对甜菜夜蛾高杀虫毒力苏云金杆菌菌株的筛选及cry2Ah5的克隆表达

doi:10.16409/j.cnki.2095-039x.2016.03.018

中国生物防治学报 ›› 2016, Vol. 32 ›› Issue (3): 414-420.DOI: 10.16409/j.cnki.2095-039x.2016.03.018

• 研究简报 • 上一篇

对甜菜夜蛾高杀虫毒力苏云金杆菌菌株的筛选及cry2Ah5的克隆表达

张雅昆¹, 赵丹¹, 郭巍², 王伟¹, 陆秀君¹, 李瑞军¹

1. 河北农业大学植物保护学院, 保定 071001;
2. 北京农学院植物科学技术学院, 北京 102206

收稿日期:2015-09-17 出版日期:2016-06-08 发布日期:2016-06-07
通讯作者: 郭巍
作者简介:张雅昆(1988-),女,博士研究生,E-mail:yakun_0830@126.com
基金资助:
国家自然科学基金(31471775);现代农业产业技术体系(CARS-14)

Screening of Bacillus thuringiensis Strains with High Toxicity against Spodoptera exigua and Identification of a New cry2Ah5 Gene

ZHANG Yakun¹, ZHAO Dan¹, GUO Wei², WANG Wei¹, LU Xiujun¹, LI Ruijun¹

1. College of Plant Protection, Agricultural University of Hebei, Baoding 071001, China;
2. College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China

Received:2015-09-17 Online:2016-06-08 Published:2016-06-07

摘要/Abstract

摘要： 利用生物活性测定方法从本实验室已分离保存的Bt菌株中筛选出了5株对甜菜夜蛾具有较高杀虫毒力的菌株。该5株Bt菌株对甜菜夜蛾初孵幼虫的LC₅₀值为0.556~0.914mg/mL胞晶混合物,其中GS3菌株杀虫活性最高,LC₅₀值为0.556mg/mL胞晶混合物;包含晶体形态主要是球形、大菱形、小菱形等;所含基因类型主要是cry1Aa、cry1Ab、cry1La、cry1Ia、cry1Ib、cry2Ab、cry2Ad、cry2Ah、cry7Ab、cry9Ba等;5株菌株均表达约60kD和130~150kD蛋白,其中2株还表达较弱的80kD蛋白。从GS3菌株中克隆得到一种新的cry2A基因,GenBank登录号为KT692984,由Bt基因国际命名委员会命名为cry2Ah5。该基因开放阅读框为1899bp,编码632个氨基酸,编码蛋白分子量为70.8kD,等电点pI为8.18,与其他Cry2Ah蛋白序列相似性为97%~99%。该基因在大肠杆菌BL21(DE3)中表达约70kD蛋白,与预测分子量相符。本研究筛选得到5株对甜菜夜蛾具有较高杀虫毒力的Bt菌株,克隆表达了一种新的cry2A基因cry2Ah5,为甜菜夜蛾的生物防治提供了新的菌株及基因资源。

关键词: 苏云金杆菌, 甜菜夜蛾, cry基因, cry2Ah5

Abstract: According to the results of bioassay, five Bacillus thuringiensis strains with high toxicity against Spodoptera exigua were obtained. The median lethal concentrations (LC₅₀) of these strains ranged from 0.556 to 0.914 mg/mL spores and crystals mixture, and the strain GS3 had the highest toxicity with a LC₅₀of 0.556 mg/mL. These strains contained cry1Aa, cry1Ab, cry1La, cry1Ia, cry1Ib, cry2Ab, cry2Ad, cry2Ah, cry7Ab and cry9Ba genes, forming globular or bipyramidal in different sizes crystals. All of them expressed 60 kD and 130-150 kD proteins, and two of them also lowly expressed 80 kD proteins. A new cry2A gene identified from the strain GS3 with an accession number of KT692984 was designated cry2Ah5 by the International Nomenclature Committee of Bt δ-endotoxin genes. Sequence analysis showed that cry2Ah5 contained a 1899 bp open reading frame, which encoded a 70.8 kD protein composing of 632 amino acids. The pI of this protein was 8.18. The Cry2Ah5 protein showed 97%-99% sequence identity with other Cry2Ah. The cry2Ah5 gene expressed a 70 kD protein in Escherichia coli BL21 (DE3) corresponding to the predict molecular weight. Our research provided more strains and genetic resources for the biological control of S. exigua.

Key words: Bacillus thuringiensis, Spodoptera exigua, cry gene, cry2Ah5

中图分类号:

S433
S476.11

张雅昆, 赵丹, 郭巍, 王伟, 陆秀君, 李瑞军. 对甜菜夜蛾高杀虫毒力苏云金杆菌菌株的筛选及cry2Ah5的克隆表达[J]. 中国生物防治学报, 2016, 32(3): 414-420.

ZHANG Yakun, ZHAO Dan, GUO Wei, WANG Wei, LU Xiujun, LI Ruijun. Screening of Bacillus thuringiensis Strains with High Toxicity against Spodoptera exigua and Identification of a New cry2Ah5 Gene[J]. journal1, 2016, 32(3): 414-420.

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对甜菜夜蛾高杀虫毒力苏云金杆菌菌株的筛选及cry2Ah5的克隆表达

Screening of Bacillus thuringiensis Strains with High Toxicity against Spodoptera exigua and Identification of a New cry2Ah5 Gene

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