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苏云金芽胞杆菌cry1Ie新基因的克隆、表达与生物活性测定

引用本文：余宗兰,贺利业,龚莉,黄刚辉,李平,郑爱萍.苏云金芽胞杆菌cry1Ie新基因的克隆、表达与生物活性测定.植物保护学报,2016,43(2):201-206

DOI：10.13802/j.cnki.zwbhxb.2016.02.004

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作者	单位	E-mail
余宗兰	四川农业大学水稻研究所, 温江 611130 四川农业大学, 西南作物基因资源与遗传改良教育部重点实验室, 雅安 625014
贺利业	四川农业大学水稻研究所, 温江 611130 四川农业大学, 西南作物基因资源与遗传改良教育部重点实验室, 雅安 625014
龚莉	四川农业大学水稻研究所, 温江 611130 四川农业大学, 西南作物基因资源与遗传改良教育部重点实验室, 雅安 625014
黄刚辉	四川农业大学水稻研究所, 温江 611130 四川农业大学, 西南作物基因资源与遗传改良教育部重点实验室, 雅安 625014
李平	四川农业大学水稻研究所, 温江 611130 四川农业大学, 西南作物基因资源与遗传改良教育部重点实验室, 雅安 625014	liping6575@163.com
郑爱萍	四川农业大学水稻研究所, 温江 611130 四川农业大学, 西南作物基因资源与遗传改良教育部重点实验室, 雅安 625014	apzh0602@gmail.com

中文摘要:为获得新的鳞翅目害虫杀虫基因,根据已知cry1I类基因编码区设计简并引物,采用直接克隆法,以苏云金芽胞杆菌(Bacillus thuringiensis,Bt)菌株BN23-5质粒DNA为模板进行扩增,并对得到的基因进行鉴定和分析。结果表明:克隆得到一个完整的cry1Ie基因,全长2160bp,由719个氨基酸组成,该氨基酸序列与已知的4种Cry1Ie蛋白不同,与Cry1Ie2和Cry1Ie3的氨基酸序列同源性最高,为95.4%,被国际Bt杀虫晶体蛋白基因命名委员会命名为Cry1Ie5(登录号为KJ710646)。将该基因插入表达载体pET-28a,转化大肠杆菌BL21(DE3),IPTG低温诱导成功表达,SDS-PAGE电泳验证其大小为81kD,与预测的分子量相符合。生物活性测定表明,Cry1Ie5表达的包涵体蛋白对小菜蛾和亚洲玉米螟具有杀虫活性,LC₅₀分别为0.43μg/mL和48.39μg/mL;对棉铃虫的致死率不高,但能明显抑制其生长;对甜菜夜蛾没有杀虫活性。

中文关键词:苏云金芽胞杆菌 cry1Ie5基因鳞翅目害虫克隆

Cloning, expression, and biological activity of a new cry1Ie gene from Bacillus thuringiensis strain

Author Name	Affiliation	E-mail
Yu Zonglan	Rice Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China Key Laboratory of Southwest Crop Gene Resource & Genetic Improvement of Ministry of Education, Sichuan Agricultural University, Ya'an 625014, Sichuan Province, China
He Liye	Rice Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China Key Laboratory of Southwest Crop Gene Resource & Genetic Improvement of Ministry of Education, Sichuan Agricultural University, Ya'an 625014, Sichuan Province, China
Gong Li	Rice Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China Key Laboratory of Southwest Crop Gene Resource & Genetic Improvement of Ministry of Education, Sichuan Agricultural University, Ya'an 625014, Sichuan Province, China
Huang Ganghui	Rice Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China Key Laboratory of Southwest Crop Gene Resource & Genetic Improvement of Ministry of Education, Sichuan Agricultural University, Ya'an 625014, Sichuan Province, China
Li Ping	Rice Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China Key Laboratory of Southwest Crop Gene Resource & Genetic Improvement of Ministry of Education, Sichuan Agricultural University, Ya'an 625014, Sichuan Province, China	liping6575@163.com
Zheng Aiping	Rice Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan Province, China Key Laboratory of Southwest Crop Gene Resource & Genetic Improvement of Ministry of Education, Sichuan Agricultural University, Ya'an 625014, Sichuan Province, China	apzh0602@gmail.com

Abstract:In order to obtain novel lepidopteran insecticidal genes, PCR method was used for identification of cry1I-type gene from Bacillus thuringiensis(Bt) strains BN23-5. According to the NCBI open cry1I-type gene encoding area, a pair of degenerate primers was designed. A full-length cry1Ie gene was cloned by PCR amplification with B. thuringiensis strain BN23-5 as template. It contained an open reading frame of 2 160 bp nucleotides encoding a protein of 719 amino acids. Compared with other Cry1Ie protein, it shared 95.4% amino acid homology with Cry1Ie2 and Cry1Ie3. It was designated as Cry1Ie5 by Bacillus thuringiensis Insecticidal Crystal Proteins Nomenclature Committee and the NCBI accession number was KJ710646. It was constructed into the recombinant plasmid pET28a, and transformed into Escherichia coliBL21 (DE3). The result of SDS-PAGE indicated that cry1Ie5 could be expressed as 81 kD protein in E.coli BL21 (DE3) strain induced by IPTG. The bioassay results indicated that the Cry1Ie5 protein was highly toxic to the larvae of Plutella xylostella and Ostrinia furnacalis with a LC₅₀ value of 0.43 μg/mL and 48.39 μg/mL, respectively; and had low activity against cotton bollworm, but could obviously inhibit the growth of it; it had no activity against beet armyworm.

keywords:Bacillus thuringiensis cry1Ie5 Lepidoptera pest clone

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