Recombined baculovirus of insect, preparation method and application as insecticide
A virus and insecticidal crystal protein technology, applied in the field of genetic engineering, can solve problems such as weak virulence, failure to meet the requirements of agricultural production, and slow insecticidal speed
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Embodiment 1
[0141] Example 1 Cloning of the truncated cry1A(c) gene of Bacillus thuringiensis subspecies Kustak HD-73
[0142] Bacillus thuringiensis subsp. Kustak (BTK) HD-73 strain, plasmid vectors pXI93 (containing cry1Ab gene) and pHT3101 were provided by the China Center for Type Culture Collection, and the plasmid vector pBluescriptSK(+) was purchased from Stratagene. Dicer, random primer labeling kit was purchased from Promega Company, T4DNA ligase was purchased from GIBCO-BRL Company, α- 32 PdATP was purchased from Beijing Furui Company.
[0143] Construction of BTK HD-73 plasmid gene library: Bacillus thuringiensis (Bt) insecticidal crystal protein gene cry1A(c) was located on its largest 47Mda plasmid. In order to clone the full-length cry1A(c) gene, HindIII was used to completely digest Thuringiensis All the plasmids of Bacillus subsp. Kustak (BTK) HD-73 were cloned with pBluescriptSK(+) as vector, and the plasmid gene library of BTK HD-73 was constructed (Fig. 2).
[0144] L...
Embodiment 2
[0147] Example 2 Construction of transposable vector pFPhBc
[0148] The plasmid vector pBluePh was provided by China Type Culture Collection, and the transposable vector pFastBacl was purchased from GIBCO / BRL. pFastBacl has a mini-Tn7 transposon with a Gm inserted between its left and right arms rGene, AcNPV polyhedron promoter, multiple cloning sites and SV40 poly(A) termination structure.
[0149] Clone the polyhedrin gene (ph) to pFastBacl: From the vector pBluePh with the complete polyhedrin gene (ph) cloned, use Pst I / Sal I double enzyme digestion and recover the complete ph gene, insert it into the same Pst I / Sal I The transposition vector pFastBacl was double digested to construct the transposition vector pFPh. Positive clones were identified by Pst I / Sal I double digestion of pFPh to release a 1.3 kb ph fragment ( Figure 10 ).
[0150] The truncated cry1A(c) gene was cloned into pFPh: using the artificially synthesized DNA sequences GCGTCGACTATGGATAACAATCCGAACATC...
Embodiment 3
[0151] Example 3 Construction of recombinant Bacmid and acquisition of recombinant virus vAcPhBc:
[0152] Escherichia coli DH10Bac (containing the shuttle plasmid Bacmid and helper plasmids constructed based on the whole genome of Autographa californica nuclear polyhedrosis virus AcNPV) was purchased from GIBCO / BRL company, Sf-9 cells (Spodoptera frugiperda cells) Provided by China Center for Type Culture Collection.
[0153] Extract transposable vector pFPhBc DNA, transform Escherichia coli DH10Bac competent cells, under the action of the trans-acting factor produced by the helper plasmid, the Gm on pFPhBc r The / Pph / cry1A(c) / ph / SV40 polyA expression cassette is transposed to the mini-att site of Bacmid (Tn7 transposition contact site) by the Tn7 left and right flanking sequences on its 3' and 5' sides to obtain a recombinant Bacmid, Since the LacZ (galactosidase gene, which can decompose galactose and its analogues) of the recombinant Bacmid is insertionally inactivated, i...
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