家蚕bmo-miR-3385-3p抑制丝素轻链基因BmFib-L的表达

Repression of Bmo-miR-3385-3p on Fibroin Light Chain Gene of Bombyx mori

为了探究家蚕中miRNA对丝素蛋白轻链基因(BmFib-L)表达的调控作用,以BmFib-L 3'UTR序列为靶序列,用RNAhybrid软件在线预测miRBase数据库中对靶基因具有调控作用家蚕miRNA,筛选到1个候选miRNA bmo-miR-3385-3p;采用荧光定量方法分析miR-3385-3p在幼虫5龄第1~7天后部丝腺和5龄第3天不同组织及BmFib-L在5龄第1~7天后部丝腺的表达水平;构建miR-3385-3p表达载体pcDNA3.0(ie1-egfp-pre-miR-3385-3p-SV40)和含萤火虫荧光素酶报告基因的BmFib-L 3'UTR重组质粒pGL3.0(A3-luc-BmFib-L-3'UTR-SV40),并人工合成miR-3385-3p的模拟物(mimic)和抑制物(inhibitor),以海肾荧光素酶表达载体pRL-CMV为内参,共转染BmN细胞,利用双荧光素酶检测系统验证miR-3385-3p对BmFib-L表达的调控作用。同时,对预测的BmFib-L 3'UTR上miR-3385-3p的靶位点进行了突变,在BmN细胞中进行了验证。进一步采用丝腺离体培养瞬时表达和5龄幼虫体腔注射转染物的方法,通过荧光定量分析后部丝腺靶基因表达水平的变化,验证miR-3385-3p在家蚕体内对BmFib-L表达的调控作用。结果表明,miR-3385-3p和BmFib-L在后部丝腺都有较高的表达水平,miR-3385-3p具备调控BmFib-L的可能性;在BmN细胞中miR-3385-3p对BmFib-L的表达具有显著抑制作用,预测的靶位点突变后miR-3385-3p对BmFib-L基因的表达没有抑制作用。在离体培养丝腺中和幼虫体内miR-3385-3p都显著下调BmFib-L基因的表达。由此证明,BmFib-L是miR-3385-3p的靶基因之一,miR-3385-3p可抑制BmFib-L的表达。

To study the regulatory function of Bombyx mori miRNAs on the expression of fibroin light chain gene(BmFib-L),BmFib-L 3'UTR sequence was used as the target for online prediction of miRNAs from miRBase by RNAhybrid software,and one candidate miRNA,bmomiR-3385-3p,was screened out which has potential regulatory function on BmFib-L.Real-time fluorescence quantitative PCR(qPCR)was employed to analyze the expression levels of bmo-miR-3385-3p and BmFib-L in posterior silk gland of the 5th instar larvae on the 1st to 7th day and the bmo-miR-3385-3p in different tissues of the 5th instar larvae on the 3rd day.The miR-3385-3p expression vector pc-DNA3.0(ie1-egfp-pre-miR-3385-3p-SV40)and recombinant plasmid of BmFib-L 3'UTR fused with a firefly luciferase reporter gene,pGL3.0(A3-luc-Bm-Fib-L-3'UTR-SV40),were constructed,and the mimic and inhibitor of miR-3385-3p were artificially synthesized and cotransfected into BmN cells taking Renilla reniformis luciferase expression vector pRLCMV as internal reference.Therefore,regulatory function of miR-3385-3p on BmFib-L was validated by dual luciferase assay system.Meanwhile,the predicted target site of miR-3385-3p on the 3'UTR of BmFib-L was mutated and validation experiment was carried out in BmN cells.Furthermore,expression change of target gene in posterior silk gland of 5th instar larvae was analyzed by qPCR to validate regulatory function of miR-3385-3p on BmFib-L after transient expression of in vitro cultured silk gland and injection of transfection solution into the silkworm.The results showed that miR-3385-3p and BmFib-L were highly expressed in posterior silk gland,indicating that it is possible for miR-3385-3p to regulate Bm-Fib-L.Meanwhile,miR-3385-3p significantly inhibited the expression of BmFib-L in BmN cells.However,miR-3385-3p had no inhibitory effect on BmFib-L after mutation of the target site.Besides,miR-3385-3p significantly down-regulated the expression of BmFib-L both in the in vitro cultured silk gland and in the body.These results proved that BmFib-L is one of the target genes of miR-3385-3p,and miR-3385-3p could down-regulate the expression of BmFib-L.