siRNA与dsRNA在白纹伊蚊体内的干扰效果

RNA interference effects triggered by dsRNA and siRNA in Aedes albopictus

目的探究siRNA(小干扰RNA)和dsRNA(双链RNA)2种干扰RNA在白纹伊蚊体内作用效果以及对蚊虫生存的影响,为蚊虫基因功能研究、新型杀虫剂研发、媒介蚊虫控制与虫媒病防控提供方法参考与技术支持。方法针对感兴趣的白纹伊蚊基因,设计合成目的基因的siRNA交由公司合成,dsRNA则通过扩增目的基因片段、体外转录和纯化方式获取,最终通过琼脂糖凝胶电泳检测验证。利用胸腔显微注射技术将干扰RNA注入白纹伊蚊体内,通过荧光定量聚合酶链反应(quantitative polymerase chain reaction,qPCR)法检测注射后第3天的蚊虫样本基因表达量,验证干扰RNA的干扰效果,同时记录蚊虫生存曲线。结果针对10个基因的22对siRNA干扰实验中,与对照组相比,仅有SR-C-2 siRNA、ENR-1 siRNA 2对siRNA成功干扰靶基因表达,干扰率分别为58%和33%,其余siRNA无干扰效果,甚至出现靶基因表达量升高的情况。dsRNA干扰实验中,Mesh dsRNA在3×10^(11)个RNA分子/只浓度下可显著降低靶基因的表达,干扰效果可持续5 d,注射后第3天的干扰率在55%~60%,注射后第5天的干扰率为34%。dsRNA注射后蚊虫3 d内生存率显著高于siRNA注射组。结论羽化后第5日的白纹伊蚊最适宜接受胸腔注射操作。dsRNA较siRNA干扰稳定,注射后生存率更高,更适用于蚊虫基因功能的深入研究。基于siRNA的基因干扰方法更简便易行,但需注重siRNA的纯化方式以及与该蚊种其他基因的同源性情况,避免由于结合蚊虫体内非靶标基因而出现非特异性实验结果。

Objective To study the interference effects of siRNA and dsRNA in Aedes albopictus and their impact on mosquito survival,providing reference and technical support for the study of mosquito gene function,the research and development of new insecticides,the control of vector mosquitoes,and the interruption of arboviruses transmission.Methods For genes of interest in Ae.albopictus,siRNAs targeting the desired genes were designed and commercially synthesized,while dsRNAs were obtained by amplifying the target gene fragments,followed by in vitro transcription and purification,with verification by agarose gel electrophoresis.The interfering RNA was injected into Ae.albopictus by thoracic microinjection.Then the expression of targeted genes on the 3rd day post-microinjection was detected by quantitative PCR to verify the interfering effect,and the survival curve of the mosquitoes was recorded at the same time.Results In the siRNA interference experiments targeting 10 genes with 22 siRNA pairs,only two siRNA pairs,SR-C-2 siRNA and ENR-1 siRNA,successfully interfered with the target gene expression compared to the control group,with interference rates of 58%and 33%,respectively,while the remaining siRNAs did not show interference effects,with some even causing increased expression of the target gene.In the dsRNA interference experiments,Mesh dsRNA significantly reduced the expression of mesh gene at the concentration of 3×10^(11) RNA molecules per mosquito,with an interference effect lasting 5 days and an interference rate of 55%-60%on the third day and 34%on the fifth-day post-injection.Mosquito survival within 3 days post-dsRNA injection was significantly higher than in the siRNA injection group.Conclusions Ae.albopictus on the fifth-day post-emergence is the most suitable to receive thoracic microinjection.dsRNA is more stable than siRNA for interference,with higher postinjection survival rates,making it more suitable for in-depth studies of mosquito gene function.Although siRNA-based gene interference is simpler and more feasible,attention should be paid to the purification method of siRNA and its homology with other genes in the mosquito species to avoid non-specific experimental results due to the off-target combinations.