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RNA干扰(RNA interference,RNAi)是一种调控基因表达的方法,其通过体外合成一段与内源靶基因同源的双链RNA(dsRNA)或siRNA,导入生物体内,使内源靶基因中同源mRNA降解,从而达到阻抑基因表达的目的。类钙粘蛋白(cadherin-likeprotein)是位于昆虫中肠刷状缘膜囊(brush border membrane vesicles,BBMV)上与钙粘蛋白(cadherin)结构相似的物质,是多种昆虫体内Bt杀虫蛋白的受体。本研究利用基因特异引物通过RT-PCR扩增了小菜蛾类钙粘蛋白基因的2个片段(CAD1和CAD2),合成相对应的双链RNA(double-stranded RNA,dsRNA);并将dsRNA通过显微注射导入小菜蛾3龄幼虫体内,测定了不同靶位点、不同剂量、不同检测时间对目的基因mRNA表达量的影响。结果表明:将70nLCAD1对应的dsRNA注射到幼虫体内48h后,基因表达量显著下降,72h后恢复。免疫印迹检测结果表明,类钙粘蛋白在注射dsRNA48h后幼虫BBMV中的含量明显下降。本实验成功实现了小菜蛾类钙粘蛋白基因的沉默,该体系的成功建立为利用RNAi技术分析小菜蛾及其他鳞翅目昆虫基因的功能提供了参考。
RNA interference (RNAi) is a method of regulating gene expression by introducing a double-stranded RNA (dsRNA) or siRNA homologous to an endogenous target gene in vitro into an organism so that the endogenous target gene is homologous The source mRNA degradation, so as to achieve the purpose of suppressing gene expression. Cadherin-likeproteins are similar to cadherin in the brush border membrane vesicles (BBMV) of insects and are found in many insect Bt insecticidal proteins Receptor. In this study, two fragments (CAD1 and CAD2) of cadherin gene of Plutella xylostella were amplified by RT-PCR using gene-specific primers to synthesize double-stranded RNA (dsRNA) The third instar larvae of Plutella xylostella were introduced by microinjection to determine the effect of different target sites, different doses and different detection time on the mRNA expression of the target gene. The results showed that the expression of 70nLCAD1 dsRNA was significantly decreased after 48h injection into larvae, and recovered after 72h. Immunoblotting results showed that cadherin significantly decreased in BBMV larvae 48h after dsRNA injection. In this study, we successfully silence the cadherin gene of Plutella xylostella. The successful establishment of the system provided a reference for the analysis of the function of Plutella xylostella and other lepidopteran insects using RNAi technology.