细胞色素P450对褐飞虱致害性变异的影响及表达调控的研究

The brown planthopper (BPH), Nilaparvata lugens (St?l) is the most important pest on paddy rice in China. Cultivating and planting resistant rice varieties are the most economical and effective method to control the BPH. However, the variation of BPH's virulence makes this great tool failure in the field condition. Thus, it is important to study the variation of the BPH's virulence. By a two dimensional electrophoresis (2-DE) system and MALDE-TOF/TOF-MS analysis we found that cytochrome P450 would be related to the variation of the BPH's virulence in rice varieties. The expression levels of P450 in N. lugens rearing on a moderately resistant rice and a susceptible variety were monitored by using real time quantitative PCR. In order to detect the effect of P450 activity on the vaniation of BPH's virulence, double stranded RNA (dsRNA) was injected in the BPH and the expression levels of P450 in N. lugens were monitored by using real time quantitative PCR. At the same time, the honeydew measurement and electrical penetration graph (EPG) of BPH population with different virulence were also studied. In order to study the mechanisms underlying regulation of P450, functional analysis of the promoters of P450 was performed in this study. Luciferase reporter plasmids containing serially truncated promoter fragments of the P450 gene. The promoter activity was measured with dual-luciferase reporter assay system.

褐飞虱Nilaparvata lugens (St?1)是我国水稻上的首要害虫，培育和种植抗虫品种是控制褐飞虱危害最经济有效的方法。然而，褐飞虱致害性的变异使得抗性品种的种植面临巨大困难，致害性变异问题已成为褐飞虱防治的主要难题之一。项目组前期工作中应用双向电泳和质谱技术发现细胞色素P450氧化酶（P450）在褐飞虱不同致害性种群中存在差异表达。为深入了解褐飞虱致害性变异的分子机制，本项目拟通过实时荧光定量PCR技术分析P450基因在不同致害性褐飞虱种群组织器官及发育阶段的特异表达；运用注射法RNAi结合蜜露测定、EPG和实时荧光定量PCR研究P450基因对褐飞虱致害性变异的影响；通过构建荧光素酶报告基因和启动子不同长度缺失片段的重组质粒，用双荧光素酶报告基因检测系统检测不同致害性褐飞虱种群P450基因启动子活性，为明确昆虫致害性变异的分子机制，探索基于基因水平的害虫防治新策略奠定基础。

褐飞虱Nilaparvata lugens (Stå1) 是我国和许多亚洲国家当前水稻的首要害虫，每年给亚洲各国农业造成大量损失。细胞色素P450可以代谢多种内源毒素和外源化合物，在植食性昆虫抵御逆境中发挥着一定的作用。本课题在已有的研究基础上，挑选10个细胞色素P450基因进行研究，发现这些细胞色素P450基因主要在2个褐飞虱不同致害性种群（TN1种群和MUDGO种群）的中肠表达，少部分在马氏管和血淋巴表达，在褐飞虱3、4龄表达较高，而在卵这一阶段相对表达较少。将褐飞虱敏感种群（TN1）接种到MUDGO水稻上进行抗性胁迫，发现CYP6CW1，CYP6AY1，CYP4C61在进行胁迫后基因表达量快速升高。同时在细胞水平上，利用 RNA 干扰手段进行功能验证，干扰CYP4C61和CYP6AY1基因均可使褐飞虱的蜜露分泌量（衡量褐飞虱取食量的标准）降低。表明CYP6AY1和CYP4C61可通过影响褐飞虱的取食行为在褐飞虱致害性变异中发挥一定的作用。同时对比分析了褐飞虱吡蚜酮抗性品系中细胞色素家族基因的表达变化，其中CYP4C61，CYP6CS1和CYP4CE1基因的表达水平显著高于敏感品系。另外，培育了红眼褐飞虱TN1敏感品系和红眼褐飞虱MUDGO抗性品系，为找到致害性，抗药性和眼色相关的基因，应用红眼这个显著的标志基因，对褐飞虱的抗药性和致害性进行检测提供基础。同时克隆了褐飞虱CYP6AY1和CYP4C61的启动子，为后续的研究提供重要的支持。