关键词: 家蚕； miRNA, bmo-miR-375-3p； 丝素轻链基因； P25基因； 基因表达调控

Abstract: 【Aim】 To explore the regulatory function of Bombyx mori microRNAs (bmo-miRNAs) on the expression of fibroin light chain gene BmFib-L and P25 protein gene BmP25. 【Methods】 BmFib-L and BmP25 mRNA 3′UTRs were used as targets to predict the potential bmo-miRNAs with target sites on BmFib-L and BmP25 by the online RNAhybrid software from the previously obtained 29 differentially expressed bmo-miRNAs in the posterior silk gland of the 4th and 5th instar larvae of B. mori. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) method was employed to analyze the expression levels of the potential bmo-miRNAs and their target genes BmFib-L and BmP25 in the posterior silk gland of the 4th and 5th instar larvae and in different tissues of the 5th instar day-3 larvae of B. mori. The regulatory function of potential bmo-miRNAs on the expression of BmFib-L and BmP25 was validated by using the dual-luciferase reporter assay system in BmN cells. 【Results】 One bmo-miRNA, miR-375-3p (previously named bmo-miR-375*), was predicted to have target sites at the mRNA 3′UTRs of both BmFib-L and BmP25. The bmo-miR-375-3p and its potential targets BmFib-L and BmP25 all showed comparatively high expression levels in the posterior silk gland, suggesting that in the posterior silk gland there are the spatial-temporal conditions for bmo-miR-375-3p to regulate the expression of BmFib-L and BmP25. When bmo-miR-375-3p recombinant expression vector co-transfected with expression vector of BmFib-L 3′UTR fused firefly luciferase report gene (luc) in BmN cells, the expression level of luc was significantly down-regulated. When bmo-miR-375-3p expression vector co-transfected with expression vector of BmP25 3′UTR fused luc report gene in BmN cells, however, the expression level of luc was slightly but not significantly down-regulated. 【Conclusion】 Bmo-miR-375-3p down-regulates the expression of BmFib-L gene significantly, but has no significant effect on the expression of BmP25.

Key words: Bombyx mori, miRNA, bmo-miR-375-3p, BmFib-L, BmP25, gene expression regulation