Method for improving rice blast resistance by inhibiting expression of OsTRXh1 gene in rice

Abstract

The invention relates to the technical field of biology, and discloses a method for improving rice blast resistance by inhibiting expression of an OsTRXh1 gene in rice, which comprises the following steps: inhibiting expression of the OsTRXh1 gene in receptor rice to obtain target rice of which the rice blast resistance is higher than that of the receptor rice; and the OsTRXh1 gene is a gene for coding an OsTRXh1 protein. An inoculation experiment shows that compared with receptor rice, the resistance of an OsTRXh1 knockout rice strain to rice blast is enhanced, and the resistance of an OsTRXh1 overexpression rice strain to rice blast is weakened, so that the OsTRXh1 is a gene related to plant disease resistance. The method disclosed by the invention is simple to operate and low in cost, greatly accelerates the breeding process, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to a method for improving rice blast resistance by inhibiting the expression of OsTRXh1 gene in rice in the field of biotechnology. Background Art

[0002] Rice blast is an important fungal disease in rice production, which seriously affects rice yield and quality and threatens food security. Breeding disease-resistant varieties is the most economical and effective measure to prevent and control rice blast. The R gene confers high resistance to rice and has always been a hot topic for scientists. However, there are also some problems with R genes in breeding. For example, most R genes have a narrow resistance spectrum. Under high selection pressure, small species mutation accelerates, causing resistant varieties to lose resistance within a few years. Although the superposition of multiple R genes can increase the resistance spectrum and reduce the probability of being quickly overcome, it usually causes compensatory effects on yield or quality. Therefore, it is necessary to tap into genetic resources with a wide resistance spectrum and less impact on yield.

[0003] In recent years, CRISPR / Cas9 gene editing technology has developed rapidly. This system can use a short RNA (single-guide RNA, sgRNA) to guide the endonuclease Cas9 to cut the target sequence and achieve mutation of the target gene, which greatly expands researchers' research on gene function and was rated by Science as one of the top ten scientific and technological advances in 2013. At present, this system has been successfully applied to food crops such as rice, rice, sorghum, and corn, providing new opportunities for ensuring food security and creating non-GMO products.

[0004] In the process of interaction between plants and pathogens, the effector proteins secreted by pathogens "hijack" or "use" plant genes in different ways, and ultimately achieve further infection of plants. Such plant genes are called susceptibility genes (S genes). If the effector proteins are conserved in different pathogens, they may regulate different diseases. Using CRISPR / Cas9 gene editing technology to mutate such S genes can make plants resistant to a variety of pathogens. In most cases, these genes do not affect the key agronomic traits of crops, which has also become a new method for breeding disease-resistant varieties. At present, multiple susceptibility genes have been reported, such as rice TaPsIPK1, which is a susceptibility gene. CRISPR / Cas9-mediated TaPsIPK1 gene editing mutants can enhance rice's broad-spectrum resistance to stripe rust without affecting agronomic traits. In addition, maize ZmNANMT, rice ROD1, and tomato SlDMR6-1 have also been proven to be susceptibility genes. These genes can confer resistance to different pathogens after mutation. In summary, discovering more new disease-susceptible genes and analyzing related action pathways can provide candidate gene resources for obtaining new disease-resistant plant varieties through gene editing technology. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to improve the resistance of rice to rice blast.

[0006] In order to solve the above technical problems, the first object of the present invention is to provide a method for improving rice blast resistance, comprising the following steps: inhibiting the expression of the OsTRXh1 gene in a recipient rice to obtain a target rice with higher resistance to rice blast than the recipient rice; the OsTRXh1 gene is a gene encoding an OsTRXh1 protein; the OsTRXh1 protein is a protein of the following A1, A2 or A3:

[0007] A1, the amino acid sequence is the protein shown in SEQ ID No.2 in the sequence list;

[0008] A2, a protein related to rice blast resistance obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 2 in the sequence list, having an identity of more than 80% with the protein shown in A1;

[0009] A3: A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1 or A2.

[0010] In the above method, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc. The amino acid sequences of some of the available tags are shown in Table 1.

[0011] Table 1 Tag sequences

[0012] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHQ c-myc 10 EQKLISEEDL

[0013] In the above method, SEQ ID No. 2 consists of 122 amino acid residues.

[0014] In the above method, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the identity value (%) can be obtained.

[0015] In the above method, the above 80% identity can be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0016] In the above method, the improvement of rice blast resistance can be specifically manifested in that the lesion length (lesion area) on the target rice leaves after infection with the rice blast pathogen is shorter (smaller) than that of the recipient rice, and the biomass of the rice blast pathogen on the target rice leaves is less than that of the recipient rice.

[0017] In the above method, the OsTRXh1 gene may specifically be the gene shown in E1 or E2 below:

[0018] E1, the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 1;

[0019] E2. The nucleotide sequence is the DNA molecule at positions 3001-4990 of SEQ ID No.3.

[0020] In the above method, the inhibition of the expression of the OsTRXh1 gene in rice is achieved by gene editing the OsTRXh1 gene in the recipient rice. The gene editing is achieved with the help of the CRISPR / Cas9 system.

[0021] In the above method, the CRISPR / Cas9 system includes a vector expressing Cas9 and sgRNA, and the target sequence of the sgRNA is SEQ ID No. 3, positions 3119-3138 and SEQ ID No. 3, positions 4330-4349 in the sequence list.

[0022] In order to solve the above technical problems, the present invention also provides a plant agent for resisting rice blast, wherein the active ingredient of the agent is a substance that inhibits the expression of the gene encoding the OsTRXh1 protein, reduces the abundance of the OsTRXh1 protein, and / or knocks out the gene encoding the OsTRXh1 protein.

[0023] In the above-mentioned agent for resisting rice blast, the substance contains the following F1, F2 or F3:

[0024] F1, sgRNA, siRNA, shRNA, miRNA or antisense RNA targeting the gene;

[0025] F2, a DNA molecule that produces an sgRNA targeting the gene, a DNA molecule that produces an siRNA targeting the gene, a DNA molecule that produces an shRNA targeting the gene, a DNA molecule that produces an miRNA targeting the gene, or a DNA molecule that produces an antisense RNA targeting the gene;

[0026] F3. an expression vector that produces an sgRNA targeting the gene, an expression vector that produces an siRNA targeting the gene, an expression vector that produces an shRNA targeting the gene, an expression vector that produces an miRNA targeting the gene, or an expression vector that produces an antisense RNA targeting the gene.

[0027] The active ingredients of the above-mentioned anti-rice blast agent may also contain other biological components and / or non-biological components. Those skilled in the art may determine the other active ingredients of the above-mentioned agent according to the disease resistance effect of plants.

[0028] The present invention also provides an OsTRXh1 protein, wherein the OsTRXh1 protein is the following protein A1, A2 or A3:

[0029] A1, the amino acid sequence is the protein shown in SEQ ID No.2 in the sequence list;

[0030] A2, a protein related to rice blast resistance obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 2 in the sequence list, having an identity of more than 80% with the protein shown in A1;

[0031] A3: A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1 or A2.

[0032] The present invention also provides a biological material related to the OsTRXh1 protein, which is any one of the following B1 to B5:

[0033] B1, a nucleic acid molecule encoding the protein OsTRXh1;

[0034] B2, an expression cassette containing the nucleic acid molecule described in B1;

[0035] B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2;

[0036] B4, a recombinant microorganism containing the nucleic acid molecule described in B1, or a recombinant microorganism containing the expression cassette described in B2, or a recombinant microorganism containing the recombinant vector described in B3;

[0037] B5. A transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule described in B1, or a transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the expression cassette described in B2.

[0038] The nucleic acid molecule may be a DNA molecule, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be an RNA molecule, such as mRNA or hnRNA.

[0039] In the above biological material, the nucleic acid molecule described in B1 is the gene shown in E1 or E2 below:

[0040] E1, the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 1;

[0041] E2. The nucleotide sequence is the DNA molecule at positions 3001-4990 of SEQ ID No.3.

[0042] The present invention also protects a CRISPR / Cas9 system, which includes a vector expressing Cas9 and sgRNA, wherein the target sequence of the sgRNA is positions 3119-3138 of SEQ ID No. 3 and positions 4330-4349 of SEQ ID No. 3 in the sequence list.

[0043] Inoculation experiments showed that compared with recipient rice, OsTRXh1 knockout rice lines had enhanced resistance to rice blast, while OsTRXh1 overexpression rice lines had weakened resistance to rice blast, indicating that OsTRXh1 is a gene related to plant disease resistance. The method of the present invention is simple to operate, low in cost, greatly accelerates the breeding process, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is the gene-edited form of OsTRXh1 in the wild-type Nipponbare (NPB) and OsTRXh1 knockout plants (ostrxh1-1 and ostrxh1-3) in Example 1 of the present invention.

[0045] Figure 2The relative expression levels of the OsTRXh1 gene in the wild-type Nipponbare (NPB) and the OsTRXh1 overexpressing plants (OsTRXh1-OE5 and OsTRXh1-OE31) in Example 2 of the present invention, where ** represents the result of the significant difference analysis of P<0.01.

[0046] Figure 3 These are the resistance test results of the wild-type Nipponbare (NPB), two knockout strains (ostrxh1-1 and ostrxh1-3) and two overexpression strains (OsTRXh1-OE5 and OsTRXh1-OE31) in Example 3 of the present invention after inoculation with rice blast fungus. Figure 3 Figure A shows the phenotypes of wild-type Nipponbare (NPB), two knockout strains (ostrxh1-1 and ostrxh1-3), and two overexpression strains (OsTRXh1-OE5 and OsTRXh1-OE31) after inoculation with the P131 strain. Figure 3 Figure B is Figure 3 Statistical results of the lesion area in Figure A. Figure 3 The C diagram is Figure 3 The detection results of rice blast fungus biomass in Figure A. In the figure, ** represents the result of significant difference analysis with P < 0.01.

[0047] Figure 4 The ROS burst levels in the wild-type Nipponbare (NPB), knockout strain (ostrxh1-1) and overexpression strain (OsTRXh1-OE5) at different time points after chitin treatment in Example 4 of the present invention.

[0048] Figure 5 The MAPKs activation levels in the wild-type Nipponbare (NPB), knockout strain (ostrxh1-1) and overexpression strain (OsTRXh1-OE5) at different time points after chitin treatment in Example 5 of the present invention.

[0049] Figure 6 The relative expression levels of the genes related to the course of disease in the wild-type Nipponbare (NPB), the knockout strain (ostrxh1-1) and the overexpression strain (OsTRXh1-OE5) after chitin treatment in Example 6 of the present invention. In the figure, * represents the result of the significant difference analysis as P<0.05, and ** represents the result of the significant difference analysis as P<0.01. The genes related to the course of disease are OsPR1b and OsAOS2. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0051] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0052] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0053] The rice variety Nipponbare in the following examples is a hybrid variety obtained by crossing "Yamabiko" and "Xingfeng" at the Aichi Prefecture Agricultural Experiment Station in Japan in 1957, and was introduced to China from Japan by the Chinese Academy of Agricultural Sciences in 1967. It was approved by Shandong in 1985 and numbered: Jingyin 153. The public can obtain it from China Agricultural University (i.e., the applicant) to repeat the experiment of this application.

[0054] The vector pYL-HU-U3-CCDB in the following examples is a product of Wuhan Boyuan Biotechnology Co., Ltd., which is recorded in the document "Zhao W, Zheng S, Ling H Q. An efficient regeneration system and Agrobacterium-mediated transformation of Chinese upland rice cultivar Handao 297. Plant Cell Tissue & Organ Culture. 2011, 106 (3): 475." The public can obtain it from China Agricultural University (i.e., the applicant) to repeat the experiments of this application.

[0055] The vector pUbi1305 in the following example is recorded in the non-patent document "Liu Y, Zhang X, Yuan G, Wang D, Zheng Y, Ma M, Guo L, Bhadauria V, Peng YL, Liu JA designer rice NLR immune receptor confers resistance to the rice blast fungus carrying noncorresponding avirulence effectors. Proc Natl Acad Sci US A. 2021Nov;118(44):e2110751118.doi:10.1073 / pnas.2110751118.", which can be obtained by the public from China Agricultural University (i.e., the applicant) to repeat the experiments of this application.

[0056] The Magnaporthe oryzae P131 strain in the following examples is a rice blast pathogen, which is recorded in the non-patent literature "Liu H, Lu X, Li M, Lun Z, Yan X, Yin C, Yuan G, Wang X, Liu N, Liu D, Wu M, Luo Z, Zhang Y, Bhadauria V, Yang J, Talbot NJ, Peng YL. Plant immunity suppression by an exo-β-1,3-glucanase and an elongation factor 1α of the rice blast fungus. Nat Commun. 2023 Sep 7; 14 (1): 5491. doi:

[0057] 10.1038 / s41467-023-41175-z.”, the public can obtain it from China Agricultural University (i.e. the applicant) to repeat the experiments of this application.

[0058] The components and concentrations of MS medium used in the following examples are shown in Table 2.

[0059] Table 2 MS medium composition and concentration used

[0060]

[0061]

[0062] The data in the following examples were processed using GraphPad Prism 9 statistical software, and the experimental results were expressed as mean ± standard deviation. The t-test was used for the analysis, and P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated a very significant difference.

[0063] Example 1. Establishment and identification of OsTRXh1 gene knockout strain

[0064] The genomic sequence of the OsTRXh1 gene is shown in SEQ ID No.3, wherein positions 1-3000 are its own promoter, positions 3099-3188 are the first exon, positions 4289-4411 are the second exon, and positions 4493-4648 are the third exon. The CDS sequence of the OsTRXh1 gene is shown in SEQ ID No.1, and the protein it encodes is named OsTRXh1, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0065] SEQ ID No.1

[0066] ATGGCCCGCCGAGGAGGGAGTCGTGATCGCCTGCCACAACAAGGACGAGTTCGACGCCCAGATGACCAAGGCCAAGGAGGCCGGCAAAGTGGTCATAATTGACTTCACTGCTTCCTGGTGTGGCCCTTGCCGCTTCATCGCCCCAGTGTTCGCTGAATACGCCAAAAAGTTCCCTGGTGCTGTCT TCCTGAAGGTTGATGTTGATGAGCTGAAGGAAGTTGCTGAAAAGTACAATGTCGAGGCAATGCCGACCTTCCTATTCATCAAGGATGGTGCTGAGGCTGACAAGGTCGTTGGCGCCAGGAAGGATGACCTCCAGAACACATCGTGAAGCACGTCGGTGCCACTGCTGCATCTGCTTCTGCCTAA

[0067] SEQ ID No.2

[0068] MAAEEGVVIACHNKDEFDAQMTKAKEAGKVVIIDFTASWCGPCRFIAPVFAEYAKKFPGAVFLKVDVDELKEVAEKYNVEAMPTFLFIKDGAEADKVVGARKDDLQNTIVKHVGATAASASA

[0069] SEQ ID No.3

[0070]

[0071] 1. A short sequence on the OsTRXh1 gene was artificially synthesized as a guide RNA (SgRNA), and the target sequences of the SgRNA were CGTGATCGCCTGCCACAACA (SEQ ID No. 3, positions 3119-3138, referred to as target 1) and CGAACACTGGGGCGATGAAG (the reverse complementary sequence of SEQ ID No. 3, positions 4330-4349, referred to as target 2).

[0072] 2. Use restriction endonuclease BsaI to digest vector pYL-HU-U3-CCDB and recover the vector backbone. Connect the coding sequence of SgRNA to the vector backbone of vector pYL-HU-U3-CCDB by T4 ligase to obtain the recombinant vector pYL-HU-U3-OsTRXh1. The structure of the recombinant vector pYL-HU-U3-OsTRXh1 is described as follows: insert the coding sequence of SgRNA into the restriction endonuclease BsaI cleavage site of vector pYL-HU-U3-CCDB, keep the other sequences of vector pYL-HU-U3-CCDB unchanged, and obtain the recombinant vector. The recombinant vector pYL-HU-U3-OsTRXh1 expresses SgRNA and Cas9.

[0073] 3. The recombinant vector pYL-HU-U3-OsTRXh1 was used for Agrobacterium-mediated rice transformation. When the callus tissue of the rice variety Nipponbare was infected, the SgRNA guided the nuclease Cas9 to the target sequence in the transgenic rice cells, completed the gene editing of the target sequence, and the T0 generation transformants were obtained by hygromycin screening.

[0074] 4. Add 60 μL M5 Hiper Ultralight Mix to a 2 mL centrifuge tube containing 5 mm diameter steel beads to directly amplify the best partner. Cut 1 cm of leaves of T0 generation transformants into a 2 mL tube and crush them with a grinder.

[0075] 5. Heat at 65°C for 10 minutes to extract rice DNA, centrifuge at 12000 rpm for 1 minute, take 0.5 μL of supernatant for PCR amplification, and the primers used are as follows:

[0076] ostrxh1-ba1-F:AGAACGCAATCTCATCGCAAG;

[0077] ostrxh1-ba1-R:TCGATCTCCTCGCGATTTCAC.

[0078] ostrxh1-ba2-F: GTATGAAATTCTGAACTAGTGGTACTTATGC;

[0079] ostrxh1-ba2-R: GCAACTTCCTGTTGGATAAAGAAA.

[0080] 6. Compare the sequence of the PCR product with the wild type to identify the gene editing form of the OsTRXh1 knockout rice material. Select several knockout homozygous lines, of which two homozygous lines are numbered ostrxh1-1 and ostrxh1-3. The results are shown in Figure 1 :

[0081] ostrxh1-1: Compared with the wild-type Nipponbare (NPB), the OsTRXh1 gene mutated in the genome of the ostrxh1-1 mutant plant. At target site 2, the two homologous chromosomes lacked the CATC at positions 4333-4336 (located in the second exon) of SEQ ID No.3, a total of 4bp was missing, resulting in a frameshift mutation in the OsTRXh1 gene, which was unable to encode the protein OsTRXh1 with the amino acid sequence of SEQ ID No.2, ultimately causing the loss of OsTRXh1 function, thereby knocking out the OsTRXh1 gene.

[0082] ostrxh1-3: Compared with the wild-type Nipponbare (NPB), the OsTRXh1 gene in the ostrxh1-3 mutant plant genome mutated. At target site 2, the two homologous chromosomes lacked the C at position 4333 (located in the second exon) of SEQ ID No.3, a total of 1bp was missing, resulting in a frameshift mutation in the OsTRXh1 gene, which was unable to encode the protein OsTRXh1 with the amino acid sequence of SEQ ID No.2, ultimately causing the loss of OsTRXh1 function, thereby knocking out the OsTRXh1 gene.

[0083] Example 2: Establishment and identification of OsTRXh1 overexpression strain

[0084] 1. Using the cDNA of the rice variety Nipponbare as a template, the open reading frame sequence of the OsTRXh1 gene was obtained by PCR amplification, and the PCR product was recovered. The primers used are as follows:

[0085] HA-OsTRXh1-Flag-F:5'-GTTCCAGATTACGCTTCT AAGCTT ATGGCCGCCGAGGAGGGA-3' (positions 25-42 are identical to positions 1-18 of SEQ ID No. 1, and the underlined sequence is the Hind III enzyme recognition site sequence);

[0086] HA-OsTRXh1-Flag-R:5'-ATGGTCTTTGTAGTCAGA AAGCTTGGCAGAAGCAGATGCAGCA-3' (positions 25-43 thereof are reverse complementary to positions 348-366 of SEQ ID No. 1, and the underlined sequence is the Hind III enzyme recognition site sequence).

[0087] The PCR product was recovered to obtain the OsTRXh1 gene fragment.

[0088] 2. Cut the vector pUbi1305 with Hind III to obtain the cut vector pUbi1305, connect the OsTRXh1 gene fragment to the cut vector pUbi1305, and obtain the recombinant expression vector pUbi1305-OsTRXh1. After verification by PCR and sequencing of the bacterial solution, the structure of the recombinant expression vector pUbi1305-OsTRXh1 is described as follows: insert the DNA molecule shown in positions 1-366 of SEQ ID NO.1 into the restriction endonuclease Hind III cutting site of the vector pUbi1305, and keep the other sequences of the vector pUbi1305 unchanged to obtain the recombinant expression vector. In the recombinant expression vector pUbi1305-OsTRXh1, the Ubi promoter drives the expression of the OsTRXh1 gene.

[0089] 3. The recombinant expression vector pUbi1305-OsTRXh1 obtained in step 2 was introduced into the Agrobacterium strain EHA105 to prepare the recombinant Agrobacterium EHA105-OsTRXh1 and infect the callus of the rice variety Nipponbare. T0 generation transformants were obtained by hygromycin screening.

[0090] 4. Cut the leaves of wild-type Nipponbare and T0-generation transformants and extract DNA respectively. Use Hyg-F and Hyg-R primers to amplify the DNA of wild-type Nipponbare and T0-generation transformants as templates. The specific sequences of Hyg-F and Hyg-R are as follows:

[0091] Hyg-F: 5'-TTGGCGACCTCGTATTGGGAA-3';

[0092] Hyg-R: 5'-CAAAGATCGTTATGTTTATCGGCACT-3'.

[0093] The obtained PCR fragments were subjected to electrophoresis detection, and the plants that amplified a hygromycin fragment of about 500 bp were OsTRXh1 transgenic positive plants of the T0 generation (ie, OsTRXh1 overexpressing plants).

[0094] 5. Transplant the OsTRXh1 transgenic positive plants of the T0 generation into the soil, self-pollinate to obtain T1 generation seeds, and then self-pollinate to obtain T2 generation seeds after further planting, and analyze the segregation of hygromycin resistance in the T2 generation. Select several overexpression homozygous strains, of which two homozygous strains are numbered OsTRXh1-OE5 and OsTRXh1-OE31.

[0095] 6. Total RNA was extracted from rice, reverse transcribed to obtain cDNA, and qRT-PCR was performed. Using the OsUBQ10 gene as an internal reference, the expression level of the OsTRXh1 gene in the wild-type Nipponbare (NPB) and each overexpression homozygous strain (OsTRXh1-OE5 and OsTRXh1-OE31) was detected by fluorescence quantitative PCR instrument (ABI 7500, USA).

[0096] The primer sequences used are shown in Table 3, and the experiment was repeated three times.

[0097] Table 3 Primer sequences

[0098] Gene name Forward primer 5'-3' Reverse primer 5'-3' OxY ATTGACTTCACTGCTTCCTGGT GCTCATCAACATCAACCTTCAGG OsUBQ10 TGGTCAGTAATCAGCCAGTTTGG GCACCACAAATACTTGACGAACAG

[0099] Results Figure 2 It can be seen that the expression of OsTRXh1 gene in transgenic rice lines was significantly upregulated.

[0100] Example 3: Analysis of the resistance phenotype of rice lines to rice blast in knockout and overexpression of OsTRXh1

[0101] In order to detect the resistance of OsTRXh1 knockout and overexpression rice lines to rice blast fungus, the phenotype was observed by scratch inoculation. The specific steps are as follows:

[0102] 1. The seeds of wild-type Nipponbare, two knockout strains (ostrxh1-1 and ostrxh1-3) and two overexpression strains (OsTRXh1-OE5 and OsTRXh1-OE31) were placed in a 37°C incubator and soaked in water for two days for germination. The germinated seeds were then transferred into small pots with a diameter of 9 cm and cultured in a 30°C culture room for about 20 days until the four-leaf one-heart stage. The greenhouse maintained a relative humidity of 70%, 14 hours of light and 10 hours of darkness per day.

[0103] 2. Activate the Magnaporthe oryzae P131 strain and grow it in an incubator at 28°C for about 7 days.

[0104] 3. Take the penultimate leaf of the rice plant at the four-leaf, one-heart stage in step 1. Take at least 5 leaves from each strain. Cut into 6 to 7 cm lengths and use an insect pin to lightly scratch about 1 mm. 2Three wounds were made on the main vein of each leaf. The scratched leaves were placed in a 10 cm × 10 cm culture dish, and moist filter paper was placed in the culture dish in advance to maintain humidity.

[0105] 4. Rinse the culture dish in step 2 with 0.025% Tween water by volume to collect the spores of strain P131 and adjust the spore solution to 1.5×10 5 A P131 spore suspension was prepared at a concentration of spores / mL.

[0106] 5. Before inoculation, spray the surface of the leaves treated in step 3 evenly with 0.025% Tween water by volume to form a mist to increase the adhesion of the spore suspension. Inoculate 10 μL of P131 spore suspension (see step 4) at each wound on the leaves. Keep the inoculated leaves moisturized in a petri dish and place them in a dark environment for 24 to 36 hours.

[0107] 6. After the dark treatment, move the culture dish to the light condition, observe the lesions and take pictures after 3 to 4 days. The phenotypes of the wild-type Nipponbare, two knockout strains (ostrxh1-1 and ostrxh1-3), and two overexpression strains (OsTRXh1-OE5 and OsTRXh1-OE31) after inoculation with the P131 strain are shown in Figure 3 Figure A.

[0108] 7. Statistical analysis of the length of the lesions is as follows:

[0109] The 12 lesion areas of the wild-type Nipponbare, two knockout lines (ostrxh1-1 and ostrxh1-3) and two overexpression lines (OsTRXh1-OE5 and OsTRXh1-OE31) infected with the P131 strain were measured, and the average values ​​were calculated and the significant differences were calculated using the Student's t test. The results are shown in Figure 3 Figure B.

[0110] 8. Quantification of bacterial biomass in rice leaves:

[0111] For specific implementation methods, please refer to "Yoji Kawano, Akira Akamatsu, Keiko Hayashi, Yusuke Housen, Jun Okuda, Ai Yao, Ayako Nakashima, Hiroki Takahashi, Hitoshi Yoshida, HannLing Wong, Tsutomu Kawasaki, Ko Shimamoto. Activation of a Rac GTPase by the NLRfamily disease resistance protein Pit plays a critical role in rice innateimmunity, Cell Host Microbe,2010,7(5):362-75" article.

[0112] After inoculation with the P131 strain, leaves with a size of 2×1 cm including lesions were taken, and DNA was extracted using the conventional CTAB method. The DNA concentration was measured and quantified to 3 μg.

[0113] Using real-time fluorescence quantitative PCR technology, according to the instructions provided by the manufacturer (Genestar), SYBR green I fluorescent dye was added to the PCR system, and the expression of the rice blast fungus MoPot2 gene was detected in a fluorescence quantitative PCR instrument (ABI 7500, USA), with the rice Ubiquitin gene as an internal reference. The primer sequences used are shown in Table 4. The experiment was repeated three times, and the results are shown in Figure 3 Figure C.

[0114] Table 4 Primer sequences

[0115] Gene name Forward primer 5'-3' Reverse primer 5'-3' MoPot2 ACGACCCGTCTTTACTTATTTGG AAGTAGCGTTGGTTTTGTTGGAT OsUbiqintin TTCTGGTCCTTCCACTTTCAG ACGATTGATTTAACCAGTCCATGA

[0116] Figure 3 The results showed that compared with the wild type, the OsTRXh1 knockout strain had enhanced resistance to rice blast, while the overexpression rice strain had weakened resistance to rice blast, specifically, the area of ​​rice blast lesions on the leaves of rice in the OsTRXh1 knockout strain became smaller and the biomass of rice blast fungus decreased, while the area of ​​rice blast lesions on the leaves of rice in the OsTRXh1 overexpression strain became larger and the biomass of rice blast fungus increased.

[0117] Example 4. ROS burst levels in OsTRXh1 knockout and overexpression rice lines during chitin treatment

[0118] To detect the ROS burst levels in OsTRXh1 knockout and overexpression rice lines when treated with chitin, the specific method is as follows:

[0119] 1. The seeds of wild-type Nipponbare, two knockout strains (ostrxh1-1 and ostrxh1-3) and two overexpression strains (OsTRXh1-OE5 and OsTRXh1-OE31) were placed in a 37°C incubator and soaked in water for two days for germination. The germinated seeds were then transferred into small pots with a diameter of 9 cm and cultured in a 30°C culture room for about 20 days until the four-leaf one-heart stage. The greenhouse maintained a relative humidity of 70%, 14 hours of light and 10 hours of darkness per day.

[0120] 2. Take rice heart slices with good growth condition, make small discs with a 4mm diameter puncher, and transfer them to sterile water to recover for 12-18 hours.

[0121] 3. Add 50 μL of sterile water to each well of the 96-well plate and use a white pipette tip to pick up a small disc and place it into the well.

[0122] 4. Prepare the reaction solution, including 0.02 mM luminol, 20 μg / ml horseradish peroxidase, 10 μg / ml chitin or sterile water, and add it to eight rows of PCR tubes.

[0123] 5. Use a pistol to draw 50 μL of the reaction solution and quickly add it to the hole with the small disc. Place the test plate in the ELISA reader and read the readings every 1 minute for 20-40 minutes until the curve drops gently. The results are shown in the table. Figure 4 .

[0124] Figure 4 The results showed that compared with the wild type, the ROS burst level in the OsTRXh1 knockout rice line ostrxh1-1 was significantly increased, while the ROS burst level in the OsTRXh1 overexpression rice line OsTRXh1-OE5 was significantly decreased.

[0125] Example 5. MAPKs activation levels in OsTRXh1 knockout and overexpression rice lines during chitin treatment

[0126] To detect the MAPKs activation levels in OsTRXh1 knockout and overexpression rice lines under chitin treatment, the specific method is as follows:

[0127] 1. Treat the seeds of wild-type Nipponbare, knockout strain ostrxh1-1 and overexpression strain OsTRXh1-OE5 with 70% alcohol for 3 minutes, wash with 30% laundry detergent for 45 minutes, and then rinse the seeds with plenty of sterile water in a clean bench until there is no foam and dry them.

[0128] 2. Spread the seeds on solid 1 / 2MS medium, culture at 28℃ for 5 to 7 days, and then transfer to liquid 1 / 2MS medium and culture for 2-3 days.

[0129] 3. Treat the seedlings of wild type, OsTRXh1 knockout and overexpression strains with ultrapure water and 10 μg / mL chitin suspension (chitin suspension is prepared with water and stored in a -20°C refrigerator after ultrasound, and the mother solution concentration is 1 mg / mL), and take samples at 0, 15, 30, and 60 minutes, respectively.

[0130] 4. To extract total protein from the sample obtained in step 3, add the phosphatase inhibitor PhosSTOP to the protein extraction buffer. TM .

[0131] 5. The protein content of MAPK3 and MAPK6 and the internal reference protein Actin in the above protein samples was detected by western blotting, using Phospho-p44 / 42 antibody (for detecting the level of phosphorylated MAPKs) and Actin antibody (both are commercial antibodies, Phospho-p44 / 42 antibody catalog number: Cell Signaling Technology 4370; Actin antibody catalog number: EASYBIO BE0027-100).

[0132] Figure 5 The results showed that compared with the wild type, the activation levels of MAPK3 and MAPK6 in the OsTRXh1 knockout rice line ostrxh1-1 were significantly increased, while the activation levels of MAPK3 and MAPK6 in the OsTRXh1 overexpression rice line OsTRXh1-OE5 were significantly decreased.

[0133] Example 6. Expression levels of pathogenesis-related genes in OsTRXh1 knockout and overexpression rice lines during chitin treatment

[0134] To detect the expression levels of pathogenesis-related genes in OsTRXh1 knockout and overexpression rice lines under chitin treatment, the specific steps were as follows:

[0135] 1. Treat the seeds of wild-type Nipponbare, knockout strain ostrxh1-1 and overexpression strain OsTRXh1-OE5 with 70% alcohol for 3 minutes, wash with 30% laundry detergent for 45 minutes, and then rinse the seeds with plenty of sterile water in a clean bench until there is no foam and dry them.

[0136] 2. Spread the seeds on solid 1 / 2MS medium, culture at 28℃ for 5 to 7 days, and then transfer to liquid 1 / 2MS medium and culture for 2-3 days.

[0137] 3. Treat the seedlings of wild-type, OsTRXh1 knockout and overexpression strains with ultrapure water and 10 μg / mL chitin suspension (chitin suspension is prepared with water and stored in a -20°C refrigerator after ultrasound, with a mother solution concentration of 1 mg / mL), respectively, and take samples 6 hours after treatment.

[0138] 4. Extract total RNA from the sample obtained in step 3 and reverse transcribe it to obtain cDNA.

[0139] 5. The expression levels of the disease-related genes OsPR1b and OsAOS2 in the above cDNA samples were detected by fluorescent quantitative PCR instrument (ABI 7500, USA), with OsUBQ10 as the internal reference gene (the detection gene and primer sequences are shown in Table 5), and the sequences are shown in Table 5.

[0140] Table 5

[0141] Gene name Forward primer 5'-3' Reverse primer 5'-3' OsPR1b TACGCCAGCCAGAGGAGC GCCGAACCCCAGAAGAGG OsAOS2 AAGCTGCTGCAATACGTGTACTGG CGACGAGCAACAGCCTTCCG OsUBQ10 TGGTCAGTAATCAGCCAGTTTGG GCACCACAAATACTTGACGAACAG

[0142] Figure 6 The results showed that compared with the wild type, the induction of pathogenesis-related genes in the OsTRXh1 knockout line rice ostrxh1-1 was significantly increased, while the induction of pathogenesis-related genes in the OsTRXh1 overexpression line rice OsTRXh1-OE5 was significantly decreased.

[0143] Based on the above results, knocking out the OsTRXh1 gene can significantly improve the rice resistance to rice blast, while overexpressing the OsTRXh1 gene can significantly reduce the rice resistance to rice blast.

[0144] 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for improving rice blast resistance, characterized in that: The method comprises the following steps: inhibiting the expression of the OsTRXh1 gene in the recipient rice to obtain the target rice with higher resistance to rice blast than the recipient rice; the OsTRXh1 gene is a gene encoding the OsTRXh1 protein; the OsTRXh1 protein is a protein of A1, A2 or A3 as follows: A1, a protein having an amino acid sequence as shown in SEQ ID No. 2 in the sequence list; A2, a protein related to rice blast resistance obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 2 in the sequence list, having an identity of more than 80% with the protein shown in A1; A3: A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1 or A2.

2. The method according to claim 1, characterized in that: The OsTRXh1 gene is the gene shown in E1 or E2 below: E1, the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 1; E2. The nucleotide sequence is the DNA molecule at positions 3001-4990 of SEQ ID No.

3.

3. The method according to claim 1 or 2, characterized in that: The inhibition of the expression of the OsTRXh1 gene in rice is achieved by gene editing of the OsTRXh1 gene in the recipient rice; the gene editing is achieved with the aid of the CRISPR / Cas9 system.

4. The method according to claim 3, characterized in that: The CRISPR / Cas9 system includes a vector expressing Cas9 and sgRNA, wherein the target sequence of the sgRNA is positions 3119-3138 of SEQ ID No. 3 and positions 4330-4349 of SEQ ID No. 3 in the sequence list.

5. An agent for resisting rice blast, characterized in that: The active ingredient of the reagent is a substance that inhibits the expression of the gene encoding the OsTRXh1 protein, reduces the abundance of the OsTRXh1 protein, and / or knocks out the gene encoding the OsTRXh1 protein.

6. The reagent according to claim 5, characterized in that: The substance contains the following F1, F2 or F3: F1, sgRNA, siRNA, shRNA, miRNA or antisense RNA targeting the OsTRXh1 gene of claim 1 or 2; F2. A DNA molecule for producing an sgRNA targeting the OsTRXh1 gene of claim 1 or 2, a DNA molecule for producing an siRNA targeting the OsTRXh1 gene of claim 1 or 2, a DNA molecule for producing an shRNA targeting the OsTRXh1 gene of claim 1 or 2, a DNA molecule for producing an miRNA targeting the OsTRXh1 gene of claim 1 or 2, or a DNA molecule for producing an antisense RNA targeting the OsTRXh1 gene of claim 1 or 2; F3. An expression vector for producing sgRNA targeting the OsTRXh1 gene of claim 1 or 2, an expression vector for producing siRNA targeting the OsTRXh1 gene of claim 1 or 2, an expression vector for producing shRNA targeting the OsTRXh1 gene of claim 1 or 2, an expression vector for producing miRNA targeting the OsTRXh1 gene of claim 1 or 2, or an expression vector for producing antisense RNA targeting the OsTRXh1 gene of claim 1 or 2.

7. The OsTRXh1 protein according to claim 1 or 2.

8. The protein-related biomaterial according to claim 7, characterized in that: Any one of B1 to B5 below: B1. A nucleic acid molecule encoding the protein according to claim 7; B2, an expression cassette containing the nucleic acid molecule described in B1; B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2; B4, a recombinant microorganism containing the nucleic acid molecule described in B1, or a recombinant microorganism containing the expression cassette described in B2, or a recombinant microorganism containing the recombinant vector described in B3; B5. A transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule described in B1, or a transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the expression cassette described in B2.

9. The biomaterial according to claim 8, characterized in that: The nucleic acid molecule B1 is the gene shown in E1 or E2 below: E1, the coding sequence of the coding strand is a DNA molecule of SEQ ID No. 1; E2. The nucleotide sequence is the DNA molecule at positions 3001-4990 of SEQ ID No.

3.

10. CRISPR / Cas9 system, characterized in that: The CRISPR / Cas9 system includes a vector expressing Cas9 and sgRNA, wherein the target sequence of the sgRNA is positions 3119-3138 of SEQ ID No. 3 and positions 4330-4349 of SEQ ID No. 3 in the sequence list.

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