家蚕bmo-miR-0031-3p体内下调丝素轻链基因BmFib-L的表达

Bmo-miR-0031-3p down-regulates the expression of Bombyx mori fibroin light chain gene BmFib-L in vivo

为了研究家蚕微RNA(microRNA, miRNA)对丝素轻链基因BmFib-L表达的调控作用,以BmFib-L mRNA的3′非翻译区(3′untranslated region, 3′UTR)为靶标,通过RNAhybrid软件分析,筛选出种子序列与BmFib-L 3′UTR完全互补的家蚕miRNA——bmo-miR-0031-3p(简称"miR-0031-3p")。分别构建miR-0031-3p表达载体pcDNA3.0[ie1-egfp-pre-miR-0031-3p-SV40]和BmFib-L 3′UTR融合萤光素酶报告基因重组表达质粒pGL3.0[A3-luc-Fib-L-3′UTR-SV40],以海肾萤光素酶表达载体pRL-CMV为内参,共转染BmN细胞,通过检测双萤光素酶活性验证miR-0031-3p的功能;人工合成miR-0031-3p的模拟物(mimic)和抑制物(inhibitor),再进一步验证miR-0031-3p对BmFib-L的调控功能。结果显示,在BmN细胞中,miR-0031-3p显著抑制BmFib-L的表达。为了进一步验证miR-0031-3p在家蚕体内对BmFib-L表达的调控作用,在5龄第2天幼虫体腔内注射转染物,分别在体内过表达和抑制内源性miR-0031-3p,荧光定量分析靶基因表达水平。结果显示,miR-0031-3p在幼虫体内能够下调BmFib-L的表达。该研究结果有利于阐明家蚕miRNA功能和蚕丝蛋白表达调控的分子机制。

To study the regulatory function of Bombyx mori microRNAs(bmo-miRNAs)on expression of the fibroin light chain gene(BmFib-L),the 3'untranslated region(3'UTR)of BmFib-L mRNA was used as the target for screen of bmo-miRNAs.By using RNAhybrid software,the bmo-miR-0031-3p(abbreviated as"miR-0031-3p")was screened out to completely bind the target gene with the seed sequence.A miR-0031-3p expression plasmid pcDNA3.0[ie1-egfp-pre-miR-0031-3p-SV40]and a BmFib-L 3'UTR fused luciferase report plasmid pGL3.0[A3-luc-Fib-L-3'UTR-SV40]were constructed,respectively.BmN cells were cotransfected with the above mentioned plasmids,and the pRL-CMV(contains a Renilla luciferase gene)was served as an intrinsic plasmid to validate the regulatory function of miR-0031-3p on BmFib-L by assay of dual luciferase activities,as well as artificially synthesized the mimic and inhibitor of miR-0031-3p.The results revealed that the miR-0031-3p significantly down-regulated the expression of BmFib-L in the BmN cells.To validate the regulatory function of miR-0031-3p in vivo,the day-2 5th instar larvae were injected with a transfection solution for overexpression and inhibition of endogenous expression analysis,and the BmFib-L expression was analyzed by quantitative reverse transcription polymerase chain reaction(RT-PCR)using total RNAs extracted from silk glands.The results showed that the miR-0031-3p significantly down-regulated the expression of BmFib-L in individuals.These findings are beneficial to clarify the molecular mechanism of miRNAs in regulating B.mori silk protein biosynthesis.