Bmo-miR-2771对家蚕丝胶蛋白基因BmSer-1表达的调控作用  

Regulation of Bmo-miR-2771 on Expression of Bombyx mori Sericin Gene BmSer-1

作　　者：钱平[1,2] 蒋涛[1,2] 王欣[1,2] 宋菲[1,2] 陈晨[1,2] 范洋洋[1,2] 唐顺明[1,2] 沈兴家[1,2] 

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、农业部蚕桑遗传改良重点开放实验室,江苏镇江212018

出　　处：《蚕业科学》2016年第5期826-831,共6页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(No.31402143,31172266)

摘　　要：微小RNA(miRNA)可通过抑制mRNA转录或使RNA降解的方式在转录后水平调节靶基因的表达。研究与家蚕丝胶蛋白基因1(BmS er-1)转录后表达有关的miRNA(Bmo-miR)及其作用方式,以丰富丝胶蛋白基因表达的精细调控信息。首先从miRBase 21数据库中获得全部成熟体Bmo-miR,通过生物信息学分析筛选到一个对BmS er-1有潜在调控作用的BmomiR,命名为Bmo-miR-2771。设计茎环引物,采用半定量RT-PCR方法检测Bmo-miR-2771及其预测靶基因BmS er-1在家蚕5龄3 d幼虫头部、脂肪体、前部丝腺、中部丝腺、后部丝腺、中肠等组织器官以及血淋巴细胞中的表达水平,结果显示Bmo-miR-2771特异性地在中部丝腺中表达,并且靶基因BmS er-1在中部丝腺的相对表达量也明显高于其他组织。将构建的表达BmomiR-2771的重组质粒pcD NA3.0(ie1-egfp-pri-miR-2771-SV40)和表达BmS er-1 3'-UTR的重组质粒pG L3(A3-luc-Ser-1 3'UTRSV40)共转染家蚕卵巢培养细胞BmN,并以共转染pcD NA3.0(ie1-egfp-SV40)和pG L3(A3-luc-Ser-1 3'UTR-SV40)的BmN细胞为阳性对照,以海肾荧光素酶表达载体pRL-CMV为内参,检测荧光素酶活性,结果显示实验组的荧光素酶活性相对于阳性对照组显著降低(P<0.05),从细胞水平证实了Bmo-miR-2771对BmS er-1基因表达具有负调控作用。MicroRNAs（ miRNAs） exert a post-transcriptional regulation on gene expression through inhibiting translation of target mRNA or degrading target mRNA molecule. Understanding the regulatory function of Bombyx mori miRNAs on Bombyx mori sericin gene 1（ BmS er-1） would enrich information on fine regulation of sericin gene expression. Firstly,we downloaded whole maturated Bmo-miRs from database miRBase 21. Then we identified Bmo-miR-2771 as the candidate miRNA regulating the expression of BmS er-1 gene. The expression profile of Bmo-miR-2771 and its predicted target gene BmSer-1 in 7 different tissues including head,fat body,anterior silk gland（ ASG）,middle silk gland（ MSG）,post silk gland（ PSG）,midgut and hemolymph from the5 th instar day 3 larvae of silkworm was explored by semiquantitative RT-PCR with the designed stem-loop primers.The determined results show that Bmo-miR-2771 specifically expressed in MSG,and the expression level of Bm S-er-1 in MSG was significantly higher than that in other tissues. The recombinant plasmids pc DNA3. 0（ ie1-egfp-pri-miR-2771-SV40） and p GL3（ A3-luc-Ser-1 3＇UTR-SV40） were constructed and then applied in the co-transfection of BmN cell line followed by luciferase assay with Renilla luciferase expression vector pRL-CMV as the internal reference,and BmN cells co-transfected with pc DNA3. 0（ ie1-egfp-SV40） plus p GL3（ A3-luc-Ser-1 3＇ UTR-SV40） were used as the positive control. The determined results showed that the luciferase signal level was significantly decreased by Bmo-miR-2711 compared with the control group（ P〈0. 05）,thus showing that Bmo-miR-2771 has negative regulatory function on expression of BmS er-1 gene.

关 键 词：家蚕 丝胶蛋白基因Ser-1 微小RNA Bmo-miR-2771 转录后调控 半定量RT-PCR 双荧光素酶报告基因系统 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q74[农业科学—畜牧兽医]