烟粉虱细胞色素P450 CYP6CX4与吡虫啉的相互作用

Imidacloprid is the most important neonicotinoid pesticides. Since 1991，it has been widely used to control sucking pests. However,variety of insects quickly evolved resistance to imidacloprid due to the continuous application, non-standard application as well as abuse.Research shows that high level of resistance to imidacloprid in China whitefly populations is due to over-expression of cytochrome P450 CYP6CX4, leading to enhanced detoxification ability, and may be it is the only mechanism. We will use homology modeling to construct three-dimensional structure of Bemisia tabaci CYP6CX4, using human cytochrome P450 CYP3A4 crystal structure as the model.Molecular docking and dynamic simulations will be used to analyze interactions of imidacloprid with the the CYP6CX4 enzyme. The binding mode with the lowest energy in the enzyme active site, the key amino acids involved will be predicted.After that we will use the Spodoptera frugiperda ovarian Sf9 cell line to express wild-type and mutant CYP6CX4 protein.Determination of kinetic constants of each protein in vitro metabolism of imidacloprid will be used to validate the reliability calculating model.After that, the QM/MM method will be used to further research the molecular mechanism of imidacloprid metabolism in vitro.Our research will lay molecular basis of P450 based synergist design.

吡虫啉被广泛用于害虫防治，然而其抗性问题日趋严重。烟粉虱是一种重要的农业害虫，申请者前期研究发现烟粉虱对吡虫啉的高水平抗性是由于体内细胞色素P450 CYP6CX4过量表达，使得解毒代谢能力增强引起。因此，本项目旨在揭示烟粉虱CYP6CX4与吡虫啉的结合模式，从而为以CYP6CX4为靶标的抑制剂的设计奠定基础。项目计划利用杆状病毒介导的表达系统表达CYP6CX4蛋白，对吡虫啉进行代谢，测定代谢过程的酶动力学常数以及代谢产物成分；以人细胞色素CYP3A4的晶体结构为模板构建CYP6CX4的三维结构，通过计算化学的方法来预测CYP6CX4与吡虫啉可能的结合模式；根据计算化学数据，对CYP6CX4进行定点突变，表达突变体蛋白，并测定突变体体外代谢吡虫啉的动力学常数的变化。以期寻找到催化能力最弱的CYP6CX4突变体蛋白；培养此突变体与吡虫啉复合物的晶体结构，以进一步验证二者的作用。

吡虫啉被广泛用于害虫防治，然而其抗性问题日趋严重。烟粉虱是一种重要的农业害虫，申请者前期研究发现烟粉虱对吡虫啉的高水平抗性是由于体内细胞色素P450CYP6CX4 过量表达，使得解毒代谢能力增强引起。因此，本项目旨在揭示烟粉虱CYP6CX4 与吡虫啉的结合模式，从而为以CYP6CX4 为靶标的抑制剂的设计奠定基础。项目按照计划利用利用杆状病毒介导的表达系统表达CYP6CX4 蛋白，对吡虫啉进行代谢，测定了代谢过程的酶动力学常数以及代谢产物成分；以人细胞色CYP3A4 的晶体结构为模板构建CYP6CX4 的三维结构，通过计算化学的方法来预测CYP6CX4 与吡虫啉可能的结合模式；根据计算化学数据，对CYP6CX4进行定点突变，表达突变体蛋白，并测定了突变体体外代谢吡虫啉的动力学常数的变化。项目成功找到了催化能力最弱的CYP6CX4 突变体蛋白；培养了突变体与吡虫啉复合物的晶体结构，.进一步验证了二者的作用，项目共发表学术论文3篇，其中两篇为SCI源论文。