Pan Zhizhen, Xu Lian, Zhu Yujing, Shi Huai, Chen Zheng, Chen Meichun, Chen Qingxi, Liu Bo
Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, 350003, China.
World J Microbiol Biotechnol. 2014 Oct;30(10):2655-62. doi: 10.1007/s11274-014-1689-x. Epub 2014 Jun 19.
Bacillus thuringiensis (Bt) strain FJAT-12 was a novel Bt strain isolated by Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Science. In this study, a new cry2Ab gene was cloned from Bt strain FJAT-12 and named as cry2Ab30 by Bt delta-endotoxin Nomenclature Committee. The sequencing results showed there were two mutations in conservative sites which led to two amino acids modification. Homology modeling indicated that the two changes were located in β-sheet of Domain II. A prokaryotic expression vector pET30a-cry2Ab30 was constructed and the expressed protein was analyzed by western blot using Cry2Ab antibody. The expression conditions including IPTG concentration, revolution and temperature were optimized to get the highest expression level by SDS-PAGE and BandScan. The bioassay results also showed that the Cry2Ab30 toxin had high insecticidal activity against Plutella xylostella and the LC50 value was 0.0103 μg.mL(-1). The two mutations in β-sheet of Domain II might contribute to insecticidal activity of Cry2Ab30 toxin against Plutella xylostella.
苏云金芽孢杆菌（Bt）菌株FJAT-12是福建省农业科学院农业生物资源研究所分离得到的一株新型Bt菌株。在本研究中，从Bt菌株FJAT-12中克隆出一个新的cry2Ab基因，被Btδ-内毒素命名委员会命名为cry2Ab30。测序结果表明，保守位点存在两个突变，导致两个氨基酸发生改变。同源建模表明，这两个变化位于结构域II的β-折叠中。构建了原核表达载体pET30a-cry2Ab30，并使用Cry2Ab抗体通过蛋白质免疫印迹法对表达的蛋白质进行分析。通过SDS-PAGE和BandScan对IPTG浓度、转速和温度等表达条件进行优化，以获得最高表达水平。生物测定结果还表明，Cry2Ab30毒素对小菜蛾具有高杀虫活性，LC50值为0.0103μg·mL-1。结构域II的β-折叠中的两个突变可能有助于Cry2Ab30毒素对小菜蛾的杀虫活性。
