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

作　　者：张雅昆[1] 赵丹[1] 郭巍[2] 王伟[1] 陆秀君[1] 李瑞军[1] 

机构地区：[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

出　　处：《中国生物防治学报》2016年第3期414-420,共7页Chinese Journal of Biological Control

基　　金：国家自然科学基金(31471775);现代农业产业技术体系(CARS-14)

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0.556～0.914 mg/mL胞晶混合物,其中GS3菌株杀虫活性最高,LC50值为0.556 mg/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60 kD和130～150 kD蛋白,其中2株还表达较弱的80 kD蛋白。从GS3菌株中克隆得到一种新的cry2A基因,Gen Bank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1899 bp,编码632个氨基酸,编码蛋白分子量为70.8 kD,等电点p I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70 kD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。According to the results of bioassay, five Bacillus thuringiensis strains with high toxicity against Spodoptera exigua were obtained. The median lethal concentrations （LCs0） 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 LCs0 of 0.556 mg/mL. These strains contained cry l Aa, cry l Ab, cry l La, cry l la, cry lIb, cry2A b, cry2Ad, cry2Ah, cry TA b 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 8-endotoxin genes. Sequence analysis showed that cry2Ah5 contained a lg99 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.

关 键 词：苏云金杆菌 甜菜夜蛾 CRY基因 cry2Ah5 

分 类 号：S433[农业科学—农业昆虫与害虫防治] S476.11[农业科学—植物保护]