Chilo suppressalis Bt protein receptor V-ATPase A subunit gene and application thereof
A technology for the diploid borer and bt protein, which is applied in the field of genetic engineering, can solve the problems of cumbersome methods, difficult to detect early resistance frequency resistance, agricultural production threat, etc., and achieves high detection sensitivity and precision, accurate and reliable detection results, and economical savings. The effect of labor and resources
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Embodiment 1
[0025] The cloning of embodiment 1 gene
[0026] 1. According to SMARTer TM The RACE cDNA Amplification Kit requires designing PCR primers for cloning the full-length cDNA of the Bt protein receptor V-ATPase A subunit gene of Chipotle borer:
[0027] GSP1 = 5'-CGGTGGCATCAACATTTTGTGCTTCACCAG-3';
[0028] GSP2 = 5'-CGAAGAGACATCGGGTGTGACCGTAGG-3'.
[0029] 2. Extraction of total RNA from the midgut of Chilo suppressalis:
[0030] The sensitive strains of Chilo borer were reared indoors on artificial feed for multiple generations without contact with any Bt preparation or protein.
[0031] ①The glassware used for RNA extraction was treated to remove RNase (180°C, baked for 4h);
[0032] ②Prepare 12-15 4th instar larvae of Chilo borer, dissect their midguts on ice, rinse them in pre-cooled 0.7% NaCl, dry the buffer with filter paper, and put them into a glass homogenizer;
[0033] ③ Add 1ml Trizol, fully homogenize on ice, transfer to a 1.5ml centrifuge tube, and incubate at ...
Embodiment 2
[0086] Example 2 Verification of the protein encoded by the V-ATPase A subunit gene as a Bt protein receptor
[0087] 1. Construction of prokaryotic expression vector and induced expression in Escherichia coli
[0088] Expression primers were designed according to the open reading frame sequence of the V-ATPase A subunit gene of Chipotle borer:
[0089] V-ATPase A subunit-F=5'-TGGTCGACAAATGGCGACGAAATCGGGTCTGA-3',
[0090] V-ATPase A subunit-R=5'-TAGCGGCCGCAATCCTCGAGGTTGCGGAAGGC-3', the upstream primer added a Sal1 restriction site, and the downstream primer added a Not1 restriction site. PCR amplification was performed with expression primers (V-ATPase A subunit-F, V-ATPase A subunit-R), and the amplified fragment was connected to the pMD19-T vector. The V-ATPase A subunit gene and the expression vector were double-digested respectively, and the results were as follows: figure 2 As shown (the left side of the Marker is the double enzyme digestion of the V-ATPase A subunit ...
Embodiment 3
[0103] Example 3 V-ATPase A subunit gene is used to detect the resistance of Chilo suppressalis to Bt insecticidal protein
[0104] 1. The extraction of the genomic DNA of the stem borer, the steps are as follows:
[0105] ①Collect the larvae of C. borer directly from the field or use high-pressure mercury lamps to lure the adults of C. barer, soak the obtained test insects in 95% ethanol and bring them back to the laboratory, and randomly select 100 larvae or adults of C. Gene frequency detection;
[0106] ② Genomic DNA was extracted using the AxyPrep Genomic DNA Mini-Extraction Kit from Aixin;
[0107] ③ Take 30 mg of the abdominal tissue of the 3-4 instar larvae or adults of Chilo borer in a 1.5 ml centrifuge tube, add 350 ml of phosphate buffer and 0.9 μl of RNaseA (50 mg / ml) solution and grind evenly. Collect 350μl tissue homogenate in a 2ml centrifuge tube;
[0108] ④ Add 150 μl lysate and 20 μl proteinase K (15 mg / ml) solution, vortex for 1 minute, centrifuge briefly, ...
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