新型vip3Aa基因的克隆与杀虫活性的测定

为了给生物防治提供更多的基因资源，以从黑龙江省分离的100株苏云金芽胞杆菌进行PCR vip3类基因鉴定，对vip3类基因进行克隆表达及生物活性测定，为vip基因储备奠定基础。利用vip3类通用引物进行基因鉴定并进行全长基因克隆表达和生物活性测定。结果表明，菌株LB73经Blast对比后发现含有vip3Aa类基因，克隆基因全长序列为2370 bp，编码789个氨基酸残基，分子量为88 kD，并在国际基因库GenBank中登记，其登录号为JQ228436，由Bt δ-内毒素基因国际命名委员会正式命名为vip3Aa46。构建原核表达系统，并进行杀虫活性的测定。vip3Aa46甜菜夜蛾LC50 4.02 μg/g，棉铃虫LC50 387.72 μg/g。生测结果表明，vip3Aa46表达可溶性蛋白对甜菜夜蛾幼虫具有高毒力；对棉铃虫幼虫具有一定毒力。

Cloning and Expression for Novel vip3Aa Gene and Its Bioassay

In order to provide more gene resources for biological control, identify vip3 gene-types of 100Bacillus thuringiensis isolates collected from Heilongjiang Province by PCR, perform cloning, expressaon and bioassay of vip3 gene for cry gene reserves. Identification used universal primer of vip3 gene, perform cloning, expression and bioassay of full-length gene. The results showed that, LB73 containsed vip3Aa gene. The gene full-length sequence was 2370 bp, coding 789 amino acid residues, MW 88 kD. Registered in GenBank with accession number JQ228436 and was designated vip3Aa46 by International Nomenclature Committee ofBacillus thuringiensis. LCso of vip3Aa46 against Spodoptera exigua was 4.02 μg,/g; LC50 of vip3Aa46 against Helieoverpa armigera was 387.72 μg/g. The results of bioassay showed soluble protein of vip3Aa46 had high activities for Spodoptera exigua and certain activities for Helieoverpa armigera.