【作者】 韩凤桐；

【导师】 刘娣；

【作者基本信息】 东北农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 Sry(sex-determining region of the Y chromosome)基因主导哺乳动物雄性性别决定和睾丸的起始发育。转基因鼠证明Sry可以诱导XX型小鼠向雄性发育。Sry蛋白含有一段可以结合DNA的区域,被称为HMG-box。HMG-box在哺乳动物不同物种间相对保守,人SRY HMG-box区域相应位置的点突变可以引起性别反转。对人SRY的亚细胞定位研究结果表明其分布在Sertoli细胞细胞核内,推测其发挥转录因子作用。目前,Sry的表达调控机制依然不清楚。鼠Sry在XY型胚胎生殖嵴Sertoli前体细胞中表达,并受到严格的时空限制,但在其他物种并不十分严格,如人、羊在成年雄性的睾丸内依然可以检测到其表达。虽然已证明Sry具有诱导睾丸发育的功能,但其下游目的基因仍然无法确定。Sox9是Sry可能的下游基因之一。Sf1 (Steroidogenic fator-1)、Gata4 (GATA binding protein 4)、Wt1 (Wilms’tumor gene)、Sox9(Sry-related HMG box-9)、Amh (Anti Mullerian Hormone)和Dax1 (Dosage sensitive sex reversal locus-1)也被证明在性别决定过程中有重要作用。目前关于哺乳动物Sry基因的大量研究主要集中在人和鼠上,在其他动物上的研究较少。牛作为重要的经济动物,在畜牧业生产中具有重要价值,奶牛繁殖时产生的半数雄性牛,经济价值很低,目前采用的性别控制技术,主要是依据X精子和Y精子DNA含量上的微小差别,以流式细胞技术分离,存在价格昂贵,分离效率低,及所分离的精子受精率低等问题。阐明牛性别控制相关基因的调控模式,有希望在分子水平上干扰性别决定的调控网络,可以为奶牛性别控制提供新的思路;也可为哺乳动物性别分化的比较生物学研究填补牛相关数据的空白。本试验对牛Sry基因进行了结构与功能的研究,主要内容包括:克隆了牛Sry基因包含5’侧翼和编码区在内的1838bp序列;原代分离培养牛生殖嵴细胞及卵巢颗粒细胞,并对生殖嵴细胞进行性别鉴定及特征鉴定;利用生物信息学方法预测牛Sry 5’侧翼1066bp内潜在转录起始位点TSS及转录因子结合位点,比较10个物种SRY/Sry蛋白同源性及趋异分化程度,并进行物种进化分析,比较牛及人SRY蛋白的三维结构;构建不同长度的缺失牛Sry 5’侧翼序列调控的报告基因载体,利用荧光素酶双报告基因分析系统在生殖嵴细胞内研究牛Sry核心启动子区域位置;构建了pcDNA3.1-Sry表达载体,在生殖嵴细胞和卵巢颗粒细胞中进行了Sry过表达,利用半定量RT-PCR对性别控制相关基因在Sry过表达前后的表达水平变化进行检测,包括Sf1、Gata4、Wt1、Sox9、Amh和Dax1;构建pEGFP-N1-Sry亚细胞定位表达载体,其可以表达融合蛋白(Sry-GFP),在牛生殖嵴细胞和卵巢颗粒细胞中进行了Sry亚细胞定位研究。主要结论如下:1.克隆了牛Sry基因包括5’侧翼及编码区(ORF)在内的1838bp序列,通过测序及序列比对,结果和GenBank中牛Sry序列(No. AB039748.1)完全一致。2.原代分离培养牛生殖嵴细胞,对其进行鉴定表明,此细胞为处于性别分化时期的雄性生殖嵴细胞,在体外生长状态良好,可以稳定传代,为后继研究奠定了基础。3.原代分离培养牛卵巢颗粒细胞,经过在体外传代观察,细胞形态均一,在体外生长状态良好,可以稳定传代,为后继研究奠定了基础。4.对牛Sry5’端1066bp序列的生物信息学分析发现,分别在-93、-419、-722(ATG中A为0点)位置存在三个潜在的转录起始点(TSS),而且这段区域含有非常丰富的转录因子结合位点信息,这和Sry基因表达的特异性、复杂性相一致。5. 10个物种SRY/Sry蛋白序列比较结果表明,牛和山羊Sry蛋白同源性最高,达到93.5%;牛和小鼠Sry蛋白趋异分化程度最高,达60.4%。6.进化分析表明牛与羊在亲缘关系上最为接近,归为一类,在比较的10个物种中是处于进化最远的亚类,表明偶蹄目动物Sry基因在进化上受自然选择的影响有较大的变异。7.比较牛和人SRY蛋白结构,仅在保守区HMG-box处有较高的相似性,但是空间构象却比较相似,都是通过结合DNA螺旋小沟来识别7个碱基的核心靶序列,作用方式也很相似,说明虽然SRY/Sry蛋白在物种间序列差异较大,但蛋白的结构和作用方式却是很保守的。8.在生殖嵴细胞内,利用双荧光素酶报告基因检测系统,用13个缺失表达载体,对牛Sry5’侧翼1056bp启动子区域进行了细致扫描,结果表明牛Sry核心启动子位于翻译起始位点ATG(A为0点)上游-599~-565bp一段35bp大小的区域内。9.利用pcDNA3.1-Sry表达载体在牛生殖嵴细胞内过量表达Sry,用RT-PCR检测性别发育相关基因Sry过表达前后表达水平变化,包括Sf1、Gata4、Wt1、Sox9、Amh和Dax1,结果证明牛Sry对Sox9表达有正调控作用,而其他基因不受Sry过表达影响。10.利用pEGFP-N1-Sry融合蛋白表达载体(Sry-GFP)在牛生殖嵴细胞和卵巢颗粒细胞中研究Sry的细胞内分布,结果证明牛Sry蛋白定位于细胞核内,进一步证实其作为转录因子发挥作用。创新点:1.首次利用生物信息学方法对牛Sry基因作全面的生物信息学分析,如结论4-7条,预测分析对牛Sry基因启动子潜在转录起始位点和转录因子结合位点;比较10个物种Sry蛋白序列同源性和分化程度;利用10个物种的Sry序列,对牛Sry基因进行进化分析;对比牛和人的SRY蛋白空间结构。2.首次细致分析了牛Sry基因启动子区域,并将核心启动子定位于翻译起始位点ATG(A为0点)上游-599~-565bp一段35bp大小的区域内。3.首次证明牛Sry基因对Sox9基因有正调控作用,证明牛Sry基因可能直接或间接调控Sox9表达。4.首次利用实验证实牛Sry蛋白定位于细胞核。更多还原

【Abstract】 In mammals, male sex determination and initiating testis development are under the control of the Sry (sex-determining region of the Y chromosome). In 1991, Koopman reported that Sry gene can direct male development in an XX transgenic mouse. Sry protein contains a DNA binding domain known as the HMG-box. The HMG box is the singular part of Sry conserved between species, and point mutations in the corresponding region of the SRY gene cause sex reversal in humans. In human, SRY protein was detected in the nuclei of Sertoli cells, and was speculated that it worked as a transcriptional factor.Although the testis-inducing function of Sry has been clearly demonstrated, its direct target gene or genes still await identification. As one likely Sry target, the Sry-related gene Sox9 has emerged. Most other genes which are involved in this complex process and play important roles in male sex differentiation have been identified, including Sf1 (Steroidogenic fator-1), Gata4 (GATA binding protein 4), Wt1 (Wilms’tumor gene), Sox9 (Sry-related HMG box-9), Amh (Anti Mullerian Hormone), and Dax1 (Dosage sensitive sex reversal locus-1). At present, almost all the researches about mammals are focus on human and mouse, and there are fewer researches on other animals.Cattle is thought to be an important economic animal, it is great valuable in animal husbandry. The male calves are valueless. At present, the sex control technologies being used in cow production is separation of X and Y sperms. Its disadvantages are expensive, low efficiency, reduction of fertility rate, and so on. Researches on molecular regulation mechanism about sex determination, finding the method to interfere the regulation net of sex determination can provide a new means for cow sex control. The researches among different species could also help us to further understand the mechanisms of sex determination and the evolutionary rules in mammalian sex determination.In this research, we cloned the 1838bp Sry sequences, including the 5’flanking and ORF. Bovine genital ridge cells and ovary granulosa cells were isolated and cultured in vitro. We identified the sex and characteristic of the genital ridge cells. The information of Sry 5’flanking and ORF were analyzed by bioinformatics method. The potential transcriptional start site and transcriptional factor binding sites in Sry 5’flanking 1066bp were predicted. To analyzing the evolution of bovine Sry, we compared Sry protein sequences among 10 species. We also predicted and compared the three-dimensional structure SRY/Sry between human and bovine. To find the core promoter of bovine Sry, We constructed 13 deletion recombinants with different length of Sry 5’ flanking, promoter activity analysis was performed in bovine genital ridge cells by the dual-luciferase reproter assay system. Sry gene was overexpressed by pcDNA3.1-Sry in genital ridge cells and granulosa cells, after that six genes which were identified as important genes in sex determination in mammals, including Sf1, Gata4, Wt1, Sox9, Amh and Dax1 were examined by semi-quantitative RT-PCR to observe their expression change influenced by Sry. Subcellular localization was also performed to analyze the disposition of Sry protein in genital ridge cells and granulosa cells. We got the conclusion below:1. The 1838bp Sry sequences, including the 5’flanking and ORF, were cloned and secquenced. Blast anslysis indicated that the sequences were consistent with bovine Sry sequences in GenBank(No. AB039748.1).2. Bovine genital ridge cells were isolated and cultured in vitro, and it were identified that it came from a male genital of sex differentiation period; they could grow normally in vitro, and be passaged stablely.3. Bovine ovary granulosa cells .were isolated cultured in vitro, they could grow normally in vitro, and be passaged stablely.4. The bovine Sry 5’flanking sequences of 1066bp were analyzed by bioinformatics method, it were indicated that there were 3 potential transcriptional start sites at -93、-419、-722(ATG, A was 0 point), and the transcriptional factor binding sites (TFBS) prediction indicated that there were copious potential TFBS in the fragment, which was consistent with the special complicated regulation mechanism of Sry.5. Comparison of 10 species SRY/Sry sequences showed that bos taurus was more homologous to capra hircus(93.5%); bos taurus was more divergent from mus musculus (60.4%).6. Evolution analysis of SRY/Sry sequences of 10 species showed bos taurus and capra hicus were more relative, and belonged to a group, they are reach to the distal end of the cladogram, indicated that artiodactylous animals has greater variation than others by means of natural selection.7. Comparison of SRY/Sry primary structure of human and bovine, the HMG-box was the singular conserved part, but they were similar in the three-dimensional structure. They both can recognize the 7bp conserved binding site sequences. It indicated that although the Sry primary structure has greater variation among different species, the three-dimensional structure and mode of action were similar.8. We constructed 13 deletion recombinants with luciferase reporeter, the promoter acativity of bovine Sry 5’flanking 1056bp was analyzed by dual-luciferase reproter assay system in genital ridge cells, the core promoter of bovine Sry was located at the -599~-565bp upstream of Sry translation start site ATG (A is 0 site.), a fragment of 35bp. 9. Sry was overexpressed in genital ridge cells by pcDNA3.1-Sry, after that six genes which were identified as important genes in sex determination in mammals, including Sf1, Gata4, Wt1, Sox9, Amh and Dax1 were examined by semi-quantitative RT-PCR to observe their expression change influenced by Sry. The results indicated that Sry can up-regulate the Sox9 expression.10. Subcellular localization was performed to analyze the disposition of Sry protein in genital ridge cells and granulosa cells. Bovine Sry protein localizated in the cell nuclei, it confirmed that bovine Sry working as transcription factor.更多还原