麦二叉蚜对氟吡呋喃酮的抗性选育及细胞色素P450介导的抗药性机理分析

Resistance selection of spring-grain aphid Schizaphis graminum to flupyradifurone and cytochrome P450-mediated resistance mechanisms

中文关键词:麦二叉蚜  氟吡呋喃酮  抗药性  细胞色素P450  RNA干扰

keywords:Schizaphis graminum  flupyradifurone  insecticide resistance  cytochrome P450  RNA interference

中文摘要:为探究麦蚜对新型新烟碱类杀虫剂氟吡呋喃酮的抗性发展潜力及其分子机制，在室内对麦二叉蚜Schizaphis graminum进行抗性选育，通过增效剂试验和解毒酶活性测定初步解析其对氟吡呋喃酮的抗性生化机制，采用实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)技术分析细胞色素P450（简称P450）基因的表达水平，并利用RNA干扰（RNA interference,RNAi）技术对上调表达的P450基因进行功能验证。结果显示：经氟吡呋喃酮连续汰选20代，麦二叉蚜的抗性倍数上升为26.06倍，达到中等抗性水平；该抗性种群对吡虫啉、噻虫嗪、高效氯氟氰菊酯、氰戊菊酯、联苯菊酯、溴氰虫酰胺、阿维菌素、虫螨腈均无明显交互抗性，但对溴氰菊酯存在低水平的交互抗性。P450酶抑制剂增效醚对氟吡呋喃酮具有明显的增效作用，而且与敏感种群相比，抗性种群P450酶活性显著升高。4个P450基因CYP380C6、CYP4CJ1、CYP4C1和CYP4CJ5在抗性种群中的表达量显著高于敏感种群；沉默这4个基因后，麦二叉蚜对氟吡呋喃酮的敏感性显著增加。表明P450酶活性增强及CYP380C6、CYP4CJ1、CYP4C1、CYP4CJ5基因过量表达是麦二叉蚜对氟吡呋喃酮产生代谢抗性的重要机制。

Abstract:To explore the potential for resistance development in spring-grain aphid Schizaphis graminum to the novel neonicotinoid insecticide flupyradifurone and its molecular mechanisms, laboratory resistance selection was performed. Synergist assays and detoxification enzyme activity assays were carried out to preliminarily explore the biochemical mechanism of resistance. Furthermore, real-time quantitative PCR(RT-qPCR) was used to analyze the expression levels of cytochrome P450(P450) genes, and the functions of upregulated P450 genes were further verified by RNA interference(RNAi). The results showed that after 20 generations of continuous selection with flupyradifurone, the resistance ratio of S. graminum increased to 26.06-fold, reaching a moderate resistance level. The resistant population showed no obvious cross-resistance to imidacloprid, thiamethoxam, lambda-cyhalothrin, fenvalerate, bifenthrin, cyantraniliprole, abamectin, and chlorfenapyr, but showed low-level cross-resistance to deltamethrin. The P450 inhibitor piperonyl butoxide had a significant synergistic effect on flupyradifurone, and P450 activity significantly higher in the resistant population than in the susceptible population. The expression levels of four P450 genes, CYP380C6, CYP4CJ1, CYP4C1 and CYP4CJ5, were significantly higher in the resistant population than in the susceptible population. After these four genes were silenced, the sensitivity of S. graminum to flupyradifurone increased significantly. These results indicate that the enhanced P450 activity and overexpression of CYP380C6, CYP4CJ1, CYP4C1, CYP4CJ5 genes are important mechanisms underlying metabolic resistance to flupyradifurone in S. graminum.