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Bacillus thuringiensis（Bt）is one of the most widely used insecticidal microbe which can produce insecticidal crystal proteins（ICPs）during its sporulation.As one of the biopesticides,most traditional Bt products have some similar drawbacks,such as narrow host range,low toxicity target pest, being resisted by insect,etc.So it is a hot topic study to build a recombinant strain with highly insecticidal activity and widely host range by biotechnology.In this work,the cry1Ac gene and p74 gene were recombinanted and constructed into XBUO01 to enhance the toxicity of crystal proteins,and a new effective recombinant strains,named XBU-H1Acp74,maybe gained.First,the p74 gene was amplified from the genosome of Autographa californica multicapsid nucleopolyhedrovirus by using primer p74-F/p74-R,the cry1Ac gene and the terminator gene of cry1Ac,named cry1Act,were amplified from the plasmid of Bt 4.0718 strain which was stored in our lab by using primer Ac-F/Ac-R and Act-F/Act-R.Three T vectors,named pTp74,pT1Ac and pT1Act,which held the aimed gene p74,cry1Ac and cry1Act respectively,were builted by using pMD18-T.The two T vectors, pTp74 and pT1Act,were digested by SmaⅠand NcoⅠ,then the two fragments were extracted and linked by T4 DNA ligase,and the middle-vector pTp74Act which held the p74-cryAct fusing gene was built.The other T vectors,pT1Ac,was digested by SmaⅠand NcoⅠas well as the middle-vector pTp74Act,and the two fragments were also extracted and linked by T4 DNA ligase,so the other middle-vector pT1Acp74 which held the cry1Ac-p74- cry1Act fusing gene was built.And then pT1Acp74 and the shuttle plasmid PHT304 were digested by SalⅠ/XmaⅠ,the two fragments were extracted and linked by T4 DNA ligase similarly,and an expressing-vector pH1Acp74 was built.Finally,pH1Acp74 was transformed into the acrystalliferous strain XBU001 and the aimed recombinant strain XBU-H1Acp74 would be gotten.The expression of Bt transformant XBU-H1Acp74 was analyzed by SDS-PAGE,which showed XBU-H1Acp74 could produce 130kD Cry1Ac protein and 50kDP74 protein.The observation of Atomic Force Microscopy demonstrated that the recombinant crystals appeared bipyramidal crystals.The insecticidal activity of transformant against Helicoverpa armigera Hubner was evaluated comparing with the contrast strains HTX-42（only cry1Ac gene was transformed into XBU001）after autolysis.The LC50of HTX-42 was higher than the XBU-H1Acp74’s,which implyed that P74 could increase the efficacy and range of Bt Cry toxins in insect control.This work constructed the fusion gene of cry1Ac and p74 successfully,which made a good ground for constructing the fusion genes of Bt cry gene and other foreign genes.
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