家蚕miR-0047对丝胶蛋白基因BmSer-1表达的调控作用研究

发布时间：2018-06-06 02:25

本文选题：家蚕 + 微小RNA　；参考：《蚕业科学》2017年06期

【摘要】：对家蚕中部丝腺小RNA高通量测序获得若干新miRNA,生物信息学分析发现Bmo-miR-0047可能对家蚕丝胶蛋白基因Bm Ser-1有调控作用。设计特异性茎环引物,利用半定量RT-PCR检测分析家蚕5龄3 d幼虫丝腺(包括前、中、后部丝腺)、头部、脂肪体、表皮、中肠、马氏管、精巢/卵巢、血淋巴细胞和气管等12个器官组织中Bmo-miR-0047的表达情况。结果显示,中部丝腺中Bmo-miR-0047的相对表达量明显高于其他组织,说明在家蚕5龄幼虫期Bmo-miR-0047对Bm Ser-1基因的表达调控存在时空特异性。为了进一步分析Bmo-miR-0047对Bm Ser-1基因表达的调控作用,构建重组表达载体pc DNA3.0(ie1-egfp-pri-miR-0047-SV40)和p GL3(A3-luc-Bm Ser-1 3'UTR-SV40),并以海肾荧光素酶报告质粒pRL-CMV为内参,利用家蚕卵巢培养细胞Bm N进行共转染。转染后检测发现实验组Bm N细胞中的荧光素酶活性与对照组相比显著减弱,表明Bmo-miR-0047在体外培养细胞中对Bm Ser-1基因的表达具有负调控作用。
[Abstract]:A number of new miRNAs were obtained by high-throughput sequencing of the small silkgland RNA in the central silkworm. Bioinformatics analysis showed that Bmo-miR-0047 might regulate the silkworm fibroin gene BM Ser-1. Specific stem ring primers were designed and semi-quantitative RT-PCR was used to detect and analyze the silk glands (including anterior, middle and posterior silk glands, head, fat body, epidermis, midgut, Markov duct, spermary / ovary) of the 5th instar larvae of Bombyx mori (Bombyx mori). Expression of Bmo-miR-0047 in 12 organs such as blood lymphocytes and trachea. The results showed that the relative expression of Bmo-miR-0047 in the central silk gland was significantly higher than that in other tissues, indicating that the regulation of Bm Ser-1 gene expression by Bmo-miR-0047 in the 5th instar larvae of Bombyx mori was space-time specific. In order to further analyze the regulatory effect of Bmo-miR-0047 on the expression of BM Ser-1 gene, the recombinant expression vector pc DNA3.0 Ie1-egfp-pri-miR-0047-SV40) and p GL3(A3-luc-Bm Ser-1 3 UTR-SV40G were constructed. The eukaryotic expression vector pRL-CMV was used as the internal reference, and the BMN was cotransfected with silkworm ovary culture cells. After transfection, the luciferase activity of BmN cells in the experimental group was significantly decreased compared with that of the control group, indicating that Bmo-miR-0047 had a negative regulatory effect on the expression of Bm Ser-1 gene in cultured cells in vitro.
【作者单位】：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;中国农业科学院蚕业研究所农业部蚕桑遗传改良重点开放实验室;
【基金】：国家自然科学基金项目(No.31402143,31372376) 江苏省自然科学基金项目(No.BK20141285) 江苏科技大学博士科研启动项目(No.1732931702)
【分类号】：S881.26

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