Cyp19a1和Foxl2基因在中华鳖早期卵巢分化中的功能研究
发布时间：2018-08-29 16:59
【摘要】：芳香化酶作为雌激素合成的限制酶,在非哺乳类脊椎动物的早期雌性性别分化中起重要的调控作用,Cyp19a1基因作为芳香化酶的编码基因,参与了龟鳖类的雌性性别分化过程。Foxl2基因是叉头框转录因子家族成员之一,在雌性早期性腺分化和卵巢功能维持中扮演重要角色。现有研究结果表明,Foxl2基因位于Cyp19a1的上游,调控芳香化酶基因的表达,二者共同作用参与雌性的性腺分化过程。本研究分析了芳香化酶编码基因Cyp19a1在中华鳖成体不同组织及胚胎发育不同时期雌雄性腺中的表达情况;利用芳香化酶抑制剂和慢病毒介导的RNA干扰/过表达技术,着重进行Cyp19a1和Foxl2基因的功能验证研究。具体研究结果如下:(1)Cyp19a1的功能研究半定量PCR和实时荧光定量PCR结果显示Cyp19a1基因在中华鳖成体卵巢组织中高度特异性表达,睾丸和心、肝、脾、肺、肾、肌、肠中表达微弱或不表达。同时我们发现,Cyp19a1基因在第18期就呈现雌性性腺特异性表达,早于性腺分化启动时间(第19期)。功能缺失实验表明,经过AI及Cyp19a1-shRNA处理后,ZW胚胎性腺皮质区退化,髓质区高度发育,形成原始性索结构,呈现雌性向雄性性逆转现象;同时雌性标记基因Foxl2的表达量下调,雄性标记基因Amh的表达量则显著上调。功能获得实验则表明,Cyp19a1过表达后的ZZ型性腺皮质区高度发育,髓质区退化,形成空洞结构,呈典型雌性特征,同时Foxl2基因的表达量上升,而Amh的表达量显著降低。免疫组化结果显示,雄性标记蛋白Sox9在对照组ZZ雄性性腺的髓质区大量表达;在对照组ZW雌性性腺中未见表达。而在Cyp19a1敲低后的ZW性腺中,Sox9蛋白在髓质区大量表达;相反,Cyp19a1过表达后的ZZ性腺中则未见Sox9蛋白表达。(2)Foxl2的功能研究我们还通过慢病毒载体介导的过表达系统,进行了中华鳖Foxl2基因的功能验证分析。结果表明,Foxl2的过表达导致了ZZ雄性性腺向雌性性腺逆转,皮质区高度发育,但未见明显的卵泡或生殖细胞,髓质区退化形成空洞结构,性腺呈典型雌性性腺特征。荧光定量PCR结果表明,经过表达处理后的ZZ性腺,雄性标记基因Dmrt1和Sox9的表达急剧下调,雌性特异性基因Cyp19a1的表达显著上调。免疫组化结果显示,Foxl2过表达后的ZZ性腺中,Sox9蛋白信号显著降低,甚至未见表达。本文揭示了Cyp19a1基因在性腺分化过程中的表达规律,发现了Cyp19a1的敲低和过表达以及Foxl2的过表达能导致性逆转的发生,明确了Cyp19a1和Foxl2在中华鳖雌性性腺分化过程中是必需的,是早期卵巢分化的关键调控因子。本研究开辟了龟鳖类动物基因功能研究的新途径,为中华鳖性别决定和性腺分化的机理研究奠定基础;同时为中华鳖全雄苗种培育提供了一个新策略,即通过过表达Cyp19a1或Foxl2获得ZZ型雌性个体,ZZ假雌与ZZ雄性个体进行交配产生全雄子代。
[Abstract]:Aromatase, as a restriction enzyme for estrogen synthesis, plays an important role in regulating early female sex differentiation in non-mammalian vertebrates. Cyp19a1 gene is the encoding gene of aromatase. Foxl2 gene is one of the members of the forked frame transcription factor family, which plays an important role in the early female gonadal differentiation and ovarian function maintenance. The results show that the Foxl2 gene is located upstream of Cyp19a1 and regulates the expression of aromatase gene. Both of them are involved in the process of female gonadal differentiation. In this study, the expression of aromatase encoding gene Cyp19a1 in female and male gonads in different tissues of Chinese soft-shelled turtle (Trionyx sinensis) and in different stages of embryonic development was analyzed, and RNA interference / overexpression mediated by aromatase inhibitor and lentivirus was used. The functional verification of Cyp19a1 and Foxl2 genes was emphasized. The results were as follows: (1) the functional study of Cyp19a1 showed that the expression of Cyp19a1 gene was highly specific in the adult ovarian tissues of Trionyx sinensis, testis and heart, liver, spleen, lung, kidney, muscle, testis and heart, liver, spleen, lung, kidney, muscle, testis, heart, liver, spleen, lung, kidney and muscle. The expression is weak or not in the intestine. At the same time, we found that Cyp19a1 gene was specifically expressed in female gonad at the 18th stage, earlier than the initiation time of gonad differentiation (phase 19). After AI and Cyp19a1-shRNA treatment, the gonadal cortex of ZW embryos degenerated, the medulla developed highly, and the primitive cord structure was formed, which showed the reverse phenomenon of female to male sex, and the expression of Foxl2, the female marker gene, was down-regulated. The expression of male marker gene Amh was significantly up-regulated. The results of functional acquisition showed that the ZZ type gonadal cortex was highly developed after overexpression of Cyp19a1, the medulla region degenerated, forming a typical female structure, and the expression of Foxl2 gene increased, while the expression of Amh decreased significantly. Immunohistochemical results showed that the male marker protein Sox9 was highly expressed in the medulla of the male gonad of the control group, but not in the female gonad of the control group. However, in the ZW gonads after Cyp19a1 knockout, the expression of Sox9 protein was found in the medullary region, but not in the ZZ gonads after the overexpression of Cyp19a1. (2) the function of Foxl2 was also studied by the lentivirus vector mediated overexpression system. The functional analysis of Foxl2 gene in Trionyx sinensis was carried out. The results showed that the overexpression of Foxl 2 resulted in the reversal of male gonad to female gonad in ZZ, the development of cortical area was high, but there were no obvious follicles or germ cells, the medullary region degenerated into cavity structure, and the gonad was typical female gonad. The results of fluorescence quantitative PCR showed that the expression of male marker gene Dmrt1 and Sox9 was down-regulated sharply and the expression of female specific gene Cyp19a1 was significantly up-regulated in the ZZ gonad after expression treatment. The results of immunohistochemistry showed that the signal of Sox9 protein in the gonad of ZZ after overexpression of Foxl 2 was significantly decreased, even no expression was found. The expression of Cyp19a1 gene in the process of gonadal differentiation was revealed. It was found that the low and overexpression of Cyp19a1 and the overexpression of Foxl2 could lead to sexual reversal. It was clear that Cyp19a1 and Foxl2 were necessary in the process of female gonad differentiation of Trionyx sinensis. It is a key regulator of early ovarian differentiation. This study opened up a new way to study the gene function of turtle and turtle, which laid a foundation for the study of sex determination and gonadal differentiation of Trionyx sinensis, and provided a new strategy for the breeding of the whole male species of Trionyx sinensis. By overexpression of Cyp19a1 or Foxl2, ZZ pseudofemales of ZZ type were obtained to mate with ZZ males to produce full male offspring.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
[Abstract]:Aromatase, as a restriction enzyme for estrogen synthesis, plays an important role in regulating early female sex differentiation in non-mammalian vertebrates. Cyp19a1 gene is the encoding gene of aromatase. Foxl2 gene is one of the members of the forked frame transcription factor family, which plays an important role in the early female gonadal differentiation and ovarian function maintenance. The results show that the Foxl2 gene is located upstream of Cyp19a1 and regulates the expression of aromatase gene. Both of them are involved in the process of female gonadal differentiation. In this study, the expression of aromatase encoding gene Cyp19a1 in female and male gonads in different tissues of Chinese soft-shelled turtle (Trionyx sinensis) and in different stages of embryonic development was analyzed, and RNA interference / overexpression mediated by aromatase inhibitor and lentivirus was used. The functional verification of Cyp19a1 and Foxl2 genes was emphasized. The results were as follows: (1) the functional study of Cyp19a1 showed that the expression of Cyp19a1 gene was highly specific in the adult ovarian tissues of Trionyx sinensis, testis and heart, liver, spleen, lung, kidney, muscle, testis and heart, liver, spleen, lung, kidney, muscle, testis, heart, liver, spleen, lung, kidney and muscle. The expression is weak or not in the intestine. At the same time, we found that Cyp19a1 gene was specifically expressed in female gonad at the 18th stage, earlier than the initiation time of gonad differentiation (phase 19). After AI and Cyp19a1-shRNA treatment, the gonadal cortex of ZW embryos degenerated, the medulla developed highly, and the primitive cord structure was formed, which showed the reverse phenomenon of female to male sex, and the expression of Foxl2, the female marker gene, was down-regulated. The expression of male marker gene Amh was significantly up-regulated. The results of functional acquisition showed that the ZZ type gonadal cortex was highly developed after overexpression of Cyp19a1, the medulla region degenerated, forming a typical female structure, and the expression of Foxl2 gene increased, while the expression of Amh decreased significantly. Immunohistochemical results showed that the male marker protein Sox9 was highly expressed in the medulla of the male gonad of the control group, but not in the female gonad of the control group. However, in the ZW gonads after Cyp19a1 knockout, the expression of Sox9 protein was found in the medullary region, but not in the ZZ gonads after the overexpression of Cyp19a1. (2) the function of Foxl2 was also studied by the lentivirus vector mediated overexpression system. The functional analysis of Foxl2 gene in Trionyx sinensis was carried out. The results showed that the overexpression of Foxl 2 resulted in the reversal of male gonad to female gonad in ZZ, the development of cortical area was high, but there were no obvious follicles or germ cells, the medullary region degenerated into cavity structure, and the gonad was typical female gonad. The results of fluorescence quantitative PCR showed that the expression of male marker gene Dmrt1 and Sox9 was down-regulated sharply and the expression of female specific gene Cyp19a1 was significantly up-regulated in the ZZ gonad after expression treatment. The results of immunohistochemistry showed that the signal of Sox9 protein in the gonad of ZZ after overexpression of Foxl 2 was significantly decreased, even no expression was found. The expression of Cyp19a1 gene in the process of gonadal differentiation was revealed. It was found that the low and overexpression of Cyp19a1 and the overexpression of Foxl2 could lead to sexual reversal. It was clear that Cyp19a1 and Foxl2 were necessary in the process of female gonad differentiation of Trionyx sinensis. It is a key regulator of early ovarian differentiation. This study opened up a new way to study the gene function of turtle and turtle, which laid a foundation for the study of sex determination and gonadal differentiation of Trionyx sinensis, and provided a new strategy for the breeding of the whole male species of Trionyx sinensis. By overexpression of Cyp19a1 or Foxl2, ZZ pseudofemales of ZZ type were obtained to mate with ZZ males to produce full male offspring.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
【参考文献】
相关期刊论文 前10条
1 蔡晗;孙伟;刘豪彬;包海声;张海艳;钱国英;葛楚天;;中华鳖Amh基因的克隆、表达及在雄性分化中的功能初探[J];中国科学:生命科学;2016年12期
2 张一;张海艳;张辉皇;江航;王银瑜;俞罗浩;钱国英;葛楚天;;Dmrt1基因在红耳龟温度依赖型性别决定中的功能研究[J];中国科学:生命科学;2016年06期
3 孙伟;史思瑞;蔡晗;宓猛冬;钱国英;葛楚天;;中华鳖Dmrt1基因在雄性性别分化中的功能分析[J];中国科学:生命科学;2015年09期
4 唐伟;洪p喎，
本文编号：2211836
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2211836.html
最近更新
教材专著
