RNA (Ribose Nucleic Acid) with high TuMV (Turnip Mosaic Virus) resistance and RNAi (RNA interfere) vector for coding same
A technology for expressing vectors and encoding genes, which is applied in the field of RNA and RNAi vectors encoding the RNA, and can solve problems such as ineffective effects and environmental pollution
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Embodiment 1
[0056] Embodiment 1, the preparation of RNAi carrier
[0057] (1) Preparation of CI Gene Conserved Fragment
[0058] According to the conservation of 118 TuMV strains registered in GenBank, the 882-1247th nucleotide sequence of CI gene with a total of 366 bp conserved fragment was selected as the RNA interference target.
[0059] Extract the total RNA of the TuMV-C4 strain, and reverse transcribe it into cDNA, use the cDNA as a template, and use the following CI366F and CI366R (the underlined parts are restriction sites Xho I, BamH I, EcoR I, Hind III) as primers, PCR amplification with high-fidelity enzyme Fastpfu DNA Polymerase:
[0060] CI366 F 5'-cc ctcgagggatcc TGGAACACCTAGCAAGAAGCACT-3';
[0061] CI366 R 5'-cg gaattcaagctt CGTGCCTGCTTTGCCGTTAC-3'.
[0062] PCR amplification program: 94°C for 5min; 30 cycles of 94°C for 30s, 56°C for 30s, 72°C for 1min; 72°C for 7min.
[0063] The PCR product was purified by agarose electrophoresis and recovered. The obtained PCR...
Embodiment 2
[0079] Example 2, RNA with high resistance to TuMV and functional verification of the RNAi vector encoding the RNA
[0080] (1) Genetic transformation of Arabidopsis and screening of positive seedlings
[0081] The pBBBTu-CI vector was transformed into Agrobacterium GV3101 (pMP90) by electric shock method, and Arabidopsis was infected by Agrobacterium-mediated inflorescence dipping method. Arabidopsis thaliana to be harvested 0 The generation seeds were sown in culture trays filled with nutrient soil, and placed in an artificial climate chamber at 22°C (day) / 18°C (night) with a photoperiod of 16h (light) / 8h (dark). After the seedlings grow two cotyledons, spray water to dilute the glufosinate-ammonium herbicide 500 times by volume (the glufosinate-ammonium herbicide is a commercial herbicide purchased in the market, and the concentration of glufosinate-ammonium is 200mg / L) for screening Positive seedlings.
[0082] After being sprayed 2-3 times with glufosinate-ammonium her...
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