本文選題：雄性黃顙魚(yú) + 超雄黃顙魚(yú)��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：黃顙魚(yú)(Pelteobagrus fulvidraco)是我國(guó)一種重要的淡水經(jīng)濟(jì)魚(yú)類,其分布廣泛。雖然全雄黃顙魚(yú)已大規(guī)模運(yùn)用于生產(chǎn),但其性別分化、精巢發(fā)育等生物學(xué)機(jī)制尚不明確。XY普通雄性和YY超雄黃顙魚(yú)可以作為研究魚(yú)類雄性性別決定以及精巢發(fā)育的理想材料。本研究通過(guò)使用Illumina平臺(tái)對(duì)雄性黃顙魚(yú)(XY)和超雄黃顙魚(yú)(YY)精巢進(jìn)行高通量測(cè)序。測(cè)序并組裝得到78148個(gè)Unigenes,1146個(gè)Unigenes在YY精巢中高表達(dá),1235個(gè)Unigenes在XY精巢中高表達(dá)。其中286個(gè)YY精巢中高表達(dá)的Unigenes以及319個(gè)XY精巢中高表達(dá)的Unigenes(共605個(gè)Unigenes)被注釋到1604 GO條目中。KEGG富集分析發(fā)現(xiàn),312個(gè)差異表達(dá)基因被注釋到了252個(gè)信號(hào)通路上,部分差異表達(dá)基因注釋到了PI3K-AKT以及G protein-coupled receptor(GPCR)信號(hào)通路。熒光定量PCR檢測(cè)了12個(gè)差異表達(dá)基因,結(jié)果與高通量測(cè)序一致。此外,miR-141/429被認(rèn)為可能在精巢發(fā)育中起重要作用,通過(guò)生物信息學(xué)預(yù)測(cè)及雙熒光素酶報(bào)告系統(tǒng)驗(yàn)證確認(rèn),PI3K-AKT以及GPCR信號(hào)通路中的一些因子是miR-141/429的靶基因。我們的研究對(duì)XY和YY黃顙魚(yú)精巢進(jìn)行了轉(zhuǎn)錄組比較分析,并揭示了miR-141/429和靶基因的相互作用在精巢發(fā)育與精子生成過(guò)程中發(fā)揮作用。有趣的是,我們通過(guò)生物信息學(xué)預(yù)測(cè)發(fā)現(xiàn)黃顙魚(yú)rbmx基因是miR-141/429的靶基因。熒光定量PCR分析發(fā)現(xiàn),rbmx基因在超雄黃顙魚(yú)(YY)精巢中的表達(dá)量顯著高于普通雄性黃顙魚(yú)(XY),與miR-141/429表達(dá)趨勢(shì)恰好相反。已有研究表明,RBMX及其逆基因在哺乳動(dòng)物性別分化、精巢發(fā)育以及精子生成過(guò)程中發(fā)揮著重要的作用。由于黃顙魚(yú)繁殖周期較長(zhǎng),且基因組信息匱乏,我們?cè)诎唏R魚(yú)中進(jìn)行rbmx基因功能研究。哺乳動(dòng)物在進(jìn)化早期產(chǎn)生了RBMX逆基因,而我們的進(jìn)化分析也顯示,哺乳動(dòng)物RBMX逆基因已在進(jìn)化早期發(fā)生了功能分化,并伴隨性染色體分化形成了RBMX和RBMY基因,同時(shí)哺乳動(dòng)物RBMX基因所在分支存在強(qiáng)烈的正向選擇。然而,在魚(yú)類中rbmx基因只存在單一拷貝,其時(shí)空表達(dá)及功能仍不清楚。時(shí)空表達(dá)分析發(fā)現(xiàn)成體斑馬魚(yú)rbmx基因主要集中在腦部和肝臟中表達(dá),在胚胎發(fā)育時(shí)期,rbmx主要在腦部表達(dá)。我們使用CRISPR/Cas9系統(tǒng)構(gòu)建了rbmx敲除體系,通過(guò)篩選,最終得到了rbmx“缺失2堿基”純合突變斑馬魚(yú)。同時(shí),我們發(fā)現(xiàn)rbmx突變體后代雄性比例要明顯大于雌性,可能是突變體發(fā)育過(guò)程中發(fā)生了雌性-雄性性逆轉(zhuǎn)。此外,rbmx突變體精子活力也較野生型斑馬魚(yú)低。研究結(jié)果表明rbmx在魚(yú)類性別分化和精子成熟過(guò)程中扮演著重要的角色。我們的研究為魚(yú)類性腺發(fā)育及配子生成研究提供了重要的生物學(xué)活體材料,同時(shí)也為進(jìn)一步探明這些生物學(xué)過(guò)程提供了新的研究思路。
[Abstract]:Pelteobagrus fulvidraco (Pelteobagrus fulvidraco) is an important freshwater economic fish in China. Although fully realgar Pelteobagrus fulvidraco has been widely used in production, its biological mechanisms, such as sex differentiation, spermary development and so on, are not clear. XY ordinary males and YY super-realgar Pelteobagrus fulvidraco can be used as ideal materials for studying male sex determination and spermary development of fish. In this study, high throughput sequencing was carried out in the testis of male Pseudobagrus fulvidraco (XY) and Realgar Pelteobagrus fulvidraco (YY) by using Illumina platform. 78,148 Unigenes' 1146 Unigenes were highly expressed in YY testis and 1235 Unigenes were highly expressed in XY testis. In 286 YY testis and 319 XY testis, the highly expressed Unigenes (605 Unigeneses) were annotated into the 1604 go entry .KEGG enrichment analysis showed that 312 differentially expressed genes were annotated to 252 signaling pathways. Some of the differentially expressed genes were annotated into the PI3K-AKT and G protein-coupled receptor GPCR signaling pathways. Twelve differentially expressed genes were detected by fluorescence quantitative PCR and the results were consistent with high throughput sequencing. In addition, miR-141 / 429 is thought to play an important role in the development of spermary. Bioinformatics prediction and double luciferase reporting system confirm that PI3K-AKT and some factors in GPCR signaling pathway are the target genes of miR-141/429. Our results show that the interaction between miR-141/429 and target genes plays an important role in the development of spermatozoa and spermatogenesis of Pelteobagrus fulvidraco XY and YY. Interestingly, we found that the rbmx gene of Pelteobagrus fulvidraco was the target gene of miR-141/429 through bioinformatics prediction. Fluorescence quantitative PCR analysis showed that the expression of rbmx gene in the testis of Pelteobagrus fulvidraco was significantly higher than that in the normal male, which was contrary to the trend of miR-141/429 expression in the testis of Pelteobagrus fulvidraco. Studies have shown that RBMX and its reverse genes play an important role in mammalian sex differentiation, spermatogenesis and spermatogenesis. Due to the long reproductive cycle and the lack of genomic information, we studied the function of rbmx gene in zebrafish. Mammalian RBMX reverse gene was produced in the early stage of evolution, and our evolutionary analysis also showed that mammalian RBMX reverse gene had been functionally differentiated in the early evolutionary stage, and accompanied by sex chromosome differentiation to form RBMX and RBMY genes. At the same time, there is a strong positive selection in the branch of mammalian RBMX gene. However, there is only a single copy of rbmx gene in fish, and its temporal and spatial expression and function are still unclear. Spatio-temporal expression analysis showed that the rbmx gene of adult zebrafish was mainly expressed in brain and liver, and rbmx was mainly expressed in brain during embryonic development. We constructed the rbmx knockout system using CRISPR/Cas9 system, and finally obtained rbmx homozygous mutant zebrafish by screening. At the same time, we found that the ratio of male to male of rbmx mutant was obviously larger than that of female, which may be due to the female male sex reversal during the development of the mutant. In addition, the sperm motility of the rbmx mutant was lower than that of wild zebrafish. The results show that rbmx plays an important role in the sex differentiation and sperm maturation of fish. Our research provides important biological living materials for the study of gonadal development and gametogenesis in fish, and also provides new ideas for further study of these biological processes.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4

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本文編號(hào)：1828763