Application of rice methyltransferase OsMTA1 in rice virus resistance

Abstract

The invention relates to a rice transcription factor OsMTA1 and application of the rice transcription factor OsMTA1 in resisting rice viruses, and after an overexpression OsMTA1 vector is transferred into plant rice, a transgenic plant with stable inheritance is obtained. The result shows that compared with the control Nipponbare rice, the transgenic plant shows higher disease resistance.

Description

Technical Field

[0001] This invention relates to a methyltransferase OsMTA1 and its application in rice resistance to viral diseases, belonging to the fields of plant genetic engineering technology and disease control. Background Technology

[0002] Rice is one of the world's most important food crops, occupying a central position in global food production in terms of yield and cultivated area. With global population growth and dwindling arable land resources, rice production faces immense pressure. According to the FAO's projections, global food production needs to increase by 70% by 2050 to feed an additional 2.3 billion people. Therefore, increasing rice yield and its resilience is crucial for global food security.

[0003] In rice production, both biotic and abiotic stresses severely impact its growth and development. Among these, rice viral diseases, due to their rapid spread, difficulty in control, and severe damage, have become a significant factor affecting stable and high rice yields. Currently, the most damaging rice viruses include Rice Stripe Virus (RSV), Southern Rice Black Streaked Dwarf Virus (SRBSDV), and Rice Stripe Mosaic Virus (RSMV). These viruses are primarily transmitted by insect vectors such as planthoppers and can cause symptoms such as yellowing, striping, and deformity of rice leaves, as well as stunted growth, leading to large-scale crop failure in severe cases.

[0004] Currently, the main control measures for rice viral diseases include pesticide control of vector insects, planting disease-resistant varieties, and adjusting cultivation systems. However, due to the diversity of virus types, rapid mutation rates, and complex transmission methods, existing control measures have limited effectiveness. Especially in double-cropping and continuous-cropping rice areas, the prevalence of viral diseases continues to rise. Therefore, elucidating the molecular-level defense mechanisms of rice against viral infection and exploring new antiviral genetic resources and regulatory pathways are important directions for current research.

[0005] In recent years, studies have shown that plants, in response to biotic and abiotic stresses, can dynamically respond to external stimuli not only through traditional gene transcription and signal transduction pathways but also through epigenetic modifications. Epigenetic regulation mainly includes DNA methylation, RNA modification, histone modification, chromatin remodeling, and non-coding RNA-mediated regulation. These modifications can affect gene transcriptional activity or post-transcriptional expression levels without altering the DNA sequence, thereby regulating plant growth, development, and stress resistance.

[0006] N6-methylation of adenosine monophosphate (m6A) is the most common epigenetic modification in eukaryotic mRNA and has been widely reported to play a crucial role in regulating RNA virus infection in animals and viral replication. m6A modification is catalyzed by methyltransferases, removed by demethylases, and ultimately recognized by m6A reading proteins. In tobacco and tomato, the HAKAI methyltransferase inhibits PepMV infection by methylating the RNA. In wheat, the methyltransferase TaMTB promotes wheat infection by modifying PepMV RNA with m6A. OsMTA1, a methyltransferase in rice, remains unclear regarding its involvement in rice's resistance to viral infection and its specific antiviral mechanism, requiring further investigation. Summary of the Invention

[0007] This invention relates to a rice methyltransferase OsMTA1 gene and its application in the breeding of virus-resistant gramineous food crops.

[0008] On the one hand, this application relates to an OsMTA1 gene. In order to improve its protein expression level, we have optimized its codons. The original nucleotide sequence (SEQ ID NO.1), the optimized nucleotide sequence (SEQ ID NO.2), and the amino acid sequence (SEQ ID NO.3) are shown below.

[0009] The original nucleotide sequence of OsMTA1:

[0010] SEQ ID NO.1

[0011] ATGTTCTTGCAGTTGGCTGACTATCATTTGAGAATGGACTCACGATCACCAA

[0012] AAATTCCAAGGAGAAGTCCTGATTCCAAGGATAAGGACTCTGATAGGAAC

[0013] AAGGAAAGAGATGCCAAGAATGATTGGGACTCATCAAGAGCATATGGCTCT

[0014] GAGACTGATTGCAAGGAGGAAATGTGTGATAGCAATAAGAGGAAGGGTTTT

[0015] GACCATGGGGGAAATTGTTGTTGACAATAGCAGATCAGTGGATAGTTGTCA

[0016] TGAAACTGAATTGCATGTGTTAAGAGATGATCGGCAGGATAAATCTGTAGA

[0017] GATAAAAGATATTTTGCATGATGGAGTTGCAAAAAGTGACTATGCTCAACG

[0018] CCAGATAGACTTGGATAGTGAAAGAAGGAATGGTACGGGAGATAACTCAA

[0019] GGGTGGATGTCCTGAGAGATGACAAATTAGACAGCGGAAGGGACAGAAAC

[0020] TGGAGCGACAGAACACGGGAGCCTGAAGGATCTAAGGACTATGTGAGGAA

[0021] TCGTCAGTGGCAAGATTCTAAGGAGGCAAATGATTCTGAATGGAAGAATGC

[0022] ACATGAAAGACTAGATGGTGGGAGTTTCCATGGGCGAGCTGGTTACCGGC

[0023] GAGATTCTAGGGGTAGATCTGAAAGTATTAGAGGTTCTTCAACCTATGGAG

[0024] GTCGGTATGACAGTTCTGATTCAATAGAGATCCGACCAAATAACAATCTTGA

[0025] TTTTGGACGAGAGGGCTCTGTTTCTGGAAGAAGATATGATGTGGGAGCTCA

[0026] TCGGGATGCAACACCTGGAACGAATGGTGATAAATCCGCCAATCCTGAAGC

[0027] AGATCAAAGTGGTAGTACCAGCACGATTTCTCAATTTCCTCAACATGGTCCT

[0028] AAAGGTGATAGATCCTCAAGAGGAAGAGGTAGACCAAATAGCAGGGATTC

[0029] TCAAAGAGTTGGAGGCACATTACCAATTATGCCCCCTCCTTTTGGTCCTCTT

[0030] GGTCTGCCACCAGGACCAATGCAACATATTGGGCCAAATATTCCCCACTCT

[0031] CCAGGCCCTCTTCTACCTGGTGTTTTTGTGCCGCCATTCCCTGGGCCTCTTG

[0032] TGTGGCCTGGGGCACGAGGTGTTGATGTGAATATGCTTTCTGTTCCACCCA

[0033] ATCTTCCCATCCCTCCAGTTGCTGCTGAACATAGATTCGCTCCAAGTATGGG

[0034] AGCTGGGCCTGGTCATAATATTCACCTAAACCAAATCGGCTCTGGAATAGGT

[0035] GCTCCGACAAATGTGTCTGGATTGAGTTTCCATCAATTGGGCACGCAAAGT

[0036] CGTGAAATGGCACATGATAAACCGCCTGCTGGTGGTGGTTGGACGCCACAT

[0037] AGAAACAGTGGACCAACCAGAAAAGCTCCTTCAAGGGGTGAGCAGAATG

[0038] ATTACTCACAGAACTTTGTGGACACTGGCATGCGACCTCAAAATTTCATTA

[0039] GAGAACTGGATCTTACAAGTGTAGCAGAGGACTATCCAAAATTGAGAGAA

[0040] CTCATCCAGAGGAAAGATGAAATTGTTGCCAATTCTGCTTCACCACCAATG

[0041] TATTATAAGTGTGATCTGAGGCAACATGTTCTTTCTCCAGAATTTTTTGGTAC

[0042] AAAGTTTGATGTTATTCTTGTGGATCCTCCATGGGAGGAGTATGTTCACCGG

[0043] GCACCAGGAATCACAGATCACATAGAGTATTGGAATGCTGAGGAGATTATG

[0044] AATTTAAAGATTGAGGCTATAGCAGATACTCCATCTTTTGTCTTCCTTTGGGT

[0045] TGGTGATGGTGTAGGTCTTGAGCAGGGCCGTCAATGCTTAAAGAAGTGGG

[0046] GATTTCGTAGATGTGAGGATGTTTGCTGGGTAAAAACCAACAAGAAAAATG

[0047] CAACACCCAGTCTGCGGCATGACTCTCATACAATACTGCAACACTCAAAGG

[0048] AGCATTGCTTGATGGGTATTAAAGGAACAGTGCGACGTAGCACTGATGGAC

[0049] ATGTTATCCACGCTAATATTGACACAGATATAATAATAGCTGAAGAACCAAC

[0050] CGATGGCTCAACTAAAAAGCCTGAAGACATGTACAGAATAATTGAGCATTT

[0051] CGCTCTTGGAAAGAGGCGCCTTGAACTCTTTGGAGAGGATCATAACATTCG

[0052] TCCTGGTTGGCTTACTCTCGGGAAAGGTTTATCGTATTCCAACTTCAACAAA

[0053] GAGGCTTATATCAAGAACTTTGCAGACAAGGATGGGAAAGTATGGCAGGG

[0054] AGGCGGTGGACGAAATCCTCCTCCGGAGGCTCCCCATCTAGTTGTGACGAC

[0055] ACCTGAGATTGAGGGCCTCAGGCCAAAGTCACCCCCACAGAAGAACTAAOsMTA1 optimized nucleotide sequence:

[0056] SEQ ID NO.2

[0057] ATGTTTTTACAACTAGCTGATTATCACTTGCGCATGGATTCGCGCTCCCCGA

[0058] AAATTCCGCGTCGTTCGCCGGATTCCAAGGATAAAGATTCTGACCGCAACA

[0059] AGGAGCGCGACGCGAAAAATGATTGGGATAGCAGCAGAGCGTATGGCTCG

[0060] GAAACCGACTGTAAAGAGGAGATGTGTGATTCTAATAAGCGTAAGGGTTTG

[0061] ACCATGGGTGAAATCGTGGTTGATAATTCTCGTTCTGTGGACAGCTGCCATG

[0062] AAACGGAACTGCATGTGCTCCGCGACGACCGTCAGGACAAGTCCGTTGAA

[0063] ATTAAAGACATTCTGCATGACGGTGTTGCTAAAAGCGATTATGCCCAGCGTC

[0064] AAATCGATCTGGACAGTGAGCGTCGTAACGGTACGGGTGACAACAGCCGC

[0065] GTGGATGTCCTGCGCGACGATAAGCTGGATTCCGGCAGAGACCGCAACTG[[ID=~]]

[0066] GTCAGACCGCACCCGTGAGCCGGAGGGTAGCAAGGACTACGTTCGTAACC

[0067] GTCAATGGCAGGACAGTAAGGAGGCCAACGACTCCGAGTGGAAAAACGC

[0068] CCACGAAAGACTGGACGGCGGTAGCTTCCATGGTCGCGCTGGCTATCGTCG

[0069] TGATAGCCGTGGTCGGTCGGAAAGCATTCGCGGTAGCAGCACATACGGCGG

[0070] CCGTTACGACTCATCCGACTCCATCGAGATCCGCCCGAACAACAATCTGGA

[0071] CTTCGGCCGTGAGGGCAGCGTAAGCGGCCGTCGTTACGATGTCGGTGCGC

[0072] ACCGGGATGCGACCCCGGGCACGAACGGCGACAAGTCTGCGAACCCGGA

[0073] AGCGGACCAAAGCGGTTCTACAAGCACGATCAGCCAGTTTCCGCAGCACG

[0074] GTCCAAAGGGTGATAGGTCCAGCCGTGGTCGTGGTCGTCCGAACAGCAGG

[0075] GACTCTCAACGCGTGGGTGGTACTCTGCCGATTATGCCGCCACCGTTTGGT

[0076] CCGCTGGGTTTGCCTCCAGGCCCGATGCAGCACATCGGTCCGAATATCCCG

[0077] CATTCTCCTGGTCCGCTGCTGCCGGGCGTGTTCGTCCCACCATTCCCGGGT

[0078] CCGCTGGTGTGGCCTGGTGCGCGTGGGGTGGACGTTAATATGCTGAGCGTT

[0079] CCGCCGAACCTCCCGATCCCGCCGGTTGCGGCAGAGCACCGTTTTGCTCCG

[0080] AGCATGGGCGCCGGTCCGGGCCACAACATCCATCTGAATCAGATTGGCTCG

[0081] GGTATAGGCGCGCCCACCAACGTGAGCGGCCTCTCCTTCCACCAGCTGGGC

[0082] ACGCAGTCCCGCGAAATGGCACACGATAAGCCGCCAGCAGGCGGCGGATG

[0083] GACCCCGCACCGCAACTCTGGCCCGACCCGTAAAGCGCCGTCGAGAGGTG

[0084] AGCAGAACGACTACAGCCAAAATTTCGTGGATACCGGTATGCGTCCGCAAA

[0085] ACTTCATTCGTGAGTTGGATTTGACCAGCGTTGCGGAAGATTACCCGAAAC

[0086] TGCGCGAGCTGATTCAGCGTAAGGACGAGATCGTTGCAAATAGCGCGAGC

[0087] CCGCCGATGTATTACAAATGCGACCTTCGCCAGCATGTCTTGAGCCCGGAG

[0088] TTTTTCGGCACTAAGTTCGATGTGATCCTGGTTGATCCGCCTTGGGAAGAAT

[0089] ACGTGCATCGCGCACCGGGCATTACCGACCACATTGAATATTGGAATGCGG

[0090] AAGAGATCATGAATCTGAAAATCGAGGCGATCGCAGATACCCCAAGCTTTG

[0091] TTTTTCTGTGGGTAGGTGATGGCGTGGGTCTGGAACAAGGCCGTCAGTGCC

[0092] TGAAGAAATGGGGTTTTCGTCGTTGCGAGGACGTCTGTTGGGTTAAAACCA

[0093] ATAAAAAGAACGCAACTCCGAGCCTGCGGCACGATAGCCATACCATTCTTC

[0094] AGCACAGCAAAGAGCACTGCTTAATGGGTATTAAAGGTACAGTTCGTCGCT

[0095] CGACCGACGGCCATGTCATCCACGCCAACATTGACACCGACATCATCATCG

[0096] CTGAGGAACCGACCGATGGCAGTACCAAAAACCGGAAGATATGTACCGT

[0097] ATTATCGAACACTTTGCACTGGGCAAGCGCCGTCTGGAATTGTTTGGTGAA

[0098] GACCATAATATTCGTCCAGGCTGGCTGACCCTGGGTAAGGGCTTAAGCTATT

[0099] CCAATTTCAACAAAGAGGCGTATATCAAGAACTTCGCTGATAAGGACGGTA

[0100] AAGTGTGGCAAGGTGGCGGCGGCCGTAACCCGCCCCCTGAAGCGCCGCAC

[0101] TTGGTTGTTACCACCCCGGAGATCGAGGGATTACGTCCGAAGTCTCCGCCG

[0102] CAAAAAAACTAA

[0103] OsMTA1 Report:

[0104] SEQ ID NO.3

[0105] MFLQLADYHLRMDSRSSPKIPRRSPDSKDKDSDRNKERDAKNDWDSSRAYGS

[0106] ETDCKEEMCDSNKRKGLTMGEIVVDNSRSVDSCHETELHVLRDDRQDKSVEI

[0107] KDILHDGVAXDYAQRQIDLDSERRNGTGDNSRVDVLRDDKLDSGRDRNWS

[0108] DRTREPEGSKDYVRNRQWQDSKEANDSEWKNAHERLDGGSFHGRAGYRRD

[0109] SRGRSESIRGSSTYGGRYDSSDSIEIRPNNNLDFGREGSVSGRRYDVGAHRDAT

[0110] PGTNGDKSANPEADQSGSTSTISQFPQHGPKGDRSSRGRGRPNSRDSQRVGGT

[0111] LPIMPPPFGPLGLPPGPMQHIGPNIPHSPGPLLPGVFVPPFPGPLVWPGARGVD

[0112] VNMLSVPPNLPIPPVAAEHRFAPSMGAGPGHNIHLNQIGSGIGAPTNVSGLSFH

[0113] QLGTQSREMAHDKPPAGGGWTPHRNSGPTRKAPSRGEQNDYSQNFVDTGMR

[0114] PQNFIRELDLTSVAEDYPKLRELIQRKDEIVANSASPPMYYKCDLRQHVLSPEF

[0115] FGTKFDVILVDPPWEEYVHRAPGITDHIEYWNAEEIMNLKIEAIADTPSFVFLW

[0116] VGDGVGLEQGRQCLKKWGFRRCEDVCWVKTNKKNATPSLRHDSHTILQHSK

[0117] EHCLMGIKGTVRRSTDGHVIHANIDTDIIIAEEPTDGSTKKPEDMYRIIEHFALG

[0118] KRRLELFGEDHNIRPGWLTLGKGLSYSNFNKEAYIKNFADKDGKVWQGGGGNRNPPPEAPHLVVTTPEIEGLRPKSPPQKN*

[0119] In some embodiments, the biomaterials related to the rice gene OsMTA1 encoded protein provided by the present invention are any one of the following A1) to A12):

[0120] A1) A nucleic acid molecule encoding the OsMTA1 gene protein;

[0121] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0122] A3) A recombinant vector containing the nucleic acid molecules described in A1);

[0123] A4) A recombinant vector containing the expression cassette described in A2);

[0124] A5) Recombinant microorganisms containing the nucleic acid molecules described in A1);

[0125] A6) Recombinant microorganisms containing the expression cassette described in A2);

[0126] A7) Recombinant microorganisms containing the recombinant vector described in A3);

[0127] A8) Recombinant microorganisms containing the recombinant vector described in A4);

[0128] A9) Transgenic plant cell lines containing the nucleic acid molecules described in A1);

[0129] A10) A transgenic plant cell line containing the expression cassette described in A2);

[0130] A11) Transgenic plant cell lines containing the recombinant vector described in A3);

[0131] A12) Transgenic plant cell lines containing the recombinant vector described in A4).

[0132] On the other hand, the present invention relates to the application of the rice methyltransferase OsMTA1 gene in the breeding of gramineous food crops resistant to Tenuivirus, Fiji virus and Cytorhabdovirus.

[0133] In some embodiments, the Tenuivirus genus includes Ricestripe virus (RSV) and Maize stripe virus (MSpV), with Ricestripe virus (RSV) being preferred.

[0134] In some embodiments, the Fiji virus genus includes Maizerough dwarf virus (MRDV), Rice black streaked dwarf virus (RBSDV), and Southern rice black streaked dwarf virus (SRBSDV); Southern rice black streaked dwarf virus (SRBSDV) is preferred.

[0135] In some embodiments, the genus Cytorhabdovirus includes Barley Yellow Striate Mosaic Virus (BYSMV), Northern Rice Mosaic Virus (NCMV), and Rice Stripe Mosaic Virus (RSMV); preferably, Rice Stripe Mosaic Virus (RSMV).

[0136] In some embodiments, the preferred gramineous food crop is rice, maize, wheat, oats, and barley; more preferably rice, and most preferably Nipponbare (Oryza);

[0137] On the other hand, this invention relates to a method for preparing transgenic plants resistant to rice stripe virus / southern rice black-streaked dwarf virus / rice stripe mosaic virus, the steps of which include:

[0138] (1) Cloning of the rice OsMTA1 gene;

[0139] (2) Construction of overexpression vectors;

[0140] (3) Agrobacterium-mediated transformation and callus induction culture;

[0141] (4) Positive identification of transgenic plants;

[0142] In some embodiments, the cloning steps of the rice OsMTA1 gene are as follows:

[0143] Primers OsMTA1-F and OsMTA1-R were designed based on the open reading frame (ORF) of OsMTA1. The primer sequences are as follows:

[0144] OsMTA1-F ATGTTTTTACAACTAGCTGATTATCACT(SEQ ID NO.4);

[0145] OsMTA1-RTTAGTTTTTTTGCGGCGGAGACTTCGG(SEQ ID NO.5);

[0146] PCR reaction was performed, and the PCR product was recovered, ligated into the pMD18-T vector, single clones were selected, and sent for testing to obtain the correct pMD18-T-OsMTA1 recombinant plasmid.

[0147] In some implementations, the construction steps of the overexpression vector are as follows:

[0148] Primers with restriction enzyme sites were designed for the construction of the OsMTA1 gene binary expression vector PCV1300. The primer sequences are as follows:

[0149] PCV-OsMTA1-F

[0150] CGACGACAAGACCGTCACCATGTTTTTACAACTAGCTGATTATCACT

[0151] (SEQ ID NO.6);

[0152] PCV-OsMTA1-R

[0153] GAGGAGAAGAGCCGTCGGTTTTTTTGCGGCGGAGACTTCGGA

[0154] (SEQ ID NO.7);

[0155] Using cDNA obtained by reverse transcription of Nipponbare RNA as a template, the OsMTA1 gene was amplified and the PCR product was recovered; then the pCV1300 vector was double-digested with ApaI enzyme and the digested vector was recovered.

[0156] The above PCR product and vector were ligated, transformed into Escherichia coli DH5α, positive clones were selected and sequenced to confirm that the pCV1300-OsMTA1 overexpression vector had been successfully constructed.

[0157] In some embodiments, the Agrobacterium transformation and callus induction culture steps include:

[0158] (1) Take an appropriate amount of the constructed overexpression vector plasmid and add it to Agrobacterium competent cells. Mix well and add it to an electrode cup pre-cooled at 4℃. Transform with a voltage of 2200V. Add 800μL of LB liquid medium without resistance. Incubate at 28℃ in a shaker for 2-3h. Centrifuge at 5000rpm for 1min. Discard the supernatant. Spread the remaining precipitate on solid medium containing 50μg / ml Kan and 50μg / ml Rif resistance. Incubate at 28℃ for 3d. Dehull the rice seeds to be transformed. Soak in 75% alcohol for 5min. Rinse twice with sterile water. Soak in 30% sodium hypochlorite solution for 30min. Soak in sterile water for 30min. Place the seeds in the induction medium with tweezers. Incubate at 28℃ in a light incubator for 3-4 weeks. Transfer the callus tissue that grows to the subculture medium with pre-sterilized tweezers. Subculture at 28℃ in a light incubator for 1 week.

[0159] (2) Select a single colony of Agrobacterium GV3101 and inoculate it into LB liquid medium, and culture the bacterial solution to the OD concentration. 600 = Approximately 0.6; Immerse the induced callus in Agrobacterium suspension culture for 5 minutes; Remove the callus and place it in a symbiotic culture medium, and culture in a 26℃ light incubator for 2-3 days; Then transfer the callus to a culture medium containing hygromycin and grow for 30-45 days; Finally, place the selected callus in a rooting medium and culture under light until the callus roots, and continue culture for 2 weeks to obtain transgenic plants.

[0160] In some embodiments, the induction medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, pH 5.8.

[0161] The subculture medium contains: N6 medium 24.1 g / L, 2 mg / L 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, pH=5.8.

[0162] The co-culture medium comprises: 24.1 g / L N6 medium, 2 mg / L 2,4-D, 200 μmol / L acetosyringone, pH = 5.2.

[0163] The rooting medium contains: 1 / 2 MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, pH=5.8.

[0164] On the other hand, the present invention also relates to a method for detecting resistance to rice stripe virus / southern rice black-streaked dwarf virus / rice stripe mosaic virus in rice, the steps of which include:

[0165] 1) Total RNA was extracted from the test rice and control Nipponbare rice using the TRIzol method, and cDNA was obtained by reverse transcription of the RNA; the relative expression level of the OsMTA1 gene was detected using quantitative primers, which are as follows:

[0166] qRT-OsMTA1-F GGGCCACAACATCCATCTGA (SEQ ID NO.8);

[0167] qRT-OsMTA1-RTAATCTTCCGCAACGCTGGT(SEQ ID NO.9);

[0168] OsUBQ5-F ACCACTTCGACCGCCACTACT(SEQ ID NO.10);

[0169] OsUBQ5-R ACGCCTAAGCCTGCTGGTT (SEQ ID NO. 11);

[0170] Based on the above gene expression level detection results, it is determined whether the rice plant to be tested is resistant rice. If the relative expression level of the OsMTA1 gene in the rice plant to be tested is significantly higher than that in the control, then it is resistant rice.

[0171] This invention utilizes plant transgenic technology to transform the full-length OsMTA1 gene into rice, and then screens for transgenic lines with high expression levels. Experiments show that after RSV / SRBSDV / RSMV infection of rice, the OsMTA1-overexpressing transgene exhibits significantly increased resistance to viral infection compared to the wild-type control. OsMTA1 plays a positive regulatory role in rice's antiviral resistance process.

[0172] This invention focuses on OsMTA1 in rice. A recombinant expression vector, pCV1300-OsMTA1, for the OsMTA1 gene was constructed. This vector was then transformed into mature rice Nipponbare embryos using Agrobacterium-mediated genetic transformation, followed by callus induction. Agrobacterium strain GV3101 was used. After obtaining transgenic rice seedlings, individual transgenic plants were identified. The expression level of the OsMTA1 gene in the transgenic plants was detected using quantitative real-time PCR (qRT-PCR), and stable T3 generation transgenic rice was obtained after screening. Resistance to OsMTA1 transgenic rice was assessed using artificial inoculation with RSV / SRBSDV / RSMV.

[0173] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0174] This invention constructs the pCV1300-OsMTA1 vector and introduces it into the rice variety (Nipponbare) using Agrobacterium-mediated transformation, obtaining two stably heritable transgenic rice varieties, named OsMTA1-ox#1 and OsMTA1-ox#2, respectively. RSV, SRBSDV, and RSMV resistance analyses were performed on these transgenic rice varieties, contributing to a richer understanding of the molecular mechanisms of interactions between rice viruses and plant host factors, and laying the foundation for further research on disease resistance theories. Attached Figure Description

[0175] Figure 1 Experimental results of the relative expression level of OsMTA1 in transgenic rice overexpressing OsMTA1.

[0176] Figure 2 Comparison of disease symptoms between OsMTA1 overexpressing transgenic strains and wild-type control strains after RSV infection.

[0177] Figure 3 Results of experiments on the viral load of OsMTA1 overexpressing transgene and wild-type control after RSV infection.

[0178] Figure 4 Comparison of disease symptoms between OsMTA1 overexpressing transgenic strains and wild-type control strains after SRBSDV infection.

[0179] Figure 5 Results of experiments on the viral load of OsMTA1 overexpressing transgene and wild-type control after SRBSDV infection.

[0180] Figure 6 Comparison of disease symptoms between OsMTA1 overexpressing transgenes and wild-type control after RSMV infection.

[0181] Figure 7 Results of experiments on the viral load of OsMTA1 overexpressing transgene and wild-type control after RSMV infection. Detailed Implementation

[0182] Example 1: Construction of OsMTA1 overexpression vector in rice

[0183] RNA was extracted from Nipponbare RNA, and the cDNA obtained by reverse transcription was used as a template to amplify the OsMTA1 gene. The PCR product was recovered. Primers with restriction enzyme sites were designed based on the open reading frame (e.g., sequence SEQ ID NO.2) for the construction of the OsMTA1 gene binary expression vector PCV1300. The primer sequences are as follows:

[0184] PCV-OsMTA1-F

[0185] CGACGACAAGACCGTCACCATGTTTTTACAACTAGCTGATTATCACT

[0186] (SEQ ID NO.6);

[0187] PCV-OsMTA1-R

[0188] GAGGAGAAGAGCCGTCGGTTTTTTTGCGGCGGAGACTTCGGA

[0189] (SEQ ID NO.7);

[0190] PCR amplification system: total volume 50 μL, including 25 μL of 2×PCR Buffer for KOD FX Neo, 1.5 μL each of forward and reverse primers (10 μM), 5 μL of dNTP Mix (2.5 mM), 1 μL of cDNA template, 1 μL of KOD FX Neo, and 15 μL of ddH2O.

[0191] PCR amplification program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 3 min, 35 cycles; 72℃ final extension for 10 min.

[0192] The PCR product was recovered, and an A-terminus was added. The pCV1300 vector was double-digested with ApaI enzyme, and the digested vector was recovered and a T-terminus was added. The recovered product with the A-terminus was ligated to the pCV1300 vector with the T-terminus. Ligation system: Total volume 10 μL, including 7 μL of the recovered product with the A-terminus and 3 μL of the pCV1300 vector with the T-terminus. Ligation reaction program: 75℃ for 20 s, 37℃ for 30 min.

[0193] A / T termination system: Total volume 50 μL, including 38.5 μL of recovered PCR product, 1.5 μL of T4 DNA polymerase, 2.15 μL of 10×NEB Buffer, and 100 Mm dATP / dTTP. A-termination reaction program: 37℃ for 20 min, 75℃ for 20 min.

[0194] After ligation, 10 μL of the ligation product was transformed into DH5α, and after standing on ice for 30 min, it was heat-shocked at 42℃ for 45 s. After the heat shock, it was quickly placed on ice, and 500 μL of antibiotic-free LB liquid medium was added to a clean bench. The plate was placed in a shaker at 37℃ and cultured at 200 rpm for 1 h. After centrifugation at 5000 rpm for 1 min, part of the supernatant was discarded, and the remaining precipitate was evenly spread on LB plates containing kanamycin sulfate resistance. The plate was incubated overnight at 37℃. Single clones were picked, and colony PCR was performed followed by sequencing to obtain the correct PCV1300-OsMTA1 recombinant plasmid.

[0195] Example 2: Genetic transformation of rice

[0196] (1) Take an appropriate amount of the constructed overexpression vector plasmid and add it to Agrobacterium competent cells. Mix well and add it to an electrode cup pre-cooled at 4℃. Transform with a voltage of 2200V. Add 800μL of LB liquid medium without resistance. Incubate at 28℃ in a shaker for 2-3h. Centrifuge at 5000rpm for 1min. Discard the supernatant. Spread the remaining precipitate on solid medium containing 50μg / ml Kan and 50μg / ml Rif resistance. Incubate at 28℃ for 3d. Dehull the rice seeds to be transformed. Soak in 75% alcohol for 5min. Rinse twice with sterile water. Soak in 30% sodium hypochlorite solution for 30min. Soak in sterile water for 30min. Place the seeds in the induction medium with tweezers. Incubate at 28℃ in a light incubator for 3-4 weeks. Transfer the callus tissue that grows to the subculture medium with pre-sterilized tweezers. Subculture at 28℃ in a light incubator for 1 week.

[0197] (2) Select a single colony of Agrobacterium GV3101 and inoculate it into LB liquid medium, and culture the bacterial solution to the OD concentration. 600 = Approximately 0.6; Immerse the induced callus in Agrobacterium suspension culture for 5 minutes; Remove the callus and place it in a symbiotic culture medium, and culture in a 26℃ light incubator for 2-3 days; Then transfer the callus to a culture medium containing hygromycin and grow for 30-45 days; Finally, place the selected callus in a rooting medium and culture under light until the callus roots, and continue culture for 2 weeks to obtain transgenic plants.

[0198] Example 3: Positive identification of transgenic plants

[0199] Total RNA was extracted from positive transgenic plants and reverse transcribed into cDNA, using the rice OsUBQ5 gene as an internal control; the quantitative primers for OsMTA1 are shown in SEQ ID No. 8-11. The relative expression levels of the transgenic lines OsMTA1-ox#1 and OsMTA1-ox#2, which overexpress the OsMTA1 gene, were significantly higher than those of the control. Figure 1 ).

[0200] qRT-OsMTA1-F GGGCCACAACATCCATCTGA (SEQ ID NO.8);

[0201] qRT-OsMTA1-RTAATCTTCCGCAACGCTGGT(SEQ ID NO.9);

[0202] OsUBQ5-F ACCACTTCGACCGCCACTACT(SEQ ID NO.10);

[0203] OsUBQ5-R ACGCCTAAGCCTGCTGGTT (SEQ ID NO. 11);

[0204] Example 4: Artificial inoculation of RSV

[0205] OsMTA1 transgenic and control Nipponbare rice seeds were soaked in a 37℃ constant temperature incubator for 2-3 days. After the seeds germinated, they were sown into 1L plastic cups with 25-30 rice seedlings per cup and 3 biological replicates. The cups were then placed in an artificial climate chamber at 25℃ with 16 hours of light and 8 hours of darkness for cultivation.

[0206] First- and second-instar non-toxic planthoppers acquire RSV infection on rice seedlings for 3-5 days, then transfer them to healthy rice seedlings and cycle for 10-12 days. At the end of the cycle, the virus-carrying rate of the planthoppers is measured, and the number of inoculated insects per seedling is calculated based on the virus-carrying rate. Virus-carrying / non-virus-carrying planthoppers are then inoculated onto rice seedlings at the three- to four-leaf stage (approximately 15 days old). Three days after inoculation, all insects are removed, and the rice seedlings are planted in the field for growth.

[0207] After approximately 20 days of field growth, plant symptoms were observed and the incidence rate was statistically analyzed to determine the disease status of rice. Two overexpressing transgenic lines of OsMTA1, OsMTA1-ox#1 and OsMTA1-ox#2, showed resistance to RSV, such as... Figure 2 As shown. Further testing of viral load revealed that the viral load in the infected transgenic strain was significantly lower than that in the wild type, such as... Figure 3 As shown.

[0208] Example 5: Artificial inoculation with SRBSDV

[0209] OsMTA1 transgenic and control Nipponbare rice seeds were soaked in a 37℃ constant temperature incubator for 2-3 days. After germination, the seeds were sown in 1L plastic cups with 25-30 seedlings per cup and 3 biological replicates. The cups were then placed in an artificial climate chamber at 25℃ with 16 hours of light and 8 hours of darkness for cultivation.

[0210] 1st-2nd instar non-toxic white-backed planthoppers acquire SRBSDV infection on rice seedlings for 3-5 days, then transfer them to healthy rice seedlings and cycle for 10-12 days. At the end of the cycle, the virus-carrying rate of the white-backed planthoppers is measured, and the number of inoculated insects per seedling is calculated based on the virus-carrying rate. Virus-carrying / non-virus-carrying white-backed planthoppers are then inoculated onto rice seedlings at the three- to four-leaf stage (approximately 15 days old). Three days after inoculation, all insects are removed, and the rice seedlings are planted in the field for growth.

[0211] After approximately 30 days of growth in the field, plant symptoms were observed, and the disease incidence was determined by statistical analysis of the disease rate. Two overexpressing transgenic lines of OsMTA1, OsMTA1-ox#1 and OsMTA1-ox#2, showed resistance to SRBSDV. Figure 4 As shown. Further testing of viral load revealed that the viral load in the infected transgenic strain was significantly lower than that in the wild type, such as... Figure 5 As shown.

[0212] Example 6: Artificial inoculation of RSMV

[0213] OsMTA1 transgenic and control Nipponbare rice seeds were soaked in a 37℃ constant temperature incubator for 2-3 days. After germination, the seeds were sown in 1L plastic cups with 25-30 seedlings per cup and 3 biological replicates. The cups were then placed in an artificial climate chamber at 25℃ with 16 hours of light and 8 hours of darkness for cultivation.

[0214] First- and second-instar non-toxic leafhoppers were infected with RSMV on rice seedlings for 3-5 days, then transferred to healthy rice seedlings and cycled for 10-12 days. At the end of the cycle, the leafhopper carrying rate was measured, and the number of leafhoppers inoculated per seedling was calculated based on the carrying rate. Infected / non-infected leafhoppers were then inoculated onto rice seedlings at the three- to four-leaf stage (approximately 15 days old). Three days after inoculation, all the leafhoppers were removed, and the rice seedlings were planted in the field for growth.

[0215] After approximately 30 days of field growth, plant symptoms were observed, and the disease incidence was determined by statistical analysis of the disease rate. Two overexpressing transgenic lines of OsMTA1, OsMTA1-ox#1 and OsMTA1-ox#2, exhibited resistance to RSMV, such as... Figure 6 As shown. Further testing of viral load revealed that the viral load in the infected transgenic strain was significantly lower than that in the wild type, such as... Figure 7 As shown.

[0216] In summary, these results indicate that, compared with the control Nipponbare, transgenic rice plants overexpressing OsMTA1 significantly enhanced rice resistance to RSV / SRBSDV / RSMV infection (including symptoms and viral load in infected plants), demonstrating that OsMTA1 positively regulates rice resistance to RSV / SRBSDV / RSMV. These findings not only clarify that OsMTA1 can influence the infection efficiency of RSV / SRBSDV / RSMV, but also lay the foundation for research on rice-virus interactions.

Claims

1. Application of rice transcription factor OsMTA1 in the breeding of virus-resistant gramineous food crops.

2. The application according to claim 1, wherein the virus comprises Tenuivirus, Fiji virus, and Cytorhabdovirus; and the nucleotide sequence of the OsMTA1 gene is shown in any one of SEQ ID NO.1-SEQ ID NO.

2.

3. The application according to any one of claims 1-2, wherein the amino acid sequence of the OsMTA1 gene is shown in SEQ ID NO.

3.

4. The application according to any one of claims 2-3, wherein the genus Tenuivirus includes Rice Stripe Virus (RSV) and Maize Stripe Virus (MSpV); preferably Rice Stripe Virus; the genus Fiji Virus includes Maize Rough Dwarf Virus (MRDV), Rice Black Streaked Dwarf Virus (RBSDV), and Southern Rice Black Streaked Dwarf Virus (SRBSDV); preferably Southern Rice Black Streaked Dwarf Virus; the genus Cytorhabdovirus includes Barley Yellowstriate Mosaic Virus (BYSMV), Northern Cereal Mosaic Virus (NCMV), and Rice Stripe Mosaic Virus (RSMV); preferably Rice Stripe Mosaic Virus.

5. The application according to any one of claims 1-4, wherein the gramineous food crop is selected from rice, corn, wheat, oats and barley; preferably rice, more preferably Huai Dao 5, Nipponbare, Wuyujing 3, and most preferably Nipponbare.

6. A method for preparing resistant transgenic rice, comprising the following steps: transferring an overexpression vector of the OsMTA1 gene into wild-type rice to obtain resistant transgenic rice, wherein the resistant transgenic rice, compared with wild-type rice, exhibits resistance to rice stripe virus, southern rice black-streaked dwarf virus, and rice stripe mosaic virus, and the amino acid sequence of the OsMTA1 gene is shown in SEQ ID NO.

3.

7. A method for identifying rice resistant to rice stripe virus and / or southern rice black-streaked dwarf virus and / or rice stripe mosaic virus, comprising the following steps: extracting total RNA from the rice plant to be tested, reverse transcribing it into cDNA, using the rice OsUBQ5 gene as an internal reference, using OsMTA1 gene and OsUBQ5 gene-specific primers for detection, and determining whether the rice plant to be tested is resistant based on the gene expression level detection results.

8. The method of claim 7, wherein the specific primer sequence is as follows: qRT-OsMTA1-F GGGCCACAACATCCATCTGA (SEQ ID NO.8); qRT-OsMTA1-RTAATCTTCCGCAACGCTGGT(SEQ ID NO.9); OsUBQ5-F ACCACTTCGACCGCCACTACT(SEQ ID NO.10); OsUBQ5-R ACGCCTAAGCCTGCTGGTT (SEQ ID NO. 11).

9. The method according to any one of claims 7-8, wherein the determination method is: if the relative expression level of the OsMTA1 gene in the rice plant to be tested is significantly higher than that in the control, then it is resistant rice.

10. The method according to any one of claims 7-9, wherein the amino acid sequence of the OsMTA1 gene is shown in SEQ ID NO.3.

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