XY和YY黄颡鱼精巢转录组分析以及rbmx基因在鱼类性腺发育中的功能研究

发布时间：2018-05-01 09:30

本文选题：雄性黄颡鱼 + 超雄黄颡鱼　；参考：《华中农业大学》2017年博士论文

【摘要】：黄颡鱼(Pelteobagrus fulvidraco)是我国一种重要的淡水经济鱼类,其分布广泛。虽然全雄黄颡鱼已大规模运用于生产,但其性别分化、精巢发育等生物学机制尚不明确。XY普通雄性和YY超雄黄颡鱼可以作为研究鱼类雄性性别决定以及精巢发育的理想材料。本研究通过使用Illumina平台对雄性黄颡鱼(XY)和超雄黄颡鱼(YY)精巢进行高通量测序。测序并组装得到78148个Unigenes,1146个Unigenes在YY精巢中高表达,1235个Unigenes在XY精巢中高表达。其中286个YY精巢中高表达的Unigenes以及319个XY精巢中高表达的Unigenes(共605个Unigenes)被注释到1604 GO条目中。KEGG富集分析发现,312个差异表达基因被注释到了252个信号通路上,部分差异表达基因注释到了PI3K-AKT以及G protein-coupled receptor(GPCR)信号通路。荧光定量PCR检测了12个差异表达基因,结果与高通量测序一致。此外,miR-141/429被认为可能在精巢发育中起重要作用,通过生物信息学预测及双荧光素酶报告系统验证确认,PI3K-AKT以及GPCR信号通路中的一些因子是miR-141/429的靶基因。我们的研究对XY和YY黄颡鱼精巢进行了转录组比较分析,并揭示了miR-141/429和靶基因的相互作用在精巢发育与精子生成过程中发挥作用。有趣的是,我们通过生物信息学预测发现黄颡鱼rbmx基因是miR-141/429的靶基因。荧光定量PCR分析发现,rbmx基因在超雄黄颡鱼(YY)精巢中的表达量显著高于普通雄性黄颡鱼(XY),与miR-141/429表达趋势恰好相反。已有研究表明,RBMX及其逆基因在哺乳动物性别分化、精巢发育以及精子生成过程中发挥着重要的作用。由于黄颡鱼繁殖周期较长,且基因组信息匮乏,我们在斑马鱼中进行rbmx基因功能研究。哺乳动物在进化早期产生了RBMX逆基因,而我们的进化分析也显示,哺乳动物RBMX逆基因已在进化早期发生了功能分化,并伴随性染色体分化形成了RBMX和RBMY基因,同时哺乳动物RBMX基因所在分支存在强烈的正向选择。然而,在鱼类中rbmx基因只存在单一拷贝,其时空表达及功能仍不清楚。时空表达分析发现成体斑马鱼rbmx基因主要集中在脑部和肝脏中表达,在胚胎发育时期,rbmx主要在脑部表达。我们使用CRISPR/Cas9系统构建了rbmx敲除体系,通过筛选,最终得到了rbmx“缺失2碱基”纯合突变斑马鱼。同时,我们发现rbmx突变体后代雄性比例要明显大于雌性,可能是突变体发育过程中发生了雌性-雄性性逆转。此外,rbmx突变体精子活力也较野生型斑马鱼低。研究结果表明rbmx在鱼类性别分化和精子成熟过程中扮演着重要的角色。我们的研究为鱼类性腺发育及配子生成研究提供了重要的生物学活体材料,同时也为进一步探明这些生物学过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.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S917.4

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