苏云金芽胞杆菌cry1Ia基因的克隆、表达与活性研究  

Cloning, Expression and Activity of cry1Ia Gene from Bacillus thuringiensis Isolate

作　　者：窦黎明[1] 韩岚岚[1] 张杰[1] 何康来[1] 赵奎军[2] 黄大昉[3] 宋福平[1] 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100094 [2]东北农业大学农学院,哈尔滨150030 [3]中国农业科学院生物技术研究所,北京100081

出　　处：《农业生物技术学报》2007年第6期1053-1057,共5页Journal of Agricultural Biotechnology

基　　金：国家重大基础研究发展规划(973)项目(No.2003CB114201);国家高技术研究发展计划(863)项目(No.2006AA02Z189和2006AA10A212)资助。

摘　　要：根据cry1Ia类基因的全长序列设计引物,以苏云金芽胞杆菌(Bacillus thuringiensis)菌株Btc008的总DNA为模板扩增出片段长为2.1kb的cry1Ia的全长基因,插入大肠杆菌(Escherichia coli)表达载体pET-21b,转化大肠杆菌BL21(DE3)菌株,诱导表达出81kD的蛋白。该蛋白由719个氨基酸组成,推导的分子量为81.2kD。该蛋白的氨基酸序列不同于已知的12种Cry1Ia蛋白,是一种新的Cry1Ia蛋白,该基因已被国际基因命名委员会正式命名为cry1Ia8。杀虫活性测定结果表明:Cry1Ia8对亚洲玉米螟(Ostrinia furnacalis)和小菜蛾(Plutella xylostella)有很强的杀虫活性,LC50分别为0.268μg/g和2.227μg/mL,其杀虫效果与Cry1Ab和Cry1Ac相当。对大豆食心虫(Leguminivora glycinivorella)也有较好的活性,但对鞘翅目叶甲科害虫榆兰叶甲(Pyrrhalta aenescens)没有活性。该基因的获得将为我国抗虫转基因作物和工程菌的研制提供新的基因来源,为筛选延缓昆虫抗性产生的基因组合提供了重要依据。A Full-length crylla gene fragment, which obtained by PCR amplification with a pair of primers designed according to cryIa-type gene sequences and DNA from Bacillus thuringiensis Btc008 as template, was introduced into expression vector pET-21b and transformed into Escherichia coli BL21 （DE3）. Molecular weight of the induced express product was 81 kD. The encoded protein was composed of 719 amino acid residues and the predicted MW was 81.2 kD. The amino acid sequence of the Crylla was very different from those of 12 known Crylla-type proteins. This gene with accession number AF373207 was designated as crylla8 by International Bt Insecticidal Gene Nomenclature Committee. The bioassay results indicated that the Cry 11a toxin protein showed distinctly insecticidal activity against Ostrinia furaacalis and Plutella xylostella with LC50 of 0.268 μg/g and 2.227 μg/mL, respectively, while it also had insecticidal activity against Leguminivora glycinivorella, but no activity against Pyrrhalta aenescens. The novel cryl Ia8 gene will be new resource for the construction of genetically engineered bacterium and transgenic plant for biocontrol of insect pests. It is also available for screening gene stacks to delay the resistance produce of the pests.

关 键 词：苏云金芽胞杆菌 cry1Ia基因 克隆 表达 杀虫活性测定 

分 类 号：S188[农业科学—农业基础科学]