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miRNA不仅广泛参与了蚊生长、发育的调控,还参与了媒介与病原体的相互作用,因此miRNAs可能作为蚊虫防制与蚊媒病原体控制的靶点,但目前尚无有效的转导系统。为研究白纹伊蚊浓核病毒-3(AalDV-3)是否可以作为有效的载体,我们利用内含子内表达miRNA海绵的策略,以埃及伊蚊内源性miR-210为靶基因,构建了重组病毒AalDV-anti-210。使用跨内含子引物通过RT-PCR验证了内含子可在蚊体内外的剪接效果,通过qPCR方法对埃及伊蚊Aag2细胞与埃及伊蚊幼虫的感染后的miR-210的表达水平进行检测。结果证实,含有miRNA海绵的内含子可以有效的蚊体内外剪接,重组病毒可以在蚊体内外高效的抑制靶miR-210的表达水平。该结果为探索AalDV-3在蚊虫基因功能研究及生物杀虫剂方面的潜在价值提供了理论依据。
Although miRNAs are widely involved in the regulation of mosquito growth and development and are also involved in the interaction of vectors with pathogens, miRNAs may serve as targets for mosquito control and mosquito-borne pathogen control. However, no effective transduction system is currently available. In order to investigate whether AalDV-3 can be an effective vector, we constructed a miRNA-sponge-based intron strategy using endogenous miR-210 as a target gene The recombinant virus AalDV-anti-210. Introns were tested for their ability to splicing in and out of mosquitoes by RT-PCR using intronic primers and qPCR to detect the expression of miR-210 after infection with Aedes aegypti Aag2 cells and Aedes aegypti larvae . The results confirmed that intron containing miRNA sponge can effectively splicing both in vitro and in vivo. Recombinant virus can effectively inhibit target miR-210 expression both inside and outside the mosquito. The results provide a theoretical basis for exploring the potential value of AalDV-3 in the research of mosquito gene function and biopesticide.