CN110791487B - Rice receptor kinase gene LOC _ Os11g47290, and coding protein and application thereof - Google Patents

Rice receptor kinase gene LOC _ Os11g47290, and coding protein and application thereof Download PDF

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CN110791487B
CN110791487B CN201911164723.4A CN201911164723A CN110791487B CN 110791487 B CN110791487 B CN 110791487B CN 201911164723 A CN201911164723 A CN 201911164723A CN 110791487 B CN110791487 B CN 110791487B
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翟文学
周永力
李全林
史晓荣
卢家玲
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Institute of Genetics and Developmental Biology of CAS
Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
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Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
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Abstract

The invention discloses a rice receptor kinase gene LOC _ Os11g47290, and a coding protein and application thereof. The invention finds the application of the rice receptor kinase gene LOC _ Os11g47290 and the coding protein thereof in regulating and controlling the disease resistance of plants, and the resistance of rice to bacterial blight can be obviously improved by destroying the biological function of the coding protein of the LOC _ Os11g47290 gene. The invention realizes efficient LOC _ Os11g47290 gene site-directed knockout by using CRISPR/Cas9 technology, and the lesion length is obviously shortened after rice is subjected to site-directed knockout and inoculated with Xanthomonas oryzae GD1358 and V. The new function of the LOC _ Os11g47290 gene provided by the invention provides a new method for disease-resistant breeding of plants, and has very important application value in agricultural production.

Description

Rice receptor kinase gene LOC _ Os11g47290, and coding protein and application thereof
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a rice receptor kinase gene LOC _ Os11g47290, and a coding protein and application thereof.
Background
Bacterial blight caused by Xanthomonas oryzae pv. oryzae is an important bacterial disease restricting rice production, and has serious harm to rice planting industry, and can generally reduce the yield of rice by about 20-30% and seriously reach 50%. The most economic and effective measure for preventing and treating the bacterial blight of rice is to culture and plant disease-resistant varieties by using the resistance genes. However, most of the currently reported 44 rice bacterial leaf blight resistance genes/loci (http:// www.shigen.nig.ac.jp/rice/oryzae base/gene/list) show the problems of narrow resistance spectrum or difficult utilization, and only the genes Xa3, Xa4, Xa21, Xa23 and the like are widely applied in production.
Since Xanthomonas oryzae rice pathogenic varieties are easy to mutate, the resistance of varieties is easy to lose due to the co-evolution of rice and Xanthomonas oryzae (Xanthomonas oryzae rice pathogenic varieties). 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
In order to solve the problems in the prior art, the invention aims to provide rice receptor kinase LOC _ Os11g47290 and application of a protein coded by the same in regulation and control of plant disease resistance.
The invention provides application of any one of the following substances A-C in regulation and control of plant disease resistance;
A. LOC _ Os11g47290 protein;
B. a nucleic acid encoding the LOC _ Os11g47290 protein;
C. an expression cassette, a recombinant vector or a recombinant microorganism comprising said nucleic acid.
The invention also provides application of substances shown as b1 or b2 in improving the disease resistance of plants: b1, substances that inhibit or reduce the activity or content of LOC _ Os11g47290 protein in plants; b2, a substance that inhibits or reduces expression in a plant of a nucleic acid encoding a LOC _ Os11g47290 protein.
In the above use, the LOC _ Os11g47290 protein is any one of (a1) to (a4) below:
(a1) Protein shown as a sequence 1 in a sequence table;
(a2) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);
(a3) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the (a1) and is related to plant disease resistance;
(a4) and (b) a protein which has 98% or more identity to (a1) and is involved in plant disease resistance.
In the above application, the nucleic acid encoding LOC _ Os11g47290 protein is any one of the following (b1) - (b 3):
(b1) the coding region is a DNA molecule shown in a sequence 2 in a sequence table;
(b2) a DNA molecule having 95% or more identity to (b1) and encoding said protein;
(b3) a DNA molecule which hybridizes with the nucleotide sequence defined in any one of (b1) or (b2) under stringent conditions and encodes the protein.
The amino acid sequence shown as the sequence 1 is a coding protein sequence of the rice LOC _ Os11g47290 gene, and a person skilled in the art can substitute, delete and/or add one or more amino acids according to the amino acid sequence disclosed by the invention, conservative substitution of the amino acids and other conventional technical means in the field without influencing the activity of the amino acid sequence, so as to obtain a mutant with the same activity as the coding protein of the rice LOC _ Os11g47290 gene disclosed by the invention.
The nucleotide sequence shown in the sequence 2 is the nucleotide sequence of the rice LOC _ Os11g47290 gene. The rice LOC _ Os11g47290 gene provided by the invention can be any nucleotide sequence capable of coding the coding protein of the rice LOC _ Os11g47290 gene. Considering the degeneracy of codons and the preference of codons of different species, the skilled person can use codons suitable for the expression of a particular species as required.
In the application, the disease resistance is bacterial blight resistance.
In the above application, the substance represented by b1 or b2 is LOC _ Os11g47290 protein or an inhibitor of a nucleic acid encoded by LOC _ Os11g47290 protein, and the inhibitor can be a nucleic acid, and can also exist in the form of an expression cassette, a vector, a recombinant bacterium or a host cell containing the nucleic acid; the inhibition factors are specifically as follows:
1) interfering RNA;
2) CRISPR/Cas9 system;
in the CRISPR/Cas9 system, the target sequence of the sgRNA is a nucleotide sequence in a form of XXXGG in the nucleic acid encoding the LOC _ Os11g47290 protein, wherein XXX is a nucleic acid sequence of any 19-20bp in the nucleic acid encoding the LOC _ Os11g47290 protein, and N is any one base in A, T, G, C.
The CRISRP/Cas9 system can cut the XXXNGG form nucleotide sequence in the rice LOC _ Os11g47290 gene at the upstream 3-4bp of NGG to generate DNA double-strand break, thereby introducing the insertion deletion of the nucleotide sequence, further causing the translation of the gene to terminate in advance or the protein conformation to change, and finally destroying the biological function of the coding protein of the gene; wherein XXXNGG is nucleotide sequence, wherein XXX is nucleic acid sequence 19-20bp, N is any one base of A, T, G, C.
Preferably, XXX is a nucleic acid sequence of any 19-20bp in the first exon in a nucleic acid (genome) encoding a LOC _ Os11g47290 protein; in embodiments of the invention, more preferably, the sgRNA is sgRNA1 and/or sgRNA 2; the target sequence of the sgRNA1 is sequence 3 (from 367 th to 386 th of the rice LOC _ Os11g47290 gene); the target sequence of the sgRNA2 is sequence 4 (from 952 th to 971 th of the rice LOC _ Os11g47290 gene).
In embodiments of the invention, the pYLCRISPR/Cas9 system includes any one of the following recombinant vectors (CRISRP/Cas9 gene editing plasmid):
the recombinant expression vector pYLCRISPR/Cas9Pubi-H-47290-T1 contains genes encoding U6a-47290-sgRNA1 and Cas9, U6a-47290-sgRNA1 has a target sequence region consisting of 20 nucleotides, and the corresponding target sequence of the target sequence region on LOC _ Os11g47290 gene is 47290-T1 shown in sequence 3; the specific construction method of the recombinant expression vector is shown in the embodiment;
the recombinant expression vector pYLCRISPR/Cas9Pubi-H-47290-T2 contains genes encoding U6b-47290-sgRNA2 and Cas9, U6b-47290-sgRNA2 has a target sequence region consisting of 20 nucleotides, and the corresponding target sequence of the target sequence region on LOC _ Os11g47290 gene is 47290-T2 shown in sequence 4; the specific construction method of the recombinant expression vector is shown in the examples.
The recombinant expression vector pYLCRISPR/Cas9Pubi-H-47290 contains U6a-47290-sgRNA-1, U6b-47290-sgRNA-2 and Cas9 encoding genes, U6a-47290-sgRNA-1 has a target sequence region consisting of 20 nucleotides, and a target sequence corresponding to the target sequence region on LOC _ Os11g47290 gene is 47290-T1 shown in sequence 3; u6b-47290-sgRNA-2 has a target sequence binding region consisting of 20 nucleotides, and the target sequence region corresponds to a target sequence shown as sequence 4 in sequence 47290-T2 on LOC _ Os11g47290 gene; the specific construction method of the recombinant expression vector is shown in the examples.
In the application, the genetic breeding is to construct a transgenic plant resistant to bacterial blight.
The improvement of the disease resistance can be expressed as a reduction in the length of lesion of bacterial blight of plants.
Another object of the present invention is to provide a method for improving disease resistance of plants, which is any one of the following methods 1) to 3), and the method can improve the disease resistance of plants;
1) the method comprises the following steps: inhibiting or reducing the activity or content of LOC _ Os11g47290 protein in a target plant;
2) the method comprises the following steps: inhibiting or reducing expression of a nucleic acid encoding a LOC _ Os11g47290 protein in a plant of interest;
3) the method comprises the following steps: performing gene editing on a nucleic acid encoding the LOC _ Os11g47290 protein in a target plant;
The invention also provides a method for preparing the transgenic plant with high disease resistance, which is any one of the following methods 1) to 3),
1) the method comprises the following steps: inhibiting or reducing the activity or content of LOC _ Os11g47290 protein in a target plant to obtain a transgenic plant;
2) the method comprises the following steps: inhibiting or reducing the expression of LOC _ Os11g47290 protein coding nucleic acid in a target plant to obtain a transgenic plant;
3) the method comprises the following steps: carrying out gene editing on LOC _ Os11g47290 protein coding nucleic acid in a target plant to obtain a transgenic plant;
the disease resistance in the transgenic plant is higher than that of the target plant;
the target plant is a wild-type plant or a recipient plant.
The method also comprises the following steps: the plant with the edited LOC _ Os11g47290 protein coding nucleic acid is selected from the transgenic plant or the plant after gene editing, and is the transgenic plant with high disease resistance.
The method for selecting a plant with an edited LOC _ Os11g47290 protein-encoding nucleic acid from a transgenic plant or a gene-edited plant comprises 1) directly amplifying a vector fragment containing a nucleic acid inhibitor encoding the LOC _ Os11g47290 protein; 2) and amplifying and sequencing the edited genome segment containing the LOC _ Os11g47290 protein coding nucleic acid target sequence in the plant.
In the embodiment of the invention, the amplification primers used in the amplified and sequenced edited genome segment containing the LOC _ Os11g47290 protein coding nucleic acid target sequence in the plant are specifically a primer consisting of a single-stranded DNA molecule shown in a sequence 11 and a single-stranded DNA molecule shown in a sequence 12.
In the above method, the disease resistance is resistance to bacterial blight.
In the above method, the inhibiting or reducing the activity or content of LOC _ Os11g47290 protein in the plant, or the inhibiting or reducing the expression of a nucleic acid encoding LOC _ Os11g47290 protein in the plant is effected by gene editing of a nucleic acid encoding LOC _ Os11g47290 protein.
In the method, the gene editing is realized by means of a CRISPR/Cas9 system;
in the CRISPR/Cas9 system, the target sequence of the sgRNA is a nucleotide sequence in a form of XXXGG in the nucleic acid for coding the LOC _ Os11g47290 protein, wherein XXX is a nucleic acid sequence of any 19-20bp in the nucleic acid for coding the LOC _ Os11g47290 protein, and N is any one base in A, T, G, C;
preferably, XXX is a nucleic acid sequence of any 19-20bp in the first exon in a nucleic acid (genome) encoding a LOC _ Os11g47290 protein; in embodiments of the invention, more preferably, the sgRNA is sgRNA1 and/or sgRNA 2; the target sequence of the sgRNA1 is sequence 3 (from 367 th to 386 th of the rice LOC _ Os11g47290 gene); the target sequence of the sgRNA2 is sequence 4 (from 952 th to 971 th of the rice LOC _ Os11g47290 gene).
In the method, gene editing specifically comprises the steps of firstly constructing a CRISRP/Cas9 gene editing plasmid containing a sgRNA target sequence binding region shown in sequence 3 or/and sequence 4, and then transferring the CRISRP/Cas9 gene editing plasmid into a plant to realize gene editing. Wherein, the CRISRP/Cas9 gene editing plasmid is a II type CRISPR system. In embodiments of the invention, the pYLCISPR/Cas 9 system includes any one of the following recombinant vectors (pYLCISRP/Cas 9 gene editing plasmid): the recombinant expression vector pYLCISPR/Cas 9Pubi-H-47290-T1, the recombinant expression vector pYLCISPR/Cas 9Pubi-H-47290-T2 or the recombinant expression vector pYLCISPR/Cas 9 Pubi-H-47290.
Still another object of the present invention is to provide a specific sgRNA or an expression cassette, a vector, a host cell, an engineered bacterium or a transgenic plant cell line containing a gene encoding the sgRNA.
The specific sgRNA provided by the invention is sgRNA1 and/or sgRNA 2;
the target sequence of the sgRNA1 is a sequence 3; the target sequence of the sgRNA2 is sequence 4.
In the present invention, the plant may be a monocotyledonous plant or a dicotyledonous plant, preferably a host plant of the species blight bacterium, including but not limited to rice.
Experiments prove that the rice plant with LOC _ Os11g47290 gene mutation can be efficiently obtained by using the sgRNA to perform CRISRP/Cas 9-mediated gene editing, and the biological function of the encoding protein of the rice LOC _ Os11g47290 gene can be damaged by insertion or deletion from 367 th to 386 th or/and from 952 th to 971 th of the sequence shown in the sequence 2, so that the rice shows the property of improving the bacterial leaf blight resistance level, and the breeding efficiency of disease-resistant plants based on the LOC _ Os11g47290 gene mutation is effectively improved.
The invention has the beneficial effects that:
(1) the invention discovers that the rice receptor kinase gene LOC _ Os11g47290 and the coding protein thereof participate in regulating and controlling the immune response of rice to bacterial blight bacteria, and the resistance of the rice to the bacterial blight can be obviously improved by destroying the biological function of the coding protein of the LOC _ Os11g47290 gene. The experiment proves that: the LOC _ Os11g47290 is knocked out at fixed points to inoculate bacterial blight of rice, the length of inoculated bacterial blight V leaf spots is shortened by 14.4%, and the length of inoculated bacterial blight GD1358 leaf spots is shortened by 48.1%, so that the mutation of a nucleotide sequence of the LOC _ Os11g47290 gene to prepare the rice material for resisting bacterial blight has very important application value in agricultural production.
(2) The invention utilizes CRISRP/Cas9 technology to modify LOC _ Os11g47290 gene in a genome targeting way, and realizes efficient site-directed knockout of LOC _ Os11g 47290. The invention discovers that the site-directed knockout of LOC _ Os11g47290 can be efficiently realized by using nucleotide sequences from 367 th to 386 th or/and from 952 th to 971 th in a rice LOC _ Os11g47290 gene as a target sequence, and the biological function of a protein coded by the rice LOC _ Os11g47290 gene can be damaged by insertion or deletion from 367 th to 386 th or/and from 952 th to 971 th in the sequence shown in a sequence 2, so that the rice shows a trait of improving the bacterial leaf blight resistance level, and the breeding efficiency of disease-resistant plants based on the mutation of the LOC _ Os11g47290 gene is effectively improved.
Drawings
FIG. 1 is a sequencing peak diagram for vector activity detection based on pYRCISPR/Cas 9 technology rice receptor kinase LOC _ Os11g47290 site-directed knockout method provided in example 1 of the present invention.
FIG. 2 shows the mutation type of the nucleotide sequence of LOC _ Os11g47290 gene in Cas9-47290 homozygous mutant plant under Nipponbare 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 shows the mutation type of the LOC _ Os11g47290 gene amino acid sequence in the Cas9-47290 homozygous mutant plant under Nipponbare background of rice provided in example 2 of the present invention.
FIG. 4 is a statistical chart of lesion phenotype after inoculating Cas9-47290 homozygous mutant plants with Klebsiella planticola GD1358 and Klebsiella planticola V respectively under Nipponbare background of rice provided in example 2 of the present invention, wherein P is < 0.05.
Detailed Description
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 following examples are illustrated by Nipponbare (Oryza sativa ssp. japonica; non-patent documents describing this material are Yongqing Jian, Yonghong Wang, Dawei Xue, Jung Wang, Meixian Yan, Guifu Liu, Guojun Dong, Dali Zeng, Zefu Lu, Xudong Zhu, Qian Qian and Jianyang Li.Regulation of OsL 14 by OsmiR156 define ideal plant architecture in Nature Genetics, 2010,42, 541-544; publicly available from the Applicant).
Bacterial blight strain V: the resistance response to 5 races of southern east rice blight fungus was described in "Ying Xianhua, Wushang faithful. IRBB21(Xa 21.) plant protection journal, 2002,29(2): 97-100", which was obtained from the applicant after the consent of the first teacher of Guangdong academy of agricultural sciences.
Bacterial blight of rice yue 1358(GD 1358): described in "the research of pathogenic type of bacterial blight of rice in china, plant pathology newspaper, 1990, 20 (2): 81-88 ", the public is available from the applicant.
Example 1 site-directed knockout method of rice receptor kinase gene LOC _ Os11g47290 based on pYLCRISPR/Cas9 system
Sequence analysis and target sequence screening of rice receptor kinase gene LOC _ Os11g47290
The nucleotide sequence of the rice receptor kinase gene LOC _ Os11g47290 is shown as a sequence 2, and the amino acid sequence of the encoded protein is a sequence 1 in a sequence table. Sequence analysis shows that the gene comprises 7 exons, which are respectively the 1 st-1696 th site (first exon), the 2042 nd and 2941 th site (second exon), the 3130 and 3278 th sites (third exon), the 3677 th and 3890 th sites (fourth exon), the 6492 and 6611 th sites (fifth exon), the 6976 and 7019 th sites (sixth exon) and the 7163 and 7171 th sites (seventh exon) of the sequence 2 sequence.
The sequence on the first exon of the rice receptor kinase gene LOC _ Os11g47290 is the 47290-T1 and 47290-T2 target sequences of the site-directed knockout method of the rice receptor kinase gene LOC _ Os11g47290 based on pYLCRISPR/Cas9 system.
Through a large number of screens, the antisense strand from 367 to 386 of the first exon of the rice LOC _ Os11g47290 gene and the sense strand from 952 to 971 of the first exon are targeted by using the pYLCRISPR/Cas9 technology to be used as target sequences 47290-T1 and 47290-T2 respectively, wherein the target sequences are shown as a sequence 3 and a sequence 4.
pYLCRISPR/Cas9 System vectors (including pYLsgRNA-OsU6a, 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/85CRISPR/9 System for Con elementary, High-efficiency sample edition single electron oligo meter and plant 1274.8. After the subtropical agriculture biological resource protection of the university of south China's college of Life sciences and the consent of the national focus laboratory Liu dazzling teacher, the public can obtain the carrier from the applicant.
Design of pYLCRISPR/Cas9 system vector primer and construction of recombinant expression vector thereof
1. Design and synthesis of pYLCISPR/Cas 9 technology target sequence primer
Designing a target sequence primer targeting LOC _ Os11g47290 gene based on pYLCRISPR/Cas9 technology, wherein sequences of 47290-T1 target sequence primers 47290-gRT1 and 47290-U6aT1 are shown as sequence 5 and sequence 6 respectively; 47290-T2 target sequences primer 47290-gRT2 and primer 47290-U6bT2 are shown as sequence 7 and sequence 8 respectively;
relevant primers for 47290-T1 and 47290-T2, based on pYLCRISPR/Cas9 technology, were synthesized separately.
Sequence 5: 47290-gRT 1: 5'-TGCAGCCTTCCTATAACACCgttttagagctagaaat-3'
And (3) sequence 6: 47290-U6aT 1: 5'-GGTGTTATAGGAAGGCTGCACggcagccaagccagca-3'
And (3) sequence 7: 47290-gRT 2: 5'-CATGTGCCGGAATGGTTAGCgttttagagctagaaat-3'
And (2) sequence 8: 47290-U6bT 2: 5'-GCTAACCATTCCGGCACATGCaacacaagcggcagca-3' are provided.
2. Construction of pYLCRISPR/Cas9 technical recombinant expression vector
1) Construction of recombinant expression vectors containing 47290-T1 or 47290-T2 alone
The recombinant expression vector pYLCRISPR/Cas9Pubi-H-47290-T1 contains encoding genes of U6a-47290-sgRNA1 and Cas9, U6a-47290-sgRNA1 has a target sequence region consisting of 20 nucleotides, and the corresponding target sequence of the target sequence region on LOC _ Os11g47290 gene is 47290-T1 shown in sequence 3; 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 47290-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 47290-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 named U6a-47290-sgRNA 1;
then, U6a-47290-sgRNA1 and pYRCISPR/Cas 9Pubi-H are subjected to enzyme digestion by BsaI enzyme, and a vector pYRCISPR/Cas 9Pubi-H-47290-T1 is obtained in a way of enzyme digestion and side ligation.
The recombinant expression vector pYLCRISPR/Cas9Pubi-H-47290-T2 contains genes encoding U6b-47290-sgRNA2 and Cas9, U6b-47290-sgRNA2 has a target sequence region consisting of 20 nucleotides, and the corresponding target sequence of the target sequence region on LOC _ Os11g47290 gene is 47290-T2 shown in sequence 4; 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 47290-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 47290-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 named U6b-47290-sgRNA 2;
Then BsaI enzyme is used for simultaneously carrying out enzyme digestion on the U6b-47290-sgRNA2 and the pYLCRISPR/Cas9Pubi-H vector, and the vector pYLCRISPR/Cas9Pubi-H-47290-T2 is obtained in a mode of enzyme digestion and connection.
2) Construction of recombinant expression vectors containing 47290-T1 and 47290-T2
The pYRCISPR/Cas 9Pubi-H-47290 contains genes encoding U6a-47290-sgRNA-1, U6b-47290-sgRNA-2 and Cas9, U6a-47290-sgRNA-1 has a target sequence region consisting of 20 nucleotides, and the corresponding target sequence of the target sequence region on the LOC _ Os11g47290 gene is 47290-T1 shown in a sequence 3; u6b-47290-sgRNA-2 has a target sequence region of 20 nucleotides, and the target sequence region corresponds to a target sequence of 47290-T2 shown in sequence 4 on LOC _ Os11g47290 gene. The specific construction method comprises the following steps: 1) u6aT1 and gRT1 are connected together by nested PCR by using primers Pps-GGL (5'-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG-3') and Pgs-GG2(5 ' -AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3), and are named as U6 a-47290-sgRNA-1; 2) u6bT2 and gRT2 are connected together by nested PCR by using primers Pps-GG2 (5'-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3') and Pgs-GGR (5'-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC-3'), and are named as U6 b-47290-sgRNA-2; 3) the BsaI enzyme simultaneously carries out enzyme digestion on U6a-47290-sgRNA-1, U6b-47290-sgRNA-2 and pYLCRISPR/Cas9Pubi-H, and the vector pYLCRISPR/Cas9Pubi-H-47290 is obtained in a mode of enzyme digestion and connection.
3. Activity assay of recombinant expression vectors
The recombinant expression vectors pYLCRISPR/Cas9Pubi-H-47290-T1 and pYLCRISPR/Cas9Pubi-H-47290-T2 prepared in the above 2 are introduced into Nipponbare protoplasts of rice through PEG mediation respectively (see https:// bio-protocol. org/bio101/e1010125), and the protoplasts of plasmids pYLCRISPR/Cas9Pubi-H-47290-T1 and pYLCRISPR/Cas9Pubi-H-47290-T1 are obtained after 16 hours.
Genomic DNAs of protoplasts transiently transduced with plasmids pYLCRISPR/Cas9Pubi-H-47290-T1 and pYLCRISPR/Cas9Pubi-H-47290-T2 were extracted, respectively, a partial nucleotide sequence of LOC _ Os11g47290 gene was amplified using sequence 11 and sequence 12 in the sequence listing, and sequencing was performed.
The results are shown in FIG. 1, FIG. 1 is a sequencing peak diagram for vector activity detection based on a fixed-point knockout method of rice receptor kinase gene LOC _ Os11g47290 by pYLCRISPR/Cas9 technology, and it can be seen that the pYLCRISPR/Cas9 technology can induce the mutation of LOC _ Os11g47290 gene at target sequences 47290-T1 and 47290-T2.
4. Obtaining of recombinant Agrobacterium tumefaciens
And (3) carrying out heat shock transformation on the recombinant expression vector pYLCRISPR/Cas9Pubi-H-47290 obtained in the step (2) to obtain the recombinant agrobacterium containing the recombinant expression vector pYLCRISPR/Cas9Pubi-H-47290, which is named as EH105-Cas 9-47290.
Agrobacterium tumefaciens EHA 105: BioIVectror NTCC type culture Collection, commercially available.
Example 2 application of pYLCISPR/Cas 9 technology-based site-specific knockout method in rice variety
First, pYLCRISPR/Cas9 technology site-directed knockout of LOC _ Os11g47290 gene
Infecting a rice variety Nipponbare (hereinafter referred to as wild rice) mature embryo induced callus by recombinant agrobacterium EH105-Cas9-47290, and respectively naming obtained rice transformation plants as NIP-Cas 9-47290; the specific method of the experiment is as follows:
1. the recombinant Agrobacterium obtained in example 1 was inoculated into 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 mature seeds of Nipponbare of a rice variety, soaking in 75% ethanol for 1min, then sterilizing in NaClO solution (mixed with water at a ratio of 1:2, and adding 1 drop of Tween 20) for 20min by oscillation, and repeating for 2 times. Washing with sterile water for several times until no foreign odor exists, inoculating sterilized Nipponbare seed of rice to 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), removing the rice material after 20-30min, inoculating the rice material 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 ℃.
4. And (4) inoculating the callus co-cultured in the step (3) into a screening culture medium (the hygromycin concentration is 50mg/L and the pH value is 5.8), screening and culturing for 12 days under a dark condition at the temperature of 28 ℃, 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 regenerated seedlings to 1/2MS culture medium for root induction; and after the plantlets are thrilled, moving the plantlets into an artificial climate chamber for nutrient solution cultivation to obtain a regenerated plant NIP-Cas 9-47290.
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 self primer sequence 9 and the sequence 10 of the recombinant expression vector pYLCRISPR/Cas9Pubi-H-47290 to screen a positive transformation plant, and amplifying the plant with a 1225bp strip, namely 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, namely the positive rate (%) are counted, and the results are shown in table 1.
Table 1. positive rate detection result of pYRCISPR/Cas 9Pubi-H-47290 transformed rice variety
Figure BDA0002287118260000091
Figure BDA0002287118260000101
7. PCR amplification was carried out using the genome of the number of positive transformed plants as a template, and a sequence shown by sequence 11 for a specific primer LOC _ Os11g47290-TF for rice receptor kinase gene LOC _ Os11g47290 and sequence 12 for LOC _ Os11g47290-TF (a fragment containing 2 target sequences in amplification gene LOC _ Os11g 47290). Nipponbare was used as a control.
Sequencing and verifying the obtained 1096bp amplification product, and recording the mutation of the amplification product as the number of the mutant transformation plants compared with Nipponbare; the sequencing verification results are shown in table 2. The number of the regenerated plants, the number of the plants transformed with mutation and the percentage of the number of the plants transformed with mutation to the number of the regenerated plants, i.e., the mutation efficiency (%) were counted, and the results are shown in table 2.
Table 2. detection results of pYLCRISPR/Cas9Pubi-H-47290 induced mutation of rice receptor kinase gene LOC _ Os11g47290
Figure BDA0002287118260000102
Secondly, site-directed knockout of 47290 gene mutation transformed plant phenotype by pYLCRISPR/Cas9 technology
And collecting seeds of the 39 obtained mutation-transformed plants, and sowing to obtain T1 generation plants.
Extracting genome DNA of 200T 1 generation plants, amplifying by using a primer shown in a sequence 11 and a primer shown in a sequence 12 to obtain a PCR product, sending to sequence, selecting a homozygous mutant strain to obtain 1 homozygous mutant type, wherein the mutant type is 40.
The mutant forms of the homozygous mutant types are as follows: both target sequences 47290-T1 and 47290-T2 were mutated and base T was replaced with base A at position 371 and base G was replaced with base A at position 380 in target sequence 1; insertion of base T between positions 968 and 969 of the target sequence 2 leads to premature termination of translation of the gene LOC _ Os11g 47290. The nucleotide sequence of this homozygous mutant type is shown in FIG. 2, and the amino acid sequence is shown in FIG. 3. The plants with this mutant form were designated as T1 generation Cas9-47290 mutant plants.
T1 generation Cas9-47290 mutant plants (Cas9-47290) are inoculated with the white leaf blight bacteria GD1358 and V. Wild type nipponlily was used as a control. 15 strains are inoculated to each strain; the experiment was repeated 3 times and the results averaged.
After 14 days of inoculation with the bacterial blight strain GD1358 and V, leaf phenotype is observed.
The length of inoculated leaf spots was counted, and as a result, as shown in FIG. 4, the Cas9-47290 mutant exhibited a phenotype in which bacterial blight spots were shortened, the length of leaf spots inoculated with Bacillus subtilis GD1358 was shortened by 48.1%, and the length of leaf spots inoculated with Bacillus subtilis V was shortened by 14.4%, compared to the wild type (Nipponbare on the abscissa of the bar chart).
The results show that the Cas9-47290 mutant created by the CRISPR/Cas9 technology enhances the disease resistance of the rice to the white leaf blight bacteria GD1358 and V.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> institute of genetics and developmental biology, institute of academy of agricultural sciences, China
<120> rice receptor kinase gene LOC _ Os11g47290, and coding protein and application thereof
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 1044
<212> PRT
<213> Artificial sequence
<400> 1
Met Gly Val Gly Pro His Cys Thr Thr Ser Leu Leu Ile Ile Leu Ala
1 5 10 15
Val Val Ile Thr Ser Ser Leu Leu Thr Thr Thr Ile Lys Ala Asp Glu
20 25 30
Pro Ser Asn Asp Thr Asp Ile Ala Ala Leu Leu Ala Phe Lys Ala Gln
35 40 45
Phe Ser Asp Pro Leu Gly Phe Leu Arg Asp Gly Trp Arg Glu Asp Asn
50 55 60
Ala Ser Cys Phe Cys Gln Trp Ile Gly Val Ser Cys Ser Arg Arg Arg
65 70 75 80
Gln Arg Val Thr Ala Leu Glu Leu Pro Gly Ile Pro Leu Gln Gly Ser
85 90 95
Ile Thr Pro His Leu Gly Asn Leu Ser Phe Leu Tyr Val Leu Asn Leu
100 105 110
Ala Asn Thr Ser Leu Thr Gly Thr Leu Pro Gly Val Ile Gly Arg Leu
115 120 125
His Arg Leu Glu Leu Leu Asp Leu Gly Tyr Asn Ala Leu Ser Gly Asn
130 135 140
Ile Pro Ala Thr Ile Gly Asn Leu Thr Lys Leu Glu Leu Leu Asn Leu
145 150 155 160
Glu Phe Asn Gln Leu Ser Gly Pro Ile Pro Ala Glu Leu Gln Gly Leu
165 170 175
Arg Ser Leu Gly Ser Met Asn Leu Arg Arg Asn Tyr Leu Ser Gly Leu
180 185 190
Ile Pro Asn Ser Leu Phe Asn Asn Thr Pro Leu Leu Gly Tyr Leu Ser
195 200 205
Ile Gly Asn Asn Ser Leu Ser Gly Pro Ile Pro His Val Ile Phe Ser
210 215 220
Leu His Val Leu Gln Val Leu Val Leu Glu His Asn Gln Leu Ser Gly
225 230 235 240
Ser Leu Pro Pro Ala Ile Phe Asn Met Ser Arg Leu Glu Lys Leu Tyr
245 250 255
Ala Thr Arg Asn Asn Leu Thr Gly Pro Ile Pro Tyr Pro Ala Glu Asn
260 265 270
Gln Thr Leu Met Asn Ile Pro Met Ile Arg Val Met Cys Leu Ser Phe
275 280 285
Asn Gly Phe Ile Gly Arg Ile Pro Pro Gly Leu Ala Ala Cys Arg Lys
290 295 300
Leu Gln Met Leu Glu Leu Gly Gly Asn Leu Leu Thr Asp His Val Pro
305 310 315 320
Glu Trp Leu Ala Gly Leu Ser Leu Leu Ser Thr Leu Val Ile Gly Gln
325 330 335
Asn Glu Leu Val Gly Ser Ile Pro Val Val Leu Ser Asn Leu Thr Lys
340 345 350
Leu Thr Val Leu Asp Leu Ser Ser Cys Lys Leu Ser Gly Ile Ile Pro
355 360 365
Leu Glu Leu Gly Lys Met Thr Gln Leu Asn Ile Leu His Leu Ser Phe
370 375 380
Asn Arg Leu Thr Gly Pro Phe Pro Thr Ser Leu Gly Asn Leu Thr Lys
385 390 395 400
Leu Ser Phe Leu Gly Leu Glu Ser Asn Leu Leu Thr Gly Gln Val Pro
405 410 415
Glu Thr Leu Gly Asn Leu Arg Ser Leu Tyr Ser Leu Gly Ile Gly Lys
420 425 430
Asn His Leu Gln Gly Lys Leu His Phe Phe Ala Leu Leu Ser Asn Cys
435 440 445
Arg Glu Leu Gln Phe Leu Asp Ile Gly Met Asn Ser Phe Ser Gly Ser
450 455 460
Ile Ser Ala Ser Leu Leu Ala Asn Leu Ser Asn Asn Leu Gln Tyr Phe
465 470 475 480
Tyr Ala Asn Asp Asn Asn Leu Thr Gly Ser Ile Pro Ala Thr Ile Ser
485 490 495
Asn Leu Ser Asn Leu Asn Val Ile Gly Leu Phe Asp Asn Gln Ile Ser
500 505 510
Gly Thr Ile Pro Asp Ser Ile Met Leu Met Asp Asn Leu Gln Ala Leu
515 520 525
Asp Leu Ser Ile Asn Asn Leu Phe Gly Pro Ile Pro Gly Gln Ile Gly
530 535 540
Thr Pro Lys Gly Met Val Ala Leu Ser Leu Ser Gly Asn Asn Leu Ser
545 550 555 560
Ser Tyr Ile Pro Asn Gly Gly Ile Pro Lys Tyr Phe Ser Asn Leu Thr
565 570 575
Tyr Leu Thr Ser Leu Asn Leu Ser Phe Asn Asn Leu Gln Gly Gln Ile
580 585 590
Pro Ser Gly Gly Ile Phe Ser Asn Ile Thr Met Gln Ser Leu Met Gly
595 600 605
Asn Ala Gly Leu Cys Gly Ala Pro Arg Leu Gly Phe Pro Ala Cys Leu
610 615 620
Glu Lys Ser Asp Ser Thr Arg Thr Lys His Leu Leu Lys Ile Val Leu
625 630 635 640
Pro Thr Val Ile Val Ala Phe Gly Ala Ile Val Val Phe Leu Tyr Leu
645 650 655
Met Ile Ala Lys Lys Met Lys Asn Pro Asp Ile Thr Ala Ser Phe Gly
660 665 670
Ile Ala Asp Ala Ile Cys His Arg Leu Val Ser Tyr Gln Glu Ile Val
675 680 685
Arg Ala Thr Glu Asn Phe Asn Glu Asp Asn Leu Leu Gly Val Gly Ser
690 695 700
Phe Gly Lys Val Phe Lys Gly Arg Leu Asp Asp Gly Leu Val Val Ala
705 710 715 720
Ile Lys Ile Leu Asn Met Gln Val Glu Arg Ala Ile Arg Ser Phe Asp
725 730 735
Ala Glu Cys His Val Leu Arg Met Ala Arg His Arg Asn Leu Ile Lys
740 745 750
Ile Leu Asn Thr Cys Ser Asn Leu Asp Phe Arg Ala Leu Phe Leu Gln
755 760 765
Phe Met Pro Asn Gly Asn Leu Glu Ser Tyr Leu His Ser Glu Ser Arg
770 775 780
Pro Cys Val Gly Ser Phe Leu Lys Arg Met Glu Ile Met Leu Asp Val
785 790 795 800
Ser Met Ala Met Glu Tyr Leu His His Glu His His Glu Val Val Leu
805 810 815
His Cys Asp Leu Lys Pro Ser Asn Val Leu Phe Asp Glu Glu Met Thr
820 825 830
Ala His Val Ala Asp Phe Gly Ile Ala Lys Met Leu Leu Gly Asp Asp
835 840 845
Asn Ser Ala Val Ser Ala Ser Met Leu Gly Thr Ile Gly Tyr Met Ala
850 855 860
Pro Val Phe Glu Leu Gly Leu Leu Cys Ser Ala Asp Ser Pro Glu Gln
865 870 875 880
Arg Thr Ala Met Ser Asp Val Val Val Thr Leu Lys Lys Ile Arg Lys
885 890 895
Asp Tyr Val Lys Leu Met Ala Thr Thr Arg Pro Gly Lys Lys Leu Met
900 905 910
Ala Thr Thr Ala Asn Arg Thr Ser Lys Gly Pro Gln Asp Asn Arg Val
915 920 925
Phe Arg Glu His Ile Phe Arg Leu Ser Cys Thr Gln Lys Arg Ser Asp
930 935 940
Ser Gln Met Arg Ala Gly Thr Ile Thr Gly Ser His Leu Ser Lys Thr
945 950 955 960
Asn Glu Ala Val Gly Gln Arg Arg Lys Lys Val Val Gly Thr Gly Asp
965 970 975
Asn Ser Arg Pro Ala Leu His Glu Leu Gln Gly Arg Glu Lys Gly Lys
980 985 990
Glu Glu Gly Gly Arg Gly Gly Ser Asp Trp Ile Gly Gly Ala Gly Asp
995 1000 1005
Arg Trp Glu Arg Ser Pro Ala His Gln Ala Tyr Pro Leu Met Gly
1010 1015 1020
Pro Glu Arg Lys Glu His Ser Lys Glu Arg Lys Gly Arg Lys Arg
1025 1030 1035
Asn Gly Glu Glu Asn Phe
1040
<210> 2
<211> 7171
<212> DNA
<213> Artificial sequence
<400> 2
atgggtgttg gtcctcattg tactactagt ctgctaataa tactggccgt cgtcatcacg 60
tcgtctttgc tcacgacgac gatcaaggca gatgagccga gcaatgatac cgacatcgcc 120
gcgctgcttg ccttcaaggc acagttctct gaccctctgg gtttcctccg tgacggctgg 180
agggaggaca atgcatcctg cttctgccag tggatcggcg tgtcgtgcag ccgccgccgg 240
cagcgcgtca ccgccctgga gctgccgggc attcccctgc aagggtcgat cacccctcac 300
ctcggtaacc tctctttcct ctacgtcctc aacctcgcca acaccagcct cacggggaca 360
ctcccgggtg ttataggaag gctgcatcgc ctggagctcc ttgatcttgg ctacaatgcc 420
ctgtcaggta acatcccagc caccatagga aacctcacca aacttgagct tcttaatctc 480
gagtttaacc agctatctgg tccaatccca gcagagctgc agggcctgcg aagccttggc 540
agtatgaatc tccgtaggaa ctatctcagt ggcttgattc ccaacagtct attcaacaac 600
accccattgt taggttatct cagcattggc aacaacagct tgtcagggcc aataccgcac 660
gtgatattct cgttgcacgt gctgcaggtc cttgttctag agcacaatca attgtccggc 720
tcactgcccc cagccatctt caacatgtcc agacttgaaa agctgtatgc cactcgaaac 780
aatctcactg gacctatccc atacccagct gaaaaccaga ccttgatgaa catccccatg 840
attcgggtga tgtgtctctc tttcaacgga ttcataggcc gaattccacc tgggcttgcg 900
gcatgccgga aactccagat gcttgagtta ggtgggaatc tcttgacgga tcatgtgccg 960
gaatggttag cgggcttgtc cctgctaagc accttggtta taggtcagaa tgagcttgtc 1020
ggttcgatcc cagttgtgct aagcaatctc accaagctca ccgtgcttga tctgtcatct 1080
tgcaagctaa gtggaatcat tccattggaa ctaggaaaga tgacacaact caacatcttg 1140
cacctctcat ttaatcgcct aactggtcct tttcctacct cccttggtaa cttgacaaaa 1200
ttatcttttc taggattaga atctaacctg ctgaccggac aagtacctga gacccttggg 1260
aacctcaggt ctctatactc ccttggtatt ggaaagaatc atctacaagg gaaacttcac 1320
ttctttgccc ttctctccaa ttgtagggaa ctccaattcc tcgacatagg aatgaattct 1380
ttctcaggga gcatttctgc gagtttacta gcaaacctct ctaacaactt acaatatttt 1440
tatgcaaatg ataacaactt aactggcagt attcctgcta ccatatcaaa tctgtctaac 1500
ctgaatgtaa taggcctttt tgacaaccaa ataagcggca caattccaga ttctataatg 1560
ctaatggata atctacaggc attggacctc tctataaaca atttgtttgg accaatccca 1620
ggacaaattg gtaccccaaa aggaatggtc gcattatctc tcagtggcaa caatctttct 1680
agttacatcc ctaacggtgt tggcaatcta agcacgttgc agtactgatt tctgtcatat 1740
aataggttgt catcagttat acccgcaagc ttagttaatc ttagtaatct tctccaacta 1800
gatatttcta ataataactt aactggttca ttgccttctg atctcagttc cttcaaagta 1860
ataggcctaa tggacatctc agcaaataat ttggtcggta gcctcccaac ttcgttggga 1920
tagctccaac tgtcaagcta cctgaattta tctcaaaaca cattcaatga ttcaattcca 1980
gactctttca aaggtctaat taatttagaa acattggatc tgtctcataa caatctttca 2040
ggaggcatac caaaatactt ttccaactta acctatctta cttctttgaa cctctccttt 2100
aacaatctac aaggtcagat accaagtgga ggtatttttt caaacatcac tatgcaatct 2160
ttaatgggaa atgctggact ctgtggtgct ccacgtctgg gatttcctgc atgtctggag 2220
aagtccgact cgactagaac aaaacacttg ctgaagattg tgctccctac tgtcattgtg 2280
gcgtttggtg ccattgttgt gttcctatac ctaatgattg caaagaaaat gaaaaatcca 2340
gatattacgg cttcttttgg catagcagat gcgatttgcc acaggctagt gtcctaccaa 2400
gaaatcgttc gcgctaccga aaatttcaat gaggacaacc tacttggagt tggaagtttt 2460
ggcaaagttt tcaagggtcg gctggatgac ggtttggtgg ttgcaatcaa aatcctcaac 2520
atgcaggttg aacgagctat taggagtttt gatgcagagt gccatgtctt gcggatggcc 2580
agacatcgca acctgataaa gatactaaat acatgttcca acttggattt cagagcactg 2640
tttcttcagt tcatgcccaa tggaaacttg gagtcatact tgcactctga aagcaggcct 2700
tgtgtgggat cattcctcaa aaggatggag attatgctag atgtgtcaat ggctatggaa 2760
tatctgcatc atgaacacca tgaggttgtc ctgcattgtg atttgaaacc tagcaacgtg 2820
ctatttgatg aagagatgac tgcacatgta gcagactttg gcatagcaaa gatgttgtta 2880
ggggatgaca attcagcagt ttcagcaagc atgctaggca caattgggta catggcacca 2940
ggtacttaca ctagccaatc taaagccaca tacatttttt gttggagttg ttacaaattc 3000
acttcaagat attggccggg cttctgacga agcggagcgg aggcccgtcg ccggaggctt 3060
gggccggagc cagtgggttg gtgtatcgtg tagccgccgc aggtaacatg catggcttcc 3120
ttgtgccagt gttcgagctg ggcttgctct gttcggctga ctcccccgag caaaggacgg 3180
cgatgagcga tgtggtcgtg acactgaaga agattagaaa ggactatgtc aaattgatgg 3240
caaccacccg acccggcaaa aaattgatgg caaccacagt aagcgttgtg cagcagtgat 3300
tcatcgctct atcgttgtat atgagcgaat gaaatacata tcatttgcat cattttcttc 3360
ttctgcatta ggaatagcat cagtgatcga ttaccctttg tttgtatttg tgtatggttg 3420
aattgaatat atatatatat atatatatat atatataaca atttcgttgg tgtaaatatg 3480
tgattgaacc gctggtcaat aaatttgcat cacgaaaatg ggagtagatg atgtgctact 3540
tatgttttct tatttctggc caaaataaat aaataaaaaa ggaatattat cggcacagca 3600
tcacaactcc ggctcgttca gccttaaaca accacaatta acagtcctaa gcagagaaac 3660
ttaacaagct tttcaggcta acagaacatc aaaaggtcca caagacaaca gggtcttcag 3720
agagcacatc ttcaggctgt catgcacgca aaaaagatca gacagccaga tgagagcggg 3780
tacaataacc ggtagccatc tcagtaaaac taacgaggct gtaggacaga gaagaaaaaa 3840
ggtagtagga acgggcgaca atagtcggcc agctctacat gagctccaag gtgaaaaagc 3900
ctacaagata ggaattttca atttttaaaa atggccatgg gcccacaagt agtgagatgc 3960
atgcaatgaa agatgctagt cagttgggtc ccacaagaaa aagaaataca tctcgggatt 4020
aactgctagc ttactctagt taatatggac atggatggag ccggcagcca gcaatactat 4080
tgaacctgct taagagcaag tgcaatagta ggctatatac cagctataaa catactttaa 4140
agagataaag gaagagagag aggaatagca gattacagca cggattacaa gacgtaatat 4200
gtgtataaca tgtgagacca gatattaata gtatagtaag caactattgt atgaactagg 4260
aaggaagccc gcgcagatgc gcaggcatct aatttattgt tttatgattt atgaaataat 4320
gctaaaagat gtcatgtctc atctttttta tgctgtgaga taaactgact ttaatatact 4380
tagtaaatga taagaatgtc tccaagatta aaagaaaatc agtctgttcc gattacttat 4440
aaattctcct attgtcacca caacttatta ttattgtttt aaaagttatg atctataaat 4500
agataaaact ggatatgtac caataaaata agcctaatac atcattgata cactaaaata 4560
ttcactactt atctaagtgt tcatgtttca cttatttccg aatcaatata taaatatttt 4620
aaaaatacca cttataccaa ttaatgaaat aacaccgtta aagataaaca tgaatgcccc 4680
tctaacgggt gaagcccatc gtagccacca agactttctt tctctgttga gttttctaaa 4740
aaaaacgaaa ataaaatttt tgataaatta atccaaaaaa ttaaaactaa attggattta 4800
cttaaaaaat agactaaatt gttttttatc attttttaca tttgtagatt taaattagtg 4860
ttgtatattt atcaagtgat attagaaatc ctaaaatata atgtgaaact atttgaaaat 4920
taccacaaca ttaattatta tatgttggtc ccatgtgtca tgatgtattt aggaccataa 4980
tatacctttt ataagacata agtatttcat ttaattaaag catgaaacta atcacaaatt 5040
gaatataatt gttgcttact atactattaa tatatggtcc cacctgtcat acgcatattg 5100
cgtcttgtag tccgtgctgc agctggctac aaatctgtag cccgctgctc ttctctctca 5160
tcgtttatct cattaaaata tatttgtagc tggctaatag cttgctattt tacttgctct 5220
aagataacag tcctaccaat ttaaataaaa attaatgatg agattagaca aagtcacagg 5280
gtggcctaca agagttggta gaaatggcac ataagataaa atatgatata gtagtaaaaa 5340
tatagctaat atagctcagg aattggtgat ttaattaaaa aaacctacag ctgctaaagt 5400
gtatgcgtag atcgtctata aaaataattg tttatttaca acaccagatc acttctattg 5460
taaattccta gcgtttgcgc attcacactc agattactct ctatgaaaaa actgatctaa 5520
taatttatga taataaatta tgatgtacat gtatcatgta atttgaaaaa agaagaaaaa 5580
aaggagaatt aggatctgga tggcatcaat cgagctcgac gaggcagatc agtctccatg 5640
gaaatgataa caaattatga tgcacatgta ttgtgtaatt agaaataaaa ggagagaggg 5700
agaatccaga tatggatggc atcaagctaa cagaagcata agtatgtacg tacgtactta 5760
tgggcggaaa atctaatggg cgatcaatcg ctaccgcgat ctggtagcga tcgcatcccc 5820
tcccccctcc ctccttccac gtttcctttt ctggcaccgc attacttttc tattttagta 5880
aaatttatgc acctaaagtt tatacaccta aagtttatag acccaaagtt tagagaccca 5940
aagtttataa atcaaaagtt tatatatccg attcaaattt aaatttgaat tcaaatattt 6000
tttatatata gtatttatat acatctaaag tttatacacc taaagtttat agacccaaag 6060
tttataaatc aaaagtttat ataccctatt caaatttgaa tttgaaatat attcgattca 6120
aaatttgaat ttgaattcaa atatttttta tatatagtat ttagatacat ctaaagttta 6180
tacacctaaa gtttatagac ccaaagttta gagacccaaa gtttacatac ccaattcaaa 6240
tttgaatttg aattgtatcc gattcaaatt taaatttgaa ttcaaatatt tttatatata 6300
gtatttctat acatctaaag tttatacacc taaagtttat agaccgaaag tttttagtaa 6360
aaagtttata tacccgattc aaatttgaat ttgaattcaa atattagttt atagatccaa 6420
agtttataag tcaaaaattt acataaccgt ttcaattctg aatttaaatt taaatattta 6480
tggtgtagta ggaagagaaa aaggaaagga ggaagggggg agaggaggga gcgactggat 6540
aggaggggcg ggtgatcgct gggagcgatc acccgcccat caggcatacc cacttatggg 6600
tccggagaga aagggtgaga aagatttagt tttgatccaa cggttaatat tattgggtcc 6660
accactttaa ataaaaactt acggtcagat atttttcttt ttctcagaat attatttaaa 6720
ttattagagc gccatgtggc gacttaggag cgtttatata taggagtctc acgtggtagc 6780
ttgagagcgt acgtagaaag tttaatgaac ttttagtata taaataatag atagatagat 6840
ttaggatttt ctctaatttg ctagaacgcc acgtggtggc ctaggatcgt tattggtcca 6900
aataatatgt aattacttat ttatataaat aatatgtaat ttagaagtat acttattcaa 6960
tatgtattgt tgcagaacat agtaaagaga ggaaggggag gaaaagaaac ggagaggagg 7020
tactcataca tgacgcacgg caggattcct tctttgtttt ttagaaagta agagaataaa 7080
ccaatctaat ctaacggctt ggagtaacgg gcccactaat tttaatgaaa attaatggct 7140
agatgttttg ctttttttat agaatttcta g 7171
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence
<400> 3
tgcagccttc ctataacacc 20
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<400> 4
catgtgccgg aatggttagc 20
<210> 5
<211> 37
<212> DNA
<213> Artificial sequence
<400> 5
tgcagccttc ctataacacc gttttagagc tagaaat 37
<210> 6
<211> 37
<212> DNA
<213> Artificial sequence
<400> 6
ggtgttatag gaaggctgca cggcagccaa gccagca 37
<210> 7
<211> 37
<212> DNA
<213> Artificial sequence
<400> 7
catgtgccgg aatggttagc gttttagagc tagaaat 37
<210> 8
<211> 37
<212> DNA
<213> Artificial sequence
<400> 8
gctaaccatt ccggcacatg caacacaagc ggcagca 37
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence
<400> 9
gcggtgtcat ctatgttact ag 22
<210> 10
<211> 22
<212> DNA
<213> Artificial sequence
<400> 10
ccgacataga tgcaataact tc 22
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence
<400> 11
atgagccgag caatgatacc 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence
<400> 12
ccaagggagg taggaaaagg 20

Claims (5)

1. The application of substances for inhibiting or reducing the expression of LOC _ Os11g47290 protein coding nucleic acid in rice in improving the bacterial leaf blight resistance of the rice or improving the bacterial leaf blight resistance of the rice:
the LOC _ Os11g47290 protein is any one of the following (a 1) - (a 2):
(a1) protein shown as a sequence 1 in a sequence table;
(a2) A fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);
the substances are as follows:
1) interfering RNA;
2) CRISPR/Cas9 system;
in the CRISPR/Cas9 system, the target sequence of sgRNA is the nucleotide sequence of XXXGG form in the encoding nucleic acid of LOC _ Os11g47290 protein, wherein XXX is the nucleic acid sequence of any 19-20 bp in the encoding nucleic acid of LOC _ Os11g47290 protein, and N is any one base in A, T, G, C.
2. Use according to claim 1, characterized in that:
the LOC _ Os11g47290 protein coding nucleic acid is a DNA molecule with a coding region shown as a sequence 2 in a sequence table.
3. Use according to claim 2, characterized in that:
the sgRNA is sgRNA1 and/or sgRNA 2;
the target sequence of the sgRNA1 is a sequence 3; the target sequence of the sgRNA2 is sequence 4.
4. A method for improving the bacterial leaf blight resistance of rice is any one of the following methods 1) to 2), and the bacterial leaf blight resistance of rice is improved;
1) the method comprises the following steps: inhibiting or reducing the expression of LOC _ Os11g47290 protein-encoding nucleic acid in target rice;
2) the method comprises the following steps: carrying out gene editing on LOC _ Os11g47290 protein coding nucleic acid in target rice;
Or, a method for producing a transgenic rice plant having high resistance to bacterial blight, which is any one of the following 1) to 2),
1) the method comprises the following steps: inhibiting or reducing the expression of LOC _ Os11g47290 protein coding nucleic acid in target rice to obtain transgenic rice;
2) the method comprises the following steps: carrying out gene editing on LOC _ Os11g47290 protein coding nucleic acid in target rice to obtain transgenic rice;
the bacterial leaf blight resistance of the transgenic rice is higher than that of the target rice;
the LOC _ Os11g47290 protein is any one of the following (a 1) - (a 2):
(a1) protein shown as a sequence 1 in a sequence table;
(a2) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);
the inhibition or reduction of the expression of the nucleic acid encoding the LOC _ Os11g47290 protein in the rice is realized by performing gene editing on the nucleic acid encoding the LOC _ Os11g47290 protein, wherein the gene editing is realized by virtue of a CRISPR/Cas9 system;
in the CRISPR/Cas9 system, the target sequence of sgRNA is the nucleotide sequence of XXXGG form in the encoding nucleic acid of LOC _ Os11g47290 protein, wherein XXX is the nucleic acid sequence of any 19-20 bp in the encoding nucleic acid of LOC _ Os11g47290 protein, and N is any one base in A, T, G, C.
5. The method of claim 4, wherein:
the sgRNA is sgRNA1 and/or sgRNA 2;
the target sequence of the sgRNA1 is a sequence 3; the target sequence of the sgRNA2 is sequence 4.
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