Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China.
Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China.
PLoS One. 2021 Dec 16;16(12):e0261391. doi: 10.1371/journal.pone.0261391. eCollection 2021.
To study the regulatory function of Bombyx mori (B. mori) miRNAs (bmo-miR) on the expression of fibroin light chain gene (BmFib-L), the 3'UTR of BmFib-L mRNA was used as the target for online prediction of miRNAs from miRBase using RNAhybrid Software, and miR-2845 was screened out. First, the expression profiles of miR-2845 and BmFib-L in larvae of the 5th instar were analyzed by Real-time quantitative PCR (RT-qPCR). Then recombinant plasmids (pcDNA3.0-pre-miR-2845 and pGL3.0-BmFib-L) were constructed to use for the expression of miR-2845 and BmFib-L 3'UTR, respectively. Cellular-level functional verification of miR-2845 on BmFib-L was carried out using multiple experimental methods (including dual luciferase reporter vectors, artificially synthesized mimics and inhibitors, and target site mutations). Finally, in vivo functional verification was performed by injecting the recombinant vector in 5th instar larvae. BmFib-L expression levels were detected using RT-qPCR in the posterior silk glands (PSG) of the injected larvae. Results showed that the expression of miR-2845 increased between the 1st and 5th day in 5th instar larvae, but began to decline on the 5th day, while the expression of the target gene BmFib-L increased sharply. This suggests that miR-2845 and BmFib-L expression levels show opposing trends, implying a negative regulatory relationship. In BmN cells, miR-2845 significantly down-regulated the expression of BmFib-L; the inhibitory effect of miR-2845 on BmFib-L was disappeared after mutation of the targeting site on 3'UTR of BmFib-L; in individuals, miR-2845 significantly down-regulated BmFib-L expression levels. Our results provide new experimental data for clarifying the molecular regulation mechanism of silk protein expression.
为研究家蚕（Bombyx mori）miRNA（bmo-miR）对丝素轻链基因（BmFib-L）表达的调控功能，利用 RNAhybrid 软件对 miRBase 中的 miRNA 进行在线预测，以 BmFib-L mRNA 的 3'UTR 为靶标，筛选出 miR-2845。首先，通过实时定量 PCR（RT-qPCR）分析 5 龄幼虫中 miR-2845 和 BmFib-L 的表达谱。然后构建重组质粒（pcDNA3.0-pre-miR-2845 和 pGL3.0-BmFib-L），分别用于 miR-2845 和 BmFib-L 3'UTR 的表达。采用双荧光素酶报告载体、人工合成的模拟物和抑制剂、靶位突变等多种实验方法，对 miR-2845 对 BmFib-L 的细胞水平功能进行验证。最后，通过向 5 龄幼虫注射重组载体，进行体内功能验证。注射幼虫后，在后丝腺（PSG）中通过 RT-qPCR 检测 BmFib-L 的表达水平。结果表明，5 龄幼虫第 1 天至第 5 天 miR-2845 表达量增加，第 5 天开始下降，而靶基因 BmFib-L 表达量急剧增加。这表明 miR-2845 和 BmFib-L 的表达水平呈相反趋势，提示存在负调控关系。在 BmN 细胞中，miR-2845 显著下调 BmFib-L 的表达；BmFib-L 3'UTR 靶位突变后，miR-2845 对 BmFib-L 的抑制作用消失；在个体中，miR-2845 显著下调 BmFib-L 的表达水平。本研究为阐明丝蛋白表达的分子调控机制提供了新的实验数据。
