Cry3a杀虫蛋白基因的合成优化及在JK-SH007中高效表达  

Gene Synthesis of Bacillus thuringiensis Cry3Aa and High-level Expression in Burkholderia pyrrocinia JK-SH007 as a Biopesticide

作　　者：李阳[1] 吴酬飞[2] 吴小芹[1] 叶建仁[1] 张立钦[1] 

机构地区：[1]南京林业大学林学院,南京210037 [2]湖州师范学院,湖州313000

出　　处：《中国生物工程杂志》2017年第6期70-77,共8页China Biotechnology

基　　金：国家社科基金(16@ZH005);国家自然科学基金(31550004)资助项目

摘　　要：人工合成优化Cry3a杀虫蛋白基因,并从大肠杆菌BL21(DE3)中克隆T7表达系统,通过同源重组克隆进PHKT2表达载体,将此表达载体导入吡咯伯克霍尔德氏菌JK-SH007,通过诱导表达,成功指导合成T7 RNA聚合酶且高效表达分子量为70k Da的Cry3a蛋白。通过粗提液对二龄榆蓝叶甲进行生物毒杀试验,结果表明粗提液具有高毒性,致死中浓度(LC_(50)=0.63(0.48-0.82)g/L)。成功构建的T7表达系统,可高效表达毒性的Cry3Aa蛋白,为进一步开发杀虫生防菌剂,建立外源基因表达技术平台奠定了基础。To develop an anti-Pyrrhalta larva biopesticide. The T7 RNA pol for T7 DNA-directed RNA polymerase was cloned from BI21 （DE3） into the Burkholderia pyrrocinia expression vector pHKT2. The insecticidal cry3Aa gene from Bacillus thuringiensis was optimized, synthesized, and cloned to the modified T7 expression system. The expression of the cry3Aa gene in wild-type endophyte B. pyrrocinia JK-SH007 was induced with temperature shifting, resulting in high-level production of Cry3Aa protein with a molecular weight of about 70 kDa. Toxic mortality bioassay was conducted on 15 P. aenescens （Fairmaire） larvae in the 2 instar in the laboratory. The crude engineered strain was highly toxic [LCs0 =0.63 （0.48 -0.82） g/L] to Pyrrhalta aenescens （Fairmaire） larvae grown in culture dishes on elm leaves. In conclusion, a novel T7 expression vector for the effective expression of a cry3Aa gene for a protein toxic to beetle larvae was devised, and its impactful delivery can make it a possible candidate for a biopesticide.

关 键 词：苏云金芽孢杆菌 Cry3a基因 吡咯伯克霍尔德氏菌 杀虫活性 表达 

分 类 号：Q786[生物学—分子生物学]