中文摘要：

【目的】探究家蚕Bombyx mori miRNAs对丝素蛋白轻链基因（fibroin light chain gene,Bm FibL）和P25蛋白基因Bm P25表达的调控作用。【方法】分别以Bm Fib-L和Bm P25 mRNA 3＇UTR为靶序列,应用在线预测软件RNAhybrid从前期家蚕4和5龄幼虫后部丝腺（posterior silk gland）sRNA高通量测序获得的29个差异表达bmo-miRNAs中,筛选对Bm Fib-L和Bm P25具有调控作用的候选bmo-miRNAs;采用半定量RT-PCR方法在mRNA水平分析候选bmo-miRNAs及其靶基因Bm Fib-L和Bm P25在4和5龄幼虫期以及5龄第3天幼虫不同组织的表达水平;利用双荧光报告基因检测系统在家蚕细胞Bm N中验证候选bmo-miRNAs对Bm Fib-L和Bm P25表达的调控作用。【结果】筛选到一个在Bm Fib-L和Bm P25 mRNA 3＇UTR上都有靶位点的bmo-miR-375-3p（曾称bmo-miR-375＊）。在后部丝腺中bmo-miR-375-3p和其预测靶基因Bm Fib-L和Bm P25都有较高的表达水平,提示bmo-miR-375-3p具备调控Bm Fib-L和Bm P25表达的时空条件。miR-375-3p重组表达载体与融合了Bm Fib-L 3＇UTR的萤火虫荧光素酶（luciferase）报告基因（luc）表达载体共转染Bm N细胞,报告基因luc的表达水平显著下降;而在miR-375-3p重组表达载体与融合了Bm P25 3＇UTR的luc表达载体共转染Bm N细胞中报告基因luc的表达水平下降不显著。【结论】miR-375-3p显著下调Bm Fib-L基因的表达,而对Bm P25基因的表达无明显调控作用。

英文摘要：

【Aim】To explore the regulatory function of Bombyx mori microRNAs（ bmo-miRNAs） on the expression of fibroin light chain gene Bm Fib-L and P25 protein gene Bm P25. 【Methods】Bm Fib-L and Bm P25 mRNA 3＇ UTRs were used as targets to predict the potential bmo-miRNAs with target sites on Bm Fib-L and Bm P25 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. Semiquantitative reverse transcription polymerase chain reaction（ RT-PCR） method was employed to analyze the expression levels of the potential bmo-miRNAs and their target genes Bm Fib-L and Bm P25 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 Bm Fib-L and Bm P25 was validated by using the dual-luciferase reporter assay system in Bm N 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 Bm Fib-L and Bm P25. The bmo-miR-375-3p and its potential targets Bm Fib-L and Bm P25 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 Bm Fib-L and Bm P25. When bmo-miR-375-3p recombinant expression vector co-transfected with expression vector of Bm Fib-L 3＇UTR fused firefly luciferase report gene（ luc） in Bm N cells,the expression level of luc was significantly down-regulated. When bmo-miR-375-3p expression vector co-transfected with expression vector of Bm P25 3＇UTR fused luc report gene in Bm N cells,however,the expression level of luc was slightly but not significantly down-regulated. 【Conclusion 】 Bmo-miR-375-3p down-regulates the expression of Bm Fib-L gene significantly,but has no significant effect on the expression of Bm P25.