本文關(guān)鍵詞： 黃河鯉 轉(zhuǎn)錄物組 卵巢分化 內(nèi)分泌干擾物 notch NF-κB TPT　出處：《河南師范大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：生殖是生命有機(jī)體非常重要的生物特征,而卵巢的發(fā)育分化是有性生殖的基本前提。魚(yú)類(lèi)是脊椎動(dòng)物中種類(lèi)最多的類(lèi)群,具有重要的經(jīng)濟(jì)價(jià)值。魚(yú)類(lèi)卵巢分化發(fā)育的類(lèi)型眾多,影響其分化發(fā)育的遺傳因素和環(huán)境因素繁雜。研究魚(yú)類(lèi)卵巢分化發(fā)育的分子機(jī)制不僅有利于漁業(yè)生產(chǎn)的發(fā)展,對(duì)于人們了解生物性別決定機(jī)制也具有重要意義。黃河鯉(Cyprinus carpio)是河南省乃至全國(guó)重要的淡水養(yǎng)殖魚(yú)類(lèi),其生長(zhǎng)具有明顯的性別二態(tài)性,雌性生長(zhǎng)較雄性快。目前鯉魚(yú)卵巢分化發(fā)育的機(jī)制研究較少,影響其分化發(fā)育的遺傳因素和環(huán)境因素也不清楚。為了全面研究黃河鯉卵巢分化發(fā)育的調(diào)控機(jī)制,尋找調(diào)控卵巢分化發(fā)育的分子機(jī)制和關(guān)鍵基因,本研究首先從組織學(xué)水平上確定了黃河鯉原始性腺向卵巢分化的關(guān)鍵時(shí)間,通過(guò)分析卵巢不同發(fā)育階段的轉(zhuǎn)錄物組信息,找到了與卵巢分化發(fā)育過(guò)程密切相關(guān)的候選基因和信號(hào)通路,通過(guò)研究這些基因結(jié)構(gòu)與時(shí)空表達(dá)模式,確定了其在卵巢分化發(fā)育中的作用;同時(shí)研究了信號(hào)通路對(duì)卵巢分化的影響及其作用機(jī)制。本文還對(duì)環(huán)境內(nèi)分泌干擾物TPT對(duì)黃河鯉性腺分化的影響進(jìn)行了分析,通過(guò)分析TPT處理后性腺的轉(zhuǎn)錄物組信息,發(fā)現(xiàn)了其影響卵巢分化的分子機(jī)制。獲得以下創(chuàng)新結(jié)果:1.確定了黃河鯉性腺分化的關(guān)鍵時(shí)間。通過(guò)組織學(xué)切片,原始性腺在幼魚(yú)40 dpf時(shí)可以從顯微鏡下分離,其在55 dpf之前保持未分化狀態(tài),在70 dpf出現(xiàn)了卵巢的分化。我們對(duì)性腺發(fā)育相關(guān)基因的表達(dá)水平從原始性腺到幼稚卵巢期間進(jìn)行檢測(cè),發(fā)現(xiàn)在多數(shù)基因在50 dpf發(fā)生顯著變化。根據(jù)原始性腺分化為精卵巢的關(guān)鍵時(shí)間早于性腺發(fā)生的組織結(jié)構(gòu)變化,我們認(rèn)為黃河鯉性腺分化的關(guān)鍵時(shí)期在45-50 dpf。2.分析比較了原始性腺、幼稚卵巢和成熟卵巢的轉(zhuǎn)錄物組,發(fā)現(xiàn)與卵巢分化高度相關(guān)的差異表達(dá)基因和信號(hào)通路。將45 dpf原始性腺,80 dpf幼稚卵巢和排卵前的成熟卵巢進(jìn)行轉(zhuǎn)錄物組測(cè)序。在原始性腺、幼稚卵巢和成熟卵巢中分別發(fā)現(xiàn)了1788,1681和1256個(gè)特異表達(dá)的基因。研究發(fā)現(xiàn)原始性腺和幼稚卵巢的差異基因有29679個(gè),原始性腺和成熟卵巢的基因間表達(dá)差異的基因有31150個(gè),幼稚卵巢和成熟卵巢間有21201個(gè)差異基因。通過(guò)對(duì)差異表達(dá)基因(degs)進(jìn)行g(shù)o和kegg分析,得到卵巢分化發(fā)育密切相關(guān)的基因,如foxl2、dax1、lhx8、dnmt1等基因。對(duì)4個(gè)與卵巢早期發(fā)育相關(guān)的候選基因foxl2、dax1、lhx8、dnmt1進(jìn)行了詳細(xì)的基因結(jié)構(gòu)和表達(dá)模式分析。4個(gè)基因均為首次在黃河鯉報(bào)道其結(jié)構(gòu)和表達(dá)模式。這些基因與卵巢分化有著密切的關(guān)系,推測(cè)其可能參與黃河鯉性腺分化和精卵巢早期的分化調(diào)控。通過(guò)degs的kegg顯著富集分析,發(fā)現(xiàn)notch、nf-κb、范可尼貧血、類(lèi)固醇生物合成,孕酮介導(dǎo)的卵母細(xì)胞成熟、ppar等信號(hào)通路參與了卵巢的分化。3.研究notch和nf-κb信號(hào)通路在黃河鯉卵巢發(fā)育中的作用,發(fā)現(xiàn)抑制notch信號(hào)可以促進(jìn)卵巢分化,抑制nf-κb信號(hào)則抑制卵巢分化。利用notch信號(hào)通路抑制劑dapt對(duì)原始性腺進(jìn)行短期體外處理和對(duì)幼魚(yú)為期30天的水體暴露試驗(yàn)。短期處理結(jié)果顯示發(fā)現(xiàn)dapt可以抑制原始性腺中的notch信號(hào)和amh,dmrt1,sox9a,sf1,dax1基因的表達(dá),增強(qiáng)foxl2,gdf9,nobox基因表達(dá)。長(zhǎng)期暴露實(shí)驗(yàn)顯示,dapt可以抑制notch信號(hào),并促進(jìn)原始性腺向卵巢的分化,這也是首次證明該信號(hào)通路參與調(diào)控魚(yú)類(lèi)的性腺分化,抑制該通路可以顯著促進(jìn)卵巢分化。利用nf-κb信號(hào)通路激活劑doc和抑制劑nai,在黃河鯉卵巢分化的關(guān)鍵時(shí)期,對(duì)原始性腺分別短期處理和長(zhǎng)期水體暴露試驗(yàn)。結(jié)果發(fā)現(xiàn)激活nf-κb信號(hào)通路,誘導(dǎo)了il-8,tnf-α、抗凋亡基因xiap和雌性發(fā)育相關(guān)基因zp2,foxl2表達(dá)上調(diào),凋亡相關(guān)基因casp3a,smac基因表達(dá)下調(diào),雌性比例增加。抑制nf-κb信號(hào)通路,則nf-κb,tnf-α,il8,xiap基因表達(dá)下調(diào),并提高了促進(jìn)了amh,sox9a,dax1基因,凋亡基因smac和casp3a的表達(dá),降低了雌性的性別比例。說(shuō)明該通路參與黃河鯉卵巢的早期分化發(fā)育。另外zp2,foxl2、amh,sox9a,sf1,dax1基因在處理后表達(dá)顯著變化,說(shuō)明這些基因受到nf-κb信號(hào)通路的調(diào)控,從而在性腺分化中發(fā)揮作用。這一結(jié)果不僅驗(yàn)證了黃河鯉性腺分化的關(guān)鍵時(shí)間,同時(shí)還發(fā)現(xiàn)了新的信號(hào)通路可能通過(guò)調(diào)控炎癥分子和細(xì)胞調(diào)亡參與黃河鯉卵巢分化。4.分析了tpt對(duì)黃河鯉幼體的長(zhǎng)期體內(nèi)處理和不同時(shí)期性腺的短期體外處理結(jié)果,發(fā)現(xiàn)tpt可以顯著改變幼體性腺分化的比例,其可能的分子機(jī)制是通過(guò)改變?cè)夹韵僦姓{(diào)控卵巢分化的關(guān)鍵基因和信號(hào)通路尤其是類(lèi)固醇激素生物合成通路而實(shí)現(xiàn)的。綜上所述,本研究確定了黃河鯉性腺發(fā)育的關(guān)鍵時(shí)間,發(fā)現(xiàn)了與卵巢分化早期的調(diào)控基因和信號(hào)通路,詳盡研究了這些基因和信號(hào)通路在卵巢發(fā)育過(guò)程中的作用;同時(shí)發(fā)現(xiàn)了與TPT影響黃河鯉性腺分化有關(guān)的基因和信號(hào)通路,揭示了TPT作為性激素類(lèi)似物的分子機(jī)理。這些結(jié)果為揭示魚(yú)類(lèi)的卵巢分化的分子機(jī)理奠定了堅(jiān)實(shí)基礎(chǔ),對(duì)于水產(chǎn)養(yǎng)殖有著重要的理論意義和實(shí)踐意義。
[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.

【學(xué)位授予單位】：河南師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S917.4
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本文編號(hào)：1448590