Insect-resistant fusion gene, protein encoded by insect-resistant fusion gene, as well as expression vector and application of insect-resistant fusion gene
A technology of fusing genes and insect-resistant genes, applied in the field of genetic engineering, can solve the problems of low insect resistance, low expression of toxic proteins, and unstable expression products of transgenic plants, achieve broad application space and market prospects, and overcome gene silencing. Phenomenon, effect of excellent insecticidal activity
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Embodiment 1
[0046] Example 1: Anti-insect gene Cry1Ab and VIP3A(a) transformation
[0047] The inventor based on a large number of long-term experimental research and practical experience, in the original Cry1Ab (GenBank serial number is AY847289.1) A substantial improvement was made on the basis of the insect-resistant gene, mainly including removing part of the redundant base sequence, leaving only the nucleotide sequence containing the functional domain, codon modification, confirmation And exclude multiple AT-enriched regions such as ATTTA and AATGAA in the original insect-resistant gene DNA sequence and inverted repeat sequences in the gene sequence, and remove some commonly used restriction endonuclease recognition site sequences ( Xba I. Sac I), the sequence of the intron of the undefined eukaryotic DNA sequence and the sequence that may cause the premature termination of the gene transcription or the instability of the mRNA are reduced, and the stop codon at the 3' end is ...
Embodiment 2
[0054] Example 2: Anti-insect fusion gene CryAb-VIP3A build
[0055] Optimized insect-resistant fusion gene CryAb-VIP3A ( CryAb-VIP3A-MR ), transformed from Example 1 CryAb Gene sequence, connecting sequence L and embodiment one transformation VIP3A(a) Gene sequence composition. Cry1Ab After the stop codon at the 3 end of the gene is removed, the reading frame of the gene can be continuously expressed until the optimally modified vip3A(a) Gene, formed after optimized transformation Cry1Ab gene and vip3A fusion gene. The nucleotide sequence of the connecting sequence L is shown in SEQ ID NO.4; the nucleotide sequence of the anti-insect fusion gene is shown in SEQ ID NO.5, and the encoded amino acid sequence is shown in SEQ ID NO.6.
[0056] To detect modified CryAb-VIP3A The prokaryotic expression vector of the fusion gene was constructed according to the in vitro expression of the gene and the toxicity to corn borer and other pests. According to the needs ...
Embodiment 3
[0062] Example 3: Anti-insect fusion gene CryAb-VIP3A Expressed in E. coli
[0063] 1. Construction of recombinant E. coli
[0064] Transform the recombinant plasmid pET-CryAb-VIP3A into Escherichia coli E. coli BL21(DE3), positive transformants were screened out. After extracting the plasmid and verifying digestion, select positive transformants and inoculate them into resistant medium, culture overnight at 37°C, transfer with 2% inoculum, and culture to OD 600 The value is about 0.5-0.6, and it is stored at 4°C for later use.
[0065] 2. Detection of recombinant bacterial expression products Indoor insecticidal effect test
[0066] (1) The experimental treatments were divided into 4 groups:
[0067] Escherichia coli containing recombinant pET-CryAb-VIP3A was induced by IPTG, then ultrasonically disrupted, and the collected supernatant was used as the test group; cultured under the same conditions containing the mCryAb Gene sequence of recombinant pET28b vector E....
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