CN100342012C - Rice disease-resistant related gene OsDR3 - Google Patents

Abstract

The present invention relates to the field of plant biotechnology, particularly to the separation clone, the function verification and the application of a DNA fragment of paddy rice. The DNA fragment comprises relevant genes OsDR3 for paddy rice to resist diseases and code WRKY proteinoid, which enable plants to resist diseases caused by pathogenic bacteria. If the fragment and an extraneous regulating sequence thereof are directly transferred into the plants, the resistivity of transgenic plants to the pathogenic bacteria is obviously enhanced.

Description

Paddy disease-resistant related gene OsDR3

Technical field

The present invention relates to plant biotechnology field.Be specifically related to the segmental separating clone of a kind of paddy DNA, functional verification and application.Described dna fragmentation comprises paddy disease-resistant related gene OsDR3, and it can significantly improve the ability of plant opposing disease.Will this segmental complete coding region (coding sequence) with directly change susceptible plant materials over to after ubiquitin (ubiquitin) promotor in the corn combines, the resistance against diseases of transfer-gen plant significantly improves.

Background technology

Plant is subjected to the infringement of multiple pathogen in the process of growth.The phytopathy original of a great variety comprises virus, bacterium, mould and nematode etc.The pathogen invaded plants causes two kinds of results: breeding in host plant of (1) pathogenic agent success causes relevant illness; (2) host plant produces disease resistance response, kills pathogen or stops its growth.Utilizing the disease resistance of resistant gene resource improvement plant, is the basic outlet that pre-disease prevention is protected environment simultaneously again.

The disease resistance response of plant is the complex process that polygene participates in regulation and control.The gene that participates in the plant disease-resistant reaction is divided into two classes:

(1) disease-resistant gene claims R (resistance) gene and (2) disease-resistant related gene again.

According to the understanding of present people's enantiopathy gene function, the product of this genoid mainly is as acceptor, and direct or indirect and cause of disease protein-interacting starts plant intravital disease-resistant signal conducting path (Tang etc., 1996, Science 274:2060-2063; Baker etc., 1997, Science 276:726-733; Jia etc., 2000, EMBO is J.19:4004-4014; Dangl and Jones, 2001, Nature 411:826-833; Nimchuk etc., 2001, Curr.Opin.Plant Biol.4:288-294).The disease resistance response strong resistance of disease-resistant gene mediation is good genetic resources.But owing to following reason, make and utilize disease-resistant gene improvement plant resistance to environment stress to be restricted: the resource-constrained of (1) disease-resistant gene, disease-resistant gene as the important disease bacterial leaf-blight of opposing paddy rice known at present is less than 30, and the disease-resistant gene of resisting the important disease-rice blast of another paddy rice also has only about 40; (2) disease-resistant gene has cause of disease kind and cause of disease physiological strain specificity, disease-resistant being limited in scope; (3) because the quick sudden change of cause of disease, the effect of a disease-resistant gene has often just been lost after several years or more than ten years.

Disease-resistant related gene is meant that all participate in the gene of disease resistance response except that disease-resistant gene, and their coded product participates in disease-resistant signaling molecule in the synthetic plant materials, participates in the signal conduction or participates in defense response etc.The common feature of this genoid is that their expression amount of pathogeny evoked back raises or reduction, therefore people can be according to the difference of pathogeny evoked front and back expression of gene amount plant identification disease-resistant related gene (Maleck etc. on a large scale, 2000, Nature Genet.26:403-410; Schenk etc., 2000, Proc.Natl.Acad.Sci.USA 97:11655-11660; Zhou etc., 2002, Science in China 45:449-467).At present, the understanding of people's enantiopathy genes involved is limited.According to existing report, most of disease-resistant related genes are made the resistance capacity of time spent separately may be littler than disease-resistant gene.But according to following reason, they are to be worth the genetic resources of exploitation energetically: (1) because the product of most disease-resistant related genes does not need directly and pathogen interacts, this genoid is the genetic resources with durable resistance; (2) disease resistance response that participates in of most of disease-resistant related genes does not have the cause of disease specificity, so they are the genetic resourceses with resistance of wide spectrum; (3) aboundresources of this genoid.

The disease resistance response of plant can be divided into two big classes.Investigators have given different titles to this two big class disease resistance response for a long time, as vertical resistance and horizontal resistance (Van Der Plank etc., 1968, Disease Resistance in Plants, Academic, New Youk), quality resistance and quantity resistance (Ou etc., 1975, Phytopathology 65:1315-1316) or complete resistance and partial resistance (Parlevliet, 1979, Annu.Rev.Phytopathol.1:203-222).Quality resistance (or vertical resistance or complete resistance) is the disease resistance response of disease-resistant gene mediation.Quantity resistance (or horizontal resistance or partial resistance) is by quantitative trait locus (quantitative trait locus, QTL) Tiao Kong disease resistance response, it is considered to not have the cause of disease specificity, and resistance lasting (Roumen, 1994, Rice Blast Disease, Zeigler etc. write, CAB International, Cambridge, UK, pp.245-265).At present, people are not clear to the gene essence of plant disease-resistant QTL.Therefore, though identified a large amount of disease-resistant QTL in the paddy rice, as resisting bacterial leaf-blight QTL (Li etc., 1999, Mol.Gen.Genet.261:58-63), blast resisting QTL (Wang etc., 1994, Genetics 136:1421-1434; Chen etc., 2003, Proc.Natl.Acad.Sci.USA100:2544-2549), anti-banded sclerotial blight QTL (Li etc., 1995, Theor.Appl.Genet.91:382-388) and viral diseases QTL (Albar etc., 1998, Theor.Appl.Genet.97:1145-1154) etc., but these resistances QTL is not performed well in the improvement of rice varieties disease resistance.

In recent years the chromosome position of discovering a lot of disease-resistant related genes is corresponding with disease-resistant QTL, and pointing out these disease-resistant related genes may be exactly corresponding QTL.Corresponding this phenomenon with disease-resistant QTL of the chromosome position of disease-resistant related gene all is observed in various plants, comprises paddy rice (Xiong etc., 2002,45:518-526; Ramalingam etc., 2003, Mol.Plant-Microbe Interact.16:14-24; Wen etc., 2003, Mol.Gen.Genomics 269:331-339; Chu etc., 2004, Mol.Gen.Genomics 217:111-120), wheat (Faris etc., 1999, Theor.Appl.Genet.98:219-225), beans (Geffroy etc., 2000, Mol.Plant-Microbe Interact.13:287-296) and potato (Trognitz etc., 2002, Mol.Plant-Microbe Interact.15:587-597).These results separate, clone and utilize the gene of disease-resistant QTL that foundation is provided for adopting candidate gene strategy.

The separating clone disease-resistant related gene is a prerequisite of utilizing this genoid improvement disease resistance of plant.Simultaneously, compare with the application of disease-resistant gene, the application of disease-resistant related gene can provide plant more wide spectrum and long lasting resistance.Carry out the improvement of variety of crops by the overexpression disease-resistant related gene, will further strengthen the disease resistance of plant, widen the anti-spectrum of plant; These aspects are that employing conventional plant breeding and improving technology institute are inaccessiable.

Summary of the invention

The dna fragmentation that the objective of the invention is a disease-resistant related gene complete coding region of separating clone section from paddy rice utilizes this improvement of genes paddy rice or other plant to resist the ability of disease.This gene is named as OsDR3 (Oryza sativa defenseresponsive 3).

The present invention relates to separate and use a kind of dna fragmentation of the OsDR3 of comprising gene, this fragment is given plant to producing disease resistance response by bacterial leaf spot pathogenic bacteria (Xanthomonas oryzae pv.oryzae) and the caused disease of Pyricularia oryzae (Pyricularia grisea).Wherein, described fragment perhaps is equivalent to the dna sequence dna shown in the SEQ ID NO:1 basically shown in sequence table SEQ ID NO:1, and perhaps its function is equivalent to the subfragment of sequence shown in the SEQ ID NO:1.

The protein of OsDR3 genes encoding has the conserved domain of WRKY proteinoid, shows that it belongs to WRKY proteinoid family.

Can adopt the OsDR3 gene of having cloned to make probe, screening obtains gene of the present invention or homologous gene from cDNA and genomic library.Equally, adopt PCR (polymerase chain reaction) technology, also can from genome, mRNA and cDNA, increase obtain OsDR3 gene of the present invention and any interested section of DNA or with its homologous section of DNA.Adopt above technology, can separate the sequence that obtains comprising the OsDR3 gene, this sequence is connected with suitable carriers, can change vegetable cell over to, produce transgenic plant.

The present invention provides a kind of new method for strengthening plant to bacillary and resistance fungal disease.This method comprise with the OsDR3 gene with change susceptible plant over to after overexpression, constitutive promoter (as the corn ubiquitin promoter) are connected, widen and strengthen the resistance of plant by overexpression OsDR3 gene pathogenic bacteria.

Change clone's disease-resistant related gene over to susceptible plant, help to produce new disease-resistant plants.Particularly can be with genetic transformation technology a plurality of resistant genes that in plant, add up, and can not produce the linked gene group sequence of following appearance in the traditional breeding technology.The clone of disease-resistant related gene overcomes traditional breeding method can not shift the disease-resistant related gene problem between plant species prerequisite.

Disease-resistant transfer-gen plant that the present invention can further provide or the above-mentioned dna fragmentation of applications exploiting obtains and corresponding seed, and with gene of the present invention or based on the recombinant chou plants transformed of this gene or the seed that obtains by this class plant.Can gene of the present invention be changed over to other plant with the mode of sexual hybridization.

In the embodiments of the invention part, we have set forth separation, functional verification and the application process of OsDR3 gene and the characteristics of this gene.

Description of drawings

The coding region sequence of the OsDR3 gene of sequence table SEQ ID No:1. separating clone of the present invention.

Fig. 1 .OsDR3 gene is positioned at No. 1 karyomit(e) of paddy rice, its chromosome position and two blast resisting QTL (Wang etc., 1994, Genetics 136:1421-1434 that independent studies is identified; Chen etc., 2003, Proc.Natl.Acad.Sci.USA100:2544-2549) corresponding.

Fig. 2. adopt the expression characteristic of RT-PCR (reverse transcription-PCR) technical Analysis OsDR3 gene behind different rice material inoculation pathogenic bacterias or water receiving (control, contrast).(A) OsDR3 expression of gene behind the inoculation Pyricularia oryzae; (B) OsDR3 expression of gene behind the inoculation bacterial leaf spot pathogenic bacteria; 1,2,3,4,5,6,7 and 8 behind representative inoculation or the water receiving 2 hours, 4 hours, 8 hours, 16 hours, 1 day, 3 days, 5 day and 7 days respectively.Actin is a beta-actin.

Fig. 3. cover the overlay chart of the Shotgun subclone of OsDR3 constant gene segment C in the rice varieties bright extensive 63.The position of arrow and length represent that the direction and the length that check order, sequence number represents Shotgun subclone numbering.Obtain the sequence of a long 2946bp by sequence assembly.

Fig. 4. adopt GENSCAN (http://genes.mit.edu/GENSCAN.html) gene structure forecasting software, with reference to Arabidopis thaliana as the partial results of analyzing the gene that is comprised in the template analysis subclone 7G7H14 sequence.The structure of showing the OsDR3 gene of prediction among the figure.Gn represents the gene numbering; Ex represents exon; Init represents gene initiating terminal exon; Term represents gene clearing end exon; Prom represents basic promotor; PlyA represents PolyA; S representation DNA chain, wherein the dna sequence dna chain of importing in "+" expression analytic process; Begin represents exon, promotor or the PolyA zero position in the input dna sequence dna; End represents exon, promotor or the PolyA final position in the input dna sequence dna; Len represents the sequence length (bp) of exon, promotor or PolyA; Frame (every dna sequence dna has 3 translations to read frame) is read in Fr representative translation; I/Ac represents 3 ' shearing site score value; Do/T represents 5 ' shearing site score value; CodRg representative translation distinguishing value; P represents the exon probability; Tscr represents the exon score value.

Fig. 5. with the intron of RT-PCR methods analyst OsDR3 gene.M represents 2kb ladder (6 stripe size from top to bottom are respectively 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp) among the figure, the 1st, with bright extensive 63 total DNA amplified production (359bp) that is template (contrast), the 2nd, be the RT-PCR amplified production (264bp) of template with the bright extensive 63RNA after the inoculation cause of disease.

The structure of Fig. 6 .OsDR3 gene and be used for the dna fragmentation length and the structure of overexpression genetic transformation vector construction.Lines are represented intron; The post bar is represented exon (encoding sequence); The base number of each structure of numeral.Long arrow is represented the position of segmental length of genetic transformation paddy DNA (2879bp) and corresponding gene structure." ATG " and " TGA " is respectively translation initiation password and termination codon.

The aminoacid sequence of the WRKY proteinoid of Fig. 7 .OsDR3 genes encoding.Single underscore place shows the conserved amino acid sequence of WRKY structural domain; The double underline place shows conservative halfcystine (C) and Histidine (H) site in the zinc fingers.

Fig. 8. (A) structure of genetic transformation carrier pU1301; (B) structure of genetic transformation carrier pCAMBIA1301.

Fig. 9. the T of transgenic rice plant D11UM8-47 ₁Separate for showing anti-sense behind the plant inoculation Pyricularia oryzae bacterial strain V86013.Bright extensive 63 is disease-resistant contrast; CO39 is susceptible contrast; Mudanjiang is for No. 8 genetic transformation acceptor material (contrast).

Figure 10. it is all high than No. 8, genetic donor bright extensive 63 and genetic recipient Mudanjiang to detect the OsDR3 expression of gene amount that shows in the bacterial leaf spot resistant germ ability enhanced transfer-gen plant with the RNA hybridization technique.RNA is hybridized the part fragment from the OsDR3 gene cDNA with probe system.

Embodiment

Previous research work result of the present invention shows that deriving from bright extensive 63 the cDNA clone of rice varieties EI12I1 is the cDNA fragment of OsDR3 gene, and OsDR3 is a disease-resistant related gene.Main according to the following aspects is arranged: (1) adopts the analysis of cDNA chip technology to find that cDNA clone EI12I1 expression amount behind rice strain IRBB10 inoculation bacterial leaf-blight bacteria strain PXO86 has increased by 2.9 times, expression amount has increased by 7.4 times of (Zhou etc. behind rice strain C101A51 inoculation Pyricularia oryzae bacterial strain V86013, The defense-responsive genes showing enhanced and repressed expression afterpathogen infection in rice (Oryza sativa L.), 2002, Science in China 45:449-467).Insertion fragment to the EI12I1 clone checks order, and obtains the cDNA sequence of a long 822bp.Sequential analysis shows that its coded product and WRKY proteinoid homology degree are very high.In view of EI12I1 is cloned in pathogenic bacteria inoculation front and back expression amount notable difference and sequence signature are arranged, we think that cDNA clones the disease resistance response regulation and control of the gene involved in plant of EI12I1 representative, be a disease-resistant related gene (Zhou etc., 2002, Science in China 45:449-467).(2) adopt BLAST analytical procedure (Altschul etc., 1997, Nucleic Acids Res.25:3389-3402) with the rice genome sequence comparing analysis of known chromosome position among this cDNA sequence and the common core thuja acid database GenBank (http://www.ncbi.nlm.nih.gov), with the OsDR3 assignment of genes gene mapping in No. 1 karyomit(e) of paddy rice, its chromosome position and known blast resisting QTL (Wang etc., 1994, Genetics136:1421-1434; Chen etc., 2003, Proc.Natl.Acad.Sci.USA 100:2544-2549) corresponding (Fig. 1), prompting OsDR3 may be exactly the gene (Wen etc. of corresponding disease-resistant QTL, Three types of defense-responsive genesare involved in resistance to bacterial blight and fungal blast diseases in rice, 2003, Mol.Gen.Genomics 269:331-339).(3) utilize 12 kinds of rice materials-pathogenic bacteria combinatory analysis OsDR3 expression of gene pattern (table 1) (Wen etc., 2003, Mol.Gen.Genomics 269:331-339).In the analyzed rice material, C101A51, C101LAC and CO39 are blast resisting near isogenic line (Mackell and Bonman, 1992, Phytopathology82:746-749), IRBB4, IRBB13 and IR24 are near isogenic line resisting bacterial leaf-blight (Ogawa etc., 1988, Rice Genet.Newslett.5:106-107).In RT-PCR (reverse transcription-polymerase chain reaction) analyzed, OsDR3 gene specific PCR primer EI12I1F (5 '-CAGTAGCTCCAAGGGGTGTC-3 ') and EI12I1R (5 '-TTAAAGTTGGGGTTCCCATTC-3 ') were according to the sequences Design of the cDNA fragment cloning EI12I1 of this gene; In addition, the standard of measuring as sample RNA with the amplified production of the PCR primer actinF (5 '-TATGGTCAAGGCTGGGTTCG-3 ') of paddy rice beta-actin (actin) and actinR (5 '-CCAT GCTCGATGGG-GTACTT-3 ').The expression amount of OsDR3 gene in all disease resisting rice materials all increased after RT-PCR analytical results (Fig. 2) showed inoculation Pyricularia oryzae or bacterial leaf spot pathogenic bacteria, and promptly pathogenic bacteria can be induced and strengthen OsDR3 genetic expression.This is induced and occurs in cause of disease the earliest and infect back 8 hours (Fig. 2).And the OsDR3 expression of gene is behind susceptible rice material inoculation pathogenic bacteria and the back no changes of all rice material water receivings (contrast).These results confirm that further OsDR3 is a disease-resistant related gene, and it not only participates in the regulation and control of rice bacterial blight resistance reaction, also participate in the regulation and control (Wen etc., 2003, Mol.Gen.Genomics 269:331-339) of blast resisting reaction.

Table 1. is used for rice material and the pathogenic bacteria combination that RT-PCR analyzes

Rice material 1	The disease-resistant gene that carries	Non-affinity pathogenic bacteria 2	The affinity pathogenic bacteria 2
				C101A51 CO39 C101LAC CO39 bright extensive 63	The susceptible unnamed blast resistant gene of the susceptible blast resistant gene Pi1 of blast resistant gene Pi2	Pyricularia oryzae V86013 Pyricularia oryzae IK81-3 Pyricularia oryzae V86013	Pyricularia oryzae V8601 Pyricularia oryzae IK81-3

Precious Shan 97 IRBB4 IR24 IRBB13 IR24 bright extensive 63 precious Shans 97

Xa26 is susceptible for the susceptible bacterial leaf spot resistant ospc gene of susceptible bacterial leaf spot resistant ospc gene Xa4 susceptible bacterial leaf spot resistant ospc gene xa13

Bacterial leaf spot pathogenic bacteria PXO61 bacterial leaf spot pathogenic bacteria PXO99 bacterial leaf spot pathogenic bacteria JL691

Pyricularia oryzae V86013 bacterial leaf spot pathogenic bacteria PXO61 bacterial leaf spot pathogenic bacteria PXO99 bacterial leaf spot pathogenic bacteria JL691

¹Rice material bright extensive 63 and precious Shan 97 are two parents of China hybrid rice commonly used; Other rice material is bacterial leaf-blight commonly used in the world and rice blast expert evidence (Ogawa etc., 1988, Rice Genet.Newslett.5:106-107; Mackell and Bonman, 1992, Phytopathology82:746-74), be so kind as to give by International Rice Research Institute (IRRI).

²Bacterial leaf-blight bacteria strain JL691 is Chinese bacterial strain (Yang etc., 2003, Theor.Appl.Genet.106:1467-1472), other bacterial leaf-blight bacteria strain and all Pyricularia oryzae bacterial strains are that bacterial leaf-blight commonly used in the world and rice blast are identified bacterial strain (Wen etc., Three types ofdefense-responsive genes are involved in resistance to bacterial blight and fungal blast diseases in rice, 2003, Mol.Gen.Genomics 269:331-339), be so kind as to give by the International Rice Research Institute.

Following examples further define the present invention, and have described the method for the present invention's separating clone OsDR3 gene and checking OsDR3 gene function on above-mentioned previous research work result's basis.According to following description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification, so that its suitable various uses and condition to the present invention.

Embodiment 1: separating clone OsDR3 gene

1. separating clone OsDR3 gene from disease resisting rice kind bright extensive 63

The present invention makees probe with above-mentioned cDNA clone EI12I1, from bright extensive 63BAC library (Peng etc., ABAC libraryconstructed to the rice cultivar ' Minghui 63 ' for cloning genes of agronomic importance, 1998, ActaBot.Sinica 40:1108-1114) screens a BAC clone 7G7 in.With restriction enzyme Hind III digestion BAC clone 7G7, endonuclease bamhi is connected with the pUC19 carrier (available from U.S. Amersham Biosciences company) that Hind III handles, electricity transformed into escherichia coli DH10B (available from American I nvitrogen company), the preparation subclone.Subclone obtains one and comprises the segmental subclone 7G7H14 of target gene through with the PCR primer EI12I1F of the sequences Design of cDNA clone EI12I1 (5 '-CAGTAGCTCCAAGGGGTGTC-3 ') and EI12I1R (5 '-TTAAAGTTGGGGTTCCCATTC-3 ') augmentation detection; The external source of this subclone is inserted fragment and is about 3.6kb.

2. sequencing analysis subclone 7G7H14

The present invention adopts the method for Shotgun check order (Sambrook and Russell, 2001, Molecular Cloning, Cold Spring Harbor Laboratory Press).7G7H14 interrupts with ultrasonic wave with subclone, separates the dna fragmentation of about 1kb by 1% agarose gel electrophoresis, mends flat terminal behind the purifying with the T4-DNA polysaccharase; These fragments are connected with the pUC18 carrier (available from U.S. Amersham Biosciences company) that restriction endonuclease sma I handles, and electric transformed into escherichia coli DH10B chooses the clone and detects the insertion clip size.Selecting to insert the clone of fragment about 1kb checks order.

Adopt the sequencing kit (Big Dye Kit) of M13-R and M13-F universal primer (giving birth to worker bio-engineering corporation), U.S. Perkin Elmer company available from Chinese Shanghai, respectively from the two ends order-checking of each subclone.Altogether 17 subclones have been carried out check order (Fig. 3).Use Sequencher 4.1 softwares (U.S. Gene Codes Corporation) splicing sequence.Automatically remove relatively poor sequence of end sequencing and pUC18 carrier sequence with this software.Carry out sequence assembly greater than the consistence of 40bp, overlap under greater than 95% condition in overlap length, obtain the sequence of a segment length 2946bp.Each base of target gene section is all determined with reference to a plurality of Shotgun fragments sequence that overlap this site.

Embodiment 2:OsDR3 gene structure is analyzed

1. the extracting of total RNA

TRIzol Reagent (available from American I nvitrogen company) is adopted in the extracting of total RNA.Working method is undertaken by the test kit specification sheets that company provides.

2. gene intron determines

Adopt predictive genes software GenScan (http://genes.mit.edu/GENSCAN.html) that the sequence (2946bp) of subclone 7G7H14 is carried out the gene structure forecast analysis.Analytical results demonstration OsDR3 gene is long 1195bp from translation initiation password to termination codon, comprises 3 exons (exon) and 2 introns (intron), the long 203bp of initial exon, the long 150bp of middle exon, the long 598bp of terminal exon; The intron that between initial exon and middle exon, has a long 95bp, the 2nd the long 149bp of intron (Fig. 4) between middle exon and terminal exon.Therefore, the long 951bp of the reading frame of the OsDR3 gene of prediction (open reading frame).

In order to verify said gene structure prediction result's exactness, the sequence (822bp) of the cDNA fragment cloning EI12I1 of encoding sequence and the OsDR3 gene of the 951bp of prediction is done the BLAST analysis, relatively find the consensus nucleic acid sequence of both overlaps, the exon sequence of the both sides, insertion site of an intron of close 3 ' end mates fully, shows that the GenScan analysis software is correct to the prediction of this intron.

The long 95bp of first intron of GenScan prediction between initial exon and middle exon, and initial exon 203bp only, makes this intron very near 5 ' end of gene.Because EI12I1 is not a full-length cDNA, its 5 ' terminal disappearance has only a part of sequence and the terminal exon of middle exon, and no initial exon sequence is so can not relatively verify the existence of first intron by sequence.Exactness for definite first intron of predicting, the present invention with the prediction the 951bp encoding sequence be stencil design one couple of PCR primers WRKY2F (5 '-AGTCCGTCGACGAGCAAG-3 ', be positioned at 1177 to 1194bp places of sequence shown in the sequence table SEQ ID NO:1) and WRKY2R (5 '-GGTAGGGAGAGC CCTTGATG-3 ' is positioned at 1535 to 1516bp places of sequence shown in the sequence table SEQ ID NO:1); Be the WRKY2F primer be positioned at initial exon and the WRKY2R primer be positioned in the middle of on the exon, just cross over the intron of first prediction.With bright extensive 63 RNAs of inoculation after the cause of diseases of rice varieties is that template is RT-PCR and is that template is done pcr amplification with bright extensive 63 genomic dnas, and both differ about 100bp at the amplified fragments size, just is the length (Fig. 5) of first intron of predicting.Working instructions according to pGEM-T Vector System 1kit test kit (available from U.S. Promega company), the RT-PCR product is connected on the pGEM-T carrier, transformed into escherichia coli DH10B (available from American I nvitrogen company) obtains positive colony by blue hickie screening.Positive colony is checked order the long 264bp of sequence.The encoding sequence of this sequence and GenScan software prediction is compared, and both sequences are in full accord.This result confirms that GenScan software also is correct (Fig. 6) to the prediction of first intron.

The structural analysis of embodiment 3:OsDR3 gene product

According to the BLAST analytical results, the protein of OsDR3 genes encoding is made up of 316 amino acid, has the sequence C-X of typical WRKY conservative domain aminoacid sequence WRKYGQK of WRKY proteinoid family and characteristic zinc fingers ₅-C-X ₂₃-H-X-H (wherein C represents halfcystine, and H represents Histidine, and X represents arbitrary amino acid) (Fig. 7).(The WRKY superfamily of plant transcription factors such as Eulgem, 2000, Trends.Plant Sci.5:199-206) comprise the number of WRKY structural domain according to the WRKY proteinoid and the feature of zinc finger print body is divided three classes WRKY albumen: the I class comprises two WRKY structural domains and C-X _4-5-C-X _22-23-H-X-H zinc fingers, II class comprise a WRKY structural domain and same C-X _4-5-C-X _22-23-H-X-H zinc fingers, III class comprise a WRKY structural domain, and its zinc fingers is different from above C ₂-H ₂Pattern, but C ₂-HC pattern (C-X ₇-C-X ₂₃-H-X-C).According to above classification, the WRKY proteinoid of OsDR3 genes encoding can be included into the II class.

The functional verification and the application of embodiment 4:OsDR3 gene

1. the structure of genetic transformation carrier

Used carrier is the pU1301 that our laboratory makes up.PU1301 is plant genetic conversion carrier pCAMBIA 1301 (Sun etc. commonly used in the world, 2004, Xa26, a gene conferring resistance to Xanthomonas oryzae pv.oryzae in rice, encoding a LRR receptor kinase-like protein.Plant Journal.37:517-527) reconstructs on the basis, carry the agriculture bacillus mediated genetic transformation carrier (Fig. 8) of corn ubiquitin composing type, overexpression promotor.The pCAMBIA1301 carrier is so kind as to give by Australian CAMBIA laboratory (Center for the Application of MolecularBiology to International Agriculture).

Adopt the method for restriction enzyme BamHI digestion, the OsDR3 gene is gone up enzyme from recombinant plasmid 7G7H14 scale off, reclaim external source fragment (Fig. 6).Simultaneously, cut the genetic transformation carrier pU1301 that carries the corn ubiquitin promoter with the BamHI enzyme; Enzyme cuts complete, uses chloroform: primary isoamyl alcohol (24: 1) extracting, purifying enzyme is cut product.Spending Starch phosphorylase Alk then cuts product to the enzyme of purifying and carries out dephosphorylation and handle.Do ligation with endonuclease bamhi that comprises the OsDR3 gene and dephosphorylized carrier.Cut screening positive clone by enzyme, and cut the positive colony that screening external source fragment forward inserts by the KpnI enzyme.The recombinant plasmid vector that obtains is named as D11U.

Adopt agriculture bacillus mediated genetic transforming method (Hiei etc., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, 1994, PlantJournal 6:271-282) D11U is imported susceptible rice varieties Mudanjiang No. 8 (Oryza sativa ssp.japonica).Agriculture bacillus mediated genetic transformation key step and reagent are as follows:

(1) reagent and solution abbreviation

6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin, Pyocianil); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indolylacetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4 dichlorophenoxyacetic acid); AS (Acetosringone, Syringylethanone); CH (CaseinEnzymatic Hydrolysate, caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); N6max (a large amount of composition solution of N6); N6mix (N6 is composition solution in a small amount); MSmax (a large amount of composition solution of MS); MSmix (MS is composition solution in a small amount)

(2) main solution formula

1) N6max mother liquor [10 times of concentrated solutions (10X)]

Saltpetre (KNO ₃) 28.3g

Potassium primary phosphate (KH ₂PO ₄) 4.0g

Ammonium sulfate ((NH ₄) ₂SO ₄) 4.63g

Sal epsom (MgSO ₄7H ₂O) 1.85g

Calcium chloride (CaCl ₂2H ₂O) 1.66g

Dissolving is settled to 1000ml under the room temperature then one by one.

2) N6mix mother liquor [100 times of concentrated solutions (100X)]

Potassiumiodide (KI) 0.08g

Boric acid (H ₃BO ₃) 0.16g

Manganous sulfate (MnSO ₄4H ₂O) 0.44g

Zinc sulfate (ZnSO ₄7H ₂O) 0.15g

Dissolving and be settled to 1000ml under the room temperature.

3) Fe2EDTA stock solution (100X)

In a big triangular flask, add 300ml distilled water and ferric sulfate (FeSO ₄7H ₂O) 2.78g

In another big triangular flask, add 300ml distilled water and be heated to 70 ℃, add b diammonium disodium edta (Na then ₂EDTA2H ₂O) 3.73g

Mix after they all dissolve, kept 2 hours in 70 ℃ of water-baths, be settled to 1000ml, 4 ℃ of preservations are standby.

4) VITAMIN stock solution (100X)

Nicotinic acid (Nicotinic acid) 0.1g

VITMAIN B1 (Thiamine HCl) 0.1g

Vitamin B6 (Pyridoxine HCl) 0.1g

Glycine (Glycine) 0.2g

Inositol (Inositol) 10g

Add water and be settled to 1000ml, 4 ℃ of preservations are standby.

5) MSmax mother liquor (10X)

Ammonium nitrate (NH ₄NO ₃) 16.5g

Saltpetre 19.0g

Potassium primary phosphate 1.7g

Sal epsom 3.7g

Calcium chloride 4.4g

Dissolving and be settled to 1000ml under the room temperature.

6) MSmix mother liquor (100X)

Potassiumiodide 0.083g

Boric acid 0.62g

Manganous sulfate 0.86g

Sodium orthomolybdate (Na ₂MoO ₄2H ₂O) 0.025g

Copper sulfate (CuSO ₄5H ₂O) 0.0025g

Dissolving and be settled to 1000ml under the room temperature.

7) 2,4-D stock solution (1mg/ml)

2，4-D 100mg.

1ml 1N potassium hydroxide dissolving 5 minutes adds the 10ml dissolved in distilled water then and is settled to 100ml, room temperature preservation after fully.

8) 6-BA stock solution (1mg/ml)

6-BA 100mg.

1ml 1N potassium hydroxide dissolving 5 minutes adds the 10ml dissolved in distilled water then and is settled to 100ml, room temperature preservation after fully.

9) NAA stock solution (1mg/ml)

NAA 100mg.

1ml 1N potassium hydroxide dissolving 5 minutes adds the 10ml dissolved in distilled water then and is settled to 100ml after fully, and 4 ℃ of preservations are standby.

10) IAA stock solution (1mg/ml)

IAA 100mg.

1ml 1N potassium hydroxide dissolving 5 minutes adds the 10ml dissolved in distilled water then and is settled to 100ml after fully, and 4 ℃ of preservations are standby.

11) glucose stock solution (0.5g/ml)

Glucose 125g

Dissolved in distilled water is settled to 250ml, and the back 4 ℃ of preservations of sterilizing are standby.

12) AS stock solution

AS 0.392g

DMSO 10ml

Divide to be filled in the 1.5ml centrifuge tube, 4 ℃ of preservations are standby.

13) 1N potassium hydroxide stock solution

Potassium hydroxide 5.6g

Dissolved in distilled water is settled to 100ml, and room temperature preservation is standby.

(3) culture medium prescription

1) inducing culture

N6max mother liquor (10X) 100ml

N6mix mother liquor (100X) 10ml

Fe ²⁺EDTA stock solution (100X) 10ml

VITAMIN stock solution (100X) 10ml

2,4-D stock solution 2.5ml

Proline(Pro) (Proline) 0.3g

CH 0.6g

Sucrose (Sucrose) 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides to install to 50ml triangular flask (25ml/ bottle), seals sterilization.

2) subculture medium

N6max mother liquor (10X) 100ml

N6mix mother liquor (100X) 10ml

Fe ²⁺EDTA stock solution (100X) 10ml

VITAMIN stock solution (100X) 10ml

2,4-D stock solution 2.0ml

Proline(Pro) 0.5g

CH 0.6g

Sucrose 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides to install to 50ml triangular flask (25ml/ bottle), seals sterilization.

3) pre-culture medium

N6max mother liquor (10X) 12.5ml

N6mix mother liquor (100X) 1.25ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

2,4-D stock solution 0.75ml

CH 0.15g

Sucrose 5g

Agar powder (Agarose) 1.75g

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, seals sterilization.

Use preceding heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (25ml/ ware) in the culture dish poured in packing into.

4) be total to substratum

N6max mother liquor (10X) 12.5ml

N6mix mother liquor (100X) 1.25ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

2,4-D stock solution 0.75ml

CH 0.2

Sucrose 5g

Agar powder 1.75g

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, seals sterilization.

Use preceding heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (25ml/ ware) in the culture dish poured in packing into.

5) suspension culture base

N6max mother liquor (10X) 5ml

N6mix mother liquor (100X) 0.5ml

Fe ²⁺EDTA stock solution (100X) 0.5ml

VITAMIN stock solution (100X) 1ml

2,4-D stock solution 0.2ml

CH 0.08g

Sucrose 2g

Adding distil water is regulated pH value to 5.4 to 100ml, divides to install in the triangular flask of two 100ml, seals sterilization.

Add 1ml glucose stock solution and 100 μ l AS stock solutions before using.

6) select substratum

N6max mother liquor (10X) 25ml

N6mix mother liquor (100X) 2.5ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

2,4-D stock solution 0.625ml

CH 0.15g

Sucrose 7.5g

Agar powder 1.75g

Adding distil water is regulated pH value to 6.0 to 250ml, seals sterilization.

The dissolving substratum adds 250 μ l HN and 400ppm CN before using, and (25ml/ ware) in the culture dish poured in packing into.

7) break up substratum in advance

N6max mother liquor (10X) 25ml

N6mix mother liquor (100X) 2.5ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

6-BA stock solution 0.5ml

KT stock solution 0.5ml

NAA stock solution 50 μ l

IAA stock solution 50 μ l

CH 0.15g

Sucrose 7.5g

Agar powder 1.75g

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.9, seals sterilization.

The dissolving substratum adds 250 μ l HN and 200ppm CN before using, and (25ml/ ware) in the culture dish poured in packing into.

8) division culture medium

N6max mother liquor (10X) 100ml

N6mix mother liquor (100X) 10ml

Fe ²⁺EDTA stock solution (100X) 10ml

VITAMIN stock solution (100X) 10ml

6-BA stock solution 2ml

KT stock solution 2ml

NAA stock solution 0.2ml

IAA stock solution 0.2ml

CH 1g

Sucrose 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 6.0.

Boil and be settled to 1000ml, divide to install to 50ml triangular flask (50ml/ bottle), seal sterilization.

9) root media

MSmax mother liquor (10X) 50ml

MSmix mother liquor (100X) 5ml

Fe ²⁺EDTA stock solution (100X) 5ml

VITAMIN stock solution (100X) 5ml

Sucrose 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.8.

Boil and be settled to 1000ml, divide to install to (25ml/ pipe) in the pipe of taking root, seal sterilization.

(4) agriculture bacillus mediated genetic transformation step

3.1 callus of induce

(1) sophisticated rice paddy seed is shelled, used 70% Ethanol Treatment then successively 1 minute, 0.15% mercury chloride (HgCl2) 15 minutes;

(2) sterilization washing seed is 4-5 time;

(3) seed is placed on the inducing culture;

(4) place dark place to cultivate 4 weeks, 25 ± 1 ℃ of temperature.

3.2 callus subculture

Select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in dark 2 weeks, 25 ± 1 ℃ of the temperature of cultivating down on the subculture medium.

3.3 pre-the cultivation

Select the embryo callus subculture of consolidation and relatively dry, be put in dark 2 weeks, 25 ± 1 ℃ of the temperature of cultivating down on the pre-culture medium.

3.4 Agrobacterium is cultivated

(1) on the LA substratum that has corresponding resistance selection, cultivated Agrobacterium EHA105 two days in advance, 28 ℃ of temperature;

(2) Agrobacterium is transferred in the suspension culture base, cultivated 2-3 hour on 28 ℃ of shaking tables.

3.5 Agrobacterium is infected

(1) pre-incubated callus is transferred in the good bottle of sterilization;

(2) regulate the suspension of Agrobacterium to OD600 0.8-1.0;

(3) callus was soaked in agrobacterium suspension 30 minutes;

(4) shifting callus blots to the good filter paper of sterilization; Be placed on then on the common substratum and cultivated temperature 19-20 ℃ 3 days.

3.6 callus washing and selection are cultivated

(1) aqua sterilisa washing callus is to cannot see Agrobacterium;

(2) be immersed in the aqua sterilisa that contains 400ppm Pyocianil (CN) 30 minutes;

(3) shifting callus blots to the good filter paper of sterilization;

(4) shift callus to selecting to select 2-3 time each 2 weeks on the substratum.(hygromycin selection concentration is 400ppm for the first time, is 250ppm later on for the second time)

3.7 differentiation

(1) kanamycin-resistant callus tissue is transferred to dark place cultivation 5-7 week on the pre-differentiation substratum;

(2) callus that shifts pre-differentiation cultivation is to division culture medium, and illumination is cultivated down, 26 ℃ of temperature.

3.8 take root

(1) cuts the root that differentiation phase produces;

(2) then it is transferred to following 2-3 week, 26 ℃ of the temperature of cultivating of illumination in the root media.

3.9 transplant

Wash the residual substratum on the root off, the seedling that will have good root system changes the greenhouse over to, divides moistening at initial several Tian Bao water holding simultaneously.

The transgenic rice plant that obtains is named as D11UM8 (wherein D11U is the genetic transformation container name, and M8 represents rice varieties Mudanjiang No. 8).The present invention obtains independent transgenic rice plant 47 strains altogether.Adopt leaf-cutting method (Kauffman etc., An improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae, 1973, Plant Dis.Rep.57:537-541), all transfer-gen plants are being become stage strain phase inoculation bacterial leaf-blight bacteria strain PXO61 (Sun etc. commonly used in the world, 2004, Xa26, a gene conferring resistance to Xanthomonas oryzae pv.oryzae inrice, encoding a LRR receptor kinase-like protein.Plant Journal.37:517-527), find significantly (P≤0.05) enhancing (table 2) of bacterial leaf spot resistant germ infringement ability of 13 strain transgenic rice plants; Compare for No. 8 with genetically modified acceptor rice varieties Mudanjiang, the lesion area of these transfer-gen plants has reduced 28.2-60.5%.

Table 2. part is carried the reaction of the transgenic rice plant of OsDR3 gene to bacterial leaf spot pathogenic bacteria strain PXO61

Transfer-gen plant	Lesion area (%) (1)	The P value
			No. 8 (susceptible contrast) IRBB4 in Mudanjiang (disease-resistant contrast) D11UM-1 D11UM-3 D11UM-7 D11UM-13 D11UM-16 D11UM-18 D11UM-20 D11UM-23 D11UM-31 D11UM-35 D11UM-39 D11UM-41 D11UM-44	61.8 11.6 33.3 33.4 24.4 40.2 44.4 33.6 36.1 38.2 44.0 37.3 34.5 39.0 42.8	0.007 0.050 0.001 0.011 0.014 0.003 0.005 0.010 0.033 0.008 0.007 0.017 0.024

⁽¹⁾5 leaves of every strain transfer-gen plant inoculation, three all " Invest, Then Investigate " scabs and sick leaf length, each data comes from the mean value of 5 leaves.

Transgenic rice plant also strengthens the resistance of Pyricularia oryzae.T to transfer-gen plant D11UM8-47 ₁For plant in vitro inoculation Pyricularia oryzae bacterial strain V86013 (Wen etc. commonly used in the world, Three types of defense-responsive genes areinvolved in resistance to bacterial blight and fungal blast diseases in rice, 2003, Mol.Gen.Genomics 269:331-339) observes, find that these plant show the anti-sense separation of expectation.Compare 23 strain T for No. 8 with adjoining tree Mudanjiang ₁For there being 9 strains performance resistance obviously to improve (Fig. 9) in the plant.

The present invention adopts composing type, overexpression (corn ubiquitin) promoter regulation OsDR3 expression of gene, the influence that therefore the OsDR3 expression of gene can not be infected by cause of disease in transgenic rice plant.Whether relevant with the OsDR3 expression of gene amount that changes over to for the resistance enhancing of verifying transfer-gen plant, the present invention adopts the RNA hybridization technique that OsDR3 expression of gene in the part transgenic rice plant is detected.The result shows that the resistance of OsDR3 expression of gene amount and plant is closely related.Basically all the OsDR3 expression of gene can not be detected in transgene receptor rice varieties under not having the cause of disease infection condition (No. 8, Mudanjiang), genetic donor (bright extensive 63) and the susceptible transgenic rice plant, and OsDR3 expression of gene (Figure 10) can be detected in the resistance enhanced transfer-gen plant.These results prove that further the OsDR3 gene participates in the regulation and control of paddy disease-resistant reaction, and overexpression OsDR3 gene can strengthen the disease resistance of paddy rice.

Sequence table SEQ ID NO:1

＜110〉Hua Zhong Agriculture University

＜120〉paddy disease-resistant related gene OsDR3

<130>

<141>2004-07-23

<160>2

<170>PatentIn version 3.1

<210>1

<211>2946

<212>DNA

<213>Oryza sativa

<220>

<221>Intron

<222>(1295)..(1389)

<223>

<220>

<221>CDS

<222>(1092)..(1294)

<223>

<220>

<221>CDS

<222>(1390)..(1539)

<223>

<220>

<221>CDS

<222>(1689)..(2283)

<223>

<220>

<221>Intron

<222>(1540)..(1688)

<223>

<220>

<221>misc_feature

<222>(5)..(5)

＜223〉a, c, g or t

<220>

<221>misc_feature

<222>(16)..(16)

＜223〉a, c, g or t

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉a, c, g or t

<220>

<221>misc_feature

<222>(37)..(37)

＜223〉a, c, g or t

<220>

<221>msc_feature

<222>(41)..(41)

＜223〉a, c, g or t

<220>

<221>misc_feature

<222>(52)..(52)

＜223〉a, c, g or t

<220>

<221>misc_feature

<222>(88)..(88)

＜223〉a, c, g or t

<220>

<221>misc_feature

<222>(108)..(108)

＜223〉a, c, g or t

<220>

<221>misc_feature

<222>(114)..(114)

＜223〉a, c, g or t

<220>

<221>misc_feature

<222>(2922)..(2922)

＜223〉a, c, g or t

<220>

<221>misc_feature

<222>(2937)..(2937)

＜223〉a, c, g or t

<400>1

tcagngggtt tggccncctg ncgtgaaccc ccgtganatg ngattattcg tnaggtagat 60

catcgatcca agttttttca cgtgtccnct tgtagtaggg atcctccncg gttngatcga 120

gtccrrcgcc cmgacggatt gctamcaaac aatacataat cttctctttt tttgttgttg 180

agatatatat gatcacgtca cagggcggca ctggttctgg aggttcgtct gtgaattaaa 240

ccacgctaaa gtacagggta tccgtatagt attccttctc atgtacgatc tggtgtgtat 300

tgtgtttcca cggggatagc agcagaagca cggggatgat tggccagatc gggttaggga 360

gggggtcgcc atcgccgcct gcactagatt aatcgctcgc cccggcccag aacgtcgctc 420

tctccgtccg tccccggctc gccaccatct cctcctccaa gacgcaactt cgagagtgag 480

agagagagag aatcgagaga tccaatccag tttgcgcaac gagtggcatg gcatgcaacc 540

gatctagaac gtcaaataaa aacaacgcca tgcgtacata cacgttcatg tgccgcgcgc 600

cggtgtagcg cagcgtagag agagagagag agagagagta tggtagtctt cctacgctcg 660

gtcagtgaca gcgtcgcggc gcataaagaa aggcatccac tagcaggagc cagcatacgc 720

tgccgcatat acaaacacgc atgtagccaa ggcgaggaga gagagagaga gagagagaga 780

gagggagatc attgaaagct gcacccatcc gtttgttcgc caccatttcc atcccctcca 840

tcatgagctc caattagtcg cacaccgatc ccctcctctt cttccccccc ttctcctctc 900

tcctcttttt ttcctcatcc gtgcgcgata ctgcgcttag tttgtgggtg gggggagtga 960

tcatcaccaa agccacaaag ggggcgcgga gagatattat atattctttg ggaaagcgtt 1020

ggattagttt ttctcgcttt gggcttttta tccggagttg gagtgtgtgt gcgccgggag 1080

tggtggtggt g atg gcg gca gga gag gag gtg atg gat cgg tcg acg tcg 1130

Met Ala Ala Gly Glu Glu Val Met Asp Arg Ser Thr Ser

1 5 10

gcc gag gac ggg tac tgc agc gcc ggc acg gac tcg ccg cgg gcg gag 1178

Ala Glu Asp Gly Tyr Cys Ser Ala Gly Thr Asp Ser Pro Arg Ala Glu

15 20 25

tcc gtc gac gag caa ggg gcc gcc gag gag tcg tcg ccc cgg gga ggg 1226

Ser Val Asp Glu Gln Gly Ala Ala Glu Glu Ser Ser Pro Arg Gly Gly

30 35 40 45

cag aag cgg gag ctc ccc tcg ccg tcg gct tcg ccg tcg tcc ccg ctg 1274

Gln Lys Arg Glu Leu Pro Ser Pro Ser Ala Ser Pro Ser Ser Pro Leu

50 55 60

cca cct gcc gcg aag cgc ag gtacggataa tattatggct cggtctgcga 1324

Pro Pro Ala Ala Lys Arg Ser

65

ttgattaaag ccctaggtag gatttaggga tagaggattg attggtgttt ggttggtttg 1384

agcag c cgg agg tca gtg gag aag cgg gtg gtg tcc gtg ccg atc gcg 1432

Arg Arg Ser Val Glu Lys Arg Val Val Ser Val Pro Ile Ala

70 75 80

gag tgc ggc gac cgg ccg aag ggc gcc ggg gag ggg ccg ccg ccg tcg 1480

Glu Cys Gly Asp Arg Pro Lys Gly Ala Gly Glu Gly Pro Pro Pro Ser

85 90 95

gac tcg tgg gcc tgg cgc aag tac ggc cag aag ccc atc aag ggc tct 1528

Asp Ser Trp Ala Trp Arg Lys Tyr Gly Gln Lys Pro Ile Lys Gly Ser

100 105 110

ccc tac cca ag gtaattaggc tttatattca gtttggttga tattcttgag 1579

Pro Tyr Pro Arg

115

cctgttgatt agttgccgtg ctgattaatc actgcaatta gatcagttaa acggttgttg 1639

aagtggcaag atttaatttg ggtttgttgc ttgtgtttat tatgctcag g ggg tac 1695

Gly Tyr

120

tac agg tgc agt agc tcc aag ggg tgt ccg gcg agg aag cag gtg gag 1743

Tyr Arg Cys Ser Ser Ser Lys Gly Cys Pro Ala Arg Lys Gln Val Glu

125 130 135

cgc agc cgc gcc gac ccc acc gtg ctg ctc gtc acc tac tcc ttc gag 1791

Arg Ser Arg Ala Asp Pro Thr Val Leu Leu Val Thr Tyr Ser Phe Glu

140 145 150

cac aac cac ccg tgg ccg cag ccc aag agc agc agc tgc cac gcg agc 1839

His Asn His Pro Trp Pro Gln Pro Lys Ser Ser Ser Cys His Ala Ser

155 160 165

aag tcg tct ccc cgg tcg acg gcg cca aag cct gag ccg gcg gct gac 1887

Lys Ser Ser Pro Arg Ser Thr Ala Pro Lys Pro Glu Pro Ala Ala Asp

170 175 180

gga cag cag ccc gag cca gcg gag aac gaa tcg tcg gcg tcg gcg gag 1935

Gly Gln Gln Pro Glu Pro Ala Glu Asn Glu Ser Ser Ala Ser Ala Glu

185 190 195 200

ctg gag gta ccg gag ccg gag ccg gag cag gaa tct gag ccg gtt gtg 1983

Leu Glu Val Pro Glu Pro Glu Pro Glu Gln Glu Ser Glu Pro Val Val

205 210 215

aag cag gag gag gag cag aag gag gaa cag aag gct gtg gtt gag ccg 2031

Lys Gln Glu Glu Glu Gln Lys Glu Glu Gln Lys Ala Val Val Glu Pro

220 225 230

gcg gcg gtc aca acc acg gtg gcg ccg gcg ccg gcg gtc gag gag gag 2079

Ala Ala Val Thr Thr Thr Val Ala Pro Ala Pro Ala Val Glu Glu Glu

235 240 245

gat gag aac ttc gac ttc gga tgg atc gac cag tac cac ccg acg tgg 2127

Asp Glu Asn Phe Asp Phe Gly Trp Ile Asp Gln Tyr His Pro Thr Trp

250 255 260

cac cgg tcg tac gcg ccg ctg ctg ccg ccg gag gag tgg gag cgc gag 2175

His Arg Ser Tyr Ala Pro Leu Leu Pro Pro Glu Glu Trp Glu Arg Glu

265 270 275 280

ctg caa ggg gac gac gcg ctg ttc gcg ggg ctc ggc gag ctc ccg gag 2223

Leu Gln Gly Asp Asp Ala Leu Phe Ala Gly Leu Gly Glu Leu Pro Glu

285 290 295

tgc gcc gtc gtg ttc ggc cgc cgg cgc gag ctc ggc ctg gcg gcc acc 2271

Cys Ala Val Val Phe Gly Arg Arg Arg Glu Leu Gly Leu Ala Ala Thr

300 305 310

gcg ccg tgc tcc tgatgatcac ctcctctttt ctttttcctc cccttttaca 2323

Ala Pro Cys Ser

315

atttcccttt ttttttttgc ttttctttag ttctccttgg agtaatttgg attggagatt 2383

agtacatagc ttgggtgatc aatgggagtg agtggcttaa cttgttcaag agagcctcct 2443

attcccactc tccaaattgc gtcgcgtgtt cgtcatccaa agcccgtcgt gccgtttccg 2503

aagtggttgg cgacgtgatt ccaacgacaa aaaagcagcg aaaggtgggc attttcaggc 2563

gtttttcctc gccgtctgtg tcgaatagac gcgcacatcg atcgctccaa ggcccatccc 2623

gccgagtgag tgatgagtga gttaggtttc tccgcacgca cgcacgcacg tccccaccgg 2683

ctgcggcttg caagtggggg cctctcgtct cgtccgatgc ggaatccaac ggtaagcggc 2743

actggcatta gccgggcaag cgttttcggc gtggtagttg gaacttggaa ggatgcgcgc 2803

gcacgaggcc acatgcccgg gtagtgtggc gctgccacgg atccacccgc gggacgcgtg 2863

ccgcgccttc ccgccttgcc ggccacggcc cacggccggc tgccgccgcc ccggtccgng 2923

ggcgctacgc ctangcgcga gcg 2946

<210>2

<211>316

<212>PRT

<213>Oryza sativa

<400>2

Met Ala Ala Gly Glu Glu Val Met Asp Arg Ser Thr Ser Ala Glu Asp

1 5 10 15

Gly Tyr Cys Ser Ala Gly Thr Asp Ser Pro Arg Ala Glu Ser Val Asp

20 25 30

Glu Gln Gly Ala Ala Glu Glu Ser Ser Pro Arg Gly Gly Gln Lys Arg

35 40 45

Glu Leu Pro Ser Pro Ser Ala Ser Pro Ser Ser Pro Leu Pro Pro Ala

50 55 60

Ala Lys Arg Ser Arg Arg Ser Val Glu Lys Arg Val Val Ser Val Pro

65 70 75 80

Ile Ala Glu Cys Gly Asp Arg Pro Lys Gly Ala Gly Glu Gly Pro Pro

85 90 95

Pro Ser Asp Ser Trp Ala Trp Arg Lys Tyr Gly Gln Lys Pro Ile Lys

100 105 110

Gly Ser Pro Tyr Pro Arg Gly Tyr Tyr Arg Cys Ser Ser Ser Lys Gly

115 120 125

Cys Pro Ala Arg Lys Gln Val Glu Arg Ser Arg Ala Asp Pro Thr Val

130 135 140

Leu Leu Val Thr Tyr Ser Phe Glu His Asn His Pro Trp Pro Gln Pro

145 150 155 160

Lys Ser Ser Ser Cys His Ala Ser Lys Ser Ser Pro Arg Ser Thr Ala

165 170 175

Pro Lys Pro Glu Pro Ala Ala Asp Gly Gln Gln Pro Glu Pro Ala Glu

180 185 190

Asn Glu Ser Ser Ala Ser Ala Glu Leu Glu Val Pro Glu Pro Glu Pro

195 200 205

Glu Gln Glu Ser Glu Pro Val Val Lys Gln Glu Glu Glu Gln Lys Glu

210 215 220

Glu Gln Lys Ala Val Val Glu Pro Ala Ala Val Thr Thr Thr Val Ala

225 230 235 240

Pro Ala Pro Ala Val Glu Glu Glu Asp Glu Asn Phe Asp Phe Gly Trp

245 250 255

Ile Asp Gln Tyr His Pro Thr Trp His Arg Ser Tyr Ala Pro Leu Leu

260 265 270

Pro Pro Glu Glu Trp Glu Arg Glu Leu Gln Gly Asp Asp Ala Leu Phe

275 280 285

Ala Gly Leu Gly Glu Leu Pro Glu Cys Ala Val Val Phe Gly Arg Arg

290 295 300

Arg Glu Leu Gly Leu Ala Ala Thr Ala Pro Cys Ser

305 310 315

Claims (3)

1, give the dna fragmentation of plant to bacterial leaf spot pathogenic bacteria (Xanthomonas oryzae pv.oryzae) and Pyricularia oryzae (Pyriculariagrisea Sacc) generation resistance, described fragment is shown in sequence table SEQ ID NO:1.

2, a kind of method that increases plant to bacterial leaf spot pathogenic bacteria and Pyricularia oryzae resistance, this method comprise that suitable promotor is gone up in the dna fragmentation connection of claim 1 changes plant materials over to, increases the resistance of plant to pathogenic bacteria.

3, the application of the described dna fragmentation of claim 1 in producing transgenic plant.

Images