| 对粉纹夜蛾高毒力cry9Ea基因的克隆及表达 |
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| 引用本文： | 孙永祥,刘廷辉,郭巍,王立光,孙伟明.对粉纹夜蛾高毒力cry9Ea基因的克隆及表达[J].微生物学报,2010,50(5):601-605. |
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| 作者姓名： | 孙永祥 刘廷辉 郭巍 王立光 孙伟明 |
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| 作者单位： | 河北农业大学生命科学学院，保定 071001;河北农业大学植物保护学院，河北省农作物病虫害生物防治工程技术中心，保定 071001;河北农业大学生命科学学院，保定 071001;河北农业大学植物保护学院，河北省农作物病虫害生物防治工程技术中心，保定 071001;河北农业大学生命科学学院，保定 071001 |
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| 基金项目： | 国家“973项目”（2009C13118900）；国家自然科学基金（30771447）；农业厅科技项目 |
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| 摘 要： | 【目的】从本实验室分离的Bt4菌株中克隆cry9Ea基因,并研究其表达和杀虫活性。【方法】以PCR-RFLP方法鉴定Bt4菌株含有cry9基因,然后以菌株Bt4的质粒为模板,利用全长引物F9EA/R9EA进行PCR扩增全长基因。【结果】将目的片段插入到表达载体pET21b,得到大肠杆菌重组表达质粒pETcry9Ea。转化E.coli BL21(DE3),诱导后表达130kDa的蛋白,再将cry9Ea7基因连接到穿梭载体pSXY422b,电激转化HD73-(cry-),得到工程菌BioHD9Ea7,提取Cry9Ea7晶体蛋白,并进行生物活性测定。生物活性测定结果显示Cry9Ea7蛋白对粉纹夜蛾(Trichoplusia ni)初孵幼虫具有高毒力,LC50为0.044μg/mL,而对甜菜夜蛾(Spodoptera exigua)和棉铃虫(Helicoverpa armigera)初孵幼虫未显示活性。【结论】克隆和表达了一个对粉纹夜蛾高毒力的基因cry9Ea7,并成功构建了工程菌BioHD9Ea7。
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| 关 键 词： | 关键词：苏云金杆菌 cry9Ea7基因 基因克隆 基因表达 杀虫活性 |
| 收稿时间： | 2009/10/14 0:00:00 |
| 修稿时间： | 2010/1/22 0:00:00 |
Cloning and characterization of a novel gene cry9Ea7 encoding crystal protein with high toxicity against Trichoplusia ni |
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| Yongxiang Sun,Tinghui Liu,Wei Guo,Liguang Wang and Weiming Sun.Cloning and characterization of a novel gene cry9Ea7 encoding crystal protein with high toxicity against Trichoplusia ni[J].Acta Microbiologica Sinica,2010,50(5):601-605. |
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| Authors: | Yongxiang Sun Tinghui Liu Wei Guo Liguang Wang and Weiming Sun |
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| Institution: | College of Life Science, Agricultural University of Hebei, Baoding 071001, China;College of Plant Protection, Agricultural University of Hebei, Biological Control Center of Plant Diseases and Plant Pests of Hebei Province, Baoding 071001, China);College of Life Science, Agricultural University of Hebei, Baoding 071001, China;College of Plant Protection, Agricultural University of Hebei, Biological Control Center of Plant Diseases and Plant Pests of Hebei Province, Baoding 071001, China);College of Life Science, Agricultural University of Hebei, Baoding 071001, China |
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| Abstract: | Objective]We cloned a novel cry9Ea gene encoding a Cry9Ea protein with a high insecticidal activity against Trichoplusia ni.Methods]We identified a cry9-type gene from Bt strain (Bt4) by PCR-RFLP.The full length cry9Ea gene was amplified by PCR with a pair of primers F9EA/R9EA.Results]We cloned the complete cry9Ea7 gene into pET21b.The SDS-PAGE results showed that the 130 kDa Cry9Ea7 protein was expressed in E.coli BL21(DE3).We also constructed an engineering strain BioHD9Ea7 by transforming a shuttle ve... |
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| Keywords: | Keyword: Bacillus thuringiensis cry9Ea7 gene gene cloning gene expression insecticidal activity |
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