Cry毒素结合蛋白V-ATPase A亚基的受体功能研究及其互作分析

Research on action mechanism of Cry toxins, especially on the receptor proteins, is the theoretical foundation of resistance management. Many binding proteins of Cry toxins has been identified, but a few of them have been validated as functional receptors. Our previous work found the binding of Cry1Ac with subunit A of V-ATPase from Plutella xylostella. But whether subunit A is the receptor of Cry toxins or not needs to be further validated. Hence, in this project, based on the analysis of gene expression changing of subunit A induced by Cry toxins and the test of enzyme activity of V-ATPase impacted by Cry toxins, prokaryotic expression and molecular docking will be used to analyze the binding sites of subunit A and Cry toxins. In addition, cell expression system and cytotoxicity will be carried out to verify the function of subunit A during the process of Cry toxins action,. Furthermore, RNAi will be used to study the susceptible changing of P. xylostella to toxins through down-regulation of subunit A expression. In general, the receptor function of subunit A will be identified by validation at genetic and celluar level as well as in vitro and vivo, which will provide us the theoretical basis for explanation the mode action of Cry toxins and insect resistance management.

对Cry毒素作用机理尤其是受体蛋白的研究是其抗性治理的理论基础。已报道的Cry毒素结合蛋白有多种，但进行系统证实为受体蛋白的仍不多。前期工作中，申请人发现小菜蛾V-ATP酶A亚基与Cry1Ac具有结合作用，但其是否为Cry毒素的受体蛋白并不清楚。本项目拟以小菜蛾V-ATP酶A亚基为研究对象，在分析A亚基基因在Cry毒素作用下表达量变化和体外测定毒素对V-ATPase酶活影响的基础上，利用原核表达及分子对接分析A亚基与Cry毒素作用的结合位点；并利用真核细胞表达结合细胞毒性进行功能分析，明确A亚基的作用；采用RNA干扰下调A亚基基因来探讨小菜蛾对Cry毒素的敏感性变化，分别从基因、体外、细胞和活体水平分析A亚基作为Cry毒素受体蛋白的功能，旨在为阐明Cry毒素作用机理及其抗药性治理提供理论支持。

随着Bt毒素的大规模使用和转Bt作物的大面积种植，靶标害虫抗性的出现成为必然，抗性综合治理成为目前急需解决的问题，而抗性问题治理的基础则是作用机理的明确，毒素作用机理研究中受体蛋白又是重要部分。本研究首先通过PCR克隆获得小菜蛾幼虫V-ATPase A亚基基因全长CDS区序列，采用实时荧光定量PCR技术研究小菜蛾幼虫不同龄期及取食Cry1Ac毒素后V-ATPase A亚基基因的表达丰度差异，结果表明在幼虫2龄期A亚基表达量最高，且在取食Cry1Ac毒素后表达量呈先下降再上升的趋势。接着，利用大肠杆菌原核表达系统表达并纯化获得V-ATPase A亚基蛋白，经配体结合试验鉴定，其与Cry1Ac、Cry2A毒素均有结合，与Cry2A的结合能力弱于Cry1Ac。通过分子模拟及对接实验表明，Cry1Ac可与A亚基的22-85位氨基酸、113-126位氨基酸、254-286位氨基酸、 318-326位氨基酸以及356位、359位、367位和440位氨基酸结合，删除A亚基1-85位氨基酸后，其与Cry1Ac的结合活性减弱。随后，利用Bac-to-Bac表达系统，在Sf9昆虫细胞中表达A亚基，细胞毒性实验表明Cry1Ac对表达有A亚基的Sf9细胞具有细胞毒性。最后，经RNAi干扰降低小菜蛾3龄幼虫的A亚基表达量后，小菜蛾幼虫对Cry1Ac的敏感性降低，但不显著。以上研究内容表明，小菜蛾幼虫体内V-ATPase A亚基是Cry1Ac毒素的潜在受体蛋白，该结果对完善Cry毒素作用机理，及其抗性综合治理提供参考和支撑。