Application of a protein related to rice blast resistance and its coding gene in rice blast resistance

Abstract

The present invention discloses the application of a protein related to rice blast resistance and its encoding gene in rice blast resistance, which relates to the technical fields of cell biology and genetic engineering. The amino acid sequence of the protein is shown as SEQ ID NO.4; the nucleotide sequence of the encoding gene is shown as SEQ ID NO.3. The present invention isolated and cloned a protein OsVPS20 located in the multivesicular body from rice, and by using the artificial small RNA-mediated gene interference method, by designing and expressing an artificial small RNA specifically targeting the OsVPS20 gene in rice to inhibit the expression of the OsVPS20 gene, the technical effect of significantly improving the blast resistance of rice was achieved. The present invention provides a new method for improving the blast resistance of rice and has important agricultural application value.

Description

Technical Field

[0001] The present invention relates to the technical fields of cell biology and genetic engineering, and particularly relates to the application of a protein related to rice blast resistance and its coding gene in rice blast resistance. Background Art

[0002] Rice blast seriously endangers rice production. Exploring rice disease-resistant or disease-resistance-related genes, analyzing the molecular mechanism of disease resistance, and cultivating disease-resistant varieties based on this are effective strategies for preventing and controlling rice blast. Researchers at home and abroad have carried out research on plant disease resistance, cloned a series of disease-resistant or disease-resistance-related genes (such as Pigm, Pib, rod1, and bsr-d1, etc.), analyzed the molecular regulatory mechanism of disease resistance, and established and continuously improved the molecular model of plant disease resistance. Applying disease-resistant genes and the theory of disease-resistant molecules to the resistance improvement of crops such as rice, a number of new crop varieties with excellent resistance have been successfully cultivated, making positive contributions to ensuring food security.

[0003] At present, the understanding of the molecular mechanism of crop disease resistance is not yet fully clear. Coupled with the rapid evolution and frequent replacement of the dominant pathogenic populations of pathogens, disease-resistant varieties usually lose their resistance after 3 to 5 years of planting. Therefore, it is still necessary to strengthen the exploration and research of new rice disease-resistant or disease-resistance-related genes to further enrich and improve the molecular regulatory theory of rice disease resistance and guide rice disease-resistant breeding. Multivesicular body trafficking plays an important regulatory role in the disease resistance response of plants, but the role of this process in rice disease resistance is still unclear. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of a protein related to rice blast resistance and its coding gene in rice blast resistance to solve the problems existing in the above-mentioned prior art. The invention research found a protein OsVPS20 located in the multivesicular body. By inhibiting the expression of the OsVPS20 gene, the resistance of rice to rice blast can be significantly improved.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] A protein related to rice blast resistance, the amino acid sequence is as shown in SEQ ID NO.4.

[0007] The present invention also provides the coding gene of the above protein, the nucleotide sequence is as shown in SEQ ID NO.3.

[0008] The present invention also provides an artificial small RNA for inhibiting the expression of the above coding gene, the nucleotide sequence is as shown in SEQ ID NO.12.

[0009] The present invention also provides a precursor sequence of artificial small RNA for inhibiting the expression of the above-mentioned coding gene, and the nucleotide sequence is as shown in SEQ ID NO.11.

[0010] The present invention also provides a recombinant vector comprising the above-mentioned precursor sequence of artificial small RNA.

[0011] Furthermore, the recombinant vector is a recombinant pCAMBIA2300-35S vector.

[0012] The present invention also provides a recombinant host cell comprising the above-mentioned recombinant vector.

[0013] Furthermore, the recombinant host cell is recombinant Agrobacterium.

[0014] The present invention also provides the application of a biological material for inhibiting the expression level of the above-mentioned coding gene in rice in improving the blast resistance of rice, and the biological material is the above-mentioned precursor sequence of artificial small RNA, recombinant vector or recombinant host cell.

[0015] The present invention also provides a method for improving the blast resistance of rice, comprising the step of transforming a biological material for inhibiting the expression level of the above-mentioned coding gene in rice into a rice plant to obtain a transgenic rice with the expression of the coding gene inhibited.

[0016] The present invention discloses the following technical effects:

[0017] The present invention isolated and cloned a protein OsVPS20 located in the multivesicular body from rice. By using the gene interference method mediated by artificial small RNA, by designing and expressing an artificial small RNA specifically targeting the OsVPS20 gene in rice to inhibit the expression of the OsVPS20 gene, the technical effect of significantly improving the blast resistance of rice is achieved, and the technical problems of the lack of understanding of the regulation of blast resistance by the multivesicular body protein in rice and how to use the multivesicular body protein to improve the blast resistance of rice are solved.

[0018] The present invention provides a new method for improving the blast resistance of rice and has important agricultural application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1To observe the results of subcellular localization of the OsVPS20 protein; among them, A is a schematic diagram of the protein expression vector 35S-OsVPS20-eGFP fusing OsVPS20 with eGFP constructed in the present invention; B is a laser confocal microscopy observation result diagram of co-expressing the OsVPS20-eGFP green fluorescent protein and the multivesicular body marker protein ARA6-RFP red fluorescent protein in tobacco, and the white arrows indicate some punctate fluorescence of co-localization; C is a laser confocal microscopy observation result diagram of co-expressing the eGFP green fluorescent protein and the multivesicular body marker protein ARA6-RFP red fluorescent protein in tobacco; the scale bars in B and C are both 20 μm;

[0021] Figure 2 Schematic diagram for constructing the interference vector of the OsVPS20 gene; among them, A is a schematic diagram of the template and primer distribution for constructing the precursor of the artificial small RNA amiROsVPS20; B is a schematic diagram of the DNA fragment obtained by 3 times of PCR amplification using primers; C is a schematic diagram of obtaining the precursor sequence of the artificial small RNA amiROsVPS20 by overlapping PCR using primers; D is a schematic diagram of the target plasmid 35S-amiROsVPS20 obtained by constructing the precursor sequence of the artificial small RNA amiROsVPS20 into the plant expression vector through homologous recombination;

[0022] Figure 3 Phenotype observation diagram (A) of transgenic rice and statistical chart (B) of the expression level of the OsVPS20 gene; the scale bar in A is 10 cm; ** in B indicates extremely significant difference compared with the control Kitaake (T test, P≤0.01);

[0023] Figure 4 Results of the identification of blast resistance of transgenic rice with inhibited OsVPS20 gene expression; among them, A is a diagram of the blast disease phenotype of the control Kitaake and 3 transgenic lines 6 days after inoculation by puncturing; B is a statistical chart of the lesion length of blast disease of the control Kitaake and 3 transgenic lines 6 days after inoculation by puncturing, and ** indicates extremely significant difference compared with the control Kitaake (T test, P≤0.01). Detailed implementation manners

[0024] The various exemplary implementation manners of the present invention will be described in detail below. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0025] It should be understood that the terms used in this invention are only for describing specific embodiments and are not intended to limit the invention. Additionally, for the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0028] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0029] The culture medium formulations used in the following examples are as follows:

[0030] LB liquid medium: Tryptone 10 g / L, Yeast extract 5 g / L, and Sodium chloride 10 g / L.

[0031] LB solid medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride 10 g / L, and Agar powder 13 g / L.

[0032] The formulations of the respective culture media obtained above are as follows:

[0033] Basic medium: MS and N6 powder (produced by Phytotechnology Lab).

[0034] Callus induction medium (1 L): N6 (4 g), Sucrose (30 g), Inositol (0.1 g), Acid-hydrolyzed casein (0.3 g), Proline (0.5 g), Glutamine (0.5 g), 2,4-D (2 mg), Phytagel (4 g), adjust pH = 5.8.

[0035] Infection solution (1 L): Yeast extract (3 g), Tryptone (5 g). After autoclaving, add 40 mg of acetosyringone and adjust the pH to 5.5.

[0036] Co-culture medium (1 L): MS (4.4 g), Sucrose (30 g), 2,4-D (2 mg), D-Sorbitol (50 g), Phytagel (4 g). Adjust the pH to 5.8. After autoclaving, add 40 mg of acetosyringone.

[0037] Selection medium (1 L): MS (4.4 g), Sucrose (30 g), 2,4-D (2 mg), Phytagel (4 g). Adjust the pH to 5.8. After autoclaving, add 400 mg of carbenicillin and 50 mg of Geneticin (G418).

[0038] Pre-differentiation medium (1 L): MS (4.4 g), Sucrose (30 g), D-Sorbitol (50 g), 6-BA (3 mg), NAA (0.5 mg), Phytagel (4 g). Adjust the pH to 5.8. After autoclaving, add 200 mg of carbenicillin and 25 mg of G418.

[0039] Re-differentiation medium (1 L): MS (4.4 g), Sucrose (30 g), D-Sorbitol (50 g), 6-BA (2 mg), NAA (0.05 mg), Phytagel (4 g). Adjust the pH to 5.8. After autoclaving, add 200 mg of carbenicillin and 25 mg of G418.

[0040] Rooting medium (1 L): MS (2.2 g), Sucrose (30 g), Phytagel (4 g). Adjust the pH to 5.8. After autoclaving, pour it into tissue culture bottles.

[0041] The vector pCAMBIA2300-35S-eGFP used in the following examples has been disclosed in the literature "A phosphofructokinase B-type carbohydrate kinase family protein, PFKB1, is essential for chloroplast development at early seedling stage in rice".

[0042] Example 1

[0043] 1. Isolate and purify the DNA fragment of the complete coding region of the OsVPS20 gene

[0044] 1.1 Extraction of total RNA from rice leaves (Trizol method)

[0045] First, pour alcohol into a mortar, ignite it, and remove RNAase in the mortar at high temperature. After the mortar cools down, add liquid nitrogen to freeze and grind the sample. Take 100 mg of the ground leaf sample of Oryza sativa ssp. japonica cv. Nipponbare and put it into a 1.5 mL EP tube, then add 1 mL of Trizol reagent. After the sample powder is fully mixed with the Trizol solution, let it stand at room temperature for 5 min to completely separate the nucleoprotein complex. Add 0.2 mL of chloroform, shake vigorously for 20 s, and let it stand at room temperature for 3 min; centrifuge at 12000 g at 4 °C for 15 min. The RNA is completely dissolved in the upper aqueous phase. Transfer the upper aqueous phase to a new 1.5 mL EP tube (about 0.6 mL); add an equal volume of chloroform again, shake for 20 s, centrifuge at 12000 g at 4 °C for 15 min, and transfer the upper aqueous phase to a new 1.5 mL EP tube; add an equal volume of isopropanol, let it stand at room temperature for 10 min, centrifuge at 12000 g at 4 °C for 10 min to precipitate the RNA; remove the supernatant. The RNA adsorbs to the bottom of the tube. Add 0.75 mL of 70% ethanol to wash the precipitate, gently flick it by hand to fully wash the precipitate, centrifuge at 7000 g at 4 °C for 5 min, and remove the supernatant; repeat this step; place the EP tube in a fume hood to let the alcohol evaporate completely. The precipitate becomes transparent. Add 30 μL of RNAase-free water and place it in a 4 °C environment to fully dissolve it for standby.

[0046] 1.2 PCR Amplification and Product Recovery of OsVPS20 Gene Sequence

[0047] In order to obtain the complete DNA fragment encoded by the OsVPS20 gene, the present invention reversely transcribes the total RNA extracted above according to the operation instructions of the SuperScript TM III Reverse Transcriptase Kit of Invitrogen Corporation to obtain cDNA as the template for PCR amplification. The full-length primers for amplifying the coding region of OsVPS20 are designed according to the reference genome as follows:

[0048] OsVPS20-F: 5’- GAGAGGACAGGGTACCCGGGGATCC ATGGGGAACATCTTCGTGAAG AAGCC-3’ (SEQ ID NO.1, the underlined sequence is the upstream homologous sequence for recombination with the subcellular localization vector);

[0049] OsVPS20-R: 5’- TGCTCACCATGGTACTAGTGTCGAC TGCAGGTAGGGGTTCTTCCAAA ACTT-3’ (SEQ ID NO.2, the underlined sequence is the upstream homologous sequence for recombination with the subcellular localization vector).

[0050] Using the Max Super-Fidelity DNA Polymerase to perform PCR amplification of the OsVPS20 gene sequence (the specific operation steps are carried out according to the instructions provided by the kit). After the amplification is completed, the PCR products are separated by 1.0% agarose gel electrophoresis, and the agarose gel of the target fragment size is cut out under ultraviolet light with a clean scalpel. Recover using an agarose gel DNA recovery kit (Thermo Fisher) to obtain the isolated and purified OsVPS20 gene product.

[0051] 1.3 Gene sequence of OsVPS20

[0052] Send the above-mentioned recovered product to a sequencing company for sequencing, and the nucleotide sequence of the OsVPS20 gene obtained is (SEQ ID NO.3):

[0053] 5’-ATGGGGAACATCTTCGTGAAGAAGCCCAAGGTGACGGACGTGGACAGGGCCAT CCTCACCCTCAAGACGCAGCGCCGGAAGCTCGCGCAGTTCCAGCAGCAGCTTGAGAAGGTGATTGAAGCAGAAAAGGAAGCTGCACGGCAGCTAGTTCAACAAAAGAAGAAAGACAGAGCTTTGATTGCACTGAAGAAGAAGAAAGCACAGGAAGAGCTGCTGAAACAAGTTGACACATGGCAAATGAATGTCGAACAACAGCTGTCAGACATTGAACTAGCAAGTAAACAGAAGGCTGTATTTGACAGCCTAAAAGCTGGAAATGCTGCTCTTAAGTCTATCCAGAATGAGATAAATATTGATGATGTCCAAAAGTTGATGGACGACACAGCTGAGGCCAAGGCTTACCAAGATGAGATAAATGCTGCTCTGGGTGAGCAGCTATCTGCTGAGGATGAGGAGGCTGTTATGGCTGAATTTGAGAACTTGGAAGCACAGCTTGCCGTTGAGTCTCTACCAGATGCTCCAGTTACTGAAGTACGGCCTGAGGAGAAATCAGAAACACCTGCTGTCACTGAGGCTGCAGAAGACATTGACGAGGTTATTGAGCTTCCTGATGTACCCACTAAGGCACCAGAGAGACCAGAAGCTGCAGAAAAAACAAAAGTTTTGGAAGAACCCCTACCTGCATAG-3’

[0054] The amino acid sequence of the protein encoded by the OsVPS20 gene predicted according to the above sequence is (SEQ ID NO.4):

[0055] MGNIFVKKPKVTDVDRAILTLKTQRRKLAQFQQQLEKVIEAEKEAARQLVQQKKKDRALIALKKKKAQEELLKQVDTWQMNVEQQLSDIELASKQKAVFDSLKAGNAALKSIQNEINIDDVQKLMDDTAEAKAYQDEINAALGEQLSAEDEEAVMAEFENLEAQLAVESLPDAPVTEVRPEEKSETPAVTEAAEDIDEVIELPDVPTKAPERPEAAEKTKVLEEPLPA。

[0056] 2. Construction of OsVPS20 Subcellular Localization Vector

[0057] The plasmid vector pCAMBIA2300-35S-eGFP was double digested with the fast-cutting enzymes BamH I and Sal I (purchased from Thermo Fisher, and the specific usage and dosage refer to the corresponding product manual). The OsVPS20 gene fragment isolated in 1.2 was homologously recombined with the above double-digested vector using a recombinase (ClonExpressII One Step Cloning Kit from Vazyme). The recombinant product was transferred into Escherichia coli DH5α (purchased from Chengdu Qingke Biotechnology Co., Ltd.) by heat shock transformation. Add 600 μL of LB liquid medium to resuscitate for 1 h, and spread it on an LB solid medium plate containing 50 μg / mL kanamycin. Incubate in a 37 °C incubator for 16 h. Pick monoclonal colonies for colony PCR detection, expand the positive colonies for culture and extract the plasmids, and send the plasmids to a sequencing company for sequencing. The vector with correct sequencing was named 35S-OsVPS20-eGFP( Figure 1 in A).

[0058] 3. Observation of Subcellular Localization of OsVPS20 Protein

[0059] To observe the subcellular localization of the OsVPS20 protein, the OsVPS20 subcellular localization vector 35S-OsVPS20-eGFP constructed in 2 and the empty vector pCAMBIA2300-35S-eGFP (35S-eGFP) were respectively transformed into Agrobacterium tumefaciens EHA105 (purchased from Chengdu Qingke Biotechnology Co., Ltd.). The reported multivesicular body localization protein ARA6-RFP was used as a multivesicular body marker to determine whether the OsVPS20-eGFP protein was localized to the multivesicular body.

[0060] The Agrobacterium transformation process of the plasmid vector is as follows: The plasmid vector was transferred into Agrobacterium tumefaciens EHA105 by the liquid nitrogen freeze-thaw method respectively, resuscitated with 600 μL of LB medium for 1 h, and spread on an LB solid medium plate containing 50 μg / mL of kanamycin and rifampicin antibiotics, and cultured in an incubator at 28 °C for 48 h.

[0061] Identification of positive clones and preparation of bacterial liquid for injection: Single colonies on the above-mentioned plate were picked for colony PCR detection, and the positive clones were inoculated into 2 mL of LB liquid medium containing 50 μg / mL of kanamycin and rifampicin antibiotics for enlarged culture for 24 h. The bacterial cells were collected by centrifugation at 4000 g for 2 min at room temperature, and then the bacterial cells were resuspended with 10 mM MgCl2 solution (added with acetosyringone to a final concentration of 200 μM) and adjusted to OD 600 to 0.8, and left standing at room temperature for 3 h for standby.

[0062] The combinations for transient protein expression by injecting tobacco leaves were as follows: In the experimental group, Agrobacterium tumefaciens containing 35S-OsVPS20-eGFP and 35S-ARA6-RFP plasmids were injected respectively; in the control group, Agrobacterium tumefaciens containing 35S-eGFP and 35S-ARA6-RFP plasmids were injected respectively.

[0063] Samples were taken 48 h after injection and observed for protein co-localization under a laser confocal microscope (Leica STELLARIS STED, Germany) (operated according to the microscope operation instructions) and photographed and recorded. The results are shown in Figure 1 Figure B. The results showed that the OsVPS20 protein was localized to the multivesicular body.

[0064] 4. Construction of an interfering vector for inhibiting the expression of the OsVPS20 gene

[0065] In the present invention, the artificial microRNA (amiRNA)-mediated gene interference method was used to inhibit the expression of the OsVPS20 gene. The artificial microRNA amiROsVPS20 specifically inhibiting the OsVPS20 gene was: UUGUUCGACAUUCACUUGCUA (SEQ ID NO.12).

[0066] The mature sequence of miRNA528 in the miRNA528 precursor was replaced with the designed artificial microRNA amiROsVPS20, and then the precursor sequence after the replacement of the mature sequence was cloned into the pCAMBIA2300-35S vector. The specific steps are as Figure 2 shown:

[0067] Using Nipponbare rice cDNA as a PCR template, using the Max Super-Fidelity DNA Polymerase was used to replace the mature sequence of miRNA528 in the miRNA528 precursor to construct the amiROsVPS20 expression precursor sequence. The replacement was carried out based on PCR amplification (the specific operation steps were performed according to the instructions provided by the kit). First, three target DNA fragments with sizes of 121 bp, 88 bp, and 122 bp were amplified using primer pairs amiROsVPS20-F / amiROsVPS20-II, amiROsVPS20-I / amiROsVPS20-IV, and amiROsVPS20-III / amiROsVPS20-R, respectively. Subsequently, using the above three DNA fragments as templates, overlapping PCR amplification was performed with the primer pair amiROsVPS20-F / amiROsVPS20-R to obtain the precursor sequence of the artificial small RNA amiROsVPS20.

[0068] amiROsVPS20-F: 5'- CAGGGTACCCGGGGATCC CAGCAGCAGCCACAGCAAA-3' (SEQ ID NO.5, the underlined sequence is the upstream homologous sequence for recombination with the target vector);

[0069] amiROsVPS20-II: 5'-TG TAGCAAGTGAATGTCGAACAA CTGCTGCTGCTACAGCC-3' (SEQ ID NO.6, the underlined sequence is the introduced amiROsVPS20 design);

[0070] amiROsVPS20-I: 5'-AG TTGTTCGACATTCACTTGCTA CAGGAGATTCAGTTTGA-3' (SEQ ID NO.7, the underlined sequence is the introduced amiROsVPS20 design);

[0071] amiROsVPS20-IV: 5'-AA TTGTTCGACATACACATGCTA AGAGAGGCAAAAGTGAA-3' (SEQ ID NO.8, the underlined sequence is the introduced amiROsVPS20 design);

[0072] amiROsVPS20-III: 5'-CT TAGCATGTGTATGTCGAACAA TTCCTGCTGCTAGGCTG-3' (SEQ ID NO.9, the underlined sequence is the introduced amiROsVPS20 design);

[0073] amiROsVPS20-R: 5'- GCCCTGGCATGCCTGCAGGCTGCTGATGCTGATGCCAT-3' (SEQ ID NO.10, the underlined sequence is the downstream homologous sequence for recombination with the target vector).

[0074] Precursor sequence of artificial small RNA amiROsVPS20 (SEQ ID NO.11):

[0075] (The single-underlined sequence is the artificially designed small RNA amiROsVPS20 specifically targeting OsVPS20, and the double-underlined sequence is the sequence paired with amiROsVPS20).

[0076] The plasmid vector pCAMBIA2300-35S-eGFP was double-digested with the fast-cutting enzymes BamH I and Pst I to remove the eGFP sequence; the obtained precursor sequence of artificial small RNA amiROsVPS20 was subjected to homologous recombination with the above double-digested vector using a recombinase (Vazyme's ClonExpress II One Step Cloning Kit). The recombinant product was transformed into Escherichia coli by the method in 2 and positive clones were detected by colony PCR. After correct sequencing, the plasmid was amplified and extracted to obtain the artificial small RNA interference vector 35S-amiROsVPS20.

[0077] 5. Construction of rice plants with inhibited OsVPS20 gene expression

[0078] The artificial small RNA interference vector 35S-amiROsVPS20 obtained in 4 was transferred into Agrobacterium tumefaciens EHA105 (purchased from Chengdu Tsingke Biotechnology Co., Ltd.) by the liquid nitrogen freeze-thaw method. Single colonies were picked for colony PCR detection, and the positive clones were stored at -80 °C for later use.

[0079] Using the Agrobacterium-mediated genetic transformation method, the above positive clone Agrobacterium tumefaciens (carrying 35S-amiROsVPS20) was transferred into japonica rice Kitaake to inhibit the expression of the OsVPS20 gene. The Agrobacterium-mediated rice genetic transformation method is as follows:

[0080] 1) Inducing callus: Shell the seeds, weigh about 24 g of seeds, treat them with 50 mL of 75% alcohol for 2 min, and wash them twice with sterile distilled water; disinfect them with 2.5% sodium hypochlorite for 20 min, then try to wash off the sodium hypochlorite on the seed surface with sterile distilled water, and try to suck off the liquid on the seed surface with sterile filter paper; place the disinfected seeds evenly on the callus induction medium with forceps, and then put them in an incubator at 30 °C for dark culture. It takes about 10 d. Then peel the callus from the seeds for the next operation;

[0081] 2) Agrobacterium tumefaciens infecting rice callus: The Agrobacterium tumefaciens carrying the recombinant plasmid taken out from the -80 °C refrigerator is streaked on the YM solid medium (containing Kan / Rif / Gm), cultured at 28 °C for 2 d, and then a small amount of bacteria is picked into 3 mL of YM liquid medium (containing Kan / Rif / Gm), cultured overnight at 28 °C and 200 rpm; suck 1 mL of the bacterial liquid into 50 mL of the infection medium, culture at 8 °C and 200 rpm, and the OD does not exceed 0.1; soak the peeled callus in the infection liquid, culture at 28 °C and 140 rpm for 30 min; pour out the infection liquid, transfer the callus to sterile filter paper, and try to dry the infection liquid on the callus surface; then transfer the callus to the co-culture medium and culture it in the dark at 22 °C for 3 d.

[0082] 3) Screening and differentiation: Transfer the callus to the screening medium, culture it in the dark at 30 °C for at least 30 d, and change the medium once in the middle; then transfer the callus to the pre-differentiation medium, culture it under light at 30 °C for 15 d, and then transfer the callus to the re-differentiation medium, culture it under light at 30 °C until green buds emerge;

[0083] 4) Rooting: Transfer the differentiated buds to the rooting medium, culture them under light at 30 °C. When the seedlings grow to about 10 cm, harden them off, and then transplant them into the soil.

[0084] The obtained plants are amplified for the G418 resistance gene carried by the 35S-amiROsVPS20 plasmid by PCR to determine whether the plants are positive transformants. Further, the expression level of OsVPS20 in the transgenic materials is detected by fluorescence real-time quantitative PCR (qRT-PCR). The specific method is as follows:

[0085] Referring to the method described in [1], total RNA was extracted from 35S-amiROsVPS20 transgenic rice and reverse-transcribed into cDNA. After the reverse transcription was completed, the obtained cDNA was diluted 5-fold for standby. The expression level of OsVPS20 was detected using the specific quantitative primers for OsVPS20, OsVPS20RT_F / OsVPS20RT_R, with the rice housekeeping gene OsUbi (LOC_Os03g13170) as the internal reference, and a fluorescence quantitative detection kit RT 2 Green qPCR Master mixes (purchased from QIAGEN) was used for detection, and the reaction system was prepared according to the following table:

[0086] Table 1 Reaction system

[0087]

[0088]

[0089] Reaction conditions: 95°C, 3 min; 95°C, 15 s; 60°C, 20 s; 40 cycles, and finally 72°C, 20 s. Using the rice gene OsUbi as the internal reference gene, with three biological replicates, the expression level was calculated by the 2 -△△Ct method.

[0090] The primer sequence information used in this step is as follows:

[0091] OsVPS20RT_F: 5’-TGAAGTACGGCCTGAGGAGA-3’ (SEQ ID NO.13);

[0092] OsVPS20RT_R: 5’-TCTCTGGTGCCTTAGTGGGT-3’ (SEQ ID NO.14);

[0093] OsUbi_F: 5’-GCCCAAGAAGAAGATCAAGAAC-3’ (SEQ ID NO.15);

[0094] OsUbi_R: 5’-AGATAACAACGGAAGCATAAAAGTC-3’ (SEQ ID NO.16).

[0095] As Figure 3 shown, the present invention successfully constructed 3 transgenic lines (#1, #2, #3) with inhibited expression of the OsVPS20 gene. The qRT-PCR detection results showed that the expression of the OsVPS20 gene in these 3 transgenic lines was significantly inhibited.

[0096] 6. Identification of Blast Resistance of Rice Plants with Inhibited OsVPS20 Gene Expression

[0097] The wild-type rice cultivation materials and genetically transformed seedlings involved in the present invention were all planted in the rice cultivation greenhouse of the Transgenic Base of Sichuan Agricultural University. Three transgenic lines (#1, #2, #3) with inhibited OsVPS20 gene expression were selected for blast resistance testing.

[0098] The blast resistance test was carried out by the method of punching and dripping bacteria on excised leaves. The specific steps were as follows: 15 penultimate leaves of the wild-type Kitaake and the transgenic lines (#1, #2, #3) with inhibited OsVPS20 gene expression that had grown for 3 weeks were taken respectively. The leaves were cut into the same length. Two points with the same distance were punched on each leaf using a 10 μL pipette tip. The leaves were placed in a petri dish containing 0.1% 6-benzylaminopurin (6-BA, pH = 7.0) solution to float on the liquid surface. A 10 μL pipette was used to suck the prepared Magnaporthe oryzae spore suspension (5×10 5 mL -1 spores of Magnaporthe oryzae Zhong10-8-14) and dropped on the punched places, 4 μL of the suspension for each point. After inoculation, it was cultured in the dark at 28 °C for 24 h, and then cultured alternately with 12 h of light / 12 h of darkness at 28 °C for 5 days. After a total of 6 days, the lesion lengths were measured and counted to compare and identify the blast resistance differences between the transgenic rice and the control.

[0099] The results of the blast resistance identification of the transgenic rice with inhibited OsVPS20 gene expression are shown in Figure 4 . The results showed that compared with the control, the blast lesion lengths of the three transgenic lines were significantly shortened, indicating that the blast resistance of rice can be improved by inhibiting the expression of the OsVPS20 gene.

[0100] The embodiments described above are only for describing the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An artificial small RNA for inhibiting the expression of a gene encoding rice blast resistance, characterized in that: The artificial small RNA nucleotide sequence is shown in SEQ ID NO.12; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

3.

2. An artificial small RNA precursor sequence for inhibiting the expression of a coding gene related to rice blast resistance, characterized in that: The nucleotide sequence of the artificial small RNA precursor sequence is shown in SEQ ID NO.11; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

3.

3. A recombinant vector, characterized in that: Comprising the artificial small RNA precursor sequence of claim 2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector is a recombinant pCAMBIA2300-35S vector.

5. A recombinant host cell, characterized in that Comprising the recombinant vector according to claim 3 or 4.

6. The recombinant host cell according to claim 5, characterized in that The recombinant host cell is a recombinant Agrobacterium.

7. Use of a biological material for inhibiting the expression of a gene encoding rice resistance to rice blast in improving rice resistance to rice blast, characterized in that: The biological material is the artificial small RNA precursor sequence according to claim 2, the recombinant vector according to claim 3 or 4, or the recombinant host cell according to claim 5 or 6.

8. A method for improving rice resistance to rice blast, characterized in that: The method comprises the steps of transforming a biological material for inhibiting the expression of a coding gene related to rice blast resistance into a rice plant to obtain a transgenic rice in which the expression of the coding gene is inhibited; The biological material is the artificial small RNA precursor sequence according to claim 2, the recombinant vector according to claim 3 or 4, or the recombinant host cell according to claim 5 or 6.

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