核型多角体病毒p74基因在苏云金芽胞杆菌中的表达

 【目的】利用核型多角体病毒（NPV）的p74基因与苏云金芽胞杆菌（Bt）的cry1Ac基因构建融合基因cry1Ac-p74，构建具有广谱杀虫作用的工程菌。【方法】从Bt4.0718菌株中克隆cry1Ac基因及其终止子cry1Act，从苜蓿丫纹夜蛾核型多角体病毒（AcMNPV）中克隆p74基因，以pMD-T作为大肠杆菌亚克隆载体，分别构建含有目的基因片段的T载体pT1Ac、pTp74、pT1Act以及中间载体pT1Act、pTp74Act，获得携带有目的融合基因cry1Ac-p74的表达载体pH1Acp74;该表达载体电转化至Bt无晶体突变株XBU001，得到目的重组菌株XBU-H1Acp74。【结果】XBU-H1Acp74可表达130 kD的Cry1Ac蛋白和50 kD的P74蛋白，生测显示P74蛋白可以协同Cry1Ac的杀虫效果。【结论】本研究成功构建了cry1Ac基因与p74基因融合的Bt工程菌，为进一步研究和构建新型生物杀虫剂的工程菌株，研制出高效、广谱、安全的杀虫剂提供了新的技术途径。

Study on the Expression of p74 Gene of Nuclear Polyhedrosis Virus in Bacillus thuringiensis

【Objective】 This study was conducted to build a recombinant strain with highly insecticidal activity and a wide host range by using the cry1Ac gene and p74 gene. 【Method】 Firstly, the p74 gene was amplified from the genosome of Autographa californica multicapsid nucleopolyhedrovirus, the cry1Ac gene and the terminator gene of cry1Ac, named cry1Act, were amplified from the plasmid of Bt 4.0718 strain. Three T vectors, named pTp74, pT1Ac and pT1Act which held the aimed gene p74, cry1Ac and cry1Act, respectively, and two middle vectors, named pTp74Act and pT1Acp74 which held the aimed fusion gene p74-cry1Act and cry1Ac-p74, respectively, were built by using pMD18-T. Then pT1Acp74 and the shuttle plasmid were digested and linked, and an expressing-vector pH1Acp74 was built. Finally, pH1Acp74 was transformed into the acrystalliferous strain XBU001 and the aimed recombinant strain XBU-H1Acp74 were obtained. 【Result】 The expression of Bt transformant XBU-H1Acp74 was analyzed by SDS-PAGE, which showed XBU-H1Acp74 could produce 130 kD Cry1Ac protein and 50 kD P74 protein. The insecticidal activity of transformant against Helicoverpa armigera Hubner was evaluated compared with the contrast strains HTX-42 (only cry1Ac gene was transformed into XBU001) after autolysis. The LC50 of HTX-42 was higher than the XBU-H1Acp74’s, which implied that P74 could increase the efficacy and range of Bt Cry toxins in insect control. 【Conclusion】 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.