segmental primer of amplification promotor that contain promotor of the present invention.

Cotton tissue specific of the present invention and pathogenic bacterium inducing promotor pGHNBS, Arabidopis thaliana transgenosis proof is found that pGHNBS can give among the guard cell of the vein of the phloem of gus gene at roots of plants, flower bud, stem, blade and pore and is efficiently expressed, show organ specifically expressing pattern, further pGHNBS promotor specifically expressing in the companion cell cell of phloem is confirmed in research.And transfer-gen plant is subjected to SA, and MeJA, Ethylene, wilt Fusarium oxysporum Schl.f.sp.vasinfectum and pseudomonas syringae DC3000 induce back GUS activity that remarkable rising is all arranged, and illustrate that pGHNBS contains the corresponding responsing reaction factor.The expression characteristic of pGHNBS in model plant shows that it is a promotor that organ is special and cause of disease is relevant.The functional study of pGHNBS can be the expression regulation mechanism and the concrete function that disclose GHNBS and lays the first stone, and also can be applicable in the genetically engineered improvement of cotton disease resistance, anti insect gene engineering and resistance.

Beneficial effect

1. the present invention has obtained a brand-new cotton pathogenic bacterium inducing promotor.Cotton is the important cash crop in the world, and the fungal disease of cotton, Micobial Disease and nematode all can cause output to reduce and quality descends.The clone of cotton disease resistance gene and the separation of promotor can make us understand the mutual work of host and pathogenic bacteria better and lay the foundation for genetically engineered improves.But up to the present, also do not isolate the promotor of any cotton disease resistance gene.The promotor of the GHNBS gene that the present invention obtains is a brand-new promotor, and BLAST shows does not have homology.And by the arabidopsis thaliana transformation analysis, find this promotor be subjected to pathogenic bacteria and SA etc. induce the back expression amount significantly raise, illustrate that this promotor is a pathogenic bacterium inducing promotor.

2. the present invention has obtained a cotton pathogenic bacteria related tissue specific expression promoter first.Since the strain transgenic plant were come out from nineteen eighty-three first, obtain suitable effective promotor was engineered research focus always, selects the promotor of suitable and effective expression to establish solid basis for engineered research.The organizing specific promotor can make expression of exogenous gene only occur in some specific organ or tissue position, and often shows the characteristic of growing adjusting.That use is maximum in the present crop transgene improvement is the promotor p35S that derives from cauliflower mosaic virus, though this class promotor can make goal gene efficiently express, but the foreign gene that they start is nonspecific continuing in recipient plant, efficiently express, not only cause waste, and might produce counter productive (Song P, Heinen JL., BurnsTH., Allen RD.Expression of Two Tissue-Specific Promoters in Transgenic Cotton Plants.The Journal of Cotton Science 2000; 4:217-223).And the promotor of specifically expressing or abduction delivering can overcome these defectives, and satisfies the demand of some needs foreign gene specifically expressing.Promotor pGHNBS among the present invention is the cotton promotor of an organizing specific expression.Organizing specific expression and pathogenic bacterium inducing promotor can be applied to agro-ecology breeding and genetically engineered to improve the improvement of crop disease-resistant, pest-resistant proterties and other production shapes.

3. express effectively.Have 3 and strengthen element on promoter sequence, they may strengthen the expression of goal gene.GUS dyeing and determination of activity also result show that the expression amount of the goal gene that this promotor starts is higher, can satisfy the requirement of genetically engineered improvement crop character.

4. better understand the mechanism of action of disease-resistant gene and be used for breeding for disease resistance.Though there are some disease-resistant genes to be cloned out at present, the understanding of antagonism mechanism also is not very deep.Disease-resistant gene generally is subjected to extraneous regulating and expressing, and the plant gene regulation and control are mainly carried out on transcriptional level, are subjected to the mutual coordinative role of multiple cis-acting elements and trans-acting factor.The promotor of plant gene is important cis-acting elements, can instruct holoenzyme to combine with the correct of masterplate, the activation RNA polymerase, and make it to have the form of initial specific transcriptional, and decision direction and the efficient and the employed RNA polymerase type of transcribing, be the center of transcriptional control.By promotor is analyzed, can understand the expression pattern of disease-resistant gene even relevant trans-acting factor, and then understand the mechanism of action and the mechanism of disease-resistant gene.And the recombinant vectors that makes up can be conveniently used in the crop disease-resistant breeding.

Four, description of drawings

Fig. 1-1559bp～-GUS coloration result behind the 28bp promoter region arabidopsis thaliana transformation plant.A: the GUS coloration result of whole plant under the anatomical lens; B: the GUS coloration result of root under the anatomical lens; C: the GUS coloration result of anatomical lens inferior lobe; D: the cross-sectional view that stem expresses under the anatomical lens; E: the coloration result of microscopically blade; F: the further amplification result of blade, can find out obviously that GUS expresses in the guard cell of pore; G: the GUS coloration result of bud under the anatomical lens.

Fig. 2-1559bp～-the 28bp promoter region changes stem's paraffin cross-sectional view of Arabidopis thaliana plant.Gus gene is mainly expressed in companion cell.

Fig. 3 promotor-1559bp～-GUS determination of activity after the 28bp section changes the Arabidopis thaliana plant and is subjected to pathogenic bacteria etc. and induces.CK is contrast; SA is a Whitfield's ointment; MJ is a methyl jasmonate; Ethylene is an ethene; DC3000 is the bacteria pathogeny bacterium pseudomonas syringae of tomato; Fusarium is the blight pathogenic bacterium disease pathogen bacterium Fusarium oxysporum Schl.f.sp.vasinfectum of cotton.

Five, embodiment

Method therefor is ordinary method if no special instructions in the following embodiment, and the primer sequence is synthetic by Shanghai Bo Ya Bioisystech Co., Ltd, and described percentage composition is the quality percentage composition.Used upland cotton (Gossypium.hirsutum) kind of this experiment be Chang Kangmian (land Hao Sheng, Pu is big new, Huang Fuguohua, Yin Huiyu wears new. high anti-defoliation verticillium new variety-Chang Kangmian. Chinese cotton 1997; 11:26-27.), the Arabidopis thaliana kind is Colombia's type (Lin X, Kaul S, Rounsley S, Shea TP, Benito MI, Town CD, Fujii CY, Mason T, BowmanCL, Barnstead M, Feldblyum TV, Buell CR, Ketchum KA, Lee J, Ronning CM, Koo HL, Moffat KS, Cronin LA, Shen M, Pai G, Van Aken S, Umayam L, Tallon LJ, Gill JE, AdamsMD, Carrera AJ, Creasy TH, Goodman HM, Somerville CR, Copenhaver GP, Preuss D, Nierman WC, White O, Eisen JA, Salzberg SL, Fraser CM, Venter JC.Sequence and analysisof chromosome 2 of the plant Arabidopsis thaliana.Nature 1999; 16; 402 (6763): 761-8.).Cotton and Arabidopis thaliana are all grown in incubator.Intensity of illumination is 130 μ mol photons m ^-2s ^-1, humidity is 65%.

One: clone and the sequential analysis of cotton GHNBS gene promoter pGHNBS

According to a cotton resistance related gene GHNBS with CC-NBS-LRR structural domain (the GENBANK number of landing is: DQ785169) designed 3 reverse primers (5 '-AATTGCAGCTCATCCTGAAGTGATTG-3 '; 5 '-CCACGAATCAACTGAACTTGTTTTGC-3 '; 5 '-CGGTTATTATAGATACAATGGCTTCC-3 ') be used for obtaining 5 ' end of gene.Method (Paterson AH with reference to Paterson etc., Brubaker CL, Wendel JF.A rapid method for extraction cotton (Gossypium spp.) Genomic DNA suitable for RFLP or PCR analysis.Plant Mol Biol Rep1993; 11:122-7.) extract the nuclear gene group DNA of Chang Kangmian blade, with restriction enzyme HindIII, DraI the nuclear gene group DNA that is extracted is carried out enzymolysis respectively.Clone the promoter sequence of cotton resistance related gene GHNBS then with chromosome walking method.Adopt the GenomeWalker of clontech company ^TMTest kit is also operated by the test kit specification sheets, and concrete grammar is: at first the dna fragmentation with the above-mentioned different lengths that produces behind different restriction enzyme enzymolysis connects corresponding separately joint, obtains having the genome dna library of joint; Then with the genome dna library that contains joint as template, under the guiding of lateral joint primer AP1:5 '-GTAATACGACTCACTATAGGGC-3 ' and outside gene specific primer GSP1:5 '-AATTGCAGCTCATCCTGAAGTGATTG-3 ', carry out first round pcr amplification; Be template with first round pcr amplification product again, under the guiding of inner contact primer AP2:5 '-ACTATAGGGCACGCGTGGTC-3 ' and inboard gene specific primer GSP2:5 '-CCACGAATCAACTGAACTTGTTTTGC-3 ', carry out second pcr amplification of taking turns.Second takes turns after pcr amplification finishes, DNA with the special clean company of dimension reclaims test kit recovery and purifying amplified fragments, dna fragmentation with purifying is connected to (Promega company) among the carrier pGEM-T Easy then, Transformed E .coli JM109 (the precious biotech firm in Dalian) competent cell, select positive colony upgrading grain, order-checking is finished (Applied Biosystem Inc., Foster City by the ABI310DNA sequenator, TX, USA).The result is a template with the cotton nuclear gene group dna fragmentation storehouse of DraI enzymolysis, behind the two-wheeled pcr amplification, obtains the dna fragmentation that length is 1.6kb, has the dna sequence dna of SEQ ID NO.1 in the sequence table.Compare with the cDNA sequence of the GHNBS that has cloned, the dna fragmentation of this 1.6kb contains 69 Nucleotide of GHNBS cDNA sequence 5 ' end coding region as a result, promptly overlap in 5 ' end coding region, show that the length of being cloned into is that the dna fragmentation of 1.6kb is exactly the promoter sequence of target gene GHNBS, with this promotor called after pGHNBS.And with the above-mentioned recombinant vectors called after pGEM-T Easy-pGHNBS that contains pGHNBS.First base of the GHNBS cDNA sequence that will be separated to 5 '-RACE method amplification is defined as transcription initiation site, promptly in the sequence table SEQ ID NO.1 from the 575th bit base of 5 ' end.

With PLACE (Higo K, Ugawa Y, Iwamoto M, Korenaga T.Plant cis-acting regulatoryDNA-elements (PLACE) .Nucl Acids Res 1999; 27:297-300.) and PlantCARE (Lescot M, D é hais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouz é P, Rombauts S.PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools in silico analysis ofpromoter sequences.Nucl Acids Res 2002; 30:325-7.) software carries out sequential analysis to the nucleotide sequence (the SEQ ID NO.1 in the sequence table) of the promotor of cotton resistance related gene GHNBS.With above-mentioned software the cis-acting elements among the GHNBS gene promoter pGHNBS is searched for, predicted, contain the homologous sequence of numerous and known Eukaryotic cis element as a result in this promotor.Wherein, first base of the GHNBS cDNA sequence that will be separated to 5 '-RACE method amplification is defined as transcription initiation site (be designated as+1), be in the sequence table SEQ IDNO.1 from 5 ' end the 1532nd bit base, wherein the TATA frame is positioned at transcription initiation site upstream-39 to-34 bit bases, and CAAT box is positioned at-136 to-139 bit bases, these elements are basic promoter element in transcribing.From the-1460 to-1455 at 5 ' end,-1360 to-1355 bit bases are Induced by Salicylic Acid or photoinduction element ASF1 (DespresC, Chubak C, Rochon A, Clark R, Bethune T, Desveaux D, Fobert PR.The Arabidopsis NPR1disease resistance protein is a novel cofactor that confers redox regulation of DNA bindingactivity to the basic domain/leucine zipper transcription factor TGA1.Plant Cell 2003; 15:2181-2191.); From the-1424 to-1419 at 5 ' end,-566 to-561,-550 to-545 bit bases are different Expression element OSE1ROOTNODULE (the Vieweg MF of Gent, Fruhling M, Quandt HJ, Heim U, BaumleinH, Puhler A.Kuster H, Andreas MP.The promoter of the Vicia faba L.leghemoglobin geneVfLb29 is specifically activated in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots from different legume and nonlegume plants.Mol PlantMicrobe Interact 2004; 17:62-69.); From the-1052 at 5 ' end ,-767 ,-441,-420 ,-186 ,-121,-1149 ,-875 ,-349,-1200 ,-881 ,-469,-333,-100 bit bases are Whitfield's ointment or photoinduction element GT1CONSENSUS (Villain P, Mache R, Zhou DX Themechanism of GT element-mediated cell type-specific transcriptional control.J Biol Chem1996; 271:32593-32598); From the-1168 at 5 ' end,-920,-860,-531 bit bases are enhancer element EECCRCAH1 (Kucho K, Yoshioka S, Taniguchi F, Ohyama K, Fukuzawa H.Cis-actingelements and DNA-binding proteins involved in CO2-responsive transcriptional activation ofCah1 encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii.Plant Physiol2003; 133:783-793.); From the-1052 at 5 ' end,-767,-100 bit bases are pathogenic bacterium inducing Expression element GT-1 (Park HC, Kim ML, Kang YH, Jeon JM, Yoo JH, Kim MC, Park CY, Jeong JC, MoonBC, Lee JH, Yoon HW, Lee SH, Chung WS, Lim CO, Lee SY, Hong JC, Cho MJ.Pathogen-and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1 box thatinteracts with a GT-1-like transcription factor.Plant Physiol 2004; 135:2150-2161.); From 5 ' end, the-1065 bit bases is disease-resistant gene Expression element MYB1LEPR (Chakravarthy S, Tuori RP, DAscenzoMD, Fobert PR, Despres C, Martin GB The tomato transcription factor Pti4 regulates defence-related gene expression via GCC box and non-GCC box cis elements Plant Cell 2003; 15:3033-3050); From the-1503 at 5 ' end,-1160 ,-1144 ,-926,-905,-672 ,-238 is ABA abduction delivering element (Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-ShinozakiK.Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activatorsin abscisic acid signaling.Plant Cell 2003; 15:63-78); From the-1248 at 5 ' end,-181 ,-919 ,-530 bit bases are sugared response element WBOXHVISO1 (Sun C., Palmqvist S., Olsson H., Boren M., Ahlandsberg S., Jansson C.A novel WRKY transcription factor, SUSIBA2, participates insugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter, Plant Cell 2003; 15:2076-2092); From the-612 at 5 ' end,-182,-1465,-1359,-918,-234 bit bases are Induced by Salicylic Acid element WBOXATNPR1 (Yu D, Chen C, Chen Z Evidence for animportant role of WRKY DNA binding proteins in the regulation of NPR1 gene expression.Plant Cell 2001; 13:1527-1540).The The sequencing results of GHNBS promotor pGHNBS shows that the GHNBS gene is subjected to the regulation and control of complicated factor in cotton resistance and stress-inducing process.

Controlling element analysis in the table 1GHNBS promoter sequence

Sequence ①	The position	Title and function	Reference
				TGACG	-1460(-)，-1360(-)	ASF1, Induced by Salicylic Acid or photoinduction	Despres C，Chubak C，Rochon A，Clark R，Bethune T，Desveaux D.Fobert PR.The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1.Plant Cell 15：2181-2191(2003)
AAAGAT	-1419(+)，-561(+)，-545(-)	OSEIROOTNODULE, root knot specifically expressing element	Vieweg MF，Fruhling M，Quandt HJ，Heim U，Baumlein H， Puhler A.Kuster H，Andreas MP.The promoter of the Vicia faba L.leghemoglobin gene VfLb29 is specifically activated in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots from different legume and nonlegume plants.Mol Plant Microbe Interact. 17：62-69(2004)
				GGATA	-934(+)，-249(+)-1200(-)，-474 (-)	MYBST1, transcriptional activation	Baranowskij N，Frohbetg C，Prat S，Willmilzer L A novel DNA binding protein with homology to Myb oncoproteins conraining only one repeat can function as a transcriptional activator EMBO J 13：5383-5392(1994)
GRWAA W	-1052(+)，-767(+)，-441(+)，- 420(+)，-186(+)，-121(+)，- 1149(-)，-875(-)，-349(-)，- 1200(-)，-881(-)，-469(-)，-333(- )，-100(-)	GT1CONSENSUS, Induced by Salicylic Acid or photoinduction	Villain P，Mache R，Zhou DX The mechanism of GT element- mediated cell type-specific transcriptional control，J Biol Chem 271：32593-32598(1996)
				GANTTN C	-1168(+)，-920(-)，-860(-)，-531(-)	EECCRCAH1, enhanser	Kucho K，Yoshioka S，Taniguchi F，Ohyama K，Fukuzawa H. Cis-acting elements and DNA-binding proteins involved in CO2- responsive transcriptional activation of Cahl encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol.133：783-793(2003).
GAAAAA	-1052(+)，-767(+)，-100(-)	GT-1motif. pathogenic bacterium inducing	Park HC，Kim ML，Kang YH，Jeon JM，Yoo JH，Kim MC，Park CY，Jeong JC，Moon BC，Lee JH，Yoon HW，Lee SH，Chung WS，Lim CO，Lee SY，Hong JC，Cho MJ，Pathogen-and NaCl- induced expression of the SC aM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor Plant Physiol.135：2150-2161(2004)
				CNGTTR	-1228(+)，-614(+)，-452(+)，- 379(+)，-429(-)，-1282(-)	MYBCORE，	Urao T.Yamaguchi-Shinozaki K，Urao S.Shinozaki K An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence Plant Cell 5：1529-1539(1993)
GTTAGT T	-1065(+)	MYBILEPR, the disease-resistant gene Expression element	Chakravarthy S.Tuori RP，DAscenzo MD，Fobert PR，Despres C.Martin GB The tomato transcription factor Pti4 regulates defence-related gene expression via GCC box and non-GCC box

			cis elements Plant Cell 15：3033-3050(2003)
				CANNTG	-1503(+)，-1160(+)，-1144(+)，-926 (+)，-905(+)，-672(+)，-238(+)	MYCCONSENSUSAT, ABA induces	Ahe H，Crao T.I to T.Seki M.Shinozaki K， Yamaguchi-Shi nozaki K.Arabidopsis AtMYC2(bliLll)and AtMYB2(MYB)function as transcriptional activators in abscisic acid signaling.Plant Cell 15：63-78 (2003)
ATATT	-1314(+)，-1195(+)，-1059(+)，- 1039(+)，-1005(+)，-296(+)，- 1356(-)，-1315(-)，-1006(-)，- 657(-)，-279(-)，-206(-)	ROOTMOTIFTAPOX1, Gent is different	Elmayan T.Tepfcr M Evaluation in tobacco of the organ speeifieity and strength of the rol D promoter，domain A of the 35S promoter and the 35S 2 promoter Transgenic Res 4：388-396(1995
				WAACC A	-1068(-)，-316(-)	MYBIAT, the ABA recognition site	Abe H，Urao T.Ito T.Seki M.Shinozaki K， Yamaguchi-Shinozaki Arahidopsis AtMYC2(bliLll)and AtMYB2(MYB)function as transcriptional activators in abscisic acid signaling Plant Cell 15：63-78 (2003)
TGACT	-1248(+)，-181(+)，-919(-)，-530(-)	WBOXHVISOI, sugared response element	C.Sun，S.Palmqvist，H Olsson.M.Boren，S.Ahlandsberg，C Jansson.A novel WRKY transcription factor，SUS1BA2. participates in sugar sigmaling in barley by binding to the sugar- responsive elements of the isol promoter，Plant Cell 15(2003) 2076-2092
				TTGAC	-612(+)，-182(+)，-1465(-)，-1359(-)，- 918(-)，-234(-)	WBOXATNPR1, Induced by Salicylic Acid	Yu D，Chen C，Chen Z Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression Plant Cell 13：1527-1540(2001)

1. N represents A, C, G or T; R represents A or G; W represents A or T;

Two. make up recombinant vectors and arabidopsis thaliana transformation that GHNBS promoter sequence and gus gene merge

Is being template for detecting promotor with Chang Kangmian DNA, the usefulness forward primer (5 '-CG AAGCTTATACGACACACTTAAAAGGAATG-3 ' band underscore base is a restriction enzyme HindIII recognition site) and reverse primer (5 '-CC GGATCCACTAATTTGCCTTGTTATTTCAGA-3 ', band underscore base is a restriction enzyme BamHI recognition site) the amplification promoter fragment, obtain fragment-1559bp of sequence SEQID NO.1～-the 28bp zone, and on two sections of sequences are added respectively the recognition site of restriction enzyme HindIII and BamHI.After reaction finishes, the PCR product is carried out 1.0% agarose gel electrophoresis to be detected, reclaim and purifying purpose fragment, the resulting PCR product of sequence verification, the PCR product carries out enzyme with HindIII and BamHI to be cut, and is connected through the plant expression vector pBI101.1 of same enzyme double digestion (Clontech company).Upstream 5 ' the end that the promoter fragment of above acquisition is connected β-Portugal's polyacetals acid enzyme (GUS) gene obtains recombinant vectors, and called after pBI-pGHNBS::GUS transforms Agrobacterium LBA4404 with freeze-thaw method with recombinant vectors.With flower dip-dye method (Clough S J, Bent A F.FIoral dip:A Simplified Method for Agrobacterium-Mediated Transformation ofArabidopsis thaliana.Plant J 1998; 16:735-743) arabidopsis thaliana transformation divides individual plant results Arabidopis thaliana seed.All seeds are being contained the enterprising row filter of MS substratum of 40mg/L kantlex, selecting green plant and transplant to nutrition soil and grow.Further detect transfer-gen plant with PCR method.Results transgenic engineering plant seed.

The transgenic engineering plant seed is seeded in the MS substratum that contains the 40mg/L kantlex.Selecting green plant transplants to the nutrition soil growth and is used for promoter function and The Characteristics.The transgenic line plant is carried out histochemical stain to detect the expression position and the feature of gus gene.

Three: active Histochemical localization analysis of GUS and fluorescent quantitative measurement

The active Histochemical localization analytical procedure of GUS is:

Get the pBI-pGHNBS::GUS Arabidopis thaliana transgenosis seedling complete stool that step 2 is grown in the greenhouse, the plant root is cleaned.Fluorescence histochemistry's localization method (Jefferson RA with reference to Jefferson etc., Kavanagh TA, Bevan MW.GUS fusions:p-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J 1987; 6:3901-7.) measure the GUS activity, concrete grammar is: the GUS histochemical stain with 5-bromo-4-chloro-3-indolyl β-Dglucuronic acid (X-Gluc) as substrate.Whole strain Arabidopis thaliana is fully decoloured after the processing with 90% acetone, be immersed in the GUS reaction solution and (contain 50mmol/L sodium phosphate buffer (pH 7.0), 10mmol/L EDTA, the 2mmol/L Tripotassium iron hexacyanide, the 2mmol/L yellow prussiate of potash, 2.0mmol/L X-gluc and 0.2%Triton X-100), 37 ℃ of incubations 1～3 day are until the painted sufficient intensity that reaches.Again with the Arabidopis thaliana plant with 70-100% series ethanol decolorization to remove chlorophyll, microscopy under the last anatomical lens.The result of GUS histochemical stain as shown in Figure 1, in transfer-gen plant, gus gene also all has expression at the expression of root very strong (Figure 1B) in stem, flower bud and blade, wherein the expression in blade is confined to (Fig. 1 C) in the vein, shows as vascular-specific expression.Further observe the cross section of transfer-gen plant stem, find that gus gene is only in phloem expression (Fig. 1 D); Examine under a microscope, find in blade, gus gene is also expressed in pore (Fig. 1 E) except expressing in vein; Further amplify, find that gus gene is to express (Fig. 1 F) in the guard cell of pore; Expression in flower bud also is confined to (Fig. 1 G) in the vascular bundle.Above-mentioned GUS fluorescence histochemistry positioning result has shown that the GHNBS gene is at the intravital tissue specific expression that is expressed as of plant.The element that has 9 specifically expressings in root on the GHNBS promotor, they may the different basis that efficiently expresses of gus gene Gent.

Utilize fluorescent quantitation to measure the gus gene activity, gus gene produces the product 4-methyl umbelliferone (4-MU) that fluorescence is arranged with 4-methylumbelliferylglucuronide as the substrate that acts on, and detects the active size of gus gene by the fluorescence intensity that detects product.The GUS activity is a unit with the pmol 4-MU that every milligram of soluble proteins per minute produces.

Concrete grammar is: will use liquid nitrogen grinding about about 0.2 gram of transgenic arabidopsis blade, then homogenate is suspended in GUS extraction damping fluid and (contains 50mM sodium phosphate buffer (pH 7.0), 0.1%Triton X-100,10mM B-mercaptoethanol, 10mM EDTA and 0.1%SDS) in, 12, collect supernatant behind 000g, 4 ℃ of centrifugal 10min.Get an amount of supernatant sample and join abundant mixing in the reaction buffer (containing 50mM sodium phosphate buffer (pH 7.0), 0.1%Triton X-100,10mM beta-mercaptoethanol, 10mM EDTA, 0.1%SDS, 5mM 4-MUG), in 37 ℃ of incubations.Use 1M Na ₂The CO3 termination reaction.Content with 4-MU in the SHIMADZU RF-5301PC fluorescent spectrophotometer measuring product.Method (Bradford MM.A rapid and sensitivemethod for the quantification of microgram quantities of protein utilizing the principle ofprotein-dye binding.Anal Biochem 1976 with reference to Bradford; 72:248-54.) that the protein in the extracting solution is carried out concentration is quantitative, (BSA) makes typical curve with bovine serum albumin.

Four: the paraffin section of GUS dyeing back stem

The vascular bundle tissue comprises xylem and phloem two portions, and the main ingredient of phloem comprises screen casing and companion cell.In order further to confirm the expression position of gus gene, after transgenic arabidopsis plant GUS histochemical stain, its stem is carried out paraffin section observe.Because water and paraffin can not dissolve each other, so the moisture in the tissue must be removed.With 70%, 80%, 90%, 95% and 100% ethanolic solns at different levels each 30min that dewaters.Because ethanol and paraffin are immiscible, and dimethylbenzene can be dissolved in ethanol and can be dissolved in paraffin, so also will pass through the dimethylbenzene transition after dewatering.With straight alcohol and dimethylbenzene equivalent mixed liquor and pure each 30min of xylene solution transparent processing, change dimethylbenzene again one time.Ooze wax and embedding with fresh vasoliniment.Hand microtome section is dyeed to present whole stem transverse cross-sectional area to lignified cell walls and nucleus with 1% sarranine at last.The microscopically microscopy detects.The blueness that found that xylem and screen casing not to have the gus gene expression and present, the blue dyeing zone is arranged in companion cell (Fig. 2), and red zone is by painted lignified cell walls of sarranine and nucleus.The promotor of these explanations GHNBS gene is mainly expressed in the companion cell of phloem.

Five: detect the induced reaction of pGHNBS transfer-gen plant to pathogenic bacteria and plant hormone

The T2 that is taken at about 3 weeks of growth in the greenhouse does various processing for pBI-pGHNBS::GUS Arabidopis thaliana transgenosis seedling.Use pathogenic bacterium pseudomonas syringae DC3000 (the Buell CR of tomato respectively, Joardar V, Lindeberg M, etal.The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonassyringae pv.tomato DC3000.Proc Natl Acad Sci U S A 2003:2; 100 (18): 10181-6), cotton fungoid blight pathogenic bacteria Fusarium oxysporum Schl.f.sp.vasinfectum (Fusarium oxysporum f.sp.vesinfectum) (Wu Aimin, Gu Benkang, Fu just holds up, Xia Zhengjun. cotton variety (being) resists withered, verticillium evaluation. 29～30) and this three kind of plant HORMONE TREATMENT Arabidopis thaliana seedling of Whitfield's ointment SA, methyl jasmonate and ethene China seed industry 1999 (1):, and be provided with and be untreated and injury reason contrast.Detect the active size of different transgenic line different treatment front and back GUS.DC3000 incubated overnight in containing the LB liquid nutrient medium of 50mg/L Rifampin wherein; The blight pathogenic bacteria was cultivated 3 days for last 25 ℃ at potato glucose substratum (5% potato soup, 1% glucose), and centrifugal collection thalline is also resuspended with distilled water, adopted to hinder revulsion and connect bacterium, got plant leaf analysis behind the inoculation pathogenic bacteria in 3 days; The treatment process of hormone is: with concentration is that the SA of 200uM directly sprays the Arabidopis thaliana seedling; With concentration is that the ethene of 200PPM directly sprays the Arabidopis thaliana seedling.The treatment process of methyl jasmonate is: in the container of an airtight 20L, place a cotton ball that dips in 200ul 0.5% (w/v) methyl jasmonate.All HORMONE TREATMENT times are 36h.

Through the GUS activation analysis results of the transgenic plant of above-mentioned different treatment as shown in Figure 3, the GUS activity of the transfer-gen plant after wherein DC3000 handles is untreated 6.55 times.The blight pathogenic bacteria is handled back transgenic line GUS expression amount increases by 3 times, has the pathogenic bacterium inducing element in this explanation promotor.SA, the GUS activity of the transfer-gen plant after MJ handles is respectively untreated 4.25 and 5.02 times.Ethene is handled 3.51 times of the active increases of back GUS.

These presentation of results GHNBS promotor is to be subjected to pathogenic bacteria, SA (Whitfield's ointment), MeJA (methyl jasmonate), and there is corresponding response element in Ethylene (ethene) inductive on it.It is a promotor that organ is special and cause of disease is relevant.Sequential analysis shows 2 AS-1 enhancer elements of existence on it.

The expression pattern that we find quite a few pathogenic bacteria associated protein (PR) is similar to GHNBS's: i.e. vascular-specific expression and be subjected to the plant hormone abduction delivering.As the PRB-1b that derives from tobacco shows as vascular-specific expression, and expression amount has increased by 20 times of (Eyal Y after being subjected to ethene and inducing, Meller Y, Lev-Yadun S, FluhrR.A basic-type PR-1 promoter directs ethylene responsiveness, vascular and abscission zone-specific expression.Plant J1993,4 (2): 225～234).The PR-1b gene of potato is the guard cell of pore, specifically expressing (Erika Hoegen, Anita in the vascular bundle tissue of glandular hairs and blade

UllaPihlgren and Erich Kombrink.Primary structure and tissue-specific expression of thepathogenesis-related protein PR-1b in potato.Molecular Plant Pathology 20023:5,329-345).Arabidopis thaliana AtPRB1 only expresses in floral organ, stem and root, does not express in mature leaf.And after ethene and methyl jasmonate are induced, expression amount (the Santamaria M. that rises rapidly, Thomson C J., Read N D., Loake G J.The promoter of a basic PR1-like gene, AtPRB1, from Arabidopsis establishes anorgan-specific expression pattern and responsiveness to ethylene and methyl jasmonate.PlantMolecular Biology 2001,47:641-652.).The promotor nematode that sucrose is transported sub-AtSUC2 is induced and is mainly expressed in companion cell.(Hoth S，Schneidereit A，Lauterbach C，Scholz-Starke J，Sauer N.Nematode infection triggers the de novo formation of unloading phloem that allowsmacromolecular trafficking of green fluorescent protein into syncytia.Plant Physiol 2005，138(1)：383-92.)。

Sequence table

＜110〉Jiangsu Province Agriculture Science Institute

＜120〉cotton tissue specific and pathogenic bacterium inducing promotor and application thereof

＜130〉specification sheets

<140>00

<141>2006-06-20

<160>1

<170>PatentIn version 3.1

<210>1

<211>1600

<212>DNA

＜213〉Gossypium hirsutum (upland cotton)

<220>

<221>gene

<222>(1)..(1536)

<223>

<400>1

agtggagtag caaaggaaag gaacagatgg agggacctgt catacgacac acttaaaagg 60

aatgcaacgt caatggcgat gcctaattgt tacttacagc aggttccaaa gatttgttcc 120

accacgcgtg aaggaatcag attcgactgc tcccaactta catgcaacgt caatatcaat 180

cctcatataa agctgattaa aagttgattt ttaatattaa gaatagcatc ccattcacct 240

cccataacag tgatcatttt gtaacatttt aaagttaagt gactaaaaca taaatttact 300

gttaatttag tgatcttgga tgtaatttat ccatatttat gttacatgca cggtgaagac 360

ttcctgcatt tgggaacatt ttcacttgtc tttctttact tcattcttgt actttaatct 420

atctcaacca agattaagat tctgtatgtt gagaactttg gttagtttat atttgaaaaa 480

aattattgat attattaaaa aaaataacta taaatttaaa aaatattaaa aacatgtaca 540

accaaaacat ctattttaaa tgaagcattt cgaaagcgaa tttgattaaa aaaggatagc 600

catgtgaaag tcaactttga gcatgtgaac aacttattga aatcatttat cattttcaat 660

tagttgaaag tcctaaattt attttttaaa aatcggtttg tttaaaacta aggtgttgat 720

agaataagta tttaattaaa tgcattattt ttagtgtaag aaaaataata attcagtatg 780

ccatgtaatt gatgtggtac gattaagttc gagcaacaaa tcgaaaggag gcggtttttg 840

ggcatgctat agggcatttg gttccgtaca aatatgcaaa taagtttaaa tgtaattttg 900

atggggccct ctctgttgac agtgggtatt cggggtgaat ttgtgcggtg catctcgagt 960

tttataaaga tcgcaaagtc catctttctg cggtggaagt catgtctact catcattgct 1020

ttattgaaag caaactactt tgaatgtatt tcttatcctg ttttatcatt catactgtta 1080

atgttgataa attgttacaa caggatgaaa ataagatagg acttagaaaa gggtgtattg 1140

ggtaagccgg ttgatttgga tttaatctgt accacttatt ttcattttgt tttcttttta 1200

tcagttttac tggtttaatg aatttgattt tatatttaaa aaaaatcaaa tataaatgtt 1260

tagttgccat cagcattagg atatcaacca catgtcaagc atgattgctt aatttgattt 1320

aaatatatat aataataaat gaaaattgac tttttggttg gatgataaca tcacttatgt 1380

aagctaaaat tgcaatttaa aatcagaaaa tgaaatcctc gagttctttt tcattcatac 1440

tttcataaat tcaactatag ttaataatag atcataaatg cagtccagtt tcttataata 1500

aaaaaacaag gtaggtctta aattcttata aatgcaagca gtccagtttc tgaaataaca 1560

aggcaaatta gttttctgag tgactaaaaa gttagggagc 1600

Claims (4)

1. cotton tissue specific and pathogenic bacterium inducing promotor are the dna sequence dnas shown in the SEQ ID NO.1 in the sequence table.

2. promotor according to claim 1 is characterized in that, this promotor can make goal gene locate to express the phloem companion cell cell of root, flower bud, stem, the vein of blade and the guard cell of pore.

3. promotor according to claim 1 is characterized in that this promotor can be activated by pathogenic bacteria, perhaps can be activated with Whitfield's ointment, ethene, methyl jasmonate treatment the time.

4. the application of the described promotor of one of claim 1～3 in plant quality improvement and plant resistance to environment stress improvement.

Images