水牛miR-16b过表达腺病毒载体构建及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2020.08.004

摘要/Abstract

摘要： 试验旨在对水牛pri-miR-16b基因进行克隆并构建腺病毒表达载体，且对水牛miR-16b及其预测靶基因进行生物信息学分析，为研究其在水牛卵母细胞成熟过程中的作用奠定基础。利用RT-PCR技术从水牛卵巢基因组中扩增pri-miR-16b基因，采用同源重组的方法构建pDC316-mCMV-EGFP-pri-miR-16b质粒；利用BLAST程序进行同源性分析，Mega 7.0构建系统进化树，ViennaRNA Web Services预测pre-miR-16b的二级结构。对腺病毒质粒与腺病毒骨架质粒pBHGloxdelE13cre进行包装，经过3次扩增获得含有pri-miR-16b的腺病毒颗粒，将获得的病毒颗粒命名为Ad-miR-16b，并进行病毒滴度测定；利用Ad-miR-16b感染水牛卵丘细胞，实时荧光定量PCR检测miR-16b在卵丘细胞中的表达情况。利用TargetScan对miR-16b进行靶基因预测，对预测的靶基因进行KEGG通路富集。结果显示，试验成功克隆水牛pri-miR-16b序列，通过比对发现与牛的序列相似性分别为100%，与其他物种同源性较高，和黄牛的亲缘关系最近。ViennaRNA Web Services预测结果显示，pre-miR-16b的二级结构具有典型的单一茎环结构。试验成功获得Ad-miR-16b腺病毒颗粒，病毒滴度为3.367×10¹⁰ GFU/mL，感染卵丘细胞后，实时荧光定量PCR检测发现miR-16b的表达量极显著升高（P<0.01）。对miR-16b预测的1 394个靶基因进行KEGG通路富集分析，发现miR-16b可能主要通过调控卵丘细胞中MAPK、TGF-β及PI3K/AKT等74条信号通路进而在卵母细胞成熟过程中发挥作用。

关键词: miR-16b; 腺病毒; 卵丘细胞; 生物信息学分析

Abstract: This study was aimed to clone and construct the adenovirus vector of pri-miR-16b gene in buffaloes,meanwhile the bioinformatics analysis of miR-16b and its predicted target genes were carried out to provide a basis for studying its role in oocyte maturation of buffaloes.pri-miR-16b gene was amplified from the ovary genome in buffaloes by RT-PCR,and then the pDC316-mCMV-EGFP-pri-miR-16b plasmid was constructed by homologous recombination.The homology and phylogenetic tree were analyzed and constructed by BLAST program and Mega 7.0,respectively,and the secondary structure of pre-miR-16 were predicted by ViennaRNA Web Services.The constructed vector was packaged with the adenovirus backbone plasmid pBHGloxdelE13cre to obtain the adenoviral particles named Ad-miR-16b,then three times of amplifications were performed and the virus titer was also determined.Ad-miR-16b adenovirus particles infected buffalo cumulus cells,and the expression of miR-16b in cumulus cells was detected by Real-time quantitative PCR.The target genes of miR-16b were predicted by TargetScan,and the KEGG pathway was enriched by the DAVID website for the predicted target genes.The results showed that the pri-miR-16b sequence in buffaloes was successfully cloned,and the sequence similarity with the bovine was found to be 100%.The high homology of pre-miR-16b in buffaloes with other species and the closest relationship with Bos taurus.The ViennaRNA Web Services prediction results showed that the secondary structure of pre-miR-16b had a typical single stem-loop structure,Ad-miR-16b adenoviral particles were obtained with a virus titer of 3.367×10¹⁰ GFU/mL.After the cumulus cells were infected,the expression of miR-16b was extremely significantly increased by Real-time quantitative PCR (P<0.01).The KEGG pathway enrichment analysis was conducted on 1 394 target genes predicted by miR-16b,and it was found that miR-16b might play a role in oocyte maturation mainly by regulating 74 signaling pathways such as MAPK,TGF-β and PI3K/AKT in cumulus cells.

Key words: miR-16b; adenovirus; cumulus cells; bioinformatics analysis

中图分类号:

王露露, 沈朋雷, 吴飞, 叶升, 陈维丽, 赵鑫, 俸云, 邓彦飞, 蒋建荣, 石德顺, 陆凤花. 水牛miR-16b过表达腺病毒载体构建及生物信息学分析[J]. 中国畜牧兽医, 2020, 47(8): 2368-2377.

WANG Lulu, SHEN Penglei, WU Fei, YE Sheng, CHEN Weili, ZHAO Xin, FENG Yun, DENG Yanfei, JIANG Jianrong, SHI Deshun, LU Fenghua. Construction of Overexpression Adenovirus Vector and Bioinformatics Analysis of miR-16b in Buffaloes[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(8): 2368-2377.

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