注射dsRNA对飞蝗Knickkopf基因在mRNA和蛋白水平的沉默效率  

Silence efficiency of mRNA and protein expression of Knickkopf in Locusta migratoria(Orthoptera: Acrididae) by dsRNA injection

作　　者：于荣荣[1,2] 丁国伟[1] 杨美玲[3] 马恩波[1] 张建珍[1] 

机构地区：[1]山西大学应用生物学研究所,太原030006 [2]山西大学生命科学学院,太原030006 [3]中国科学院动物研究所,北京100101

出　　处：《昆虫学报》2016年第12期1395-1400,共6页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31272380;31672364);山西省科技基础条件平台项目(2015091010)

摘　　要：【目的】本研究旨在评估dsRNA对飞蝗Locusta migratoria Knickkopf(Knk)基因mRNA和蛋白表达的沉默效率,以期为基于RNAi技术探索飞蝗体内基因功能的研究提供基础资料。【方法】选择飞蝗2龄第1天的若虫,将体外合成的飞蝗Knk基因dsRNA注射进入飞蝗体腔内,注射后48,72和96 h分别解剖试虫的腹部表皮,一半用于RT-q PCR方法检测其mRNA表达;另一半用于Western blot法检测其蛋白的表达量。【结果】dsRNA注射进入飞蝗体腔内48,72和96 h后,与对照相比,Knk mRNA水平相对表达量分别降低78.3%,96.2%和95.1%;Knk蛋白表达量分别降低61.2%,33.3%和52.9%。【结论】注射ds Knk能显著沉默飞蝗Knk基因mRNA水平和蛋白水平的表达,但mRNA水平的沉默效率高于蛋白水平。[Aim] To assess the silence efficiency of Knickkopf （Knk） gene at the mRNA and protein levels in the migratory locust, Locusta migratoria, by double-stranded RNA （dsRNA）, so as to provide basic information for the gene function analysis in the migratory locust by using RNAi technology. [ Methods] dsRNA was synthesized and injected into the body cavity of the day-1 2nd instar nymphs, and the integuments of abdomen were dissected after dsRNA injection into the locusts at 48, 72 and 96 h, respectively. Half of abdomen integuments were collected and used for the mRNA expression detection by RT-qPCR, while the other half were prepared and used for the protein expression detection by Western blotting. [ Results ] Compared with the control, the relative expression level of Knk mRNA in the treatment group decreased by 78.3%, 96.2% and 95.1%, respectively, while the relative expression level of Knk protein decreased by 61.2%, 33.3% and 52.9%, respectively, at 48, 72 and 96 h after dsRNA injection into the locusts. [ Conclusion] The Knk expression can be significantly suppressed at both mRNA and protein levels in the migratory locust after dsKnk injection. However, the silence efficiency at the mRNA level is much stronger than that at protein level.

关 键 词：飞蝗 基因沉默 RNA干扰 DSRNA Western BLOT RT-QPCR 

分 类 号：Q966[生物学—昆虫学]