【摘要】 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。更多还原

【Abstract】 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 m RNA was used as the target for screen of bmo-miRNAs. By using RNAhybrid software, the bmo-miR-0031-3 p(abbreviated as"miR-0031-3 p") was screened out to completely bind the target gene with the seed sequence. A miR-0031-3 p expression plasmid pc DNA3.0[ie1-egfp-pre-miR-0031-3 p-SV40] and a BmFib-L 3′ UTR fused luciferase report plasmid p GL3.0[A3-luc-Fib-L-3′ UTR-SV40] were constructed, respectively. Bm N 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-0031-3 p on BmFib-L by assay of dual luciferase activities, as well as artificially synthesized the mimic and inhibitor of miR-0031-3 p. The results revealed that the miR-0031-3 p significantly down-regulated the expression of BmFib-L in the BmN cells. To validate the regulatory function of miR-0031-3 p in vivo, the day-2 5 th 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-0031-3 p 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.更多还原