苏云金芽孢杆菌vip3A基因的鉴定、克隆及活性分析

Identification and Cloning of vip3A Genes from Isolates of Bacillus thuringiensis and Their Bioactivity Analysis

【目的】鉴定我国自行分离的苏云金芽孢杆菌(Bacillus thuringiensis,Bt)菌株中vip3A基因的分布和基因型,从其中对鳞翅目幼虫表现高毒力的Bt菌株中克隆vip3Aa基因。【方法】利用PCR-RFLP方法确定vip3A基因的分布和鉴定基因型,利用PCR方法克隆vip3A全长基因。【结果】171株野生型Bt菌株中共鉴定出63株含有vip3A基因,均与vip3Aa1类基因相似。从TF9和Bt16菌株中克隆得到2个vip3Aa基因,分别构建了携带vip3Aa基因的表达载体p30vip-26和p30vip-27,SDS-PAGE和Western Blot分析表明,IPTG诱导后均可表达88 kDa左右的Vip3A蛋白,蛋白可溶性分析表明约10%可溶。这两种基因序列已被国际Bt基因命名委员会分别正式命名为vip3Aa26和vip3Aa27。生物测定结果显示,Vip3Aa27蛋白对粉纹夜蛾(Trichoplusia ni)、甜菜夜蛾(Spodoptera exigua)和棉铃虫(Helicoverpa armigera)3种重要鳞翅目害虫初孵幼虫的毒力较高,LC50值分别为0.125μg/mL,0.238μg/mL和9.238μg/mL。而Vip3Aa26蛋白仅对粉纹夜蛾有活性,LC50值为4.423μg/mL。【结论】本研究中的Vip3Aa27蛋白对粉纹夜蛾、甜菜夜蛾和棉铃虫幼虫均能表现高杀虫活性,而Vip3Aa26蛋白仅对粉纹夜蛾幼虫有活性,实验结果表明Vip3A蛋白个别氨基酸的变化对其杀虫活性影响很大。

[ Objective ] To survey the distribution of vip3 A-type genes from isolates of Bacillus thuringiensis in China and to clone novel vip3A genes encoding Vip3A proteins with high insecticidal activity against Lepidopteran insect larvae. [ Methods] We applied PCR-RFLP method to identify vip3A-type genes from 171 isolates and cloned novel vip3Aa genes. [ Results] The vip3A-type genes appeared in 63 of 117 B. thuringiensis isolates. We cloned 2 novel vip3Aa genes from isolates of TF9 and Bt16. Then, we subcloned vip3Aa26 and vip3Aa27 into vector pQE30 and transformed into Escherichi coli M15, respectively. The results of SDS-PAGE and Western blot analyses showed that an 88 kDa peptide was expressed in E. coli M15 with lmmol/L of IPTG induction at 37 ℃, respectively. The International Nomenclature Committee of Bt nominated these two genes as the novel rip genes of vip3Aa26 and vip3Aa27, respectively. The bioassay results indicated that the Vip3Aa27 proteins were highly toxic to Trichoplusia hi, Spodoptera exigua and Helicoverpa armigera larvae and the LC50 values were 0. 125μg/mL, 0.238μg/mL and 9.238μg/mL, respectively. However, the Vip3Aa26 protein only possessed toxicity to T. ni larvae. [ Conclusions] The novel Vip3Aa27 protein had higher activity to Lepidopteran insect larvae compared with that for Vip3Aa26 protein. The results demonstrated that some amino acid changes had remarkable effect on the insecticidal activity.