Use of osga2 protein, encoding gene or biological material related thereto in modulating rice blast resistance in plants

Abstract

The present application relates to the technical field of gene editing, and particularly relates to application of OsGA2 protein, coding gene or biological material related to the OsGA2 protein in regulating rice blast resistance. The present application researches and finds that the OsGA2 protein or the biological material related to the OsGA2 protein has the function of regulating the rice blast resistance, through knocking out the OsGA2 gene, the expression amount of the OsGA2 protein is reduced, the rice blast resistance of rice can be improved, and through overexpressing the OsGA2 gene, the expression amount of the OsGA2 protein is improved, the rice blast resistance of rice can be reduced. Through identification and application of the rice blast resistance gene OsGA2 of rice, a breeder can develop rice varieties with rice blast resistance suitable for different rice planting areas in different regions, which will provide an important guarantee for increasing the yield of rice.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and in particular to the application of OsGA2 protein, its encoding gene, or related biological materials in regulating plant resistance to rice blast. Background Technology

[0002] Rice is one of my country's main food crops and is widely cultivated throughout the country. However, as a populous nation, my country needs to increase its rice production by more than 40% by 2030 to meet its growing food demand. [1] This challenge must be overcome by developing high-yielding rice varieties resistant to biotic and abiotic stresses. [2] Among biological stresses, rice blast is the most harmful threat to high rice yields. [3,4] Rice blast is one of the most serious fungal diseases affecting rice production. It is a common disease in rice fields, characterized by its long duration of infection, multiple infection sites, and diverse symptoms. [5] Rice blast easily affects the growth process of rice, leading to reduced yield and quality. Rice blast can occur throughout the entire rice growth cycle, and depending on the stage and location of infection, it can be classified into seedling blast, leaf blast, node blast, neck blast, and grain blast, among others. Leaf blast and neck blast are the most common and have the most severe impact on rice yield. The pathogen of rice blast multiplies rapidly under humid conditions of 26-28℃, while sunlight inhibits its reproduction. When rice is infected with blast, the affected plants are usually brown with a large amount of gray mold. If measures are not taken in time, the seedlings will gradually curl and die. [6] .

[0003] The Gadd45 gene is a novel gene discovered in rice, previously unstudied in plants, but its animal counterpart has been extensively studied. The animal Gadd45 gene was first identified on an induced basis after exposure to various stresses associated with growth arrest, and thus named a growth-inhibiting and DNA-damage-inducible gene. [7] The gadd gene was first cloned from Chinese hamster ovary (CHO) cells. As a subset of transcription factors that are persistently upregulated after exposure to ultraviolet (UV) radiation, the gadd gene acts as a growth arrest and DNA damage repair gene in many cases, functioning as a growth arrest signal along with other DNA damaging agents, including methyl methanesulfonate (MMS), hydrogen peroxide, and N-acetoxy-2-acetaminofluorobutadiene. [8]Gadd45a is the 45th member of this collection of over 100 cDNA clones. Gadd45a responds to a variety of drugs involved in DNA damage, apoptosis, cell cycle checkpoint control, cell injury, and other growth regulatory processes. The Gadd45 protein is also involved in various cellular processes, typically associated with stress signals and other growth regulatory pathways. [9] Gadd45a has been reported to induce kinase activity in human cells after exposure to ionizing radiation (IR) in a TM-dependent and protein kinase-independent manner.

[10] This induction is regulated by p53.

[11] In fact, Gadd45a was the first stress gene discovered to be regulated by p53 transcription.

[12] Gadd45b was initially cloned as a gene expressed after IL-6-induced terminal differentiation and growth arrest in M1D+ myeloid precursor cells. Gadd45g was initially cloned as an early IL-2 response gene in T cells. All three members respond to a variety of environmental factors associated with growth control. These three proteins are highly conserved in metazoans, although only one Gadd45 gene exists in insects, similar to Gadd45g, suggesting that this may be an ancestral gene. These proteins are all small (18 kDa), highly negatively charged, and located in the cell nucleus.

[13] .

[0004] The Gadd45 gene has multiple functions as a stress-related gene in animals, but the function of its homology OsGA2 in rice has not been reported in plants.

[0005] References:

[0006] [1]Khush GS(2005)What it will take to feed 5.0 billion rice consumers in 2030.Plant Mol Biol 59:1–6.

[0007] [2] Selvaraj CI, Nagarajan P, Thiyagarajan K, Bharathi M, Rabindran R (2011) Studies on heterosis and combining ability of well known blastresistant rice genotypes with high yielding varieties of rice (Oryza sativaL.). Int J Plant Breed Genet 5(2):111–129.

[0008] [3]Kwon JO,Lee SG(2002)Real-time micro-weather factors ofgrowingfield to the epidemics of rice blast.Res Plant Dis 8:199–206(in Korean,Englishabstract).

[0009] [4]Li YB, Wu CJ, Jiang GH, Wang LQ, He YQ (2007) Dynamic analyzes of riceblast resistance for the assessment of genetic and environmental effects. Plant Breeding 126:541–547.

[0010] [5] Zheng Shengyou. Integrated control technology and promotion strategy for rice blast disease [J]. Southern Agriculture, 2022, 16(4): 66-68.

[0011] [6] He Xiaoling, Qin Feifeng. Occurrence and control of rice sheath blight and rice blast [J]. Guangdong Sericulture, 2021, 55(9): 15-16.

[0012] [7] Fornace AJ Jr, Nebert DW, Hollander MC, Luethy JD, Papathanasiou M, Fargnoli J, Holbrook NJ (1989) Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. Mol Cell Biol 9:4196–4203.

[0013] [8] Fornace AJJ, Alamo IJ, Hollander MC (1988) DNA damage-inducible transcripts in mammalian cells. Proc NatlAcad Sci USA 85:8800–8804.

[0014] [9]Gao M,Guo N,Huang C,Song L(2009)Diverse roles ofGADD45alpha instress signaling.CurrProteinPept Sci 10:388–394.

[0015]

[10] Papathanasiou MA,Kerr NC,Robbins JH,McBride OW,Alamo IJ,BarrettSF,Hickson ID,Fornace AJJ(1991)Induction by ionizing radiation of thegadd45gene in cultured human cells:lack ofmediation by protein kinase C.MolCell Biol11:1009–1016.

[0016]

[11] Kastan MB,Zhan Q,el-Deiry WS,Carrier F,Jacks T,Walsh WV,PlunkettBS,Vogelstein B,Fornace AJJ(1992)A mammalian cell cycle checkpoint pathwayutilizingp53 and GADD45 is defective in ataxia-telangiectasia.Cell 71:587–597.

[0017]

[12] Hollander MC,Fornace AJ Jr(2002)Genomic instability,centrosomeamplifi cation,cell cycle checkpoints and Gadd45a.Oncogene 21:6228–6233.

[0018]

[13] CretuA,Sha X,Tront J,Hoffman B,Liebermann DA(2009)Stress sensorGadd45 genes as thera-peutic targets in cancer.CancerTher 7:268–276. Summary of the Invention

[0019] To address the aforementioned problems, this invention provides the application of the OsGA2 protein, its encoding gene, or related biological materials in regulating resistance to rice blast disease. The OsGA2 protein provided by this invention has a regulatory effect on rice blast disease resistance.

[0020] To achieve the above objectives, the present invention provides the following technical solution:

[0021] This invention provides an OsGA2 protein that regulates plant resistance to rice blast, the OsGA2 protein comprising a protein with an amino acid sequence as shown in SEQ ID NO.1.

[0022] The present invention also provides an OsGA2 gene encoding the OsGA2 protein described in the above scheme, wherein the CDS sequence of the OsGA2 gene includes the sequence shown in SEQ ID NO.2.

[0023] This invention also provides the application of the OsGA2 protein or related biological materials described in the above scheme in regulating plant resistance to rice blast.

[0024] Preferably, the regulation includes increasing plant resistance to rice blast by negatively regulating the expression level of OsGA2 protein, or decreasing plant resistance to rice blast by positively regulating the expression level of OsGA2 protein.

[0025] Preferably, the biomaterial comprises one or more of the following:

[0026] 1) The OsGA2 gene mentioned in the above scheme;

[0027] 2) A recombinant vector containing the OsGA2 gene described in the above scheme;

[0028] 3) Negative regulation of OsGA2 gene expression using sgRNA or recombinant vectors as described in the above scheme.

[0029] Preferably, the target sequence of the sgRNA includes one or more of the target sequences shown in SEQ ID NO.3 and SEQ ID NO.4.

[0030] Preferably, the recombinant vector described in 3) includes a target sequence and a base vector; the target sequence includes the target sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

[0031] Preferably, the base vector includes pYLCRISPR-Cas9PUbi-H.

[0032] Preferably, the plant includes rice.

[0033] The present invention also provides the application of the OsGA2 protein or related biological materials described in the above scheme in the cultivation of transgenic plants with improved resistance to rice blast.

[0034] Beneficial effects:

[0035] This invention provides an OsGA2 protein that regulates rice blast resistance. The OsGA2 protein comprises a protein with the amino acid sequence shown in SEQ ID NO. 1. This invention has found that the OsGA2 protein plays a role in regulating rice blast resistance. Knocking out the OsGA2 gene and reducing the expression level of the OsGA2 protein can increase rice blast resistance, while overexpressing the OsGA2 gene and increasing the expression level of the OsGA2 protein can decrease rice blast resistance. Through the identification and application of the rice blast resistance gene OsGA2, breeders can develop blast-resistant rice varieties suitable for different rice-growing regions, which will provide an important guarantee for increasing rice yields. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0037] Figure 1 The preparation of osga2 mutants and screening of Cas9-free plants are shown in the diagrams. A represents the OsGA2 genome sequence diagram and the locations of the two target sites within the OsGA2 genome sequence; B represents the Sanger sequencing results near the target sites in two homozygous osga2 mutants with different editing methods; C represents the two homozygous Cas9-free mutants, osga2-1 and osga2-2, obtained using hygromycin screening without the CRISPR / Cas9 vector; D represents the detection of OsGA2 protein expression levels in the complemented lines using Western blot with the FLAG antibody; the "-" in -60bp indicates the length of the 60bp gene bar.

[0038] Figure 2 The lesion phenotypes of osga2 mutants inoculated with rice blast S5 strain are shown in Figure A, where A represents the enhanced resistance phenotype of osga2 mutants to rice blast S5, and B represents the statistical analysis of lesion area using Adobe Photoshop CS6 Extended 64bit software.

[0039] Figure 3Preparation of OsGA2OE overexpressing plants: A represents the expression level of OsGA2 in wild-type (Kitaake) and three independent OsGA2 overexpressing plants (OsGA2OE-1, OsGA2OE-2, and OsGA2OE-3) detected by Western blot; B represents the expression level of OsGA2 in wild-type (Kitaake) and overexpressing plants (OsGA2OE-1, OsGA2OE-2, and OsGA2OE-3) identified by real-time quantitative PCR (RT-qPCR).

[0040] Figure 4 The lesion phenotypes of OsGA2OE overexpressing plants inoculated with rice blast S5 strain were shown. In the figure, A represents the phenotype of weakened resistance of OsGA2OE overexpressing plants to rice blast S5 strain, and B represents the lesion area statistics obtained using Adobe Photoshop CS6 Extended 64bit software. Detailed Implementation

[0041] This invention provides an OsGA2 protein that regulates resistance to rice blast disease in plants. The OsGA2 protein comprises a protein with the amino acid sequence shown in SEQ ID NO.1, and the specific amino acid sequence shown in SEQ ID NO.1 is as follows:

[0042] MAEETPVEAPPAPVLGEPMDLMTALQLVMKKSSAHDGLVKGLREAAKAIEKHAAQLCVLAEDCDQPDYVKLVKALCAEHNVHLVTVPSAKTLGEWAGLCKIDSEGKARKVVGCSCVVVKDFGEESEGLNIVQDYVKSH.

[0043] The OsGA2 protein described in this invention plays a role in regulating rice blast resistance. By knocking out the OsGA2 gene and reducing the expression level of the OsGA2 protein, the resistance to rice blast can be improved, while overexpressing the OsGA2 gene and increasing the expression level of the OsGA2 protein can reduce the resistance to rice blast.

[0044] The present invention also provides an OsGA2 gene encoding the OsGA2 protein described in the above scheme, wherein the CDS sequence of the OsGA2 gene includes the sequence shown in SEQ ID NO.2. The nucleotide sequence shown in NO.2 is as follows: ATGGCGGAAGAGACTCCAGTTGAGGCCCCACCTGCCCCGGTTCTTGGAGAGCCGATGGACCTGATGACTGCTCTGCAGCTTGTGAATGAAAGTCAAGCGCTCATGATGGGCTCGTGAAGGGGCTCCGTGAGGCGGCCAAGGCCATTGAGAAGCATGCCGCTCAGCTTTGCGTGCTTGCTGAGGACTGTGACCAGCCTGA TTATGTCAAGTTGGTCAAGGCACTCTGCGCTGAGCACAATGTTCACCTCGTTACCGTGCCTAGTGCTAAAACTCTTGGCGAGTGGGCAGGGCTTTGCAAGATTGATTCTGAGGGCAAGGCGAGGAAGGTCGTAGGCTGCTCCTGCGTCGTTGTCAAGGACTTCGGTGAAGAGTCAGAGGGCCTCAACATAGTCCAGGACTATGTCAAGTCCCACTAG.

[0045] This invention provides the application of the OsGA2 protein or related biological materials described in the above-described scheme in regulating plant resistance to rice blast. In this invention, the plant preferably includes rice.

[0046] In this invention, the regulation preferably includes increasing plant resistance to rice blast by negatively regulating the expression level of OsGA2 protein, or decreasing plant resistance to rice blast by positively regulating the expression level of OsGA2 protein.

[0047] In this invention, the biomaterial preferably includes one or more of the following:

[0048] 1) The OsGA2 gene mentioned in the above scheme;

[0049] 2) A recombinant vector containing the OsGA2 gene described in the above scheme;

[0050] 3) Negative regulation of OsGA2 gene expression using sgRNA or recombinant vectors as described in the above scheme.

[0051] In this invention, the recombinant vector for the OsGA2 gene in the above-described scheme preferably includes a recombinant vector capable of overexpressing the OsGA2 gene. The recombinant vector capable of overexpressing the OsGA2 gene preferably includes a base vector and the coding sequence of the OsGA1 protein. The base vector preferably includes a pCsV1300-3×FLAG vector, and the coding sequence of the OsGA1 protein is preferably located between the XbaI and BamHI sites of the pCsV1300-3×FLAG vector. The recombinant vector capable of overexpressing the OsGA2 gene provided by this invention can increase the expression level of the OsGA2 protein and reduce resistance to rice blast.

[0052] The pCsV1300-3×FLAG vector of the present invention is preferably constructed by inserting a 3×FLAG sequence after the BamHI site of the pCsV1300 vector. The specific method preferably includes: designing homologous primers based on the FLAG tag sequence (ATGGACTACAAAGACCATGATGGAGACTATAAGGATCACGACATCGATTACAAGGACGATGACGATAAG, SEQ ID NO.11): FLAG-F: 5′-ctcggtaccggatccATGGACTACAAAGACCATGATGGAGACTATAAGGATCACGACATCGATTACAAGGACGATGACGATAAG-3′, SEQ ID NO.12 and FLAG-R: 5′-gaacgatcgggaattCTTATCGTCATCGTCCTTGTAATCGATGTCGTGATCCTTATAGTCTCCATCATGGTCTTTGTAGTCCAT-3′, SEQ ID NO.13, wherein the lowercase sequence in the two primer sequences is the homologous arm. 1 μL each of primers FLAG-F and FLAG-R (both working concentrations 100 μM) were placed in 48 μL of ddH2O, mixed, and then placed in boiling water until the boiling water slowly cooled to room temperature, thus annealing the single-stranded primers into a double-stranded short DNA fragment with homologous arms. The obtained double-stranded short DNA fragment was then homologously recombinated with the pCsV1300 vector obtained by BamHI digestion to obtain the pCsV1300-3×FLAG vector. The pCsV1300 vector described in this invention has been reported in the prior art; see (ADNA Methylation Reader–Chaperone Regulator–Transcription Factor Complex Activates OsHKT1;5 Expression during SalinityStress, 2020.09).

[0053] In this invention, the target sequence of the sgRNA preferably includes the target sequence shown in SEQ ID NO.3 and / or SEQ ID NO.4. The nucleotide sequence of the target sequence shown in SEQ ID NO.3 is as follows: CCGATGGACCTGATGACTGCTC (the last three bases are PAM sites); the nucleotide sequence of the target sequence shown in SEQ ID NO.4 is as follows: GCTCGTGAAGGGGCTCCGTGAGG (the last three bases are PAM sites).

[0054] In this invention, the recombinant vector for negatively regulating the expression of the OsGA2 gene in the above-mentioned scheme preferably includes a target sequence and a base vector; the target sequence preferably includes the target sequence shown in SEQ ID NO.3 or SEQ ID NO.4; the base vector preferably includes pYLCRISPR-Cas9PUbi-H.

[0055] The sgRNA or recombinant vector that negatively regulates OsGA2 gene expression provided by this invention can reduce the expression of the OsGA2 gene and the expression level of OsGA2 protein, thereby improving rice blast resistance. Through the identification and application of the rice blast resistance gene OsGA2, breeders can develop blast-resistant rice varieties suitable for different rice-growing regions, which will provide an important guarantee for increasing rice yields.

[0056] This invention also provides the application of the OsGA2 protein or related biological materials described in the above scheme in the cultivation of transgenic plants with enhanced resistance to rice blast.

[0057] In this invention, the transgenic plant preferably includes transgenic rice.

[0058] To further illustrate the present invention, the application of the OsGA2 protein, encoding gene, or related biological materials provided by the present invention in regulating plant resistance to rice blast is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] OsGA2 gene mutant constructed using CRISPR / Cas9 gene editing technology

[0061] Using the website http: / / skl.scau.edu.cn / , two different target sites were selected based on the OsGA2 gene sequence. The target site locations are as follows: Figure 1As shown in A, the nucleotide sequence of target 1 is: CCGATGGACCTGATGACTGCTC (SEQ ID NO.3, the first three bases are PAM sites); the nucleotide sequence of target 2 is: GCTCGTGAAGGGGCTCCGTGAGG (SEQ ID NO.4, the last three bases are PAM sites).

[0062] Candidate target sequences target1 and target2 were inserted into the intermediate vector pYLsgRNA-LacZ-OsU6a, respectively. Subsequently, the fragments were inserted into the final vector pYLCRISPR-Cas9PUbi-H using the BsaI restriction site via Golden Gate cloning (for detailed construction steps, refer to: 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. Molecular Plant, 2015, 8: 1274-1284.).

[0063] The successfully constructed CRISPR / Cas9 vector was transformed into wild-type rice Kitaake callus. Positive transgenic plants were obtained by hygromycin screening. Specifically, plants that could grow on a medium containing hygromycin were considered positive transgenic plants (transformation method and screening process reference: Toki S, Hara N, Ono K, et al. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant Journal, 2006, 47: 969-976.).

[0064] 2. Molecular identification of the editing patterns of the positive transgenic plants obtained above.

[0065] Using genomic DNA from rice leaves as a template, PCR amplification was performed with the following identification primers. The amplified products were then subjected to Sanger sequencing to determine the mutant editing patterns.

[0066] The primers for identifying the osga2 mutant are: OsGA2-crispr-Check-F (SEQ ID NO.5): GCGAGTTGTTCTGGCTGAG; OsGA2-crispr-Check-R (SEQ ID NO.6): CGCCTTGCCCTCAGAAT.

[0067] The PCR amplification system was prepared according to the instructions for use of 2×Es TaqMasterMix (Dye), specifically: 10 μL of 2×Es TaqMasterMix (Dye), 0.8 μL of OsGA2-crispr-Check-F (working concentration 10 μM), 0.8 μL of OsGA2-crispr-Check-R (working concentration 10 μM), 1 μg of DNA template, and ddH2O to a final volume of 20 μL.

[0068] The PCR reaction system was as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 15 s, for 35 cycles; 72℃ extension for 2 min.

[0069] The results are as follows Figure 1 As shown in B, two independent CRISPR homozygous mutant lines of osga2 were obtained, named osga2-1 and osga2-2, respectively. In osga2-1, a four-base deletion (GACC) occurs 58–61 bp downstream of the OsGA2 gene start codon ATG, resulting in a frameshift mutation and premature formation of the stop codon at 58–60 bp. In osga2-2, a one-base C deletion occurs 126 bp downstream of the OsGA2 gene start codon ATG, resulting in a frameshift mutation and premature formation of the stop codon at 331–333 bp.

[0070] To eliminate the influence of the Cas9 gene in progeny plants, seeds from homozygous mutant plants were screened on 1 / 2 MS medium containing 50 mg / L hygromycin. Seeds that could not germinate on this medium were considered Cas9-free homozygous mutants without the CRISPR / Cas9 vector and were used as experimental materials for subsequent experiments. Figure 1 As shown in C.

[0071] 3. Constructing complemented lines Com#2-1 and Com#2-2 with an osga2 mutant background.

[0072] To further confirm whether the osga2 mutant phenotype is caused by the deletion of the OsGA2 gene, the OsGA2 genome information was retrieved from https: / / phytozome-next.jgi.doe.gov / . The region 1–2000 bp upstream of the ATG start codon of the OsGA2 gene (OsKitaake07g080700.1) was selected as the promoter to drive the expression of the CDS sequence of the OsGA2 gene fused with the FLAG tag. A recombinant plasmid OsGA2pro::2×FLAG-OsGA2 (OsGA2pro represents the selected promoter) was constructed using pCAMBIA1302 as the vector backbone. This plasmid was then transformed into the Cas9-free homozygous mutant osga2-1 callus. Callus that grew on 1 / 2 MS medium containing 50 mg / L hygromycin was considered successfully transformed and further differentiated into complement lines with the OsGA2 mutant as the background, numbered Com#2-1 and Com#2-2, respectively. Subsequently, Western blot analysis was performed using FLAG antibody to detect the protein expression level of OsGA2 in the recombinant line plants, with α-H3 as a control.

[0073] The results are as follows Figure 1 As shown in D, the experimental results showed that the target band of FLAG-OsGA2 could be detected in the recombinant line plants Com#2-1 and Com#2-2.

[0074] 4. Disease phenotypes of wild-type (Kitaake), mutants (osga2-1 and osga2-2), and replacement lines (Com#2-1 and Com#2-2) inoculated with rice blast disease strain S5.

[0075] Rice blast-S5 bacterial suspension was inoculated into wild-type Kitaake, mutants osga2-1 and osga2-2, and reintroduced lines Com#2-1 and Com#2-2, respectively. Three wounds were made on each leaf of each material using sterile forceps, and 10 μL of bacterial suspension was added to each wound. The viable bacterial count in the suspension was 2 × 10⁻⁶. 51 spore / mL, 3 plants of each material, observe the symptoms of rice blast 7 days after inoculation (the rice blast S5 mentioned is referred to in the literature: Lu F, Wang H, Wang S, et al. Enhancement of innate immune system in monocot rice by transferring the dicotyledonous elongation factor Tureceptor EFR. Journal of Integrative Plant Biology. 2015, 57, 641–652.).

[0076] The results are as follows Figure 2 As shown in Figure A, the leaves of wild-type Kitaake rice inoculated with rice blast S5 showed small areas of yellowing lesions. The leaves of mutants osga2-1 and osga2-2 showed almost no yellowing lesions at the inoculation site. The leaves of the replacement lines Com#2-1 and Com#2-2 also showed small areas of yellowing lesions at the inoculation site. The same results were obtained in two independent inoculation experiments using two independent mutant lines. The image shows several randomly selected leaves from the results.

[0077] 5. Statistics on the lesion area of ​​wild-type (Kitaake), mutants (osga2-1 and osga2-2), and replacement lines (Com#2-1 and Com#2-2) inoculated with rice blast disease strain S5.

[0078] Images of rice leaves showing yellow spots after inoculation with the rice blast virus strain S5 were opened using Adobe Photoshop CS6 Extended 64-bit. The magnetic lasso tool was used to select the locations of the yellow spots. The histogram panel was opened in the window, and the pixels of the selected area were calculated. Using the formula: 1 square inch = 72 * 72 pixels = 5184 pixels, 1 square inch = 0.00064516 square meters, the area of ​​the selected yellow spots was calculated. The statistical results of the disease area proportion were obtained from data of three leaves for each material. Data are presented as the experimental mean ± SD, with three biological replicates, and a t-test. ** represents P < 0.01. Statistical results are shown below. Figure 2 As shown in B and Table 1.

[0079] Table 1. Proportion of lesion area (%) using different materials

[0080] Group Leaf 1 Leaf 2 Leaf 3 Kitaake 16.17 14.81 15.43 osga2-1 5.13 4.27 4.86 osga2-2 4.93 3.75 4.38 Com#2-1 15.13 14.52 14.79 Com#2-2 14.61 16.32 15.79

[0081] Depend on Figure 2 As shown in B and Table 1, the leaf lesion area of ​​mutants osga2-1 and osga2-2 was significantly smaller than that of wild-type Kitaake and replenishment line materials Com#2-1 and Com#2-2.

[0082] Example 2

[0083] Constructing OsGA2OE plants overexpressing the OsGA2 gene

[0084] RNA was extracted from leaves of the rice variety Kitaake and reverse transcribed into cDNA. Using this cDNA as a template, amplification was performed using primers SEQ ID NO.7 (5′-ATGGCGGAAGAGACTCCAGTTGA-3′) and SEQ ID NO.8 (5′-GTGGGACTTGACATAGTCCTGGACTA-3′). The PCR amplification system was prepared according to the 2×PlantaMasterMix instruction manual: 25 μL 2×PlantaMasterMix, 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 400 ng cDNA template, and ddH2O to a final volume of 50 μL. The PCR reaction was performed as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 72℃ extension for 60 s, 32 cycles; and 72℃ final extension for 5 min, yielding a 414 bp PCR product. This PCR product contains nucleotides 1 to 414 of the sequence shown in SEQ ID NO.2.

[0085] Using the recovered product as a template, a second PCR amplification was performed using homologous recombination primers (SEQ ID NO.9: 5′-tctatcgattctagaATGGCGGAAGAGACTCCAGTTGA-3′ and SEQ ID NO.10: 5′-gtagtccatggatccGTGGGACTTGACATAGTCCTGGACTA-3′). The second round PCR amplification system was prepared according to the 2×PlantaMasterMix instruction manual, specifically: 12.5 μL of 2×PlantaMasterMix, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 10 ng of template, and ddH2O to a final volume of 25 μL. The second round PCR reaction system was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 72℃ extension for 60 s (if the primer Tm value is ≥72℃, the annealing step can be omitted, and the extension step can be performed directly), 32 cycles; and 72℃ final extension for 5 min.

[0086] After recovering the second-round PCR product, homologous recombination was performed with the pCsV1300-3×FLAG vector backbone obtained by XbaI and BamHI digestion. The pCsV1300-3×FLAG vector was obtained by modifying the pCsV1300 vector backbone by adding a 3×FLAG sequence after the original BamHI site. The specific method is as follows: Homologous primers were designed based on the FLAG tag sequence (SEQ ID NO.11): FLAG-F (SEQ ID NO.12) and FLAG-R (SEQ ID NO.13). 1 μL each of primers FLAG-F and FLAG-R (both working concentrations 100 μM) were placed in 48 μL ddH2O, mixed, and then placed in boiling water until the boiling water slowly cooled to room temperature, thus annealing the single-stranded primers into double-stranded short DNA fragments with homologous arms. The obtained double-stranded short DNA fragment was homologously recombinated with the pCsV1300 vector obtained by BamHI digestion to obtain the pCsV1300-3×FLAG vector. The pCsV1300 vector described in this invention has been reported in the prior art, see (A DNA Methylation Reader–Chaperone Regulator–Transcription Fact or Complex Activates OsHKT1;5 Expression during Salinity Stress, 2020.09).

[0087] After obtaining the recovered product from the second round of PCR, the PCR product was inserted between the XbaI and BamHI sites of the linearized pCsV1300-3×FLAG vector using 2×ClonExpress Mix recombinase to obtain the final vector pCsV1300-OsGA2-3×FLAG.

[0088] The homologous recombination reaction system consisted of: 18 ng of the recovered product from the second PCR, 60 ng of the linearized pCsV1300-3×FLAG vector (the molar ratio of the recovered product from the second PCR to the linearized pCsV1300-3×FLAG vector was 3:1), 5 μL of 2×ClonExpress Mix, and ddH2O to a final volume of 10 μL.

[0089] Sequencing revealed that the recombinant vector was an OsGA2 overexpression vector, in which nucleotides from position 1 to position 414 of the sequence described in SEQ ID NO.2 were inserted between the XbaI and BamHI restriction sites of the pCsV1300-3×FLAG vector.

[0090] The successfully constructed pCsV1300-OsGA2-3×FLAG overexpression vector was transformed into wild-type rice Kitaake callus. Positive transgenic plants were obtained by hygromycin screening. Specifically, plants that could grow on a medium containing hygromycin were considered positive transgenic plants (transformation method and screening process reference: Toki S, Hara N, Ono K, et al. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant Journal, 2006, 47: 969-976.).

[0091] 2. Molecular identification of the positive transgenic plants obtained above.

[0092] The third fresh, young leaf from the top of the plant was cut and placed in a pre-chilled 2mL centrifuge tube containing steel balls. The tube was then immediately placed in liquid nitrogen to freeze and ground thoroughly into powder using a grinder. The ground sample was then added to 200μL of IP buffer for lysis. After complete lysis, the sample was centrifuged and boiled. Western blot analysis of the OsGA2 protein expression level in wild-type (Kitaake) and overexpressing plants (OsGA2OE) was performed using α-FLAG antibody, with α-H3 as a control.

[0093] The results are as follows Figure 3 As shown in Figure A, using wild-type (Kitaake) as a negative control, three overexpression lines (OsGA2OE-1, OsGA2OE-2, and OsGA2OE-3) with high OsGA2 protein expression levels were screened. Furthermore, the expression level of OsGA2 in transgenic plants was identified using real-time quantitative PCR (RT-qPCR). Figure 3 As shown in B and Table 2, the expression level of OsGA2 in all three transgenic lines was significantly increased compared to wild-type Kitaake.

[0094] Table 2. OsGA2 expression levels in different strains

[0095] Group Plant 1 Plant 2 Plant 3 Kitaake 1.008 0.995 1.037 OsGA2OE-1 24.853 25.256 25.126 OsGA2OE-2 31.192 30.757 30.653 OsGA2OE-3 26.783 27.515 27.128

[0096] 3. Observe the disease phenotype of rice blast disease strain S5 after OsGA2 overexpression lines are inoculated.

[0097] Wild-type (Kitaake) and overexpression (OsGA2OE) seeds, harvested from the same year and location, were placed in sterilized Erlenmeyer flasks. An appropriate amount of sodium hypochlorite solution (available chlorine ≥8%) was added, and the seeds were soaked for 10 minutes. After rinsing 4–5 times with distilled water, the cleaned seeds were placed in glass petri dishes lined with moist filter paper in an incubator (the filter paper was kept moist throughout the experiment). The culture conditions were: 28℃, 65% relative humidity, 10 h light, 14 h darkness, and a light intensity of 200 μM photons·m. -2 ·s -1 .

[0098] To further investigate the function of OsGA2 in rice blast resistance, three independent overexpression lines of OsGA2 (OsGA2OE-1, OsGA2OE-2, and OsGA2OE-3) were cultured to the three-leaf-one-heart stage. Rice blast S5 bacterial suspension was inoculated onto the leaf surface of wild-type Kitaake rice and the overexpression lines (inoculation method and dosage refer to step 4 in Example 1). The symptoms of rice blast were observed 7 days after inoculation.

[0099] like Figure 4 As shown in Figure A, wild-type Kitaake leaves inoculated with rice blast virus S5 showed small areas of yellowing lesions, while leaves of overexpression lines (OsGA2OE-1, OsGA2OE-2, and OsGA2OE-3) showed large areas of yellowing lesions at the inoculation site. The same results were obtained in two independent inoculation experiments using three independent overexpression lines. The image shows several randomly selected leaves from the results.

[0100] 4. Statistics on the lesion area of ​​rice blast disease S5 strain inoculated with OsGA2 overexpressing lines.

[0101] Images of rice leaves showing yellow lesions after inoculation with rice blast fungus strain S5 using OsGA2 overexpression lines were opened in Adobe Photoshop CS6 Extended 64-bit. The magnetic lasso tool was used to select the locations of the yellow lesions. The histogram panel was opened in the window, and the pixels of the selected area were calculated. Using the formula: 1 square inch = 72 * 72 pixels = 5184 pixels, 1 square inch = 0.00064516 square meters, the area of ​​the selected yellow lesion region was calculated. The statistical results of the disease area proportion were obtained from data of three leaves for each material. Data are presented as experimental mean ± SD, with three biological replicates, and a t-test. ** represents P < 0.01. Statistical results are as follows: Figure 4 As shown in B and Table 3.

[0102] Table 3. Proportion of lesion area (%) using different materials

[0103] Group Leaf 1 Leaf 2 Leaf 3 Kitaake 14.1 15.2 14.9 OsGA2OE-1 89.6 88.1 88.7 OsGA2OE-2 92.3 93.8 92.8 OsGA2OE-3 91.6 94.3 93.5

[0104] Depend on Figure 4 As shown in B and Table 3, the leaf lesion area of ​​the OsGA2 overexpression lines (OsGA2OE-1, OsGA2OE-2 and OsGA2OE-3) was significantly larger than that of the wild-type Kitaake material.

[0105] In summary, this invention demonstrates that the OsGA2 protein or related biological materials play a role in regulating rice blast resistance. Knocking out the OsGA2 gene and reducing its expression level can increase rice blast resistance, while overexpressing the OsGA2 gene and increasing its expression level can decrease it. Through the identification and application of the rice blast resistance gene OsGA2, breeders can develop blast-resistant rice varieties suitable for different rice-growing regions, which will provide an important guarantee for increasing rice yields.

[0106] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of OsGA2 protein-related biomaterials in regulating plant resistance to rice blast; wherein the regulation is achieved by negatively regulating the expression level of OsGA2 protein to enhance plant resistance to rice blast; the amino acid sequence of the OsGA2 protein is shown in SEQ ID NO. 1; the plant includes rice; The biomaterials include: negative regulation OsGA2 The sgRNA or recombinant vector for gene expression; OsGA2 The CDS sequence of the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The target sequence of the sgRNA is shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. The application according to claim 1, characterized in that, The recombinant vector includes a target sequence and a base vector; the target sequence is shown in SEQ ID NO.3 or SEQ ID NO.

4.

4. The application according to claim 3, characterized in that, The underlying vector includes pYLCRISPR-Cas9PUbi-H.

5. Application of OsGA2 protein-related biomaterials in the cultivation of transgenic plants with enhanced resistance to rice blast; said biomaterials include: negative regulation OsGA2 sgRNA or recombinant vector for gene expression; The OsGA2 The CDS sequence of the gene is shown in SEQ ID NO.2; the plant includes rice.

6. The application according to claim 5, characterized in that, The target sequence of the sgRNA is shown in SEQ ID NO.3 or SEQ ID NO.

4.

7. The application according to claim 5, characterized in that, The recombinant vector includes a target sequence and a base vector; the target sequence is shown in SEQ ID NO.3 or SEQ ID NO.

4.

8. The application according to claim 7, characterized in that, The underlying vector includes pYLCRISPR-Cas9PUbi-H.

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