Anti-magnaporthe oryzae paddy gene OsWRKY19 and application thereof
A rice blast fungus and gene technology, which is applied in the field of rice blast fungus-resistant rice genes to achieve the effect of improving disease resistance
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Embodiment 1
[0020] Embodiment 1, the cloning of gene
[0021] (1) Cloning of OsWRKY19 gene cDNA sequence:
[0022] The genomic DNA sequence, cDNA sequence and amino acid sequence of the OsWRKY19 gene were obtained from the MSU / TIGR rice genome database ( http: / / rice.plantbiology.msu.edu / ).
[0023] Design gene-specific PCR primers according to the cDNA coding sequence of the OsWRKY19 gene:
[0024] F1: 5'-ATGGTGGAGCTCTGCGGC-3' (SEQ ID No: 4);
[0025] R1: 5'-CAGATTCTGAATCTCCGATT-3' (SEQ ID No: 5).
[0026] The cDNA coding sequence of the OsWRKY19 gene was cloned from the cDNA library of the rice japonica (Oryza sativa L. ssp. Japonica) near-isogenic line variety IRBL22 (bred by International Rice) using these primers. Since the GC content in some regions of the cDNA coding sequence of the OsWRKY19 gene is as high as 80%, it cannot be cloned with common high-fidelity enzymes. After many experiments, the inventors used GC buffer I (TaKaRa) and Pfu high-fidelity enzymes to amplify The f...
Embodiment 2
[0029] Embodiment 2, the acquisition of transgenic rice constitutively expressing the OsWRKY19 gene
[0030] (1) Rice callus induction:
[0031] Select plump Taipei 309 (TP309) rice seeds, peel off the seed coat, sterilize and wash, evenly spot on sterilized NB solid medium with 2 mg / L 2,4-D, and keep light at 32°C for 5 days Induce callus formation.
[0032] (2) Agrobacterium transformation:
[0033] At the same time, the pWM101 vector carrying the OsWRKY19 gene was transformed into Agrobacterium EHA105 competent cells by the heat shock method, spread on solid LB medium with 50 μg / L kanamycin, and cultured in the dark at 28°C for 2 days. Positive clones were screened with specific primers F1 and R1. The obtained positive clones were cultured in the dark at 28° C. for 3 days in solid AB medium containing 50 mg / L kanamycin and used for rice transformation.
[0034] (3) Rice callus transformation:
[0035] Wash the Agrobacterium from the AB medium with filter-sterilized liq...
Embodiment 3
[0040] Example 3. Constitutive expression of OsWRKY19 gene enhances resistance of rice to rice blast
[0041] (1) Detection of OsWRKY19 gene expression level in transgenic rice:
[0042] Cut the leaves of transgenic rice seedlings, extract the total plant RNA with Trizol reagent (Invitrogen), treat and digest the DNA with DNase I (TaKaRa), and perform reverse transcription with the reverse transcription kit of Invitrogen to obtain the cDNA of the transgenic plants. Gene-specific real-time quantitative PCR primers realF and realR were designed according to the cDNA sequence of OsWRKY19 gene, and the expression level of OsWRKY19 gene in transgenic plants was detected by real-time quantitative PCR. The internal reference gene is the UBQ gene of rice (primers use realF2 and realR2), and the sequences of each primer are as follows:
[0043] realF: 5'-GCCAGTTCAGCAGCGACTTC-3' (SEQ ID No: 6);
[0044] realR: 5'-GTGCATTCGGCCACCTACAG-3' (SEQ ID No: 7);
[0045] realF2: 5'-GTGGCCAGTAA...
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