利用RNA干扰技术沉默稻纵卷叶螟几丁质合成酶A基因

稻纵卷叶螟(Cnaphalocrocis medinalis)是一种主要的水稻害虫,几丁质合成酶是昆虫体内几丁质合成通路中的一个关键酶,在昆虫生长发育中具有重要作用,是控制害虫的理想靶标。本文采用RNA干扰(RNAi)技术,以稻纵卷叶螟几丁质合成酶A基因(CmCHSA)为靶标,设计两个靶位点CmCHSA-I和CmCHSA-II,在体外分别合成其双链RNA(dsRNA),用显微注射法导入3龄幼虫体内,然后通过表型观察和荧光定量PCR(RT-q PCR)检测干扰效应。结果表明,注射两种dsRNA后纵卷叶螟对水稻的取食明显下降,生长发育阻滞,虫体变小变黑和畸形,有的出现死亡。RT-q PCR表明,注射CmCHSA-I和CmCHSA-II两种dsRNA在96 h后,CmCHSA的表达量相比对照分别下降了59%和64%。注射CmCHSA-I和CmCHSA-II两种dsRNA 7 d后,稻纵卷叶螟的死亡率分别为80%和83.33%,与对照组相比,分别增加了66.67%和70%。本研究用注射法RNAi技术成功实现了对稻纵卷叶螟几丁质合成酶A基因的沉默,为利用该技术研究昆虫基因的功能,以及通过转基因RNAi技术防治稻纵卷叶螟奠定了基础。

Using RNA interference technique to silence chitin synthase A Gene in the rice leaf folder,Cnaphalocrocis medinalis (Lepidoptera：Pyralidae)

The rice leaf folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) is a chief pest of rice. Chitin synthase is a key enzyme in the pathway of insect chitin synthesis and plays an important role in insect growth and development. Thus, it is believed that this enzyme is an ideal target for pest control. In this study, we designed two target sites (CmCHSA-I and CmCHSA-II) specific to chitin synthase A (CmCHSA) in C. medinalis for RNA interference (RNAi) . CmCHSA-I and CmCHSA-II double-stranded RNAs (dsRNAs) were synthesized in vitro and were separately intro-duced into the 3rd instar larvae through microinjection. RNAi effects were detected by phenotype observation and real-time quantitative PCR (RT-qPCR). After injection of dsRNA, the larvae developed loss of appetite, growth retardation,and malformation; some died. RT-qPCR showed that at the 96th h after injection of CmCHSA-I and CmCHSA-II dsR-NAs, the mRNA expression of CmCHSA decreased by 59% and 64%, respectively, in comparison with the control. At the 7th d after injection of CmCHSA-I and CmCHSA-II dsRNAs, the larval mortality rates were 80% and 83. 33%, re-spectively, increased by 66. 67% and 70%, compared to the control. CmCHSA was successfully silenced using RNAi by injection, which will lay the foundation for elucidating the function of insect genes as well as for controlling C. medinalis by transgenic RNAi.