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

作　　者：陈艳花 蒋涛 王雪珍[1,2] 钱平 唐顺明 沈兴家[2] CHEN Yanhua;JIANG Tao;WANG Xuezhen;QIAN Ping;TANG Shunming;SHEN Xingjia(Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu, China;Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, Jiangsu, China)

机构地区：[1]江苏科技大学生物技术学院江苏省蚕桑生物学与生物技术重点实验室,江苏镇江212018 [2]中国农业科学院蚕业研究所农业农村部蚕桑遗传改良重点实验室,江苏镇江212018

出　　处：《浙江大学学报（农业与生命科学版）》2019年第2期229-236,共8页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金(31672490;31172266);江苏省自然科学基金(BK20151322);江苏省高校自然科学研究重大项目(15KJA180001)

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。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.

关 键 词：微RNA 家蚕 bmo-miR-0031-3p 丝素轻链基因 转录后调控 

分 类 号：S881.2[农业科学—特种经济动物饲养]