Protein related to insect resistance and encoding gene and application thereof
An encoding gene and insect resistance technology, applied in the field of insect resistance-related proteins and their encoded genes and applications, can solve problems such as the rampant pest resistance, environmental pollution, and pesticide residues.
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Embodiment 1
[0034] Example 1. Cloning of target gene and acquisition of overexpressed SlNAC072 transgenic tomato
[0035] 1. Cloning of the target gene
[0036] The total RNA of tomato (Solanum lycopersicon) variety M82 was extracted, and its reverse transcription product was used as a template for PCR amplification under the guidance of primers 1 and 2.
[0037] Primer 1: 5′-TCT GGTACC ATGGGTGTTCAAAGAAAAAAGATCCAC-3' (upstream primer, the base underlined is the Kpn I recognition site)
[0038] Primer 2: 5′-CAG CTGCAG CTACTGCTTGAACCCGAGATTTAAC-3' (downstream primer, the base underlined is the Pst I recognition site)
[0039] After the reaction, the PCR product was purified and connected to the pMD18-T vector, transformed into Escherichia coli DH5α, and positive clones were selected, and the positive clones were cultured in liquid, and the positive plasmid was extracted for sequencing. The result was inserted into pMD18-T with The genes shown in nucleotides 28-1077 in SEQ ID NO: 1 of ...
Embodiment 2
[0079] Example 2, Verification of the transcriptional activation activity of SlNAC072
[0080] 1. Cloning of the target gene
[0081] The vector pGBKT7 was purchased from Clontech, USA, the product catalog number is 630442. pGADT7 was purchased from Clontech, USA, the product catalog number is 630443.
[0082] According to the cDNA sequence of SlNAC072 and the suitable restriction site of the vector pGBKT7, primers were designed to amplify the full-length coding sequence of SlNAC072, the N-terminal coding sequence and the C-terminal coding sequence. The primer sequences are as follows:
[0083] Primer 5: 5'- CATATG GGTGTTCAAGAAAAAGA-3' (full-length coding sequence upstream primer, the underlined part of the base is the Nde I recognition site)
[0084] Primer 6: 5'- CTGCAG CTACTGCTTGAACCCGAGAT-3' (full-length coding sequence downstream primer, the underlined part of the base is the Pst I recognition site)
[0085] Primer 7: 5'- CATATG GGTGTTCAAGAAAAAGA-3' (N-terminal c...
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