CN101062944A - Vegetable disease-resistant protein and its coding gene and application - Google Patents

Abstract

The invention discloses a plant disease-resistant protein, coding gene and appliance, which is characterized by the following: comprising (a) and (b) protein; choosing amino acid residue sequence from sequences 3 in sequence table as the protein (a); replacing or deleting or adding amino acid residue sequence from sequences 3 trough one or several amino acid residue sequence; relating to active oxygen kill; deriving from the protein (a) as the protein (b). This invention possesses important meaning in increasing agronomic crop output.

Description

A kind of disease resistance of plant albumen and encoding gene and application

Technical field

The present invention relates to a kind of disease resistance of plant albumen and encoding gene and application.

Background technology

Plant and various potential pathogenic micro-organism coexist at nature, and these microorganisms comprise fungi, bacterium, nematode and virus or the like, and plant has formed signal sensing and the defense mechanism meticulous to pathogenic micro-organism in long-term coevolution process.Congenital immunity is the important defense mechanism of plant antagonism pathogen infection, difference according to the plant resistance to environment stress reaction, congenital immunity can be divided into resistance two classes of basic resistance and disease-resistant gene mediation, though this two classes resistance has evident difference, might share some common molecular mechanisms.The basis resistance is meant pattern recognition acceptor (the pattern recognition receptors of plant, PRRs) total pathogen-associated molecular pattern (the pathogen-associated molecular patterns of the multiple pathogenic bacteria of identification, PAMPs), then by MAPK (mitogen-activated protein kinases, MAPKs) or other signal pathway start defense response.The resistance pattern of disease-resistant gene mediation is to be proposed when the disease resistance of flax rust is carried out genetic analysis by Flor at first, suppose that different pathogenic bacteria microspecies carry different nontoxic genes, the nontoxic gene product of the disease-resistant gene product identification specific pathogen bacterium of plant, and by signal conduction startup defense response, this pattern can be explained the disease resistance difference of crop different varieties in the agriculture production well, and can instruct in the breeding process disease-resistant gene with disease-resistant variety to change susceptible variety over to obtain resistance.Just because of this, the resistance of enantiopathy gene mediated has been carried out a large amount of research in decades, a lot of disease-resistant genes have been cloned at present from different plants, mainly can fall into 5 types according to their proteic constructional features, wherein Nucleotide Binding Site-Leucine Rich Repeat (NBS-LRR) is a maximum class disease-resistant gene family, and find that up to the present the NBS-LRR gene is main relevant with disease-resistant function, so NBS-LRR albumen might be the core component of plant immunization system.

Up to the present 6 bacterial leaf spot resistant ospc genes and 6 blast resistant genes from paddy rice, have been cloned, 6 bacterial leaf spot resistant ospc genes are respectively Xa21, Xa1, Xa26, xa5, Xa27 and xa13, wherein have only Xa1 to belong to the NBS-LRR gene family, Xa21 and Xa26 belong to the similar kinases of acceptor family, and xa5, xa13 and Xa27 are the disease-resistant genes of special construction, can not be referred in the typical 5 class disease-resistant genes; 6 blast resistant genes are respectively Pib, Pita, Pi9, Pid2, Pi2 and Pizt, and they are from 4 different sites, and wherein Pi9, Pi2 and Pizt are the not isoalleles of same gene locus.Except similar kinases of silk Threonine acceptor of Pid2 coding, other 5 blast resistant genes belong to the NBS-LRR gene family all, derive from the granule wild-rice except Pi9 in these 6 blast resistant genes of having cloned in addition, other 5 genes all derive from long-grained nonglutinous rice.From present clone's paddy disease-resistant gene, the bacterial leaf spot resistant ospc gene adheres to separately a plurality of different classes of, and blast resistant gene mainly is the NBS-LRR gene.

Paddy rice is divided into Xian, two subspecies of round-grained rice, japonica rice is mainly planted in the Japan and the Korea peninsula that are positioned at the north temperate zone, be positioned at the South East Asia and the South Asian nation in the torrid zone, as main plantation long-grained nonglutinous rice such as India, Philippines, Indonesia, Thailand, the Yangtze valley of China is considered to the main source region of paddy rice, and the northern rice district of China is main plants japonica rice and the main plantation in southern rice district long-grained nonglutinous rice.Ling Zhong specially waits and utilizes 7 Japan to represent fungus strain that the rice varieties of China is carried out the disease-resistant gene analysis, discovery can be inferred the genotype of the northern most of japonica rice variety of China, but be difficult to determine the genotype of southern china rice variety, wherein all performance is disease-resistant to all 7 fungus strains for the many susceptible rice variety of southern china, and this explanation can not be infected the rice variety of southern china from the fungus strain of Japanese japonica rice growing area.In addition the blast resistant gene of rice variety is carried out genetic analysis and also show, compare the contained blast resistant gene of most rice varieties with most of japonica rice variety complicated more, needs to make up near isogenic line usually or RIL just can carry out the assignment of genes gene mapping.The experiment that the indica rice Cultivar connects the dientification of bacteria is shown also from the Pyricularia oryzae in Chinese North Japonica Rice district and southern long-grained nonglutinous rice district under Pekinese's condition, the resistance of most long-grained nonglutinous rices is significantly better than the japonica rice variety in the north respectively in our laboratory utilization.We also find that to the resistance analysis from the Pyricularia oryzae of different areas most rice varieties are better than the bacterium to the long-grained nonglutinous rice district to the resistance of japonica rice district bacterium to Xian, japonica rice variety, and most japonica rice variety is better than the bacterium to the japonica rice district to the resistance of long-grained nonglutinous rice district bacterium.Hao Zhongna etc. did similar analysis in the Zhejiang area of southern china, had also obtained same conclusion.This illustrates that most of Xian, japonica rice variety there are differences the resistance of the Pyricularia oryzae of planted in different ecological areas.

Along with fine complete genomic order-checking makes progress to long-grained nonglutinous rice 93-11 and japonica rice Japan, a plurality of study group analyze the NBS-LRR gene of the full genome encoding of paddy rice, the result has shown the paddy gene group coding nearly 500 NBS-LRR genes, account for 1% of paddy rice whole genome encoding gene greatly, approaching with ratio shared in the arabidopsis gene group.The genome encoding of Arabidopis thaliana Col-0 149 NBS-LRR genes, according to difference by their encoded protein N end structures, the NBS-LRR gene of Arabidopis thaliana can be divided into the TNL (TIR-NBS-LRR) that contains Toll/Interliukin-1 acceptor similar structures and the CNL (CC-NBS-LRR) that contains the Coiled-coil structure two big class.The NBS-LRR gene that compares paddy rice and Arabidopis thaliana, maximum difference is that paddy rice does not have the TNL genoid, and the TNL genoid is in the great majority in Arabidopis thaliana, and the major part in the paddy rice NBS-LRR gene is in the unknown structure of N end coding, and portion gene is held the Coiled-coil structure of having encoded at N.In addition, in the proteic leucine iteron of the NBS-LRR of paddy rice genes encoding bigger variation is arranged, its size can not wait by from 350 to 700 amino acid; Though whole zone is to be rich in leucicly, most leucine repeating structure is all imperfect, does not have clear repeating unit of debating.

Rice blast is the important disease of paddy rice, has a strong impact on output when paddy fields is popular, causes financial loss.The disease-resistant gene that utilizes paddy rice self is cost-effectively to prevent rice blast popular method, and can overcome the environmental hazard of using chemical bactericide to bring.The utilization of succeeding in agriculture production of a plurality of blast resistant genes has been arranged at present, but the big area of single disease-resistant gene is promoted and often morbific Pyricularia oryzae is caused selection, therefore and popular disease-resistant variety susceptibleization in several years that cause of pathogenic bacterium provides new anti-source, the new blast resistant gene of clone extremely important to the paddy disease-resistant breeding.

Summary of the invention

The purpose of this invention is to provide a kind of disease resistance of plant albumen and encoding gene and application.

Disease resistance of plant albumen provided by the present invention, name is called PID3, and derive from paddy rice and belong to paddy rice (Oryzasativa var.Lansheng), be following (a) or protein (b):

(a) protein of forming by the amino acid residue sequence of sequence in the sequence table 3;

(b) with the amino acid residue sequence of sequence in the sequence table 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have disease-resistant correlation function by (a) deutero-protein.

Wherein, the sequence in the sequence table 3 is made up of 924 amino-acid residues.The amino acid 158-466 amino acids of the sequence 3 NBS structural domain of having encoded in sequence table, wherein comprise four conservative motif, their sequence is respectively GMGGIGKTA (the amino acid 202-210 amino acids of sequence 3 in sequence table), KRYVLVLDDVW (the amino acid 280-290 amino acids of sequence 3 in sequence table), IGRIILTSRNYDV (the amino acid 307-319 amino acids of sequence 3 in sequence table), GLPIAI (the amino acid 373-378 amino acids of sequence 3 in sequence table), these four conservative motif represent kinase 1a (p-loop) respectively, kinase 2, kinase 3a and GLPL motif; Proteic C end has predicted that 13 LRR repeating units, its aminoacid sequence are respectively PNVSSLHSLPKSVKLLSVLDLTDSSVDRLP (the amino acid 565-594 amino acids of sequence 3 in sequence table), KEVFGLFNLRFLGLRRTKISKLP (the amino acid 595-617 amino acids of sequence 3 in sequence table), SSIGRLKNLLVLDAWKCKIVKLP (the amino acid 618-640 amino acids of sequence 3 in sequence table), LAITKLQKLTHLIVTSKAVVVSKQFVPSVGVPAP (the amino acid 641-674 amino acids of sequence 3 in sequence table), LRICSMTTLQTLLLMEASS (the amino acid 675-693 amino acids of sequence 3 in sequence table), QMVHHLGSLVELRTFRISKVRSCHCE (the amino acid 694-719 amino acids of sequence 3 in sequence table), QLFMAITNMIHLTRLGIQADSSQEV (the amino acid 720-744 amino acids of sequence 3 in sequence table), LHLESLKPPPLLQKLFLQGTLSHESL (the amino acid 745-770 amino acids of sequence 3 in sequence table), PHFVSVSNLNNLTFLRLAGSRIDE (the amino acid 771-794 amino acids of sequence 3 in sequence table), NAFLNLEGLQQLVKLQLYDAFDGMNIY (the amino acid 795-821 amino acids of sequence 3 in sequence table), FHENSFPKLRILKIWGAPHLNEIKMTK (the amino acid 822-848 amino acids of sequence 3 in sequence table), GAVASLTHLKFLLCPNLKQLP (the amino acid 849-869 amino acids of sequence 3 in sequence table), CGIEHVRTLEELTLDHTAEELVDRV (the amino acid 870-894 amino acids of sequence 3 in sequence table); Conservative MHD motif:MHDILRV (the amino acid 502-508 amino acids of sequence 3 in sequence table) and NBS LRR linker motif:EQNFCIVVNHS (the amino acid 516-526 amino acids of sequence 3 in sequence table) are arranged between NBS structural domain and LRR zone in addition.

In order to make PID3 in (a) be secreted in cell pericentral siphon or the substratum or to make its function-stable, proteinic N end that can the amino acid residue sequence of sequence 3 is formed in by sequence table connects signal peptide sequence, for the PID3 in (a) is convenient to purifying, proteinic N end or C end that can the amino acid residue sequence of sequence 3 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	11	EQKLISEEDL

Above-mentioned (b) but in the PID3 synthetic, also can synthesize its encoding gene earlier, carry out biology according to following method again and express and to obtain.The encoding gene of PID3 in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna of sequence in the sequence table 1 or sequence 2, and/or carry out the missense mutation of one or several base pair, and/or at the encoding sequence of its 5 ' end attach signal peptide, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The proteic encoding gene of above-mentioned disease resistance of plant (pid3) also belongs to protection scope of the present invention.

The proteic genomic gene of above-mentioned disease resistance of plant can have one of following nucleotide sequence:

1) dna sequence dna of sequence 1 in the sequence table;

2) polynucleotide of protein sequence shown in the sequence 3 in the code sequence tabulation;

3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with sequence in the sequence table 1.

Wherein, sequence 1 is made up of 6235 deoxynucleotides in the sequence table, and its reading frame is the 3010th to the 5781st Nucleotide of 5 ' end from sequence 1.

The proteic cDNA gene of above-mentioned disease resistance of plant can have one of following nucleotide sequence:

1) dna sequence dna of sequence 2 in the sequence table;

2) polynucleotide of protein sequence shown in the sequence 3 in the code sequence tabulation;

3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with sequence in the sequence table 2.

Wherein, sequence 2 is made up of 2969 deoxynucleotides in the sequence table, from 3 ' and the 63-2834 position Nucleotide of end is encoding sequence (ORF).

The rigorous condition of above-mentioned height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.

The expression vector, transgenic cell line and the host bacterium that contain above-mentioned plant disease resistance genes all belong to protection scope of the present invention.

Blast resistant gene Pid3 provided by the present invention can utilize any carrier that can guide foreign gene to express in plant, will import vegetable cell, obtains disease-resistant or disease-resistant enhanced transgenic cell line and transfer-gen plant.Gene of the present invention can add any general promotor, strengthen promotor or inducible promoter in being building up to plant expression vector the time before its transcription initiation nuclear former times acid.For the ease of transgenic plant or transgenic plant cells being identified and being screened, can process employed carrier, as add the alternative mark gus gene of plant, luciferase gene etc.) or antibiotic marker thing (gentamicin, kantlex etc.) with resistance.For the security that transgenic plant discharge, when making up plant expression vector, also can not carry any marker gene, directly screen in seedling stage with the Pyricularia oryzae inoculation.

Contain the expression vector of Pid3 of the present invention can be by using conventional biological method transformed plant cells or tissues such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, agriculture bacillus mediated or particle gun, and the plant transformed tissue cultivating become plant.By the plant transformed host both can be monocotyledons, also can be dicotyledons, as: paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass, clover etc.

Disease-resistant protein of the present invention and encoding gene thereof can be used for cultivating the disease-resistant plants kind, particularly cultivate the blast resisting plant variety; To enlarging the crop-planting scope, it is significant to improve crop yield.

Description of drawings

Fig. 1 is the sequential analysis of Pid3 full-length cDNA

Fig. 2 is the proteic aminoacid sequence comparison of NBS-LRR in Pid3 and wheat, barley, the corn

Fig. 3 is the structure diagram of pCAM-Pid3

Fig. 4 shows resistance detected result for plant to rice blast fungi isolates Zhong-10-8-14 for changeing pCAM-Pid3 T0

Fig. 5 detects and the PCR detection for the resistance to Zhong-10-8-14 for changeing pCAM-Pid3 plant T1

Embodiment

The experimental technique of mentioning among the following embodiment is ordinary method if no special instructions.

The acquisition of embodiment 1, disease-resistant (rice blast) property albumen and encoding gene thereof

Utilizing high-fidelity PCR strategy, is template with disease-resistant rice variety ground paddy (available from Inst. of Paddy Rice, Sichuan Agriculture Univ.) genomic dna, with ACC GaattcCACACATTGTACACCTACGACCAC and ACC GtcgacGAACGACAAGTGCGACATGATTG is that primer (adds EcoRI and SalI restriction enzyme site respectively, underscore is represented the restriction enzyme site that adds), amplification system is Pfu enzyme 2U, genomic dna template 0.1 μ g, 10 * PCR reaction buffer, 5 microlitres, concentration is dNTP 2 microlitres of 2.5mmol/L, concentration is upstream primer and each 1 microlitre of downstream primer of 5 μ mol/L, sterilized water is supplied 50 microlitres, and the PCR reaction is undertaken by following program: pre-94 ℃ of 5min of sex change; 94 ℃ of 45S then, 56 ℃ of 45S, 72 ℃ of 1.45min, totally 35 circulations; Be incubated 72 ℃ of 10min at last.Amplification obtains the dna fragmentation of 6235 bp, show that through order-checking this fragment has the nucleotide sequence of sequence 1 in the sequence table, its reading frame is the 3010th to the 5781st Nucleotide of 5 ' end from sequence 1, with this unnamed gene is Pid3, the aminoacid sequence of sequence 3 in the tabulation of Pid3 code sequence.

In order to obtain the full-length cDNA fragment of Pid3, carried out 5 ' RACE, 3 ' RACE and RT-PCR and analyzed.Extract total RNA of rice seedling blade, reverse transcription obtains the first chain cDNA, according to the SMART of Clontech company ^TMRACE (Rapid Amplification of cDNA Ends) test kit specification sheets carries out the full-length cDNA fragment of 5 ' RACE and 3 ' RACE amplification Pid3.

5 ' RACE amplification: with 5 ' the joint primer that provides in the test kit, and CCATCTTGCACATCCTCTTGAGTG is a gene specific primer, with the above-mentioned first chain cDNA is template, carry out 5 ' RACE amplification, amplification system is Pfu enzyme 2U, template 0.1 μ g, 10 * PCR reaction buffer, 5 microlitres, concentration is dNTP 2 microlitres of 2.5mmol/L, concentration is upstream primer and each 1 microlitre of downstream primer of 5 μ mol/L, sterilized water is supplied 50 microlitres, and PCR reaction is undertaken by following program: 94 ℃ of pre-sex change 5 minutes; Then 94 ℃ 45 seconds, 56 ℃ 45 seconds, 72 ℃ 2 minutes, repeat 30 circulations; Last 72 ℃ 10 minutes.

3 ' RACE amplification: with 3 ' the joint primer that provides in the test kit, and ACATCTTATTGGAGAAGCAC is a gene specific primer, with the above-mentioned first chain cDNA is template, carry out 3 ' RACE amplification, amplification system is Pfu enzyme 2U, template 0.1 μ g, 10 * PCR reaction buffer, 5 microlitres, concentration is dNTP 2 microlitres of 2.5mmol/L, concentration is upstream primer and each 1 microlitre of downstream primer of 5 μ mol/L, sterilized water is supplied 50 microlitres, and PCR reaction is undertaken by following program: 94 ℃ of pre-sex change 5 minutes; Then 94 ℃ 45 seconds, 56 ℃ 45 seconds, 72 ℃ 2 minutes, repeat 30 circulations; Last 72 ℃ 10 minutes.

With intragenic special upstream primer GAAGCTAGCAAGCTATGGCGGAG of Pid3 and downstream primer ACGTCACAAATCATTCGCTC, with the above-mentioned first chain cDNA is template, carry out pcr amplification, amplification system is Pfu enzyme 2U, template 0.1 μ g, 10 * PCR reaction buffer, 5 microlitres, concentration is dNTP 2 microlitres of 2.5mmol/L, concentration is the upstream primer of 5 μ mol/L and each 1 microlitre of downstream primer, and sterilized water is supplied 50 microlitres, and the PCR reaction is undertaken by following program: 94 ℃ of pre-sex change 5 minutes; Then 94 ℃ 45 seconds, 56 ℃ 45 seconds, 72 ℃ 5 minutes, repeat 30 circulations; Last 72 ℃ 10 minutes.Order-checking shows the nucleotide sequence that fragment that above 5 ' RACE, 3 ' RACE and RT-PCR obtain has sequence 2 in the sequence table.The nucleotide sequence total length 2969bp of sequence 2 in the sequence table, its transcription initiation position is at 63bp place, ATG upstream (sequence 25 ' end the 1st Nucleotide), and (5 ' of sequence 2 is held the 2969th Nucleotide) (Fig. 1) in position, 135bp place, TAA downstream for the polyA tailing.

Wherein, sequence 1 is made up of 6235 deoxynucleotides in the sequence table, and its reading frame is 5 ' end the 3010th to the 5781st bit base from sequence 1; Sequence 2 is made up of 2969 deoxynucleotides in the sequence table, from 3 ' and the 63-2834 position Nucleotide of end is encoding sequence (ORF).

The NBS-LRR albumen of a Pid3 and a non-TIR class of cDNA sequence encoding thereof is with its called after PID3; PID3 has the amino acid residue sequence of sequence 3 in the sequence table.

At protein structure analyzing web site SMART (http://smart.embl-heidelberg.de/) and pfam (http://pfam.wustl.edu/) each structural domain of the protein sequence of Pid3 is analyzed, the result shows the aminoterminal 158-466 amino acids of sequence 3 in the sequence table NBS structural domain of having encoded, wherein comprise four conservative motif, their sequence is GMGGIGKTA (the amino acid 202-210 amino acids of sequence 3 in sequence table), KRYVLVLDDVW (the amino acid 280-290 amino acids of sequence 3 in sequence table), IGRIILTSRNYDV (the amino acid 307-319 amino acids of sequence 3 in sequence table), GLPIAI (the amino acid 373-378 amino acids 373-378aa of sequence 3 in sequence table) represents kinase 1a (p-loop) respectively, kinase 2, kinase 3a and GLPL motif.The analysis revealed that the NBS-LRR albumen of arabidopsis gene group coding is carried out have in the LRR zone L * * L * * L * * L * L * * multiple structural unit like this, but proteic analysis finds that the LRR repeating unit has bigger variation on size and sequence to the NBS-LRR of paddy gene group coding, be difficult to the accurately position of prediction repeating unit, we have predicted 13 LRR repeating units at the proteic C end of Pid3, repeating structure is not very complete, their sequence is respectively PNVSSLHSLPKSVKLLSVLDLTDSSVDRLP (the amino acid 565-594 amino acids of sequence 3 in sequence table), KEVFGLFNLRFLGLRRTKISKLP (the amino acid 595-617 amino acids of sequence 3 in sequence table), SSIGRLKNLLVLDAWKCKIVKLP (the amino acid 618-640 amino acids of sequence 3 in sequence table), LAITKLQKLTHLIVTSKAVVVSKQFVPSVGVPAP (the amino acid 641-674 amino acids of sequence 3 in sequence table), LRICSMTTLQTLLLMEASS (the amino acid 675-693 amino acids of sequence 3 in sequence table), QMVHHLGSLVELRTFRISKVRSCHCE (the amino acid 694-719 amino acids of sequence 3 in sequence table), QLFMAITNMIHLTRLGIQADSSQEV (the amino acid 720-744 amino acids of sequence 3 in sequence table), LHLESLKPPPLLQKLFLQGTLSHESL (the amino acid 745-770 amino acids of sequence 3 in sequence table), PHFVSVSNLNNLTFLRLAGSRIDE (the amino acid 771-794 amino acids of sequence 3 in sequence table), NAFLNLEGLQQLVKLQLYDAFDGMNIY (the amino acid 795-821 amino acids of sequence 3 in sequence table), FHENSFPKLRILKIWGAPHLNEIKMTK (the amino acid 822-848 amino acids of sequence 3 in sequence table), GAVASLTHLKFLLCPNLKQLP (the amino acid 849-869 amino acids of sequence 3 in sequence table), CGIEHVRTLEELTLDHTAEELVDRV (the amino acid 870-894 amino acids of sequence 3 in sequence table) is with the Pib that has cloned in the paddy rice, the situation of Pita and Pi9/Pi2/Pizt is similar.Conservative MHD motif:MHDILRV (the amino acid 502-508 amino acids of sequence 3 in sequence table) and NBS LRR linker motif:EQNFCIVVNHS (the amino acid 516-526 amino acids of sequence 3 in sequence table) are arranged between NBS structural domain and LRR zone in addition.Paddy rice does not have TIR class NBS-LRR albumen, most gene is in the unknown structure of N end coding, portion gene is held the Coiled-coil structure of having encoded at N, at the N of PID3 end one section conservative motif:RSLALSIEDVVD (the amino acid 78-89 amino acids of sequence 3 in sequence table) is arranged, but do not find the Coiled-coil structure, so Pid3 is the NBS-LRR gene that belongs to the non-TIR class.

Be positioned on the 1st karyomit(e) with the highest NBS-LRR gene of Pid3 similarity in the paddy rice, full length cDNA sequence (Kome clone name:J023059I04, Genbank accession number:AK111827) arranged, both amino acid sequence similarities 67.1%.The Pid3 amino acid sequence coded is BlastP to be analyzed in the NCBI website, at wheat, the disease-resistant albumen of homologous is all arranged in barley and the seeding corn and other crops, the HV1LRR1 of Pid3 and barley (Genbank accession number:AAD46469) similarity 40.5% wherein, LRR14 and LRR19 (Genbank accession number:AAK20742 with wheat, AAK20736) similarity is respectively 42.8% and 43.5%, RXO1 (Genbank accession number:AAX31149) similarity 32.5% with corn, Pid3 and this several proteic aminoacid sequences are carried out the homology comparison, as shown in Figure 2, though their amino acid sequence homology is only about 40%, but proteic one-piece construction is very similar, and the conservative motif of NBS structural domain is very consistent with conservative leucic position, LRR zone.Compare with several blast resisting NBS-LRR albumen of having cloned in the paddy rice, the similarity of Pid3 and Pib (Genbank accession number:AB013449) has only 19.0%, with the similarity of Pita (Genbank accession number:AF207842) be 20.5%, with the similarity of Pi9 (Genbankaccession number:DQ285630) be 25.4%.

The transgenosis functional verification of embodiment 2, blast resisting albumen and encoding gene Pid3 thereof

In order further to confirm the blast resisting function of Pid3, we have carried out paddy rice and have changeed the experiment of Pid3 gene.

Utilizing high-fidelity PCR strategy, is template with disease-resistant rice variety ground paddy genomic dna, with ACC GaattcCACACATTGTACACCTACGACCAC and ACC GtcgacGAACGACAAGTGCGACATGATTG is that primer (adds EcoRI and SalI restriction enzyme site respectively, underscore is represented the restriction enzyme site that adds), amplification system is Pfu enzyme 2U, genomic dna template 0.1 μ g, 10 * PCR reaction buffer, 5 microlitres, concentration is dNTP 2 microlitres of 2.5mmol/L, concentration is upstream primer and each 1 microlitre of downstream primer of 5 μ mol/L, sterilized water is supplied 50 microlitres, and PCR reaction is undertaken by following program: 94 ℃ of pre-sex change 5 minutes; Then 94 ℃ 45 seconds, 56 ℃ 45 seconds, 72 ℃ 6 minutes, repeat 30 circulations; Be incubated at last 72 ℃ 10 minutes.The dna fragmentation of amplification 6235bp shows that through order-checking this fragment is the Pid3 gene fragment, comprises the complete coding region sequence of Pid3, the promoter region of initiator codon ATG upstream 3010bp and terminator codon TAA downstream 451bp sequence.The fragment that pcr amplification is obtained, cut with EcoRI and SalI enzyme, be inserted between the EcoRI and SalI restriction enzyme site of double base conversion carrier pCAMBIA1300, will show the recombinant vectors called after pCAM-Pid3 (structure diagram as shown in Figure 3) that contains the Pid3 gene fragment through order-checking.PCAM-Pid3 is changed among the Agrobacterium LBA4404, then to the susceptible japonica rice variety TP309 of paddy rice (this kind does not have resistance to Pyricularia oryzae Zhong-10-8-14) (Chen, X., Shang, J., Chen, D., Lei, C., Zou, Y., Zhai, W., Liu, G., Xu, J., Ling, Z., Cao, G., et al. (2006) .A B-lectin receptor kinase geneconferring rice blast resistance.Plant J 46, mature embryo evoked callus 794-804) carries out Agrobacterium-mediated Transformation, the callus after transforming is transferred to common culture medium (NB minimum medium+100uM Syringylethanone) go up in 26 ℃ of dark cultivations 2 days.Thoroughly wash callus several times with sterilized water then, to contain in the sterilized water of Reflin 500mg/L 120rpm vibration washing 2 hours at callus then, callus after the washing is layered on last cultivation of the selection substratum (NB minimum medium+2mg/L 2,4-D+500mg/L Reflin+50mg/L Totomycin) that contains Totomycin 50mg/L and carries out the 1st selection 3 weeks.Transfer to the last continuation cultivation of new selection substratum (NB minimum medium+2mg/L 2,4-D+500mg/L Reflin+50mg/L Totomycin) then and carry out the 2nd selection 2-3 week.To after cultivating, twice selection go up in 26 ℃ of dark cultivations 2-3 week at pre-differentiation substratum (NB minimum medium+5mg/L ABA+2mg/L NAA+50mg/L Totomycin) by eugonic callus, transfer to division culture medium (NB minimum medium+0.5mg/L NAA+1.0mg/L IAA+50mg/L Totomycin then,) the following 26 ℃ of thoughtful differentiation and seedling emergences of continuation cultivation 2-3 of last continuous light, be transplanted in the soil when high when regrowth grows to 10cm, in the greenhouse, grow to maturation.The result obtains 21 T0 altogether for changeing the pCAM-Pid3 plant.

Utilize Pyricularia oryzae Zhong-10-8-14

(Zheng, X., Chen, X., Zhang, X., Lin, Z., Shang, J., Xu, J., Zhai, W., and Zhu, L. (2004) .Isolation and identification of a gene in response to rice blast disease in rice.Plant Mol Biol 54,99-109) spore suspension carries out rice blast resistance detection to changeing pCAM-Pid3 T0 for plant and rice varieties TP309 (contrast) with the method for spray inoculation, the concrete grammar that rice blast resistance detects is: paddy rice is cultured to tri-leaf period, with sterilized water configuration 5 * 10 ⁴The Zhong-10-8-14 spore suspension of spore/milliliter, the Tween20 of adding 0.02%, even spraying kept 100% humidity 24 hours in 26 ℃ of shadings to rice seedling, move on to then in 25～28 ℃ of greenhouses of spraying plant, carried out the resistance investigation after the week.The result shows with check variety TP309 and compares, change pCAM-Pid3 T0 and obtained resistance rice blast fungi isolates Zhong-10-8-14 for plant, rice varieties TP309 (contrast) then shows seriously susceptible (Fig. 4, A shows that transgenic line 15 connects the bacterium rear blade and occurs without any scab among Fig. 4, and B shows the susceptible scab of check variety TP309 blade among Fig. 4).

T0 is T1 for changeing the pCAM-Pid3 plant for the seed of commentaries on classics pCAM-Pid3 plant selfing generation and the plant that grows up to thereof.Utilize the method for magnaporthe grisea spore suspension spray inoculation equally, 100 strain T1 are carried out resistance evaluation and transgenosis Molecular Identification for changeing the pCAM-Pid3 plant, the transgenosis Molecular Identification is carried out the PCR detection according to conversion carrier sequences Design primer GACGGTGTCGTCCATCACAGTTT and ACTCACCGCGACGTCTGTCGAGAA, the band of 495bp of increasing is promptly positive, the result of the blast resisting evaluation of part PCR evaluation and corresponding plant as shown in Figure 5, A is a rice blast fungus resistance detected result among Fig. 5, B is the PCR evaluation amplified band of corresponding plants among Fig. 5, among Fig. 5 among the B swimming lane M be molecular weight standard, swimming lane 4,6,9,13,14 are the amplified production electrophoresis result that PCR identifies negative plant, and all the other are the amplified production electrophoresis result that PCR identifies the male plant.The result shows can both the increase band of 495bp of plant of all performance resistances, and susceptible plant can not be increased and obtained this band, and the resistance that shows transgenic progeny is the result who changes the Pid3 gene over to.

Sequence table

<160>3

<210>1

<211>6235

<212>DNA

＜213〉paddy rice belongs to paddy rice (Oryza sativa var.Lansheng)

<400>1

cacacattgt acacctacga ccaccgttca ccgttcagag actaagacta tggtggtgtt 60

tggatccagg gactaaattt tagtgcctga catatcggat gtttgaacac taattagaag 120

tattaaatgt agactaataa taaaactaat tgcataaata agagctaatt cgcaagaaat 180

ttttaagcct aattaatcca taactagcac atgtttacta tagcatcaca taggctaatt 240

atggattaat tagtctcaat agattcgcct cgcgaattag tccaaagtta tggaatgggt 300

tttgtcatta gtctacattt gatacttctc attagcgtct aaacatccga tgtgatttta 360

gtccctgatc taatcagccc ctggctggca aattctatga atgatgaagt aacaacacac 420

aattcactat caacgagcat atcgtctgcc acttcatttt aggtataata ttcaaattta 480

attctatcat aaaccttatt aacgttgatt gccttgctga atctgattaa tgtctttatg 540

tttataataa actttattga tgctgattgc attgctagcc ctggctgaga aagggtatat 600

atatgaattg tctttatctg atctaacaaa atattaaacg atgaggttag tccatgcatt 660

ttttatatta atttgtcctc tcttcacaaa gtaaataagc taatgctaac aatacatatc 720

gatatatctc aattcatgca aattaagatc gaaatatggc aatcgagtta gctataattc 780

ccgttataaa ttccaacaag atactgtctc cgttccaaaa taagtgcagc catggatatc 840

cgtgtaccgt ccgtctgatt tgaaaaattt ataatttttt aaaaaattag tcacacataa 900

agtactattc atgttttata acctaataca ataaaaatac taatcatgaa aaaaattcaa 960

ataagacgaa tggtcaaacg ctggatatga atttttataa aactacactt attttgggac 1020

gaaaggagta catatttttc ctttgttacc gttagcacgt tttttaaact ggtaaatggt 1080

gtcttcgagt gaaaactttc ttcatagaag ttgctataga aaacaaataa atccatttat 1140

taagtttttt ataattaaaa ttcaattaat catgtgctaa tgacttatct cgttttgtgt 1200

gcgtacttaa tcttcatatt ccatcagctt caaacgggcc ttaaatcttt ttacatgagt 1260

ttatttaatc ctctagggtt aatttaattt tggtgacgta taataattgt tttaatattt 1320

atgcctttaa gggtcagcaa attaattttt tcttgatctg aattagaata aatttaattt 1380

tattagtcca taaatttagg tttgaataac acataacgat tgccatatat atttctgtgt 1440

tatatctatg tttgcaaaat ttggtcttta aaaaccttaa ttggatcatt atttaatttt 1500

aaactattaa atacactatt taaaaacgtg aaataaagtt tattgatgct tttggggata 1560

tttaaaaatt cagagaacgt aataatttct agagattggg catgagtaaa caaatgtagt 1620

agtaggttta tatttcatta ggtaaggaca ttccctgcga ttgtttttct atggaactat 1680

gcacccatga cctatagtaa aattttactc taaaggtggc cccgctaaaa agggaggaca 1740

agcatgatgg atagattaat cagcatttag cagtaaatct tttcaagatt tcttttaaaa 1800

aaagagagaa tgaacattca attaaaaaaa ggaaacatct tactttcaat ttaaatttaa 1860

ttcgttataa ttgtttttgg aacaatctag tacacatgcc cgcgcgaatg catgggctat 1920

tttcctagtt taacaaattt taagtaaaaa catggctgtg tttagttcac gccaaaatta 1980

aaagcttggt tgaaattgga acgatgtgat ggaaaagttg aaagtttgtg tgtgtaggaa 2040

agttttgatg tgatgaaaaa attgtaagtt tgaagaaaaa ctttggatct aaacacggcc 2100

cataaataaa aacaaataat tttgtatgat tgctagccgt gtaattaggt aggtcaccca 2160

tctagtttta attagacaac caactcttca gggcctgttt agttcgcgaa ataaaaattt 2220

ttgggtgtca cgtcggacgt ttgataggat gttggaaggg gttttcggac acgaacaaaa 2280

aaaactaatt tcataactcg cttggaaacc gcgagacgaa tcttttgggc ctaattaatc 2340

caccattagc acatgtggga taatgtagca cttatggcta attatggact aattagattc 2400

aaaagattcg tttcacgatt ttctccctaa ctgtgcaatt agttttcttt taatctacat 2460

ttaatgctcc atgcatgtgt caaagattcg atttaatttt tttggaaaaa atttttggga 2520

ctaaactgct tgttgaaaag atcaacccac ggaatcttag atataaacat cacagcttca 2580

aaccgtacaa cgaaagattg aacatccttt agtaatatca agggtgtatt tagtttatgc 2640

taaaattgaa agcttggttg taattggaag gatgtgctgg aaaaattgaa agtttatatg 2700

tgtagaaaag ttttgatgtg atggaaaagt tggaacttcg aaaaaaagtt ttggaactaa 2760

actcggccca agttgcaatc gtggaacagg aatcagtatt atcatatatc ggcaccaatg 2820

caagttgctt ccttttttaa cacgaaactg acgaagcgtc agaagaccct gcgttttatg 2880

aaaggggtga agatgttcct tttgggggga aaaaggaaac ataatggatc aagaccagtt 2940

agataccaag cgagaaggaa gtaacaccca aggataggat agcttagttg aagttgaagc 3000

tagcaagcta tggcggaggg tgttgtgggc tcactaatcg tcaagcttgg ggatgccctg 3060

gcgagtgaag cagtggaggt cgccaagtcc ttgcttgggc tagagggatc tgctctgaag 3120

cgcctctttt ctgagatcgg ggaggtgaag ggtgagctgg agagcatcca tgccttcttg 3180

caggcagccg agcggttcaa ggacgccgac gagacaacct ccgcgttcgt caagcaggtg 3240

aggagcctcg ccctgagcat cgaggacgta gtcgatgagt tcacctacga gctgggggaa 3300

ggagacggcc ggatggggat ggcagtggca ctcaagagga tgtgcaagat gggcacatgg 3360

tctcgcctgg ctggcaatct gcaggacatc aaggtcaacc tgaaaaacgc tgccgagagg 3420

aggattaggt atgatctgaa gggcgtcgag agaggcgcga aatcgacagc gggaaggagg 3480

agctcaaact ggagatctga ctctgtactc ttcaagaggg aggatgagct tgttggtatc 3540

gagaagaaga gggatttgct gatgaaatgg gtgaaagatg aggagcagcg ccgcatggtg 3600

gtcagtgtgt ggggaatggg tggaatcggc aagacggcac tggttgctaa tgtttacaat 3660

gctatcaagg ctgactttga cacctgtgct tggatcactg tgtctcagag ctacgaggct 3720

gacgatttgc ttaggcgaac tgctcaagag tttcgcaaga atgatcggaa gaaagacttc 3780

ccagttgacg ttgatatcac aaattacaga ggcttggtcg aaaccacccg ctcttacctg 3840

gagaacaaaa ggtatgtcct tgtcctagat gatgtatgga atgcaaatgt ttggttcgat 3900

agcaaagatg catttgaaga tggcaatatt ggccgaataa ttcttacatc aaggaattat 3960

gatgtggcgt tacttgcgca tgaaacccat ataattaatt tgcagccgtt ggagaaacac 4020

cacgcgtggg atctgttctg taaagaggca ttttggaaga atgagataag gaattgtccg 4080

ccggagttgc agccctgggc taataatttt gttgacaagt gcaatggctt gccaatcgca 4140

attgtgtgca ttggacgcct tctgtcattt caagggtcaa cttattcaga ctgggaaaag 4200

gtgtataaaa atcttgagat gcaactaaca aacaactcta tcatggacat gatgaacatt 4260

atcttgaaga ttagtttgga agatctaccg cacaacatta agaactgctt cctttattgc 4320

tccatgtttc ctgaaaatta tgtgatgaag aggaagtccc tagtacgact ctgggttgct 4380

gaaggattta ttgaagaaac tgagcataga acacttgagg aagtggcaga gcattacttg 4440

actgaacttg tgaaccgatg tcttctgttg ctggtgaaga gaaatgaggc tggacatgtt 4500

catgaagtcc aaatgcacga tatcctccgt gttttggctc tttccaaggc tcgtgaacaa 4560

aatttttgca ttgtcgttaa ccactcgagg agtacacatc ttattggaga agcacgccgt 4620

ttatcaattc agagagggga ttttgcacaa cttgcagacc atgcaccaca tcttcgatcc 4680

ttgctgcttt tccaaagttc acccaatgtc agttcgcttc attcattacc aaagtctgtg 4740

aagttgttgt ctgttttgga tctaactgat agttcagttg ataggctgcc aaaggaagtg 4800

tttggcttgt tcaacttgcg ttttctgggt ctcaggcgta ctaaaatctc caagcttcca 4860

agctccattg gaaggctaaa aaatctgctg gtgttggacg cttggaagtg taaaattgta 4920

aagcttccat tggcgattac aaaacttcaa aagctaacac atcttattgt aacttcgaaa 4980

gcagtcgttg tttctaagca atttgttcct tctgttggtg tgccagcacc tttgcgtatc 5040

tgctccatga caacccttca gacattacta ctcatggaag ctagttctca aatggttcat 5100

cacctaggct ctcttgtgga gttaagaacc tttcgtatca gcaaggtgcg aagttgccat 5160

tgtgagcagt tgttcatggc catcactaat atgattcatc ttacccgtct tgggatccag 5220

gcagacagta gtcaagaagt gctgcatctt gaatcactta aacctcctcc tctacttcag 5280

aaacttttct tgcaaggtac attatctcat gaatcattac ctcatttcgt gtctgtaagc 5340

aatctgaata acctcacgtt tctacgtctt gctgggtcaa gaattgacga aaatgcattc 5400

cttaatcttg agggattaca gcagttggta aagctacagc tttatgatgc atttgatgga 5460

atgaatatat acttccatga gaactcattt ccaaagctca gaatactgaa aatatggggt 5520

gccccacacc tgaatgaaat taagatgaca aaaggagctg tggcaagcct aacacatctg 5580

aagttcctgc tctgtccaaa cctgaagcag ttgccttgcg gtattgaaca tgtgaggact 5640

cttgaggagc tcactctgga tcatacagca gaagagcttg tggatagagt ccgacggaaa 5700

aaagagcgaa tgatttgtga cgtccagaga gtttatgttg ggttcatcag aaatggtgtg 5760

ttggctgcag aaaggattca ataagttggt ttcagtcagg acattcattc tttatcctgc 5820

atcacatagt caatgttcaa ttttactttc aatagtattt tcccattatg tcttattcat 5880

atttttttcg ataatagaat cttaattaag cgtctacatc cgggatgaat catgatcacc 5940

atctgaaatt gactttcaat tttaacttgt tccataccct caatattgta tattcaaata 6000

tatatatctt tttggcaaaa tttgccacag tacatccaaa aattcgtgta attattagct 6060

gggagataaa aaaaacacta cactgccgat gtacaccaca aaatttatat aattggctgt 6120

aggacttcca aggcaatatt ttttatcatt ttcaaaagga cataaatgcc cttggcctcg 6180

atagcatgta aaaatgtatc cccaactaga cccaatcatg tcgcacttgt cgttc 6235

<210>2

<211>2969

<212>DNA

＜213〉paddy rice belongs to paddy rice (Oryza sativa var.Lansheng)

<400>2

aagcgagaag gaagtaacac ccaaggatag gatagcttag ttgaagttga agctagcaag 60

ctatggcgga gggtgttgtg ggctcactaa tcgtcaagct tggggatgcc ctggcgagtg 120

aagcagtgga ggtcgccaag tccttgcttg ggctagaggg atctgctctg aagcgcctct 180

tttctgagat cggggaggtg aagggtgagc tggagagcat ccatgccttc ttgcaggcag 240

ccgagcggtt caaggacgcc gacgagacaa cctccgcgtt cgtcaagcag gtgaggagcc 300

tcgccctgag catcgaggac gtagtcgatg agttcaccta cgagctgggg gaaggagacg 360

gccggatggg gatggcagtg gcactcaaga ggatgtgcaa gatgggcaca tggtctcgcc 420

tggctggcaa tctgcaggac atcaaggtca acctgaaaaa cgctgccgag aggaggatta 480

ggtatgatct gaagggcgtc gagagaggcg cgaaatcgac agcgggaagg aggagctcaa 540

actggagatc tgactctgta ctcttcaaga gggaggatga gcttgttggt atcgagaaga 600

agagggattt gctgatgaaa tgggtgaaag atgaggagca gcgccgcatg gtggtcagtg 660

tgtggggaat gggtggaatc ggcaagacgg cactggttgc taatgtttac aatgctatca 720

aggctgactt tgacacctgt gcttggatca ctgtgtctca gagctacgag gctgacgatt 780

tgcttaggcg aactgctcaa gagtttcgca agaatgatcg gaagaaagac ttcccagttg 840

acgttgatat cacaaattac agaggcttgg tcgaaaccac ccgctcttac ctggagaaca 900

aaaggtatgt ccttgtccta gatgatgtat ggaatgcaaa tgtttggttc gatagcaaag 960

atgcatttga agatggcaat attggccgaa taattcttac atcaaggaat tatgatgtgg 1020

cgttacttgc gcatgaaacc catataatta atttgcagcc gttggagaaa caccacgcgt 1080

gggatctgtt ctgtaaagag gcattttgga agaatgagat aaggaattgt ccgccggagt 1140

tgcagccctg ggctaataat tttgttgaca agtgcaatgg cttgccaatc gcaattgtgt 1200

gcattggacg ccttctgtca tttcaagggt caacttattc agactgggaa aaggtgtata 1260

aaaatcttga gatgcaacta acaaacaact ctatcatgga catgatgaac attatcttga 1320

agattagttt ggaagatcta ccgcacaaca ttaagaactg cttcctttat tgctccatgt 1380

ttcctgaaaa ttatgtgatg aagaggaagt ccctagtacg actctgggtt gctgaaggat 1440

ttattgaaga aactgagcat agaacacttg aggaagtggc agagcattac ttgactgaac 1500

ttgtgaaccg atgtcttctg ttgctggtga agagaaatga ggctggacat gttcatgaag 1560

tccaaatgca cgatatcctc cgtgttttgg ctctttccaa ggctcgtgaa caaaattttt 1620

gcattgtcgt taaccactcg aggagtacac atcttattgg agaagcacgc cgtttatcaa 1680

ttcagagagg ggattttgca caacttgcag accatgcacc acatcttcga tccttgctgc 1740

ttttccaaag ttcacccaat gtcagttcgc ttcattcatt accaaagtct gtgaagttgt 1800

tgtctgtttt ggatctaact gatagttcag ttgataggct gccaaaggaa gtgtttggct 1860

tgttcaactt gcgttttctg ggtctcaggc gtactaaaat ctccaagctt ccaagctcca 1920

ttggaaggct aaaaaatctg ctggtgttgg acgcttggaa gtgtaaaatt gtaaagcttc 1980

cattggcgat tacaaaactt caaaagctaa cacatcttat tgtaacttcg aaagcagtcg 2040

ttgtttctaa gcaatttgtt ccttctgttg gtgtgccagc acctttgcgt atctgctcca 2100

tgacaaccct tcagacatta ctactcatgg aagctagttc tcaaatggtt catcacctag 2160

gctctcttgt ggagttaaga acctttcgta tcagcaaggt gcgaagttgc cattgtgagc 2220

agttgttcat ggccatcact aatatgattc atcttacccg tcttgggatc caggcagaca 2280

gtagtcaaga agtgctgcat cttgaatcac ttaaacctcc tcctctactt cagaaacttt 2340

tcttgcaagg tacattatct catgaatcat tacctcattt cgtgtctgta agcaatctga 2400

ataacctcac gtttctacgt cttgctgggt caagaattga cgaaaatgca ttccttaatc 2460

ttgagggatt acagcagttg gtaaagctac agctttatga tgcatttgat ggaatgaata 2520

tatacttcca tgagaactca tttccaaagc tcagaatact gaaaatatgg ggtgccccac 2580

acctgaatga aattaagatg acaaaaggag ctgtggcaag cctaacacat ctgaagttcc 2640

tgctctgtcc aaacctgaag cagttgcctt gcggtattga acatgtgagg actcttgagg 2700

agctcactct ggatcataca gcagaagagc ttgtggatag agtccgacgg aaaaaagagc 2760

gaatgatttg tgacgtccag agagtttatg ttgggttcat cagaaatggt gtgttggctg 2820

cagaaaggat tcaataagtt ggtttcagtc aggacattca ttctttatcc tgcatcacat 2880

agtcaatgtt caattttact ttcaatagta ttttcccatt atgtcttatt catatttttt 2940

tcgataatag aatcttaatt aagcgtcta 2969

<210>3

<211>924

<212>PRT

＜213〉paddy rice belongs to paddy rice (Oryza sativa var.Lansheng)

<400>3

Met Ala Glu Gly Val Val Gly Ser Leu Ile Val Lys Leu Gly Asp Ala

1 5 10 15

Leu Ala Ser Glu Ala Val Glu Val Ala Lys Ser Leu Leu Gly Leu Glu

20 25 30

Gly Ser Ala Leu Lys Arg Leu Phe Ser Glu Ile Gly Glu Val Lys Gly

35 40 45

Glu Leu Glu Ser Ile His Ala Phe Leu Gln Ala Ala Glu Arg Phe Lys

50 55 60

Asp Ala Asp Glu Thr Thr Ser Ala Phe Val Lys Gln Val Arg Ser Leu

65 70 75 80

Ala Leu Ser Ile Glu Asp Val Val Asp Glu Phe Thr Tyr Glu Leu Gly

85 90 95

Glu Gly Asp Gly Arg Met Gly Met Ala Val Ala Leu Lys Arg Met Cys

100 105 110

Lys Met Gly Thr Trp Ser Arg Leu Ala Gly Asn Leu Gln Asp Ile Lys

115 120 125

Val Asn Leu Lys Asn Ala Ala Glu Arg Arg Ile Arg Tyr Asp Leu Lys

130 135 140

Gly Val Glu Arg Gly Ala Lys Ser Thr Ala Gly Arg Arg Ser Ser Asn

145 150 155 160

Trp Arg Ser Asp Ser Val Leu Phe Lys Arg Glu Asp Glu Leu Val Gly

165 170 175

Ile Glu Lys Lys Arg Asp Leu Leu Met Lys Trp Val Lys Asp Glu Glu

180 185 190

Gln Arg Arg Met Val Val Ser Val Trp Gly Met Gly Gly Ile Gly Lys

195 200 205

Thr Ala Leu Val Ala Asn Val Tyr Asn Ala Ile Lys Ala Asp Phe Asp

210 215 220

Thr Cys Ala Trp Ile Thr Val Ser Gln Ser Tyr Glu Ala Asp Asp Leu

225 230 235 240

Leu Arg Arg Thr Ala Gln Glu Phe Arg Lys Asn Asp Arg Lys Lys Asp

245 250 255

Phe Pro Val Asp Val Asp Ile Thr Asn Tyr Arg Gly Leu Val Glu Thr

260 265 270

Thr Arg Ser Tyr Leu Glu Asn Lys Arg Tyr Val Leu Val Leu Asp Asp

275 280 285

Val Trp Asn Ala Asn Val Trp Phe Asp Ser Lys Asp Ala Phe Glu Asp

290 295 300

Gly Asn Ile Gly Arg Ile Ile Leu Thr Ser Arg Asn Tyr Asp Val Ala

305 310 315 320

Leu Leu Ala His Glu Thr His Ile Ile Asn Leu Gln Pro Leu Glu Lys

325 330 335

His His Ala Trp Asp Leu Phe Cys Lys Glu Ala Phe Trp Lys Asn Glu

340 345 350

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

355 360 365

Asp Lys Cys Asn Gly Leu Pro Ile Ala Ile Val Cys Ile Gly Arg Leu

370 375 380

Leu Ser Phe Gln Gly Ser Thr Tyr Ser Asp Trp Glu Lys Val Tyr Lys

385 390 395 400

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

405 410 415

Ile Ile Leu Lys Ile Ser Leu Glu Asp Leu Pro His Asn Ile Lys Asn

420 425 430

Cys Phe Leu Tyr Cys Ser Met Phe Pro Glu Asn Tyr Val Met Lys Arg

435 440 445

Lys Ser Leu Val Arg Leu Trp Val Ala Glu Gly Phe Ile Glu Glu Thr

450 455 460

Glu His Arg Thr Leu Glu Glu Val Ala Glu His Tyr Leu Thr Glu Leu

465 470 475 480

Val Asn Arg Cys Leu Leu Leu Leu Val Lys Arg Asn Glu Ala Gly His

485 490 495

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

500 505 510

Lys Ala Arg Glu Gln Asn Phe Cys Ile Val Val Asn His Ser Arg Ser

515 520 525

Thr His Leu Ile Gly Glu Ala Arg Arg Leu Ser Ile Gln Arg Gly Asp

530 535 540

Phe Ala Gln Leu Ala Asp His Ala Pro His Leu Arg Ser Leu Leu Leu

545 550 555 560

Phe Gln Ser Ser Pro Asn Val Ser Ser Leu His Ser Leu Pro Lys Ser

565 570 575

Val Lys Leu Leu Ser Val Leu Asp Leu Thr Asp Ser Ser Val Asp Arg

580 585 590

Leu Pro Lys Glu Val Phe Gly Leu Phe Asn Leu Arg Phe Leu Gly Leu

595 600 605

Arg Arg Thr Lys Ile Ser Lys Leu Pro Ser Ser Ile Gly Arg Leu Lys

610 615 620

Asn Leu Leu Val Leu Asp Ala Trp Lys Cys Lys Ile Val Lys Leu Pro

625 630 635 640

Leu Ala Ile Thr Lys Leu Gln Lys Leu Thr His Leu Ile Val Thr Ser

645 650 655

Lys Ala Val Val Val Ser Lys Gln Phe Val Pro Ser Val Gly Val Pro

660 665 670

Ala Pro Leu Arg Ile Cys Ser Met Thr Thr Leu Gln Thr Leu Leu Leu

675 680 685

Met Glu Ala Ser Ser Gln Met Val His His Leu Gly Ser Leu Val Glu

690 695 700

Leu Arg Thr Phe Arg Ile Ser Lys Val Arg Ser Cys His Cys Glu Gln

705 710 715 720

Leu Phe Met Ala Ile Thr Asn Met Ile His Leu Thr Arg Leu Gly Ile

725 730 735

Gln Ala Asp Ser Ser Gln Glu Val Leu His Leu Glu Ser Leu Lys Pro

740 745 750

Pro Pro Leu Leu Gln Lys Leu Phe Leu Gln Gly Thr Leu Ser His Glu

755 760 765

Ser Leu Pro His Phe Val Ser Val Ser Asn Leu Asn Asn Leu Thr Phe

770 775 780

Leu Arg Leu Ala Gly Ser Arg Ile Asp Glu Asn Ala Phe Leu Asn Leu

785 790 795 800

Glu Gly Leu Gln Gln Leu Val Lys Leu Gln Leu Tyr Asp Ala Phe Asp

805 810 815

Gly Met Asn Ile Tyr Phe His Glu Asn Ser Phe Pro Lys Leu Arg Ile

820 825 830

Leu Lys Ile Trp Gly Ala Pro His Leu Asn Glu Ile Lys Met Thr Lys

835 840 845

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

850 855 860

Leu Lys Gln Leu Pro Cys Gly Ile Glu His Val Arg Thr Leu Glu Glu

865 870 875 880

Leu Thr Leu Asp His Thr Ala Glu Glu Leu Val Asp Arg Val Arg Arg

885 890 895

Lys Lys Glu Arg Met Ile Cys Asp Val Gln Arg Val Tyr Val Gly Phe

900 905 910

Ile Arg Asn Gly Val Leu Ala Ala Glu Arg Ile Gln

915 920

Claims (9)

1, a kind of disease resistance of plant albumen is following (a) or protein (b):

(a) protein of forming by the amino acid residue sequence of sequence in the sequence table 3;

(b) with the amino acid residue sequence of sequence in the sequence table 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the disease resistance function by (a) deutero-protein.

2, conversion-resisting resisting related protein according to claim 1 is characterized in that: described disease resistance of plant albumen, its amino acid residue sequence are sequences 3 in the sequence table.

3, claim 1 or the proteic encoding gene of 2 described disease resistance of plant.

4, encoding gene according to claim 3 is characterized in that: its encoding sequence of the proteic encoding gene of described disease resistance of plant be in the sequence table sequence 1 from 5 ' the 3010th to the 5781st Nucleotide of end.

5, encoding gene according to claim 4 is characterized in that: the proteic genomic gene of described disease resistance of plant has one of following nucleotide sequence:

1) dna sequence dna of sequence 1 in the sequence table;

2) polynucleotide of sequence 3 protein sequences in the code sequence tabulation;

3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with sequence in the sequence table 1.

6, encoding gene according to claim 4, it is characterized in that: the proteic cDNA gene of described disease resistance of plant has one of following nucleotide sequence:

1) dna sequence dna of sequence 2 in the sequence table;

2) polynucleotide of sequence 3 protein sequences in the code sequence tabulation;

3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with sequence in the sequence table 2.

7, the recombinant expression vector, transgenic cell line or the host bacterium that contain any proteic encoding gene of described disease resistance of plant among the claim 3-6.

8, any described disease resistance of plant albumen has application in the plant that disease resistance or disease resistance improve in cultivation among the claim 3-6.

9, application according to claim 8 is characterized in that: described disease resistance is the resistance to rice blast fungus; Described plant is a paddy rice.

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