CN111116725B - Application of gene Os11g0682000 and its encoded protein in regulating rice bacterial blight resistance - Google Patents

Abstract

The invention discloses the application of gene Os11g0682000 and its encoded protein in regulating the resistance of rice bacterial blight. The present invention firstly discloses the application of the following proteins in regulating the bacterial blight resistance of plants: A1) a protein whose amino acid sequence is SEQ ID No. 1; A2) an amino acid sequence at the N-terminus or A fusion protein obtained by linking a tag with the C-terminal; A3) The amino acid sequence shown in SEQ ID No. 1 is obtained by substituting and/or deleting and/or adding one or several amino acid residues to the fusion protein shown in A1). Proteins are proteins that are more than 90% identical and have the same function. Further disclosed is a method for cultivating mutant plants with enhanced bacterial blight resistance. The invention provides an efficient breeding method for creating bacterial blight resistant rice based on the gene Os11g0682000, and has important application value.

Description

Gene Os11g0682000 and application of protein coded by same in regulation and control of bacterial leaf blight resistance of rice

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a gene Os11g0682000 and application of a protein coded by the gene in regulation and control of rice bacterial leaf blight resistance.

Background

Bacterial blight caused by gram-negative bacterium Xanthomonas oryzae pathovar oryzae (Xanthomonas oryzae pv. oryzae) is the most serious and wide-ranging bacterial disease in rice production. The rice yield can be reduced by 20-30% in general, can reach 50% in serious cases, and sometimes even can be harvested absolutely. Practice proves that the disease-resistant variety cultivated and planted by utilizing the resistance gene is the most economic and effective measure for preventing and treating the bacterial blight of rice. However, most of the 45 rice bacterial leaf blight resistance genes/loci (http:// www.shigen.nig.ac.jp/rice/oryzae base/gene/list) reported at present show the problems of narrow resistance spectrum or difficult utilization, and only the Xa3, Xa4, Xa21, Xa23 and other genes are widely applied in production.

As the Xanthomonas oryzae rice pathogenic variety is easy to mutate, the co-evolution of rice and Xanthomonas oryzae (Xanthomonas oryzae rice pathogenic variety) leads to the easy loss of variety resistance. Therefore, the disease resistance of rice varieties is improved by identifying and knocking out the bacterial leaf blight susceptibility gene, and the method has important application value for rice disease resistance breeding.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the bacterial leaf blight resistance of rice.

In order to solve the technical problems, the invention firstly provides a protein, named as protein Os11g0682000, which is derived from rice (Oryza sativa L.) and is any one of the following proteins:

A1) protein with an amino acid sequence of SEQ ID No. 1;

A2) a fusion protein obtained by connecting labels at the N end or/and the C end of the amino acid sequence shown in SEQ ID No. 1;

A3) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.1, has more than 90 percent of identity with the protein shown in A1), and has the same function.

Wherein SEQ ID No.1 consists of 624 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

Among the above proteins, protein-tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

The invention also provides application of the protein Os11g0682000 in regulation and control of bacterial leaf blight resistance of plants.

The protein Os11g0682000 related biological material also belongs to the protection scope of the invention, and the invention also provides a new application of the protein Os11g0682000 related biological material.

The protein Os11g0682000 related biological material is applied to regulation and control of bacterial leaf blight resistance of plants, and the related biological material is any one of the following materials:

C1) a nucleic acid molecule encoding the protein Os11g 0682000;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2);

C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector;

C5) a transgenic plant cell line comprising C1) the nucleic acid molecule, or a transgenic plant cell line comprising C2) the expression cassette, or a transgenic plant cell line comprising C3) the recombinant vector;

C6) transgenic plant tissue comprising C1) the nucleic acid molecule, or transgenic plant tissue comprising C2) the expression cassette, or transgenic plant tissue comprising C3) the recombinant vector;

C7) a transgenic plant organ containing C1) said nucleic acid molecule, or a transgenic plant organ containing C2) said expression cassette, or a transgenic plant organ containing C3) said recombinant vector;

C8) a transgenic plant containing C1) the nucleic acid molecule, or a transgenic plant containing C2) the expression cassette, or a transgenic plant containing C3) the recombinant vector;

C9) a tissue culture produced from regenerable cells of the transgenic plant of C8);

C10) protoplasts produced from the tissue culture of C9);

C11) a nucleic acid molecule which disrupts the expression level of the gene of the protein Os11g0682000 and/or inhibits the activity of the protein Os11g0682000 and/or reduces the content of the protein Os11g 0682000;

C12) an expression cassette, a recombinant vector or a recombinant microorganism comprising the nucleic acid molecule according to C11).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above-mentioned related biological material, C1) the nucleic acid molecule is any one of:

B1) DNA molecule shown in SEQ ID No. 2;

B2) the coding sequence is a DNA molecule shown in SEQ ID No. 5;

B3) hybridizes with the DNA molecule defined by B1) or B2) under strict conditions and encodes the protein Os11g 0682000.

Wherein, SEQ ID No.2 consists of 5563 nucleotides, the coding sequence is shown as SEQ ID No.5, and consists of 1875 nucleotides, and encodes the protein shown as SEQ ID No. 1.

The stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS and 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS.

In the above-mentioned related biological materials, the expression cassette described in C2) refers to a DNA molecule capable of expressing the protein Os11g0682000 in a host cell, and the DNA molecule may include not only a promoter for initiating transcription of the Os11g0682000 gene but also a terminator for terminating transcription of the Os11g 0682000. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: the promoter of the Os11g0682000 gene itself, constitutive promoters, tissue-, organ-and development-specific promoters and inducible promoters. PromotersExamples of (c) include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No.5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-Bus3053). they can be used alone or in combination with other plant promoters. all references cited herein are incorporated herein in their entirety suitable transcription terminators include, but are not limited to, the Os11g0682000 gene' S own terminator, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example, Odell et al (I patent 200710099169.7))⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

In the above-mentioned related biological materials, C3) the recombinant vector may contain a DNA molecule for encoding the protein Os11g0682000 as shown in SEQ ID No. 5.

The existing plant expression vector can be used for constructing a recombinant vector containing the protein Os11g0682000 gene or the protein Os11g0682000 gene expression cassette. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector and the like, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA 1391-Xb. When the Os11g0682000 is used for constructing the recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV)35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like can be added in front of the transcription initiation nucleotide, and can be used alone or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In the above-mentioned related biological material, the nucleotide sequence of the nucleic acid molecule of C11) is a DNA fragment, i.e., a target sequence, targeting the nucleic acid molecule of C1). Specifically, the target sequence is positioned on any nucleotide sequence of an Os11g0682000 gene including XXXNGG; wherein XXX is any nucleic acid sequence of 19-20bp in the coding sequence of the protein Os11g0682000, and N is any base of A, T, G, C.

More specifically, the target sequence is SEQ ID No.3 (corresponding to position 753-772 of SEQ ID No. 2) and/or SEQ ID No.4 (corresponding to position 3935-3954 of SEQ ID No. 2).

In the related biological material, C12) the recombinant vector can be a recombinant vector which is prepared by using CRISPR/Cas9 technology and can destroy the expression quantity of the protein Os11g0682000 gene and/or inhibit the activity of the protein Os11g0682000 and/or reduce the content of the protein Os11g 0682000.

In a specific embodiment of the present invention, the recombinant vector comprises any one of the following (i.e., CRISRP/Cas9 gene editing plasmids):

the recombinant vector pYLCRISPR/Cas9Pubi-H-0682000-T1 contains U6a-0682000-sgRNA1 and Cas9 encoding genes, the U6a-0682000-sgRNA1 has a target sequence 0682000-T1 consisting of 20 nucleotides and shown in SEQ ID No.3, and the position corresponding to the Os11g0682000 gene is 753-772 th position of SEQ ID No. 2;

the recombinant vector pYLCRISPR/Cas9Pubi-H-0682000-T2 contains U6b-0682000-sgRNA2 and Cas9 encoding genes, U6b-0682000-sgRNA2 has a target sequence 0682000-T2 consisting of 20 nucleotides and shown in SEQ ID No.4, and the position corresponding to the Os11g0682000 gene is 3935-3954 th of SEQ ID No. 2;

the recombinant vector pYLCRISPR/Cas9Pubi-H-0682000 contains U6a-0682000-sgRNA-1, U6b-0682000-sgRNA-2 and Cas9 encoding genes, U6a-0682000-sgRNA1 has a target sequence 0682000-T1 shown in SEQ ID No.3 consisting of 20 nucleotides, and the position corresponding to the Os11g0682000 gene is 753-772 th position of SEQ ID No. 2; u6b-0682000-sgRNA2 has a target sequence 0682000-T2 shown in SEQ ID No.4 consisting of 20 nucleotides, and the position corresponding to the Os11g0682000 gene is 3935-3954 th of SEQ ID No. 2.

In the related biological material, the recombinant microorganism can be yeast, bacteria, algae and fungi; the bacterium may be Agrobacterium EHA105, for example.

In the above related biological material, the transgenic plant organ may be root, stem, leaf, flower, fruit and seed of the transgenic plant.

In the above related biological materials, the tissue culture may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

In the related biological material, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation materials.

The invention further provides a product for regulating and controlling bacterial blight resistance of plants, which contains the protein Os11g0682000 or related biological materials thereof.

The application of the protein Os11g0682000 or the related biological materials thereof in any one of the following is also within the protection scope of the invention:

D1) the application in cultivating gene mutation plants with enhanced bacterial leaf blight resistance;

D2) the application in preparing and cultivating gene mutation plant products with enhanced bacterial leaf blight resistance;

D3) application in breeding gene mutation plants with reduced bacterial blight resistance;

D4) application in preparing and cultivating gene mutation plant products with reduced bacterial leaf blight resistance;

D5) application in plant breeding.

Among the above applications, the plant breeding application may specifically be to cross a plant in which the expression of the protein Os11g0682000 gene is disrupted and/or the activity of the protein Os11g0682000 is inhibited and/or the content of the protein Os11g0682000 is reduced with another plant to perform plant breeding.

The present invention also provides a method for breeding a genetically mutant plant having enhanced resistance to bacterial blight.

The method for cultivating the gene mutation plant with enhanced bacterial blight resistance comprises the steps of destroying the expression quantity of the protein Os11g0682000 gene in a target plant and/or inhibiting the activity of the protein Os11g0682000 in the target plant and/or reducing the content of the protein Os11g0682000 to obtain the gene mutation plant; the gene mutant plant has a higher bacterial blight resistance than the target plant.

In the method, the method for destroying the expression level of the protein Os11g0682000 gene in the target plant and/or inhibiting the activity of the protein Os11g0682000 in the target plant and/or reducing the content of the protein Os11g0682000 in the target plant is realized by knocking out or inhibiting or changing the protein Os11g0682000 gene in the target plant.

In the invention, the aim of enhancing the bacterial blight resistance can be achieved by using any biotechnology to destroy the expression quantity of the protein Os11g0682000 gene and/or inhibit the activity of the protein Os11g0682000 and/or reduce the content of the protein Os11g 0682000.

In a specific embodiment of the invention, the method for disrupting the expression level of the protein Os11g0682000 gene in the target plant and/or inhibiting the activity of the protein Os11g0682000 in the target plant and/or reducing the content of the protein Os11g0682000 in the target plant is realized by knocking out the protein Os11g0682000 gene in the target plant by using a CRISPR/Cas9 technology. Wherein the protein Os11g0682000 gene is a DNA molecule shown in SEQ ID NO.2 or a DNA molecule shown in SEQ ID NO. 5.

The invention utilizes the CRISRP/Cas9 system to cut the XXXNGG form nucleotide sequence in the rice Os11g0682000 gene at the upstream 3-4bp of NGG to generate DNA double-strand break, thereby introducing the insertion or deletion of the nucleotide sequence, further causing the translation of the gene to be terminated early or the protein conformation to be changed, and finally destroying the biological function of the coding protein of the gene.

In a specific embodiment of the invention, the target sequence in the CRISPR/Cas9 technology is SEQ ID No.3(SEQ ID No.2, position 753-772) and/or SEQ ID No.4(SEQ ID No.2, position 3935-3954), namely the invention selects representative SEQ ID No.3(SEQ ID No.2, position 753-772) and/or SEQ ID No.4(SEQ ID No.2, position 3935-3954) as the target sequence, verifies that the insertion or deletion of the XXXNGG form (XXX is any nucleic acid sequence of 19-20bp in the coding sequence of the protein Os11g0682000, and N is any base in A, T, G, C) can destroy the normal expression of plant genes and show the character of improved blight resistance.

In the method, the CRISPR/Cas9 technology is specifically that CRISRP/Cas9 gene editing plasmid containing a target sequence binding region shown by SEQ ID No.3 (corresponding to the 753-772 site of SEQ ID No. 2) and/or SEQ ID No.4 (corresponding to the 3935-3954 site of SEQ ID No. 2) is firstly constructed; the agrobacterium tumefaciens containing the CRISRP/Cas9 gene editing plasmid is transferred into rice, so that insertion or deletion is carried out at the 753-772 site of SEQ ID No.2 or/and the 3935-3954 site of SEQ ID No.2 in the rice, the biological function of the coding protein of the Os11g0682000 gene of the rice is damaged, the rice shows the character of improving the bacterial leaf blight resistance level, and the experiment proves that: the length of the leaf spot of the bacterial blight IV inoculated to the Os11g0682000 site-directed knockout gene mutation plant is shortened by 42.7 percent, which shows that the rice bacterial leaf blight resistant material is prepared by mutating the nucleotide sequence of the Os11g0682000 gene, and the breeding efficiency of the disease resistant plant based on the Os11g0682000 gene mutation is effectively improved.

In a specific embodiment of the invention, the CRISPR/Cas9 technology comprises any one of the following CRISRP/Cas9 gene editing plasmids:

the recombinant vector pYLCRISPR/Cas9Pubi-H-0682000-T1 contains U6a-0682000-sgRNA1 and Cas9 encoding genes, the U6a-0682000-sgRNA1 has a target sequence 0682000-T1 consisting of 20 nucleotides and shown in SEQ ID No.3, and the position corresponding to the Os11g0682000 gene is 753-772 th position of SEQ ID No. 2;

the recombinant vector pYLCRISPR/Cas9Pubi-H-0682000-T2 contains U6b-0682000-sgRNA2 and Cas9 encoding genes, U6b-0682000-sgRNA2 has a target sequence 0682000-T2 consisting of 20 nucleotides and shown in SEQ ID No.4, and the position corresponding to the Os11g0682000 gene is 3935-3954 th of SEQ ID No. 2;

the recombinant vector pYLCRISPR/Cas9Pubi-H-0682000 contains U6a-0682000-sgRNA-1, U6b-0682000-sgRNA-2 and Cas9 encoding genes, U6a-0682000-sgRNA1 has a target sequence 0682000-T1 shown in SEQ ID No.3 consisting of 20 nucleotides, and the position corresponding to the Os11g0682000 gene is 753-772 th position of SEQ ID No. 2; u6b-0682000-sgRNA2 has a target sequence 0682000-T2 shown in SEQ ID No.4 consisting of 20 nucleotides, and the position corresponding to the Os11g0682000 gene is 3935-3954 th of SEQ ID No. 2.

In the present invention, the plant is M1) or M2) or M3) or M4):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) plants of the genus oryza;

m4) rice.

In the present invention, the rice may be specifically Nipponbare and other rice varieties having the same Os11g0682000 allele as Nipponbare.

In the present invention, the enhancement of bacterial blight resistance is embodied in a reduction in lesion length of plant bacterial blight.

In the present invention, the bacterial leaf blight resistance is resistance to bacterial blight disease IV.

In the invention, the CRISPR/Cas9 technology is a II-type CRISPR system, in particular to a pYLCRISPR/Cas 9-based system.

The invention discovers that the rice gene Os11g0682000 and the coding protein thereof participate in regulating and controlling the immune response of rice to the bacterial blight, the gene Os11g0682000 is targeted and modified by the CRISRP/Cas9 technology, the high-efficiency fixed-point knockout of the gene Os11g0682000 is realized, the biological function of the coding protein of the gene Os11g0682000 is destroyed, the resistance of the rice to the bacterial blight is obviously improved, the breeding efficiency of disease-resistant plants based on the gene Os11g0682000 mutation is effectively improved, the rice material for resisting the bacterial blight can be obtained by mutating the gene Os11g0682000, and the invention has very important application value in agricultural production.

Drawings

FIG. 1 is a sequencing peak diagram of vector activity detection of a rice gene Os11g0682000 site-directed knockout method based on pYLCRISPR/Cas9 technology.

FIG. 2 shows the mutation type of the nucleotide sequence of the Os11g0682000 gene in a Cas9-0682000 homozygous mutant plant under the Japanese sunny background of rice provided in example 2 of the present invention; underlined nucleotide sequences are target sequences and replaced or inserted nucleotide sequences are in black boxes.

FIG. 3 is a statistical chart of lesion phenotype of a mutant plant homozygous for Cas9-0682000 and a wild type Nipponbare inoculated with P.albugineus IV respectively under the Nipponbare background of rice provided in example 2 of the present invention, wherein P is < 0.05.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The sources of materials used in the following examples are as follows:

pYLCRISPR/Cas9 System vectors (including pYLSRgRNA-OsU 6a, pYLsgRNA-OsU6b, and pYLCRISPR/Cas9Pubi-H vectors) non-patent literature describing this material is "Ma X., Zhang Q., Zhu Q., Liu W., Chen Y., Qiu R., Wang B., Yang Z., Li H., Lin Y., Xie Y., Shen R., Chen S., Wang Z., Cheng Z., Chen Y., Guo J., Chen L., Zhao X., Dong Z., and Liu Y. -G. (2015.), A Robust/9 System for CONE I, High efficiency Diffy edition expression vector and plant 1274. plant 1278. After the protection of subtropical agricultural biological resources of the national institute of agricultural science of south China university and the consent of national key laboratory Liu dazzling teachers, the public can obtain the carrier from the institute of agricultural science and crop science.

Non-patent literature describing this material is Yongqing Jiao, Yonghong Wang, Dawei Xue, Jung Wang, Meixian Yan, Guifu Liu, Guojun Dong, Dali Zeng, Zefu Lu, Xudong Zhu, Qian Qian and Jianying Li.Regulation of OsSPL14 by OsmiR156 define ideal plant 544, 2010,42, 541 one.

Agrobacterium tumefaciens EHA 105: purchased from the BioIVector NTCC type culture Collection.

Bacterial blight of rice strain IV: the resistance reaction of IRBB21(Xa21) to 5 races of the bacterial blight of Guangdong rice, Huangshaohua, Wushangzhi, plant protection journal, 2002,29(2):97-100, was obtained from the institute of crop science, Guangdong academy of agricultural sciences, with the consent of the former teacher, Guangdong academy of agricultural sciences.

Agrobacterium tumefaciens EHA 105: purchased from the BioIVector NTCC type culture Collection.

Example 1 Rice gene Os11g0682000 site-specific knockout method based on pYLCRISPR/Cas9 system

Sequence analysis and target sequence screening of rice gene Os11g0682000

The nucleotide sequence of the rice gene Os11g0682000 is shown in SEQ ID No.2, and the amino acid sequence of the coded protein is SEQ ID No. 1. Sequence analysis shows that the gene comprises 2 exons, namely 636-.

According to the invention, a sequence on a first exon of a rice gene Os11g0682000 is a 0682000-T1 target sequence of a rice gene Os11g0682000 site-directed knockout method based on a pYLCRISPR/Cas9 system, and a sequence on a second exon of the rice gene Os11g0682000 is a 0682000-T2 target sequence of the rice gene Os11g0682000 site-directed knockout method based on the pYLCRISPR/Cas9 system.

Through a large number of screens, the pYLCRISPR/Cas9 technology is used for targeting 118 th to 137 th positions (AAGAAGAACTTGTACCGGAT, namely SEQ ID No.3, corresponding to 753-772 th positions of the antisense strand of the first exon of the rice Os11g0682000 gene) as target sequences 0682000-T1, and the 225 th to 244 th positions (CAACATCACCATGATGACTA, namely SEQ ID No.4, corresponding to 3935-3954 th positions of the antisense strand of the second exon) as target sequences 0682000-T2 respectively.

Design of pYLCRISPR/Cas9 system vector primer and construction of recombinant expression vector thereof

1. Design and synthesis of pYLCRISPR/Cas9 technology target sequence primer

Related primers for targeting an Os11g0682000 gene are designed based on a pYLCRISPR/Cas9 technology, wherein the 0682000-T1 target sequence primers 0682000-gRT1 and 0682000-U6aT1 sequences are respectively shown as SEQ ID No.7 and SEQ ID No. 8; 0682000-T2 target sequence primers 0682000-gRT2 and 0682000-U6bT2 have sequences shown in SEQ ID No.9 and SEQ ID No.10, respectively:

0682000-gRT1：5′-AAGAAGAACTTGTACCGGATgttttagagctagaaat-3′(SEQ ID No.7)

0682000-U6aT1：5′-ATCCGGTACAAGTTCTTCTTCggcagccaagccagca-3′(SEQ ID No.8)

0682000-gRT2：5′-CAACATCACCATGATGACTAgttttagagctagaaat-3′(SEQ ID No.9)

0682000-U6bT2：5′-TAGTCATCATGGTGATGTTGCaacacaagcggcagc-3′(SEQ ID No.10)。

relevant primers 0682000-T1 and 0682000-T2, based on pYLCRISPR/Cas9 technology, were synthesized separately.

2. Construction of pYLCRISPR/Cas9 technical recombinant expression vector

1) Construction of recombinant expression vectors containing 0682000-T1 or 0682000-T2 alone

a. The recombinant expression vector pYLCRISPR/Cas9Pubi-H-0682000-T1 contains U6a-0682000-sgRNA1 and Cas9 encoding genes, U6a-0682000-sgRNA1 has a target sequence 0682000-T1 consisting of 20 nucleotides and shown in SEQ ID No.3, and the position corresponding to the Os11g0682000 gene is 753-772 th position of SEQ ID No. 2; the specific construction method of the recombinant expression vector is as follows:

using pYLsgRNA-OsU6a vector as template, using primers UF (5'-CTCCGTTTTACCTGTGGAATCG-3') and 0682000-U6aT1 to perform PCR amplification, and naming the correct sequence as U6aT 1; using pYLsgRNA-OsU6a vector as template, using primers gR-R (5'-CGGAGGAAAATTCCATCCAC-3') and 0682000-gRT1 to perform PCR amplification, and naming the correct sequence as gRT 1; the two fragments were ligated together by means of nested PCR using primers Pps-GGL (5'-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG-3') and Pgs-GGR (5'-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC-3') and designated U6a-0682000-sgRNA 1;

then BsaI enzyme is used for simultaneously carrying out enzyme digestion on U6a-0682000-sgRNA1 and pYRCISPR/Cas 9Pubi-H, and the vector pYRCISPR/Cas 9Pubi-H-0682000-T1 is obtained in a mode of enzyme digestion and connection.

b. The recombinant expression vector pYLCRISPR/Cas9Pubi-H-0682000-T2 contains U6b-0682000-sgRNA2 and Cas9 encoding genes, U6b-0682000-sgRNA2 has a target sequence 0682000-T2 consisting of 20 nucleotides and shown in SEQ ID No.4, and the position corresponding to the Os11g0682000 gene is 3935-3954 th of SEQ ID No. 2; the specific construction method of the recombinant expression vector is as follows:

using pYLsgRNA-OsU6b vector as template, using primers UF (5'-CTCCGTTTTACCTGTGGAATCG-3') and 0682000-U6bT2 to perform PCR amplification, and naming the correct sequence as U6bT 2; using pYLsgRNA-OsU6b vector as template, using primers gR-R (5'-CGGAGGAAAATTCCATCCAC-3') and 0682000-gRT2 to perform PCR amplification, and naming the correct sequence as gRT 2; the two fragments were ligated together by means of nested PCR using primers Pps-GGL (5'-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG-3') and Pgs-GGR (5'-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC-3') and designated U6b-0682000-sgRNA 2;

then BsaI enzyme is used for simultaneously carrying out enzyme digestion on the U6b-0682000-sgRNA2 and the pYLCRISPR/Cas9Pubi-H vector, and the vector pYLCRISPR/Cas9Pubi-H-0682000-T2 is obtained in a mode of enzyme digestion and connection.

2) Construction of recombinant expression vectors containing 0682000-T1 and 0682000-T2

The pYLCRISPR/Cas9Pubi-H-0682000 contains genes encoding U6a-0682000-sgRNA-1, U6b-0682000-sgRNA-2 and Cas9, the U6a-0682000-sgRNA1 has a target sequence 0682000-T1 shown in SEQ ID No.3 consisting of 20 nucleotides, and the position corresponding to the Os11g0682000 gene is 753-772 th position of SEQ ID No. 2; u6b-0682000-sgRNA2 has a target sequence 0682000-T2 shown in SEQ ID No.4 consisting of 20 nucleotides, and the position corresponding to the Os11g0682000 gene is 3935-3954 th of SEQ ID No. 2. The specific construction method comprises the following steps: 1) u6aT1 and gRT1 are connected together by means of nested PCR by using primers Pps-GGL (5'-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG-3') and Pgs-GG2(5 ' -AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3), and are named as U6 a-0682000-sgRNA-1; 2) u6bT2 and gRT2 are connected together by means of nested PCR by using primers Pps-GG2 (5'-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3') and Pgs-GGR (5'-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC-3'), and are named as U6 b-0682000-sgRNA-2; 3) the BsaI enzyme simultaneously carries out enzyme digestion on U6a-0682000-sgRNA-1, U6b-0682000-sgRNA-2 and pYLCRISPR/Cas9Pubi-H, and the vector pYLCRISPR/Cas9Pubi-H-0682000 is obtained in a mode of enzyme digestion and connection.

3. Activity assay of recombinant expression vectors

The recombinant expression vectors pYLCRISPR/Cas9Pubi-H-0682000-T1 and pYLCRISPR/Cas9Pubi-H-0682000-T2 prepared in the above 2 are introduced into protoplasts of Nipponbare (hereinafter referred to as wild type Nipponbare) of rice varieties by PEG mediation respectively (see https:// bio-protocol. org/bio101/e1010125), and the protoplasts transiently transduced with pYLCRISPR/Cas9Pubi-H-0682000-T1 and pYLCRISPR/Cas9Pubi-H-0682000-T1 plasmids are obtained after 16 hours respectively.

Genomic DNAs of protoplasts transiently transduced with pYLCISPR/Cas 9Pubi-H-0682000-T1 and pYLCISPR/Cas 9Pubi-H-0682000-T2 plasmids were extracted, respectively, and partial nucleotide sequences of the Os11g0682000-T1(Os11g0682000-TF 1: 5'-ATCGTCTTCATCGTCCCAAC-3' and Os11g0682000-TR 1: 5'-AGGCTAGGCATGGCATTAGA-3') and Os11g0682000-T2(Os11g0682000-TF 2: 5'-ACAACAATGATCTAGACAAGCCT-3' and Os11g0682000-TR 2: 5'-AAGTGAGTGTTGCAGCCAAG-3') were amplified with specific primers Os11g0682000-T1 of Oryza sativa gene, respectively, and sequencing and verification was performed.

The result is shown in FIG. 1, FIG. 1 is a sequencing peak diagram of vector activity detection of a rice gene Os11g0682000 site-directed knockout method based on pYLCRISPR/Cas9 technology, and it can be seen that pYLCRISPR/Cas9 technology can induce the mutation of the Os11g0682000 gene at target sequences 0682000-T1 and 0682000-T2.

4. Obtaining of recombinant Agrobacterium tumefaciens

And (3) carrying out heat shock transformation on the agrobacterium EHA105 by using the recombinant expression vector pYLCRISPR/Cas9Pubi-H-0682000 obtained in the step (2) to obtain the recombinant agrobacterium containing the recombinant expression vector pYLCRISPR/Cas9Pubi-H-0682000, which is named as EH105-Cas 9-0682000.

Example 2 application of pYLCRISPR/Cas9 technology-based site-specific knockout method in rice variety

pYLCRISPR/Cas9 technology site-directed knockout of Os11g0682000 gene

Infecting the wild type Nipponbare mature embryo induced callus with recombinant agrobacterium EH105-Cas9-0682000, and respectively naming the obtained rice transformation plants as NIP-Cas 9-0682000; the specific method of the experiment is as follows:

1. the recombinant Agrobacterium EH105-Cas9-0682000 obtained in example 1 was inoculated in YEB liquid medium (containing 50. mu.g/ml kanamycin and 20. mu.g/ml rifampicin), and shake-cultured at 28 ℃ and 200rpm to OD600 of 0.6-0.8; centrifuging at 5000rpm and 4 deg.C for 5min, and resuspending thallus precipitate with AAM liquid culture medium (acetosyringone concentration of 200 μ M/L, pH 5.2) to OD600 of 0.6-0.8.

2. Respectively removing glumes of wild type mature seeds of Nipponbare, soaking in 75% ethanol for 1min, then sterilizing in NaClO solution (mixed with water 1:2, and adding 1 drop of Tween 20) for 20min under shaking, and repeating for 2 times. Washing with sterile water for several times until no foreign odor exists, inoculating sterilized wild type Nipponbare seed on NBD2 culture medium to induce callus, culturing in dark at 26 deg.C for 8-10 days, cutting off root and residual endosperm, and subculturing for 10 days to obtain mature embryo callus.

3. And (3) respectively soaking the mature embryo callus obtained in the step (2) in the recombinant agrobacterium tumefaciens resuspension obtained in the step (1), transferring the mature embryo callus after 20-30min, inoculating the mature embryo callus on a co-culture medium (the concentration of the acetosyringone is 100 mu M/L and the pH value is 5.2) containing two layers of filter paper, and co-culturing for 3 days under the dark condition at the temperature of 26 ℃ to obtain the co-cultured callus.

4. Inoculating the callus co-cultured in the step 3 into a screening culture medium (hygromycin concentration is 50mg/L and pH is 5.8), screening and culturing for 12 days at 28 ℃ in the dark to obtain resistant callus, and transferring the resistant callus to a selection culture medium containing 50mg/L Hyg for continuous screening.

5. After repeated screening for 2 times, transferring the resistant callus to a differentiation medium (24 hours of illumination/day) for induced differentiation; when new rootless seedlings are generated, transferring the regenerated seedlings to 1/2MS culture medium for inducing rooting; and after the plantlets are strong, moving the plantlets into an artificial climate chamber for nutrient solution cultivation to obtain regenerated plants NIP-Cas 9-0682000.

6. After the obtained regeneration plant is transplanted to survive, extracting the total DNA of the leaves of the regeneration plant, carrying out PCR amplification on the basis of self primers (5'-GCGGTGTCATCTATGTTACTAG-3' and 5'-CCGACATAGATGCAATAACTTC-3') of a recombinant expression vector pYLCRISPR/Cas9Pubi-H-0682000 to screen a positive transformation plant, and obtaining the plant with a 1225bp band as the positive transformation plant.

The number of the detected regenerated plants, the number of the positive transformed plants and the percentage of the number of the positive transformed plants to the number of the detected regenerated plants are counted, namely the positive rate (%), and the results are shown in table 1, wherein the positive rate reaches 67.7%.

Table 1. result of positive rate detection of pYLCRISPR/Cas9Pubi-H-0682000 transformed rice variety

Regenerated plant	Number of regenerated plants	Number of positive transformed plants	Positive rate (%)
				Nip-Cas9-0682000	65	44	67.7

7. The genome of positive transformed plant is taken as a template, and PCR amplification is carried out by using specific primers Os11g0682000-T1(Os11g0682000-TF 1: 5'-ATCGTCTTCATCGTCCCAAC-3' and Os11g0682000-TR 1: 5'-AGGCTAGGCATGGCATTAGA-3') and Os11g0682000-T2(Os11g0682000-TF 2: 5'-ACAACAATGATCTAGACAAGCCT-3' and Os11g0682000-TR 2: 5'-AAGTGAGTGTTGCAGCCAAG-3') of rice gene Os11g0682000 (fragments of 2 target sequences in the gene Os11g0682000 are respectively amplified). Wild type nipponlily was used as a control.

Sequencing and verifying the obtained 521bp and 519bp amplification products, and comparing the amplification products with wild Nipponbare, wherein the mutation of the amplification products is recorded as the number of the mutant transformation plants; the sequencing verification results are shown in table 2. The number of positive transformed plants, the number of mutated plants and the percentage of the number of mutated plants to the number of positive transformed plants to be detected, i.e., the mutation efficiency (%) were counted, and the results are shown in table 2, which revealed that 34 positive transformed plants had a total of gene Os11g0682000 that had been mutated, and the mutation efficiency reached 77.3%.

Table 2. detection result of pYLCRISPR/Cas9Pubi-H-0682000 induced rice gene Os11g0682000 mutation

Regenerated plant	Number of positive transformed plants	Number of plants transformed by mutation	Efficiency of mutation (%)
				Nip-Cas9-0682000	44	34	77.3

Secondly, the phenotype of the transformed plant of the 0682000 gene mutation is knocked out by pYLCRISPR/Cas9 technology in a fixed point way

Collecting seeds of the 34 mutant transformed plants obtained in the step one, and sowing to obtain T₁And (5) plant generation.

Extraction of T₁The genome DNA of the generation plant is amplified by using rice gene Os11g0682000 specific primers Os11g0682000-T1(Os11g0682000-TF 1: 5'-ATCGTCTTCATCGTCCCAAC-3' and Os11g0682000-TR 1: 5'-AGGCTAGGCATGGCATTAGA-3') and Os11g0682000-T2(Os11g0682000-TF 2: 5'-ACAACAATGATCTAGACAAGCCT-3' and Os11g0682000-TR 2: 5'-AAGTGAGTGTTGCAGCCAAG-3') respectively to obtain PCR products, the PCR products are sent for sequencing, homozygous mutant strains are selected to obtain 1 homozygous mutant type, and 40T mutant types are obtained₁And (5) plant generation.

The mutant forms of the homozygous mutant types compared to wild-type nipponlily are as follows: both target sequences 0682000-T1 and 0682000-T2 are mutated and a base G is inserted between positions 755 and 756 of the target sequence 0682000-T1; the insertion of the base T between position 3937 and position 3938 of the target sequence 0682000-T2 results in a geneOs11g0682000 translation was terminated early. The nucleotide sequence of the homozygous mutation type is schematically shown in figure 2, and the amino acid sequence is shown in SEQ ID NO. 6. The plant with this mutant form was designated as T₁And (3) generation Cas9-0682000 mutant plants.

For wild type Nipponbare, T₁The method for identifying the bacterial blight resistance of the mutant plant (Cas9-0682000) of the generation Cas9-0682000 comprises the following specific steps: adopting leaf-cutting method to treat wild Nipponbare and T in tillering stage₁The mutant plants (Cas9-0682000) of the generation Cas9-0682000 are inoculated with the blight bacteria IV, wild type Nipponbare or T₁The generation Cas9-0682000 mutant plants (Cas9-0682000) were inoculated with 15 independent plants and each plant was inoculated with 3 leaves; after the lesion length is stabilized (generally 14 days after inoculation with B.albicans), the lesion length of each leaf is measured and T is added₁And comparing the average lesion length of the generation Cas9-0682000 mutant plant (Cas9-0682000) with that of the wild Nipponbare to complete the resistance identification of the mutant.

The length of the inoculated leaf lesion is counted, and the result is shown in FIG. 3. after inoculation of the bacterial blight IV, the length of the wild type Japanese fine lesion is 8.21cm, T₁The lesion length of the generation Cas9-0682000 mutant plant is 4.67cm, and the lesion length is obviously shortened by 42.7%.

The results show that the Cas9-0682000 mutant created by the CRISPR/Cas9 technology enhances the disease resistance of rice to the bacterial blight IV.

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

SEQUENCE LISTING

<110> institute of crop science of Chinese academy of agricultural sciences

<120> gene Os11g0682000 and application of protein coded by same in regulation of rice bacterial leaf blight resistance

<130> GNCFY200036

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 624

<212> PRT

<213> Rice (Oryza sativa L.)

<400> 1

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

1 5 10 15

Asn Pro Pro Gly Gly Phe Trp Arg Ser Thr Glu Ala Ala Gly Asp Pro

20 25 30

Val Leu Pro Gly Leu His Pro Ile Arg Tyr Lys Phe Phe Phe Phe Ser

35 40 45

Asn Ala Ile Ser Phe Ile Ala Ser Leu Leu Ala Ile Thr Phe Asn Val

50 55 60

Tyr Tyr Glu Lys Leu Asp Leu Lys Arg Ile Lys Phe Pro Leu Tyr Gly

65 70 75 80

Leu Thr Val Thr Ala Ile Leu Gly Leu Gly Gly Ala Tyr Ala Ala Gly

85 90 95

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

100 105 110

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

115 120 125

Ala Ile Ser Leu Pro Phe Gly Thr Arg Gly Ser Lys Gln Tyr Asn Asn

130 135 140

Asp Leu Asp Lys Pro Cys Glu Leu Ile Gln Leu Leu Ala Ile Leu Ala

145 150 155 160

Ala Ile Val Ala Tyr Gln Ala Gly Ile Asp Pro Pro Gly Gly Val Trp

165 170 175

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

180 185 190

His Pro Arg Arg Tyr Lys Val Phe Phe Tyr Phe Asn Ser Val Ala Phe

195 200 205

Val Ala Ser Leu Val Ile Met Val Met Leu Gln Asn Glu Phe Leu Val

210 215 220

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

225 230 235 240

Ile Gly Ala Tyr Ala Val Gly Ser Cys Arg Asp Thr Ser Thr Ser Ile

245 250 255

Tyr Thr Val Ala Leu Ala Gly Gly Val Leu Ile Tyr Val Val Ile His

260 265 270

Ile Leu Phe Ser Thr Leu Glu Lys Lys Ser Asp Lys Gln Gly Glu Glu

275 280 285

Asp Lys Ile Lys Glu His Gln Leu Glu Lys Lys Arg Glu Leu Leu Leu

290 295 300

Leu Val Glu Ile Leu Ala Ala Thr Leu Thr Tyr Gln Ala Gly Leu Thr

305 310 315 320

Pro Pro Gly Gly Phe Trp Glu Asn Asp Glu Phe Gly His Arg Ala Gly

325 330 335

Phe Pro Val Leu Leu Asp Lys Phe Pro Ile Arg Tyr Lys Ala Phe Phe

340 345 350

Tyr Cys Asn Ala Ala Ser Phe Met Ala Ser Val Ala Leu Ile Ile Leu

355 360 365

Leu Leu Asn Arg Asn Leu Tyr Gly Pro Gly Ile Lys Cys Tyr Ala Leu

370 375 380

Phe Val Cys Met Val Ala Gly Met Phe Gly Leu Ile Asp Ala Tyr Ala

385 390 395 400

Ala Gly Ser Ser Met His Leu Arg Thr Ser Ile Val Val Leu Ile Leu

405 410 415

Val Thr Val Val Phe Ala Ala Val Val Tyr Val Ala Ile Ile Gly Arg

420 425 430

Gly Gln Arg Ala Asn Ile Asn Gln His Gln Ser Lys Gln Thr Gln Asn

435 440 445

Gln Gln Thr Asn Lys Glu Asp Gly Met Met Asp Thr Pro Arg Gln Thr

450 455 460

Gln Asp Gln Gln Glu Ala Asp Met Lys Lys Lys Ala Asp Met Met Ala

465 470 475 480

Lys Tyr Leu Met Leu Ala Gly Ile Leu Ala Ala Ser Val Ala Tyr Leu

485 490 495

Thr Gly Leu Lys Pro Pro Gly Gly Leu Trp Arg Asp Glu Gly Asn Gly

500 505 510

His Ser Ala Gly Asn Pro Val Leu Tyr Asp Ile Asp Lys Arg Arg Tyr

515 520 525

Asn Ala Phe Phe Tyr Ser Asn Ser Thr Ser Phe Met Ala Ser Ile Thr

530 535 540

Val Ile Val Leu Leu Leu Arg Arg Met Thr Lys Gly Asp Glu His Lys

545 550 555 560

Leu Pro Leu Trp Pro Met His Thr Ala Met Leu Leu Asp Met Leu Ala

565 570 575

Leu Leu Gly Ala Tyr Ala Ala Gly Ser Thr Arg Asn Trp Cys Thr Phe

580 585 590

Lys Asp Ala Ile Leu Leu Leu Leu Pro Val Leu Gly Phe Val Val Ile

595 600 605

Leu Phe Phe Trp Lys Lys Gly Gly Glu Glu Lys His Asp Gly Ser Lys

610 615 620

<210> 2

<211> 5563

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 2

gtattattgc tcaaagattc acctcgatcg atcacccgat caacagtagc agatcaatgg 60

cggctccggc ggtggccggt ggaagtagcc agcaaaacct cacttcttcc agcggcagca 120

gcgacgacaa gcagccgtcg tcgtcgtcgc caatggagtg ccagctgaag aagtacctgg 180

tgctgctggc caccctggtg gcgacggtga cctacgcggc ggggctgaac ccgccgggtg 240

gctcgtggct ggaggacggc ggcggcggcg gccggtggca gctggccggc gacgccatcc 300

tacaggacac caactactgg cgctacatcg tcttctactg gttcaacgcc atctccttcg 360

cggcgtcgct ggtggtggtc agcctcctct tcctcctcct gcacaagggc cggccaccac 420

cagcaccagg ggaccaagct gctcacgctc acccgggcgg tgatggtggt cgacctgctc 480

gccctcatgg gcgcctacgc cgccgggacc agccatgaca agttcaccac catcggcgcc 540

gccgcgctcg tctccgccac cgccgcctat gtcatcgtct tcatcgtccc aaccctcacg 600

gcgaaaacga caccgccgcc gccgtcggga tcctgatgtt gatcgccatc ttcgtggcga 660

ccgacgccta tgtagctggg ctcaacccac ccggcggctt ctggcggagc accgaggccg 720

ccggcgaccc ggtgctgccg ggcctccatc caatccggta caagttcttc ttcttctcca 780

acgccatctc cttcatcgcg tccctgctcg ccatcacgtt caacgtgtac tacgagaagc 840

ttgacctcaa gcgcatcaag ttcccgctgt acgggctcac cgtcacggcg atcttgggac 900

tcggcggcgc ctacgccgcc gggagctgca gggacagcag gcacaccggc tatgttcttg 960

gcctgattgt cccggttctt gtctgcatct ttctccagtg gtttcttgta ggatctaatg 1020

ccatcagtct cccttttggt accaggtacg cagtattcat cttcatcatg catttctaat 1080

gccatgccta gccttttttt aatttttttt atcttttctt gttacttttt taaaaaaaag 1140

tcacaaccgc gcgctccaaa cctgaattgc attcaaataa tttaacaaaa gtctgaaagc 1200

aaaacttctt tgtccttaat aaaaatcatt atggtatgat tagattcata tttcgaaaca 1260

ttttccaatg atgtcaattt caagatcatg agcaatataa tataaaatac aatggtcaaa 1320

gtgtaggata tataaaacca cattaggtca aaaacataca taccctacat ttttccttgc 1380

ttcatgtagt acatttatat gcaggagaga gtccaaatat caccctaaac tttgattgaa 1440

acaccaggag agaaatcacc tttaatatca gttgaaaacc gccattaatc ccgattatag 1500

caaccgtgac aaaaaatcga gactaaagat ctcgatctga accggtacta aagatgtata 1560

cccattaatt ccagttagca accgtgacaa aaaaaaatca agactaaaga accggtacta 1620

acgatgcaaa ccaagtcaaa aaaaaaatat gtgggatgtg ggaatcaaac tcaagatatc 1680

tcacttcagc cctcacgcgt gttacgatcc cacctacctt acacatttga cttgaataga 1740

gatgatttcc ttttgaacta actcaggagg catctttagt aaaaatatct tgaatatggg 1800

ttggtgttac ctactagtac taaaaataca tctttagtac cggttgataa tatcaacggg 1860

actaaagatc catcagcatt aatctcgatt ggtgctaaca ccggtactac agatgtattt 1920

ttaataccgg ttgataacac caaccggtac taaaaatgtt tctagggact tgggctttta 1980

gaaccggtac taaagagatt tagtaacgct tcttaatcca tccgaggtat ttgtatgttt 2040

ggggcattgg ataaaagatg agttctctag tagtaaaagt ctatctagca ccctaaactt 2100

cgaaaacgga catacaactc tctgaacttt atagtactat ttatataacc cgataagatg 2160

tttttaatct aatttgtata taagttgaat gagtttgatc acgtgacata tacattagat 2220

atacatgcta agatcttcac ttaaatatcc cttctttaat ttgtaccctt tattcaatct 2280

cacttacaaa atgagtatca acataataat aatatggcac cactatataa tataaattga 2340

tgtcctacta actaatcata tttaaatatg tatattagtt attcaaaacc actcgtttaa 2400

gccttcaaat atgcaaaaac accatccaaa accaccttag agagtaatat acatggtatt 2460

acaaagttta ggagaatggg tgtttgattt taaagttcaa ggtgcttgac ggatttccat 2520

ccaaagttta ggggggtatt tgaacttttt ccttatgtaa agatatatgg aaaacgatca 2580

tagatttata ttgtgtattg taggaaatga ttttacttag aaatatttga aaactgtaaa 2640

actataataa acgagcacat gctcactgag aaccattgat aggtaaagaa actggtacgg 2700

taaaagaaaa aaaaggtcca tggactcaat ggtgtcttgc tcccaaaagt atgtagacat 2760

gagccttaca aaatgagcct atcactagaa ctatttcact attaacatct atactttatt 2820

gtaccagttt tgcttgaaca tgatcataga tccacaaagc acatgctcat cgagaaccat 2880

tgatctaggc gacatgtttg ttctcccttc gaaaggctga tagcaaccga gcacgcatag 2940

cgttagtctt gtcctcgagt ctatgggttg gagtcgccaa cttgcagcgg agtgcttttt 3000

tttctttttc ctgatcatag tctcttaggg ctataatctt ataatttttt tcctgctagt 3060

ccatgccact gcacccaatg accttgcttc caatccggac tgctcatcag ctcaagctct 3120

tcacaccttg atgtgttcat aatgcccaac tacttgtgct ccacatcatt aaaatgctat 3180

cagtcgcctg ccaattttat tcccttgttt ttagcctcga cccgggcatg aggaaattct 3240

agcttgctcc atcgctgctt atcatcattg aaaaaaaata tttctattca tgctttctac 3300

ttccatgaag atagctctac gatcatccat gccgctggca tgggcataga aaggaccagt 3360

actagggggg ctgtagcatt tctaatagtt tgagtacata tcaattaaga ggaaggaaaa 3420

gaccttcgtg cccctcaatc aagacctagg gagatgtagt gattactgat tggagccatt 3480

taaggccaca tattgggcta accgaacaaa aattaatttg tttcacattg ttaactatca 3540

tttcaatcat gatgacatgg gggaccgggg gggggattga ttcggtgaga atttcttgca 3600

tttttgtaca cattttttga gtgctagaaa ttcttaattc cacatcatca atttcgagta 3660

ccaaaagaat tattctagac tgcaattgaa ctatctattt aattgtacag gggaagtaaa 3720

caatacaaca atgatctaga caagccttgt gaattaattc agctccttgc cattcttgcg 3780

gcgatcgttg cttaccaagc aggaatagat ccacctggag gcgtctgggc agacaatggg 3840

gctagccaca gtggagaccc aatactcctg acaacacacc ctaggcggta caaggtcttc 3900

ttctatttta actcggtggc ttttgtggca tccctagtca tcatggtgat gttgcagaat 3960

gaatttctag ttagaagcca tgtactggag gcagccatga tacttgattt gttttgcctc 4020

ataggcgcgt acgctgtcgg aagttgcagg gacacaagca cttccattta cacagttgcc 4080

ttggctggtg gtgtccttat ctatgtggtg attcacattc tcttctccac actagaaaag 4140

aagtcggaca agcagggtga ggaagacaag ataaaggagc atcagttgga gaagaagcgt 4200

gagttgttgc tgcttgttga aatcttggct gcaacactca cttaccaagc cggcttgacc 4260

ccaccgggtg gcttctggga aaatgatgag tttgggcacc gtgcaggctt ccctgtccta 4320

ctagacaagt tccctatccg ttacaaggca tttttttatt gcaatgcagc aagcttcatg 4380

gcatccgtgg ctctcatcat tctccttctc aaccgaaatc tgtacgggcc gggcataaag 4440

tgctatgcgc tctttgtctg catggtagcg ggcatgttcg gcctcatcga tgcctatgct 4500

gctggaagtt cgatgcatct gcgaacctcc atcgttgtct taatattggt tacggtagtt 4560

tttgcagctg tagtctatgt ggcaatcatc ggtcgtggac aacgtgcaaa cataaatcaa 4620

catcagtcaa aacaaacaca aaatcaacaa accaacaagg aagatggcat gatggacaca 4680

ccaagacaaa cacaagatca acaggaagct gacatgaaaa agaaagccga catgatggcc 4740

aagtacttaa tgctagcagg aatattggct gcgagtgtgg cctacctcac gggcctaaaa 4800

ccacctggtg gcctgtggag ggatgaaggt aatgggcact ctgctggcaa cccggtcctc 4860

tacgacatcg acaagcgtcg gtacaatgct tttttctata gcaactccac ttccttcatg 4920

gcatccatca ctgtcattgt tttgctgctt cgaaggatga ccaagggcga tgaacataaa 4980

ttgcctcttt ggccaatgca cacagccatg ttgctggaca tgcttgccct cctgggagcc 5040

tacgcggcag gcagtactcg gaattggtgc acattcaagg atgccatcct gcttctctta 5100

cctgtgttgg gctttgttgt tattcttttc ttctggaaga aaggaggtga agaaaaacat 5160

gatggctcca aatagaatcc tactatcaag tctggccctg aacatgctta aaactgatct 5220

agtgtcgaat gtctcatatg acgttcttat atagcggcgg attaattaca caggtccata 5280

ttttcaagaa tatatacaat agctgctttt gcttgattta agctttctga agttggagtc 5340

caaggtgtgt gttgagatgt cgagttgttt gctcccatat gtattctttg ttggtctata 5400

agagaaaact gaataaaaga ctttctagat gttattatat ggagccaaag tcacatatcg 5460

agtcgtctat agatctcccc atactgtaaa tggtatattt catgggtatg tatgtcaata 5520

atgaagaatc cttgtactac caagtcacaa ataaatggaa ttt 5563

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aagaagaact tgtaccggat 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caacatcacc atgatgacta 20

<210> 5

<211> 1875

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgttgatcg ccatcttcgt ggcgaccgac gcctatgtag ctgggctcaa cccacccggc 60

ggcttctggc ggagcaccga ggccgccggc gacccggtgc tgccgggcct ccatccaatc 120

cggtacaagt tcttcttctt ctccaacgcc atctccttca tcgcgtccct gctcgccatc 180

acgttcaacg tgtactacga gaagcttgac ctcaagcgca tcaagttccc gctgtacggg 240

ctcaccgtca cggcgatctt gggactcggc ggcgcctacg ccgccgggag ctgcagggac 300

agcaggcaca ccggctatgt tcttggcctg attgtcccgg ttcttgtctg catctttctc 360

cagtggtttc ttgtaggatc taatgccatc agtctccctt ttggtaccag gggaagtaaa 420

caatacaaca atgatctaga caagccttgt gaattaattc agctccttgc cattcttgcg 480

gcgatcgttg cttaccaagc aggaatagat ccacctggag gcgtctgggc agacaatggg 540

gctagccaca gtggagaccc aatactcctg acaacacacc ctaggcggta caaggtcttc 600

ttctatttta actcggtggc ttttgtggca tccctagtca tcatggtgat gttgcagaat 660

gaatttctag ttagaagcca tgtactggag gcagccatga tacttgattt gttttgcctc 720

ataggcgcgt acgctgtcgg aagttgcagg gacacaagca cttccattta cacagttgcc 780

ttggctggtg gtgtccttat ctatgtggtg attcacattc tcttctccac actagaaaag 840

aagtcggaca agcagggtga ggaagacaag ataaaggagc atcagttgga gaagaagcgt 900

gagttgttgc tgcttgttga aatcttggct gcaacactca cttaccaagc cggcttgacc 960

ccaccgggtg gcttctggga aaatgatgag tttgggcacc gtgcaggctt ccctgtccta 1020

ctagacaagt tccctatccg ttacaaggca tttttttatt gcaatgcagc aagcttcatg 1080

gcatccgtgg ctctcatcat tctccttctc aaccgaaatc tgtacgggcc gggcataaag 1140

tgctatgcgc tctttgtctg catggtagcg ggcatgttcg gcctcatcga tgcctatgct 1200

gctggaagtt cgatgcatct gcgaacctcc atcgttgtct taatattggt tacggtagtt 1260

tttgcagctg tagtctatgt ggcaatcatc ggtcgtggac aacgtgcaaa cataaatcaa 1320

catcagtcaa aacaaacaca aaatcaacaa accaacaagg aagatggcat gatggacaca 1380

ccaagacaaa cacaagatca acaggaagct gacatgaaaa agaaagccga catgatggcc 1440

aagtacttaa tgctagcagg aatattggct gcgagtgtgg cctacctcac gggcctaaaa 1500

ccacctggtg gcctgtggag ggatgaaggt aatgggcact ctgctggcaa cccggtcctc 1560

tacgacatcg acaagcgtcg gtacaatgct tttttctata gcaactccac ttccttcatg 1620

gcatccatca ctgtcattgt tttgctgctt cgaaggatga ccaagggcga tgaacataaa 1680

ttgcctcttt ggccaatgca cacagccatg ttgctggaca tgcttgccct cctgggagcc 1740

tacgcggcag gcagtactcg gaattggtgc acattcaagg atgccatcct gcttctctta 1800

cctgtgttgg gctttgttgt tattcttttc ttctggaaga aaggaggtga agaaaaacat 1860

gatggctcca aatag 1875

<210> 6

<211> 69

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

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

1 5 10 15

Asn Pro Pro Gly Gly Phe Trp Arg Ser Thr Glu Ala Ala Gly Asp Pro

20 25 30

Val Leu Pro Gly Leu His Pro Ile Ala Val Gln Val Leu Leu Leu Leu

35 40 45

Gln Arg His Leu Leu His Arg Val Pro Ala Arg His His Val Gln Arg

50 55 60

Val Leu Arg Glu Ala

65

<210> 7

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aagaagaact tgtaccggat gttttagagc tagaaat 37

<210> 8

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atccggtaca agttcttctt cggcagccaa gccagca 37

<210> 9

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caacatcacc atgatgacta gttttagagc tagaaat 37

<210> 10

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tagtcatcat ggtgatgttg caacacaagc ggcagc 36

Claims (7)

1. Application of a protein-related biological material in enhancing rice bacterial blight resistance; The protein is the following A1) or A2): A1) The protein whose amino acid sequence is SEQ ID No.1; A2) A fusion protein obtained by linking a tag at the N-terminus or/and C-terminus of the amino acid sequence shown in SEQ ID No.1; The relevant biological material is any of the following: C11) a nucleic acid molecule that disrupts the expression of the gene of the protein and/or reduces the content of the protein; C12) An expression cassette, recombinant vector or recombinant microorganism comprising the nucleic acid molecule of C11). 2. application according to claim 1, is characterized in that: described C11) described gene is as shown in any of the following: B1) DNA molecule shown in SEQ ID No.2; B2) The coding sequence is the DNA molecule shown in SEQ ID No.5. 3. Use of the relevant biological material described in claim 1 or 2 in any of the following: D1) application in breeding genetic mutant rice with enhanced bacterial blight resistance; D2) Application in the preparation of breeding mutant rice products with enhanced bacterial blight resistance. 4. A method for cultivating mutant rice with enhanced bacterial blight resistance, characterized in that: the method comprises destroying the expression of the gene of the protein described in claim 1 in the target rice and/or reducing the expression in claim 1 According to the protein content, gene mutant rice is obtained; the bacterial blight resistance of the gene mutant rice is stronger than that of the target rice. 5. The method according to claim 4, wherein the method for destroying the expression of the gene of the protein described in claim 1 in the target rice and/or reducing the content of the protein in claim 1 is by This is achieved by knocking out or inhibiting the gene of the protein of claim 1 in the target rice. 6. The method according to claim 4, wherein the method for destroying the expression of the gene of the protein described in claim 1 in the target rice and/or reducing the content of the protein described in claim 1 is to use CRISPR It is achieved by knocking out the gene of the protein in claim 1 in the target rice using the /Cas9 technology. 7. The method according to claim 6, wherein the target sequence in the CRISPR/Cas9 technology is SEQ ID NO.3 or SEQ ID NO.4.

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