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1、中国农业科学2009,42(4):1243-1251ScientiaAgriculturaSinicadoi:10.3864/j.issn.0578-1752.2009.04.015核型多角体病毒p74基因在苏云金芽胞杆菌中的表达1,21叶湘漓，夏立秋12（湖南师范大学生命科学学院微生物分子生物学湖南省重点实验室，长沙410081；湖南师范大学医学院，长沙410013）摘要：【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
2、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130kD的Cry1Ac蛋白和50kD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B
3、t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。关键词：苏云金芽孢杆菌；核型多角体病毒；cry1Ac基因；p74基因；融合基因StudyontheExpressionofp74GeneofNuclearPolyhedrosisVirusinBacillusthuringiensis1,21YEXiang-li,XIALi-qiu1(KeyLaboratoryofMicrobialBiologyofHunanProvince,CollegeofLifeScience,HunanNormalUniversity,Changsha4
4、10081;2CollegeofMedicine,HunanNormalUniversity,Changsha410013)Abstract:【Objective】Thisstudywasconductedtobuildarecombinantstrainwithhighlyinsecticidalactivityandawidehostrangebyusingthecry1Acgeneandp74gene.【Method】Firstly,thep74genewasamplifiedfromthegenosomeofAutographacalifornicamulticapsidn
5、ucleopolyhedrovirus,thecry1Acgeneandtheterminatorgeneofcry1Ac,namedcry1Act,wereamplifiedfromtheplasmidofBt4.0718strain.ThreeTvectors,namedpTp74,pT1AcandpT1Actwhichheldtheaimedgenep74,cry1Acandcry1Act,respectively,andtwomiddlevectors,namedpTp74ActandpT1Acp74whichheldtheaimedfusiongenep74-cry1Ac
6、tandcry1Ac-p74,respectively,werebuiltbyusingpMD18-T.ThenpT1Acp74andtheshuttleplasmidweredigestedandlinked,andanexpressing-vectorpH1Acp74wasbuilt.Finally,pH1Acp74wastransformedintotheacrystalliferousstrainXBU001andtheaimedrecombinantstrainXBU-H1Acp74wereobtained.【Result】TheexpressionofBttransfo
7、rmantXBU-H1Acp74wasanalyzedbySDS-PAGE,whichshowedXBU-H1Acp74couldproduce130kDCry1Acproteinand50kDP74protein.TheinsecticidalactivityoftransformantagainstHelicoverpaarmigeraHubnerwasevaluatedcomparedwiththecontraststrainsHTX-42(onlycry1Ac
