Cyp19a1和Foxl2基因在中華鱉早期卵巢分化中的功能研究
發(fā)布時(shí)間：2018-08-29 16:59
【摘要】：芳香化酶作為雌激素合成的限制酶,在非哺乳類脊椎動(dòng)物的早期雌性性別分化中起重要的調(diào)控作用,Cyp19a1基因作為芳香化酶的編碼基因,參與了龜鱉類的雌性性別分化過程。Foxl2基因是叉頭框轉(zhuǎn)錄因子家族成員之一,在雌性早期性腺分化和卵巢功能維持中扮演重要角色。現(xiàn)有研究結(jié)果表明,Foxl2基因位于Cyp19a1的上游,調(diào)控芳香化酶基因的表達(dá),二者共同作用參與雌性的性腺分化過程。本研究分析了芳香化酶編碼基因Cyp19a1在中華鱉成體不同組織及胚胎發(fā)育不同時(shí)期雌雄性腺中的表達(dá)情況;利用芳香化酶抑制劑和慢病毒介導(dǎo)的RNA干擾/過表達(dá)技術(shù),著重進(jìn)行Cyp19a1和Foxl2基因的功能驗(yàn)證研究。具體研究結(jié)果如下:(1)Cyp19a1的功能研究半定量PCR和實(shí)時(shí)熒光定量PCR結(jié)果顯示Cyp19a1基因在中華鱉成體卵巢組織中高度特異性表達(dá),睪丸和心、肝、脾、肺、腎、肌、腸中表達(dá)微弱或不表達(dá)。同時(shí)我們發(fā)現(xiàn),Cyp19a1基因在第18期就呈現(xiàn)雌性性腺特異性表達(dá),早于性腺分化啟動(dòng)時(shí)間(第19期)。功能缺失實(shí)驗(yàn)表明,經(jīng)過AI及Cyp19a1-shRNA處理后,ZW胚胎性腺皮質(zhì)區(qū)退化,髓質(zhì)區(qū)高度發(fā)育,形成原始性索結(jié)構(gòu),呈現(xiàn)雌性向雄性性逆轉(zhuǎn)現(xiàn)象;同時(shí)雌性標(biāo)記基因Foxl2的表達(dá)量下調(diào),雄性標(biāo)記基因Amh的表達(dá)量則顯著上調(diào)。功能獲得實(shí)驗(yàn)則表明,Cyp19a1過表達(dá)后的ZZ型性腺皮質(zhì)區(qū)高度發(fā)育,髓質(zhì)區(qū)退化,形成空洞結(jié)構(gòu),呈典型雌性特征,同時(shí)Foxl2基因的表達(dá)量上升,而Amh的表達(dá)量顯著降低。免疫組化結(jié)果顯示,雄性標(biāo)記蛋白Sox9在對(duì)照組ZZ雄性性腺的髓質(zhì)區(qū)大量表達(dá);在對(duì)照組ZW雌性性腺中未見表達(dá)。而在Cyp19a1敲低后的ZW性腺中,Sox9蛋白在髓質(zhì)區(qū)大量表達(dá);相反,Cyp19a1過表達(dá)后的ZZ性腺中則未見Sox9蛋白表達(dá)。(2)Foxl2的功能研究我們還通過慢病毒載體介導(dǎo)的過表達(dá)系統(tǒng),進(jìn)行了中華鱉Foxl2基因的功能驗(yàn)證分析。結(jié)果表明,Foxl2的過表達(dá)導(dǎo)致了ZZ雄性性腺向雌性性腺逆轉(zhuǎn),皮質(zhì)區(qū)高度發(fā)育,但未見明顯的卵泡或生殖細(xì)胞,髓質(zhì)區(qū)退化形成空洞結(jié)構(gòu),性腺呈典型雌性性腺特征。熒光定量PCR結(jié)果表明,經(jīng)過表達(dá)處理后的ZZ性腺,雄性標(biāo)記基因Dmrt1和Sox9的表達(dá)急劇下調(diào),雌性特異性基因Cyp19a1的表達(dá)顯著上調(diào)。免疫組化結(jié)果顯示,Foxl2過表達(dá)后的ZZ性腺中,Sox9蛋白信號(hào)顯著降低,甚至未見表達(dá)。本文揭示了Cyp19a1基因在性腺分化過程中的表達(dá)規(guī)律,發(fā)現(xiàn)了Cyp19a1的敲低和過表達(dá)以及Foxl2的過表達(dá)能導(dǎo)致性逆轉(zhuǎn)的發(fā)生,明確了Cyp19a1和Foxl2在中華鱉雌性性腺分化過程中是必需的,是早期卵巢分化的關(guān)鍵調(diào)控因子。本研究開辟了龜鱉類動(dòng)物基因功能研究的新途徑,為中華鱉性別決定和性腺分化的機(jī)理研究奠定基礎(chǔ);同時(shí)為中華鱉全雄苗種培育提供了一個(gè)新策略,即通過過表達(dá)Cyp19a1或Foxl2獲得ZZ型雌性個(gè)體,ZZ假雌與ZZ雄性個(gè)體進(jìn)行交配產(chǎn)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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4
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