黄河鲤卵巢发育早期相关基因与信号通路的鉴定和作用研究

发布时间：2018-01-20 14:48

  本文关键词： 黄河鲤 转录物组 卵巢分化 内分泌干扰物 notch NF-κB TPT　出处：《河南师范大学》2017年博士论文　论文类型：学位论文

【摘要】：生殖是生命有机体非常重要的生物特征,而卵巢的发育分化是有性生殖的基本前提。鱼类是脊椎动物中种类最多的类群,具有重要的经济价值。鱼类卵巢分化发育的类型众多,影响其分化发育的遗传因素和环境因素繁杂。研究鱼类卵巢分化发育的分子机制不仅有利于渔业生产的发展,对于人们了解生物性别决定机制也具有重要意义。黄河鲤(Cyprinus carpio)是河南省乃至全国重要的淡水养殖鱼类,其生长具有明显的性别二态性,雌性生长较雄性快。目前鲤鱼卵巢分化发育的机制研究较少,影响其分化发育的遗传因素和环境因素也不清楚。为了全面研究黄河鲤卵巢分化发育的调控机制,寻找调控卵巢分化发育的分子机制和关键基因,本研究首先从组织学水平上确定了黄河鲤原始性腺向卵巢分化的关键时间,通过分析卵巢不同发育阶段的转录物组信息,找到了与卵巢分化发育过程密切相关的候选基因和信号通路,通过研究这些基因结构与时空表达模式,确定了其在卵巢分化发育中的作用;同时研究了信号通路对卵巢分化的影响及其作用机制。本文还对环境内分泌干扰物TPT对黄河鲤性腺分化的影响进行了分析,通过分析TPT处理后性腺的转录物组信息,发现了其影响卵巢分化的分子机制。获得以下创新结果:1.确定了黄河鲤性腺分化的关键时间。通过组织学切片,原始性腺在幼鱼40 dpf时可以从显微镜下分离,其在55 dpf之前保持未分化状态,在70 dpf出现了卵巢的分化。我们对性腺发育相关基因的表达水平从原始性腺到幼稚卵巢期间进行检测,发现在多数基因在50 dpf发生显著变化。根据原始性腺分化为精卵巢的关键时间早于性腺发生的组织结构变化,我们认为黄河鲤性腺分化的关键时期在45-50 dpf。2.分析比较了原始性腺、幼稚卵巢和成熟卵巢的转录物组,发现与卵巢分化高度相关的差异表达基因和信号通路。将45 dpf原始性腺,80 dpf幼稚卵巢和排卵前的成熟卵巢进行转录物组测序。在原始性腺、幼稚卵巢和成熟卵巢中分别发现了1788,1681和1256个特异表达的基因。研究发现原始性腺和幼稚卵巢的差异基因有29679个,原始性腺和成熟卵巢的基因间表达差异的基因有31150个,幼稚卵巢和成熟卵巢间有21201个差异基因。通过对差异表达基因(degs)进行go和kegg分析,得到卵巢分化发育密切相关的基因,如foxl2、dax1、lhx8、dnmt1等基因。对4个与卵巢早期发育相关的候选基因foxl2、dax1、lhx8、dnmt1进行了详细的基因结构和表达模式分析。4个基因均为首次在黄河鲤报道其结构和表达模式。这些基因与卵巢分化有着密切的关系,推测其可能参与黄河鲤性腺分化和精卵巢早期的分化调控。通过degs的kegg显著富集分析,发现notch、nf-κb、范可尼贫血、类固醇生物合成,孕酮介导的卵母细胞成熟、ppar等信号通路参与了卵巢的分化。3.研究notch和nf-κb信号通路在黄河鲤卵巢发育中的作用,发现抑制notch信号可以促进卵巢分化,抑制nf-κb信号则抑制卵巢分化。利用notch信号通路抑制剂dapt对原始性腺进行短期体外处理和对幼鱼为期30天的水体暴露试验。短期处理结果显示发现dapt可以抑制原始性腺中的notch信号和amh,dmrt1,sox9a,sf1,dax1基因的表达,增强foxl2,gdf9,nobox基因表达。长期暴露实验显示,dapt可以抑制notch信号,并促进原始性腺向卵巢的分化,这也是首次证明该信号通路参与调控鱼类的性腺分化,抑制该通路可以显著促进卵巢分化。利用nf-κb信号通路激活剂doc和抑制剂nai,在黄河鲤卵巢分化的关键时期,对原始性腺分别短期处理和长期水体暴露试验。结果发现激活nf-κb信号通路,诱导了il-8,tnf-α、抗凋亡基因xiap和雌性发育相关基因zp2,foxl2表达上调,凋亡相关基因casp3a,smac基因表达下调,雌性比例增加。抑制nf-κb信号通路,则nf-κb,tnf-α,il8,xiap基因表达下调,并提高了促进了amh,sox9a,dax1基因,凋亡基因smac和casp3a的表达,降低了雌性的性别比例。说明该通路参与黄河鲤卵巢的早期分化发育。另外zp2,foxl2、amh,sox9a,sf1,dax1基因在处理后表达显著变化,说明这些基因受到nf-κb信号通路的调控,从而在性腺分化中发挥作用。这一结果不仅验证了黄河鲤性腺分化的关键时间,同时还发现了新的信号通路可能通过调控炎症分子和细胞调亡参与黄河鲤卵巢分化。4.分析了tpt对黄河鲤幼体的长期体内处理和不同时期性腺的短期体外处理结果,发现tpt可以显著改变幼体性腺分化的比例,其可能的分子机制是通过改变原始性腺中调控卵巢分化的关键基因和信号通路尤其是类固醇激素生物合成通路而实现的。综上所述,本研究确定了黄河鲤性腺发育的关键时间,发现了与卵巢分化早期的调控基因和信号通路,详尽研究了这些基因和信号通路在卵巢发育过程中的作用;同时发现了与TPT影响黄河鲤性腺分化有关的基因和信号通路,揭示了TPT作为性激素类似物的分子机理。这些结果为揭示鱼类的卵巢分化的分子机理奠定了坚实基础,对于水产养殖有着重要的理论意义和实践意义。
[Abstract]:Reproduction is the biological characteristics of living organisms is very important, and the development and differentiation of ovarian is the basic premise of sexual reproduction. Fish species is the most vertebrates, has an important economic value. Many types of fish ovarian differentiation, affect the differentiation and development of genetic and environmental factors are complicated. The molecular mechanism of fish ovarian differentiation development is not only conducive to the development of fishery production, also has an important significance to understand the biological mechanism of sex determination. The Yellow River carp (Cyprinus carpio) in Henan province is an important freshwater fish species, with two state sex obvious growth, female growth faster than those of the male. The Research on the mechanism of carp ovarian differentiation less the effect of genetic factors, differentiation and environmental factors is not clear. In order to study the regulation mechanism of the Yellow River carp ovarian differentiation and development of comprehensive optimization Find the key molecular mechanisms and gene regulation of ovarian differentiation, this study first from the histological level to determine the the Yellow River carp to the key time of ovarian primordial gonad differentiation, through the analysis of transcriptome information of ovary at different developmental stages, found the process of candidate genes and signaling pathways are closely related with the differentiation of ovarian development, through the study of these gene structure and expression pattern, the differentiation and development of ovarian function; at the same time on the signaling effect on ovarian differentiation and its mechanism of action. This paper also analyzes the effect of environmental endocrine disruptors TPT on gonadal differentiation of the Yellow River carp, through the analysis of the TPT treated gonad transcriptome information found the effect of ovarian differentiation mechanism. To obtain the following results: 1. innovation to determine the critical time of gonadal differentiation of the Yellow River carp. By histologic slices, the original The gonads can be isolated from under the microscope in the juvenile 40 DPF, which maintain the undifferentiated state before 55 DPF in 70, DPF appeared in ovarian differentiation. We detected during the expression level of genes related to gonadal development from primitive gonad to immature ovary, found in most genes in 50 DPF significant change. Change of organizational structure according to the original key time for fine ovarian gonadal differentiation earlier than the gonadogenesis, we believe that the key period of gonadal differentiation of the Yellow River carp in the 45-50 dpf.2. analysis and comparison of the primitive gonad transcriptome, immature ovary and mature ovary, found differentially expressed genes and signaling pathways are highly correlated with the ovarian differentiation. 45 DPF primitive gonad. The mature ovary 80 DPF naive ovarian and before ovulation by transcriptome sequencing. In primitive gonad, immature ovary and mature ovaries were found 17881681 and 1256 specific table The study found that genes. Genes differentially primitive gonad and ovary immature. There are 29679 differences in 31150 genes expression of primitive gonad and mature ovary gene between immature and mature ovary ovary between 21201 genes. The gene expression difference of (DEGs) go and KEGG analysis, get the differentiation of ovarian development closely related genes, such as FOXL2, DAX1, Lhx8, DNMT1 and so on. The early gene candidate gene FOXL2, the development of 4 DAX1 Lhx8, DNMT1 and ovarian, analyzed the gene structure and expression patterns with.4 genes were first reported in the Yellow River carp its structure and expression pattern of these genes and the ovary. Differentiation are closely related, suggesting that it may be involved in regulating the differentiation of ovarian gonadal differentiation and fine early the Yellow River carp by DEGs. KEGG significant enrichment analysis, found that Notch, nf- kappa B, Fanconi anemia, steroid biosynthesis, pregnancy Ketone mediated oocyte maturation, PPAR signaling pathway is involved in the ovarian differentiation of.3. notch and nf- B signaling pathway in the development of the Yellow River carp in the ovary, found that inhibition of Notch signaling can promote ovarian differentiation, inhibit nf- kappa B signal inhibits ovarian differentiation. The original short term in vitro and gonad juvenile 30 days water exposure test using the Notch signal pathway inhibitor dapt. The results show that short-term dapt can inhibit the primitive gonad Notch signal and AMH, Dmrt1, sox9a, SF1, DAX1 gene expression, enhanced FOXL2, GDF9, NOBOX gene expression. Long term exposure experiments show that dapt can inhibit the Notch signal. And promote the primordial gonad to ovarian differentiation, this is the first time that the signal pathway involved in the regulation of gonadal differentiation of fish, inhibiting this pathway can significantly promote ovarian differentiation. Using nf- B pathway activator Doc and inhibitor Nai in the critical period of the Yellow River carp ovarian differentiation, the original short-term and long-term water treatment respectively gonadal exposure test. The results showed that the activation of nf- B pathway, induced by IL-8, tnf- alpha, anti apoptosis gene XIAP and female development related gene ZP2, FOXL2 expression, apoptosis related gene casp3a, Smac gene expression, female ratio increased. The inhibition of nf- B signaling pathway, nf- kappa B, tnf- alpha, IL8, XIAP gene expression, and enhanced the AMH, sox9a, DAX1 gene, expression of Smac and casp3a, reduced the proportion of female gender. The early differentiation pathway participate in the the Yellow River carp ovary. The addition of ZP2, FOXL2, AMH, sox9a, SF1, DAX1 gene expression was significantly changed after the treatment, that these genes are regulated by nf- B signaling pathway, which play a role in gonadal differentiation. The results not only verify the carp of the Yellow River The key time of glandular differentiation, also found new signaling pathways by regulating inflammatory molecules and apoptosis in the Yellow River carp ovarian differentiation analysis of.4. TPT of the Yellow River carp larvae in different periods and long-term treatment of gonadal short-term in vitro processing results demonstrate that TPT can significantly change the proportion of the larval gonad differentiation, possible the molecular mechanism is the key genes and signaling pathways in regulating the differentiation of ovarian primordial gonad changes especially the steroid hormone biosynthesis pathway. In conclusion, this study identified the key time of gonad development of the Yellow River carp, and found the early differentiation of ovarian regulatory genes and signaling pathways, a detailed study of these genes and signaling pathways in the in the process of ovarian development; at the same time found and TPT effect of the Yellow River carp gonadal differentiation genes and related signaling pathways, as revealed by TPT As a molecular mechanism of sex hormone analogues, these results lay a solid foundation for revealing the molecular mechanism of fish ovarian differentiation, and have important theoretical and practical significance for aquaculture.

【学位授予单位】：河南师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S917.4
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本文编号：1448590