CN103243110B - Anti-magnaporthe oryzae paddy gene OsWRKY19 and application thereof - Google Patents

Abstract

The invention provides an anti-magnaporthe oryzae paddy gene OsWRKY19 and an application thereof. The anti-magnaporthe oryzae paddy gene provided by the invention is a plant transcription factor or derived protein with the same functions in an amino acid sequence shown in SEQ IDNo:1 of a coding sequence list. The magnaporthe oryzae resistance of paddy can be improved through expressing the gene in the paddy. The gene OsWRKY19 plays an important application role in plant disease-resistant genetic engineering, and anti-magnaporthe oryzae paddy varieties with practical production and application values can be cultured through utilizing the gene for molecular breeding.

Description

Paddy gene OsWRKY19 and the application thereof of resisting rice blast bacteria

Technical field

The invention belongs to plant gene engineering technology field, be specifically related to a kind of purposes of paddy gene and this gene of resisting rice blast bacteria.

Background technology

Paddy rice (Oryza sativa L.) is as important food crop, and particularly the grain security of Asian countries is closely bound up with the whole world, and how improving paddy disease-resistant insect pest ability is the important subject that various countries Agricultural Scientist is concerned about.Rice blast is to endanger the most serious rice disease, all has all the year round generation in various degree, can cause paddy rice underproduction 10-30%, and how preventing and treating rice blast is to be related to the especially significant problem of paddy rice main product state grain security of various countries.Cultivation disease resisting rice kind is the important means of control rice blast: by traditional hereditary means, breeding scholar both domestic and external has obtained some and have the rice varieties of better rice blast resistance.But, traditional breeding method excessive cycle and inefficiency, and due to the complicacy of physiological races of rice blast fungus heredity, pathogenic diversity, often cause disease-resistant variety resistance after popularizing planting 3-5 to disappear, cause rice blast recurrence and popular.The another kind of method that prevents rice blast is to carry out chemical prevention by spraying mycocide, but the method not only costliness but also contaminate environment, and the shortage of permanently effective sterilant also makes it to become the means of effectively preventing rice blast.So, also carry out on its basis molecular breeding by the rice blast resistance of molecular biology method Study On Rice, be expected to overcome tradition and prevent and treat all restrictions of method.

The interactional research of paddy rice-Pyricularia oryzae, as the model of research plant-fungi pathology molecular mechanism, is subject to botanist and phytopathologist's extensive concern nearly ten years.In the interaction of paddy rice-Pyricularia oryzae, study many two genoids that have: the one, the resistant gene of identification pathogenic bacteria, the 2nd, the gene relevant to defense response, this two genoid plays an important role in the defense response of paddy rice.Wherein the effect of resistant gene is the identification of mediated plant to avirulence gene of rice blast product, and causes the specific resistance of paddy rice to Pyricularia oryzae.Their Interactions Mode is followed gene pairs gene hypothesis, and host's resistant gene and the nontoxic gene of pathogenic bacteria are depended in the interaction of plant and pathogenic micro-organism, only has plant in the time that they exist simultaneously just to show resistance.After resistant gene product and nontoxic gene product are identified mutually, first invade in the vegetable cell at position and the cell of vicinity and can induce rapidly and form allergy germ, other defense response of inducing paddy rice simultaneously, as the release of active oxygen, the reinforcing of plant cell wall, synthetic and other pathogenesis-related proteins of plant protecting chemical, plant alexin, chitinase, proteinase inhibitor and the induction of defence associated protein are synthetic, to full out control or kill the germ of intrusion.The conduction of the startup of plant allergy and the defence signal that causes thereof, finally makes other tissue of host produce resistance of wide spectrum, i.e. systemic acquired resistance to pathogenic bacteria subsequently.

Except above two genoids, the gene that also has some to participate in paddy rice signal pathway also participates in the defense response of paddy rice to Pyricularia oryzae.The people such as Reyna in 2006 are studied 17 MAPK in paddy rice, and finding wherein has 9 genes can be by Pyricularia oryzae abduction delivering, illustrate that these MAPK may have certain function in the signal conduction of the defense response of paddy rice.2009, the people such as Li have studied the function of OsWAK1 gene, this gene can be also had Pyricularia oryzae abduction delivering by injury, SA, MeJA, in paddy rice, constitutive expression OsWAK1 can produce resistance to rice blast pathogenic microspecies by render transgenic plant, illustrates that this gene has vital role in paddy rice defense response.In addition, overexpression OsWRKY31 or OsWRKY53 gene in paddy rice, all can strengthen the resistance of plant to Pyricularia oryzae.These genes encodings participate in the regulation and control of paddy rice defense response signal path, and preliminary research finds that they have critical function in paddy disease-resistant process, and their concrete function and mechanism of action also require further study.

The gene of the participation paddy rice defense response finding by the whole bag of tricks all has potential production application and is worth, and can obtain rapidly a large amount of transgenic lines by ripe Transgenic Rice technology.Since the eighties in 20th century, along with the rise of biotechnology and perfect, the particularly widespread use of genetic engineering technique aspect crop improvement, provides new means for cultivating disease-resistant variety.Plant disease-resistant transgenosis becomes biotechnology personnel's study hotspot, and the gene transformation paddy rice that is established as of the rice conversion system of efficient stable has been created condition.Particularly, the genetically engineered of paddy disease-resistant is mainly carried out from resistance (R) gene and two aspects of Analysis of Defence Genes Involved.

The people such as Mackil have cultivated a set of near isogenic line with Pi-1, Pi-2, Pi-3, Pi-4a and Pi-4b rice blast resistance gene using CO39 as genetic background, experimenter carries out inoculation experiments with this cover near isogenic line, finds to carry that the disease-resistant time of the near isogenic line C104PKT of Pi-3 is long, scab number is few and also rice blast is had to very high resistance with the cumulative system of other assortments of genes; And then utilize C104PKT and the near isogenic line A57-119 hybridization with Pi-1 and Pi-2 to cultivate the near isogenic line of BL13 (carrying Pi-1, Pi-3), BL-23 (carrying Pi-2, Pi-3) and BL-123 (carrying Pi-1, Pi-2, Pi-3), introduce a fine variety at present the states such as the U.S., Vietnam, Philippines, Indonesia, Japan, all shown very high resistance through the plantation of 3 to 5 years.And chitinase gene Cht-2, the Cht-3 of paddy rice are proceeded to respectively japonica rice Nipponbare and Koshihikari by the people such as Nishizawa, find in transfer-gen plant that Cht-2 is at thin intracellular accumulation, and Cht-3 accumulates in extracellular.In the R0 of Cht-2 or Cht-3 transfer-gen plant and R1 generation, improve the resistance of the pathogenic microspecies of rice blast fungus, and the expression amount of resistance and chitinase has dependency.The I class chitinase gene Chi11 in paddy rice is proceeded to long-grained nonglutinous rice by the people such as people and Datta such as Lin, and transfer-gen plant shows certain resistance to banded sclerotial blight, and the content of chitinase is proportionate with resistance with active.Rice chitinase gene RC24 and clover beta-1,3-glucanase gene are proceeded to paddy rice by the people such as Feng simultaneously, and part R1 represents that to 5 of Magnaporthe grisea in Guangdongs the resistance that microspecies show in various degree strengthens for plant.

Existing studies have shown that, utilizes transgenic technology to carry out molecular breeding and is expected to find the Transgenic Rice with production application value.But existing transgenosis achievement is also rarely put in the middle of production application, and the key gene that participates in now rice anti-rice blast need to find, how to find that these genes and large-scale rice transformation identifys to need to further investigate.

Summary of the invention

The object of the present invention is to provide a kind of gene and proteins encoded thereof of blast resisting, for improving the disease-resistant performance of the plants such as paddy rice.

Blast resistant gene provided by the present invention, name is called OsWRKY19, derives from paddy rice (Oryza sativa), coding following proteins (i) or (ii):

(i) protein of aminoacid sequence shown in the SEQ ID No:1 in sequence table;

(ii) the SEQ ID No:1 aminoacid sequence in sequence table is through replacement, disappearance or the interpolation of one to ten amino-acid residue and derivative protein, and derivative protein has identical function.

SEQ ID No:1 in sequence table is made up of 277 amino-acid residues, and wherein the 105th to 165 is conservative WRKY structural domain.One to ten amino-acid residue of described replacement, disappearance or interpolation can be the amino-acid residue in non-conservative region, and its change can not exert an influence to the function of this albumen.The method that amino-acid residue is replaced, lacked or adds, and be all well-known to those skilled in the art to the detection of protein function, normally utilize engineered means to suddenly change to its encoding gene, and then give expression to corresponding albumen and detect its function.

The nucleotide sequence of rice blast resistant gene OsWRKY19 of the present invention can be its cDNA sequence, can be also genomic dna sequence, or has 90% above homology and the DNA sequence dna of the identical function albumen of encoding with these sequences.In sequence table shown in SEQ ID NO:2 is the cDNA sequence of OsWRKY19 gene, and wherein the 73rd to 903 being CDS sequences of proteins encoded, and shown in SEQ ID NO:3 is the genomic dna sequence of OsWRKY19 gene.

The present invention also provides the expression vector of the expression regulation sequence that comprises above-mentioned nucleotide sequence and be connected with this nucleotide sequence operability.In embodiment preferably, described expression regulation sequence comprises the regulating and controlling sequence of composing type high expression level, for example corn Ubiquitin1 promotor.

Application OsWRKY19 gene can improve the disease resistance of plant, comprise the resistance to Pyricularia oryzae, its method can be by OsWRKY19 gene transfered plant cell, tissue or organ, again the vegetable cell being converted, tissue or organ are cultivated into plant, described OsWRKY19 gene is expressed in plant, obtain the transgenic plant that rice blast resistance is improved.Wherein, OsWRKY19 gene imports vegetable cell, tissue or organ by plant expression vector.

The present invention is by nucleotide sequence overexpression in paddy rice of coding OsWRKY19 gene, and the main fungal disease of Inoculated Rice---Pyricularia oryzae, after detecting, find, OsWRKY19 gene has the function that improves disease resistance, shows the resistance of rice blast is strengthened with the transgenic paddy rice of OsWRKY19 expression casette.Therefore, OsWRKY19 gene provided by the present invention has important using value in disease resistance of plant genetically engineered, utilizes this gene to carry out molecular breeding and is expected to cultivate the anti-rice blast rice kind with production application value.

Brief description of the drawings

Fig. 1 has shown in embodiment 3 and has detected the expression of OsWRKY19 gene in part transgenic rice plant by real-time quantitative PCR.

Fig. 2 has shown that in embodiment 3, Resistance Identification and the Molecular Identification of OsWRKY19 transgenic paddy rice seedling to Pyricularia oryzae represents result, and wherein A is the phenotype of 7 days after TP309, OsWRKY19 transgenic paddy rice 6-1 strain and 6-2 strain seedling leaves inoculation Pyricularia oryzae 96-4-1a microspecies; B is for inoculating Pyricularia grisea Race after 96-4-1a7 days, OsWRKY19 gene relative expression quantity in representative blade in corresponding OsWRKY19 transgenic line and wild-type strain.

Embodiment

In following embodiment, method therefor is ordinary method if no special instructions, and concrete steps can be referring to: " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W, Molecular Cloning:A Laboratory Manual, 3 ^rdedition, 2001, NY, Cold Spring Harbor).

The clone of embodiment 1, gene

(1) clone of OsWRKY19 gene cDNA sequence:

Genomic dna sequence, cDNA sequence and the aminoacid sequence of OsWRKY19 gene from MSU/TIGR rice genome database, obtain ( http:// rice.plantbiology.msu.edu/).

PCR primer according to the cDNA encoding sequence design gene specific of OsWRKY19 gene:

F1：5′-ATGGTGGAGCTCTGCGGC-3′(SEQ?ID?No：4)；

R1：5′-CAGATTCTGAATCTCCGATT-3′(SEQ?ID?No：5)。

With this, primer is cloned the cDNA encoding sequence of OsWRKY19 gene from the cDNA library of paddy rice japonica rice (Oryza sativa L.ssp.Japonica) near isogenic line kind IRBL22 (being cultivated by international paddy rice).Because subregion GC content in the cDNA encoding sequence of OsWRKY19 gene is up to more than 80%, with common high-fidelity enzyme clone less than, after many experiments, present inventor utilizes GC damping fluid I (TaKaRa) and Pfu high-fidelity enzymatic amplification to obtain the full-length cDNA encoding sequence of OsWRKY19 gene.PCR product is connected into pBluescript carrier with T4 ligase enzyme, screening is with forward Insert Fragment (ATG of gene is near T7 sequencing primer) carrier order-checking, and the plasmid called after pBS-OsWRKY19 that checks order correct is for building the plant expression vector of OsWRKY19 gene.

(2) structure of OsWRKY19 gene plant expression vector:

In order to build the plant expression vector of OsWRKY19 gene, present inventor has transformed plant expression vector pWM101 for rice conversion.Concrete, pWM101 is done to enzyme with HindIII/KpnI and cut and reclaim digested plasmid, corn Ubiquitin1 promoter sequence clone is connected into pBluescript carrier, and the pBS-pUbi carrier HindIII/BamHI enzyme obtaining is cut, and reclaims the DNA segment with promoter sequence.Again the cDNA sequence of OsWRKY19 gene is cut from pBS-OsWRKY19 carrier B amHI/KpnI.Above three nucleic acid fragments reclaim after purifying, spend the night with 4 DEG C of connections of T4 ligase enzyme, connect product and transform bacillus coli DH 5 alpha competent cell, use gene specific primer F1 and R1 to carry out PCR screening, from the positive colony that obtains of screening, extract the pWM101 vector plasmid with OsWRKY19 gene order, with BamHI enzyme cut checking correct after for the callus conversion of paddy rice.

The acquisition of the transgenic paddy rice of embodiment 2, constitutive expression OsWRKY19 gene

(1) rice callus induction:

Choose the full Taibei 309 (TP309) rice paddy seed, peel off seed coat, after sterilizing washing, point is with 2 mg/litre 2 uniformly, the sterilizing NB solid medium of 4-D, and 5 days evoked callus of 32 DEG C of continuous lights form.

(2) Agrobacterium-mediated Transformation:

PWM101 carrier with OsWRKY19 gene is transformed to Agrobacterium EHA105 competent cell by heat shock method simultaneously, coat on the solid LB substratum with 50 micrograms per litre kantlex, 28 DEG C of dark culturing are after 2 days, with gene specific primer F1 and R1 screening positive clone.The positive colony obtaining is 28 DEG C of dark culturing of solid AB substratum that contain 50 mg/litre kantlex 3 days, for rice conversion.

(3) rice callus transforms:

Transform substratum with the liquid that contains 100 micromoles per liter Syringylethanones of filtration sterilization and from AB substratum, Agrobacterium is washed down, bacterial concentration is adjusted to OD600 in 0.08 left and right.Choose the callus that upgrowth situation is good, in bacterium liquid, soak 2 minutes, then on aseptic filter paper, dry, then callus is moved to the NB culture medium altogether that contains 100 micromoles per liter Syringylethanones, under 25 DEG C of dark, cultivate altogether 3 days.

(4) Screening of Rice callus:

Cultivate altogether after 3 days, with sterilized water washing callus 5 times, again with 200 milliliters of aseptic washings that contain 500 mg/litre carbenicillin disodiums one time, carefully remove liquid, with aseptic nipper by callus gripping to aseptic filter paper, after drying, be transferred to NB screening culture medium (containing 2 of 2 mg/litre, the damp enzyme element of 4-D, 50 mg/litre and the carbenicillin disodium of 400 mg/litre), 32 DEG C of continuous lights 2 weeks.

(5) differentiation of positive callus:

Choose the positive callus that well-grown is bright yellow, move to the pre-division culture medium of NB (the damp enzyme element that contains 1 mg/litre NAA, 5 mg/litre ABA, 2 mg/litre kinetin, 25 mg/litre and the carbenicillin disodium of 200 mg/litre) with aseptic nipper upper, 32 DEG C of continuous lights are cultivated.After 2 weeks, select eugonic callus and proceed to MS division culture medium (the damp enzyme element that contains 0.02 mg/litre NAA, 2 mg/litre kinetin, 50 mg/litre and the carbenicillin disodium of 200 mg/litre), 32 DEG C of continuous lights are cultivated.Treat that seedling out of differentiation grows to 2-5 millimeter, proceed to not containing hormone and antibiotic MS culture medium culturing 2-3 week, then move into and in soil, be placed in greenhouse growth (temperature 28-30 DEG C, 16 hours illumination/8 hour dark).

Embodiment 3, constitutive expression OsWRKY19 gene strengthen the resistance of paddy rice to rice blast

(1) detection of OsWRKY19 gene expression dose in transgenic paddy rice:

The blade of clip transgenic paddy rice seedling, with the total RNA of Trizol reagent (Invitrogen) extraction plant, DNase I (TaKaRa) processes after DNA digestion, carries out reverse transcription with the reverse transcription test kit of Invitrogen, obtains the cDNA of transfer-gen plant.According to real-time quantitative PCR primer realF and the realR of the cDNA sequences Design gene specific of OsWRKY19 gene, detect the expression level of OsWRKY19 gene in transfer-gen plant by real-time quantitative PCR.Reference gene is selected the UBQ gene (primer uses realF2 and realR2) of paddy rice, and each primer sequence is as follows:

realF：5′-GCCAGTTCAGCAGCGACTTC-3′(SEQ?ID?No：6)；

realR：5′-GTGCATTCGGCCACCTACAG-3′(SEQ?ID?No：7)；

realF2：5′-GTGGCCAGTAAGTCCTCAGC-3(SEQ?ID?No：8)；

realR2：5′-ACAATGAAACGGGACACGAC-3′(SEQ?ID?No：9)。

Real-time quantitative PCR detects the expression of OsWRKY19 gene in transgenic rice plant as shown in Figure 1; The not genetically modified Taibei of TP309 representative 309 rice plants.

(2) the rice blast inoculation experiments of transgenic paddy rice:

Select Pyricularia oryzae toxicity microspecies 96-4-1a, transgenic paddy rice seedling is carried out to Live leaf inoculation.Pyricularia oryzae 96-4-1a 28 DEG C of dark culturing about 1 week on oat medium, with aseptic washing lower surface mycelia, be applied on a new oat medium, cultivate after 1 day for 28 DEG C, scrape off gently surperficial mycelia with aseptic cotton carrier, be placed under fluorescent lamp and produce spore.After 24 hours, with spore under the aseptic washing that contains 0.02%Tween-20, calculate spore concentration with blood counting chamber, spore concentration is adjusted to 1 × 10 ⁵spore/milliliter.Get four leaf phase rice seedlings, carry out the spray inoculation of magnaporthe grisea spore, every young plant spraying 2-4mL, is coated with after spore suspension that one deck is tiny drips uniformly until the blade surface of all seedling, is placed on 100% humidity, 24h in the dark culturing case of 26 DEG C.After one day, recover the normal light cycle and cultivate, after 7 days, observe phenotype.Result as shown in Figure 2, is crossed the transgenic paddy rice seedling of expressing OsWRKY19 gene to the resistance enhancing of rice blast microspecies 96-4-1a.Can reach a conclusion thus, the constitutive expression of OsWRKY19 gene has strengthened the resistance of paddy rice to rice blast.

The present invention, by rice transformation callus, obtains the mistake express transgenic paddy rice of OsWRKY19 gene, finds that by connecing bacterium test this transgenic paddy rice strengthens the resistance of rice blast.The protein of this genes encoding is plant transcription factor, and the product of this gene can improve the resistance against diseases of plant, has the using value for cultivating anti-rice blast rice.

Claims (7)

1. Rice Os WRKY19 gene improve paddy rice to the application in the resistance of Pyricularia oryzae, the protein of aminoacid sequence shown in the SEQ ID No:1 in described OsWRKY19 gene coded sequence table.
2. 2. application as claimed in claim 1, is characterized in that, the sequence of described OsWRKY19 gene is cDNA sequence or the genomic dna sequence of this gene.
3. 3. application as claimed in claim 2, is characterized in that, the nucleotide sequence of described OsWRKY19 gene, as shown in SEQ ID No:2 in sequence table or SEQ ID No:3.
4. 4. the application as described in as arbitrary in claim 1～3, it is characterized in that, by described OsWRKY19 gene transfered plant cell, tissue or organ, again the vegetable cell being converted, tissue or organ are cultivated into plant, described OsWRKY19 gene is expressed in plant, obtain the transgenic plant that blast resistance improves.
5. 5. application as claimed in claim 4, is characterized in that, described OsWRKY19 gene imports vegetable cell, tissue or organ by plant expression vector.
6. 6. application as claimed in claim 5, is characterized in that, described plant expression vector comprises described OsWRKY19 gene order and the expression regulation sequence being connected with this gene order operability.
7. 7. application as claimed in claim 6, is characterized in that, in described plant expression vector, with the expression of OsWRKY19 gene described in corn Ubiquitin1 promoters driven.

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