家蚕miR-0047对丝胶蛋白基因BmSer-1表达的调控作用研究

Regulation of Bmo-miR-0047 on Expression of Bombyx mori Serincin Gene BmSer-1

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。

A number of novel miRNAs were obtained from high-throughput sequencing of small RNAs isolated from middle silk gland of silkworm（ Bombyx mori）. Bioinformatic analysis revealed that Bmo-miR-0047 may be associated with the regulation of silkworm sericin gene Bm Ser-1. In order to examine the expression of Bmo-miR-0047 in silk gland（ including anterior,middle and posterior silk glands）,head,fat body,epidermis,midgut,Malpighian tubule,testis/ovary,hemocyte and trachea of day 3 silkworm larvae of the 5 th instar,specific loop-stem primers were designed and semiquantitative RT-PCR analysis was carried out. The results showed that expression level of Bmo-miR-0047 in middle silk gland was significantly higher than that in other tissues,demonstrating that the regulation of Bm Ser-1 gene by Bmo-miR-0047 has spatial-temporal specificity in silkworm larvae of the 5 th instar. In order to further analyze the regulatory function of Bmo-miR-0047 on Bm Ser-1 gene expression,we constructed recombinant plasmids pc DNA3. 0（ ie1-egfp-pri-miR-0047-SV40） and p GL3（ A3-luc-Bm Ser-1 3＇UTR-SV40）,and used Renilla luciferase reporter plasmid pRL-CMV as reference to carry out co-transfection into Bm N cells. The luciferase activity in transfected Bm N cells was significantly decreased compared with the control group,indicating that Bmo-miR-0047 can negatively regulate the expression of Bm Ser-1 gene in cultured cells.