【作者】 文爱韵；

【导师】 宋林生； 尤锋；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2010， 博士

【摘要】 本文首先克隆了牙鲆(Paralichthys olivaceus)dmrt1、dmrt4和sox9基因,连同已知的P450arom,研究了这4个基因在成鱼各组织的表达差异谱,明确了它们的两性差异表达模式,进一步定量和定位分析了这些基因在性腺分化和发育过程中的表达;并在体外对dmrt1基因进行了原核重组表达,同时从表观遗传学水平上研究了从头DNA甲基化转移酶基因dnmt3基因的表达差异图式以及性腺中性别相关基因启动子CpG岛的甲基化水平与这些基因两性表达差异的相关性。研究表明:牙鲆dmrt1基因cDNA全长3373bp,其5’UTR中存在与性别相关的转录因子Sry、Sox9和Sox5的结合位点,编码蛋白含有保守的DM结构域;dmrt4基因包含2个外显子和1个内含子,其5’UTR中有性别相关的转录因子Sox9的结合位点,编码产物含有保守的DM结构域和特有的DMA结构域;sox9基因包含3个外显子和2个内含子,在其5’UTR中发现了转录因子Ftz、Foxd3和Oct1的结合位点,编码蛋白含有保守的HMG结构域。牙鲆P450arom基因5’UTR中具有性别相关的转录因子Foxl2和Sf1的结合位点以及DM结构域的结合位点。组织特异表达研究显示,牙鲆dmrt1、dmrt4、sox9和P450arom均为两性差异表达的性别相关基因。dmrt1基因在牙鲆性腺中特异表达,且在精巢中的表达强于卵巢;dmrt4和sox9基因的表达范围相对较广,在性腺、脾脏、鳃、脑和胃等组织中都有不同程度的表达,且也都是在精巢的表达较卵巢的表达强;而P450arom基因在卵巢中的表达明显强于精巢,在其它一些组织中有强弱不同的表达。根据real-time RT-PCR结果可以看出,dmrt1基因在牙鲆原始性腺和性腺分化期间的表达都很低,在分化的精巢中表达强度迅速上升,而在卵巢中的表达却依然很弱。dmrt4则在原始性腺中高表达,其后的性腺分化期间表达降到很低的水平,然后在分化的精巢中强表达。sox9基因在牙鲆原始性腺中高表达,之后在性腺分化期间的表达呈现上下浮动的模式,在分化的精巢卵巢中具有不同程度的表达。P450arom在原始性腺中开始表达,其表达量在性腺分化期间有向上向下浮动的变化趋势,其后在分化的卵巢早期发育阶段表达很强。这4个基因在牙鲆各期性腺中的表达也各有不同,dmrt1在牙鲆I期性腺中表达很弱,II期精巢中表达量迅速上升,到了IV期精巢,其表达量有所下降,V期精巢中的表达量又上升而达到峰值;dmrt1在II期到V期卵巢的表达量却都极低。dmrt4基因在I期和II期精巢中表达很强,且呈上升趋势,其后在III、IV期精巢中的表达急剧下降,到了V期精巢,其表达量又略有回升;而其在各期的卵巢中表达一直很低。sox9在牙鲆I期精巢的表达量较卵巢的低,之后精巢中的表达开始上升,II期到V期精巢中该基因的表达量均较卵巢中的高,II期卵巢达到峰值,III到V期卵巢却几乎不表达。P450arom基因在I到V期卵巢的表达量均高于同期精巢的表达量,其中I期和II期的差异较大,这两个时期卵巢中的表达量很高,其后各期卵巢中的表达有所降低,而精巢中的表达量一直处于较低水平。性腺组织切片原位杂交定位表达分析可知,dmrt1、dmrt4和sox9基因的转录本主要定位于牙鲆精巢的精母细胞和Sertoli细胞,且在精母细胞中的表达强于Sertoli细胞的表达;在卵巢中,这3个基因的杂交信号都很弱。牙鲆P450arom基因则主要在卵巢的滤泡细胞和卵母细胞中表达,且滤泡细胞的表达强于卵母细胞中的表达,在精巢的精母细胞中却弱表达。整体原位杂交定位表达研究结果显示,在牙鲆胚胎发育过程中,dmrt4在外胚层来源的嗅基板,以及神经外胚层来源的前脑、端脑和耳基板中表达。从神经胚期到孵化期的所有时期,dmrt4基因在嗅基板的表达一直很强,而在端脑中的表达则存在动态变化。在胚胎发育的较早阶段,dmrt4的转录本分布于前脑中,并从神经胚期到尾芽形成期保持强表达;在早尾芽期,表达变得相对较弱,并逐渐局限于背侧端脑第一脑室周围的一小片区域。从5-6体节期到孵化期,低水平的转录本还可以在听基板中检测到。甲基化表观遗传修饰水平研究结果显示,牙鲆dmrt1、dmrt4和P450arom基因启动子CpG位点的甲基化水平存在两性差异: dmrt1启动子CpG位点在精巢中的甲基化水平为0,卵巢中的甲基化水平为57.69%,相应的,该基因在精巢的相对表达量约为卵巢的70倍。P450arom基因启动子CpG位点在卵巢的甲基化水平为73.33%,精巢中的甲基化水平为97.5%,该基因在卵巢的相对表达量约为精巢的40倍。dmrt4启动子CpG位点的甲基化水平在精巢中的甲基化水平(2%)较卵巢中的(7%)略低,这也与该基因在精巢中的表达强于卵巢的表达的两性差异表达模式是一致的。但是,sox9基因启动子CpG岛甲基化水平在精巢卵巢中却没有差异,都是完全去甲基化的,这与其在两性性腺中的差异表达图式不存在相关性。牙鲆从头DNA甲基化转移酶基因dnmt3存在两性差异表达,在精巢中的表达强于卵巢中的表达,在肾脏、脾脏、脑和眼睛中也有不同程度的表达。该基因在原始性腺表达较高,在性腺分化期间的表达上下浮动,在分化和发育的精巢卵巢中均具有两性表达差异。应用原核表达系统对Dmrt1蛋白进行体外重组表达:将牙鲆dmrt1基因序列插入到表达质粒pProEXTMHTa上,在大肠杆菌E.coli BL-21中进行体外表达与分析。该重组蛋白分子量约为32.5KDa,存在于宿主菌中,没有分泌到细胞外。诱导的菌体经超声破碎后,在沉淀和上清的样品中都有目的蛋白的条带存在,大部分的Dmrt1重组蛋白以包涵体形式出现在沉淀中,只有少量的蛋白以可溶形式存在于上清中。当诱导时间4 h、IPTG浓度0.6mmol/L时,重组蛋白表达量最大;在诱导温度为34℃时,表达的可溶蛋白的量比例最大。更多还原

【Abstract】 In the present study, we first cloned olive flounder (Paralichthys olivaceus) dmrt1, dmrt4 and sox9 genes. Together with olive flounder P450arom gene, their expression patterns in adult tissues were analyzed using convertional RT-PCR. The result identified these four genes’sexual dimorphic expression patterns. We further studied the expression of these genes during flounder gonadal differentiation and development through real-time RT-PCR and in situ hybridization. We also expressed flounder Dmrt1 fusion protein in vitro using E.coli BL-21(DE3) expression system. Meanwhile, we studied the methylation patterns of these four genes’promoter CpG island in testis and ovary at the epigenetic level.Flounder dmrt1’s total cDNA sequence is 3373bp, and its binding sites of sex-related transcriptional factors Sry, Sox9 and Sox5 were identified in the promoter region. The encoded protein contains a conserved DM domain. Flounder dmrt4 gene included two exons and one intron, of which promoter region contained binding site of sex-related transcriptional factor Sox9. A conserved DM domain and a specific DMA domain were found in the encoded protein. Flounder sox9 gene included three exons and two introns, and binding sites of transcriptional factors Ftz, Foxd3 and Oct1 were identified in its promoter region. This gene encoded a protein containing a conserved HMG domain. The 5’UTR of flounder P450arom was cloned, in which a DM domain binding site, two steroidogenic factor 1 (Sf1) binding sites and a forkhead-responsive consensus site (Foxl2) binding site were identified.The tissue specific expression patterns of dmrt1, dmrt4, sox9 and P450arom in adult flounder indicated that these four genes were all sex-related genes with sexual dimorphic expression. In detail, flounder dmrt1’s expression was restricted to gonads, and was higher expressed in testis than that in ovary. Both dmrt4 and sox9 were stronger in testis and weaker in ovary, and were also differentially expressed in some other tissues. P450arom was higher expressed in ovary than that in testis, and was expressed in some other tissues with varied degrees too. Results of real-time RT-PCR indicated that flounder dmrt1 was scarcely expressed in primitive gonad and during following periods of gonadal differentiation. But its expression went up rapidly in differentiating testis, and remained very low in differentiating ovary. Flounder dmrt4 was strongly expressed in primitive gonad, and much lower expressed during following periods of gonadal differentiation. Then its expression became stronger in differentiating testis. The sox9 was also highly expressed in flounder primitive gonad, much lower expressed after that and had some fluctuated expression during gonadal differentiation period. And then, it differentially expressed in differentiating testis and ovary. However, P450arom expressed in primitive gonad at first, and its expression quantity went upward and downward during following periods of gonadal differentiation. In the early stage of differentiating ovary, its expression became stronger.These four genes are also differentially expressed during five developmental stages of gonad. Flounder dmrt1 was weakly expressed in the testis and ovary of stage I, and its expression quantity went up rapidly in the testis of stage II. After that, the quantity went a little down in the testis of stage IV. Then its expression quantity reached the peak in the testis of stage V. From the stage II to the stage V, the expression quantity in ovaries were all scare. The dmrt4 gene was highly expressed in the testes of stage I and II with upward trend. Then its expression quantity went down significantly in the stage III and IV, but went up a little until stage V. The expression quantities in all the five stages of ovaries were always low. Flounder sox9 had lower expression quantity in the testis of stage I than in the ovary of stage I. And then, its expression in testis began to go up. From the stage II to V, the expression quantities were higher in testes than that in ovaries. Its expression quantities in ovary reached the peak in the stage II. From the stage III to V, there was almost no expression in the ovaries. P450arom gene had higher expression quantity in the testes from stage I to V than thoes in the ovaries. The difference of quantity was bigger in the stage I and II, in which the expression quantities of ovaries were very high. And after that, the expression in ovary went down. But its expression in testis was always weak.Gonad tissue slice in situ hybridization results showed that transcripts of dmrt1, dmrt4 and sox9 were mainly localized in spermatocytes and Sertoli cells of flounder testis, and the signal in spermatocytes was stronger than that in Sertoli cells, but there was weak signal detected in ovary. On the contrary, flounder P450arom mRNA was located in follicular cells and oocytes of ovary, and the signal in follicular cells was stronger than that in oocytes, and there was only a little signal detected in spermatocytes of testis. Whole mount in situ hybridization analysis showed that during flounder embryogenesis, flounder dmrt4 is expressed in the ectodermally derived olfactory placodes, neuroectodermal forebrain and telencephalon and otic placodes during embryogenesis. The expression level of dmrt4 is always high in the olfactory placodes for all these stages from neurula stage to the hatching stage, but it is dynamic in the telencephalon. While transcripts are distributed throughout the forebrain at early stages, remaining strongly expressed from the neurula stage to tail-bud forming stage, the expression domain becomes more and more restricted to a small area around the first ventricle in the dorsal telencephalon and is relatively weak at the early tail-bud stage.Results of genes’promoter CpG islands methylation patterns analyses showed that there were sexual dimorphic differences in the CpG methylation level of flounder dmrt1、dmrt4和P450arom promoters. No CpG demethylation of flounder dmrt1 promoter in testis and its 57.69% CpG island (CGI) methyaltion in ovary corresponded to this gene’s about 70 times higher expression in testis than that in ovary. On the other hand, 97.5% CGI methylation of flounder P450arom promoter in testis and its 73.33% CGI methyaltion in ovary correlated to this gene’s about 40 times lower expression in testis than that in ovary. The levels of flounder dmrt4 promoter CGI methylation in gonads were also sexual dimorphic. There were 2% CGI methylation of this gene promoter in testis and 7% methylation in ovary, which was consistent with its sexual dimorphic expression with higher expression in testis than that in ovary. However, there was no difference to be detected in the levels of flounder sox9 promoter CGI methylation in both gonad, and its sox9 promoter CpGs in testis and ovary were all totally demethylated, which was inconsistent with this gene’s differential expression pattern with higher expression in testis than that in ovary. Flounder de novo DNA (cytosine-5)-methyltransferases 3 (dnmt3) had a sexual dimorphic expression pattern that was higher expressed in testis than that in ovary. And it was also differentially expressed in kidney, spleen, brain and eye. Dnmt3 was highly expressed in flounder primitive gonad, and had some fluctuated expression during gonadal differentiation period. It was sexually dimorphic expressed during differentiation and development of testis and ovary.Flounder dmrt1 gene (encoded 293aa protein with molecular weight of 32.5 KDa) was recombined and fused on plasmid pPROEXTMHTa, and was transferred into E.coli BL-21(DE3) for Dmrt1 expression in vitro. The result of SDS-PAGE showed that fusion protein highly expressed inside the E.coli BL-21(DE3) cell after induction. Most fusion protein expressed in the form of inclusion bodies, and only a little fusion protein expressed in the form of soluble protein. The fusion Dmrt1 protein had the highest expression quantity after 4 h induction with 0.6mmol/L IPTG. When the induction temperature was 34℃, expression quantity of the soluble protein was the maximum.更多还原