CN115677839B - Rice OsTOBP 1C protein and application of encoding gene thereof - Google Patents

Abstract

The application discloses application of rice OsTOBP 1C protein and a coding gene thereof. The application discovers that the rice OsTOBP 1C protein and the encoding gene LOC_Os03g19190 thereof are involved in regulating and controlling the resistance reaction of rice to bacterial blight, and increasing the transcription level of the LOC_Os03g19190 gene can obviously enhance the disease resistance of rice to bacterial blight. After the transgenic plant over-expressing LOC_Os03g19190 gene is infected with bacterial leaf blight Guangdong race IV, leaf spot length is obviously shortened. Meanwhile, the 521 th to 543 th bits of LOC_Os03g19190 gene are used as target sequences, so that the gene site-directed editing can be efficiently realized, and the susceptibility of rice to bacterial blight is obviously increased. The novel function of LOC_Os03g19190 gene provides a novel method for breeding disease-resistant plants, and has important application value in agricultural production.

Description

Rice OsTOBP 1C protein and application of encoding gene thereof

Technical Field

The application belongs to the technical field of plant genetic engineering, relates to application of rice OsTOBP 1C protein and a coding gene thereof, and in particular relates to application of rice OsTOBP 1C protein and a coding gene LOC_Os03g19190 thereof in regulation and control of plant disease resistance.

Background

Bacterial leaf blight caused by invasion of xanthomonas oryzae rice pathogenic variety (Xanthomonas oryzae pv. Oryzae) into rice leaf vascular bundles is one of important bacterial diseases in rice crop production, and forms a serious threat to rice yield, and can generally cause about 20% -30% of rice yield reduction, and the serious can reach 50%. The breeding and planting of disease-resistant varieties by using disease-resistant genes are the most economical and environment-friendly effective measures for preventing and treating bacterial leaf blight of rice at present. Although 46 rice bacterial leaf blight resistance genes or loci are reported at present, most bacterial leaf blight resistance genes have the problems of narrow resistance spectrum, resistance loss, difficult utilization and the like in agricultural production.

As the bacterial leaf blight bacteria (xanthomonas oryzae rice pathogenic varieties) of the rice are rapidly mutated, the resistance of the rice varieties is extremely easy to lose due to the co-evolution caused by the bacterial leaf blight bacteria, so that the identification of the bacterial leaf blight disease resistance genes is beneficial to the excavation of resistant genetic resources and the cultivation of broad-spectrum disease resistance varieties, and has important application value for rice disease resistance breeding.

Disclosure of Invention

The application aims to solve the technical problem of how to regulate and control the disease resistance of plants. Aims at providing the application of the rice OsTOBP 1C protein and the encoding gene LOC_Os03g19190 thereof in regulating and controlling plant disease resistance. The technical problems to be solved are not limited to the technical subject matter as described, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

In order to solve the above technical problems, the present application provides an application of an anti-disease related protein or a substance for regulating the activity and/or content of the protein, wherein the application can be any of the following:

d1 The use of a protein or a substance regulating the activity and/or content of said protein for regulating plant disease resistance or for increasing plant disease resistance;

d2 The use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product regulating or increasing plant disease resistance;

d3 Use of a protein or a substance regulating the activity and/or content of said protein for growing disease-resistant plants;

d4 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product for growing disease-resistant plants;

d5 Use of a protein or a substance regulating the activity and/or content of said protein in plant breeding;

the use in plant breeding may be in genetic breeding to improve plant disease resistance.

The modulation of plant disease resistance may be a decrease in disease resistance.

The disease resistance may be a disease resistance of a plant to bacterial leaf blight.

The bacterial leaf blight bacteria may be xanthomonas oryzae rice pathogenic varieties (Xanthomonas oryzae pv.

The protein is named OsTOBP 1C and can be A1), A2) or A3) as follows:

a1 A protein having an amino acid sequence of SEQ ID No. 1;

a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues in the amino acid sequence shown in SEQ ID No.1, has more than 80% of identity with the protein shown in A1) and has disease resistance function;

a3 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).

The amino acid sequence shown in SEQ ID No.1 is a protein sequence encoded by a rice LOC_Os03g19190 gene, and a person skilled in the art can substitute, delete and/or increase more than one amino acid on the premise of not affecting the activity of the protein sequence disclosed by the application according to the conventional technical means in the art such as the amino acid sequence disclosed by the application, conservative substitution of the amino acid and the like, so as to obtain a mutant with the same activity as the protein OsTOBP 1C encoded by the rice LOC_Os03g19190 gene disclosed by the application.

In order to facilitate purification or detection of the protein in A1), a tag as shown in Table 1 may be attached to the amino-terminus or the carboxyl-terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.1 of the sequence Listing, but is not limited thereto.

Table 1: tag sequence

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The OsTOBP 1C protein in the A2) can be synthesized artificially or can be obtained by synthesizing the encoding gene and then biologically expressing.

The encoding gene of the OsTOBP 1C protein in the above A2) can be obtained by deleting one or several amino acid residues in the DNA sequence shown in SEQ ID No.2 and/or performing one or several base pair missense mutation and/or ligating the encoding sequences of the tags shown in Table 1 at the 5 'end and/or the 3' end thereof.

Wherein, the DNA molecule shown in SEQ ID No.2 codes for OsTOBP 1C protein shown in SEQ ID No. 1.

In the above application, the OsTOBP 1C protein is derived from rice (Oryza sativa).

In the above application, the plant may be F1) or F2) or F3):

f1 Monocotyledonous or dicotyledonous plants;

f2 A gramineous plant;

f3 Rice.

The application also provides application of the biological material related to the OsTOBP 1C protein, wherein the application can be any one of the following:

d1 Application of biological materials related to OsTOBP 1C protein in regulating plant disease resistance or improving plant disease resistance;

d2 Application of biological material related to OsTOBP 1C protein in preparing products for regulating and controlling plant disease resistance or improving plant disease resistance;

d3 The application of biological materials related to OsTOBP 1C protein in the cultivation of disease-resistant plants;

d4 Application of biological material related to OsTOBP 1C protein in preparing products for cultivating disease-resistant plants;

d5 Use of biological material related to OsTOBP 1C protein in plant breeding;

the biomaterial may be any one of the following B1) to B7):

b1 A nucleic acid molecule encoding an OsTOBP 1C protein;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising B1) said nucleic acid molecule, or a transgenic plant cell line comprising B2) said expression cassette, or a transgenic plant cell line comprising B3) said recombinant vector;

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2).

In the above application, the nucleic acid molecule may be a DNA molecule as shown in b 1) or b 2) or b 3) below:

b1 A DNA molecule or a cDNA molecule with a coding sequence shown in SEQ ID No. 2;

b2 A DNA molecule or a cDNA molecule represented by SEQ ID No. 2;

b3 A DNA molecule or cDNA molecule having 75% or more identity to the nucleotide sequence defined in b 1) or b 2) and encoding an OsTOBP 1C protein.

The nucleotide sequence encoding the OsTOBP 1C protein of the present application can be easily mutated by one of ordinary skill in the art using a known method, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the OsTOBP 1C protein isolated by the present application are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application as long as they encode the OsTOBP 1C protein and function as the OsTOBP 1C protein.

The nucleotide sequence shown in SEQ ID NO.2 is the nucleotide sequence of the rice LOC_Os03g19190 gene. The rice LOC_Os03g19190 gene can be any nucleotide sequence capable of encoding OsTOBP 1C protein. In view of the degeneracy of codons and the preferences of codons of different species, one skilled in the art can use codons appropriate for expression of a particular species as desired.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above applications, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

In such applications, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In the above application, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be an RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

Herein, the substance regulating the activity or content of the protein may be a substance regulating the expression of a gene encoding the protein OsTOBP 1C.

In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).

The substance for regulating the expression of the genes can be specifically the biological material of any one of B1) to B3).

Herein, the vector may be a plasmid, cosmid, phage, or viral vector. The plasmid can be specifically Lb-CRISPR/Cpf1 or a vector obtained by modifying Lb-CRISPR/Cpf 1.

The plasmid may be pMDC43, or may be a vector obtained by modifying pMDC 43.

The recombinant vector can be a Lb-LOC_Os03g19190 recombinant expression vector; the Lb-LOC_Os03g19190 recombinant expression vector is obtained by inserting a DNA fragment shown as SEQ ID NO.4 into the downstream of a rice U6 promoter on the Lb-CRISPR/Cpf1 vector, and keeping other sequences of the Lb-CRISPR/Cpf1 vector unchanged.

The recombinant vector can also be pMDC43-Os03g19190; the pMDC43-Os03g19190 contains a nucleotide sequence shown in SEQ ID NO. 2.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria may be derived from Escherichia, erwinia, agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas, bacillus (Bacillus), etc.

The application also provides a method for cultivating the disease-resistant plant, which comprises the step of improving the content and/or activity of OsTOBP 1C protein in the target plant to obtain the disease-resistant plant with disease resistance higher than that of the target plant.

In the above method, the increase in the content and/or activity of OsTOBP 1C protein in the target plant is achieved by increasing the expression level of the gene encoding the protein in the target plant.

The gene encoding the OsTOBP 1C protein described herein is designated LOC_Os03g19190.

In the above method, the coding gene of the protein is a DNA molecule as shown in the following b 1) or b 2) or b 3):

b1 A DNA molecule or a cDNA molecule with a coding sequence shown in SEQ ID No. 2;

b2 A DNA molecule or a cDNA molecule represented by SEQ ID No. 2;

b3 A DNA molecule or cDNA molecule having 75% or more identity to the nucleotide sequence defined in b 1) or b 2) and encoding an OsTOBP 1C protein.

In the above method, the plant is F1) or F2) or F3):

f1 Monocotyledonous or dicotyledonous plants;

f2 A gramineous plant;

f3 Rice.

In the present application, the disease-resistant plant is understood to include not only the first generation transgenic plant obtained by transforming the LOC_Os03g19190 gene into the target plant, but also the progeny thereof. The gene may be propagated in that species, or may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The disease resistant plants include seeds, calli, whole plants and cells.

The OsTOBP 1C protein and/or the biological material are also within the scope of the application.

The application also provides application of the method in creating disease-resistant plants and/or plant breeding.

The application identifies bacterial leaf blight resistance related candidate gene LOC_Os03g19190 by carrying out Genome association analysis (Genome-wide associated study, GWAS) on the length of bacterial leaf blight bacteria IV inoculated on 196 domestic and foreign rice materials, the bacterial leaf blight resistance related candidate gene LOC_Os03g19190 is involved in the biosynthesis of rice topoisomerase II binding protein 1C (Topoisomerase II binding protein C, TOPP 1C), the coding sequence (CDS) is shown as SEQ ID NO.2, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 1. Furthermore, according to the application, bacterial blight resistance evaluation is carried out on a rice variety Nipponbare with the gene edited in a targeted manner by CRISPR/Cpf1 technology, and the gene editing is found to be capable of remarkably increasing susceptibility of rice to bacterial blight. Furthermore, according to the application, bacterial blight resistance evaluation is carried out on a rice variety Nipponbare over-expressing the nucleotide sequence of the gene coding protein, and the over-expression of the nucleotide sequence of the gene coding protein is found to be capable of obviously enhancing the disease resistance of rice against bacterial blight.

Specifically, the application provides application of a rice LOC_Os03g19190 gene, a coding protein thereof, an inhibitor of the rice LOC_Os03g19190 gene or a nucleic acid molecule containing the LOC_Os03g19190 gene coding protein in regulating plant disease resistance.

The application provides application of rice LOC_Os03g19190 gene, encoding protein thereof or inhibiting factor of rice LOC_Os03g19190 gene or nucleic acid molecule containing encoding OsTOBP 1C protein in genetic breeding for improving plant disease resistance.

Specifically, the disease resistance of the application is the disease resistance of plants against bacterial leaf blight bacteria.

Specifically, the genetic breeding is to construct transgenic plants resistant to bacterial leaf blight.

Specifically, in the above application, the disease resistance of plants is improved by increasing the expression level of the LOC_Os03g19190 gene of rice.

The improvement in disease resistance or improvement in plant disease resistance described herein may be manifested as a reduction in the length of lesions of bacterial leaf blight of rice.

The inhibiting factor of the rice LOC_Os03g19190 gene is a nucleic acid capable of disrupting the biological function of a protein encoded by the rice LOC_Os03g19190 gene.

The nucleic acid may be selected from: antisense oligonucleotides, small interfering RNAs (sirnas), micrornas (mirnas), or short hairpin RNAs (shrnas). The antisense oligonucleotide refers to a nucleic acid fragment which can be complementary to a target gene (LOC_Os 03g19190 gene), can be bound to the target gene through a base complementary principle, so as to inhibit, block or reduce the expression of the LOC_Os03g19190 gene, can be antisense DNA or antisense RNA, and can be artificially synthesized or expressed in vivo by a method well known to a person skilled in the art; the small interfering RNA (siRNA), micro RNA (miRNA) or short hairpin RNA (shRNA) refers to RNA which inhibits or reduces the expression of LOC_Os03g19190 gene by RNA interference (RNAi), and can be prepared by methods known to those skilled in the art.

In particular, the nucleic acid may be a gRNA or an interfering RNA.

Specifically, the target sequence of the gRNA is XXX in 5'-TTTNXXX-3' form nucleotide sequence in the LOC_Os03g19190 gene of the rice; wherein, 5'-TTTN-3' is PAM sequence, N is any one base in A, T, G, C, XXX is nucleic acid sequence of 22-24 bp.

More specifically, the target sequence of the gRNA is 521 th to 543 th positions of the LOC_Os03g19190 gene of the rice.

In the application, the nucleic acid molecule containing LOC_Os03g19190 gene encoding protein is a nucleotide sequence capable of encoding OsTOBP 1C protein.

Specifically, the nucleic acid sequence is CDS (coding sequence).

Specifically, the CDS is the nucleotide sequence of the rice LOC_Os03g19190 gene exon.

Specifically, the CDS is a sequence formed by connecting 80 th to 178 th, 290 th to 379 th, 462 th to 566 th, 918 th to 1019 th, 2042 nd to 2162 th, 2602 nd to 2686 th, 2794 th to 2838 th, 3051 th to 6038 th, 6205 th to 6421 th and 6559 th to 7131 th of the LOC_Os03g19190 gene of the rice.

The above-mentioned application of the rice LOC_Os03g19190 gene, its encoding protein, the inhibitor of the rice LOC_Os03g19190 gene or the nucleic acid molecule containing the protein encoded by the LOC_Os03g19190 gene may be in the form of the rice LOC_Os03g19190 gene, its encoding protein, the inhibitor of the rice LOC_Os03g19190 gene or the nucleic acid molecule itself containing the protein encoded by the LOC_Os03g19190 gene, or in the form of an expression cassette, a vector containing the rice LOC_Os03g19190 gene or the inhibitor thereof, a host cell containing the expression cassette or the vector.

Specifically, the application provides a gRNA for editing the LOC_Os03g19190 gene of rice, and the target sequence of the gRNA is 521 th to 543 th positions of the LOC_Os03g19190 gene of the rice.

Specifically, the gRNA targets the sequence shown in SEQ ID NO. 4.

The gRNA can be matched with a CRISRP/cpf1 gene editing tool to realize editing of the rice LOC_Os03g19190 gene and destroy the biological functions of encoding proteins of the rice LOC_Os03g19190 gene.

The gRNA is obtained through a large number of screening, experiments prove that the rice plant subjected to CRISPRP/cpf 1 mediated gene editing can be obtained efficiently, and insertion or deletion at 521 th to 543 rd positions of a sequence shown as SEQ ID NO.3 can cause the disruption of the biological functions of the rice LOC_Os03g19190 gene, so that the rice shows the property of reduced bacterial leaf blight resistance level.

Specifically, the application also provides a biological material comprising the gRNA for editing the LOC_Os03g19190 gene of rice, wherein the biological material comprises an expression cassette, a vector, a host cell, an engineering bacterium or a transgenic plant cell line.

In particular, the present application provides a method for regulating bacterial leaf blight resistance of plants or for growing transgenic plants, comprising: regulating the biological function of the encoding protein of the rice LOC_Os03g19190 gene in plants;

specifically, bacterial leaf blight resistance of plants is increased or decreased by enhancing or disrupting the biological function of the encoded protein of the rice loc_os03g19190 gene.

The enhancement of the biological function of the encoded protein of the LOC_Os03g19190 gene of rice can be achieved by conventional technical means in the art.

Specifically, the biological function of the rice LOC_Os03g19190 gene-encoded protein can be enhanced by using a PMDC43 plant binary expression system; the PMDC43 plant binary expression system can be a pMDC43-Os03g19190 recombinant vector, the pMDC43-Os03g19190 recombinant vector contains LOC_Os03g19190 gene CDS sequence shown in SEQ ID No.2 in a sequence table and a 35S promoter, can express the fusion protein of OsTOBP 1C protein and GFP shown in SEQ ID No.1, and the expression of the fusion protein is driven by the 35S promoter.

The cDNA of the LOC_Os03g19190 gene of the rice can be over-expressed in a target rice variety by utilizing a PMDC43 plant binary expression system, so that the biological function of the protein encoded by the gene is enhanced.

As an aspect of the present application, the method for enhancing bacterial leaf blight resistance of plants or growing transgenic plants comprises the steps of:

(1) Constructing a PMDC43 plant binary expression vector plasmid containing a sequence shown in SEQ ID NO. 2;

(2) Transferring the PMDC43 plant binary expression vector plasmid constructed in the step (1) into rice;

(3) The rice material for resisting bacterial leaf blight is obtained through screening and identification.

Specifically, in the step (1), the PMDC43 plant binary expression vector plasmid may be any plant binary expression vector system;

specifically, in the step (2), the PMDC43 plant binary expression vector plasmid constructed in the step (1) is transferred into rice varieties in an agrobacterium tumefaciens-mediated mode.

Specifically, in the step (3), the screening method may specifically be: and (3) carrying out PCR amplification by using primers aiming at hygromycin marker genes carried by PMDC43 plant binary expression vector plasmids, and if 480bp target bands are amplified, indicating that the PMDC43 plant binary expression cassettes successfully transform plants.

Specifically, in the step (3), the identification method specifically includes: detecting the transcription expression level of the LOC_Os03g19190 gene in the transformed plant by using a specific primer of the LOC_Os03g19190 gene, wherein if the transcription abundance of the nucleotide sequence shown between SEQ ID NO.15 and SEQ ID NO.16 is increased to cause the over-expression of the LOC_Os03g19190 gene, the biological function of the LOC_Os03g19190 gene of the plant is enhanced.

The above-mentioned biological functions of the encoded protein disrupting the rice LOC_Os03g19190 gene can be achieved by conventional means in the art.

Specifically, the CRISPR/cpf 1system can be used to disrupt the biological function of the protein encoded by the rice loc_os03g19190 gene; in the CRISPR/cpf 1system, the target sequence of the gRNA is 521 th to 543 th of the LOC_Os03g19190 gene of the rice.

More specifically, the gRNA targets the sequence shown in SEQ ID NO. 4.

The CRISPR/cpf 1system can be utilized to cut the 5'-TTTNXXX-3' -form nucleotide sequence of the rice LOC_Os03g19190 gene at 18-23bp far away from the PAM sequence to generate a 5nt sticky end, thereby leading the translation of the gene to be terminated in advance or the protein conformation to be changed and further destroying the biological function of the protein encoded by the gene; wherein N is any one base in A, T, G, C, XXX is a nucleic acid sequence of 22-24 bp.

As an aspect of the present application, the method for controlling bacterial leaf blight resistance of plants or for growing transgenic plants comprises the steps of:

(1) Constructing a CRISPR/cpf1 gene editing plasmid of gRNA targeting the target sequence shown in SEQ ID NO. 4;

(2) Transferring the CRISPR/cpf1 gene editing plasmid constructed in the step (1) into rice;

(3) The bacterial leaf blight susceptible rice material is obtained through screening and identification.

Specifically, in step (1) above, the CRISPR/cpf1 gene editing plasmid can be a class II V-type CRISPR/Cas vector system;

specifically, in the step (2), the CRISPR/cpf1 gene editing plasmid constructed in the step (1) is transferred into rice varieties in an agrobacterium tumefaciens-mediated mode.

Specifically, in the step (3), the screening method may specifically be: PCR amplification is performed by using primers for hygromycin marker genes carried by CRISPR/cpf1 gene editing plasmids, and if 480bp target bands are amplified, the CRISPR/cpf1 gene editing expression cassette is proved to successfully transform plants.

Specifically, in the step (3), the identification method specifically includes: amplifying and sequencing a LOC_Os03g19190 gene fragment of the genome of the transformed plant by using a specific primer of the LOC_Os03g19190 gene, wherein if the LOC_Os03g19190 gene is knocked out due to insertion or deletion mutation of a 521 th to 543 rd nucleic acid sequence of a sequence shown as SEQ ID NO.3, the biological function of the LOC_Os03g19190 gene of the plant is destroyed.

In the present application, the plant may be a monocot or dicot, and in particular may be a recipient plant of bacterial blight bacteria, including but not limited to rice.

The application has the beneficial effects that:

(1) The application discovers that the rice OsTOBP 1C protein and the encoding gene LOC_Os03g19190 thereof are involved in regulating and controlling the immune response of rice to bacterial blight, and can obviously improve the bacterial blight resistance of the rice by enhancing the expression level of the LOC_Os03g19190 gene. Experiments prove that after LOC_Os03g19190 gene is over-expressed to infect the bacterial wilt, the length of the bacterial wilt is shortened by 44.3% -52.7%, and the application value in agricultural production is very important when the expression level of the LOC_Os03g19190 gene is increased to prepare the bacterial wilt-resistant rice material.

(2) The application utilizes CRISPR/Cpf1 technology to carry out genome targeting modification of LOC_Os03g19190 gene, thereby realizing efficient fixed-point editing of LOC_Os03g19190. The application discovers that the target sequence is the nucleotide sequence from 521 rd position to 543 rd position in the rice LOC_Os03g19190 gene, the fixed-point editing of the LOC_Os03g19190 gene can be realized efficiently, and the insertion or deletion from 521 rd position to 543 rd position of the sequence shown as SEQ ID NO.3 can cause the destruction of the biological function of the rice LOC_Os03g19190 gene, so that the rice shows the property of reducing the bacterial leaf blight resistance level, namely the disease spot length after the LOC_Os03g19190 gene knockout plant infects rice bacterial wilt bacteria IV is increased by 40.5% -47.5%.

Drawings

FIG. 1 shows the type of mutation of the nucleotide sequence of LOC_Os03g19190 gene in a homozygous editing plant of cpf-Os 03g19190 of Oryza sativa in example 3, and the nucleotide sequence in black frame is the nucleotide sequence in which deletion occurs.

FIG. 2 shows the type of mutation of the amino acid sequence of LOC_Os03g19190 gene in a homozygous editing plant of cpf-Os 03g19190 against the Japanese sunny background of rice in example 3 of the present application, and the amino acid sequence in which deletion occurs is in the black frame.

FIG. 3 is a chart showing the phenotype and length statistics of the bacterial leaf blight bacterium IV inoculated on a homozygous editing plant of cpf-Os 03g19190 under the Japanese sunny background of rice in example 2 of the present application; wherein, in FIG. 3A is cpf1-Os03g19190 gene expression level detection of LOC_Os03g19190 in homozygous editing plants; FIG. 3B shows the plaque phenotype of cpf1-Os03g19190 homozygous edited plants inoculated with bacterial leaf blight bacterium IV; in FIG. 3, C is the comparison statistics of the lesion length of cpf1-Os03g19190 homozygous edited plants inoculated with bacterial blight bacteria IV; scale = 2 cm, × represents P <0.01.

FIG. 4 is a chart showing the phenotype and length statistics of the spot after inoculation of bacterial leaf blight bacterium IV on NIP-OE-Os03g19190 over-expressed plants in the Nipponbare background of rice in example 2 of the present application; in FIG. 4, A is detection of LOC_Os03g19190 gene expression level in NIP-OE-Os03g19190 over-expressed plants; FIG. 4B shows the plaque phenotype after inoculation of a NIP-OE-Os03g19190 homozygous editing plant with bacterial leaf blight bacterium IV; FIG. 4C shows the comparative statistics of NIP-OE-Os03g19190 homozygous edited plants inoculated with bacterial blight strain IV; NIP-OE-Os03g19190#9, NIP-OE-Os03g19190#11 and NIP-OE-Os03g19190#23 were derived from 3 different independent transformed plants, respectively, scale = 2 cm, representing P <0.01.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 Lb-CRISPR/Cpf1 System-based Rice OsTOBP 1C protein coding gene LOC_Os03g19190 fixed-point editing method

1. Sequence analysis and target sequence screening of rice OsTOBP 1C protein coding gene LOC_Os03g19190

The genome sequence of the rice DNA replication protein gene LOC_Os03g19190 is shown as SEQ ID NO.3, the coding sequence (CDS) is shown as SEQ ID NO.2, the amino acid sequence of the coding protein OsTOBP 1C is shown as SEQ ID NO.1, and sequence analysis shows that the gene comprises 10 exons which are respectively positioned at 80-178, 290-379, 462-566, 918-1019, 2042-2162, 2602-2686, 2794-2838, 3051-6038, 6205-6421 and 6559-7131 of the sequence of the SEQ ID NO. 3.

According to sequence analysis, the sequence (521-543 th bit of SEQ ID NO. 3) on the third exon of the LOC_Os03g19190 gene of the rice OsTOBP 1C protein coding gene is taken as the LOC_Os03g19190 target sequence of the LOC_Os03g19190 fixed-point editing method based on the CRISPR/Cpf1 technology.

Through a large number of screening, the application determines that the Lb-CRISPR/Cpf1 technology is utilized to target the sense strand of the third exon of the rice LOC_Os03g19190 gene, takes 521 th to 543 rd positions of SEQ ID NO.3 as the LOC_Os03g19190-T target sequence, and the LOC_Os03g19190-T target sequence is shown as SEQ ID NO. 4.

2. Lb-CRISPR/Cpf1 vector primer design and construction of recombinant expression vector thereof

(1) Design and synthesis of Lb-CRISPR/Cpf1 technical target sequence primer

Primers targeting LOC_Os03g19190-T target sequence of LOC_Os03g19190 gene are designed based on Lb-CRISPR/Cpf1 technology, and LOC_Os03g19190-T target sequence primers LOC_Os03g19190-TF and LOC_Os03g19190-TR sequences are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.

SEQ ID NO.5：LOC_Os03g19190-TF：5′-AGATGGTGGAAGCTAGCTTGGATCGGG-3′；

SEQ ID NO.6：LOC_Os03g19190-TR：5′-AAAACCCGATCCAAGCTAGCTTCCACC-3′。

Primers LOC_Os03g19190-TF and LOC_Os03g19190-TR were synthesized, respectively.

(2) Construction of Lb-CRISPR/Cpf1 recombinant expression vector

The LOC_Os03g19190-T1 target sequence primers LOC_Os03g19190-TF and LOC_Os03g19190-TR are synthesized into double-stranded target sequences by a primer annealing method, and are inserted into an Lb-CRISPR/Cpf1 vector by a BsaI-HF digestion and ligation method (the Lb-CRISPR/Cpf1 vector is also called LbCPf1 in the following documents: muguin Wang, yanfei Mao, yuming Lu, xiaoping Tao and Jian-kang Zhu. Multiple Gene Editing in Rice Using the CRISPR-Cpf1System. Molecular Plant,2017,10,1011-1013) downstream of a rice U6 promoter, so as to obtain an Lb-LOC_Os03g19190 recombinant expression vector; sequencing proves that the downstream of the rice U6 promoter of the Lb-LOC_Os03g19190 recombinant expression vector is inserted with a sequence shown as SEQ ID NO. 4.

3. Acquisition of recombinant Agrobacterium tumefaciens

The recombinant expression vector Lb-LOC_Os03g19190 constructed in the above 2 was heat-shock transformed into Agrobacterium EHA105 (Biovector NTCC collection, commercially available) to obtain recombinant Agrobacterium containing the recombinant expression vector Lb-LOC_Os03g19190, designated EHA105-Lb-LOC_Os03g19190.

Example 2 overexpression of Rice OsTOBP 1C protein coding Gene LOC_Os03g19190

1. Cloning of nucleotide sequence of protein encoded by LOC_Os03g19190 Gene

(1) Design and synthesis of amplification primer containing LOC_Os03g19190 gene coding protein nucleotide sequence

Cloning primers comprising the LOC_Os03g19190 gene CDS were designed, and the LOC_Os03g19190-cloning-F and LOC_Os03g19190-cloning-R sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

SEQ ID NO.7：LOC_Os03g19190-cloning-F：5′-GCATCAATGGCCTCTCCCAT-3′；

SEQ ID NO.8：LOC_Os03g19190-cloning-R：5′-CACGCCTGTATAGCAGCAGA-3′。

Primers LOC_Os03g19190-cloning-F and LOC_Os03g19190-cloning-R were synthesized, respectively.

(2) Design and synthesis of cloning primer of LOC_Os03g19190 gene encoding protein nucleotide sequence

The cloning over-expression primer of LOC_Os03g19190 gene CDS is designed, and the LOC_Os03g19190-OE-F and LOC_Os03g19190-OE-R sequences are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10.

SEQ ID NO.9：LOC_Os03g19190-OE-F：5′-tgaactatacaaaggcgcgccaATGGCCTCTCCCATCAGCGG-3′；

SEQ ID NO.10：LOC_Os03g19190-OE-R：5′-ctctagaactagttaattaaTCACCTTTTTCGCTTGGATAC-3′。

Primers LOC_Os03g19190-OE-F and LOC_Os03g19190-OE-R were synthesized, respectively.

(3) Cloning of LOC_Os03g19190 Gene encoding protein nucleotide sequence

Extracting total RNA of a rice variety of Japanese head or leaf sheath, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification by using the cDNA as a template and utilizing a forward primer LOC_Os03g19190-cloning-F and a reverse primer LOC_Os03g19190-cloning-R to obtain a primary amplification product; using the primary amplification product as a template, and carrying out PCR amplification by using a forward primer LOC_Os03g19190-OE-F and a reverse primer LOC_Os03g19190-OE-R to obtain a secondary amplification product; the secondary amplification product was subjected to band size identification and gel cutting purification recovery, and the recovered product was designated as OE-LOC_Os03g19190.

2. Construction of LOC_Os03g19190 Gene overexpression recombinant expression vector

The above recovered product OE-LOC_Os03g19190 was subjected to an Infusion reaction with the objective vector pMDC43 (Mark Curtis & Ueli Grossniklaus. A Gateway cloning vector set for high-throughput functional analysis of genes in plants Physiology,2003,133,462-469) treated with Pac I and Asc I enzymes to give a recombinant vector containing the correct CDS nucleotide sequence shown in SEQ ID NO.2 designated pMDC43-Os03g19190. Information reaction system: pac I and Asc I enzyme treated vector pMDC 43.75. Mu.L (50-100 ng), OE-LOC_Os03g 19190.25. Mu.L (10-30 ng), infusion enzyme mix. Mu.L.

Information reaction conditions: after incubation at 50℃for 15 minutes, the reaction system was transformed into E.coli DH 5. Alpha. And positive clones were selected to give the correct expression vector containing the nucleotide sequence of OE-LOC_Os03g19190, designated pMDC43-Os03g19190.pMDC43-Os03g19190 contains LOC_Os03g19190 gene CDS sequence shown in SEQ ID NO.2 in the sequence table and 35S promoter, can express the fusion protein of OsTOBP 1C protein shown in SEQ ID NO.1 and GFP, and the expression of the fusion protein is driven by 35S promoter.

3. Acquisition of recombinant Agrobacterium tumefaciens

And (3) carrying out heat shock transformation on agrobacterium tumefaciens EHA105 by using the recombinant expression vector pMDC43-Os03g19190 constructed in the step (2) to obtain recombinant agrobacterium containing the recombinant expression vector pMDC43-Os03g19190, and naming the recombinant agrobacterium as EHA105-pMDC43-Os03g19190.

Example 3 application of Lb-CRISPR/Cpf1 technology-based fixed point editing method or transgenic overexpression technology to rice varieties

The recombinant agrobacterium EHA105-Lb-LOC_Os03g19190 or EHA105-pMDC43-Os03g19190 is used for infecting callus induced by mature embryo of rice variety Japanese sunny, and the obtained rice transformed plants are named as NIP-Lb-LOC_Os03g19190 and NIP-OE-LOC_Os03g19190 respectively, and the specific methods are as follows:

1. the recombinant Agrobacterium EHA105-Lb-LOC_Os03g191 obtained in examples 1 and 2 was subjected to90. EHA105-pMDC43-Os03g19190 were inoculated into YEB liquid medium (containing 50. Mu.g/ml kanamycin and 20. Mu.g/ml rifampicin), respectively, and shake-cultured at 28℃and 200rpm to OD _600nm 0.6-0.8; centrifuging at 5000rpm and 4deg.C for 5min, and re-suspending the bacterial precipitate with AAM liquid culture medium (acetosyringone concentration of 200 μm, pH 5.2) to OD _600nm And 0.6-0.8 to obtain two recombinant agrobacterium resuspension solutions.

2. Mature seeds of the rice variety Nippon Temminck were glume removed, respectively, soaked in 75% ethanol solution for 1min, then sterilized in NaClO solution (1:2 mixture with water, 1 drop of Tween 20) with shaking for 20min, and repeated 2 times. Washing with sterile water for several times until no foreign odor is generated, inoculating sterilized rice Japanese seed on NBD2 culture medium to induce callus, dark culturing at 26deg.C for 8-10 days, removing root and residual endosperm, and subculturing for 10 days to obtain mature embryo callus.

3. And (2) respectively immersing the mature embryo callus obtained in the step (2) in the two recombinant agrobacterium heavy suspensions obtained in the step (1), removing rice materials after 20-30min, inoculating the rice materials on a co-culture medium (100 mu M acetosyringone concentration and pH 5.2) containing two layers of filter paper, and co-culturing for 3 days in a dark condition at 26 ℃.

4. The callus subjected to the co-culture in the step 3 is inoculated into a screening medium (hygromycin concentration is 50mg/L, pH is 5.8), the screening culture is carried out for 12 days under the dark condition of 28 ℃, and the resistant callus is transferred to a selection medium containing 50mg/L Hyg for continuous screening.

5. After 2 repeated selection, transfer of resistant calli onto differentiation medium (24 hours light/day) to induce differentiation; transferring regenerated seedlings to a 1/2MS culture medium to induce rooting when new rootless seedlings are generated; and after the seedlings grow, the seedlings are transferred into a climatic chamber for nutrient solution cultivation. Cpf1-Os03g19190 regenerated plants (plants obtained from recombinant Agrobacterium EHA105-Lb-LOC_Os03g 19190) and NIP-OE-Os03g19190 regenerated plants (plants obtained from recombinant Agrobacterium EHA105-pMDC43-Os03g19190 infected with Nipponbare) were obtained, respectively.

6. After the obtained regenerated plant is transplanted to survive, total DNA of leaves of the regenerated plant is extracted, and PCR amplification is carried out on the primers Hyg-F (the sequence is shown as SEQ ID NO. 11) based on hygromycin marker genes contained in the recombinant expression vector and the primers Hyg-R (the sequence is shown as SEQ ID NO. 12) respectively, wherein the size of amplified product fragments is 480bp, so that positive transformed plants are screened. The number of regenerated plants, the number of positive transformed plants, and the percentage (%) of the number of positive transformed plants to the number of regenerated plants detected were counted, and the results are shown in Table 2.

TABLE 2 detection result of Positive Rate of LOC_Os03g19190 Gene-transformed Rice variety

Regenerated plants	Regenerated plant number	Positive transformation plant number	Positive rate (%)
				cpf1-Os03g19190	22	22	100.0
NIP-OE-Os03g19190	32	32	100.0

7. The genome of the obtained cpf-Os 03g19190 regenerated plant is used as a template, the specific primers LOC_Os03g19190-Deaction-F (the sequence is shown as SEQ ID NO. 13) of the rice OsTOBP 1C protein coding gene LOC_Os03g19190 and LOC_Os03g19190-Deaction-R (the sequence is shown as SEQ ID NO. 14) are used for carrying out PCR amplification, and the obtained 339bp amplification product is subjected to sequencing verification. Sequencing verification results show that LOC_Os03g19190 genes of 8 transformed plants in 22 positive transformed plant species are subjected to gene editing (cpf-Os 03g19190 edited plants for short). The number of positive transformants detected, the number of occurrence-editing transformants, and the percentage of occurrence-editing transformants to the number of regeneration plants detected, that is, editing efficiency (%) were counted, and the results are shown in table 3.

TABLE 3 detection results of the editing of the gene LOC_Os03g19190 encoding OsTOBP 1C protein of rice induced by Lb-LOC_Os03g19190

Regenerated plants	Positive transformation plant number	The number of plants is edited and transformed	Editing efficiency (%)
				cpf1-Os03g19190	22	8	36.4

8. Collecting seed of cpf-Os 03g19190 editing plant, screening homozygous editing plant in autonomous isolation mode, screening to obtain 2 homozygous editing types (cpf-Os 03g19190#1 and cpf1-Os03g 19190#2), wherein the nucleotide sequence is shown in figure 1, and the amino acid sequence is shown in figure 2.

9. Total RNAs of leaves of wild type Japanese sunny plants, cpf1-Os03g19190#1 and cpf1-Os03g19190#2 were extracted respectively, and reverse transcription was performed, using forward primer LOC_Os03g19190-qRTF (sequence shown in SEQ ID NO. 15): 5'-ACCAAAGAAGTGACCGCGAA-3' and the reverse primer LOC_Os03g19190-qRTR (sequence shown in SEQ ID NO. 16): 5'-AAGCAGTCTTCCAACCACCG-3' primer pair detects the expression level of LOC_Os03g19190 gene. The internal reference is an action, a forward primer action-F (the sequence is shown as SEQ ID NO. 17): 5'-CCTGACGGAGCGTGGTTAC-3' and a reverse primer action-R (the sequence is shown as SEQ ID NO. 18): 5'-CCAGGGCGATGTAGGAAAGC-3'. As a result, as shown in FIG. 3, the expression levels of the LOC-Os 03g19190 gene in cpf1-Os03g19190#1 and cpf1-Os03g19190#2 were significantly reduced relative to the wild type Japanese sunny (NIP) and were about 0.22 and 0.09 times the wild type, respectively.

10. Total RNA of leaves of regenerated plants of wild type Japanese sunny and NIP-OE-Os03g19190 is extracted respectively, reverse transcription is carried out, and the expression level of LOC_Os03g19190 gene is detected by utilizing a primer pair consisting of a forward primer LOC_Os03g19190-qRTF and a reverse primer LOC_Os03g 19190-qRTR. The internal references are an action, a forward primer action-F and a reverse primer action-R. 3 plants with higher LOC_Os03g19190 gene expression level are selected and marked as NIP-OE-Os03g19190#9, NIP-OE-Os03g19190#11 and NIP-OE-Os03g19190#23 respectively. As shown in FIG. 4, the expression levels of the LOC_Os03g19190 gene in NIP-OE-LOC_Os03g19190#9, NIP-OE-LOC_Os03g19190#11, and NIP-OE-LOC_Os03g19190#23 were all significantly up-regulated relative to the wild type Japanese sunny (NIP), and were 121.27, 100.20, and 93.99 times that of the wild type, respectively.

11. Plants such as wild type Nippon, cpf1-Os03g19190#1, cpf1-Os03g19190#2, NIP-OE-Os03g19190#9, NIP-OE-Os03g19190#11 and NIP-OE-Os03g19190#23 were inoculated with bacterial strain IV, and resistance evaluation analysis of the LOC_Os03g19190 gene against bacterial strain of rice was performed, wherein 15 leaves were counted per plant system, and the results are shown in FIGS. 3 and 4, respectively, and the results showed that: compared with the wild type (Japanese sunny), cpf-Os 03g19190#1 and cpf-Os 03g19190#2 show a phenotype of prolonged bacterial leaf blight and increased length of IV lesions by more than 40.5%; the plants of NIP-OE-Os03g19190#9, NIP-OE-Os03g19190#11 and NIP-OE-Os03g19190#23 all show a phenotype of shortened bacterial leaf blight lesions, and the length of bacterial leaf blight bacterial IV lesions is reduced by more than 44.3 percent; the result shows that the LOC_Os03g19190 gene encoding the protein OsTOBP 1C of the rice positively regulates the disease resistance of the rice against bacterial leaf blight bacterium IV.

Japanese (Nipponbare, oryza sativa ssp. Japonica): is described in "Yongqing Jiao, yonghong Wang, dawei Xue, jing Wang, meixian Yan, guifu Liu, guojun Dong, dali Zeng, zefu Lu, xudong Zhu, qian Qian and Jiayang Li. Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in price. Nature Genetics,2010,42, 541-544".

Bacterial strain IV of rice bacterial leaf blight: described in "once listed, yellow and rare, wu Shangzhong. IRBB21 (Xa 21) has a resistance response to 5 race of Rhizoctonia solani, plant protection theory, 2002,29 (2): 97-100".

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

SEQUENCE LISTING

<110> institute of genetic and developmental biology of national academy of sciences

<120> Rice OsTOBP 1C protein and application of coding gene thereof

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 1462

<212> PRT

<213> Rice (Oryza sativa)

<400> 1

Met Ala Ser Pro Ile Ser Gly Ser Asp Asp Asp Asp Glu His Leu Phe

1 5 10 15

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

20 25 30

Gln Tyr Arg Ser Glu Met Val Arg Arg Ser Gly Val Asp Ala Gly Arg

35 40 45

Phe Gly Ser Gly Cys Thr His Val Ile Val Cys Gly Leu Val Tyr Asp

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Asp Pro Ala Cys Val Ala Ala Arg Ala Glu Gly Lys Lys Val Val Thr

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Glu Leu Trp Val Glu Ala Ser Leu Asp Arg Gly Met Leu Ala Asp Ala

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Asp Arg Val Met Tyr Trp Pro Val Arg Asp Leu Asn Gly Ile Ala Gly

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Ser Glu Ser Leu Arg Ile Cys Leu Thr Gly Tyr Gln Arg Ser Asp Arg

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

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Ser Leu Asn Pro Asp Val Val Thr His Leu Ile Cys Tyr Lys Phe Glu

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Gly Glu Lys Tyr Glu Ala Ala Lys Lys Ala Lys Leu Lys Phe Asn Phe

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Asn Ile Lys Leu Val Asn His Arg Trp Leu Glu Asp Cys Leu Lys Cys

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Trp Lys Ile Leu Pro Val Asp Asp Tyr Ser Lys Ser Ser Trp Glu Leu

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Glu Ile Met Glu Ala Gln Ala Lys Asp Ser Glu Asp Glu Glu Asp Val

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

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Lys Gly Ser Ala Gly Thr Ser Ala Asn Pro Val Val Asn Ala Pro Ile

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

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Gln Pro Asn Ile Pro Gly Glu Ile Arg Lys Ala Glu Asp Ala Val Asn

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Arg Ile His Asp Val Ala Val Gln Gly Thr Pro Arg Thr Arg Leu Ala

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Met Ser Ala Asn Thr Asp Phe Ser Thr Pro Ser Gln Ile Pro Phe Ile

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His Ser Asp Ser Arg Gly Asp Ala Ala Val Arg Asp Leu Lys Asn Ala

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Asp Gln Ile Gln Gly Asn Lys His Lys Asp Val Gly Thr Lys Thr Leu

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Asp Val Thr Ser Gly Ala Ser Gly Thr Pro Cys Ser Ser Lys Met Val

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Val Ser Ala Asn His Asn Val His Ser Leu Asn Lys Thr Asn Phe Val

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Glu Asp His Gly Asp Thr Asp Ala Ser Lys Ala Asp Leu Thr Thr Pro

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Ser Arg Glu Ile Leu Pro Ala Asn Val Leu Asp Ser Ser Asn Val Ala

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

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Ala Ala Gly Gln Ser Thr Ile Asn Asp Asn Val Thr Asn Asn Asn Ile

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

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

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

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Asp Ser Ser Ile Ser Thr Ala Arg Lys Gly Asp Lys Ile Ile Ala Asp

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Leu Ala Gly Val Gln Val Leu Lys Gly Gly Glu Asn Ile Gln Asp Glu

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Asn Val Leu Asp Gly Ala Tyr Ser Gln Lys Lys Lys Cys Ser Ile Ser

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Pro Val Leu Phe Lys Val Gln Asn Gly Asp Thr Gly Lys Glu Thr Gly

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Ala Leu Asn Ser Pro Ser Ala Asn Arg Leu Ser Asp Thr Ser Glu Pro

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

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Asp Leu Gly Lys Gln Gln Ser Gly Ser Ser Lys Ser Arg Ser Arg Thr

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Ala Leu Lys His Gly Asn Leu Val Asp Gly Ile Lys Leu Pro Glu Tyr

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

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Leu Ala Thr Ser Leu Ser Ala Thr Val His Asp Val Lys Arg Cys Pro

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Asp Phe Ser Phe Gln Asn Lys Asp Gly Asp Tyr Ala Gln Asp Ser Gly

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Val Leu Ala Lys Val Arg Thr Ser Asp Ile Ser Leu His Ser Ser Arg

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

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Asp Ala Leu Asp Ile Glu Lys Asn Lys Ala Ala Val Ala Ser Asn Cys

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Lys Pro Glu Lys Val Val Pro Asp Glu Asn Ile Lys Ala Asp Gln Leu

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Lys Asp Phe Pro Gly Thr Ser Asn Asn Val Leu Arg Gln Thr Gly Tyr

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Leu Lys Lys Val Ala Ser Arg Lys Gly Met Lys Ala Ser Thr Lys Arg

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

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Lys Thr Lys Thr Val Val Ser Glu Ser Glu Pro Asp Lys Met Ile Ala

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His Lys His Ile Gly Glu Thr Ala Lys Asp Gly Pro Asp Ser Val Asn

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

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Ala Ser Arg Ile Ser Arg Arg Leu Gln Asn Val His Thr Met Lys Asn

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Asp Arg His Ala Ala Ser Asn Leu Glu Ser Ser Lys Met Ile Ser Glu

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Glu Asn Thr Gly Ile Gly Ile Thr Pro Lys Lys Phe Val Ser Asn Ala

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Thr Thr Glu Gly His Gln Thr Asn Ser Pro Lys Met Leu Pro Asn Thr

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Ser Met Arg Asn Thr Phe Ala Lys Arg Ser Arg Val Ser Asp Thr Lys

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Ser Leu Phe Asp Asp Leu Phe Pro Ser Gln Asn Ile Asp His Pro Lys

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Lys Leu Ser Ser Ser Ala Ser Ala Asp Gly Cys Gly Ser Leu Ser Cys

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Ser Val Lys Thr Asp Gly Met Arg Asp Val Ser Gly Leu Phe Ser

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Ser Asp Thr Ser Val Ile Asp Arg Ser Glu Asn Leu Asn Asn Ser

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Lys Leu Arg Cys Ser Lys Arg Asn Lys Ser Leu Ser Leu Asp His

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Glu Lys Glu Asn Met Gln Asp Asn Gly Thr Leu Ser Ser Lys Ser

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

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Gly Asn Gly Pro Gly Thr Leu Ile Thr Pro Glu Pro Thr Cys Phe

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Ile Leu Ser Gly His Arg Gln Gln Arg Lys Asp Tyr Arg Ser Ile

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Leu Arg Cys Leu Lys Ala Arg Val Cys Arg Asp Ser His His Trp

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Ser Tyr Gln Ala Thr His Phe Ile Ala Pro Asp Pro Leu Lys Arg

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Thr Glu Lys Phe Phe Ala Ala Ala Ala Ala Gly Lys Trp Ile Leu

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Lys Thr Asp Tyr Leu Thr Ser Cys Asn Glu Val Gly Lys Leu Leu

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Asp Glu Glu Pro Phe Glu Trp Ser Gly Thr Gly Leu Asn Asp Gly

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

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Gln Gln Met Gly His Gly Ala Phe Tyr Gly Met Gln Ile Ile Val

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Tyr Gly Gln Leu Val Ser Pro Ser Leu Asp Thr Val Lys Arg Ala

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

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

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Ala Met Pro Ser Ala Asp Ala Trp Val Gln Gln Phe Ile Ser His

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Asp Ile Pro Cys Ile Thr Ala Asp Tyr Leu Val Glu Tyr Val Cys

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Lys His Gly His Pro Leu Asp Arg His Val Leu Phe Asn Thr Asn

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Asp Leu Ala Asn Lys Ser Leu Lys Lys Leu Leu Gln Asn Gln Gln

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Glu Val Ala Thr Asp Val Leu Lys Pro Gln Glu Asp Gly Asp Pro

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

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Val Met Leu Ile Cys Gly Asn Glu Asp Gly Ser Thr Gly Cys Gly

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Val Gly Met His Ile Asp Cys Cys Asp Pro Pro Leu Glu Ala Val

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Pro Glu His Asp Trp Leu Cys Pro Gln Cys Glu Met Pro Lys Ala

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Thr Lys Lys Ser Ala Ser Arg Val Ala Ser Lys Ser Arg Val Ser

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Lys Arg Lys Arg

1460

<210> 2

<211> 4389

<212> DNA

<213> Rice (Oryza sativa)

<400> 2

atggcctctc ccatcagcgg cagcgacgac gatgacgagc acctcttcgc cggcgtccgc 60

ttcttcctcg tcgggttcga ccccctctcg gagtcccagt atcggtcgga gatggtgcgg 120

cgcagcggcg tggacgccgg gaggttcggc agcgggtgca cccacgtcat cgtttgcggc 180

ctcgtctatg atgacccggc atgcgtggcg gcgcgggcgg aagggaaaaa ggtcgtcacc 240

gagctttggg tggaagctag cttggatcgg ggaatgcttg ccgatgccga tagggttatg 300

tattggccgg tgcgagattt gaatggaata gcaggcagtg aatcattacg catttgcttg 360

acgggctacc aaagaagtga ccgcgaagat ataatgagca tggtttcttt gatgggagcg 420

caattctcca aatctttgaa tccagacgta gtcactcacc ttatttgcta taaatttgaa 480

ggtgagaagt atgaggctgc taaaaaggcg aagttgaagt tcaactttaa catcaaactt 540

gttaatcacc ggtggttgga agactgctta aagtgttgga aaattcttcc agtagatgat 600

tacagcaaaa gtagttggga actagagata atggaggcgc aagccaagga ctcagaagat 660

gaagaagatg taggccaaag atcatttaga aataagattg tcagatccac tctgaaccca 720

aaaggtagcg cgggaacttc tgccaaccct gttgtgaatg caccaattcg gtcaccgact 780

atttcgagtg gtaatatagg gatggttttg gaaaaacagc cgaacattcc tggagagatc 840

aggaaagcag aagatgccgt caacaggata catgatgtcg cagtccaagg cactcctaga 900

actagattgg caatgtctgc taacactgat tttagcacac ccagtcagat tccatttatt 960

catagtgata gtagagggga tgctgcagta agagatttga aaaatgcaga tcagatccag 1020

gggaataaac acaaagatgt tggcacaaag acacttgatg ttacatccgg tgcatcaggc 1080

actccatgct caagcaagat ggttgtttct gctaaccata acgtgcattc cttaaacaag 1140

acaaattttg tggaggatca tggcgacact gatgctagca aagctgactt gaccacccca 1200

tccagagaaa ttctgcctgc aaatgtactt gattcatcta atgttgccag agggcgaagt 1260

caagaggatt atggagccac atgtactcct gatgcagcag ctggtcagtc aaccattaat 1320

gacaatgtca ccaataataa tattggtttg aagtcaggaa acaatgcttc gctcaacatc 1380

aacaacaaaa gttctttgaa acccttagag aagtcggtac tgcctgaaca atattcagct 1440

aaccgtatag gaccacttca gggggctgat gggagcatga tgagatctga ttctagtatc 1500

tcgacagcac gtaagggaga taaaataatt gctgatctag ctggcgtcca ggttctgaaa 1560

ggtggtgaaa atatacagga tgagaatgta ctggatggtg catattccca aaaaaagaag 1620

tgctccattt ccccagtttt gttcaaagtg caaaatggag atacggggaa agaaactggt 1680

gcgttgaact ctccttctgc aaataggctt agtgacacat ctgaaccagc aatttggtca 1740

tctgtaggaa caaatccaag tgaagctaat aatgttgatt tgggaaagca acagtctggc 1800

tcatctaaat caagatctag gactgcactc aagcatggta atctggttga tggaatcaag 1860

ctccctgaat attcttcaag tgagacaaat gcgcagcctc caccgaaacc aaaagaattg 1920

ctcgccacaa gcctttcagc cactgttcac gatgtaaaga gatgtccaga tttctctttt 1980

caaaacaagg atggtgatta tgcacaagat agtggtaatg cattgaatca agatggctcg 2040

ccactgatgc gcaagacaga aaacgttctt gcaaaggtca gaacatctga catatcttta 2100

cacagttcaa ggaaatcaaa acttgttccc tcttctggaa atggtgatac tgagatgtct 2160

gatgcacttg atattgaaaa aaataaggca gcagtggctt caaattgcaa gcctgagaaa 2220

gtggtacctg atgaaaatat aaaagcagac cagcttaaag actttccagg tacttcaaac 2280

aatgttctgc gccaaacagg ttatttaaag aaagtagcat ctcgtaaagg aatgaaagca 2340

agcacaaaga gacctcgtag tgctagcaag gtggttgatg agccagtagt tgacgatggt 2400

aagactaaga cagtggtttc tgagtcagag cctgataaaa tgattgctca caagcacatt 2460

ggtgaaacag ctaaagatgg ccctgatagt gtaaacgcag cagaacacag aacaaattct 2520

tcagacaaag ttcttacaga tggagcgagt aggatttcca gaagactgca gaatgttcac 2580

accatgaaaa atgatagaca tgcagcttcc aacttggaat ccagtaagat gatttctgag 2640

gaaaatactg gaattgggat aacccctaag aagtttgtga gtaatgcaac tactgaggga 2700

catcaaacaa attcaccgaa aatgttaccc aataccagta tgaggaatac atttgccaag 2760

aggtctcggg tatctgacac caaaatgact ggtgaatcat cagctgataa aactgaaaca 2820

gtggctggca agtcactatt tgatgactta tttccttccc aaaacattga tcaccctaaa 2880

aagctttcaa gtagtgcaag tgctgacggc tgtggatcac tatcctgtaa gaatgcatcg 2940

cctgccagag ttaggaatgc agttgccaag aggaaaataa aagctctaga agacaagtca 3000

gacagcaagc ttggaaaaat tggtggtgcc attgtatctg cagctaaagc tgtggcatca 3060

aggggaattg aagaaagctc atgcaacatc aacaaagtaa gttctgatca aaattctgtg 3120

aagaccgatg gaatgagaga tgtatctgga ttattttcga gtgatacttc tgtgatagac 3180

aggtcagaaa atctgaataa ctctaagttg agatgcagca aaagaaataa atctctttct 3240

ttagaccatg aaaaggagaa tatgcaagac aatggtactc tcagttctaa atccaatggt 3300

agaactgcta ttatgaattc caacttagat gcaaattcga tgaagcacgg tgcaaatatg 3360

ttcaatgagc ccaatagaat aaaaggaaac ggacctggca ccttgatcac gcctgagcct 3420

acatgtttta ttttaagtgg acatcgccaa cagagaaagg actatagatc aatacttcgg 3480

tgcctgaagg cacgagtttg cagggattca catcattggt cataccaagc gacacatttt 3540

attgccccag accctctcaa gagaactgaa aaattctttg cagctgcagc agctggcaag 3600

tggatactga aaactgatta cttgacctct tgcaatgagg ttggcaaatt gttggacgaa 3660

gaaccatttg aatggtctgg tacaggcctt aatgacgggg aaacaatcag ctttgaagct 3720

cccaagaaat ggcgtgtttt gaggcagcag atgggccatg gtgccttcta tgggatgcag 3780

ataattgtat atggacagct cgttagtcca tcgctggata cagtaaagcg tgcagtacga 3840

tctggcgatg gcaccatctt agcaacctcg ccaccgtaca cacggttctt gaactctggc 3900

gtcgacttcg ctgtggtatc ttcggccatg ccaagtgcag acgcatgggt tcaacaattc 3960

atcagtcacg atattccttg tatcactgct gattatcttg ttgagtacgt ctgcaagcat 4020

gggcatccgc tcgacaggca tgttctcttc aacacaaacg atctggccaa caagtccctc 4080

aagaaactac tgcaaaacca gcaagaggtg gccacagatg tactgaagcc acaggaggac 4140

ggagatcccg acgacctgag ctgctcggcg tgcgggagta cggaccgggg cgaggtgatg 4200

ctcatctgcg gcaacgagga tggctcgacc ggctgcggtg taggtatgca catcgactgc 4260

tgcgaccctc ccctggaagc tgtcccagaa catgactggc tgtgccccca gtgcgaaatg 4320

cccaaagcga cgaagaaatc tgcgtcacgc gtagctagca aatcgagagt atccaagcga 4380

aaaaggtga 4389

<210> 3

<211> 7338

<212> DNA

<213> Rice (Oryza sativa)

<400> 3

ggaggaggag aagtagaaat ccccaaatca aagggacaaa ccctagcccc tgctccgatc 60

cagacgcccc ctcgcatcaa tggcctctcc catcagcggc agcgacgacg atgacgagca 120

cctcttcgcc ggcgtccgct tcttcctcgt cgggttcgac cccctctcgg agtcccaggt 180

gagcgcgtag cttctccccc ccggcccgcg agctcggggg tttcgcggtg gctcgcctcg 240

gttgtctctg ggggatgatg ttttggtact tttggtggcg ttctcgcagt atcggtcgga 300

gatggtgcgg cgcagcggcg tggacgccgg gaggttcggc agcgggtgca cccacgtcat 360

cgtttgcggc ctcgtctatg tgagtcgggc ttacggttct gtgtgctgat gtgttggcga 420

cttcgttggg gggccatggt gtgattttgg tttttgggca ggatgacccg gcatgcgtgg 480

cggcgcgggc ggaagggaaa aaggtcgtca ccgagctttg ggtggaagct agcttggatc 540

ggggaatgct tgccgatgcc gatagggtcg gtctttctgc taattgtttg cgtatctttt 600

gctcctattt tcgcatcagc cacaaatttg tttgagtttt tctatagttg ttgtgagata 660

ccggaagatt gtaacttcct gtgatacacc gaaaaattta acctccaatg atgtactgaa 720

aattctagca tggcgtttct gagaatatgg tacagaaaat aaaatggtat ttcaatcgga 780

ctagaaaaac tcaagttgtt taagctcgtt taaaaagggt gcacctttca atgcagtcat 840

atggccgctt ttgtttccat atattgctgg tttaggtttt tgaagcggca ctgcagcact 900

gattgttctt tttccaggtt atgtattggc cggtgcgaga tttgaatgga atagcaggca 960

gtgaatcatt acgcatttgc ttgacgggct accaaagaag tgaccgcgaa gatataatgg 1020

taataaacaa acttgctagt tcaaaattct gctgcatacg gttttacata taatctaccg 1080

ttccttgtgt tttctgatac tattgtttat tatgtgtctt taattgattt gttgtcacac 1140

cattttgaaa ttttctgtcc tgaatcctag aatgttgttt gtggtttctt agcagcgtcc 1200

tagattatct ctgaaaatat ttgatcttac tgcagttgaa atctgagtga gtgtttcctc 1260

aacatgtaac agtaaggtta atatgcttca ggagcagatg gatatctctt tgcttagttg 1320

agtagaaaga actgcgttcc acctattaac aagtaatgaa cagctgcagt aatacattaa 1380

tattatgtaa tgcatgtttc atctatctga catatttgta caaggggcaa tggattgctt 1440

tgggtgtttg catttctttt acctcctagt ttctttagat ctttatagac ataattattg 1500

cttatttttt cctctgtaga aacccctgtt attttgagca tattgtagaa acccctgtta 1560

ttttttcctc agtgcacttg gttctgtacg ctgatgtttg tgtacatatt ttctgtggac 1620

cacatcctta agcaaatcta gagatttata aatgtcattg catttattat atgtacgctg 1680

atgtttgtgt acatattttc tgtggaccac atccttaagc aaatctagag atttataaat 1740

gtcattgcat ttattataga aaaaaatgct ggatataaga ggaatgactc tcatatagaa 1800

atttattaag agagaggtga ggctcgaacc cgggtcggtt agcccacagc ttgtggtgct 1860

agccagaaga cccatgggcc tttctctcat ttaatataga gtgaatggtg ttatatatca 1920

ggagttcaag aaagtatcac gttgcattca tacataatct ttaatattgt tggatttgtg 1980

gttgcccaat tatatttatt tatagtgaag cattcctttt tttctccttt tgtttttgca 2040

gagcatggtt tctttgatgg gagcgcaatt ctccaaatct ttgaatccag acgtagtcac 2100

tcaccttatt tgctataaat ttgaaggttt gttgctactc ttagcagtcc attggtgcta 2160

catatagata aacatacttc acattaaact tgcattgtat tgcttgtttg ttcggccagt 2220

ctttgaccat gtaacttcat aatttctctc aaatttggac catgtaactt caagcgtttt 2280

tttcattttc ttttgtttgt tatttgtgaa ccatgtagaa actaatctca tggtcatggt 2340

gttttttttt tccgtttagg catagtagta tacaaatgaa aaccttcaga aacttttgaa 2400

tgtatatcat aaaaaatgtt ttttgctggt tagaaagcat ccaagaaaat attatttcct 2460

ctagtatgtt gaaaaaatac cagggcatgc ctatcagaag gactgctaac gatcatctct 2520

ttctacacgt atatatttat gatgaaacaa aattttaccg tatctctttt catgccacat 2580

ctcagtttcc aaatgaacta ggtgagaagt atgaggctgc taaaaaggcg aagttgaagt 2640

tcaactttaa catcaaactt gttaatcacc ggtggttgga agactggtat tacacagcgt 2700

cctttcttta ccaacttcag ttgaagtaaa catgttaacc agattattat ataatttaac 2760

tgttcccttt ttgttttttt tttaaaatgg cagcttaaag tgttggaaaa ttcttccagt 2820

agatgattac agcaaaaggt atgtaatccg tgcttgcttc tttgaagatc ttttttgaac 2880

ctgtgttgta ttcctgtgtt gagttctcaa tccactgtaa ctcattgtag ggagtttgct 2940

tgttctagtt tgggaatcta tttatggatt tctcatgtac taatgctgac tatttattct 3000

ttcagtagtt gggaactaga gataatggag gcgcaagcca aggactcaga agatgaagaa 3060

gatgtaggcc aaagatcatt tagaaataag attgtcagat ccactctgaa cccaaaaggt 3120

agcgcgggaa cttctgccaa ccctgttgtg aatgcaccaa ttcggtcacc gactatttcg 3180

agtggtaata tagggatggt tttggaaaaa cagccgaaca ttcctggaga gatcaggaaa 3240

gcagaagatg ccgtcaacag gatacatgat gtcgcagtcc aaggcactcc tagaactaga 3300

ttggcaatgt ctgctaacac tgattttagc acacccagtc agattccatt tattcatagt 3360

gatagtagag gggatgctgc agtaagagat ttgaaaaatg cagatcagat ccaggggaat 3420

aaacacaaag atgttggcac aaagacactt gatgttacat ccggtgcatc aggcactcca 3480

tgctcaagca agatggttgt ttctgctaac cataacgtgc attccttaaa caagacaaat 3540

tttgtggagg atcatggcga cactgatgct agcaaagctg acttgaccac cccatccaga 3600

gaaattctgc ctgcaaatgt acttgattca tctaatgttg ccagagggcg aagtcaagag 3660

gattatggag ccacatgtac tcctgatgca gcagctggtc agtcaaccat taatgacaat 3720

gtcaccaata ataatattgg tttgaagtca ggaaacaatg cttcgctcaa catcaacaac 3780

aaaagttctt tgaaaccctt agagaagtcg gtactgcctg aacaatattc agctaaccgt 3840

ataggaccac ttcagggggc tgatgggagc atgatgagat ctgattctag tatctcgaca 3900

gcacgtaagg gagataaaat aattgctgat ctagctggcg tccaggttct gaaaggtggt 3960

gaaaatatac aggatgagaa tgtactggat ggtgcatatt cccaaaaaaa gaagtgctcc 4020

atttccccag ttttgttcaa agtgcaaaat ggagatacgg ggaaagaaac tggtgcgttg 4080

aactctcctt ctgcaaatag gcttagtgac acatctgaac cagcaatttg gtcatctgta 4140

ggaacaaatc caagtgaagc taataatgtt gatttgggaa agcaacagtc tggctcatct 4200

aaatcaagat ctaggactgc actcaagcat ggtaatctgg ttgatggaat caagctccct 4260

gaatattctt caagtgagac aaatgcgcag cctccaccga aaccaaaaga attgctcgcc 4320

acaagccttt cagccactgt tcacgatgta aagagatgtc cagatttctc ttttcaaaac 4380

aaggatggtg attatgcaca agatagtggt aatgcattga atcaagatgg ctcgccactg 4440

atgcgcaaga cagaaaacgt tcttgcaaag gtcagaacat ctgacatatc tttacacagt 4500

tcaaggaaat caaaacttgt tccctcttct ggaaatggtg atactgagat gtctgatgca 4560

cttgatattg aaaaaaataa ggcagcagtg gcttcaaatt gcaagcctga gaaagtggta 4620

cctgatgaaa atataaaagc agaccagctt aaagactttc caggtacttc aaacaatgtt 4680

ctgcgccaaa caggttattt aaagaaagta gcatctcgta aaggaatgaa agcaagcaca 4740

aagagacctc gtagtgctag caaggtggtt gatgagccag tagttgacga tggtaagact 4800

aagacagtgg tttctgagtc agagcctgat aaaatgattg ctcacaagca cattggtgaa 4860

acagctaaag atggccctga tagtgtaaac gcagcagaac acagaacaaa ttcttcagac 4920

aaagttctta cagatggagc gagtaggatt tccagaagac tgcagaatgt tcacaccatg 4980

aaaaatgata gacatgcagc ttccaacttg gaatccagta agatgatttc tgaggaaaat 5040

actggaattg ggataacccc taagaagttt gtgagtaatg caactactga gggacatcaa 5100

acaaattcac cgaaaatgtt acccaatacc agtatgagga atacatttgc caagaggtct 5160

cgggtatctg acaccaaaat gactggtgaa tcatcagctg ataaaactga aacagtggct 5220

ggcaagtcac tatttgatga cttatttcct tcccaaaaca ttgatcaccc taaaaagctt 5280

tcaagtagtg caagtgctga cggctgtgga tcactatcct gtaagaatgc atcgcctgcc 5340

agagttagga atgcagttgc caagaggaaa ataaaagctc tagaagacaa gtcagacagc 5400

aagcttggaa aaattggtgg tgccattgta tctgcagcta aagctgtggc atcaagggga 5460

attgaagaaa gctcatgcaa catcaacaaa gtaagttctg atcaaaattc tgtgaagacc 5520

gatggaatga gagatgtatc tggattattt tcgagtgata cttctgtgat agacaggtca 5580

gaaaatctga ataactctaa gttgagatgc agcaaaagaa ataaatctct ttctttagac 5640

catgaaaagg agaatatgca agacaatggt actctcagtt ctaaatccaa tggtagaact 5700

gctattatga attccaactt agatgcaaat tcgatgaagc acggtgcaaa tatgttcaat 5760

gagcccaata gaataaaagg aaacggacct ggcaccttga tcacgcctga gcctacatgt 5820

tttattttaa gtggacatcg ccaacagaga aaggactata gatcaatact tcggtgcctg 5880

aaggcacgag tttgcaggga ttcacatcat tggtcatacc aagcgacaca ttttattgcc 5940

ccagaccctc tcaagagaac tgaaaaattc tttgcagctg cagcagctgg caagtaagaa 6000

ctgcaatgtt gattcttaag attttatttt gaagtgggca gctatattag cttattattt 6060

tatatttttt gctacatatg catctggctt atattttatt tacatgttat aatgctacat 6120

gctgatgcac atgactaatg ttcctacttt actcaatagg tggatactga aaactgatta 6180

cttgacctct tgcaatgagg ttggcaaatt gttggacgaa gaaccatttg aatggtctgg 6240

tacaggcctt aatgacgggg aaacaatcag ctttgaagct cccaagaaat ggcgtgtttt 6300

gaggcagcag atgggccatg gtgccttcta tgggatgcag ataattgtat atggacagct 6360

cgttagtcca tcgctggtaa gacttgttag tccaagcaga tcatctgaga gcacaacttc 6420

acctttttct gaaagaaaaa acaataatac attgatgcgc attgtgatcc attctccatt 6480

ttaatctaat tttcaaattt ccttccattc caggatacag taaagcgtgc agtacgatct 6540

ggcgatggca ccatcttagc aacctcgcca ccgtacacac ggttcttgaa ctctggcgtc 6600

gacttcgctg tggtatcttc ggccatgcca agtgcagacg catgggttca acaattcatc 6660

agtcacgata ttccttgtat cactgctgat tatcttgttg agtacgtctg caagcatggg 6720

catccgctcg acaggcatgt tctcttcaac acaaacgatc tggccaacaa gtccctcaag 6780

aaactactgc aaaaccagca agaggtggcc acagatgtac tgaagccaca ggaggacgga 6840

gatcccgacg acctgagctg ctcggcgtgc gggagtacgg accggggcga ggtgatgctc 6900

atctgcggca acgaggatgg ctcgaccggc tgcggtgtag gtatgcacat cgactgctgc 6960

gaccctcccc tggaagctgt cccagaacat gactggctgt gcccccagtg cgaaatgccc 7020

aaagcgacga agaaatctgc gtcacgcgta gctagcaaat cgagagtatc caagcgaaaa 7080

aggtgatttt tctgcctttt ttttctgctt tcaccctgtt ttattgaaca gaataaacgg 7140

tgtgtttatt ttcgtccgtt gtaaagtttt tatcagtggc agagatgctg tctcggcatt 7200

tctgagtgta gttttttctc attctgctgc tatacaggcg tgttttcctc atggcttatg 7260

tgttcaatga ttcgttaaag cggatagcaa gaaatttgtt gattcgtcaa aatactaaaa 7320

gtcggttgat tgatgaat 7338

<210> 4

<211> 23

<212> DNA

<213> Rice (Oryza sativa)

<400> 4

ggtggaagct agcttggatc ggg 23

<210> 5

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

agatggtgga agctagcttg gatcggg 27

<210> 6

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

aaaacccgat ccaagctagc ttccacc 27

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

gcatcaatgg cctctcccat 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

cacgcctgta tagcagcaga 20

<210> 9

<211> 42

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

tgaactatac aaaggcgcgc caatggcctc tcccatcagc gg 42

<210> 10

<211> 41

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

ctctagaact agttaattaa tcaccttttt cgcttggata c 41

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

agcgagagcc tgacctattg 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

ctccatacaa gccaaccacg 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

gctgatgtgt tggcgacttc 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

ctcagaaacg ccatgctaga 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

ctcagaaacg ccatgctaga 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

ctcagaaacg ccatgctaga 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

ctcagaaacg ccatgctaga 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

ctcagaaacg ccatgctaga 20

Claims (7)

1. Use of a protein, characterized in that the use is any of the following:

d1 Application in regulating plant disease resistance or improving plant disease resistance;

d2 Application in preparing products for regulating and controlling plant disease resistance or improving plant disease resistance;

d3 Use in the cultivation of disease-resistant plants;

d4 The application of the plant to the preparation of products for cultivating disease-resistant plants;

the protein is A1) or A2) as follows:

a1 A protein having an amino acid sequence of SEQ ID No. 1;

a2 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A1);

the disease resistance is bacterial blight resistance, the disease resistant plant is bacterial blight resistance, and the plant is rice.

2. Use of a biological material related to the protein as claimed in claim 1, characterized in that: the application is any one of the following:

d1 Application in regulating plant disease resistance or improving plant disease resistance;

d2 Application in preparing products for regulating and controlling plant disease resistance or improving plant disease resistance;

d3 Use in the cultivation of disease-resistant plants;

d4 The application of the plant to the preparation of products for cultivating disease-resistant plants;

the disease resistance is bacterial blight resistance, the disease resistant plant is bacterial blight resistance, and the plant is rice;

the biomaterial is any one of the following B1) to B4):

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3).

3. The use according to claim 2, wherein the nucleic acid molecule is b 1) or b 2) as follows:

b1 A DNA molecule with a coding sequence shown in SEQ ID No. 2;

b2 The nucleotide sequence is a DNA molecule shown as SEQ ID No. 2.

4. A method of growing a disease resistant plant comprising increasing the amount and/or activity of a protein of claim 1 in a plant of interest to obtain a disease resistant plant having a disease resistance higher than that of the plant of interest; the disease resistance is bacterial blight resistance, the disease resistant plant is bacterial blight resistance, and the plant is rice.

5. The method according to claim 4, wherein the increase in the content and/or activity of the protein according to claim 1 in the plant of interest is achieved by increasing the expression level of a gene encoding the protein in the plant of interest.

6. The method according to claim 5, wherein the protein is encoded by the following b 1) or b 2):

b1 A DNA molecule with a coding sequence shown in SEQ ID No. 2;

b2 The nucleotide sequence is a DNA molecule shown as SEQ ID No. 2.

7. Use of the method according to any one of claims 4-6 for creating rice against bacterial leaf blight.