利用RNAi技术沉默稻纵卷叶螟膜结合型海藻糖酶基因

Silencing Membrane-bound Trehalase Gene in Cnaphalocrocis medinalis Through RNA Interference

【目的】明确沉默稻纵卷叶螟膜结合型海藻糖酶基因(CmTre2)对其生长发育的影响,为稻纵卷叶螟的防治奠定理论基础。【方法】基于RNA干扰(RNA interference,RNAi)技术,针对稻纵卷叶螟膜结合型海藻糖酶基因(CmTre2)设计靶位点,体外合成双链RNA(dsCmTre2),将其注射入稻纵卷叶螟3龄幼虫体内,观察试虫表型变化,用实时荧光定量PCR(RT-qPCR)检测CmTre2的表达水平,测定酶活力。【结果】注射dsCmTre2的幼虫取食量明显下降,生长发育阻滞,体型变小,出现畸形和死亡现象。注射dsCmTre2后5 d,幼虫死亡率明显高于对照组,存活幼虫体重明显轻于对照组;CmTre2和几丁质合成酶B基因(CmCHSB)的表达量较对照分别下降53.70%和34.41%;膜结合型海藻糖酶活力较对照组下降35.61%。【结论】CmTre2基因对稻纵卷叶螟的生长发育具有重要作用,该基因的沉默影响几丁质合成,引起发育障碍。该研究结果为进一步利用RNAi技术在田间防治稻纵卷叶螟奠定了基础。

【Objective】The objective of the paper is to confirm the effect of silencing membrane-bound trehalase gene(CmTre2)on growth development of Cnaphalocrocis medinalis and to lay a theoretical foundation for control of Cnaphalocrocis medinalis.【Method】Based on RNA interference(RNAi)technology and designing target site according to membrane-bound trehalase gene(CmTre2),double-stranded RNA(dsCmTre2)synthesized in vitro is injected into 3rd instar Cnaphalocrocis medinalis larvae.The phenotypic change of tested insect is observed,and the expression level of CmTre2 and the enzyme activity is determined by real-time fluorescent quantitative PCR(RT-qPCR).【Result】The larvae injected dsCmTre2 was featured with less food intake,growth retardation,smaller type,deformity and death.The mortality of larvae injected dsCmTre2 after 5 days was higher than CK significantly,and the weight of survival larvae injected dsCmTre2 after 5 days was lighter than CK obviously.The expression level of CmTre2 and chitin synthetase B gene(CmCHSB)was decreased by 53.70%and 34.41%compared with CK respectively.The activity of membrane-bound trehalase was decreased by 35.61%compared with CK.【Conclusion】CmTre2 gene plays an important role in growth development of Cnaphalocrocis medinalis.The silencing CmTre2 gene affects chitin synthesis and causes developmental disorder.The research result lays a foundation for further using RNAi technology to control of Cnaphalocrocis medinalis in field.