苏云金芽胞杆菌杀虫晶体蛋白Cry1Ba结构域Ⅱ中Loops结构与功能关系研究

Relationship between Structure and Function of Loops from Bacillus thuringiensis Insecticidal Crystal Protein Cry1Ba

摘要:

为了明确杀虫晶体蛋白中各个Loop的结构与功能的关系, 以及Loop突变对Cry1Ba蛋白杀虫活性的影响, 首先通过三维结构模拟以及同源序列分析的方法, 找到Cry1Ba蛋白三个结构域及三个Loop相对应的氨基酸片段; 然后通过重叠引物PCR将编码Cry1Ba蛋白结构域Ⅱ中的三个Loop进行了相应的突变, 共获得了5个突变体M1 (Loop1: 340WSNTR344—缺失(Cry1A))、M2 (Loop2: 402Y—G)、M3 (Loop2: 400GIYLEP405—PSAV (Cry3A))、M4 (400GIYLEPIH407—ILGS(Cry1A))、M5 (Loop3: 472LQSRV476—AGAVYTL (Cry1A))。将这些突变体在大肠杆菌BL21中进行了诱导表达, 提取蛋白, 分别对小菜蛾进行了生物活性测定。生测结果表明, Loop1的缺失突变M1的毒力, 与Cry1Ba (LC50 0.96 mg/mL)相比, 显著降低, LC50为35.51 mg/mL; 在Loop2的突变中, 单个氨基酸的突变 (Y/G)M2的毒力略有下降 (LC50为1.31 mg/mL); 而另两种突变 (M3和M4)对小菜蛾的毒力明显下降, LC50值分别为11.56 mg/mL、34.8 mg/mL; Loop3的突变M5对小菜蛾的毒力略有提高 (LC50 0.81 mg/mL), 但差异不显著。对Cry1Ba蛋白突变前后结构与功能之间关系的分析结果表明, Loop区突变对Cry1Ba蛋白的结构和功能影响非常显著; Loop1和Loop2在决定Cry1Ba对小菜蛾的毒性方面起着重要作用。

Abstract:

To indicate the relationship between structure and function of loops from Bacillus thuringiensis insecticidal crystal protein Cry1Ba, and the influence of amino acids mutation on toxicity against diamond back moth Plutella xylostella, five mutations at the loops of Cry1Ba were constructed by overlapping primer PCR, and expressed in E. coli BL21 (DE3). Bioassay results showed that the toxicity of mutation M1 (loop1: 340WSNTR344-deletion), compared with that of Cry1Ba (LC50 0.96 mg/mL), decreased significantly with LC50 35.51 mg/mL. And the toxicity of mutation M2 (402Y-G), M3 (400GIYLEP405-PSAV), M4 (400GIYLEPIH407-ILGS) was also reduced to some extent respectively. Only M5 (mutation at loop3: 472LQSRV476 - AGAVYTL) showed slightly increased activity against P. xylostella, but not significantly (LC50 0.81 mg/mL). Referring to the structures of Cry1Ba which was predicted using Swiss-Model software, and bioassay data, we can conclude that loop1 and loop2 play a important role on determining the activity of Cry1Ba against P. xylostella.