辛硫磷、灭多威和dsRNA饲喂对棉铃虫ace基因表达的影响

Effects of Phoxim,Methomyl and Feeding dsRNA on the Expression of ace Genes in Helicoverpa armigera

乙酰胆碱酯酶(ACh E)是有机磷与氨基甲酸酯类杀虫剂的作用靶标,以棉铃虫体内乙酰胆碱酯酶为对象,通过检测辛硫磷、灭多威两类农药处理和饲喂ace ds RNA表达菌对乙酰胆碱酯酶基因(ace1、ace2)转录活性的影响,为阐明棉铃虫化学防治及基于RNAi的防治提供一定的参考。实验采用实时荧光定量PCR方法,分析辛硫磷、灭多威对棉铃虫4龄幼虫诱导12 h、24 h、36 h、48 h、60 h后ace基因的转录水平,利用表达ds RNA的菌液喂食2龄棉铃虫进行RNAi后检测ace基因的沉默效果。结果显示:灭多威和辛硫磷处理后,在12 h时,ace1和ace2皆显著下调,其后,ace1表达下调而ace2上调;RNAi处理后,棉铃虫ace1、ace2基因表达量在24 h、48 h下调明显,随后表达量略有上调。推测农药处理使棉铃虫幼虫生理代谢破坏造成ace1、ace2表达降低,随后ace1基因表达持续下降,而ace2表达逐渐升高,这可能与补偿农药对Ach E酶活性的抑制有关;RNAi处理使棉铃虫生长发育受到不同程度的抑制,ace1和ace2基因都出现明显的表达抑制。以上结果为以乙酰胆碱酯酶及其编码基因为靶标的棉铃虫防治提供了参考。

Acetyl cholinesterase ACh E is a target of organophosphorus-and carbamate-type pesticides. This study focuses on theacetylcholinesterase genes of Helicoverpa armigera,including the effects of phoxim,methomyl and feeding the ace ds RNA-expressing bacteria on the transcriptional activities of acetyl cholinesterase genes,which may provide reference for the chemical control and RNAi-based control of H. armigera. The real-time quantitative PCR method was used to analyze the transcriptional levels of ace genes in phoxim-and methomylinduced 4 th instar larvae of H. armigera for 12 h,24 h,36 h,48 h,and 60 h. The 2 th instar H. armigera larvae were fed with ds RNAexpressing bacteria,and the silencing effect of ace genes after RNAi was detected. Our results showed that the transcriptional levels of ace1 and ace2 were down-regulated at 12 h after phoxim and methomyl induction;subsequently,the transcriptional level of ace1 was down-regulatedwhile the transcriptional level of ace2 was up-regulated. The transcriptional levels of ace1 and ace2 were down-regulated obviously at 24 h and48 h then increased slightly after RNAi treatment. It is inferred that the pesticide treatment leads to the physiological and metabolic damages of H. armigera and consequently the significant reduction of transcriptional levels of ace1 and ace2;subsequently,the ace2 expression was decreasing constantly while the ace1 expression was gradually increasing,which might be related to compensation for pesticides inhibition to Ach E enzyme activity. RNAi treatment caused varied inhibitions to both growth and development of H. armigera,as well as the obvious inhibitions to the transcriptions of ace1 and ace2 genes. The above results provide a reference for the control of H. armigera while using acetyl cholinesterase and its coding genes as the target.