蒙古马睾丸发育及精子生成相关piRNA的鉴定及表达调控研究

发布时间：2018-01-22 01:25

本文关键词： 蒙古马高通量测序 piRNA 睾丸组织精子生成　出处：《内蒙古农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：Piwi-interactingRNAs(piRNAs)是一类在雄性生殖细胞中特异性表达的非编码小分子RNA,主要在哺乳动物卵巢卵母细胞和睾丸精原细胞中分布。piRNA与Argonaute蛋白家族中Piwi亚家族蛋白结合调控生殖细胞和干细胞生长发育、沉默转座子、抑制异染色质的形成、维持生殖系DNA的完整性、调控基因的表达,保证睾丸的正常发育和精子的生成,从而提高繁殖性能。蒙古马是我国优秀的地方马品种资源之一,具有适应性能强、耐性强、抗病性强等特性。但如今内蒙地区蒙古马的数量急剧下降并且品种严重退化。本研究利用高通量测序、生物信息学等生物学技术鉴定蒙古马睾丸组织中的piRNA并对分布和功能进行分析,对piRNA来源基因进行GO功能和KEGG Pathway分析。同时对piRNA cluster进行差异表达分析,探索piRNA在蒙古马睾丸发育和精子生成过程中的功能机制,并筛选出与蒙古马睾丸发育和精子生成相关的差异显著相关代谢通路和piRNAs,进一步确定了 piRNA对蒙古马生殖系统和繁殖性能的影响。本研究主要获得如下结果:(1)利用IllumiaHiseq高通量测序技术,成功获得蒙古马性成熟前后睾丸组织小分子RNA,并在蒙古马性成熟前后睾丸组织样品中共鉴定了 1048575个新候选piRNAs;在总体小RNA组成上性成熟前后睾丸组织种类数目差异不明显,但是在成熟睾丸组织中的小RNA种类更加丰富。(2)对新发现的来源基因进行Gene ontology和KEGG通路富集分析发现,这些基因富集到的主要GOterm有:细胞内、代谢过程、细胞蛋白修饰、初级代谢过程、蛋白质的结合等,提示这些piRNA参与了睾丸发育或精子发生过程。KEGG富集分析结果显示:来源基因不同程度富集到粘着斑、磷脂酰环已六醇信号系统内吞、黏着连接等代谢通路。此外,还有MAPK、Axon guidance、GnRH、Wnt等与生殖系统发育相关代谢通路。(3)对piRNAcluster进行差异表达分析发现,在性成熟前后所有样品中表达的piRNAcluster共有16857,差异表达的piRNAcluster有7890,其中3016个显著上调,4874个显著下调。在性成熟前后piRNA cluster表达量存在较大差异,可推测这些差异piRNA cluster对性成熟前后睾丸组织发育有影响。(4)根据GO功能、KEGG Pathway分析结果和piRNA簇差异表达分析结果,筛选出性成熟前后差异表达并且与繁殖发育相关的部分PiRNA(uniq_2455796、uniq_2331522、uniq_2343853、uniq_2331184、uniq_2449807、uniq_2347778 和uniq_2333097等7个piRNAs)。采用实时荧光定量PCR进行表达丰度的鉴定,分析其在不同发育时期睾丸组织中的表达情况。结果表明,uniq_2455796、uniq_2331522、uniq_2343853、uniq_2331184、uniq_2449807、uniq_2347778、uniq_2333097 等7个piRNAs在性成熟前后蒙古马睾丸组织中均有表达。其中,uniq_2455796和uniq_2331522在蒙古马性成熟前后睾丸组织中的表达量差异显著(P0.05),其他5个piRNAs在蒙古马性成熟前后睾丸组织中的表达量差异不显著(P0.05)。
[Abstract]:Piwi-interacting RNAspiRNAs) is a kind of non-coding small molecule RNA that is specifically expressed in male germ cells. Mainly distributed in mammalian oocytes and spermatogonial cells. PiRNAs bind to Piwi subfamily proteins in Argonaute protein family to regulate the growth and development of germ cells and stem cells. Silencing transposons, inhibiting the formation of heterochromatin, maintaining the integrity of reproductive DNA, regulating gene expression, ensuring normal testicular development and spermatogenesis. Mongolian horse is one of the outstanding local horse breeds in China, which has strong adaptability and strong tolerance. However, the number of Mongolian horses in Inner Mongolia region has declined sharply and the variety has been degraded seriously. This study uses high-throughput sequencing. Bioinformatics and other biological techniques were used to identify piRNA in testis of Mongolian horse and to analyze its distribution and function. Go function, KEGG Pathway and differential expression of piRNA cluster were analyzed. To explore the functional mechanism of piRNA in the development of testis and spermatogenesis of Mongolian horses, and to screen out the significant differences in metabolic pathways and piRNAs related to testis development and spermatogenesis in Mongolian horses. The effects of piRNA on the reproductive system and reproductive performance of Mongolian horses were further determined. The following results were obtained: 1) IllumiaHiseq high-throughput sequencing technique was used. Small molecule RNAs of testis were successfully obtained before and after sexual maturation of Mongolian horses. 1048575 new candidates for piRNAswere identified in testis tissue samples of Mongolian horses before and after sexual maturation. There was no significant difference in the number of testicular tissues before and after sexual maturation in the overall small RNA composition. However, the small RNA species in mature testis were more abundant. 2) enrichment analysis of Gene ontology and KEGG pathway was carried out on the newly discovered source genes. The major GOterm enriched by these genes are: intracellular, metabolic, cellular protein modification, primary metabolism, protein binding and so on. These results suggest that these piRNA are involved in testicular development or spermatogenesis. The results of KEGG enrichment analysis showed that the source gene was enriched to different extent to the plaque and the phosphatidyl cyclohexanol signaling system was endocytosis. In addition, there are MAPK, Axon guidance and GnRH. The differential expression of piRNAcluster was found by Wnt and other metabolic pathways associated with reproductive system development. The expression of piRNAcluster in all samples before and after sexual maturation was 16857, and the differential expression of piRNAcluster was 7890, 3016 of which were significantly up-regulated. There were significant differences in the expression of piRNA cluster before and after sexual maturation. It can be inferred that these differences of piRNA cluster have an effect on testicular tissue development before and after sexual maturation. The results of KEGG Pathway analysis and piRNA cluster differential expression analysis. The partial PiRNA-uniq-related PiRNA-uniq-tip2331522uniq-tip2343853 was screened out before and after sexual maturation and related to reproduction and development. Uniq_2331184,uniq_2449807. Uniq_2347778 and uniq_2333097 were used to identify the expression abundance of 7 piRN Ass. The results of the analysis of their expression in testicular tissues at different developmental stages showed that the number of tippings was 2331522 / uniq _ (2343853), and the results showed that: 2455796 / uniq _ tid _ 2331 _ 1522 / uniq _ 2. Uniq_2331184,uniq_2449807,uniq_2347778. Seven piRNAs including uniq_2333097 were expressed in testis of Mongolian horse before and after sexual maturation. The expression of uniq_2455796 and uniq_2331522 in testis of Mongolian horse before and after sexual maturation was significantly different (P0.05). There was no significant difference in the expression of other five piRNAs in testis of Mongolian horse before and after sexual maturation (P 0.05).
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S821.81

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