Application of rice LOCOs06g20960 protein in regulation and control of plant disease resistance

Abstract

The invention relates to the technical field of biology, in particular to application of rice LOCOs06g20960 protein to regulation and control of plant disease resistance. The invention discloses application of a substance for protecting the protein or regulating the expression substance of the coding gene of the protein or regulating the activity or content of the protein in any one of Y1-Y5: application of Y1 in regulating the disease resistance of plants; the Y2 is applied to preparation of a product for regulating and controlling plant disease resistance; the Y3 is applied to cultivation of plants with changed disease resistance; the Y4 is applied to preparation of products for cultivating plants with changed disease resistance; the invention discloses application of Y5 in plant breeding. It is found that the rice LOCOs06g20960 protein has the function of positively regulating and controlling the bacterial blight resistance of rice, the bacterial blight resistance of the rice can be remarkably improved by transferring the LOCOs06g20960 gene overexpression sequence into the rice, and the rice LOCOs06g20960 gene overexpression sequence is of great significance to cultivation of new bacterial blight resistant rice varieties.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of rice LOC_Os06g20960 protein in regulating plant disease resistance. Background Art

[0002] Xanthomonas oryzae Xanthomonas oryzae pv. oryzae ( Xoo Rice bacterial blight, caused by the bacterial blight of rice (Oryza sativa L.), is a major bacterial disease of rice cultivation worldwide. It can cause a yield reduction of approximately 10% in typical years, and 50%-60% in severe cases. Breeding resistant varieties using resistance genes is currently the most cost-effective and effective means of combating rice bacterial blight. To date, 49 rice bacterial blight resistance genes have been reported domestically and internationally (http: / / www.shigen.nig.ac.jp / rice / oryzabase / gene / list). However, resistance genes derived from wild rice are difficult to utilize; some only confer resistance in adult plants, and most have a narrow spectrum of resistance. The pathogen of X. oryzae is complex and highly variable. Production practice has shown that widespread adoption of resistant varieties carrying a single major gene can lead to the emergence of potentially virulent races as dominant ones or the emergence of new virulent races, which can easily lead to a loss of resistance in the variety.

[0003] With the development of molecular biology techniques, disease-resistance genes can be introduced into susceptible varieties through hybridization or genetic modification, improving plant disease resistance. Alternatively, disease-susceptibility genes can be edited to improve plant disease resistance. Therefore, identifying genes that confer resistance or susceptibility to rice bacterial blight is crucial for improving rice resistance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to improve the disease resistance of rice, especially the resistance to rice bacterial blight.

[0005] In order to solve the problems existing in the prior art, the present invention provides the use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in any one of Y1-Y5: Application of Y1 in regulating plant disease resistance; Application of Y2 in the preparation of products for regulating plant disease resistance; Use of Y3 in breeding plants with altered disease resistance; Use of Y4 in the preparation of products for breeding plants with altered disease resistance; Application of Y5 in plant breeding; The protein is named LOC_Os06g20960 and is the following A1, A2 or A3 protein: A1, a protein having an amino acid sequence of SEQ ID NO: 3; A2. A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO: 3 in the sequence listing, which has more than 80% identity with the protein of A1) and is associated with resistance to bacterial blight of rice in plants; A3: A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

[0006] In the above applications, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, the blastp program can be used with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0007] In the above applications, the above 80% or greater identity may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0008] In the above applications, the protein tags include but are not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0009] In the above application, the protein may be derived from rice.

[0010] In the above application, regulating the expression of the gene encoding the protein can be at least one of the following six regulatory factors: 1) regulation at the transcriptional level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the cell nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0011] In the present invention, the substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein may be a substance that upregulates, enhances, or increases the expression of the gene encoding the protein or a substance that upregulates, enhances, or increases the activity or content of the protein. The substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein may also be a substance that downregulates, weakens, or reduces the expression of the gene encoding the protein or a substance that downregulates, weakens, or reduces the activity or content of the protein.

[0012] In the above application, the substance that upregulates, enhances or increases the expression of the gene encoding the protein or the substance that upregulates, enhances or increases the activity or content of the protein may be any one of c1-c7: c1) a nucleic acid molecule encoding the above protein; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) a transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) transgenic plant tissue containing the nucleic acid molecule described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1) or a transgenic plant organ containing the expression cassette described in c2).

[0013] In the above application, the nucleic acid molecule in c1) is any one of the following DNA molecules: d1) the nucleotide sequence is a DNA molecule shown in SEQ ID NO: 1; d2) The CDS sequence is the DNA molecule shown in SEQ ID NO: 2 in the sequence listing; d3) a DNA molecule that is 90% or more identical to the nucleotide sequence defined in d1) or d2), is derived from rice, and encodes the LOC_Os06g20960 protein; d4) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the above-mentioned LOC_Os06g20960 protein.

[0014] Optionally, the recombinant vector in c3) is a plasmid having the DNA molecule shown in SEQ ID NO: 1 or SEQ ID NO: 2, such as pMDC43-LOC_Os06g20960 prepared in the following example.

[0015] The vectors described herein are well known to those skilled in the art, including but not limited to plasmids, phages (such as lambda phage or M13 filamentous phage), cosmids (ie, cosmids), Ti plasmids or viral vectors.

[0016] Existing plant expression vectors can be used to construct a recombinant expression vector containing the LOC_Os06g20960 gene. Such plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. Such plant expression vectors may also contain the 3' untranslated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor. Examples include, but are not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the rouge synthase Nos gene) and the 3' transcribed untranslated region of plant genes (such as the soybean storage protein gene), all of which have similar functions.

[0017] When using the LOC_Os06g20960 gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription start nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the ubiquitin promoter of corn. They can be used alone or in combination with other plant promoters. In addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are extensive and can be natural or synthetic. The translation initiation region can come from a transcription initiation region or a structural gene.

[0018] To facilitate identification and screening of transgenic plant cells or plants, the plant expression vectors used can be modified to include, but are not limited to, genes encoding enzymes or luminescent compounds that can be expressed in plants (such as the GUS gene or luciferase gene), antibiotic resistance markers (such as gentamicin and kanamycin), or chemical resistance marker genes (such as herbicide resistance genes). For safety reasons, it is possible to omit any selectable marker genes and directly screen for transformed plants using stress.

[0019] The LOC_Os06g20960 gene or gene fragment provided by the present invention can be introduced into plant cells or recipient plants using any vector capable of directing exogenous gene expression in plants, resulting in transgenic cell lines and transgenic plants with altered disease resistance. Expression vectors carrying the LOC_Os06g20960 gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transfection, and the transformed plant tissues can be cultivated into plants.

[0020] In the above application, the substance that downregulates, weakens or reduces the expression of the gene encoding the protein or the substance that downregulates, weakens or reduces the activity or content of the protein may be the following F1, F2 or F3: F1, sgRNA, siRNA, shRNA, miRNA or antisense RNA targeting the gene encoding the protein; F2, a DNA molecule that produces an sgRNA targeting the gene encoding the protein, a DNA molecule that produces an siRNA targeting the gene encoding the protein, a DNA molecule that produces an shRNA targeting the gene encoding the protein, a DNA molecule that produces an miRNA targeting the gene encoding the protein, or a DNA molecule that produces an antisense RNA targeting the gene encoding the protein; F3, an expression vector that produces an sgRNA targeting the gene encoding the protein, an expression vector that produces an siRNA targeting the gene encoding the protein, an expression vector that produces an shRNA targeting the gene encoding the protein, an expression vector that produces an miRNA targeting the gene encoding the protein, or an expression vector that produces an antisense RNA targeting the gene encoding the protein.

[0021] Optionally, according to the above application, the target sequence of the sgRNA may be as shown in SEQ ID NO: 4. The encoding gene of the sgRNA may be as shown in SEQ ID NO: 6.

[0022] The above-mentioned sgRNA can work in conjunction with the CRISRP / cas9 gene editing tool to achieve efficient site-specific knockout of the rice LOC_Os06g20960 gene, thereby destroying the biological function of the rice LOC_Os06g20960 gene.

[0023] In the above application, the goal of the cultivation is to cultivate plants with high disease resistance, and the disease resistance is resistance to bacterial blight.

[0024] The aforementioned regulation of plant disease resistance may be to increase / decrease plant disease resistance, for example, manifested as shortening / increasing the length of lesions of rice bacterial blight.

[0025] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0026] The invention also provides a method for improving disease resistance of rice.

[0027] The method for improving rice disease resistance provided by the present invention comprises the following steps: promoting the expression of the protein in recipient rice and / or increasing the abundance of the protein to obtain target rice with higher disease resistance than the recipient rice; the disease resistance is resistance to rice bacterial blight.

[0028] In one embodiment of the present invention, the method for improving disease resistance of rice comprises the following steps: (1) constructing a recombinant expression vector comprising the DNA molecule shown in SEQ ID NO: 1 or the DNA encoding the LOC_Os06g20960 gene shown in SEQ ID NO: 2; (2) Transforming the recombinant expression vector constructed in step (1) into a recipient plant (such as a crop or rice); (3) Obtaining disease-resistant plants having higher disease resistance than the recipient plants through screening and identification.

[0029] Herein, the introduction can be carried out by transforming a vector carrying the DNA molecule of the present invention into a host bacterium by any known transformation method such as chemical transformation or electroporation. The introduced DNA molecule can be a single copy or multiple copies. The introduction can be carried out by integrating the exogenous gene into the host chromosome or by expressing it extrachromosomally from a plasmid.

[0030] In the present invention, the purpose of plant breeding may include cultivating disease-resistant plants. The disease resistance is the resistance of plants to bacterial blight.

[0031] The plant described herein may be any one of the following: N1) a monocot or a dicot; N2) a grass; N3) a rice; N4) rice.

[0032] The above-mentioned LOC_Os06g20960 protein, biological materials related to the LOC_Os06g20960 protein, and the above-mentioned substances that regulate the expression of the gene encoding the LOC_Os06g20960 protein may also fall within the scope of protection of the present invention.

[0033] This study discovered that the rice gene LOC_Os06g20960 positively regulates rice resistance to bacterial blight. Overexpression of the LOC_Os06g20960 gene in rice significantly improved resistance to bacterial blight. Gene editing, however, disrupted the biological function of the LOC_Os06g20960 gene, reducing resistance. The LOC_Os06g20960 gene can be used to improve rice resistance to bacterial blight and is of great significance for the development of new rice varieties resistant to bacterial blight. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 PCR identification results of the LOC_Os06g20960 gene overexpression transgenic strain in Example 4 of the present invention; wherein, M is a DNA molecular weight standard (DL2000+DNA marker), P is a positive control, N is a negative control, and OE1 and OE2 are LOC_Os06g20960 overexpression transgenic strains.

[0035] Figure 2 The relative expression analysis results for the LOC_Os06g20960 gene-overexpressing transgenic lines OE1 and OE2 in Example 4 of the present invention are shown. In the figure, Nipponbare is labeled "Nip," OE1 is labeled "OE-1," and OE2 is labeled "OE-2." ** indicates a significance level of P < 0.01, and *** indicates a significance level of P < 0.001.

[0036] Figure 3 This is the sequencing peak diagram of the LOC_Os06g20960 gene-edited strains KO1 and KO2 in Example 4 of the present invention. Nip in the figure is the wild-type Nipponbare sequence.

[0037] Figure 4 This is a nucleotide sequence comparison of the LOC_Os06g20960 gene-edited strains KO1 and KO2 and Nipponbare (Nip) in Example 4 of the present invention.

[0038] Figure 5Lesion lengths of Nipponbare (Nip) and the LOC_Os06g20960 gene-overexpressing transgenic lines OE1 and OE2, as described in Example 5 of the present invention. In the figure, Nipponbare is labeled Nip, OE1 is labeled OE-1, and OE2 is labeled OE-2. ** indicates a significance analysis result of P < 0.01, and *** indicates a significance analysis result of P < 0.001.

[0039] Figure 6 Lesion lengths of Nipponbare (Nip) and the LOC_Os06g20960 gene-edited strains KO1 and KO2, respectively, from Example 5 of the present invention. In the figure, Nipponbare is labeled Nip, KO1 is labeled KO-1, and KO2 is labeled KO-2. *** indicates a significance analysis result of P < 0.001. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0041] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0042] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0043] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

[0044] The vector pGWC in the following examples was obtained from the BioVector NTCC Type Culture Collection.

[0045] The vector pMDC43 in the following examples was obtained from the BioVector NTCC Type Culture Collection.

[0046] In the following examples, Agrobacterium tumefaciens EHA105 was obtained from the BioVector NTCC Type Culture Collection.

[0047] The pYLsgRNA-OsU6a vector and the pYLCRISPR / Cas9Pubi-H vector in the following examples are described in the non-patent document "Ma X, Zhang Q, Zhu Q. et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants, Mol Plant. 2015, 8(8): 1274-1284," which can be obtained from the applicant to repeat the experiments of the present invention.

[0048] The bacterial blight pathogen PXO99A in the following examples is described in the non-patent document "Salzberg SL, Sommer DD, Schatz MC, Phillippy AM, et al. Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. oryzae PXO99A. BMC Genomic, 2008, 9:534", which is available to the public from the applicant for repeating the experiments of the present invention.

[0049] The rice variety Nipponbare in the following examples is described in the non-patent document Wang W, Mauleon R, Hu Z, et al. Genomic variation in 3010 diverse accessions of Asian cultivated rice, Nature, 2018, 557(7703): 43-49. The public can obtain it from the applicant to repeat the experiments of the present invention.

[0050] LOC_Os06g20960 encodes a SAM-dependent carboxyl methyltransferase, designated LOC_Os06g20960. The genomic sequence is shown in SEQ ID No: 1, the CDS sequence is shown in SEQ ID No: 2, and the encoded protein sequence is shown in SEQ ID No: 3.

[0051] SEQ ID No: 1 SEQ ID NO:2 SEQ ID No: 3 MATIQIVHMNPGQGETSYARNSTIQKTAQDRMKPLIEEAVTAFCGVSVPKSMAIADLGCSSSGPNALTLISSTVDAIHRYCMECAQPPPEMCLFLNDLPSNDFNSVAKSLAEFKHSQDVSSHHVVVANMVPGSFYERLFTSDSVHFFCSSISLQWLSKAPEELAKRKIPMYDSDERLRLL NHEIVADAYARQFRKDFTLFLSLRARELVLGGRLIFSLIGRCSSNPASVSTQVWKVVSVALNDMASRGVISKEKFDTFHIPIYAPMENELNGIIEDEGSFQINKAMAHDTFLATDGVLASPNTIAAMVRAVFEPAIVQHFGFSAGIMDDFASVVERLSTTSAVEAEFPLACLCFSLTRAR Example 1 Cloning of Rice LOC_Os06g20960 Gene The coding region sequence of the rice LOC_Os06g20960 gene was obtained as follows: Total RNA was extracted from leaves of the rice variety Nipponbare and cDNA was synthesized using FastKing gDNA Dispelling RTSuperMix (Code: KR118, TIANGEN). Using this cDNA as a template, PCR amplification was performed using primers LOC_Os06g20960-CDS-F: 5'-ATGGCCACCATACAAATTGTC-3' and LOC_Os06g20960-CDS-R: 5'-TCAACGTGCTCTGGTGAG-3', using PrimeSTARGXL DNA Polymerase (Code: R050A, Takara). The amplified product, i.e., the CDS sequence of the LOC_Os06g20960 gene, was obtained, as shown in SEQ ID No: 2. The gene encodes the LOC_Os06g20960 protein, as shown in SEQ ID No: 3.

[0052] Example 2. Construction of rice disease resistance gene LOC_Os06g20960 expression vector and gene editing vector 1. Construction of LOC_Os06g20960 gene expression vector The CDS sequence of the LOC_Os06g20960 gene from Nipponbare was constructed into the expression vector pMDC43 using the Gateway system to generate the expression vector pMDC43-LOC_Os06g20960 containing the LOC_Os06g20960 gene. The steps are as follows: (1) Total RNA was extracted from the rice variety Nipponbare and reverse transcribed to obtain cDNA. The cDNA was used as a template and PCR amplified using forward primers LOC_Os06g20960-CDS-F: 5'-ATGGCCACCATACAAATTGTC-3' (SEQ ID No: 7) and LOC_Os06g20960-CDS-R: 5'-TCAACGTGCTCTGGTGAG-3' (SEQ ID No: 8). The amplified product, i.e., the CDS sequence of LOC_Os06g20960 (shown as SEQ ID No: 2), was obtained and recovered by gel excision.

[0053] (2) The gel-cleaved product obtained in step (1) was subjected to a DNA purification treatment. Specifically, 20 μL of the recovered product was mixed with 20 μL of PCR SuperMix (Code: AS111-11, TRANSGEN BIOTECH) and a PCR reaction was performed. The reaction procedure was: 95°C for 5 min, 72°C for 20 min, and storage at 4°C. The PCR product was then purified and recovered using a standard DNA product purification kit (Code: DP204-02, TIANGEN).

[0054] (3) The recovered product obtained in step (2) was ligated with the vector backbone obtained by digesting the entry vector pGWC with Eam1105 to obtain a recombinant vector with the correct sequence containing the DNA fragment shown in SEQ ID No: 2, which was named the positive entry clone plasmid pGWC-LOC_Os06g20960.

[0055] (4) The positive entry clone plasmid pGWC-LOC_Os06g20960 obtained in step (3) was subjected to LR reaction with the destination vector pMDC43 to obtain a recombinant vector with the correct sequence containing the DNA fragment shown in SEQ ID No: 2, named pMDC43-LOC_Os06g20960.

[0056] LR reaction system: pGWC-LOC_Os06g20960 1 μL (50-100 ng), vector pMDC43 1 μL (50-100 ng), LR enzyme mix 0.5 μL.

[0057] LR reaction conditions: Incubate at 25°C for 6 hours. The reaction system was transformed into Escherichia coli DH5α, and positive clones were screened to obtain the correct expression vector pMDC43-LOC_Os06g20960 containing the LOC_Os06g20960 gene. pMDC43-LOC_Os06g20960 contains the CDS sequence of the LOC_Os06g20960 gene as shown in SEQ ID No: 2 in the sequence listing and the 35S promoter. It can express the fusion protein of the LOC_Os06g20960 protein as shown in SEQ ID No: 3 and GFP, and its expression is driven by the 35S promoter.

[0058] 2. Construction of LOC_Os06g20960 gene knockout vector A recombinant vector for editing the LOC_Os06g20960 gene was constructed using the CRISPR / Cas9 method.

[0059] Target sequence: GTCCTTGAGGGCTCGAGAAC (positions 1816-1835 of SEQ ID No: 1 in the sequence listing, i.e., SEQ ID No: 4); The steps for constructing the sgRNA expression cassette are as follows: 1) Using the pYLsgRNA-OsU6a vector as a template, PCR amplification was performed using primers UF (5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID No: 9)) and U6a-LOC_Os06g20960-R (5'-GTTCTCGAGCCCTCAAGGACGGCAGCCAAGCCAGCA-3' (SEQ ID No: 10)). The DNA fragment with the correct sequence was named U6a-LOC_Os06g20960; 2) Using the pYLsgRNA-OsU6a vector as a template, PCR amplification was performed using primers gR-LOC_Os06g20960-F (5'-TCCTTGAGGGCTCGAGAACGTTTTAGAGCTAGAAAT-3' (SEQ ID No: 11)) and gR-R (5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID No: 12)). The DNA fragment with the correct sequence was named sgRNA-LOC_Os06g20960; 3) U6a-LOC_Os06g20960 and sgRNA-LOC_Os06g20960 were linked together using the overlapping PCR method. Subsequently, Pps-GGL (5'-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG-3' (SEQ ID No: 13)) and Pgs-GGR (5'-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC-3' (SEQ ID No: 14)) were used for PCR amplification and an infusion linker was added. The resulting DNA fragment with the correct sequence was named U6a-sgRNA-LOC_Os06g20960, which is the sgRNA expression cassette. The sequence of U6a-sgRNA-LOC_Os06g20960 is SEQ ID No: 5 in the sequence listing. Positions 43-490 of SEQ ID No: 5 represent the U6a promoter, and positions 491-593 represent the coding sequence of the sgRNA. U6a-sgRNA-LOC_Os06g20960 encodes an sgRNA, and the coding sequence of the sgRNA is SEQ ID No: 6 in the sequence listing.

[0060] Construction of the recombinant vector CRISPR / Cas9-LOC_Os06g20960: The above-mentioned U6a-sgRNA-LOC_Os06g20960 was subjected to homologous recombination ligation reaction with the vector backbone obtained by BsaI digestion of the pYLCRISPR / Cas9Pubi-H vector to obtain the LOC_Os06g20960 gene knockout vector CRISPR / Cas9-LOC_Os06g20960.

[0061] Homologous recombination reaction system: U6a-sgRNA-LOC_Os06g20960 (10-15 ng), pYLCRISPR / Cas9Pubi-H vector digested with BsaI (60-80 ng), 10× CutSmart Buffer 1.5 μL, 10 mM ATP 1.5 μL, BsaI-HF 10 U, T4 DNA ligase 35 U, and add ddH2O to 15 μL.

[0062] The enzyme digestion-ligation reaction protocol was as follows: 37°C for 10 min, 10°C for 5 min, and 20°C for 5 min, for 3 cycles; and 37°C for 3 min, 10°C for 5 min, and 20°C for 5 min, for 10 cycles. The reaction system was transformed into Escherichia coli DH5α, and positive clones were screened. The resulting recombinant vector with the correct sequence was named CRISPR / Cas9-LOC_Os06g20960. CRISPR / Cas9-LOC_Os06g20960 contains the sgRNA expression cassette and can express both sgRNA and Cas9.

[0063] Example 3: Obtaining transgenic rice Using pMDC43-LOC_Os06g20960 and CRISPR and Cas9-LOC_Os06g20960 from Example 2, transgenic rice was prepared using Nipponbare as the recipient plant. The rice variety Nipponbare showed moderate resistance to the Chinese bacterial blight strain PXO99A. The specific steps were as follows: (1) Take out the mature seeds of the plant, remove the shells, and select the plump, smooth and sterile seeds for disinfection.

[0064] (2) Inoculate the sterilized seeds onto the induction medium and culture them in the dark at 28°C for about 14 days. Select the callus tissue with good appearance and good growth ability.

[0065] (3) The recombinant vector pMDC43-LOC_Os06g20960 constructed in Example 2 was introduced into Agrobacterium tumefaciens EHA105 to obtain a recombinant bacterium named EHA105 / pMDC43-LOC_Os06g20960; the recombinant vector CRISPR / Cas9-LOC_Os06g20960 constructed in Example 2 was introduced into Agrobacterium tumefaciens EHA105 to obtain a recombinant bacterium named EHA105 / CRISPR / Cas9-LOC_Os06g20960.

[0066] (4) Take the recombinant bacteria EHA105 / pMDC43-LOC_Os06g20960 and EHA105 / CRISPR / Cas9-LOC_Os06g20960 obtained in step (3), resuspend the bacteria in infection culture medium, and obtain EHA105 / pMDC43-LOC_Os06g20960 bacterial suspension and EHA105 / CRISPR / Cas9-LOC_Os06g20960 bacterial suspension, respectively.

[0067] (5) Soak the Nipponbare callus tissue from step (2) in the bacterial suspension prepared in step (4) and infect for 20 minutes. After infection, discard the bacterial suspension, take the callus tissue, dry it with sterile filter paper, and then place it on the co-cultivation medium and incubate it in the dark at 28°C for 50-55 hours.

[0068] (6) After completing step (5), callus tissue without obvious Agrobacterium on the surface was selected and transferred to antibacterial medium and cultured in the dark at 28°C for 3-4 days.

[0069] (7) After completing step (6), the callus tissue was transferred to the screening medium and cultured in the dark at 28°C for 30 days, with subculture every 10 days.

[0070] (8) After completing step (7), fresh hygromycin-resistant callus tissue was taken and inoculated into a pre-regeneration medium. The cells were cultured in the dark at 28°C for 7 days, and then placed in a light culture room (12 h light / 12 h dark) for another 7 days. The cells were then transferred to a regeneration medium and cultured in the light until regenerated plants were grown to obtain transgenic plants.

[0071] The transgenic plants obtained using the recombinant vector pMDC43-LOC_Os06g20960 were recorded as LOC_Os06g20960 transgenic plants.

[0072] The transgenic plants obtained using the recombinant vector CRISPR / Cas9-LOC_Os06g20960 were recorded as LOC_Os06g20960 gene knockout plants.

[0073] Culture medium and formula used for genetic transformation: Induction medium: CaCl2·2H2O 440mg, KH2PO4 170mg, MgSO4·7H2O 370mg, NH4NO3 1650mg, KNO3 1900mg, KI 0.83mg, CoCl2·6H2O 0.025mg, H3BO3 6.2mg, Na2MoO4·2H2O 0.25mg, MnSO4·4H2O 22.3mg, CuSO4·5H2O 0.025mg, ZnSO4·7H2O 8.6mg, Na2-EDTA·2H2O 37.3mg, FeSO4·7H2O 27.8mg, VB1 0.1mg, VB6 0.5mg, niacin 0.5mg, inositol 100mg, glycine 2mg, 2,4-D 2mg, hydrolyzed casein 2g, maltose 30g, agar 3g, deionized water to 1L.

[0074] Infection medium: Preparation method refers to the reference: Hiei Y, Ohta S, Komari T, et al. Efficient transformation of rice (Oryza sativa, L.) mediated by Agrobacterium, and sequence analysis of the boundaries of the T-DNA [J]. Plant Journal, 1994, 6(2): 271-282. The concentration of acetosyringone in the reference was replaced with 200 μM.

[0075] Co-culture medium: acetosyringone and glucose were added to the induction medium to make the final concentration of acetosyringone in the medium be 200 μM and the final concentration of glucose in the medium be 10 g / L.

[0076] Antibacterial medium: Add cephalosporin to the induction medium to make the final concentration of cephalosporin in the medium be 500 mg / L.

[0077] Screening medium: Add hygromycin and cephalosporin to the induction medium to make the final concentration of hygromycin in the medium be 65 mg / L and the final concentration of cephalosporin in the medium be 500 mg / L.

[0078] Pre-regeneration medium: CaCl2·2H2O 440mg, KH2PO4 170mg, MgSO4·7H2O 370mg, NH4NO3 1650mg, KNO3 1900mg, KI 0.83mg, CoCl2·6H2O 0.025mg, H3BO3 6.2mg, Na2MoO4·2H2O 0.25mg, MnSO4·4H2O 22.3mg, CuSO4·5H2O 0.025mg, ZnSO4·7H2O 8.6mg, Na2-EDTA·2H2O 37.3mg, FeSO4·7H2O 27.8mg, VB1 0.1mg, VB6 0.5 mg, niacin 0.5 mg, inositol 100 mg, glycine 2 mg, hydrolyzed casein 2 g, maltose 30 g, agar 3 g, kinetin 2 mg, naphthaleneacetic acid 1 mg, deionized water to 1 L; before pouring the plate, add hygromycin to a concentration of 50 mg / L.

[0079] Regeneration medium: CaCl2·2H2O 440mg, KH2PO4 170mg, MgSO4·7H2O 370mg, NH4NO3 1650mg, KNO3 1900mg, KI 0.83mg, CoCl2·6H2O 0.025mg, H3BO3 6.2mg, Na2MoO4·2H2O 0.25mg, MnSO4·4H2O 22.3mg, CuSO4·5H2O 0.025mg, ZnSO4·7H2O 8.6mg, Na2-EDTA·2H2O 37.3mg, FeSO4·7H2O 27.8mg, VB1 0.1mg, VB6 0.5 mg, niacin 0.5 mg, inositol 100 mg, glycine 2 mg, hydrolyzed casein 2 g, maltose 30 g, agar 6 g, kinetin 2 mg, naphthaleneacetic acid 1 mg, deionized water to 1 L; before pouring the plate, add hygromycin to a concentration of 50 mg / L.

[0080] Example 4: Identification of transgenic rice 1. Screening and identification of positive LOC_Os06g20960 overexpressing transgenic plants Plants to be tested: Nipponbare (CK) and the LOC_Os06g20960 overexpressing transgenic plants obtained in Example 3.

[0081] Genomic DNA was extracted from the test plants and used as a template for PCR amplification using a primer pair consisting of pMDC43-TF (5'-GCGTGCAACTCGCTGATCAT-3' (SEQ ID No: 15)) and pMDC43-TR (5'-AATCATCGCAAGACCGGC-3' (SEQ ID No: 16)). The pMDC43-LOC_Os06g20960 plasmid was used as a positive control, and the recipient variety Nipponbare was used as a negative control.

[0082] The PCR amplification products were subjected to 1% agarose gel electrophoresis. The positive control (P) and the positive LOC_Os06g20960 overexpression transgenic plants both showed a 1378 bp band, while the negative control (N) could not amplify a positive band. Figure 1 Two positive LOC_Os06g20960 overexpression transgenic lines were selected and designated as OE1 and OE2.

[0083] 2. Identification of overexpressed transgenic rice at the RNA level Total RNA was extracted from LOC_Os06g20960 overexpressing transgenic lines (OE1 and OE2) and wild-type Nipponbare, and reverse transcribed. qRT-PCR was used to detect the expression of LOC_Os06g20960 at the RNA level. The primers used were LOC_Os06g20960-qF: 5'-CTCCTCCATCAGCTTGCAATGG-3' (SEQ ID No: 17) and LOC_Os06g20960-qR: 5'-GGCCGGGTTGCTTGAACAT-3' (SEQ ID No: 18). The internal reference gene was detected using primers UBQ-F: 5'-GCTCCGTGGCGGTATCAT-3' (SEQ ID No: 19) and UBQ-R: 5'-CGGCAGTTGACAGCCCTAG-3' (SEQ ID No: 20). Ubq ( LOC_Os03g13170 ).

[0084] The results are as follows Figure 2 As shown, the expression levels of the LOC_Os06g20960 gene in OE1 and OE2 were significantly upregulated compared with the wild-type Nipponbare (Nip).

[0085] 3. Identification of LOC_Os06g20960 gene knockout plants Plants to be tested: recipient variety Nipponbare (Nip) and LOC_Os06g20960 gene knockout plants obtained in Example 3.

[0086] Genomic DNA was extracted from the test plants and PCR amplified using the genomic DNA as a template with primers LOC_Os06g20960-TF (5'-TCCTCCATCAGCTTGCAATG-3' (SEQ ID No: 21) and LOC_Os06g20960-TR (5'-CGAAATTTCAATGCCCACCG-3' (SEQ ID No: 22). The recipient variety Nipponbare was used as a negative control.

[0087] The obtained PCR amplification product was subjected to 1% agarose gel electrophoresis, and the DNA molecular weight standard showed a band of 541 bp.

[0088] The PCR amplification products were sequenced, and the sequences of the PCR amplification products of the LOC_Os06g20960 gene knockout plants were compared with the negative control. The knockout strain KO1 had 5 nucleotides missing from positions 1723 to 1727 of SEQ ID No: 1, 4 nucleotides missing from positions 1829 to 1832, and nucleotide A inserted at positions 1828 and 1829 in the sequence list. The knockout strain KO2 had 110 nucleotides missing from positions 1723 to 1832 of SEQ ID No: 1 in the sequence list. The other sequences of the LOC_Os06g20960 gene remained unchanged. The sequencing peaks of the knockout strains are shown in FIG. Figure 3 As shown, the sequence changes as Figure 4 shown.

[0089] Example 5: Identification of bacterial blight resistance of transgenic lines The plants to be tested are: Nipponbare (Nip), positive LOC_Os06g20960 overexpression transgenic lines OE1 and OE2, and positive LOC_Os06g20960 gene knockout lines KO1 and KO2.

[0090] 1. After culturing each plant to be tested in a greenhouse for about 25 days, transplant it into a net house for planting. Plant individually, and plant 20 plants per plant.

[0091] 2. In step 1, when the rice plants are in the peak tillering stage, they are inoculated with the Chinese bacterial blight pathogen PXO99A. The rice plants are artificially inoculated using the leaf cutting method. Five leaves are inoculated per plant (the bacterial solution concentration is 1×10 9 cfu / mL), and the inoculum volume of each leaf was equal, 40 μL.

[0092] 3. About 14 days after inoculation, measure the length of the lesions on the leaves of each plant. Each leaf has a lesion along the vein. Measure the length of the lesions on three inoculated leaves of each plant and calculate the average value.

[0093] The statistical results are as follows Figure 5 and Figure 6 The lesion length of Nipponbare (Nip) was 6.0 cm. The lesion lengths of the LOC_Os06g20960-overexpressing transgenic lines OE1 and OE2 were 4.0 cm and 4.5 cm, respectively, both significantly shorter than those of Nipponbare (Nip), indicating that the LOC_Os06g20960 gene can enhance rice resistance to bacterial blight. The lesion lengths of the LOC_Os06g20960-knockout lines KO1 and KO2 were 8.2 cm and 10.5 cm, significantly longer than those of Nipponbare (Nip), indicating that knocking out the LOC_Os06g20960 gene coding region can reduce rice resistance to bacterial blight.

[0094] The above results indicate that the LOC_Os06g20960 gene can positively regulate rice resistance to bacterial blight.

[0095] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in any of Y1-Y5: Application of Y1 in regulating plant disease resistance; Application of Y2 in the preparation of products for regulating plant disease resistance; Use of Y3 in breeding plants with altered disease resistance; Use of Y4 in the preparation of products for breeding plants with altered disease resistance; Application of Y5 in plant breeding; The protein is the following A1, A2 or A3 protein: A1, a protein having an amino acid sequence of SEQ ID NO: 3; A2. A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO: 3 in the sequence listing, which has more than 80% identity with the protein of A1) and is associated with resistance to bacterial blight of rice in plants; A3: A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

2. The use according to claim 1, characterized in that: The substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein is a substance that upregulates, enhances or increases the expression of the gene encoding the protein or a substance that upregulates, enhances or increases the activity or content of the protein; the substance that upregulates, enhances or increases the expression of the gene encoding the protein or the substance that upregulates, enhances or increases the activity or content of the protein is any one of c1-c7: c1) a nucleic acid molecule encoding the protein according to claim 1; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) a transgenic plant cell line containing the nucleic acid molecule described in c1) or a transgenic plant cell line containing the expression cassette described in c2); c6) transgenic plant tissue containing the nucleic acid molecule described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1) or a transgenic plant organ containing the expression cassette described in c2).

3. The use according to claim 2, characterized in that: c1) The nucleic acid molecule is a DNA molecule shown in any of the following: d1) the nucleotide sequence is a DNA molecule shown in SEQ ID NO: 1; d2) The CDS sequence is the DNA molecule shown in SEQ ID NO: 2 in the sequence listing; d3) a DNA molecule having 90% or more identity with the nucleotide sequence defined in d1) or d2), which is derived from rice and encodes the protein of claim 1; d4) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in d1) or d2) and encodes the protein described in claim 1.

4. The use according to claim 1, characterized in that: The substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein is a substance that downregulates, weakens or reduces the expression of the gene encoding the protein or a substance that downregulates, weakens or reduces the activity or content of the protein; The substance that downregulates, weakens or reduces the expression of the gene encoding the protein or the substance that downregulates, weakens or reduces the activity or content of the protein is the following F1, F2 or F3: F1, sgRNA, siRNA, shRNA, miRNA or antisense RNA targeting the gene encoding the protein of claim 1; F2. A DNA molecule that produces an sgRNA targeting the gene encoding the protein of claim 1, a DNA molecule that produces an siRNA targeting the gene encoding the protein of claim 1, a DNA molecule that produces an shRNA targeting the gene encoding the protein of claim 1, a DNA molecule that produces an miRNA targeting the gene encoding the protein of claim 1, or a DNA molecule that produces an antisense RNA targeting the gene encoding the protein of claim 1; F3. An expression vector for producing an sgRNA targeting the gene encoding the protein according to claim 1, an expression vector for producing an siRNA targeting the gene encoding the protein according to claim 1, an expression vector for producing an shRNA targeting the gene encoding the protein according to claim 1, an expression vector for producing an miRNA targeting the gene encoding the protein according to claim 1, or an expression vector for producing an antisense RNA targeting the gene encoding the protein according to claim 1.

5. The use according to claim 4, characterized in that: The target sequence of the sgRNA is shown in SEQ ID NO:

4.

6. The use according to claim 5, characterized in that: The encoding gene of the sgRNA is shown in SEQ ID NO:

6.

7. A method for improving disease resistance of rice, characterized by: The invention promotes the expression of the protein described in claim 1 in the recipient rice and / or increases the abundance of the protein described in claim 1 to obtain the target rice with higher disease resistance than the recipient rice; the disease resistance is resistance to rice bacterial blight.

8. Protein, characterized in that: The protein is the following A1, A2 or A3 protein: The protein is the following A1, A2 or A3 protein: A1, a protein having an amino acid sequence of SEQ ID NO: 3; A2. A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO: 3 in the sequence listing, which has more than 80% identity with the protein of A1) and is associated with resistance to bacterial blight of rice in plants; A3: A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

9. The substance that upregulates, enhances or increases the expression of the gene encoding the protein or the substance that upregulates, enhances or increases the activity or content of the protein according to claim 2 or 3.

10. The substance according to any one of claims 4 to 6 that downregulates, weakens or reduces the expression of the gene encoding the protein, or the substance that downregulates, weakens or reduces the activity or content of the protein.

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