核型多角体病毒p74基因在苏云金芽胞杆菌中克隆与表达的研究
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摘要
苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是目前世界上应用范围最广的杀虫微生物,其杀虫活性主要来源于芽胞形成期产生的杀虫晶体蛋白。传统的Bt菌株均存在杀虫谱窄、毒力低等缺陷,因此,通过生物技术手段构建高效广谱的Bt工程菌一直是国内外研究的热点和方向。本研究利用Bt的cry1Ac基因与核型多角体病毒的p74基因构建融合基因,并获得新型杀虫工程菌株XBU-H1Acp74。
从苜蓿银纹夜蛾核型多角体病毒(Autographa californicamulticapsid nucleopolyhedrovirus,简称AcMNPV)中提取病毒DNA,设计引物p74-F/p74-R,以病毒DNA为模版PCR扩增p74基因;从研究室保存的菌株Bt 4.0718(CCTCC No.M200016)中提取质粒,设计引物Ac-F/Ac-R和Act-F/Act-R,以质粒为模版PCR分别扩增cry1Ac基因及cry1Ac基因的终止下游序列cry1Act;以pMD18-T作为大肠杆菌亚克隆载体,分别构建含有目的基因p74、cry1Ac、cry1Act的T载体pTp74、pT1Ac和pT1Act;用SmaⅠ和NcoⅠ双酶切pTp74和pT1Act,回收两个酶切片段并进行连接,构建中间载体质粒pTp74Act;再分别用SalⅠ和BglⅡ双酶切pT1Ac和中间载体pTp74Act,回收两个酶切片段并进行连接,构建中间载体质粒pT1Acp74;然后将中间载体pT1Acp74和穿梭载体pHT304分别用SalⅠ和XmaⅠ双酶切,回收两个酶切片段并进行连接,构建表达载体质粒pH1Acp74,将表达载体pH1Acp74电转化至Bt无晶体突变株XBU001,获得目的重组菌株XBU-H1Acp74。
SDS-PAGE分析显示,工程菌株XBU-H1Acp74可表达130 kD的Cry1Ac蛋白和50 kD的P74蛋白;原子力显微镜观察显示,工程菌株XBU-H1Acp74可产生呈菱形的晶体蛋白;生测结果显示,工程菌株XBU-H1Acp74与普通工程菌HTX-42(cry1Ac单价基因转XBU001)相比,对初孵棉铃虫在48 h的LC_(50)是47.624μg/ml,72 h的LG_(50)是29.0782μg/ml,分别低于HTX-42在48h的116.4880μg/ml和72h的90.7526μg/ml,表明P74蛋白可以协同Cry1Ac的杀虫效果。
本研究成功构建了cry1Ac基因和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。
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 LC_(50)of 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.
从苜蓿银纹夜蛾核型多角体病毒(Autographa californicamulticapsid nucleopolyhedrovirus,简称AcMNPV)中提取病毒DNA,设计引物p74-F/p74-R,以病毒DNA为模版PCR扩增p74基因;从研究室保存的菌株Bt 4.0718(CCTCC No.M200016)中提取质粒,设计引物Ac-F/Ac-R和Act-F/Act-R,以质粒为模版PCR分别扩增cry1Ac基因及cry1Ac基因的终止下游序列cry1Act;以pMD18-T作为大肠杆菌亚克隆载体,分别构建含有目的基因p74、cry1Ac、cry1Act的T载体pTp74、pT1Ac和pT1Act;用SmaⅠ和NcoⅠ双酶切pTp74和pT1Act,回收两个酶切片段并进行连接,构建中间载体质粒pTp74Act;再分别用SalⅠ和BglⅡ双酶切pT1Ac和中间载体pTp74Act,回收两个酶切片段并进行连接,构建中间载体质粒pT1Acp74;然后将中间载体pT1Acp74和穿梭载体pHT304分别用SalⅠ和XmaⅠ双酶切,回收两个酶切片段并进行连接,构建表达载体质粒pH1Acp74,将表达载体pH1Acp74电转化至Bt无晶体突变株XBU001,获得目的重组菌株XBU-H1Acp74。
SDS-PAGE分析显示,工程菌株XBU-H1Acp74可表达130 kD的Cry1Ac蛋白和50 kD的P74蛋白;原子力显微镜观察显示,工程菌株XBU-H1Acp74可产生呈菱形的晶体蛋白;生测结果显示,工程菌株XBU-H1Acp74与普通工程菌HTX-42(cry1Ac单价基因转XBU001)相比,对初孵棉铃虫在48 h的LC_(50)是47.624μg/ml,72 h的LG_(50)是29.0782μg/ml,分别低于HTX-42在48h的116.4880μg/ml和72h的90.7526μg/ml,表明P74蛋白可以协同Cry1Ac的杀虫效果。
本研究成功构建了cry1Ac基因和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。
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 LC_(50)of 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.
引文
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