Cry1Ia和Cry2Ab融合蛋白的表达
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摘要
[目的]苏云金芽孢杆菌表达的杀虫蛋白Cry1Ia和Cry2Ab对鳞翅目和鞘翅目昆虫具有较好的杀虫活性。为充分利用已有Cry蛋白,本文构建了两者的融合蛋白,并对融合蛋白的原核表达进行检测。[方法]本文将两个基因的编码基因,按照两种先后顺序拼接起来,获得cry1Ia-cry2Ab融合基因和cry2Ab-cry1Ia融合基因。另外,将Cry1Ia蛋白N端第一个α-螺旋(73个氨基酸)的编码序列删除后,接入cry2Ab基因5'端,获得第三个融合基因cry1IaD73-cry2Ab。将3个基因分别插入pHT304载体,并由cry1Ac基因的启动子和终止子调控其在Bt细胞中表达。[结果]SDS-PAGE结果显示,Cry1Ia-Cry2Ab、Cry2Ab-Cry1Ia和Cry1IaD73-Cry2Ab三个融合蛋白均能够在Bt细胞中表达,其测定分子量与预期大小一致,分别约152kDa、152kDa和144kDa。蛋白印迹法分析发现,在表达过程中,三种融合蛋白均有一定比例的降解。另外,不同温度对融合蛋白的表达也有不同程度的影响。[结论]本研究构建了3种融合杀虫基因并在Bt细胞中成功表达,为进一步解析这类融合蛋白的杀虫活性和机理奠定了基础。
[Objectives]Bacillus thuringiensis(Bt)produces the insecticidal proteins known as Cry toxins such as Cry1 Ia and Cry2 Ab which hold insecticidal activities against Lepidoptera and Coleoptera pests.In order to take full advantage of thesetoxins,the fusion proteins of the Cry1 Ia and Cry2 Ab were constructed and expressed in Bt cells in present study.[Methods]The cry1 Ia gene was fused to the 5'terminus of cry2 Ab gene to generate the cry1 Ia-cry2 Ab fusion gene.The cry2 Ab-cry1 Iafusion gene was constructed by similar method except that the cry2 Ab genewas located on the 5'terminus.In addition,a truncated gene cry1 IaD73 with 219 bp deletion in 5'end of the cry1 Ia gene,which encoding the firstα-helix of Cry1 Ia,was obtained and inserted into the 5'terminus of cry2 abgene.The resulting fusion gene was designated as cry1 IaD73-cry2 Ab.The three fusion genes were individually inserted into a pHT304 derived plasmid and were controlled by the cry1 Ac gene promoter and terminator.These vectors were used to express the three fusion proteins in Bt cells.[Results]The SDS-PAGE analysis showed that the three fusion proteins were able to express in Bt cells,and the molecular weight observed were consistent with the calculated sizes,which were 152 kDa,152 kDa,and 144 kDa for cry1 Ia-cry2 Ab,cry2 Ab-cry1 Ia,and cry1 IaD73-cry2 Ab,respectively.Some degradedmolecules of these fusion proteins were detected by western blot analysis.Yields of the target proteins were also affected by the culture temperature.[Conclusion]In this study,three fusion insecticidal genes were constructed and expressed successfully in Bt cells,whichmakes it convenient to investigate theirinsecticidal activities and mode of function in the future.
[Objectives]Bacillus thuringiensis(Bt)produces the insecticidal proteins known as Cry toxins such as Cry1 Ia and Cry2 Ab which hold insecticidal activities against Lepidoptera and Coleoptera pests.In order to take full advantage of thesetoxins,the fusion proteins of the Cry1 Ia and Cry2 Ab were constructed and expressed in Bt cells in present study.[Methods]The cry1 Ia gene was fused to the 5'terminus of cry2 Ab gene to generate the cry1 Ia-cry2 Ab fusion gene.The cry2 Ab-cry1 Iafusion gene was constructed by similar method except that the cry2 Ab genewas located on the 5'terminus.In addition,a truncated gene cry1 IaD73 with 219 bp deletion in 5'end of the cry1 Ia gene,which encoding the firstα-helix of Cry1 Ia,was obtained and inserted into the 5'terminus of cry2 abgene.The resulting fusion gene was designated as cry1 IaD73-cry2 Ab.The three fusion genes were individually inserted into a pHT304 derived plasmid and were controlled by the cry1 Ac gene promoter and terminator.These vectors were used to express the three fusion proteins in Bt cells.[Results]The SDS-PAGE analysis showed that the three fusion proteins were able to express in Bt cells,and the molecular weight observed were consistent with the calculated sizes,which were 152 kDa,152 kDa,and 144 kDa for cry1 Ia-cry2 Ab,cry2 Ab-cry1 Ia,and cry1 IaD73-cry2 Ab,respectively.Some degradedmolecules of these fusion proteins were detected by western blot analysis.Yields of the target proteins were also affected by the culture temperature.[Conclusion]In this study,three fusion insecticidal genes were constructed and expressed successfully in Bt cells,whichmakes it convenient to investigate theirinsecticidal activities and mode of function in the future.
引文
[1]Gatehouse J A.Biotechnological prospects for engineering insect-resistant plants[J].Plant Physiology,2008,146(3):881-887.
[2]Zhao J Z,Cao J,Collins H L,et al.Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants[J].Proceedings of the National Academy of Sciences,2005,102(24):8426-8430.
[3]Bohorova N,Frutos R,Royer M,et al.Novel synthetic Bacillus thuringiensiscry1Bgene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer,sugarcane borer and fall armyworm in transgenic tropical maize[J].Theoretical and Applied Genetics,2001,103(6-7):817-826.
[4]Ho N H,Baisakh N,Oliva N,et al.Translational fusion hybrid Bt genes confer resistance against yellow stem borer in transgenic elite Vietnamese rice(Oryza sativa L.)Cultivars[J].Crop Science,2006,46(2):781-789.
[5]Salm T V D,Bosch D,Honee G,et al.Insect Resistance of Transgenic Plants that Simultaneously Express Modified Bacillus thuringiensis cryIA(B)and cryIC Genes:A Resistance Management Strategy[J].Biocontrol Science and Technology,1994,26(1):51-59.
[6]Yang Y Y,Mei F,Zhang W,et al.Creation of Bt Rice Expressing a Fusion Protein of Cry1Ac and Cry1I-Like Using a Green TissueSpecific Promoter[J].Journal of Economic Entomology,2014,107(4):1674-1679.
[7]Xu C,Cheng J,Lin H,et al.Characterization of transgenic rice expressing fusion protein Cry1Ab/Vip3Afor insect resistance[J].Scientific Reports,2018,8(1):15788-15795.
[8]Bravo A,Pacheco S,Gómez I,et al.Insecticidal proteins from Bacillus thuringiensis and their mechanism of action,in Bacillus thuringiensis and Lysinibacillus sphaericus:Characterization and use in the field of biocontrol[M].Cham:Springer International Publishing,2017:53-66.
[9]Jurat Fuentes J L,Crickmore N.Specificity determinants for Cry insecticidal proteins:Insights from their mode of action[J].Journal of Invertebrate Pathology,2017,142:5-10.
[10]彭琦,周子珊,张杰.苏云金芽胞杆菌杀虫晶体蛋白研究进展[J].中国生物防治学报,2015,31(5):712-722.
[11]Adalat R,Saleem F,Crickmore N,et al.In vivo crystallization of three-domain Cry toxins[J].Toxins(Basel),2017,9(3):80-92.
[12]Ge B,Bideshi D,Moar WJ,et al.Differential effects of helper proteins encoded by the cry2A and cry11A operons on the formation of Cry2Ainclusions in Bacillus thuringiensis[J].FEMS Microbiology Letters,1998,165(1):35-41.
[13]Kostichka K,Warren G W,Mullins M,et al.Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis:the cryV-encoded protein is expressed early in stationary phase[J].Journal of Bacteriology,1996,178(7):2141-2144.
[14]Liao C Y,Heckel D G,Akhurst R.Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera(Lepidoptera:Noctuidae),major pests of cotton[J].Journal of Invertebrate Pathology,2002,80(1):55-63.
[15]Grossi-De-Sa M F,Magalhaes M Q D,Silva M S,et al.Susceptibility of Anthonomus grandis(Cotton Boll Weevil)and Spodoptera frugiperda(Fall Armyworm)to a Cry1Ia-type Toxin from a Brazilian Bacillus thuringiensis Strain[J].Journal of Biochemistry and Molecular Biology,2007,40(5):773-782.
[16]Soberon M,Pardo-Lopez L,Lopez I,et al.Engineering modified Bt toxins to counter insect resistance[J].Science,2007,318(5856):1640-1642.
[17]高建华,王思宇,黄婷婷,等.Cry1Ia和Cry2Ab杂交分子的构建和表达[J].山西农业科学,2015,43(5):521-523+531.
[18]Gao J,Zhang Y,Zhao Q,et al.Transgenic rice expressing a fusion protein of Cry1Ab and Cry9Aa confers resistance to a broad spectrum of lepidopteran pests[J].Crop Science,2011,51(6):2535-2543.
[2]Zhao J Z,Cao J,Collins H L,et al.Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants[J].Proceedings of the National Academy of Sciences,2005,102(24):8426-8430.
[3]Bohorova N,Frutos R,Royer M,et al.Novel synthetic Bacillus thuringiensiscry1Bgene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer,sugarcane borer and fall armyworm in transgenic tropical maize[J].Theoretical and Applied Genetics,2001,103(6-7):817-826.
[4]Ho N H,Baisakh N,Oliva N,et al.Translational fusion hybrid Bt genes confer resistance against yellow stem borer in transgenic elite Vietnamese rice(Oryza sativa L.)Cultivars[J].Crop Science,2006,46(2):781-789.
[5]Salm T V D,Bosch D,Honee G,et al.Insect Resistance of Transgenic Plants that Simultaneously Express Modified Bacillus thuringiensis cryIA(B)and cryIC Genes:A Resistance Management Strategy[J].Biocontrol Science and Technology,1994,26(1):51-59.
[6]Yang Y Y,Mei F,Zhang W,et al.Creation of Bt Rice Expressing a Fusion Protein of Cry1Ac and Cry1I-Like Using a Green TissueSpecific Promoter[J].Journal of Economic Entomology,2014,107(4):1674-1679.
[7]Xu C,Cheng J,Lin H,et al.Characterization of transgenic rice expressing fusion protein Cry1Ab/Vip3Afor insect resistance[J].Scientific Reports,2018,8(1):15788-15795.
[8]Bravo A,Pacheco S,Gómez I,et al.Insecticidal proteins from Bacillus thuringiensis and their mechanism of action,in Bacillus thuringiensis and Lysinibacillus sphaericus:Characterization and use in the field of biocontrol[M].Cham:Springer International Publishing,2017:53-66.
[9]Jurat Fuentes J L,Crickmore N.Specificity determinants for Cry insecticidal proteins:Insights from their mode of action[J].Journal of Invertebrate Pathology,2017,142:5-10.
[10]彭琦,周子珊,张杰.苏云金芽胞杆菌杀虫晶体蛋白研究进展[J].中国生物防治学报,2015,31(5):712-722.
[11]Adalat R,Saleem F,Crickmore N,et al.In vivo crystallization of three-domain Cry toxins[J].Toxins(Basel),2017,9(3):80-92.
[12]Ge B,Bideshi D,Moar WJ,et al.Differential effects of helper proteins encoded by the cry2A and cry11A operons on the formation of Cry2Ainclusions in Bacillus thuringiensis[J].FEMS Microbiology Letters,1998,165(1):35-41.
[13]Kostichka K,Warren G W,Mullins M,et al.Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis:the cryV-encoded protein is expressed early in stationary phase[J].Journal of Bacteriology,1996,178(7):2141-2144.
[14]Liao C Y,Heckel D G,Akhurst R.Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera(Lepidoptera:Noctuidae),major pests of cotton[J].Journal of Invertebrate Pathology,2002,80(1):55-63.
[15]Grossi-De-Sa M F,Magalhaes M Q D,Silva M S,et al.Susceptibility of Anthonomus grandis(Cotton Boll Weevil)and Spodoptera frugiperda(Fall Armyworm)to a Cry1Ia-type Toxin from a Brazilian Bacillus thuringiensis Strain[J].Journal of Biochemistry and Molecular Biology,2007,40(5):773-782.
[16]Soberon M,Pardo-Lopez L,Lopez I,et al.Engineering modified Bt toxins to counter insect resistance[J].Science,2007,318(5856):1640-1642.
[17]高建华,王思宇,黄婷婷,等.Cry1Ia和Cry2Ab杂交分子的构建和表达[J].山西农业科学,2015,43(5):521-523+531.
[18]Gao J,Zhang Y,Zhao Q,et al.Transgenic rice expressing a fusion protein of Cry1Ab and Cry9Aa confers resistance to a broad spectrum of lepidopteran pests[J].Crop Science,2011,51(6):2535-2543.