Abstract:  【Aim】 miRNAs not only regulate the metamorphosis and development of insects, but also play important roles in the reproductive regulation of insects. Throuth identifying the target gene of the miRNA, Bmo-miR-2763, related to reproductive regulation in Bombyx mori and analyzing its expression, this study aims to reveal the potential molecular mechanism of miRNAs in the regulation of the testicular and ovarian development of B. mori. 【Methods】 The target genes of Bmo-miR-2763 were predicted using bioinformatics methods. At 2, 6, 12 and 24 h after injection of 5 μg/individual of 20-hydroxyecdysone (20E) into the hemolymph of the 5th instar larvae of B. mori, the expression levels of Bmo-miR-2763 and its predicted target gene BmGRF in the fat body were detected by qRT-PCR. The expression levels of Bmo-miR-2763 and its predicted target gene BmGRF in different tissues (hemolymph, cuticle, midgut, head, fat body, silk gland, Malpighian tubules, testis and ovary) of the 5th instar larvae treated with 20E for 24 h and in the untreated normal larvae at the 4th－5th instars, pupae and adults of B. mori were also detected using qRT-PCR. The interaction between Bmo-miR-2763 and its predicted target gene BmGRF was analyzed using dual luciferase reporter assay system. 【Results】 After 20E treatment, the expression level of Bmo-miR-2763 in the fat body of the 5th instar larvae of B. mori was significantly increased， and that of the predicted target gene of Bmo-miR-2763, BmGRF, was also significantly up-regulated, as compared with those of the control group. Bmo-miR-2763 and BmGRF showed high expression levels in the testes and ovaries of the 5th instar larvae of B. mori at 24 h after 20E treatment, and their expression trends were consistent in testes, but opposite in ovaries. In the untreated normal B. mori at different developmental stages, the expression levels of BmGRF were negatively correlated with those of Bmo-miR-2763. The results of dual luciferase activity assay showed that the luciferase activity was decreased by 56% after co-transfection with Bmo-miR-2763 mimics and BmGRF 3′UTR overexpression vector, indicating that Bmo-miR-2763 could significantly inhibit the expression of luciferase reporter gene by interacting with the 3′UTR region of BmGRF. 【Conclusion】 20E can induce the expression of Bmo-miR-2763 in B. mori, and Bmo-miR-2763 mainly participates in the development regulation of B. mori ovaries by regulating the target gene BmGRF.

Key words: Bombyx mori, miRNA, testis, ovary, molting hormone, development regulation