定点突变对苏云金芽胞杆菌cry11ba蛋白结构和功能的影响微生物学专业论文.docxVIP

研究生优秀毕业论文 G147A、S149A表达的蛋白对胰酶稳定性提高，表明这4个位点氨基 酸的改变可能导致其对胰酶的消化作用敏感性降低。G144A对致倦库 G147A、S149A表达的蛋白对胰酶稳定性提高，表明这4个位点氨基 酸的改变可能导致其对胰酶的消化作用敏感性降低。G144A对致倦库 蚊和埃及伊蚊的LC50分别提高了1．84倍和3．31倍，其他8株工程菌 对致倦库蚊和埃及伊蚊的LC，。均降低。 本研究构建了13株具有潜在应用价值的突变工程菌株，深入探讨 了cryllBa基因结构变化对杀虫功能的影响，在杀蚊晶体蛋白分子结 构改变引起杀蚊功能改变方面获得了有力的佐证。同时，成功获得了 1株毒力提高的突变工程菌株G144A，为构建高效Bt基因工程菌株提 供技术支持，为后续Bt杀蚊基因功能域突变位点的选择和优化奠定了 重要基础，对微生物杀虫剂的开发和应用具有强烈的借鉴作用。 关键词：苏云金芽胞杆菌；定点突变；Cryl lBa；杀蚊活性；二硫键 万 方数据 AbstractBacillus Abstract Bacillus thuringiensis(Bt)has been used as a biopesticide in agriculture．The distinguished insecticidal characteristics of this bacterium are due to its ability for producing insecticidal crystal proteins(ICPs) during sporulation．Currently,it is important to study the molecular mechanism of insecticidal crystal in Bt for improving the yield of insecticidal crystal protein，improving the bioassay of Bt and constructing recombinant Bt strain with high efficiency and broad spectrum．However， the molecular structure and mechanism of Cry l 1 Ba needs further study． ClustalX2 and MEGA4．1 software was used to compare amino acid sequences of Cryl 1Aa and Cryl 1Ba，and DiANNA 1．1 Web Server was used to predict the position of Cryl 1Ba disulfide bond，and Swiss—Model software was used to establish a 3D structural model of Cry l 1 Ba，of which five sites on cry l 1 Ba C-terminel and nine sites on N—terminel Domain I from 1 4 1 to 1 5 0 were selected to be mutated．Site—directed mutation on Cry l 1 Ba was carried out using the technology of oligonucleotide—induced circular mutation．The mutants were transduced and expressed in acrystalliferous Bt strain 4Q7．The biochemical characteristics of the mutated Cryl 1Ba protein were studied through phase—contrast microscope， SDS—PAGE，solubility assay，trypsin stability analysis and bioassay． The five mutants in C-terminel could form oval crystals and express 8 1 kDa protoxins．Next，crystals protein of the five mutants increased gradually in buffer solution．Crystal protein of C7 1 4S was more s