利用RNAi技术沉默东方粘虫V-ATP酶H亚基研究

Silencing of V-ATPase Subunit H of Mythimna separatathrough RNA Interference

昆虫V-ATP酶(vacular-type ATPase)是以ATP为能量跨膜转运H+的质子泵,对其亚基进行干扰都会影响ATPase的活性,进而影响昆虫的生理生化指标。本研究利用特异性引物通过RT-PCR法扩增东方粘虫(Mythimna separata)V-ATPase H亚基的2个片段,合成相应dsRNA,通过显微注射导入3龄粘虫体内,观察试虫表型及存活情况,RT-PCR检测试虫体内H亚基的表达。结果表明,注射粘虫V-ATPase H亚基的dsRNA能够显著降低粘虫中肠V-ATPase H亚基的表达水平,直至导致目的基因沉默和粘虫死亡。本项研究成功实现东方粘虫体内V-ATPase H亚基基因沉默。

The insect vacuolar ATPases is an ATP-driven proton pump in the process of H＋transmembrane tran sportation.Any interference insect V-ATP enzyme synthesis of substances to insects will have a dramatic growth and development,and even result the death of the insect.In this study,two dsRNAs of the V-ATPase H were synthesized in vitro and micro-injected armyworm（Mythimna separata）larvae.Phenotype changes and the expression of V-ATPase were analyzed by RT-PCR.The results showed that the relative expression level of V-ATPAse H in M.separata which injected by the dsRNA coding sequences of V-ATPAse H were suppressed,and led to the death of M.separata.The V-ATPase subunit H was silenced successfully in this experiment and the establishment of RNAi technique can help reveal the gene function of M.separata V-ATPase H and other Lepidoptera insects.