Cry3a杀虫蛋白基因的合成优化及在JK-SH007中高效表达

doi:10.13523/j.cb.20170611

中国生物工程杂志

2017, Vol. 37

Issue (6): 70-77 DOI: 10.13523/j.cb.20170611

技术与方法

Cry3a杀虫蛋白基因的合成优化及在JK-SH007中高效表达

李阳¹, 吴酬飞², 吴小芹¹, 叶建仁¹, 张立钦¹

1. 南京林业大学林学院南京 210037;
2. 湖州师范学院湖州 313000

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

LI Yang¹, WU Chou-fei², WU Xiao-qin¹, YE Jian-ren¹, ZHANG Li-qin¹

1. College of Forest, Nanjing Forestry University, Nanjing 210037, China;
2. College of Life Sciences, Huzhou Teachers College, Huzhou 313000, China

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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70kDa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（LC₅₀=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。

关键词： 杀虫活性; 表达; Cry3a基因; 吡咯伯克霍尔德氏菌; 苏云金芽孢杆菌

Abstract:

To develop an anti-Pyrrhalta larva biopesticide. The T7 RNA pol for T7 DNA-directed RNA polymerase was cloned from BL21(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[LC₅₀=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.

Key words: Cry3a Insecticidal activity Burkholderia pyrrocinia Expression Bacillus thuringiensis

收稿日期: 2017-03-15 出版日期: 2017-06-25

ZTFLH:

Q786

基金资助:

国家社科基金（16@ZH005）、国家自然科学基金（31550004）资助项目

通讯作者: 张立钦 E-mail: zlqwz@zjhu.edu.cn

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引用本文:

李阳, 吴酬飞, 吴小芹, 叶建仁, 张立钦. Cry3a杀虫蛋白基因的合成优化及在JK-SH007中高效表达[J]. 中国生物工程杂志, 2017, 37(6): 70-77.

LI Yang, WU Chou-fei, WU Xiao-qin, YE Jian-ren, ZHANG Li-qin. Gene Synthesis of Bacillus thuringiensis Cry3Aa and High-level Expression in Burkholderia pyrrocinia JK-SH007 as a Biopesticide. China Biotechnology, 2017, 37(6): 70-77.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170611 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I6/70

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