CN102533754B - FGI1 promoter for controlling induced expression of gene at early pathogen infection stage and applications thereof - Google Patents

Abstract

The invention relates to an FGI1 promoter for controlling induced expression of a gene at early pathogen infection stage and applications thereof. According to the invention, the promoter which is capable of responding a pathogen infection signal and controlling the specific expression of a target gene is obtained by separation for the first time. The promoter is very useful for controlling the expression of the target gene so as to change the disease resistance of plants under the pathogen infection state.

Description

FGI1 promotor and application thereof that regulatory gene is expressed at the pathogen infection early evoking

Technical field

The invention belongs to biotechnology and phytology field; More specifically, the present invention relates to FGI1 promotor and the application thereof that regulatory gene is expressed at the pathogen infection early evoking.

Background technology

Plant promoter plays a key role in regulating plant genetic expression, can control plant and express in particular organization, etap and varying environment condition.Promotor is divided and can be divided into constitutive promoter, organizing specific type promotor and induce the Idiotype promotor by its function.Aspect crop disease-resistant, Main Viewpoints thinks that constitutive promoter expresses any disease-resistant gene in the world at present, capital causes plant to be in Defense response state (mode), thereby makes the plant defense system preferentially obtain energy and resources allocation affects the economic characters such as plant biomass.And this disease-resistant application that also is considered to adopt at present transgenosis means the cause for the success not yet.A rational method that addresses this problem is to adopt the inducing specific promotor, control disease-resistant gene and only express (Gurr when pathogenic bacteria is invaded, S.J., and Rushton, P.J. (2005) .Engineering plants withincreased disease resistance:how are we going to express it? Trends inBiotechnology 23,283-290).Adenylic acid (AMP) transferring enzyme (adenine nucleotide translocase, ANT) is the abundantest proteinoid complex body of content on plastosome.this proteinoid complex body is positioned on mitochondrial inner membrane, be that production capacity in organism is connected main connection with power consumption, also to form mitochondrial permeability transition pore mixture (mitochondrial permeability transition pore complex, mtPTPC) key ingredient, participate in regulating the opening of mitochondrial permeability transition pore, with the apoptosis (Shen that is closely related, Q.F., Qin, F.S., Gao, Z.Y., Cui, J., Xiao, H., Xu, Z.H., and Yang, C.L. (2009) .AdenineNucleotide Translocator Cooperates with Core Cell Death Machinery To PromoteApoptosis in Caenorhabditis elegans.Molecular and Cellular Biology 29, 3881-3893).ANT is the homodimer (Notario by 2 32kD monomer compositions of nuclear gene encoding, B., Zamora, M., Vinas, O., and Mampel, T. (2003) .All-trans-retinoic acid bindsto and inhibits adenine nucleotide translocase and induces mitochondrialpermeability transition.Molecular Pharmacology 63,224-231),, by the variation of its conformation, realize the exchange of the cross-film between ATP in ADP and plastosome in tenuigenin.4 kinds of isomer ANT1-4 have been found successively in the people, ANT1 is relevant to apoptosis, ANT2 (Barath relevant to cell proliferation, P., Luciakova, K., Hodny, Z., Li, R.G., and Nelson, B.D. (1999) .Thegrowth-dependent expression of the adenine nucleotide translocase-2 (ANT2) geneis regulated at the level of transcription and is a marker of cell proliferation.Experimental Cell Research 248,583-588).forefathers' research shows, gland nucleotidyl transferase in corn is encoded up to ANT-G1 and two genes of ANT-G2 of 98% jointly by similarity, these two genes have one section extension except the N end, with fungi and mammiferous ANT gene height homology (BathgateB, Baker A, Leaver CJ. (1989) TWo genes encode the adenine nucleotide translocatorof maize mitochondria.Isolation, characterisation and expression of the structuralgenes.Eur J Biochem.1989Aug 1, 183 (2): 303-10, Winning, B.M., Day, C.D., Sarah, C.J., and Leaver, C.J. (1991) .Nucleotide sequence oftwo cDNAs encodingthe adenine nucleotide translocator from Zea mays L.Plant Mol Biol 17,305-307), but the function of ANT-G1 and ANT-G2 and the promotor report that builds that so far there are no in the art in corn.

Summary of the invention

The object of the present invention is to provide regulatory gene expresses under pathogeny evoked situation ANT-G2 gene (in the application also referred to as the FGI1 gene) promotor and application thereof.

, in a first aspect of the present invention, provide a kind of promotor of separation, described promotor:

(a) separation is from the upstream of gland nucleotidyl transferase ANT-2 encoding gene;

(b) base length is 500-900;

(c) have and cause necessary site and the transcripting start point of transcribing; And

(d) has response pathogen infection signal and start the specific expressed function of goal gene.

In another preference, described gland nucleotidyl transferase derives from corn (Zea mays).

In another preference, described promotor contains TATA-box and CAAT-box structure.

In another preference, the base length of described promotor is 600-800.

In another preference, described promotor is:

(1) promotor of the nucleotide sequence shown in SEQ ID NO:1;

(2) nucleotide sequence and SEQ ID NO:1 have 95% above homogeny and have the promotor that responds the pathogen infection signal and start the specific expressed function of goal gene;

(3) nucleotides sequence is listed under stringent condition and can hybridizes and have the polynucleotide that respond the pathogen infection signal and start the specific expressed function of goal gene with the polynucleotide sequence of the arbitrary restriction in (1)-(2); Or

(4) promotor of the nucleotide sequence complete complementary shown in nucleotide sequence and SEQ ID NO:1.

In another aspect of this invention, provide a kind of carrier, described carrier contains the arbitrary described promotor in front.

In another preference, described carrier also contains the goal gene that is operably connected with described promotor.

In another preference, the albumen that described goal gene coding has specific function.

In another preference, described goal gene is foreign gene.

In another preference, described goal gene is antifungus protein gene, pathogenesis-related proteins gene.

In another preference, described goal gene is positioned at the downstream of described promotor, and with the direct sequence of contiguous encoding gene of described promoter region.Usually, the interval of described promotor and goal gene less than 1000bp (preferred, less than 500bp; Preferred, less than 100bp; Most preferred, less than 50bp).

In another aspect of this invention, provide a kind of genetically engineered host cell, described cell:

Contain the arbitrary described carrier in front; Or

Be integrated with the arbitrary described promotor in front of external source in its genome.

In another aspect of this invention, a kind of plant method that can respond the pathogen infection signal for preparing is provided, described method comprises: with the construction conversion of plant, the goal gene that described construction contains the arbitrary described promotor in front and with described promotor, is operably connected, described goal gene is expressed under the pathogen infection state.

In another preference, described method comprises:

(a) provide the Agrobacterium of carrying expression vector, contain construction in described expression vector, the goal gene that described construction contains described promotor and with described promotor, is operably connected;

(b) vegetable cell, tissue or organ are contacted with the Agrobacterium in step (a), thereby make described construction change vegetable cell, tissue or organ over to;

(c) select vegetable cell, tissue or the organ that has changed described construction over to; And

(d) vegetable cell, tissue or neomorph in step (c) are become plant, in described plant, goal gene is expressed under the pathogen infection state.

In another preference, described plant is grass.

In another preference, described plant is paddy rice, corn, wheat, barley, Chinese sorghum, rye.

In another aspect of this invention, provide the purposes of the arbitrary described promotor in front, described promotor is used for response pathogen infection signal and starts goal gene specific expressed.

In another preference, described pathogenic agent is fungi.

In another preference, described pathogenic agent is selected from but is not limited to: F.graminearum schw (Fusariumgraminearum) bacterium, Pyricularia oryzae (Magnaporthe oryzae), fusarium oxysporum (Fusariumoxysporum).

Other side of the present invention, due to the disclosure of this paper, is apparent to those skilled in the art.

Description of drawings

Fig. 1, pTG-19T-FGI1 promoter carrier collection of illustrative plates.In the carrier collection of illustrative plates, promotor is denoted as Fgi1promoter.

Fig. 2, pCAM-FGI1 promoter expression vector collection of illustrative plates.In the carrier collection of illustrative plates, promotor is denoted as Fgi1promoter.

Fig. 3, turn FGI1::GUS trans-genetic hybrid rice vanes F.graminearum schw and induce that rear GUS is active to be detected.A:T0 is for being infected; B:T0 contrasts for water; C:T1 is for being infected; The contrast of D:35S promoters driven GUS transfer-gen plant water; E: wild-type is infected; F: wild-type water contrast; G:35S promoters driven GUS transfer-gen plant is for infected by F.graminearum schw.Wherein arrow represents to have GUS dyeing (blueness) to locate.

Fig. 4, turn FGI1::GUS trans-genetic hybrid rice T0 in more detail and induce for the vanes F.graminearum schw that rear GUS is active to be detected.Comprise transgenic line FGI1-3, FGI1-4, FGI1-15 and wild-type (in spend 11) negative control (corresponding to table 4).Wherein arrow represents to have GUS dyeing (blueness) to locate.

Fig. 5, turn FGI1::GUS trans-genetic hybrid rice T1 in more detail and induce for the vanes F.graminearum schw that rear GUS is active to be detected.Comprise transgenic line FGI1-4, FGI1-8, FGI1-9, FGI1-11, FGI1-14, FGI1-17 (corresponding to table 4).Wherein arrow represents to have GUS dyeing (blueness) to locate.

Embodiment

The inventor, through extensive and deep research, is separated to first one and can responds the pathogen infection signal, and regulates the specific expressed promotor of goal gene.Described promotor is very useful for the expression of the gene of lowering program at the pathogen infection state with the disease resistance that changes plant.Completed on this basis the present invention.

Term

As used herein, described " plant (or crop, farm crop) " comprises the plant of any kind, as long as this plant is fit to carry out the conversion operation (transgeneic procedure) of gene, as various farm crop, flower plant or forestry plant etc.Described plant is such as being: dicotyledons, monocotyledons or gymnosperm.Such as but not limited to: paddy rice gramineous, wheat, barley, Chinese sorghum, corn, rye; Chinese cabbage, Plantula Brassicae chinensis that the Cruciferae rape belongs to; Cruciferae mouse ear mustard; Comprise in addition tobacco, melon and fruit, vegetables, rape etc.more specifically, described plant includes, but is not limited to: wheat, barley, rye, paddy rice, corn, jowar, beet, apple, pears, Lee, peach, apricot, cherry, strawberry, rasp berry, blackberry, blueberry, beans, French beans, pea, soybean, rape, mustard, opium poppy, olea, Sunflower Receptacle, coconut, the Viscotrol C plant, cocoa beans, peanut, cucurbit, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, natsudaidai, spinach, the piemarker lettuce, asparagus, cabbage, Chinese cabbage, Plantula Brassicae chinensis, Radix Dauci Sativae, onion, potato, tomato, green pepper, avocado, cassia bark, camphor, tobacco leaf, nut, coffee, eggplant, sugarcane, tealeaves, pepper, grapevine, oyster fiber crops grass, banana, natural rubber tree and ornamental plant etc.As optimal way of the present invention, described plant is grass, includes but not limited to paddy rice, wheat, barley, Chinese sorghum, corn, rye.

As used herein, unless otherwise indicated, described " pathogenic agent " refers to infect the microorganism of plant,, normally to the plant harmful microorganism, causes plant generation disease.Described pathogenic agent is for example that pathogenic fungi, pathogenetic bacteria, plant virus, epidemic disease are mould, nematode.More specifically, described pathogenic agent is selected from: pathogenic fungi F.graminearum schw (Fusarium graminearum) bacterium (herein also referred to as F.graminearum schw, Gibberella zeae Gibberella zeae is otherwise known as), Pyricularia oryzae (Magnaporthe oryzae), fusarium oxysporum (Fusarium oxysporum) etc.

As used herein, " separation " refers to that material separates (if natural substance, primal environment is namely natural surroundings) from its primal environment.There is no separation and purification as the polynucleotide under the native state in active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, for separation and purification.

As used herein, described " being operably connected " refers to functional spatial disposition of two or more nucleic acid region or nucleotide sequence.For example: promoter region is placed in the specific position with respect to the goal gene nucleotide sequence, make transcribing of nucleotide sequence be subject to the guiding of this promoter region, thereby promoter region is " operably connected " on this nucleotide sequence.

As used herein, described " promotor " or " promoter region (territory) " refers to a kind of nucleotide sequence, and the upstream (5 ' end) that it is present in the goal gene encoding sequence usually, can be transcribed into mRNA by the guiding nucleus acid sequence.Usually, promotor or promoter region provide RNA polymerase and correct initial recognition site of transcribing necessary other factors.In this article, described promotor or promoter region comprise the variant of promotor, and it, by inserting or deletion regulation and control zone, carries out random or rite-directed mutagenesis etc. and obtain.

As used herein, term " specific expressed " refers to that goal gene is in specific time and/or specifically tissue and/or specific state expression.Described " in the promotor of expressing under the pathogen-inducible situation " refers under this class promoter regulation, goal gene is specific expressed in the process of pathogen infection (comprise pathogenic agent is initial infect in), and does not express under the state of pathogen infection not having.

Usually, if at the lower mRNA of particular state (this particular state refers generally to the state of pathogen infection herein) with than lower high at least 5 times at other state (as there is no the state of pathogen infection), preferably at least 10 times, more preferably high at least 100 times, more preferably high at least 1000 times of levels are expressed, and this promotor is considered to be in specificity under specific time and/or particular state and drives destination gene expression.

As used herein, " external source " or " allos " refers to from the two or more pieces nucleic acid of different sources or the relation between protein sequence.For example, if the combination of promotor and goal gene sequence is not naturally occurring usually, promotor is external source for this goal gene.Particular sequence is " external source " for its cell that inserts or organism.

As used herein, " cis-regulating element " refers to the transcription initiation of gene and transcribes the conservative property base sequence that efficiency plays regulatory role.

As used herein, " goal gene " refers to be started or to be instructed by promotor of the present invention the gene of expression.Suitable goal gene includes but not limited to: improvement disease resistance of plant, proterties or the relevant gene of metabolism.Suitable goal gene includes but not limited to: antifungal protein encoding gene, pathogenesis-related proteins gene (pathogenesis-related genes, PR1-PR17).concrete pathogenesis-related proteins comprises PR-1 (cysteine-rich secretary protein is rich in the halfcystine secretory protein), PR-2 β-1, the 3-glucanase dextranase, the PR-3Chitinase chitinase, the PR-4Chitinas chitinase, the PR-5Thaumatin-like monellin is similar, PR-6Proteinase-inhibitor I proteinase inhibitor, the PR-7Endoproteinase protein incision enzyme, PR-8chitinase type III chitinase, the PR-9lignin-formingperoxidase peroxidase, PR-10Ribonuclease-like rnase albuminoid, the PR-11Chitinase chitinase, the PR-12Defensin alexin, the PR-13Thionin thionin, PR-14Lipid-transfer protein turns lipoprotein, PR-15Oxalate oxidase Oxalate oxidase, PR-16OxOLP Oxalate-oxidase-like Oxalate oxidase albuminoid etc., referring to van Loon, L.C., M.Rep, et al. (2006). " Significance of inducible defense-related proteins in infectedplants. " Annu Rev Phytopathol 44:135-62.Described goal gene can also be H.Wongand T.B.Ng, Gymnin, a potent defensin-like antifungal peptide from the Yunnanbean Gymnocladus chinensis Baill, Peptides 24 (2003), pp.963-968.2; BenhamouN, Broglie K, Broglie R, Chet I.Antifungal effect of bean endochitinase onRhizoctonia solani:ultrastructural changes and cytochemical aspect of chitinbreakdown.Can J Microbiol 1993; 39:318-28.3; Broekaert WF, Van Parijs J, LeynsF, Joos H, Peumans WJ.A chitin-binding lectin from stinging nettle rhizomes withantifungal properties.Science 1989; Those that report in 245:1100-2.

As used herein, described " containing ", " having " or " comprising " comprised " comprising ", " mainly by ... form ", " basically by ... form " and " by ... form "; " mainly by ... form ", " basically by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".

As used herein, described " FGI1 ", " FGI-1 " are used interchangeably.

Promotor

The invention provides a kind of promotor specific expressed under the pathogen infection state, described promotor has following characteristics: (a) separate the upstream from gland nucleotidyl transferase (herein referred to as ANT-G2 or FGI1) encoding gene; (b) base length is 500-900; (c) have and cause necessary site and the transcripting start point of transcribing; And (d) has response pathogen infection signal and start the specific expressed function of goal gene.As one embodiment of the present invention, described promotor has the promotor of the nucleotide sequence shown in SEQ ID NO:1.

The hybridization of polynucleotide is technology well known to those skilled in the art, the indication of hybridization characteristic their similarity or the identity of specific a pair of nucleic acid.Therefore, the invention still further relates to and the hybridization of the nucleotide sequence (as SEQ ID NO:1) of aforementioned appointment and two sequences between have at least 50%, preferably at least 70%, polynucleotide of (for example 85%, 90%, 95%, 96%, 97%, 98% or 99%) homogeny more preferably at least 80%.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention (as SEQ ID NO:1).

" stringent condition " (or " stringent condition ") refers to: (1) at the hybridization than under low ionic strength and comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only the homogeny between two sequences is at least 50%, preferred more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, is more preferably 95% and just hybridizes when above.And interfertile polynucleotide also have the function of response pathogen infection signal.

The present invention also comprises with any promoter sequence of the present invention having 50% or above (preferred more than 60%, more than 70%, more than 80%, more preferably more than 90%, most preferably more than 95%, as 98%, 99%) nucleic acid of homogeny, described nucleic acid also has response pathogen infection signal and starts the specific expressed function of goal gene." homogeny " refers to according to the identical per-cent in position, the similar level between two or more pieces nucleic acid (being sequence homology, similarity or identity).

Should understand, although this promotor and the function thereof that derive from corn are provided in example of the present invention, yet, the promotor that has certain homogeny (conservative property) with this promotor that derives from other similar plant is also included within scope of the present invention, as long as those skilled in the art can separate and obtain this promotor easily the information that provides according to the application after the application has been provided from other plant.

In specific embodiments of the invention, take corn gene group DNA as template, use the method for PCR to clone promoter sequence of the present invention, it is built up on plant expression vector pCAMBIA1300 with fusion gene GUS, by agriculture bacillus mediated rice transformation, obtain the FGI1promoter::GUS transfer-gen plant, the FGI1 promotor has been carried out startup activity and pathogenic bacterium inducing analysis.The GUS activity can be detected in the transgenic paddy rice callus, show that this promotor has the activity of startup.In growth of seedling period, its root and Ye Junwei detect the GUS activity, show that the FGI1 promotor does not drive downstream gene expression under general growth conditions; And after F.graminearum schw (Fusarium graminearum) bacterium was infected, the GUS activity of FGI1 promoters driven had up-regulated expression.Therefore think that FGI 1 promotor has the driving gus gene and is being subject to the startup activity of pathogen infection early evoking expression.

Start destination gene expression

Promotor of the present invention can be operatively connected on goal gene, and this goal gene can be external source (allos) for promotor.Nucleotide sequence to described goal gene has no particular limits (as a kind of functional or structural nucleotide sequence), and described goal gene optimized encoding has the albumen of specific function, and for example some has the albumen of antipathogen function.

Promotor of the present invention can also be operably connected on the goal gene sequence that is modified, and this goal gene is external source (allos) with respect to promotor.Described goal gene can be modified to produce the characteristic of various expectations.For example, goal gene can be modified to increase the content of indispensable amino acid, improves the translation of aminoacid sequence, change the modification (as phosphorylation site) after translating, outside the translation product transporte to cells, improve the stability of albumen, insert or delete cell signal etc.

In addition, promotor and goal gene can be designed to lower specific gene.This is generally that this sequence oppositely is directed with antisense by promotor is connected on the goal gene sequence and realizes.Those of ordinary skill in the art is familiar with this antisense technology.Any nucleotide sequence can be conditioned by this way.

Any aforesaid promotor and/or goal gene sequence can be comprised in recombinant vectors.

As a kind of mode, described recombinant vectors comprises promotor of the present invention, comprises multiple clone site or at least one restriction enzyme site in the downstream of described promotor.When needs are expressed goal gene, goal gene is connected in suitable multiple clone site or restriction enzyme site, thereby goal gene is operably connected with promotor.

As another kind of mode, described recombinant vectors comprises (from 5 ' to 3 ' direction): promotor, and goal gene.If necessary, described recombinant vectors can also comprise 3 ' transcription terminator, 3 ' polymerized nucleoside acidifying signal, other untranslated nucleotide sequence, transhipment and target nucleotide sequence, resistance selective marker, enhanser or operation.

Method for the preparation of recombinant vectors is well known in the art.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell is viral or other carriers.When being used for plant, it can be also a kind of carrier for expression of eukaryon.In a word, as long as it can copy and stablize in host, any plasmid and carrier are all can be adopted.The selection of some carriers is that those skilled in the art can be known.

Method well-known to those having ordinary skill in the art can be used for building the expression vector that contains promotor of the present invention and/or goal gene sequence.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of the host cell that transforms, as Tetrahydrofolate dehydrogenase, neomycin resistance, hygromycin resistance and green fluorescent protein (GFP) etc.

, except containing promotor of the present invention, also can contain one or more other promotors in recombinant vectors.Described other promotor is for example: tissue-specific, composing type or induction type.Such as the cauliflower mosaic virus 19S of mannosaminic acid synthetic enzyme and 35S (CaMV19S CaMV35S), CaMV, the tobacco RB7 etc. that strengthen.

Comprise above-mentioned suitable promotor and the carrier of goal gene, can be used for transforming suitable host cell, with can marking protein.

Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; The fungal cell; Yeast; Vegetable cell etc.Persons skilled in the art are all known carrier and the host cell that How to choose is suitable.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be processed with the CaCl2 method in exponential growth after date results, and step used is well-known in this area.Another kind method is to use MgCl2.If necessary, transforming the also method of available electroporation carries out., when the host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method such as microinjection, electroporation, liposome packing etc.Conversion of plant also can use the methods such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as Ye Panfa, rataria conversion method, bud infusion method etc.Can use ordinary method regeneration plant for the vegetable cell, tissue or the organ that transform, thereby obtain genetically modified plant.

As a kind of mode, the method for preparing transgenic plant is: the carrier that will carry promotor and goal gene (both are operably connected) changes Agrobacterium over to, and the carrier segments that Agrobacterium will contain promotor and goal gene again is incorporated on the karyomit(e) of plant.The transgene receptor plant that relates to is for example paddy rice, wheat, corn, Arabidopis thaliana, tobacco, fruit tree etc.

In example of the present invention, described recombinant vectors is the pCAMBIA carrier, it carries beta-glucosidase (GUS) gene, promotor of the present invention is building up to the upstream of gus gene in this carrier, transformed plant, promotor will activate the expression of gus gene, and described startup is subject to the regulation and control of each cis-acting elements of promoter region, simulate the situation that gene is activated in vivo and transcribes.The a series of beta-glucoside of beta-glucosidase (GUS) energy catalytic pyrolysis, produce the material with chromophoric group or fluorescence, and the methods such as available spectrophotometer, photofluorometer or histological chemistry are carried out quantitatively and the spatial positioning analysis the GUS activity.In the art, gus gene has been widely used as the reporter gene of transgenic plant, bacterium and fungi, and particularly it can be used to concrete cell and the tissue site of Study of Exogenous genetic expression.Usually following common recognition has been reached in this area: express if a kind of promotor can drive gus gene, it also can drive the expression of gus gene other goal gene in addition.

Application

According to embodiments of the invention, infect the real-time quantitative PCR result of early stage maize cell based on the F.graminearum schw that is subjected to that is obtained by the laser capture microdissection cutting technique, plastosome gland nucleotidyl transferase gene (ANT-G2) is up-regulated expression obviously, and has specificity.

According to embodiments of the invention, the blade of the transgenic paddy rice of promoters driven gus reporter gene of the present invention has no gus gene through GUS dyeing and expresses when being infected, and shows visible the expression through GUS dyeing while being infected.Drive the transgenic paddy rice of gus reporter gene with 35S promoter, show visible the expression through GUS dyeing while being infected.Promotor of the present invention has important using value in theory research and agronomy improvement.

The present invention can be widely used in plant genetic engineering: promotor can merge with target gene as important tool in plant genetic engineering, and by transgene carrier, conversion of plant also obtains transfer-gen plant.When plant is subject to pathogenic agent infringement (infecting), it (can be interested any functional gene that can change economical character that this promotor just can start the downstream targets gene, the gene of antipathogen infringement particularly) expression, control thereby reach the purpose that a certain functional gene is expressed under pathogenic agent infringement state.

The promotor that the present invention obtains can be used for transgenic crop (as paddy rice, corn etc.) and drive specific other gene (as the antipathogen gene), make this gene be subjected to the pathogen infection early expression at transfer-gen plant, protect the effect of producing thereby reach performance disease and insect resistance when having pathogenic agent to threaten, and keep closing minute orientation high yield that is conducive to energy and nutrition when disease-free the threat, tilt.Drive the plant anti-pathogen gene with startup of the present invention, can be when being subject to pathogen infection, react rapidly, infecting of defence pathogenic agent, thereby more effective in the early stage in time prevention of morbidity morbidity, be different from again simultaneously composition type expression promoter, can and don't affect the output of farm crop in the antiviral while.

Major advantage of the present invention is:

(1) promotor of the present invention belongs to the Idiotype promotor.It is subject under the state of pathogen infection plant, signal is infected in response, just start downstream gene expression, and be not subject under the state of pathogen infection expression amount plant very low or do not express, it can make foreign gene effectively play a role in plant materials, reduces again the disadvantageous effect of this expression to plant simultaneously.

(2) adopted aspect crop disease-resistant at present is the promotor of some constitutive expressions more, as cauliflower mosaic virus (CaMV) 35S promoter, corn ubiquitin promotor, the promotor that the present invention obtains is more special than these composition type expression promoters.This promotor is only expressed when crop is subject to infecting, and at the crop normal epoch, does not express.

(2) promotor of the present invention has and responds characteristic rapidly.

, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are not used in and limit the scope of the invention for explanation the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, the third edition, 2002) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.

Unless otherwise defined, the same meaning that all specialties and scientific words and the one skilled in the art who uses in literary composition is familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.

Materials and methods

Material

corn (Zea mays) kind B73 is available from The Maize Genetics Cooperation Stock Center (U.S. University of Illinois) or referring to Schnable, P.S., D.Ware, et al. (2009). " " Science 326 (5956): 1112-5 for TheB73maize genome:complexity, diversity, and dynamics., rice varieties " in spend 11 " (Oryza sativa japomca Zhonghua 11) is referring to Chinese patent 200510110629.9, F.graminearum schw (Fusarium graminearum) bacterial strain PH-1 available from FungalGenetics Stock Center (http://www.fgsc.net/) referring to Cuomo, C.A., U.Guldener, etal. (2007). " The Fusarium graminearum genome reveals a link between localizedpolymorphism and pathogen specialization. " Science 317 (5843): 1400-2, agrobacterium tumefaciens (Agrobacterium tumefaciens) bacterial strain EHA105 is referring to Hood, E.E. etc., 1993, NewAgrobacterium helper plasmids for gene transfer to plants.Transgen.Res.2:208-218, the pCAMBIA1300 plasmid is available from CAMBIA company, the pTG-19T plasmid is available from TaRaKa company.Test is kantlex (Kan) 50 μ g/ml, penbritin (Amp) 100 μ g/ml with antibiotic concentration.Restriction enzyme, T4DNA ligase enzyme, Taq archaeal dna polymerase etc. are TaRaKa company product; 5-bromo-4-chloro-3-indole glucoside acid (X-Gluc) is available from future biotechnology company; Penbritin (Amp), kantlex (Kan) are available from ancient cooking vessel state biotechnology company.

ANT-G2 genetic expression detects

Design two pairs of RT-PCR primers and detect expression on gene:

Fgi-1primerF1：CGAGGCCTGTATTTCGGACTTTACG(SEQ?ID?NO：4)；

Fgi-1primerR1：GCACCGTTGGTGATCAGCCA(SEQ?ID?NO：5)；

Fgi-1primerF2：TGATGATGACTTCTGGTGAGGGTGT(SEQ?ID?NO：6)；

Fgi-1primerR2：TTAGCACCAGCACCCTTGAACAG(SEQ?ID?NO：7)。

The clone of FGI1 promotor and sequential analysis

, according to the FGI1 gene order, designed the upstream and downstream primer of the FGI1 gene promoter that contains respectively EcoRI and BamHI restriction enzyme site:

FGI1F：5’-GAATTCTCCGTTTCTCCATAGGTG-3’(SEQ?ID?NO：8)；

FGI1R：5’-GGATCCCCTCAAAACTGTTGGCTG-3’(SEQ?ID?NO：9)。

Extract the total DNA of maize leaf with reference to " molecular cloning: lab guide " upper CTAB method.Take total corn DNA as template,, with aforementioned upstream and downstream primer PCR amplification FGI1 promoter sequence, be connected to the pTG-19T plasmid, Transformed E .coli DH5 α competent cell, blue hickie screening obtains recombinant plasmid.Deliver to rich still order-checking company and carry out sequencing.Sequencing result is compared to http://www.ncbi.nlm.nih.gov/ and http://www.maizesequence.org/.

The structure of FGI1promoter::GUS expression vector and conversion

Clone's FGI1 promoter fragment is cut with EcoRI and BamHI enzyme, be connected carrier with the EcoRI of pCAMBIA1300 with the BamHI enzyme after reclaiming and be connected, obtain the expression vector FGI1promoter::GUS that FGI1 promotor and gus gene merge; By freeze-thaw method, expression vector is changed in agrobacterium tumefaciens EHA105 bacterial strain.Get after pollination the paddy rice immature seed of 12-15 days with 70% (v/v) alcohol disinfecting 1min, then use 2% (v/v) chlorine bleach liquor to sterilize more than 90min, aseptic water washing 4-5 time; Get rataria and induced 26 ℃ of dark cultivations on inducing culture that to obtain callus standby in about 7 days.Method (Hiei with reference to Hiei etc., Y., Ohta, S., Komari, T., and Kumashiro, T. (1994) .EfficientTransformation of Rice (Oryza-Sativa L) Mediated by Agrobacterium andSequence-Analysis of the Boundaries of the T-DNA.Plant Journal 6,271-282), carry out agriculture bacillus mediated genetic transformation, transformant screening and transgenic plant regeneration.

Pathogenic bacterium inducing is processed

In the T0 of FGI1promoter::GUS transfer-gen plant generation and T1 generation, carried out the functional verification of promotor, get the approximately transgenic paddy rice blade in 8 weeks of growth, use the tip tweezers to scratch the blade back, cut off from vein, half carries out F.graminearum schw (Fusarium.graminearum) bacterial strain PH-1, and (bacterium suspension concentration is 1 * 10 ⁸Cfu/ml) inoculation is processed, second half water treatment contrast.After 48h, the rice leaf after processing is carried out the active detection of GUS, repeated experiments is set 3 times.

GUS is active to be detected

Method (Jefferson with reference to Jefferson, R.A. (1987) .Assaying chimeric genes inplants:the GUS gene fusion system.Plant Molecular Biology Reporter 5,387-405), rice tissue is dyeed and detects with GUS is active.The 8h that dyes under 37 ℃, process to negative control material and be white in color with 95% ethanol decolorization.Microscopically is observed the preservation of taking pictures.

Embodiment

Embodiment 1, FGI1 genetic expression detected result

The inventor detected endogenous FGI1 gene invaded by F.graminearum schw or not intrusion situation under expression, the results are shown in Table 1.

Table 1

Corn stem cell (the Invaded: the cell of directly by F.graminearum schw, being invaded that F.graminearum schw infected one day that is subject to by real-time reverse transcription PCR detection laser micro-dissections acquisition; Near Zone: the cell adjacent with the cell of by F.graminearum schw, being invaded, not yet directly by F.graminearum schw, invaded), compare the FGI1 gene expression dose 2.5-4.5 doubly (Exp1 and Exp2 represent the independent experiment data twice) that all rises with the similar corn stem cell through the false inoculation of water (Mock).

Corn ANT-G2 native gene is expressed and to be subjected to F.graminearum schw to induce the lower 2.5-4.5 doubly (with comparing of not being subjected to that F.graminearum schw infects) that improved herein.Adopt promotor FGI to be subjected to F.graminearum schw to infect the abduction delivering increase in subsequent embodiment more obvious.

The clone of embodiment 2, FGI1 promotor and sequential analysis

1) inventor adopts the laser capture microdissection cutting technique to obtain the sample that is infected in early days from the maize cell that infected by F.graminearum schw.Confirm as and be subjected in early days infected cell under fluorescent microscope;

2) two parts that obtain are subjected to the maize cell sample (every duplicate samples is comprised of 800 cells) that F.graminearum schw infects to obtain total RNA through specific trace quantity reagent kit extracting, through external Bioanalyzer, detect and confirm that total RNA quality keeps complete;

3) two parts are subjected to total RNA (each 1ng) of the maize cell sample that F.graminearum schw infects to obtain can be used for the aRNA (each 8 micrograms) of Real-time PCR Analysis through the two-wheeled linear amplification, and amplification gained aRNA quality detects and confirms through external Bioanalyzer;

4) utilize analysis of biological information, this gene is a gland nucleotidyl transferase gene (ANT-G2, adenine nucleotide translocator 2) gene, and is called FGI1 in the application;

5) The DNA sequence of the gene coding region is as follows (SEQ? ID? NO: 2):ATGGCGGACCAGGCTAACCAACCCACTGTCCTTCATAAGCTAGGTGGCCAGTTCCACCTGAGCTCCAGCTTCTCTGAAGGTGTACGGGCCCGTAACATCTGCCCTTCTTTCTCACCTTATGAAAGGAGATTTGCCACGAGGAACTACATGACCCAGAGCCTTTGGGGCCCTTCAATGTCTGTTAGCGGTGGCATCAATGTTCCAGTGATGCCGACTCCGCTTTTTGCCAATGCTCCAGCCGAGAAAGGTGGCAAAAACTTCATGATTGATTTCATGATGGGCGGTGTTTCAGCTGCTGTTTCGAAGACTGCAGCTGCTCCCATTGAACGAGTGAAGCTGCTTATTCAGAATCAGGATGAGATGATCAAGTCTGGTAGGCTATCAGAGCCGTACAAGGGTATTGCTGATTGCTTCAAACGTACCATCAAGGATGAGGGTTTCTCTTCCTTGTGGAGGGGTAACACTGCTAATGTTATTCGTTACTTCCCTACTCAGGTAGCCCACACATTCCATTATATTTTGTACTGAGTAACTATTGTAAATATGTTGAAACTGCATGGTCTTTCAGTACCTAAATTATTGAATGCTCATTATGGTTTTCCTGATCTTGCCGTGTGTACAAGTTACTGATTATAATTTAACTTATATATTACTAATTGTCTTAATATCTTTGTCCTAGGCTTTGAACTTTGCGTTTAAGGATACTTCAAGAGGCTGTTCAACTTCAAGAAGGACAGGGATGGTTACTGGAAGTGGTTTGCTGGCAACCTGGCCTCTGGTGGTGCTGCTGGTGCTTCCTCTTTGTTTTTTGTGTACTCCCTGGATTATGCGAGGACAAGGTTGGCCAATGATGCCAAGGCTGCCAAGGGAGGAGGCGATAGACAATTCAATGGTCTTGTGGATGTCATCCGCAAGACACTCAAATCTGATGGTATTGCTGGGCTTTACCGTGGATTTAACATATCTTGTGTTGGAATTATTGTTTATCGAGGCCTGTATTTCGGACTTTACGATTCTATCAAGCCAGTTGTCCTCACTGGCAGCCTCCAGGTTTGCATAATGTCTCTCTCTCTCTCTAATGTAATTTTGCTTTTGCCTGTGTGAGCTATAAGATGTTTCTCTCTTTTGCAGGACAACTTCTTTGCCAGTTTTGCTCTGGGTTGGCTGATCACCAACGGTGCTGGTCTTGCATCTTACCCCATTGATACTGTCCGCAGAAGGATGATGATGACTTCTGGTGAGGGTGTCAAGTACAAGAGCTCATTGGACGCATTCCAGCAGATCCTTAAGAAGGAAGGCCCCAAGTCCCTGTTCAAGGGTGCTGGTGCTAACATTCTTCGTGCCATTGCTGGCGCTGGTGTGCTTTCTGGCTACGACCAGCTCCAGATCCTCTTCTTCGGAAAGAAGTACGGCTCCGGCGGTGCTTAAATGGAGAAAAA

6) based on described sequence, the inventor has designed specific primers, and to have obtained length be 743 promotor in order to clone its promotor.Promoter sequence following (SEQ ID NO:1):

TCCGTTTCTCCATAGGTGTGTGCGTCGCAGTGGATTTAATGCTCCGTTTTTCCTGGAGCCTTGGAATCTTTAGATCTTTATGGAGTCTTTTTAGATCGTGAGATAATTGGTTGGTAATCGGGTAAGCGGGAAGAAAAAATTGTCGTATTTTGACCGCTGTTTTGTCATAGTGTCGGTGCCGCGTTAGATCTGTCTGTTATGAGCCGTGGATGGCGATCTGACGGCAGGCGACCGTCTCATTAGTACTTGTATAACTACCATTGTTTCAGTTTTTTTTTCAGTTCTTGAAAGTACTGAGTAATAGAGTGCACACCTATACTTTTGACATGCTATGATGTTCTTCGCTGGTTTTGTAATTAAATTTGTAAATCATGGTAATCTTTGAATGCTAAATTTTCTGAACTGTTTGAGCATGCTA CAATGGGCATCTGTTCTATTTTGATGATTGTTGTCAAAACTAGAGTTATGGAACATGAAA AATATAAATGTGTATGTTTGTATGATTGCATTCATTGTTTATTTCTGTCTAATAGT TTTATCTGTTAGTGTTTGCACGGAGGCTCTCTGCTTTAGTTAGATGTGTCTAGCTT TGATTTGTTCTTACTC TATATTGAAACTGTAAAA TATACTCATCCTTTATTGCACAGATGCGGAGGTCTTATTTTGTTTCTCTTTATGTAATGT CCTAAGTTTTGTGTGTTGACATTATGTTGCAGCCAACAGTTTTGAGG

In above sequence, contain prediction TATABox, CAATBox, and restriction enzyme site EcoRI: With restriction enzyme site BamHI:

The inventor is with this promotor called after FGI1promoter.

Described FGI1promoter is connected between the T/A joint of pTG-19T carrier, through screening, obtains recombinant vectors pTG-19T-FGI1promoter (Fig. 1).

One section 1116 long long promotor of FGI1 (FGI1L) of base in kind obtains also carrier construction and is used for the transgenic paddy rice promoter Analysis.(the FGI1L promotor has increased by 373 bases than FGI1 promotor SEQ ID NO:1 at 5 ' end (after being positioned at restriction enzyme site EcoRI).FGI1L order following (SEQ ID NO:3):

TTTACCCAGGAAGGCATCGCCCTCTCTTTCACAAAGTTCGGA GTCGA GGCCGCCGCCCAGACCGCCTCCCCTTCCCCGCCTCCGGTGCCCAAGGCCAG GGTGAGTCTCGTGTCCCGCCAGGCTTCTTTCCCGTCCTCTGTTCCCGATTTAG GCTCGTGTTCTCGTTCCTATTTGTGGCGATCCGTCCGGCATGCCTTCTGTTATA TAGATCTGCTCTCCCCGTCCGTGTTCGTCACGTAGATCGATTATTTCCGGTTTA GGCTTGGATTGTTCTTGTGGTAGCCTCGGCCTTTGGAGTTCATTCAGATCGGT GATTCCTGCGTCAGGTAAGGGCGGTTGATACTCTCCTAGTTCATCTATACTTGA GATCTACTCCGTTTCTCCATAGGTGTGTGCGTCGCAGTGGATTTAATGCTCCGTTTTTCCTGGAGCCTTGGAATCTTTAGATCTTTATGGAGTCTTTTTAGATCGTGAGATAATTGGTTGGTAATCGGGTAAGCGGGAAGAAAAAATTGTCGTATTTTGACCGCTGTTTTGTCATAGTGTCGGTGCCGCGTTAGATCTGTCTGTTATGAGCCGTGGATGGCGATCTGACGGCAGGCGACCGTCTCATTAGTACTTGTATAACTACCATTGTTTCAGTTTTTTTTTCAGTTCTTGAAAGTACTGAGTAATAGAGTGCACACCTATACTTTTGACATGCTATGATGTTCTTCGCTGGTTTTGTAATTAAATTTGTAAATCATGGTAATCTTTGAATGCTAAATTTTCTGAACTGTTTGAGCATGCTA CAATGGGCATCTGTTCTATTTTGATGATTGTTGTCAAAACTAGAGTTATGGAACATGAAA AATATAAATGTGTATGTTTGTATGATTGCATTCATTGTTTATTTCTGTCTAATAGT TTTATCTGTTAGTGTTTGCACGGAGGCTCTCTGCTTTAGTTAGATGTGTCTAGCTT TGATTTGTTCTTACTC TATATTGAAACTGTAAAA TATACTCATCCTTTATTGCACAGATGCGGAGGTCTTATTTTGTTTCTCTTTATGTAATGT CCTAAGTTTTGTGTGTTGACATTATGTTGCAGCCAACAGTTTTGAGG

Described FGI1L promoter is connected between the T/A joint of pTG-19T carrier, through screening, obtains recombinant vectors pTG-19T-FGI1L promoter.

The structure of embodiment 3, integrative gene expression vector and conversion

Cut recombinant plasmid pTG-19T-FGI1promoter or pTG-19T-FGI1Lpromoter with EcoRI and BamHI enzyme, reclaim cloned sequence, be connected and be connected with BamHI double digestion plasmid vector with the EcoRI of pCAMBIA1300, cut checking through conversion and enzyme, obtain FGI1promoter::GUS fusion gene plasmid pCAM-FGI1promoter (Fig. 2, in the carrier collection of illustrative plates, FGI1promoter is denoted as Fgi1promoter) or pCAM-FGI1L promoter.By freeze-thaw method, the recombinant expression vector that obtains is transformed Agrobacterium EHA105 bacterial strain, and, through the plasmid enzyme restriction analysis, proved conclusively and be transformed into Agrobacterium, the Agrobacterium of acquisition is called EHA105/pCAM-FGI1promoter.Acquired clone is correct through the DNA sequencing checking.The Agrobacterium that obtains is called EHA105/pCAM-FGI1promoter.PCAM-FGI1L promoter carrier is transformed into Agrobacterium equally, and the Agrobacterium of acquisition is called EHA105/pCAM-FGI1L promoter.

The acquisition of embodiment 4, transgenic paddy rice

Take Agrobacterium EHA105/pCAM-FGI1promoter as mediation, by described in " materials and methods ", to build in the nascent callus that the FGI1promoter::GUS mosaic gene imports " in spend 11 " immature embryo source, screening regeneration obtains 17 independently transgenic paddy rice strains.Most transfer-gen plant growths are normal, and can be solid.,, to the total DNA of its whole individual plant extracting blade, use with the primer of the special pairing in GUS coding region and analyze independently in transformation plant at each, all can amplify special product, tentative confirmation is incorporated on rice chromosome with the T-DNA of mosaic gene.Root and leaf to callus and transgenic paddy rice seedling carry out the active detection of GUS, and wherein 14 strains show when callus dyes has GUS active, at Seedling Stage (Ye Hegen), does not show that all GUS is active.As table 2.

Table 2

Take Agrobacterium EHA105/pCAM-FGI1L promoter as mediation, the same transgenic paddy rice strain L1-L7 that obtains, with active the detection seedling (Ye Hegen) of GUS, GUS active (table 3) is arranged, namely have the constructive expression when not infected by F.graminearum schw, therefore FGI1L is not suitable as inducible promoter.

Table 3

Embodiment 5, F.graminearum schw are induced transgenic paddy rice GUS expression of results

On the basis of above-mentioned analysis, transgenic paddy rice is carried out the abduction delivering of F.graminearum schw.As Fig. 3, shown in Figure 4 and Figure 5, GUS is active to be detected for carrying out when being subjected to F.graminearum schw to infect 48h for FGI1promoter transgenic paddy rice T0 generation and T1, has GUS to express near wound and conduit, and water treatment does not have GUS to express.Drive the transgenic paddy rice of gus reporter gene with 35S promoter, all show visible the expression through GUS dyeing while being infected.

Fusarium graminearum is infected abduction delivering GUS dyeing situation such as the table 4 of transgenic paddy rice blade or coleoptile tissue.Coloration result such as the table 5 of water contrast.

Table 4

Table 5

Fig. 4 has represented that transgenosis T0 is for the result of testing for the first time.

Fig. 5 has represented transgenosis T1 generation experimental result for the second time.

Discuss

The present invention has cloned the 743bp promotor of corn ANT-G2 gene (in the application also referred to as the FGI1 gene), utilize agriculture bacillus mediated method rice transformation, obtain 17 transgenic lines, wherein 14 have GUS to express in callus, all do not show GUS and express when being infected.Utilize the leaf-cutting revulsion to verify this promoter function, the result demonstration, transfer-gen plant T0 generation and T1, for as seen expressing when infected by F.graminearum schw, have no expression while not infected by F.graminearum schw, result shows that this promotor is infected by F.graminearum schw and induces, and abduction delivering strengthens approximately 10 times.Result shows, can duplicate detection in 5 FGI1promoter independence transgenic lines to the abduction delivering after infected by F.graminearum schw.Because in independent transgenic line, the insertion point of each gene on rice chromosome is generally different, can consistent abduction delivering be detected at a plurality of transgenic lines, prompting FGI1 promotor is subjected to insertion point karyomit(e) environment to affect less.When the inventor has detected coleoptile and two kinds of tissues of blade and infected by F.graminearum schw, the FGI1 promotor can abduction delivering, and this also points out FGI1 promotor abduction delivering not limit by tissue.

The long promotor of the FGI1 of 1116bp has background and expresses when being infected, be not suitable as inducible promoter.

The FGI1 gene promoter is cloned the promotor that is subjected to Fusarium graminearum early evoking cance high-expression gene that obtains can be used for specific other genes (as antifungal genes) of transgenic crop (paddy rice, corn etc.) driving, make this gene be subjected to Fusarium graminearum to infect early expression at transfer-gen plant, bring into play thereby reach when having fungal disease to threaten the effect that antibiotic guarantor produces, and keep closing minute orientation high yield that is conducive to energy and nutrition when disease-free the threat, tilt.The gland nucleotidyl transferase gene (ANT-G2) of corn is to invade the gene of the early stage high expression level of corn at pathogenic fungi, the present invention obtains the promoters driven Genes For Plant Tolerance mycosis gene of this gene, the transgenic paddy rice that obtains, can be when being subject to F.graminearum schw and infecting, make a response rapidly, inducing anti-disease genetic expression, infecting of defence Fusarium graminearum, thereby more effective in the early stage in time prevention of morbidity morbidity, be different from again simultaneously composition type expression promoter, can be in disease-resistant and don't affect the output of farm crop.

All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (7)

1. the promotor of a separation, is characterized in that, described promotor is the promotor of the nucleotide sequence shown in SEQ ID NO:1.

2. a carrier, is characterized in that, described carrier contains promotor claimed in claim 1.

3. carrier as claimed in claim 2, is characterized in that, described carrier also contains the goal gene that is operably connected with described promotor.

4. a genetically engineered host cell, is characterized in that, described cell:

Contain the arbitrary described carrier of claim 2-3; Or

Be integrated with the promotor of the claim 1 of external source in its genome.

5. one kind prepares the plant method that can respond the pathogen infection signal, it is characterized in that, described method comprises: with the construction conversion of plant, the goal gene that described construction contains promotor claimed in claim 1 and with described promotor, is operably connected, described goal gene is expressed under the pathogen infection state;

Wherein, described plant is grass; Described pathogenic agent is selected from: F.graminearum schw (Fusarium graminearum) and fusarium oxysporum (Fusarium oxysporum).

6. method as claimed in claim 5, is characterized in that, described method comprises:

(a) provide the Agrobacterium of carrying expression vector, contain construction in described expression vector, the goal gene that described construction contains promotor claimed in claim 1 and with described promotor, is operably connected;

(b) vegetable cell, tissue or organ are contacted with the Agrobacterium in step (a), thereby make described construction change vegetable cell, tissue or organ over to;

(c) select vegetable cell, tissue or the organ that has changed described construction over to; And

(d) vegetable cell, tissue or neomorph in step (c) are become plant, in described plant, goal gene is expressed under the pathogen infection state.

7. the purposes of promotor claimed in claim 1, is characterized in that, described promotor is used for response pathogen infection signal and starts goal gene specific expressed; Wherein, described pathogenic agent is selected from: F.graminearum schw (Fusarium graminearum) and fusarium oxysporum (Fusarium oxysporum).

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