Abstract:
【Aim】 To explore the role of miRNAs in regulating the expression of chemosensory protein genes in the silkworm, Bombyx mori, so as to further study the functions of miRNAs and their target genes in insect chemoreception. 【Methods】 The miRNAs that may interact with silkworm CSPs gene family were predicted and screened using bioinformatics methods, and the expression changes of candidate miR-301 and its target gene in different tissues of silkworm adults were analyzed using RT- qPCR. Dual luciferase report vector for miR-301 target gene 3′-UTR was constructed and transfected into human embryonic kidney 293 (HEK293) cells with miR-301 mimics or the negative control. The regulation of expression of the target gene by miR-301 was detected by the change of luciferase activity in dual luciferase reporter gene assay system. 【Results】 The results of bioinformatics analysis showed that csp9, a silkworm chemosensory protein gene, is a predicted target gene of miR-301, and its binding site with miR-301 is located in the 3′ -UTR region of csp9. RT-qPCR results showed that the expression of miR-301 was significantly up-regulated in male and female adult antennae and male adult head after mating, while the expression of csp9 was significantly down-regulated in these tissues. Dual luciferase assays showed that miR-301 significantly inhibited the expression of luciferase reporter gene by interacting with csp9 3′-UTR after co-transfection into HEK293 cells. 【Conclusion】In B. mori the chemosensory protein gene csp9 is a target gene of miR-301, its expression at the translational level is inhibited by miR-301 through binding to its 3′-UTR.

Key words: Bombyx mori; miRNA, chemosensory protein, target gene, RT-qPCR, dual luciferase