APPLICATION OF THE RICE GENERATION OSIDS1 IN THE REGULATION OF RICE RESISTANCE TO MAGNAPORTHE ORYZAE

Description

However, there are two significant disadvantages: First, rapid mutations of the Avr genes of pathogenic effector proteins lead to a rapid loss of disease resistance. For example, varieties with the Pi-ta gene lose their stable resistance as early as 3 to 5 years after their introduction. Second, the vertical resistance of individual R genes cannot withstand the diversity of 5 physiological races; more than 60% of the currently widespread main varieties exhibit only one or two resistance loci. Third, the genetic linkage of disease resistance traits and agronomic characteristics significantly impairs the efficiency of breeding selection. Although 10-gene pyramiding can delay the onset of resistance, growth inhibition caused by multiple genes limits the practical application of such breeding lines.For this reason, the discovery of novel regulatory factors, especially transcription factors, as well as the elucidation of the mechanisms of action of associated signaling networks, represents the most economical and effective measure for the defense against plant pathogens. CONTENT OF THE INVENTION 20 The purpose of the present invention is to provide a new application of the rice gene OsIDS1 in the regulation of rice resistance to rice smut, thereby creating a theoretical basis and technical support for the breeding of new rice varieties with 25 high rice smut resistance. Investigations have shown that the rice gene OsIDS1 plays a key role in the defense of rice against Magnaportheoryzae. Deletion of the OsIDS1 gene weakens the basic defense capacity of rice against Magnaportheoryzae, whereas overexpression of the 30 OsIDS1 gene increases the resistance of rice to rice smut BE2026 / 7285 3.To achieve the aforementioned objective of the present invention, the present invention is realized by the following technical solutions. The present invention provides an application of the rice gene OsIDS1 in regulating the resistance of rice to Magnaporthe oryzae. The expression level of the OsIDS1 gene or the activity of the protein encoded by it in rice is increased, thereby improving the resistance of rice to rice gangrene; wherein the nucleotide sequence of the OsIDS1 gene is specified by SEQIDNO:210 and the protein encoded by it has an amino acid sequence according to SEQIDNO:1. Preferably, the methods for increasing the expression level of the OsIDS1 gene or the activity of the protein encoded by it comprise the introduction of a recombinant vector with the 15 nucleotide sequence according to SEQIDNO:2 into rice.Preferably, the methods for constructing the recombinant vector comprise: using the WMV023 vector as an expression vector and inserting the nucleotide sequence according to SEQIDNO: 2 between the recognition sites of the restriction enzyme KpnIde of the WMV023 vector, thereby obtaining the recombinant vector. Preferably, rice smut is caused by the strain MagnaportheoryzaeRB22. The present invention further provides a method for breeding rice smut-resistant rice plants. The method comprises the following 25 steps: using the rice variety Zhonghua11 as a recipient, introducing the nucleotide sequence according to SEQIDNO: 2 by means of agrobacteria-mediated transformation, identifying the transgenic plants, and obtaining rice lines with overexpression of the OsIDS1 gene; where, in comparison to the rice variety Zhonghua11, the rice with 30 overexpression of the OsIDS1 gene shows a significantly improved resistance BE2026 / 7285 4 against rice gangrene.Preferably, the methods for identifying the transgenic plants comprise carrying out a detection using a forward primer with the nucleotide sequence according to SEQIDNO:11 and a reverse primer with the nucleotide sequence according to SEQIDNO:5 12. The present invention further provides a method for rice breeding. The method comprises: increasing the expression level of the OsIDS1 gene or the activity of the protein encoded therein in rice, thereby increasing the resistance of rice to rice rash. Advantageous effects are as follows: Systematic investigations within the scope of the present invention have shown that the rice gene OsIDS1 plays a crucial positive regulatory role in the defense against rice gangrene. Deletion of the OsIDS1 gene leads to a significant decrease in the resistance of rice to the strain MagnaportheoryzaeRB22.Overexpression of the OsIDS1 gene in rice significantly increases rice resistance to rice smut disease and provides a new genetic resource for the breeding of 20 new smut-resistant rice varieties. Furthermore, the procedure is straightforward and can be directly applied in rice breeding, resulting in considerable practical benefits. 25 DESCRIPTION OF THE APPENDED DRAWINGS Figure 1 shows the map view of the double target knockout vector of the OsIDS1 gene. Figure 2 shows the result of the target sequence analysis of 30 rice plants with a deletion of the OsIDS1 gene. BE2026 / 7285 5 Figure 3 shows the map view of the WMV023 vector. Figure 4 shows the phenotypic analysis of the effect of lines with a deletion of the OsIDS1 gene on the resistance of rice to rice blast disease. Figure 5 shows the phenotypic analysis of the effect of lines with 5 overexpression of the OsIDS1 gene on the resistance of rice to rice blast disease.DETAILED DESCRIPTION 10 To make the technical solutions of the present invention more understandable to the field, the present invention is explained in more detail below using examples. Example 1: Obtaining rice OsIDS1 mutants The nucleotide sequence of the rice gene OsIDS1 is specified by SEQIDNO:215. The target gene is selectively deleted using CRISPR / Cas9 technology, thereby obtaining rice OsIDS1 mutants. The steps for constructing the deletion vector are as follows: (1) Target design. Two targets with high target score, low off-target efficiency, and location within the first exon are designed via the CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The nucleotide sequence of the first target is given as SEQIDNO:3 (5'-GTCCCCGGAGTTTAGCACCGAGG-3'), and the nucleotide sequence of the second target is given as SEQIDNO:4 (5'-CTGACGCGTCACGATCCCCGAGG-3'). (2) Digestion of the vector. A quantity of 1 µg of the WMC001 vector (Weimi Biotechnology Co., Ltd.)) is digested with the restriction enzyme BsaI, the reaction takes place at 37°C over a period of one hour, and a gel extraction of the digested products follows.30 The reaction system of enzymatic digestion is given in Table 1 BE2026 / 7285 6. Table 1 WMC001 vector 1 µg (10 µL) BsaI enzyme 1 µL BsaI buffer 5 µL ddH2O3 4 µL Total volume 50 µL (3) Primer design and PCR amplification. Using a forward primer with a BsaI interface according to SEQIDNO:5 (5'-ccaatggtctcatgtgtgTCCCCGGAGTTTAGCACCGGTTTTTagagctagaaata5 g-3') and a reverse primer according to SEQIDNO:6 (5'-ATTGGGGTCTCTAAACCGGGGATCGTGACGCGTCACGCCACGGA TCATCTGCACA-3'), PCR amplification is performed using the plasmid Psg-OsU31 (Weimi Biotechnology Co., Ltd.) as a template. The PCR reaction system is given in Table 2.10 Table 2 PCR product 30 µL BsaI enzyme 1 µL BsaI buffer 5 µL ddH2O 14 µL Total volume 50 µL The PCR reaction program is designed as follows: BE2026 / 7285 7 After completion of the reaction, 3 µL of the PCR product are applied to a gel to check the correct band size of approximately 550 bp; if the size is correct, the original product mixture is extracted. 5 (4) Enzymatic treatment of the PCR products. The above PCR products are digested with BsaI, the reaction is carried out at 37°C for a period of one hour, and a gel extraction of the digested products is then carried out. The reaction system of the enzymatic digestion is given in Table 310. Table 3 PCR product 30 µL BsaI enzyme 1 µL BsaI buffer 5 µL ddH2O 14 µL Total volume 50 µL (5) Ligation reaction. After completion of a ligase program in the PCR instrument at 16°C for a period of one hour, storage in a refrigerator for three hours or overnight is carried out. 15 The ligation reaction system is given in Table 4.Table 4 BE2026 / 7285 8 Vector digestion product 2 µL PCR digestion product 6 µL T4 ligase 1 µL T4 ligase buffer 1 µL Total volume 10 µL The resulting dual-target deletion vector is shown in Figure 1. The final vector will be presented by Weimi Biotechnology Co., Ltd. After transformation into Agrobacterium, the rice is introduced into callus tissue of the rice variety Zhonghua11. The genomic DNA of the resulting gene-edited rice line ids1 is amplified using a primer pair consisting of SEQIDNO:7 (5'-GTGAGTCGTCGTCAGTCGAG-3') and SEQIDNO:8 (5'-AAGCGAGGGATTAATGGCGG-3'), and a sequence analysis of the PCR products using SEQIDNO:7 leads to the identification of the deletion lines KO27 and KO5. The target sequence analysis results of KO27 and KO5 are shown in Figure 210. Example 2: Obtaining rice lines with overexpression of the OsIDS1 gene. The WMV023 vector (Figure 3, Weimar Biotechnology Co., Ltd.) is linearized using the restriction enzyme KpnI.The OsIDS1 gene with the 15-nucleotide sequence according to SEQIDNO:2 is amplified from the genome of Zhonghua11 using the primers SEQIDNO:9 (5'-tgcagaagcttggtaccATGTTGTTGGATCTCAAT-3') and SEQIDNO:10 (5'-tggtctttgtagtccatGGCGGTTGGCGGGAAGTA-3') and integrated into the linearized WMV023 vector20 by homologous recombination, resulting in an overexpression vector for the OsIDS1 gene. The constructed plasmid is transformed into Escherichiacoli using a heat shock procedure; positive clones are selected and tested. The sequence-correct plasmid is sent to Weimar Biotechnology Co., Ltd. After transformation into Agrobacterium, the 25 BE2026 / 7285 9 Introduction into callus tissue of the rice variety Zhonghua11. Positive T0 transformants are identified using the forward primer SEQIDNO:11 (5'-TTGGCGACCTCGTATTGGGAA-3') and the reverse primer SEQ IDNO:12 (5'-CAAAGATCGTTATGTTTATCGGCACT-3'), thereby obtaining the rice lines OE16, OE43 and OE51 with overexpression of the OsIDS1 gene.Example 3: Influence of OsIDS1 on rice resistance to rice gangrene. To confirm the involvement of the OsIDS1 gene in the defense reaction of rice against Magnaportheoryzae, an investigation of the altered resistance of OsIDS1 deletion mutants and overexpression lines to rice gangrene was carried out. The specific procedures are as follows: Cultivation of the strain and preparation of a spore suspension: The MagnaportheoryzaeRB22 strain is activated on oat medium. Cultivation takes place at 25°C for three days under dark conditions and a further four days under light conditions. Sterile ddH2O is applied to the culture dish. Hyphae are carefully detached using an inoculation loop, thereby eluting Magnaportheoryzae spores from the medium. The elution fluid is filtered through a special filter cloth to remove 20 to gain a spore suspension.The spore suspension is transferred to 2 mL centrifuge tubes, centrifuged at 12,000 rpm for two minutes, the supernatant is discarded, and the spore concentration is adjusted to at least 10⁵ cells / mL by adding sterile 1 mL ddH₂O. Tween-20 is added to adjust to a final concentration of 0.01%, so that the suspension is suitable for inoculation with Magnaportheoryzae. Spray inoculation: Wild-type plants (ZH11) and the homozygous T2 deletion lines KO27 and KO5 are cultivated in rice nutrient solution for 14 days. The pre-cultured and prepared conidiospore suspension 30 is sprayed evenly onto the leaves of young rice plants. After inoculation, the seedlings undergo a 24-hour dark cultivation at a constant 25°C and a relative humidity of 90%. This is followed by alternating cultivation with 12 hours of light and 12 hours of darkness. The disease progression on the leaves is observed five days after inoculation. Each experiment is performed three times.5. Ex vivo inoculation: Wild-type plants (ZH11) and the three overexpression lines OE16, OE43, and OE51 are cultivated in rice nutrient solution up to the four-leaf stage. Leaf sections 6 cm long are taken from identical areas of the seedlings. The leaf surface is carefully punctured every 2.0–10 cm using a 10 µL pipette tip, resulting in three epidermal wounds per leaf. The leaves are transferred to culture dishes and suspended on the surface of an aqueous solution containing 10 µg / mL of 6-benzylaminopurine at a pH of 7.0. 5 µL of the prepared conidiospore suspension is applied to each of the 15 punctured wounds using a pipette. After completion of inoculation, the dishes are covered and incubated in a constant-temperature incubator. Temperature and light at 25°C. After 24 hours of dark cultivation, a five-day alternating cultivation with 12 hours of light and 12 hours of darkness is carried out before the disease course is observed. Each experiment is performed three times.Determination of hyphal biomass: Diseased leaf areas of rice are taken, and total DNA is extracted using the CTAB method. The rice gene OsUbiquitindientals is used as a reference gene (LOC_Os03g13170), the amount of the fungal gene Mopot2 (MGG_13294) from 25 Magnaportheoryzae is determined using quantitative real-time PCR, and the fungal biomass is analyzed. Relative gene expression is calculated using the 2-ΔΔCT method. The primer sequences used are as follows: SEQIDNO:13(qOsUBQ-F: 5'-AAGAAGCTGAAGCATCCAGC-3'), SEQIDNO:14(qOsUBQ-R:30 5'-CCAGGACAAGATGATCTGCC-3'),SEQIDNO:15(Mopot2-F: BE2026 / 7285 11 5'-ACGACCCGTCTTTACTTATTTGG-3') andSEQIDNO:16(Mopot2-R: 5'-AAGTAGCGTTGGTTTTGTTGGAT-3'). The results of the inoculation show that, compared to the wild type (ZH11), the lesion area and the relative fungal biomass of the deletion mutants KO27 and KO5 are significantly increased, so that their resistance to rice dermatitis is considerably reduced (Figure 4).In both overexpression lines OE16, OE43 and OE51, the lesion length and the relative fungal biomass are significantly lower than in the wild type, resulting in a significantly improved resistance to rice dermatitis.