本文關(guān)鍵詞： 蒙古馬 高通量測(cè)序 piRNA 睪丸組織 精子生成　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：Piwi-interactingRNAs(piRNAs)是一類在雄性生殖細(xì)胞中特異性表達(dá)的非編碼小分子RNA,主要在哺乳動(dòng)物卵巢卵母細(xì)胞和睪丸精原細(xì)胞中分布。piRNA與Argonaute蛋白家族中Piwi亞家族蛋白結(jié)合調(diào)控生殖細(xì)胞和干細(xì)胞生長(zhǎng)發(fā)育、沉默轉(zhuǎn)座子、抑制異染色質(zhì)的形成、維持生殖系DNA的完整性、調(diào)控基因的表達(dá),保證睪丸的正常發(fā)育和精子的生成,從而提高繁殖性能。蒙古馬是我國(guó)優(yōu)秀的地方馬品種資源之一,具有適應(yīng)性能強(qiáng)、耐性強(qiáng)、抗病性強(qiáng)等特性。但如今內(nèi)蒙地區(qū)蒙古馬的數(shù)量急劇下降并且品種嚴(yán)重退化。本研究利用高通量測(cè)序、生物信息學(xué)等生物學(xué)技術(shù)鑒定蒙古馬睪丸組織中的piRNA并對(duì)分布和功能進(jìn)行分析,對(duì)piRNA來(lái)源基因進(jìn)行GO功能和KEGG Pathway分析。同時(shí)對(duì)piRNA cluster進(jìn)行差異表達(dá)分析,探索piRNA在蒙古馬睪丸發(fā)育和精子生成過(guò)程中的功能機(jī)制,并篩選出與蒙古馬睪丸發(fā)育和精子生成相關(guān)的差異顯著相關(guān)代謝通路和piRNAs,進(jìn)一步確定了 piRNA對(duì)蒙古馬生殖系統(tǒng)和繁殖性能的影響。本研究主要獲得如下結(jié)果:(1)利用IllumiaHiseq高通量測(cè)序技術(shù),成功獲得蒙古馬性成熟前后睪丸組織小分子RNA,并在蒙古馬性成熟前后睪丸組織樣品中共鑒定了 1048575個(gè)新候選piRNAs;在總體小RNA組成上性成熟前后睪丸組織種類數(shù)目差異不明顯,但是在成熟睪丸組織中的小RNA種類更加豐富。(2)對(duì)新發(fā)現(xiàn)的來(lái)源基因進(jìn)行Gene ontology和KEGG通路富集分析發(fā)現(xiàn),這些基因富集到的主要GOterm有:細(xì)胞內(nèi)、代謝過(guò)程、細(xì)胞蛋白修飾、初級(jí)代謝過(guò)程、蛋白質(zhì)的結(jié)合等,提示這些piRNA參與了睪丸發(fā)育或精子發(fā)生過(guò)程。KEGG富集分析結(jié)果顯示:來(lái)源基因不同程度富集到粘著斑、磷脂酰環(huán)已六醇信號(hào)系統(tǒng)內(nèi)吞、黏著連接等代謝通路。此外,還有MAPK、Axon guidance、GnRH、Wnt等與生殖系統(tǒng)發(fā)育相關(guān)代謝通路。(3)對(duì)piRNAcluster進(jìn)行差異表達(dá)分析發(fā)現(xiàn),在性成熟前后所有樣品中表達(dá)的piRNAcluster共有16857,差異表達(dá)的piRNAcluster有7890,其中3016個(gè)顯著上調(diào),4874個(gè)顯著下調(diào)。在性成熟前后piRNA cluster表達(dá)量存在較大差異,可推測(cè)這些差異piRNA cluster對(duì)性成熟前后睪丸組織發(fā)育有影響。(4)根據(jù)GO功能、KEGG Pathway分析結(jié)果和piRNA簇差異表達(dá)分析結(jié)果,篩選出性成熟前后差異表達(dá)并且與繁殖發(fā)育相關(guān)的部分PiRNA(uniq_2455796、uniq_2331522、uniq_2343853、uniq_2331184、uniq_2449807、uniq_2347778 和uniq_2333097等7個(gè)piRNAs)。采用實(shí)時(shí)熒光定量PCR進(jìn)行表達(dá)豐度的鑒定,分析其在不同發(fā)育時(shí)期睪丸組織中的表達(dá)情況。結(jié)果表明,uniq_2455796、uniq_2331522、uniq_2343853、uniq_2331184、uniq_2449807、uniq_2347778、uniq_2333097 等7個(gè)piRNAs在性成熟前后蒙古馬睪丸組織中均有表達(dá)。其中,uniq_2455796和uniq_2331522在蒙古馬性成熟前后睪丸組織中的表達(dá)量差異顯著(P0.05),其他5個(gè)piRNAs在蒙古馬性成熟前后睪丸組織中的表達(dá)量差異不顯著(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).
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S821.81

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