CN101892244B - Oryza officinalis anti-Xanthomonas oryzae major gene Xa3/Xa26-2 and application for improving disease resistance of rice thereof - Google Patents

Abstract

The invention relates to the technical field of the plant genetic engineering, in particular to the isolation and cloning and functional verification of the DNA fragment of oryza officinalis anti-Xanthomonas oryzae major gene Xa3/Xa26-2. The Xa3/Xa26-2 gene is used to code the leucine-rich protein kinase protein. The Xa3/Xa26-2 gene ensures that rice can resist diseases caused by bacterial pathogen-Xanthomonas oryzae pv. oryzae. The DNA fragment and the regulatory sequence thereof are directly transferred in rice, and the resistance capability to Xanthomonas oryzae of the transgenic rice carrying Xa3/Xa26-2 is significantly enhanced.

Description

Oryza officinalis resisting bacterial leaf-blight major gene Xa3/Xa26-2 and its application in improvement paddy disease-resistant property

Technical field

The present invention relates to gene engineering technology field.Be specifically related to separating clone, functional verification and the application of a rice bacterial blight resistance major gene Xa3/Xa26-2.Described dna fragmentation can be given the paddy rice opposing by the microbial disease of bacterial leaf-blight.Directly change this fragment adjusting sequence endogenous with it over to plant materials, transgenic paddy rice can produce the defense response to bacterial leaf spot pathogenic bacteria by this gene mediated.

Technical background

Plant receives the infringement of the several diseases original in the process of growth.The phytopathy original of a great variety comprises virus, bacterium, fungi and nematode etc.The pathogen invaded plants causes two kinds of results: in host plant, breeding 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 the prevention disease 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 types: (1) disease-resistant (main effect) gene, claim R (resistance) gene and (2) disease-resistant related gene again.

According to the understanding of present people's enantiopathy gene function, the product of disease-resistant gene mainly is as acceptor, and direct or indirect and cause of disease protein-interacting starts the intravital disease-resistant signal conducting path of plant (Chisholm etc., 2006).The disease resistance response strong resistance of disease-resistant gene mediation is good genetic resources.But the resource-constrained of disease-resistant gene in the arviculture kind.From nearly edge species, excavating new disease-resistant gene, to be used for the farm crop genetic improvement be the important goal that the investigator works always.

Paddy rice is an important crops in the world, but the influence of disease usually causes the decline of its yield and quality.Bacterial blight of rice is caused by bacterial leaf spot pathogenic bacteria (Xanthomonas oryzae pv.oryzae), is in the world to one of maximum bacterial disease of rice hazard (crossing Chong Jian, 1995).The resource of disease-resistant gene is very limited in the rice cropping kind (Oryza sativa L.).As the bacterial leaf spot resistant ospc gene of knowing at present has only about 30 (Chu Zhaohui and Wang Shi are flat, 2007).Because wild-rice is in wild state for a long time, has stood the natural selection of various poor environments and disaster, has formed extremely abundant genetic diversity.Containing in the wild-rice has good disease-resistant gene resource, is the valuable source storehouse that people excavate new excellent genes.Just there are a plurality of genes from wild-rice, to import to advance in the cultivated rice in known about 30 bacterial leaf spot resistant ospc genes.Be derived from the long medicine wild-rice in West Africa (Oryza longistaminata) like resisting bacterial leaf-blight dominant gene Xa21, high anti-to most of bacterial leaf-blight bacterial strain performances; But this gene is a strain-forming period resistance, promptly only becomes the strain phase just bacterial leaf spot pathogenic bacteria to be had complete resistance (Khush etc., 1990 in paddy rice; Wang etc., 1996).The separated clone of Xa21 gene (Song etc., 1995).One as yet not separated clone's resisting bacterial leaf-blight dominant gene Xa23 be derived from common wild-rice (Oryza rufipogon), it has resistance of wide spectrum (Zhang Qi etc., 2000).Another separated clone's bacterial leaf spot resistant ospc gene Xa27 come from tetraploid granule wild-rice (Orzyaminuta), these gene bacterial leaf spot resistant germ Philippines microspecies 2,3,5 and 6 (Amante-Bordeos etc., 1992; Gu etc., 2005).In addition, separated clone's bacterial leaf spot resistant ospc gene Xa29 (t) and Xa30 (t) are not (Tan Guangxuan etc., 2004 that from oryza officinalis (Orzya officinalis) and common wild-rice, identify respectively as yet; Wang Chun company etc., 2004).

Summary of the invention

The objective of the invention is bacterial leaf spot resistant ospc gene Xa3/Xa26-2 that carries in the separating clone oryza officinalis (Oryza officinalis) and the dna fragmentation that comprises the promotor of regulating and control this gene, utilize this improvement of genes rice varieties or other plant to resist the ability of disease.

The present invention relates to separate and use the dna fragmentation that comprises the Xa3/Xa26-2 gene, its nucleotide sequence is shown in sequence table SEQ ID NO:1, and its encoding sequence is shown in 3284-6191 and 6295-6662 position among the sequence table SEQ ID NO:1.This gene (fragment) is given paddy rice to producing specific disease resistance response by the caused disease of bacterial leaf spot pathogenic bacteria (Xanthomonas oryzae pv.oryzae).This invention is applicable to the plant that all are responsive to this pathogenic bacteria.These plants comprise monocotyledons and dicotyledons.Except that above-mentioned described dna fragmentation shown in SEQ ID NO:1, the defined gene of the present invention comprises also and is equivalent to the dna sequence dna shown in the SEQ IDNO:1 basically that perhaps its function is equivalent to the subfragment of sequence shown in the SEQID NO:1.Sequence shown in the sequence table SEQ ID NO:1 is carried out bioinformatic analysis to be shown; A kind of receptor protein kinase of this dna sequence encoding; It comprises extracellular rich leucine and repeats (leucine-rich repeat, LRR) structural domain and intracellular protein kinase structural domain.

Can adopt the Xa3/Xa26-2 gene of having cloned to make probe, from cDNA and genomic library, screen gene of the present invention or homologous gene.Equally, adopt PCR (polymerase chain reaction) technology, also can from genome, mRNA and cDNA, increase obtain Xa3/Xa26-2 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 sequence of Xa3/Xa26-2 gene or comprise one section Xa3/Xa26-2 gene, this sequence is connected with suitable carriers, can change vegetable cell over to; And express the Xa3/Xa26-2 gene, produce disease-resistant transgenic plant.Adopting this transgenic technology to create disease-resistant plants is that traditional breeding technology institute is inaccessiable.

Change clone's disease-resistant gene over to susceptible plant, help to produce new disease-resistant plants.Particularly can be with genetic transformation technology a plurality of disease-resistant genes that in plant, add up, and can not produce do not follow in the traditional breeding technology appearance with the burdensome kind that needs improvement of introducing of genetic linkage.Simultaneously, the clone of disease-resistant gene can overcome traditional breeding method can not shift disease-resistant gene between plant species problem.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 can change gene of the present invention over to other plant with the mode of sexual hybridization.

The present invention provides a kind of new method for strengthening paddy rice to the resistance of bacterial leaf-blight.This method comprises Xa3/Xa26-2 gene that derives from wild-rice and its regulating DNA sequence is connected, changes over to susceptible paddy rice with the genetic transformation carrier that the improvement paddy rice is to the resistance of bacterial leaf-blight.

More detailed technical scheme sees that " embodiment " is said.

Description of drawings

Sequence table SEQ ID NO:1. is the dna sequence dna of the present invention's Xa3/Xa26-2 gene of cloning.

Fig. 1. the schema of evaluation of the present invention and separating clone rice bacterial blight resistance gene Xa3/Xa26-2 and checking Xa3/Xa26-2 gene function.

Fig. 2. the structure of Xa3/Xa26 gene and MRKa gene and PCR primer (arrow) position that is used to prepare dna probe in the rice variety bright extensive 63.

The dna probe of Fig. 3 .Xa3/Xa26 gene fragment and 9 BAC clones' results of hybridization.λ-ECoT14I is dna molecular size criteria marker (DNAladder; Available from Dalian TaKaRa company).

Fig. 4. Xa3/Xa26 gene family member's distribution and sequence homology in rice variety bright extensive 63 and the oryza officinalis.Each arrow is represented an Xa3/Xa26 family member and transcriptional orientation.Xa3/Xa26 and Xa3/Xa26-2 gene downstream have 1.4kb nucleotide sequence homology degree to reach 83% approximately.11.9kb section is to be used for the genetic transformation dna fragmentation.

Fig. 5. the structure of genetic transformation carrier pCAMBIA1301.

Fig. 6 .T ₁Be divided into for the reporter gene GUS in the genetic transformation plant and disease-resistant phenotype and leave.Explain to be divided into and leave with the closely linked Xa3/Xa26-2 gene of gus gene and disease-resistant phenotype.Contrast is paddy rice susceptible variety Mudanjiang 8 (acceptor of genetic transformation).Lesion area is the data of inoculation bacterial leaf spot pathogenic bacteria PXO61 after two weeks.

Fig. 7. compare with contrast Mudanjiang 8, the genetic transformation plant (D101OM43) of carrying the Xa3/Xa26-2 gene has all strengthened the resistance of different bacterial leaf spot pathogenic bacterias.

Fig. 8 .Xa3/Xa26-2 gene structure.Arrow shows PCR primer location and the direction that is used for the gene structure analysis." ATG " and " TGA " is respectively translation initiation password and termination codon.Numeral is shown the Nucleotide number of each structure.

Embodiment

Early-stage Study result of the present invention shows that the resisting bacterial leaf-blight dominant gene Xa3/Xa26 that derives from long-grained nonglutinous rice (Oryza sativa L.ssp.indica) kind bright extensive 63 belongs to the multigene family member; The member of this family is positioned at paddy rice No. 11 chromosomal long-armed (Yang etc., 2003 with the form that is arranged in series; Sun etc., 2004,2006; Xiang etc. 2006).In bright extensive 63, Xa3/Xa26 family is made up of 4 members (Xa3/Xa26, MRKa, MRKc and MRKd), and the dna sequence dna homology between their coding region is 60% to 78% (Sun etc., 2004).Rich leucine of Xa3/Xa26 coding repeats (leucine-richrepeat, LRR) protein kinase proteinoid (Sun etc., 2004).Target gene in according to the present invention and Xa3/Xa26 gene order homology analysis with it in the Xa3/Xa26 family of oryza officinalis (Oryza officinalis) with other members' opposite position relation; Confirm that this gene is the allelotrope of Xa3/Xa26 gene, so called after Xa3/Xa26-2 gene (looking the Xa3/Xa26 gene is Xa3/Xa26-1).

Further definition the present invention in following examples.Fig. 1 has described the flow process of evaluation and separating clone Xa3/Xa26-2 gene and checking Xa3/Xa26-2 gene function.According to following description and these embodiment; Those skilled in the art can confirm 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 to the present invention, so that its suitable various uses and condition.

Embodiment 1: separating clone Xa3/Xa26-2 gene and gene structure analysis from oryza officinalis

1. identify and carry the large fragment DNA of Xa3/Xa26 dna homolog sequence

Researchist of the present invention at first uses specific PCR primer RKb-3 ' race2 (5 '-TGGTCAAATACCGGAAGGAG-3 ') and the RKb-2R (5 '-CAGTCCACCACATGGACAAG-3 ') of Xa3/Xa26 gene and the specific PCR primer RKa-11L of Xa3/Xa26 family member MRKa gene (5 '-TTGGCTTGAACGGCTTAACT-3 ') and RKa-1R (5 '-AAGATGAAATATGCTCGGTGGT-3 ') from bright extensive 63, to amplify the dna fragmentation of Xa3/Xa26 gene and MRKa, each about 1kb (Fig. 2) of amplification length.These two sections pcr amplification products are mixed genome BAC (bacterialartificial chromosome) library (Ammiraju etc., 2006) as probe screening oryza officinalis (Oryza officinalis), identified 9 BAC clone.These 9 BAC clone (Ammiraju etc., 2006) are so kind as to give by Rod professor Wing of U.S. University of Arizona university.To screen to such an extent that BAC clones enlarged culturing, adopt standard base (SB) cracking process separation and Extraction BAC plasmid (Sambrook and Russell, 2001).With gained BAC plasmid with Hind III complete degestion, electrophoresis, be transferred to nylon membrane; Hybridize (Sambrook and Russell, 2001) with the nylon membrane that is loaded with the BAC plasmid respectively with above-mentioned Xa3/Xa26 gene fragment probe and MRKa gene fragment probe then.The results of hybridization of making probe with the Xa3/Xa26 gene fragment shows that BAC clone OOBa0120J21 has maximum hybrid belts (Fig. 3), infers that this BAC clone possibly cover Xa3/Xa26 gene family section.Decision is checked order to the OOBa0120J21 clone.

2.BAC the structure in clone's shotgun library

Researchist of the present invention has at first made up the OOBa0120J21 clone's who is used to check order shotgun library.Adopt supersonic method to make up shotgun library (Sambrook and Russell, 2001).Circular plasmids 1.6 seconds (Ultrasonic Cell Disruptor Soniprep 150 is the SANYO Company products, and concrete operations are with reference to the working instructions of this instrument) with ultrasonication OOBa0120J21 clone.Separate the dna fragmentation of about 2.0-3.5kb through 1% agarose gel electrophoresis, reclaim test kit (giving birth to worker's biotechnology ltd) purify DNA from running gel available from Chinese Shanghai with UNIQ-10 pillar DNA glue.DNA behind the purifying uses T ₄Archaeal dna polymerase is mended flat terminal; Be connected with dephosphorylized pUC19 carrier (available from the U.S. Amersham Bioscience company) flush end that the Restriction enzyme Sma I enzyme is cut; (electric conversion instrument is the eppendorf Company products to electricity transformed into escherichia coli DH10B (available from American I nvitrogen company); The present embodiment applied voltage is 1800V, and concrete operations are with reference to the working instructions of this instrument).Choose the clone and detect the insertion clip size: extract the positive colony plasmid; And carry out electrophoresis with empty pUC19 plasmid and compare; Reject false positive clone and cloning of small fragment; Or with EcoRI and HindIII double digestion recombinant plasmid, detect and insert clip size, screening is inserted the suitable clone of clip size and is used for order-checking as the shotgun library.

3.Shotgun the order-checking in library

Adopt M13-F (5 '-GTAAAACGACGGCCAGT-3 ') and M13-R 5 '-CAGGAAACAGCTATGAC-3 ') sequencing kit (BigDye Kit) of universal primer (giving birth to worker's biotechnology ltd), U.S. Perkin Elmer company available from Chinese Shanghai, according to the test kit specification sheets with the terminal cessation method of dideoxy nucleotide respectively from the shotgun library clone two ends of random choose check order.

4. the splicing of original series

Use Squencer 4.5 softwares (U.S. Gene Codes Corporation) splicing sequence.Automatically remove relatively poor sequence of end sequencing and pUC19 carrier sequence with Squencer4.5 software; Software is not removed the then deletion by hand of clean sequence.The DNA of bacteria sequence of the chip sequence of BAC carrier and pollution is removed through the method (Altschul etc., 1997) that compares with the BAC sequence or BLAST analyzes.With Squencer 4.5 softwares to the parameter that two sequences splices be: overlap length (Mini Overlap) is greater than 20bp (base pair), and the consistence of overlap (Mini Match) is greater than 85%.Each base is established a capital really will be with reference to a plurality of shotgun fragment sequences that overlap this site.For by shotgun fragment sequence institute region covered sequence verification once more, to guarantee the accuracy of base.

5.DNA sequential analysis

At first use Blastn and Blastx method (Altschul etc., 1997) to analyze institute's calling sequence.Adopt Fgenesh (http://www.softberry.ru/berry.phtml) (Salamov and Solovyev then; 2000) and GeneMark.hmm (http://exon.gatech.edu/GeneMark/eukhmm.cgi) (Besemer and Borodovsky; 2005) software analysis predicted gene structure; Analytical results further uses Blastp method (Altschul etc., 1997) to confirm.Article two, or the comparative analysis of many Nucleotide or aminoacid sequence use BLAST2 sequence method (Tatusova etc., 1999) and ClustalW method (http://www.ebi.ac.uk).

BAC clone OOBa0120J21 order-checking and sequence assembly are formed three big sequence fragments, and its length is respectively 84.1kb, 25.8kb and 3.3kb.Sequential analysis finds on the fragment of 84.1kb, to have 4 genes encoding LRR-protein kinase proteinoids; With the product of rice bacterial blight resistance gene Xa3/Xa26 coding homology is in various degree arranged, they are named as OoRKa, OoRKb1, OoRKb and OoRKf (Fig. 4).With Fgenesh, GeneMark.hmm and Blastx analysis revealed, OoRKa, OoRKb and OoRKf are 3 complete genes, and OoRKb1 is an imperfect gene (Fig. 4).The transcriptional orientation of OoRKa and OoRKb is identical, and the transcriptional orientation of OoRKf and OoRKa and OoRKb opposite (Fig. 4).

Xa3/Xa26 gene family section in this section and the paddy rice bright extensive 63 is compared analysis.The homology degree of finding the MRKa gene in the OORKa gene and bright extensive 63 in the oryza officinalis reaches 80%, the OoRKb gene in the oryza officinalis and with bright extensive 63 in Xa3/Xa26 (being called MRKb again) dna homolog degree up to 95%; And OoRKb and MRKb gene downstream have 1.4kb section homology degree to reach 83% approximately, so OoRKb is the allelotrope of Xa3/Xa26, we name this gene is Xa3/Xa26-2 (Fig. 4).

The functional verification of embodiment 2:Xa3/Xa26-2 gene

1. the structure of genetic transformation carrier

Used carrier of the present invention is pCAMBIA1301 (Fig. 5), and it is the rice genetic conversion carrier of using always (Sun etc., 2004).Go up enzyme with restriction enzyme EcoRV from BAC clone OOBa0120J21 and cut the fragment (Fig. 4) that back recovery 11.9kb comprises coding region, promotor and the afterbody sequence of Xa3/Xa26-2 gene.Simultaneously, cut genetic transformation carrier pCAMBIA1301 with the Restriction enzyme Sma I enzyme; Enzyme cuts complete, with SAP (shrimp alkaline phosphotase) dephosphorylation; Use chloroform: primary isoamyl alcohol (volume ratio 24: 1) extracting, purifying enzyme are cut product.Do ligation with enzyme switchback section of taking up that comprises the Xa3/Xa26-2 gene and the good carrier of purifying.Cut the checking positive colony through enzyme, the recombinant plasmid of acquisition is named as D101O.

2. genetic transformation and T ₀Analyze for the genetic transformation plant

Adopt agriculture bacillus mediated genetic transforming method (Lin and Zhang, 2005) that D101O is imported paddy rice susceptible variety Mudanjiang 8 (Oryza sativa ssp.japonica).The genetic transformation plant that obtains is named as D101OM (previous section of this name is that D101O is the genetic transformation container name, and M represents rice varieties Mudanjiang 8).The present invention obtains independent transformed plant 33 strains altogether.To half transformed plant inoculation bacterial leaf-blight bacterial strain PXO61 wherein, second half transformed plant inoculation bacterial leaf-blight bacterial strain PXO341.The bacterial leaf spot pathogenic bacteria inoculation adopts leaf-cutting method that the paddy rice that becomes the strain phase is inoculated (Sun etc., 2004).Bacterial leaf-blight bacterial strain PXO61 and PXO341 are so kind as to give (Sun etc., 2004 by the Philippines International Rice Research Institute; Wu etc., 2008).The method of having published (Sun etc., 2004) is followed in the cultivation of bacterial leaf spot pathogenic bacteria.Inoculate 14 days " Invest, Then Investigate " lesion areas (scab length/sick leaf length * %).Compare with contrast Mudanjiang 8 and the negative plant of genetic transformation, the resistance of the positive genetic transformation plant of the overwhelming majority significantly strengthens (table 1).

Table 1.T ₀Phenotype for genetic transformation plant (D101OM) inoculation bacterial leaf-blight bacterial strain PXO61 and PXO341

⁽¹⁾Every strain genetic transformation gene plant inoculation 3-5 sheet leaf, 14 days " Invest, Then Investigate " scabs and sick leaf length, each data is from the mean+SD of a plurality of blades.

⁽²⁾Negative transformed plant, the positive transformed plant of other plant.

3. the coseparation analysis of genetic transformation plant

For the resistance against diseases of verifying the genetic transformation plant whether strengthen with the Xa3/Xa26-2 gene change over to relevant, at T ₀T for resistance enhanced 3 strain genetic transformation plant (D101OM1, D101OM14, D101OM43) ₁Carry out genotype and resistance coseparation analysis for family.Inoculation bacterial leaf spot pathogenic bacteria PXO61 inoculates 14 days " Invest, Then Investigate " lesion areas when boot stage.The DNA extraction of taking a sample simultaneously adopts the PCR primer Gus2F (5 '-CCAGGCAGTTTTAACGATCAGTTCGC-3 ') and Gus2R (5 '-GAGTGAAGATCCCTTTCTT GTTACCG-3 ') augmentation detection positive genetic transformation plant of the entrained reporter gene GUS (GRD beta-glucuronidase) of genetic transformation carrier.Analyzing resistance enhancing and the gus gene of finding the genetic transformation plant is divided into from (Fig. 6).The resistance enhancing that the genetic transformation plant is described is the existence because of the Xa3/Xa26-2 gene, and Xa3/Xa26-2 is a bacterial leaf spot resistant ospc gene.

4. the anti-spectrum analysis of genetic transformation plant

To T ₁Inoculate different bacterial leaf-blight bacterial strains in boot stage for genetic transformation family D101OM43 and contrast Mudanjiang 8, analyze the anti-spectrum of genetic transformation plant.Above-mentioned leaf-cutting method is adopted in inoculation.Philippines bacterial leaf-blight bacterial strain PXO61, PXO71, PXO112, PXO341 are so kind as to give (Sun etc., 2004 by the Philippines International Rice Research Institute; Wu etc., 2008).Chinese white leaf blight bacterial strain is bacterial strain (Sun etc., 2004 commonly used; Li etc., 2009).Japanese strain T7174 also is a bacterial leaf-blight bacterial strain (Cao etc., 2007) commonly used.Compare with genetic transformation acceptor rice varieties Mudanjiang 8, the genetic transformation plant of carrying the Xa3/Xa26-2 gene has significantly strengthened (P＜0.01) to the resistance (Fig. 7) of different bacterial leaf-blight bacterial strains.The lesion area of genetic transformation plant just contrasts 12% to 75% of Mudanjiang 8.These presentation of results Xa3/Xa26-2 gene pairs bacterial leaf spot pathogenic bacteria has resistance of wide spectrum.

5.Xa3/Xa26-2 gene structure and coded product analysis

Researchist of the present invention utilizes transfer-gen plant (D101OM) to analyze the structure of Xa3/Xa26-2 gene.TRIzol Reagent (American I nvitrogen company) is adopted in the extracting of total RNA of transfer-gen plant, and working method is undertaken by the specification sheets that company provides.

To be used for DNA enzyme I (the American I nvitrogen company) processing of total RNA of reverse transcription with no RNA enzymic activity earlier, room temperature was placed 15 minutes, removed the DNA that possibly exist among total RNA and polluted; Again total RNA was handled deactivation DNA enzyme I 10 minutes at 65 ℃.With SuperScript III ThermoScript II (American I nvitrogen company), carry out reverse transcription by its specification sheets that provides.Adopt PCR primer RKb3F and RKb2R (table 2) amplifying genom DNA and the RNA reverse transcription product of crossing over the prediction introne position, the RT-PCR product is littler than genome amplification product, explains that there is an intron (Fig. 8) really in this section.Adopt other primer (table 2) of Xa3/Xa26-2 constant gene segment C to carry out reverse transcription (RT)-pcr analysis, do not find the existence of other intron again.

The gene terminal sequence analysis adopts the Clontech SMARTer RACE cDNA Amplification Kit of company test kit; According to the test kit specification sheets; Through RACE (rapid amplification of cDNA end) analytical procedure, confirm 5 ' and 3 ' end sequence of Xa3/Xa26-2 gene.

Table 2. is used for the gene-specific primer of RT-PCR and RACE analysis

Adopt above-mentioned dna sequencing method, the RT-PCR product is checked order.The cDNA sequence and the genome sequence of comparative analysis Xa3/Xa26-2 gene confirm that this gene is made up of 3600 Nucleotide, comprise two exons and an intron (Fig. 8).First exon is formed (the 3238-6191bp place that is positioned at sequence table SEQ ID NO:1) by 2954 Nucleotide; (untranslated region UTR) (is positioned at the 3238-3283bp place of sequence table SEQ ID NO:1) and the coding regions (being positioned at the 3284-6191bp place of sequence table SEQ ID NO:1) of 2908 Nucleotide compositions wherein to comprise 5 ' the end non-translational region be made up of 46 Nucleotide; Intron is formed (the 6192-6294bp place that is positioned at sequence table SEQ ID NO:1) by 103 Nucleotide; Second exon formed (the 6295-6837bp place that is positioned at sequence table SEQ ID NO:1) by 543 Nucleotide, wherein comprises coding region of being made up of 368 Nucleotide (being positioned at the 6295-6662bp place of sequence table SEQ ID NO:1) and 3 ' the end UTR (being positioned at the 6663-6837bp place of sequence table SEQ ID NO:1) that is made up of 175 Nucleotide.

LRR receptor kinase proteinoid of forming by 1092 amino acid of Xa3/Xa26-2 genes encoding.The proteic amino acid consistence of XA3/XA26-2 albumen and XA3/XA26 is 92%, and amino acid whose homology is 95%.

Reference

Chu Zhaohui, Wang Shi is flat, (2007), resistant gene separating clone, structure and function and molecular evolution are seen: Zhang Qi chief editor, the heredity of bacterial blight of rice resistance and improvement.Beijing: Science Press, pp.349-377.

Cross Chong Jian (1995) Crop in China disease and pest.The chief editor of Plant Protection institute, Chinese Academy of Agricultral Sciences, Chinese agriculture press, pp.14-24.

Tan Guangxuan appoints Xiang, Weng Qingmei, Shi Zhenying, Zhu Lili, He Guangcun, the location of a new gene of resisting bacterial leaf-blight of oryza officinalis transformation offspring, Acta Genetica Sinica, (2004) 31:724-729.

Wang Chunlian, Zhao Ping Yu, Zhang Qi, Zhao Kaijun, Xing Quandang, the evaluation of bacterial blight of rice new resistance source Y238 and near isogenic line thereof are cultivated, plant genetic resources journal, (2004) 5:26-30.

Zhang Qi, Zhao Ping Yu, Zhao Kaijun, the evaluation of the new gene Xa23 of anti-bacterial blight of rice of common wild-rice and molecule marker location, Acta Agronomica Sinica, (2000) 26:536-542.

Altschul SF，Madden TL，Schaffer AA，Zhang J，Zhang Z，Miller W，Lipman DJ(1997)GappedBLAST and PSI-BLAST：a new generation of protein database search programs.NucleicAcids Res.25：3389-3402.

Amante-Bordeos A，Sitch LA，Nelson R，Dalmacio RD，Oliva NP，Aswidinnoor H，Leung H(1992)Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta tocultivated rice，Oryza sativa.Theor.Appl.Genet.84：345-354.

Ammiraju JS，Luo M，Goicoechea JL，Wang W，Kudrna D，Mueller C，Talag J，Kim H，SisnerosNB，Blackmon B，Fang E，Tomkins JB，Brar D，MacKill D，McCouch S，Kurata N，Lambert G，Galbraith DW，Arumuganathan K，Rao K，Walling JG，Gill N，Yu Y，SanMiguel P，SoderlundC，Jackson S，Wing RA(2006)The Oryza bacterial artificial chromosome library resource：construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10genome types of the genus Oryza.Genome Res.16：140-147.

Besemer J，Borodovsky M(2005)GeneMark：web software for gene finding in prokaryotes，eukaryotes and viruses.Nucleic Acids Res.33：451-454.

Cao Y，Ding X，Cai M，Zhao J，Lin Y，Li X，Xu C，Wang S(2007)The expression pattern of a ricedisease resistance gene Xa3/Xa26 is differentially regulated by the genetic backgrounds anddevelopmental stages that influence its function.Genetics 177：523-533.

Chisholm ST，Coaker G，Day B，Staskawicz BJ(2006)Host-microbe interactions：shaping theevolution of the plant immune response.Cell 124：803-814.

Gu K，Yang B，Tian D，Wu L，Wang D，Sreekala C，Yang F，Chu Z，Wang GL，White FF，Yin Z.(2005)R gene expression induced by a type-III effector triggers disease resistance in rice.Nature 435：1122-1125.

Li G，Song CF，Pang XM，Yang Y，Wang JS(2009)Analysis of pathotypic and genotypic diversityof Xanthomonas oryzae pv.oryzae in China.J.Photopathol.157：208-218.

Lin YJ，Zhang Q(2005)Optimising the tissue culture conditions for high efficiency transformationof indica rice.Plant Cell Rep.23：540-547.

Khush GS，Bacalangco E，Ogawa T(1990)A new gene for resistance to bacterial blight from O.longistaminate.Rice Genet.Newslett.7：121-122.

Salamov A，Solovyev V(2000)Ab initio gene finding in Drosophila genomic DNA.Genome Res.，10：516-522.

Sambrook J，Russell D(2001)Molecular cloning：A laboratory manual.Cold Spring Harbor，NY：Cold Spring Harbor Laboratory Press.

Song WY，Wang GL，Chen LL，Kim HS，Pi LY，Holsten T.Gardner J，Wang B，Zhai WX，Zhu LH，

Fauquet C，Ronald P(1995)A receptor kinase-like protein encoded by the rice diseaseresistance gene，Xa21.Science 279：1804-1806.

Sun X，Cao Y，Yang Z，Xu C，Li X，Wang S，Zhang Q(2004)Xa26，a gene conferring resistance to

Xanthomonas oryzae pv.Oryzae in rice，encoding a LRR receptor kinase-like protein.Plant J.37：517-527.

Sun X，Cao Y，Wang S(2006)Point mutations with positive selection were a major force during theevolution ofa receptor-kinase resistance gene family of rice.Plant Physiol.140：998-1008.

Tatusova TA，Madden TL(1999)BLAST2 Sequences，a new tool for comparing protein andnucleotide sequences.FEMS Microbiol.Lett.174：247-250.

Wang GL，Song WY，Ruan DL，Sideris S，Ronald PC(1996)The cloned gene，Xa21，confersresistance to multiple Xanthomonas oryzae pv.oryzae isolates in transgenic plants.Mol.Plant-Microbe Interact.9：850-855.

Wu X，Li X，Xu C，Wang S(2008)Fine genetic mapping of xa24，a recessive gene for resistanceagainst Xanthomonas oryzae pv.oryzae in rice.Theor.Appl.Genet.118：185-191.

Xiang Y，Cao Y，Xu C，Li X，Wang S(2006)Xa3，conferring resistance for rice bacterial blight andencoding a receptor kinase-like protein，is the same as Xa26.Theor.Appl.Genet.113：1347-1355.

Yang Z，Sun X，Wang S，Zhang Q(2003)Genetic and physical mapping of a new gene for bacterialblight resistance in rice.Theor.Appl.Genet.106：1467-1472.

Sequence table

< 110>Hua Zhong Agriculture University

< 120>oryza officinalis resisting bacterial leaf-blight major gene Xa3/Xa26-2 and its application in improvement paddy disease-resistant property

<130>

<141>2010-03-17

<160>2

<170>PatentIn version 3.3

<210>1

<211>11848

<212>DNA

<213>Oryza sativa

<220>

<221>gene

<222>(3238)..(6837)

<220>

<221>5’UTR

<222>(3238)..(3283)

<220>

<221>CDS

<222>(3284)..(6191)

<220>

<221>Intron

<222>(6192)..(6294)

<220>

<221>CDS

<222>(6295)..(6662)

<220>

<221>3’UTR

<222>(6663)..(6837)

<400>1

tatcttagat atgatgagca tgtgcaccat tagacgtata ataaagataa agggcatttt 60

ataatataga ttatatcatt ctatatcatt ttataaacat acatagccaa agtcatgtgt 120

gatttacttt aaatctaact aaaaaaatca tgtctacaat aataagaatc atagaaattt 180

aaaaaacaga aaaactcaag agcatatacc ctaatagtaa ataaaaatat caatggctaa 240

tccctaggac atataaaatt ataaaggttt gttaaacgtc ctatacaacc ctcctacagt 300

gtctattaaa gtctttaaca aataagagaa aactttaaaa ttcttctata atagacaaag 360

catccaacac ttaatcctca ttaatttaat gctcccatta attttagcca ttggtcatgt 420

caatgggtat gtgtatacac acattattta ataaaaaaac catccattca agcgtgtttt 480

gtaatagaaa attatttgag atgtcaggtg aggttgtagt gacctgttgc aaggttcaca 540

gggttgtagc aagaatgacc agcatcaagt tgcaagaggt tgccagccat ctaatgtatc 600

actaaaagag cccataggag atatttgtgt tttgttgatt agagccacaa ccaaaagaag 660

gcaacaagag aaccccaaca caaataattt ttatgaataa accttctctc aactcaagta 720

tctatgtaat attatttaag tcgcatcagc ttgaaacatg cataatttga taacatggta 780

tatctagaag gctatatgtt agaacattca attaacccac cttagaaata ataatactta 840

gcaagaactc atgcctgtaa caaatttcta tctttaattt tgatatttaa ttttatcact 900

gaaaaggggg agatagagta tgtgaatatt gtccaatttg caagctaaat gtttttttaa 960

atgtgatagt tcagttggtt actgattctc aagtattatc atactattca agagtagagc 1020

aagcaataat catatccaaa acagatacaa agaacttaca tatcatagat aattttaaca 1080

tttagaaaca cgaagatgcc tgcgcaaccg cgtgggctac cttcctagtt aatataaata 1140

tgtaaaggta taacttaaga ttataattta tttgatgata aaacaagtat ttatgtatat 1200

atatatatat atatttaata aaatgaatga tcaaacgcta tacgaaaaat caacggcgtc 1260

gtgcattttg aaatggagga agtatttttt ttcgaggtaa tcgttatgtt tgaggaaatg 1320

tggcatgagt tcaccacttg ccaacttgga atataacaca tcgtatcgtt agtgttccat 1380

gcactaaaag tctctttctt tcccctttgg aatgagaaca atatagcttg gtaatactgt 1440

tcatcttatt cgcctttttc ctcctttgga aagggacagt tgagcggtgg tctggtggtt 1500

acatctaaaa catgccatga gaagctttgg cactgtgtgt cttgtgcttc aaatggctcg 1560

acattacaac ctaataaatc tgaacacttt gttccatctt ggacttcaga gcactggtac 1620

ttcagtacat gcccaagggt agcttagaag cactctgcac tcagaacaag gaaagcaatt 1680

aggctttctc cagaggttgg ttattatgct agatgtgtca atggcaatgg aatacctaca 1740

tcatgagcac tatgaggtgg tcttacacta cgatctgaag cctagcaacg tactatttga 1800

cgatgatatg atggcacatg tgggagactt tggcattgca aggttattgt taggtgatga 1860

caactccatg atctcagcta gcatgccagg aacagttggg tacatggcac caggtactta 1920

gtactagttt ttgttgtctt gctcaaacat tgcctgatat tttattatta tcaagtaggg 1980

tgcaactaat ttttggttgt ctatctttct taagcagagt atggggctct tggaaaagca 2040

tcacagaaga gcgacgtgtt cagctacggg atcatgctac tcgaagtgtt cactaggaag 2100

agatccacag atgttatgtt tgtaggagaa ctgaacatca ggcagtgggt tcaccaggcg 2160

tttcctgcag agcttgtcca tttgatggac tgccaacttc tacaggatgg ctcttcttct 2220

tcttccagta acatgcatgg cttccttgtg ccagtgttcg agctgggctt gctctgctca 2280

gctgactccc cggagcaaag gatggcgatg agcgatgtgg tcgtgacact gaagaagatt 2340

aggaaggact atgtcaaatt gatggcaacc acagggagcg ctgtgcagca atgatccatc 2400

gctctctcgt ggtatatgag cgaataaaat atatatcctt tgcatccatt tcttcttctg 2460

catcaggaat agcatcagtg catgcccagt gatcgattac cctatttgtg tacggttgaa 2520

ttgaatatat ctgtggtgct tcaggttcag caataattta gttggtgtaa aaatgtgatt 2580

gaactgttgg tcaataaatt tgcatgatga aaatgggagt agatgatgtg ctgcttatgt 2640

tttcttattt ctggccaaaa taaataaaaa aaggaatatt ctgggcacag catcacaact 2700

ccggctcaat cagccttaaa cagccacagt taacagtcct aagcagagaa acttaacaag 2760

cttttcaggc aaacaaaaca tcaaaaggtc cacaagacaa cagggtcttc agggagcaca 2820

tcttcaggct gtgatgcaaa aaggatctga cagccgtatg ataactactg aacaggtcgt 2880

gtatattgat aaggtccgcc actaaccacc caatcaacaa aagagtaagg ccgttgccgt 2940

caaattgttt gacagaaaaa aaaaatttca gtgagtgtat ggctgatgac cgagcgacac 3000

agctatcata tctagcgtgt cgtgcacacc gcgatctgct gaatatatat tttgtgatga 3060

ctttattttc cagcgtttac ttagtagtgc tgccaaatat ttatgactgg aatttgactg 3120

gagggagtat catttaagtt tctttcactt tctgagagca acagtcaagg tcgtccgaga 3180

tgttgaaagc aagctagcac tactgtgcta aataaagctc aacttgatcg tcactgtgaa 3240

gtatgatgca ctcttgttgc caatgcatca cacacaacca gac atg gcg ctt gga 3295

Met Ala Leu Gly

1

ttg cca gta tgg att ttc gtt gcg ttg ttg atc gct ttg tcc act gtg 3343

Leu Pro Val Trp Ile Phe Val Ala Leu Leu Ile Ala Leu Ser Thr Val

5 10 15 20

cct tgt gct tcc tct cta ggt ccg agc aag agt aac ggc agt gac atc 3391

Pro Cys Ala Ser Ser Leu Gly Pro Ser Lys Ser Asn Gly Ser Asp Ile

25 30 35

gac ctc gct gca ctt ttg gct tta aaa tcg cag ttc tct gat cct gat 3439

Asp Leu Ala Ala Leu Leu Ala Leu Lys Ser Gln Phe Ser Asp Pro Asp

40 45 50

aac att ctt gcc ggc aac tgg acc att ggc acg cca ttc tgc caa tgg 3487

Asn Ile Leu Ala Gly Asn Trp Thr Ile Gly Thr Pro Phe Cys Gln Trp

55 60 65

atg ggt gtc tcg tgc agc cac cgc cgg cag cgc gtc acc gcc ctg aaa 3535

Met Gly Val Ser Cys Ser His Arg Arg Gln Arg Val Thr Ala Leu Lys

70 75 80

ctg cca aac gtt cct ctc caa gga gag ctc agc tct cac ctt ggt aac 3583

Leu Pro Asn Val Pro Leu Gln Gly Glu Leu Ser Ser His Leu Gly Asn

85 90 95 100

att tct ttt ctc ttg atc ctc aac ctc acc aac acc ggc ctc aca ggc 3631

Ile Ser Phe Leu Leu Ile Leu Asn Leu Thr Asn Thr Gly Leu Thr Gly

105 110 115

ttg gtg ccg gat tat ata gga agg cta cgt cgc ctt gag atc ctt gat 3679

Leu Val Pro Asp Tyr Ile Gly Arg Leu Arg Arg Leu Glu Ile Leu Asp

120 125 130

ctc ggc cac aat gcc ttg tca ggt ggc gtc cca atc gcc ata ggg aac 3727

Leu Gly His Asn Ala Leu Ser Gly Gly Val Pro Ile Ala Ile Gly Asn

135 140 145

ctc acg agg ctt cag cta ctt aat cta cag ttt aac cag cta tat ggt 3775

Leu Thr Arg Leu Gln Leu Leu Asn Leu Gln Phe Asn Gln Leu Tyr Gly

150 155 160

cca atc cca gca gag ctg cag ggg ctg cac agt ctt gac agc atg aat 3823

Pro Ile Pro Ala Glu Leu Gln Gly Leu His Ser Leu Asp Ser Met Asn

165 170 175 180

ctc cgt cac aat tac ctc act gga tca att ccg gac aat ctg ttc aac 3871

Leu Arg His Asn Tyr Leu Thr Gly Ser Ile Pro Asp Asn Leu Phe Asn

185 190 195

aac aca tct ttg cta act tat ctc aac gtt ggt aac aat agc ctg tca 3919

Asn Thr Ser Leu Leu Thr Tyr Leu Asn Val Gly Asn Asn Ser Leu Ser

200 205 210

gga ccg ata ccg ggt tgc atc ggt tcc ttg cca atc ctc caa tac ctt 3967

Gly Pro Ile Pro Gly CysIle Gly Ser Leu Pro Ile Leu Gln Tyr Leu

215 220 225

aac ttg cag gcc aat aac tta act ggg gcg gtg cca cca gcc atc ttc 4015

Asn Leu Gln Ala Asn Asn Leu Thr Gly Ala Val Pro Pro Ala Ile Phe

230 235 240

aac atg tct aaa tta agt act att tct ctt ata tcg aat ggt tta act 4063

Asn Met Ser Lys Leu Ser Thr Ile Ser Leu Ile Ser Asn Gly Leu Thr

245 250 255 260

ggc cct atc cct ggt aat aca agt ttc agc ctc cca gtt cta caa tgg 4111

Gly Pro Ile Pro Gly Asn Thr Ser Phe Ser Leu Pro Val Leu Gln Trp

265 270 275

ttc gcc atc agt aaa aac aat ttc ttt ggt caa att cca ctg ggg ctc 4159

Phe Ala Ile Ser Lys Asn Asn Phe Phe Gly Gln Ile Pro Leu Gly Leu

280285290

gca gcg tgt cca tac ctc caa gtt att gcc ctg cct tat aat tta ttc 4207

Ala Ala Cys Pro Tyr Leu Gln Val Ile Ala Leu Pro Tyr Asn Leu Phe

295300305

gag ggt gtt ttg cca cca tgg ctg ggc aag ttg acg agt ctt aat gcc 4255

Glu Gly Val Leu Pro Pro Trp Leu Gly Lys Leu Thr Ser Leu Asn Ala

310315320

atc tcc ttg ggt tgg aat aac ctt gat gct ggc ccg atc cct act gaa 4303

Ile Ser Leu Gly Trp Asn Asn Leu Asp Ala Gly Pro Ile Pro Thr Glu

325 330 335 340

ctt agc aac ctc acc atg ctg gca gtc tta gat ttg tcg acg tgc aac 4351

Leu Ser Asn Leu Thr Met Leu Ala Val Leu Asp Leu Ser Thr Cys Asn

345 350 355

ctg aca gga aac atc cct gca gat att ggg cac cta ggc caa ctt tca 4399

Leu Thr Gly Asn Ile Pro Ala Asp Ile Gly His Leu Gly Gln Leu Ser

360 365 370

tgg ttg cat ctt gcg agg aat caa cta aca gga cct att cct gct tct 4447

Trp Leu His Leu Ala Arg Asn Gln Leu Thr Gly Pro Ile Pro Ala Ser

375 380 385

ctt ggc aac ctt tca tcg tta gca atc ctg cta ttg aaa gga aac ttg 4495

Leu Gly Asn Leu Ser Ser Leu Ala Ile Leu Leu Leu Lys Gly Asn Leu

390 395 400

ttg gat gga tca tta cca gcg aca gtt gat agc atg aac tca cta act 4543

Leu Asp Gly Ser Leu Pro Ala Thr Val Asp Ser Met Asn Ser Leu Thr

405 410 415 420

gca gtt gat gtt act gaa aac aat cta cac gga gat ctc aac ttc ctt 4591

Ala Val Asp Val Thr Glu Asn Asn Leu His Gly Asp Leu Asn Phe Leu

425 430 435

tct act gtt tcc aat tgt aga aag ctt tct acc ctt caa atg gac ttt 4639

Ser Thr Val Ser Asn Cys Arg Lys Leu Ser Thr Leu Gln Met Asp Phe

440 445 450

aat tat atc acc gga agc ctc cca gac tat gtt ggg aac ctg tcg tca 4687

Asn Tyr Ile Thr Gly Ser Leu Pro Asp Tyr Val Gly Asn Leu Ser Ser

455 460 465

cag ctg aaa tgg ttc acg tta tct aac aac aag tta act ggc acg ctt 4735

Gln Leu Lys Trp Phe Thr Leu Ser Asn Asn Lys Leu Thr Gly Thr Leu

470 475 480

cca gct acc att tca aat tta act ggt ctt gag gtg ata gat ctt tcg 4783

Pro Ala Thr Ile Ser Asn Leu Thr Gly Leu Glu Val Ile Asp Leu Ser

485 490 495 500

cat aac caa ctg cgc aat gca att cca gaa tca atc atg acg att gag 4831

His Asn Gln Leu Arg Asn Ala Ile Pro Glu Ser Ile Met Thr Ile Glu

505 510 515

aat ctc caa tgg ctt gac cta agt gga aat agc ttg tct ggc ttc atc 4879

Asn Leu Gln Trp Leu Asp Leu Ser Gly Asn Ser Leu Ser Gly Phe Ile

520 525 530

cca tcg aat act gca ctt cta agg aac att gta aaa cta ttc ctt gaa 4927

Pro Ser Asn Thr Ala Leu Leu Arg Asn Ile Val Lys Leu Phe Leu Glu

535 540 545

agc aac gaa att tct ggc tcc ata cca aag gac atg aga aac ctc act 4975

Ser Asn Glu Ile Ser Gly Ser Ile Pro Lys Asp Met Arg Asn Leu Thr

550 555 560

aat cta gag cac ctt cta ttg tct gat aac caa tta acg tca acc gtg 5023

Asn Leu Glu His Leu Leu Leu Ser Asp Asn Gln Leu Thr Ser Thr Val

565 570 575 580

cca cca agc tta ttt cat ctt gat aaa atc atc agg cta gat ctt tct 5071

Pro Pro Ser Leu Phe His Leu Asp Lys Ile Ile Arg Leu Asp Leu Ser

585 590 595

cga aac ttc ttg agt ggt gca ctg ccg gtt gat gta ggg tac ttg aag 5119

Arg Asn Phe Leu Ser Gly Ala Leu Pro Val Asp Val Gly Tyr Leu Lys

600 605 610

caa att acc atc ata gat ctc tct gac aac agc ttt tct ggc agc atc 5167

Gln Ile Thr Ile Ile Asp Leu Ser Asp Asn Ser Phe Ser Gly Ser Ile

615 620 625

cca gat tcg ata gga gaa ctt cag atg tta aca cac ctg aat cta tca 5215

Pro Asp Ser Ile Gly Glu Leu Gln Met Leu Thr His Leu Asn Leu Ser

630 635 640

gct aac gaa ttc tat gat tct gtt cca gac tct ttt ggt aat tta act 5263

Ala Asn Glu Phe Tyr Asp Ser Val Pro Asp Ser Phe Gly Asn Leu Thr

645 650 655 660

ggc ttg caa act ttg gac ata tcc cat aac aat att tct ggt acc atc 5311

Gly Leu Gln Thr Leu Asp Ile Ser His Asn Asn Ile Ser Gly Thr Ile

665 670 675

cca aac tac ttg gct aat ttt acg acc ctt gtt agc ttg aac cta tct 5359

Pro Asn Tyr Leu Ala Asn Phe Thr Thr Leu Val Ser Leu Asn Leu Ser

680 685 690

ttc aat aaa cta cat ggt caa ata ccg gaa gga ggt atc ttt gca aac 5407

Phe Asn Lys Leu His Gly Gln Ile Pro Glu Gly Gly Ile Phe Ala Asn

695 700 705

atc act tta caa tac ttg gtg ggg aac tca ggg cta tgt ggt gct gcc 5455

Ile Thr Leu Gln Tyr Leu Val Gly Asn Ser Gly Leu Cys Gly Ala Ala

710 715 720

cgt tta gga ttc cca cca tgc caa acc acc tcc ccc aag aga aat ggt 5503

Arg Leu Gly Phe Pro Pro Cys Gln Thr Thr Ser Pro Lys Arg Asn Gly

725 730 735 740

cac atg cta aaa tac ttg cta ccg act ata atc ata gta gtt gga gtt 5551

His Met Leu Lys Tyr Leu Leu Pro Thr Ile Ile Ile Val Val Gly Val

745 750 755

gta gct tgt tgc ttg tat gta atg att aga aag aaa gct aac cat caa 5599

Val Ala Cys Cys Leu Tyr Val Met Ile Arg Lys Lys Ala Asn His Gln

760 765 770

aag att tct gct ggt atg gct gac ctt atc agc cat caa ttt ctg tcc 5647

Lys Ile Ser Ala Gly Met Ala Asp Leu Ile Ser His Gln Phe Leu Ser

775 780 785

tat cat gag ctt ctt cgt gca acc gat gat ttc agt gat gat aac atg 5695

Tyr His Glu Leu Leu Arg Ala Thr Asp Asp Phe Ser Asp Asp Asn Met

790 795 800

ttg ggc ttc gga agc ttt gga aaa gtt ttt aag gga cag ttg agc aac 5743

Leu Gly Phe Gly Ser Phe Gly Lys Val Phe Lys Gly Gln Leu Ser Asn

805 810 815 820

ggt atg gtg gtt gcc ata aaa gta ata cac cag cat ctg gaa cat gcc 5791

Gly Met Val Val Ala Ile Lys Val Ile His Gln His Leu Glu His Ala

825 830 835

atg aga agc ttt gac acc gag tgt cgt gta ctc cga att gct cga cat 5839

Met Arg Ser Phe Asp Thr Glu Cys Arg Val Leu Arg Ile Ala Arg His

840 845 850

cgc aac ctg ata aag att ctg aac act tgt tcc aac ctg gac ttc aga 5887

Arg Asn Leu Ile Lys Ile Leu Asn Thr Cys Ser Asn Leu Asp Phe Arg

855 860 865

gca ctc gta ctt cag tac atg ccc aag ggt agc tta gaa gca ctc ctg 5935

Ala Leu Val Leu Gln Tyr Met Pro Lys Gly Ser Leu Glu Ala Leu Leu

870 875 880

cat tca gaa caa gga aag caa tta ggc ttt ctc gag agg ttg gat att 5983

His Ser Glu Gln Gly Lys Gln Leu Gly Phe Leu Glu Arg Leu Asp Ile

885 890 895 900

atg cta gat gtg tca atg gca atg gaa tac ctg cat cat gag cac tat 6031

Met Leu Asp Val Ser Met Ala Met Glu Tyr Leu His His Glu His Tyr

905 910 915

gag gtg gtc tta cac tgc gat ttg aag cct agc aac gta cta ttt gac 6079

Glu Val Val Leu His Cys Asp Leu Lys Pro Ser Asn Val Leu Phe Asp

920 925 930

gat gat atg acg gca cat gtg gca gac ttt ggc att gca agg ttg ttg 6127

Asp Asp Met Thr Ala His Val Ala Asp Phe Gly Ile Ala Arg Leu Leu

935 940 945

tta ggt gat gac aac tcc atg atc tca gct agc atg cca gga aca gtt 6175

Leu Gly Asp Asp Asn Ser Met Ile Ser Ala Ser Met Pro Gly Thr Val

950 955 960

ggg tac atg gca cca g gtacttagta ctagtttttg ttgtcttgct caagcattgc 6231

Gly Tyr Met Ala Pro

965

tgatctttta ttattatcaa gtagggtgcg actaattttt ggtgactaac ttttcttgag 6291

cag ag tat ggg gct cta gga aag gcg tca cgg aag agc gat gtg ttc 6338

Glu Tyr Gly Ala Leu Gly Lys Ala Ser Arg Lys Ser Asp Val Phe

975 980

agt tac ggg atc atg ttg ttt gaa gtg ttc act ggg aag aga ccc aca 6386

Ser Tyr Gly Ile Met Leu Phe Glu Val Phe Thr Gly Lys Arg Pro Thr

985 990 995 1000

gat gct atg ttt gtg gga gaa ctg aac atc agg cag tgg gtt cac 6431

Asp Ala Met Phe Val Gly Glu Leu Asn Ile Arg Gln Trp Val His

1005 1010 1015

cag gcg ttt cct gca gag ctt gtc cat gtg gtg gac tgc caa ctt 6476

Gln Ala Phe Pro Ala Glu Leu Val His Val Val Asp Cys Gln Leu

1020 1025 1030

cta cat gat ggc tct tct tcc agt aac atg cat ggc ttc ctt gtg 6521

Leu His Asp Gly Ser Ser Ser Ser Asn Met His Gly Phe Leu Val

1035 1040 1045

cca gtg ttc gag ctg ggc ttg ctc tgc tca gct gac tcc cca gac 6566

Pro Val Phe Glu Leu Gly Leu Leu Cys Ser Ala Asp Ser Pro Asp

1050 1055 1060

caa agg atg gcg atg agc gat gtg gtc gtg aca ctg aag aag att 6611

Gln Arg Met Ala Met Ser Asp Val Val Val Thr Leu Lys Lys Ile

1065 1070 1075

agg aag gac tat gtc aaa ttg atg gca acc aca gag aac gct gtg 6656

Arg Lys Asp Tyr Val Lys Leu Met Ala Thr Thr Glu Asn Ala Val

1080 1085 1090

cag cag tgattcatca ctttcttgtg gtatatgagc gaatgaaatg tatatccttt 6712

Gln Gln

gcatccattt cttcttctgc attaggaaca gcatcagtgc atgcccagtg atcgaataac 6772

ccttttgctt ctatttgtgt atggttgaat tgaatatatc tacggtgctt caggttcagc 6832

aacaatttag ttggtgtaaa aatgtgattg aactgctggt cgataaattt gcatcatgaa 6892

aatgaaaatg ggagtagatg atgtgctgct tatattttcc tatttctggc caaatatata 6952

taaaaaagga tattctcact tgaaaacaga atgaggtttg ctttgtagac attgggcctt 7012

tgtcgtgggc ttctctggta ccctgacatg ctttatagcc catgggccta tgtttgtaat 7072

gggcttttgt ttctttcgaa tgactcaagg tatattaagg cctgtttgaa cacttcggaa 7132

tattttgcgg tggtagaggt ggacactaaa cgacgacata ggtaaaatga tatatagaaa 7192

caaaattaat tcaaacgata tactcatgct ataaatttga gatatggtaa ttaataaaac 7252

ttcacatagg caacgaaccg tatatataac agttaatgca taataatggc agagatttga 7312

taatgaatcc tccaagattt actcttgtga ctctatataa acagctgcaa aatgaagcta 7372

ggtagtcagc atatgcagta gcttccttct aaaagaagat ctctcatctc atcatggagc 7432

aacttctcaa caagaaggct gcagtgttct tgttcatagc tcttatggtg atggctaccg 7492

taaatttctc atcctgtcat actacacaag gtatatacct ctggaattaa tttctacatc 7552

acatccatat atattgaaaa cagtttaact tgttctcgtt tgcaatggct ccatcacatc 7612

aatgaagcta gaataatctg atatagcctg acaagtatat gtgcatttta cattttctgt 7672

atgaactatt ttagctttct gtaaaagtcg ggtcattaac aatataatga tcttttctct 7732

aaaaacatgc attttacatg tgtatatata taggtggata tggagaaatg gattcgtgca 7792

tggtccttga acgttgcgat atgaacaagt gcatgagtgc ctgccaagtc aacaagtaca 7852

acggaggtca gtgcgacggc gagttgaacg accactgctg ctgtactgat gaggccccgc 7912

acaaataata gatttctctt gtccaaatca acgacgacgg ttgtataatc aacggcaatc 7972

ttattattga ctgatgcaga gattccataa taaacttgca gttgatgctt gtaccaattc 8032

actcagtatg tcaattagta aaaggagaat cccacgcaaa tgtgcgggca acttatttat 8092

tctttattaa aagaataaat tttatttaaa gactgtagat tatcttatgt cattaaaaaa 8152

gaaatatctc tatcctgaaa ttgatgatag actttgatac actaaactaa cttataagag 8212

tatctctcat attaaaaaaa gatcactatg tcttggttat atataaactc tcctatcaca 8272

actacttatt attctctcga aaaagctaag tacgttttta atagatagaa ttgcttatta 8332

aaaaacaaac ataatacatc attaatgcaa taaaaaaatc atctctattt cattccgaag 8392

tgcacatgtc tcacttgtgt ctgaatgtat aaatatttta aaatgccatt tctgtcaatt 8452

aatgaaagaa aaccattaaa attagatata aatacccatg taatgtgtaa agttggtcat 8512

agctgttaag gctttattct ctaatgcctt gaaatatggg tcctgcattg agccatgccc 8572

ctcaagacta cgtcgatcat aatttttaac acctagctca ctctctacac atttataaag 8632

aattgtatca gtaagtaggc tagttgagct ctacctccaa gagtgaaaga agtcataggg 8692

gattcgatgg actgatccta tgtatgtgtg tacaaacata ataacatttt agttttaaat 8752

ttgaggatta ggagggaaag attaatattt tttcaattgt acatgcatac atatattttt 8812

tctagggttt tggatgaaca gtacttattt ttctgtggta gtggtactta catgggagat 8872

taacagggaa ggattacttt aatacatcta atcaaatatt ttaaaataat gggtatattg 8932

gtttaagtat aaatcaatgg tagatgtttt gcttttttct catatttttt tatattttca 8992

ccaatttatt agagcatcag gttgttgcct aaggagcgtt tgtagagatt aggtgggagt 9052

gattagttta acagatctaa tctaataatt aaaaatattg ggtcaaccca tttaagtata 9112

atcaatcgct agatgtttta ctttttttta gaatttcttg gatttttcct aatttattag 9172

agcaccatgt gacggcttaa gattgtttgt agcagtgcca catggtggct tagaagcgtt 9232

tgtatgatgt tcaatggact tttagtatat aataatattt ttgtctcaat gattggtgga 9292

tagcactgca tatgtagaat tgatacatga gttcgagtct gacataaaat taacacataa 9352

ggtaagacat gcatgagtcg aatacattgg atcttattta ttatataccc agattaactt 9412

aagtttaaac tgcagagact gaagaggaaa agatcaacat atatgcctcc tgttactctt 9472

aattaagaca tatcgatccg gttcatttaa tcatttattt tctatagtcc tttacaatat 9532

atctagattt atattactgt gataattgat attttaaagc gaagataata ttttttaatg 9592

gtaatcacaa ttagagtatt ttagtttgga acattgtaga ttgctttaaa tattaaagtt 9652

atgatgatgt caatcatgaa agatttaagt aaagttattt tgttttgtga aaaataatat 9712

tgttttaaca aataaatatt ttagaacagt ggtattattt tttgggagtg ctaatcacaa 9772

ctaattagaa tattttattt tgtgatatta ttgagctaat ctaattatgc atgattgtga 9832

aatgatatat gatgatgtga tctacattat ctattttctt cacaaccttt tttattgttg 9892

ggatgacatg caaaaggttc ggatatgatt taaaatacta gaaggaatat tttttggaac 9952

gtcactcacg atttaagtgt ttttgttttt tttcatatct atcatattta atttatttgt 10012

gcatgtttta taaagtgata tatgatcata taatatacgt tgtcattttt ttatttaaca 10072

gcttcaattg catgcttagg ttaggatgtg atgaaaatta aatatttttt tatgagttaa 10132

agaatgtagt ggcttatatg ggtatttgaa tggttaatta agcatcatga atacaattat 10192

aaagaatgat aaaaagattt gtgtgtaatt taaagattat gattatttct ctatacggaa 10252

attatttaaa aatattattt ttaattgtaa caacttaaaa gaaaaaaaga gatgagaaga 10312

cttataggag ggggagggat acgtacatac actatgggag ggggaggggg gtcggctggt 10372

ggggggagag gggagggagg tgtattgtgg ggttttttta aaaaaaataa atctaataga 10432

taaatatagt aggcctaccg gtttattaga gagtcatatg acaacttaga agcatttata 10492

gaagccccac gtgacggctt gagaatgttt agtagaagtt taatgaactt ttagtatata 10552

atagataaat taattaatga tgatgaggtt ggcctgcctt ggcttctctc atgttgattt 10612

actattgtgt atggcccatg tatatgaaaa ggcaaaatgt agcaatgagt cggtttgact 10672

taaaacatgc gggtttaaaa tgacatgctc tgtaaccgat gagagtactc agtttaagtt 10732

aaggtaacaa atcaatatta caagcagaaa tacagatgca agtggtacga acaaacggtt 10792

ttttattcta attattttca aattcagttt tcaaagtaat agtgtttatt tgatctggaa 10852

tttcataggg atgagcaatc ttaagccaaa tatcataatc ttgagccata atgtaagatt 10912

acatcttgtc acaccaataa ggaaaatcga caccatcgaa aacatgtggc ttggttgaaa 10972

acttgttagc catgacttaa aactttagat cggtaaaaat ccaaagaaga aaacgaggct 11032

ctgataccac ttgtaggatc gaatcccaaa taccaagcca accagagggg gtgaatgtat 11092

cctattgtcc ctggcatgct ggctgtaact gcagagttgt catcacctaa cagtaatttt 11152

gcaatgccaa agtccgccac atgtgccgtg atttcctcat caaacagcac attgctaggc 11212

ttgaggtcac agtactgcaa aacaacctca gaatgatgat agtgaaggtg ctccattgcc 11272

attgatacgt ccagcataat gtcaagtctc tttaggaatc ctagtggagg gtgcccttcc 11332

ttgtgcaggt atgtctccaa gctgccattg ggcatgtact gaagcagcag tgctttgaag 11392

tctatgttgg agcatatgtt tagtatcctt atcaagttgc gatgtcgagc catgcgtaga 11452

acctgacact caacatcaaa gcttctcata gcttgttcct cttgcatgtt cagaacttta 11512

atcgcaacca ccattccatc atccaaatga cccttgaaaa ctttgccaaa acttccagtt 11572

cccaacatat tattctcatt gaaactttct gtggcacgaa caatctcttc atatgatact 11632

aacctataag aagtaggtgt agtaatatct agtttctttt taatcttctt tctagtcatt 11692

tggtataaac aaagagcaag tgcgccaact gctattgtga tggcagggag tataaacttc 11752

aggtagtggt taccattagt tgagtgtgac ttgtttgggc atggtgaaaa tcctagacga 11812

ggaagaccac acaaggcagc attgcccatc aaagat 11848

<210>2

<211>1092

<212>PRT

< 213>paddy rice (Oryza sativa)

<400>2

Met Ala Leu Gly Leu Pro Val Trp Ile Phe Val Ala Leu Leu Ile Ala

1 5 10 15

Leu Ser Thr Val Pro Cys Ala Ser Ser Leu Gly Pro Ser Lys Ser Asn

20 25 30

Gly Ser Asp Ile Asp Leu Ala Ala Leu Leu Ala Leu Lys Ser Gln Phe

35 40 45

Ser Asp Pro Asp Asn Ile Leu Ala Gly Asn Trp Thr Ile Gly Thr Pro

50 55 60

Phe Cys Gln Trp Met Gly Val Ser Cys Ser His Arg Arg Gln Arg Val

65 70 75 80

Thr Ala Leu Lys Leu Pro Asn Val Pro Leu Gln Gly Glu Leu Ser Ser

85 90 95

His Leu Gly Asn Ile Ser Phe Leu Leu Ile Leu Asn Leu Thr Asn Thr

100 105 110

Gly Leu Thr Gly Leu Val Pro Asp Tyr Ile Gly Arg Leu Arg Arg Leu

115 120 125

Glu Ile Leu Asp Leu Gly His Asn Ala Leu Ser Gly Gly Val Pro Ile

130 135 140

Ala Ile Gly Asn Leu Thr Arg Leu Gln Leu Leu Asn Leu Gln Phe Asn

145 150 155 160

Gln Leu Tyr Gly Pro Ile Pro Ala Glu Leu Gln Gly Leu His Ser Leu

165 170 175

Asp Ser Met Asn Leu Arg His Asn Tyr Leu Thr Gly Ser Ile Pro Asp

180 185 190

Asn Leu Phe Asn Asn Thr Ser Leu Leu Thr Tyr Leu Asn Val Gly Asn

195 200 205

Asn Ser Leu Ser Gly Pro Ile Pro Gly Cys Ile Gly Ser Leu Pro Ile

210 215 220

Leu Gln Tyr Leu Asn Leu Gln Ala Asn Asn Leu Thr Gly Ala Val Pro

225 230 235 240

Pro Ala Ile Phe Asn Met Ser Lys Leu Ser Thr Ile Ser Leu Ile Ser

245 250 255

Asn Gly Leu Thr Gly Pro Ile Pro Gly Asn Thr Ser Phe Ser Leu Pro

260 265 270

Val Leu Gln Trp Phe Ala Ile Ser Lys Asn Asn Phe Phe Gly Gln Ile

275 280 285

Pro Leu Gly Leu Ala Ala Cys Pro Tyr Leu Gln Val Ile Ala Leu Pro

290 295 300

Tyr Asn Leu Phe Glu Gly Val Leu Pro Pro Trp Leu Gly Lys Leu Thr

305 310 315 320

Ser Leu Asn Ala Ile Ser Leu Gly Trp Asn Asn Leu Asp Ala Gly Pro

325 330 335

Ile Pro Thr Glu Leu Ser Asn Leu Thr Met Leu Ala Val Leu Asp Leu

340 345 350

Ser Thr Cys Asn Leu Thr Gly Asn Ile Pro Ala Asp Ile Gly His Leu

355 360 365

Gly Gln Leu Ser Trp Leu His Leu Ala Arg Asn Gln Leu Thr Gly Pro

370 375 380

Ile Pro Ala Ser Leu Gly Asn Leu Ser Ser Leu Ala Ile Leu Leu Leu

385 390 395 400

Lys Gly Asn Leu Leu Asp Gly Ser Leu Pro Ala Thr Val Asp Ser Met

405 410 415

Asn Ser Leu Thr Ala Val Asp Val Thr Glu Asn Asn Leu His Gly Asp

420 425 430

Leu Asn Phe Leu Ser Thr Val Ser Asn Cys Arg Lys Leu Ser Thr Leu

435 440 445

Gln Met Asp Phe Asn Tyr Ile Thr Gly Ser Leu Pro Asp Tyr Val Gly

450 455 460

Asn Leu Ser Ser Gln Leu Lys Trp Phe Thr Leu Ser Asn Asn Lys Leu

465 470 475 480

Thr Gly Thr Leu Pro Ala Thr Ile Ser Asn Leu Thr Gly Leu Glu Val

485 490 495

Ile Asp Leu Ser His Asn Gln Leu Arg Asn Ala Ile Pro Glu Ser Ile

500 505 510

Met Thr Ile Glu Asn Leu Gln Trp Leu Asp Leu Ser Gly Asn Ser Leu

515 520 525

Ser Gly Phe Ile Pro Ser Asn Thr Ala Leu Leu Arg Asn Ile Val Lys

530 535 540

Leu Phe Leu Glu Ser Asn Glu Ile Ser Gly Ser Ile Pro Lys Asp Met

545 550 555 560

Arg Asn Leu Thr Asn Leu Glu His Leu Leu Leu Ser Asp Asn Gln Leu

565 570 575

Thr Ser Thr Val Pro Pro Ser Leu Phe His Leu Asp Lys Ile Ile Arg

580 585 590

Leu Asp Leu Ser Arg Asn Phe Leu Ser Gly Ala Leu Pro Val Asp Val

595 600 605

Gly Tyr Leu Lys Gln Ile Thr Ile Ile Asp Leu Ser Asp Asn Ser Phe

610 615 620

Ser Gly Ser Ile Pro Asp Ser Ile Gly Glu Leu Gln Met Leu Thr His

625 630 635 640

Leu Asn Leu Ser Ala Asn Glu Phe Tyr Asp Ser Val Pro Asp Ser Phe

645 650 655

Gly Asn Leu Thr Gly Leu Gln Thr Leu Asp Ile Ser His Asn Asn Ile

660 665 670

Ser Gly Thr Ile Pro Asn Tyr Leu Ala Asn Phe Thr Thr Leu Val Ser

675 680 685

Leu Asn Leu Ser Phe Asn Lys Leu His Gly Gln Ile Pro Glu Gly Gly

690 695 700

Ile Phe Ala Asn Ile Thr Leu Gln Tyr Leu Val Gly Asn Ser Gly Leu

705 710 715 720

Cys Gly Ala Ala Arg Leu Gly Phe Pro Pro Cys Gln Thr Thr Ser Pro

725 730 735

Lys Arg Asn Gly His Met Leu Lys Tyr Leu Leu Pro Thr Ile Ile Ile

740 745 750

Val Val Gly Val Val Ala Cys Cys Leu Tyr Val Met Ile Arg Lys Lys

755 760 765

Ala Asn His Gln Lys Ile Ser Ala Gly Met Ala Asp Leu Ile Ser His

770 775 780

Gln Phe Leu Ser Tyr His Glu Leu Leu Arg Ala Thr Asp Asp Phe Ser

785 790 795 800

Asp Asp Asn Met Leu Gly Phe Gly Ser Phe Gly Lys Val Phe Lys Gly

805 810 815

Gln Leu Ser Asn Gly Met Val Val Ala Ile Lys Val Ile His Gln His

820 825 830

Leu Glu His Ala Met Arg Ser Phe Asp Thr Glu Cys Arg Val Leu Arg

835 840 845

Ile Ala Arg His Arg Asn Leu Ile Lys Ile Leu Asn Thr Cys Ser Asn

850 855 860

Leu Asp Phe Arg Ala Leu Val Leu Gln Tyr Met Pro Lys Gly Ser Leu

865 870 875 880

Glu Ala Leu Leu His Ser Glu Gln Gly Lys Gln Leu Gly Phe Leu Glu

885 890 895

Arg Leu Asp Ile Met Leu Asp Val Ser Met Ala Met Glu Tyr Leu His

900 905 910

His Glu His Tyr Glu Val Val Leu His Cys Asp Leu Lys Pro Ser Asn

915 920 925

Val Leu Phe Asp Asp Asp Met Thr Ala His Val Ala Asp Phe Gly Ile

930 935 940

Ala Arg Leu Leu Leu Gly Asp Asp Asn Ser Met Ile Ser Ala Ser Met

945 950 955 960

Pro Gly Thr Val Gly Tyr Met Ala Pro Glu Tyr Gly Ala Leu Gly Lys

965 970 975

Ala Ser Arg Lys Ser Asp Val Phe Ser Tyr Gly Ile Met Leu Phe Glu

980 985 990

Val Phe Thr Gly Lys Arg Pro Thr Asp Ala Met Phe Val Gly Glu Leu

995 1000 1005

Asn Ile Arg Gln Trp Val His Gln Ala Phe Pro Ala Glu Leu Val

1010 1015 1020

His Val Val Asp Cys Gln Leu Leu His Asp Gly Ser Ser Ser Ser

1025 1030 1035

Asn Met His Gly Phe Leu Val Pro Val Phe Glu Leu Gly Leu Leu

1040 1045 1050

Cys Ser Ala Asp Ser Pro Asp Gln Arg Met Ala Met Ser Asp Val

1055 1060 1065

Val Val Thr Leu Lys Lys Ile Arg Lys Asp Tyr Val Lys Leu Met

1070 1075 1080

Ala Thr Thr Glu Asn Ala Val Gln Gln

1085 1090

Claims (2)

1. one kind isolatingly produces the gene Xa3/Xa26-2 of resistance to bacterial leaf-blight, and its nucleotide sequence is shown in sequence table SEQ IDNO:1.

2. the described gene of claim 1 is increasing paddy rice to the application in the bacterial leaf spot resistance.

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