Constructing cry genes mutant library with DNA shuffling method for screening high toxic Cry protein | 利用DNA shuffling技术构建含5个cry基因的突变体库以筛选高毒力杀虫蛋白

Chinese. 摘　　要：以改造合成的5个Bt基因（cry1C＊、cry2A＊、cry1Ab、cry1Ac和cry9C＊）为材料，利用DNA shuffling的体外分子进化技术构建1个含有1 000个重组基因克隆的大肠杆菌表达库；通过诱导表达重组蛋白，ELISA方法测定蛋白表达量，以棉铃虫（Helicoverpa armigera）为试验昆虫进行毒性试验。结果显示：重组克隆的蛋白毒性与阳性对照Cry1Ac相比，增强的有122个，持平的有38个，有毒性但毒性降低的有232个，失去毒性的有608个；对这1 000个重组克隆进行了测序，比较了重组基因与原始基因的序列同源性，除去55个克隆未能找到其原始基因来源，将其余的945个重组克隆从5个Cry蛋白的核苷酸编码序列的同源性出发，划分为源于cry1Ab，cry1Ac，cry1C＊，cry2A＊和cry9C＊的5个重组克隆类型。根据毒力变化分为毒力上升、持平、下降3组。对1 000个重组基因的氨基酸序列进行分析后发现，相对于毒力上升和持平组，5个毒力下降组的重组克隆间的氨基酸序列同源性最低。_

English. An E.coli-expression mutant library containing 1 000 cry mutant genes was constructed using DNA family shuffling method with five initial cry genes cry1Ac,cry1Ab,_cry1C＊,cry2A＊ and cry9C＊.The 1 000 clones were expressed in E.coli and their toxicity was tested upon Helicoverpa armigera （cotton bollworm） larvae.The expression level of mutation clones were quantified by ELISA.Compared with the original Cry1Ac,there were 122 clones with significantly enhanced toxicity,38 clones with similar toxicity; 232 clones with decreased toxicity; and 608 clones with lost toxicity.All 1 000 cry mutant genes in the library were sequenced and amino acid sequence homology of all cry mutant genes was analyzed by multiple comparisons.Gene sources of 945 clones were found and divided into 5 classes based on homology,55 clones were unrecognized.According to toxicity level,the 1 000 clones were further divided into 3 classes：increased toxicity,similar toxicity and decreased toxicity.Analyzing the amino acid sequences of the 1 000 clones,the homology in 5 decreased toxicity classes was lower than increased toxicity classes and similar toxicity classes._