家蚕bmo-miR-217对核型多角体病毒(BmNPV)lef-1基因表达的调控作用

Expressional Regulation of Bombyx mori Nucleopolyhedroviruslef-1 Gene by Silkworm bmo-miR-217

昆虫的单链小分子RNA(microRNAs)不仅通过与自身的靶基因互补进行转录后水平调控,从而参与重要的生命过程,并且还可以与病原体靶基因相互作用,在免疫防御方面发挥重要功能。给家蚕5龄眠起幼虫接种家蚕核型多角体病毒(Bm NPV)后72 h,收集蚕体血淋巴提取总RNA用于高通量测序,经克隆验证获得一条由Bm NPV诱导上调表达的家蚕miRNA(bmo-miR-217),用生物信息学方法预测到bmo-miR-217对应的一个病毒靶基因为lef-1。构建bmo-miR-217及Bm NPV lef-1 3'-UTR的表达载体,并共转染家蚕卵巢培养细胞Bm N,于48 h后通过检测细胞的双荧光素酶相对活性,显示出bmo-miR-217可显著下调Bm NPV lef-1基因的表达(P<0.05)。已知lef-1是Bm NPV侵染复制的必需基因之一,因此抑制其表达是家蚕先天免疫的一种有效策略。

MicroRNAs are involved in the important life process of insects through post-transcriptional regulation by comple- mentation with target genes and play an important role in insect immune defense by interacting with target genes of pathogens. At 72 h after the newly exuviated silkworm （ Bombyx mori） larvae of the 5th instar were inoculated with Bombyx mori nucleopoly- hedrovirus （BmNPV）, total RNA was extracted from hemolymph for high throughput sequencing. An up-regulated miRNA （bmo-miR-217） in silkworm, which was induced by BmNPV infection, was cloned and verified. Viral gene lef-1 is characterized to be one of the target genes of bmo-miR-217 through bioinformatical prediction. The expression vectors of bmo-miR-217 and BmNPV lef-1 3＇-UTR were constructed and co-transfected into BmN cells. Determination of relative dual-luciferase activity of BmN cells at 48 h after infection revealed that bmo-miR-217 could significantly down-regulate the expression level of BmNPV lef-1 （P〈0. 05）. lef-1 is a essential gene for BmNPV replication. Thus, inhibition of lef-1 expreesion is an effective strategy of innate immune response of silkworm.