飞蝗CncC/Keap1信号通路调控细胞色素P450基因的分子机制研究

Cytochrome P450 plays an important role in both endogenous and exogenous metabolism of insect. Insect CncC/Keap1 signal pathway involves in transcriptional regulation of cytochrome P450 genes, however there is a lack of systematic research on the regulation mechanism. Our group focused on the CncC/Keap1 signal pathway, and recently identified 3 genes putatively involved in transcriptional regulation. Based on our preliminary studies, we propose to advance our research to study the CncC/Keap1 signal pathway. The major objectives of our project are to: 1) Clone the full-length cDNA sequences of the key genes in CncC/Keap1 signal pathway by RACE-PCR, and analysis their phylogenetic relationship and mRNA expression patterns, and detect the locations of mRNA and its protein by in situ hybridization and immunohistochemistry, respectively. 2) Discover new gene by co-immunoprecipitation method, and understand the interaction among key proteins by using both GST pull-down and yeast two hybrid system. 3) Analysis on the recognition principle and response mode against exogenous chemicals in CncC/Keap1 signal pathway, and identify the modified residues in Keap1 caused by exogenous chemicals using UPLC-MS/MS. 4) Silence the key genes in CncC/Keap1 signal pathway by RNAi technology, screen the downstream P450 genes by comparative transcriptome, and then detect the sensitivity and detoxification ability against exogenous toxics in Locusta migratoria. Understand the recognition mechanism against cis-acting element in CncC/Keap1 signal pathway by Dual-Luciferase Reporter (DLR) assay and electrophoretic mobility shift assay (EMSA). The research result is expected to improve the theory of insect transcriptional regulation of cytochrome P450 gene, and has important significance on scientific prevention of Locusta migratoria.

细胞色素P450在昆虫内外源代谢中发挥重要功能。CncC/Keap1通路参与昆虫P450基因的转录调控，但调控机制尚缺乏系统研究。课题组对飞蝗CncC/Keap1通路开展系列研究，已鉴定出3个P450调控基因。本项目拟在此基础上，对飞蝗该通路开展如下研究：1）克隆该通路关键基因，进行系统发育及表达模式分析；采用原位杂交/免疫组化检测关键基因/蛋白的细胞、组织定位；2）利用Co-IP挖掘新蛋白；通过酵母双杂交和GST pull-down研究关键蛋白的互作机制；3）研究该通路对外源毒物的识别规律和应答方式；质谱法分析外源毒物对Keap1的修饰；4）RNAi技术沉默关键基因后，比较转录组筛选下游P450基因，并检测飞蝗对外源毒物的敏感性和代谢能力；利用双荧光素酶报告基因和凝胶迁移实验，研究该通路对P450顺式元件的识别机制。研究结果将完善昆虫P450基因转录调控理论，对飞蝗的科学防控有重要意义。

本项目以飞蝗和赤拟谷盗为研究对象，探讨CncC等转录因子调控解毒基因影响杀虫剂敏感性的分子机制，结果如下：..一、RNA沉默LmCncC基因，飞蝗对溴氰菊酯和吡虫啉的敏感性分别增加37.6%和25.4%。从RNAi-CncC比较转录组中筛选获得5个细胞色素P450。RNA沉默LmCYP6FD1后，飞蝗对溴氰菊酯的敏感性提高30.7%；RNA沉默LmCYP6FE1基因后，飞蝗对吡虫啉的敏感性增强25.9%。..二、UGT抑制剂磺吡酮处理，飞蝗对吡虫啉和溴氰菊酯的敏感性上升38%和66%。沉默LmCncC后，飞蝗对吡虫啉和溴氰菊酯的敏感性分别增加了42%和38%；沉默LmMaf后，飞蝗对吡虫啉和溴氰菊酯的敏感性分别增加了52%和56%。从RNAi-CncC和RNAi-Maf比较转录组和RT-qPCR结果中，筛选出3个UGT基因。LmCncC基因突变也导致上述3个UGT基因表达水平下降，并导致飞蝗对吡虫啉敏感性提升；RNA沉默LmUGT392C1或LmUGT392C1基因突变，也导致飞蝗对吡虫啉的敏感性均上升。荧光素酶报告基因结果表明LmCncC和LmMaf参与对LmUGT392C1的转录调控。酵母双杂交结果表明LmCncC和LmMaf存在蛋白相互作用。..三、RNA沉默TcAhR或TcARNT，赤拟谷盗对溴氰菊酯、氰戊菊酯和氟胺氰菊酯的敏感性上升31.25%-67.65%。P450抑制剂PBO处理后，飞蝗对溴氰菊酯、氰戊菊酯和氟胺氰菊酯的敏感性上升54.05%-58.11%。RNAi-ARNT比较转录组和实时定量PCR筛选获得唯一解毒基因TcCYP6BT1。RNA沉默TcCREB后，赤拟谷盗对辛硫磷和乐果的敏感性分别上升64.70%和36.63%；RNA沉默TcHNF4后，赤拟谷盗对辛硫磷和乐果的敏感性分别上升63.2%和28.52%。RNAi-TcCREB比较转录组和实时定量PCR筛选获得7个解毒基因，其中RNA沉默TcCYP345B1、TcATP32C提高飞蝗对有机磷类杀虫剂的敏感性；RNAi-TcHNF4比较转录组和实时定量PCR筛选获得10个解毒基因，其中RNA沉默TcCYP345B1、TcCYP6BT1、TcCYP9AB1提高飞蝗对有机磷类杀虫剂的敏感性。..本项目的研究成果对于完善昆虫解毒基因的转录调控机制，对与飞蝗和赤拟谷盗的抗性