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3.367×10^10 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1394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。
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 3.367×10^10 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 1394 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-βand PI3K/AKT in cumulus cells.
作者
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
WANG Lulu;SHEN Penglei;WU Fei;YE Sheng;CHEN Weili;ZHAO Xin;FENG Yun;DENG Yanfei;JIANG Jianrong;SHI Deshun;LU Fenghua(State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Guangxi University,Nanning 530004,China)
出处
《中国畜牧兽医》
CAS
北大核心
2020年第8期2368-2377,共10页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31560633,31760666)。
