摘要
旨在克隆牦牛miR-378的前体序列,阐明其组织表达规律,结合bta-miR-378靶基因的生物信息学预测和分析,探讨miR-378在牦牛生长发育过程中的调控功能。采用PCR方法成功克隆类乌齐牦牛miR-378前体序列,实时荧光定量PCR(RT-qPCR)检测miR-378-3p在各组织中的表达模式,结合生物信息学软件TargetScan、DAVID以及数据库NCBI、miRbase等对miR-378进行保守性分析、靶基因预测及其生物学功能分析。结果表明,miR-378在各物种间高度保守,且miR-378-3p在各组织中广泛表达,其中在臀大肌中表达水平最高,显著高于其他组织(P<0.01),在臀脂中的表达高于卵巢、大脑、乳腺和肝脏。获得的272个靶基因主要参与细胞分化、细胞发育、大分子代谢等多个生物学过程,涉及孕酮介导的卵母细胞成熟、促性腺激素释放激素(GnRH)信号通路等,由此推测,miR-378可能在卵泡发育、卵母细胞成熟过程中起关键作用,进而影响母牦牛的繁殖性能。
This work aims to clone the precursor sequence of yak miR-378,to elucidate its tissue expression pattern,and to explore theregulatory function of miR-378 in the growth and development of yak combined with bioinformatics prediction and analysis of bta-miR-378 targetgene. PCR was employed to successfully clone the miR-378 precursor sequence of leiwuqi yak,and real-time quantitative PCR(RT-qPCR)was used to detect the expression patterns of miR-378-3p in various tissues. Further,bioinformatics software and databases such as TargetScan,DAVID,NCBI,miRbase,etc were together applied to perform conservative analysis,target gene prediction and biological function analysisof miR-378. The results showed that miR-378 was highly conserved among different species and miR-378-3p was widely expressed in all tissues,the expression level was the highest in gluteus maximus,which was significantly higher than that in other tissues(P < 0.01). Expression inhip fat was higher than in ovary,brain,breast and liver. The obtained 272 target genes were mainly involved in many biological processessuch as cell differentiation,cell development,and macromolecular metabolism,involved in progesterone-mediated oocyte maturation andgonadotropin releasing hormone(GnRH)signaling pathways. It is speculated that miR-378 may play a key role in follicular development andoocyte maturation,which may affect the reproductive performance of female calves.
引文
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