绿僵菌素A对四种亲和蛋白功能的影响与机理研究

Destruxins A (DA) is a cyclopeptidic lactone secondary compound produced by the entomopathogenic fungus, Metarhizium anisopliae. It has insecticidal activity and anti-immune effects, but the molecular mechanism is unclear. In previous experiments, the applicants discovered that four silkworm's proteins had high affinity (affinity constant <1μM) with DA in vitro, which indicated that the functions of the 4 proteins were affected by DA. This proposal is designed to verify the in vivo interactions of DA with these proteins and to explore the effects of DA on their functions and the underlying mechanism. First, the qPCR technology will be used to study the effects of DA on these 4 protein expressions in different tissues/organs of various stages of Bombyx mori and Tribolium castaneum. Second, the genes of these proteins from Bombyx mori and Tribolium castaneum will be heterologously expressed in E. coli. Then, the proteins will be extracted and purified and subjected to affinity analysis with DA. Third, expression system of bimolecular fluorescence complementation will be employed to survey the effects of DA on the interactions of these proteins with other proteins. Forth, in order to find the key sites for protein binding to DA, the genetic mutation against key amino of the proteins will be used to investigate the affinity changes of DA with mutant proteins and the interactions between the mutant proteins and other proteins. The results will reveal the interactions between DA and 4 proteins, clarify the effects of DA on the functions of the proteins, and exhibit the protein-DA binding mode. It will give new insights to understanding of the molecular mechanism of destruxins, and provide new ideas for the development of new pesticides.

绿僵菌素A（DA）是虫生真菌-金龟子绿僵菌产生的一种环肽内酯类次生化合物，具有杀虫活性和抗免疫作用，但其分子作用机理尚不清楚。申请人前期发现4种家蚕蛋白在体外与DA具有高亲和性（亲和常数<1μM），显示其功能受DA影响。本项目拟在体内进一步验证DA与这些蛋白的互作关系，探索DA对其功能的影响与及其机理。采用qPCR法研究DA影响这些蛋白基因在赤拟谷盗和家蚕不同虫态与组织/器官的表达特点；异源表达并分离纯化二种昆虫的上述蛋白，分析它们与DA的亲和性差异；采用双分子荧光互补系统研究DA对上述蛋白与其他蛋白互作功能的影响；进而，点突变肽链中的关键氨基酸，考察突变蛋白与DA亲和性的变化，分析突变蛋白与其他蛋白的互作关系，发现蛋白与DA结合的关键位点。研究结果将证明DA与4种蛋白的互作关系，阐明DA对其功能的影响，了解蛋白-DA结合模式，可提高对绿僵菌素分子作用机理的认识，为新型杀虫剂研发提供思路。

绿僵菌素A（DA）是由虫生真菌金龟子绿僵菌分泌产生的次生代谢产物，具有杀虫和抗虫免疫活性，但其作用分子机理尚未解析。申请人前期在家蚕中发现了4种与DA互作较强的蛋白，本项目在赤拟谷盗和家蚕中对4种DA亲和蛋白的分布特点研究和与DA亲和力以及DA对4种亲和蛋白功能的影响进行研究，取得如下研究结果。BmPLOD3、Bm26SPRS4、BmSEC23和BmTMEM214四种蛋白在家蚕卵、3-5龄幼虫、蛹期以及成虫期均有不同程度的表达，4种蛋白在幼虫期的四种组织（表皮、消化道、头部、血细胞）中均有表达。TcPLOD1、TcSec23A、Tc26SPRS4在赤拟谷盗卵、幼虫期、蛹期、成虫期均有不同程度的表达，在蛹期或成虫期达到最高表达，3种蛋白基因在老熟幼虫的头部、脂肪体、表皮、消化道、血淋巴都有表达，在血淋巴的表达量最大。家蚕四种蛋白BmTMEM214、BmSEC23、BmPLOD3及Bm26SPRS4与DA存在较强的相互作用关系，其亲和常数KD值分别为：0.286µM、0.291µM、0.710µM和0.899µM。赤拟谷盗Tc26SPRS4、TcPLOD1、TcSec23A与DA的亲和系数KD值分别为1.083×10-5M、1.890×10-4M、3.055×10-4M。DA对家蚕BmSEC23-BmSEC13和BmTMEM214-BmSEC13-like蛋白互作的影响最为显著，DA显著影响BmSEC23蛋白的互作功能，敲除该基因发现家蚕对DA敏感性降低。DA对赤拟谷盗三组互作蛋白的影响，DA处理后，目标蛋白与互作蛋白的相互作用受到明显的抑制作用，DA浓度为0.002mg/L时，Tc26SPRS4-Tc26SPNRS14、TcSec23A-TcSEC13互作受到影响。研究结果阐明DA与4种蛋白的互作关系，提高对绿僵菌素分子作用机理的认识，为新型杀虫剂研发提供思路。