【作者】 江玲霞；

【导师】 徐银学；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 Sox基因是由众多富含HMG盒DNA绑定蛋白区域的基因家族,Sox9属于Sox家族成员之一,Sox9是与早期胚胎发育有关的基因,是与位于雄性动物Y染色体上的Sry相关的睾丸决定因子,可与DNA序列特异结合,在很多动物中都有表达,因此在进化上非常保守。在人类SOX9突变可引起CD综合征和性反转。在睾丸和卵巢中都可以检验到Sox9mRNA6勺转录本,暗示它在性腺发育的作用,显示它在猪的性腺中可能具有同样功能。对猪Sox9基因的克隆测序和初步分析,可以为研究猪Sox9基因的生物学功能和品种改良提供基础的基因信息,也为研究人的基因功能建立动物试验模型提供参考。本研究采用电子克隆的方法,结合RT-PCR和RACE技术,获得了猪Sox9基因的cDNA全序列。利用生物信息学方法预测了该蛋白的结构和功能,并进行了同源序列多重比对和分子系统发生分析。利用实时定量PCR(Real-time quantitative PCR)技术分析了猪Sox9基因mRNA在不同组织的相对表达水平,具体内容如下:1 Sox9基因的克隆测序用人的Sox9基因的cDNA全序列作为电子探针,分别从NCBI的dbEST和UniGene数据库钓取了共12条猪的EST序列,将其输入DNAstar-SeqMan程序,最后拼接出1个重叠群(Contig1)序列。在此基础上,利用Primet Premier 5.0软件手动设计引物,借助RT-PCR和RACE技术一方面验证了电子克隆的正确性;另一方面扩增出ORF和cDNA全序列。2 Sox9蛋白的生物信息学分析借助借助生物信息学网络资源和软件,猪Sox9基因定位于12p13-p11,预测出完整的ORF编码框,位于第357-1886bp核苷酸之间,开放阅读框的长度为1530bp,编码509个氨基酸残基;通过对Sox9启动子的预测,可能性高于0.8的有10个启动子,从第187bp开始到第1541bp为CpG岛,长度为1355bp。CpG位点有151个;通过对蛋白质序列相似性检索,预测出猪Sox9蛋白不合有信号肽,不含有跨膜区,定位于细胞核中,在509个氨基酸中从第104到174位共71个氨基酸残基构成HMG盒,功能区主要是HMG盒。发现有HMG、DnaJ、GIT、HSF和Prim-Zn-Ribbon结构域。推测猪Sox9蛋白是一种核蛋白,有DNA结合能力,参与染色质修饰和基因转录调控。3 Real-time quantitative PCR分析Sox9基因的组织表达谱采用实时荧光定量PCR技术,按照双标准曲线法,分析了猪Sox9不同组织中的相对表达水平,结果表明:Sox9基因在猪的不同组织中广泛表达,但表达水平存在差异。在性腺和脑组织表达水平最高,而在脾、胸腺、淋巴组织表达水平最低,说明了Sox9基因在猪性腺、脑的发育以及软骨发生的功能与作用。更多还原

【Abstract】 Sox9 is a member of the Sox family of transcription factors and contains a DNA-binding motif known as the high-mobility-group(HMG) domain.Sox9 is a related factor about early embryo development.It is an important male animal Sry-related testis-determining gene.The encoded proteins are capable of binding to DNA in a sequence-specific manner.To date,SOX9 has been identified in many different species, thereby testifying to its conserved role inevolution.It has been shown that mutations in the human SOX9 gene cause campomelic dysplasia(CD) and autosomal sex reversa.The detection of Sox9 mRNA in both the testis and ovary of human suggests that this gene might be involved in the gonad development of pig.The cDNA cloning and preliminary analysis of porcine Sox9 gene not only provides primary information for better understanding the biological functions of Sox9 in pig,but also for the establishment of animal model to investigate human gene funtion.In this study,using in silico cloning approach combined with reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(5’ RACE and 3’ RACE),we cloned the full-length cDNA of Sox9.Bioinformatics methods are adopted to predict the structure and function of Sox9 protein,and also to construct a phylogenetic tree to reveal the evolutionary relationship of various species.To check related expression levels of Sox9 mRNA in various porcine tissues,the real-time PCR was performed.The contents are as followed:1 cDNA cloning and sequencing of porcine Sox9 geneThe full-length cDNA sequence of human SOX9 gene served as an electronic probe, which was submitted to generate BLAST reciprocal best hits from dbEST and UniGene database,and about 12 EST sequences were retrieved.Using DNAstar-SeqMan program, these ESTs were assembled into one contigs.Based on the contig,the gene specific primers were designed by Primer Premier 5.0.We carried out not only to cheek the validity of in silico approach,but also to obtain ORF and the full-length sequence of porcine Sox9 cDNA by RT-PCR and RACE-PCR.2 Bioinformatics analysis of Sox9 protein Using bioinformatics network resources and relevant softwares,the full-length cDNA of porcine Sox9 gene was firstly acquired,which localized on 12p13-p11 of porcine chromosome,we predict that Sox9 cDNA has a 1530bp open reading frame(ORF) from the 357bp site to the 1886bp site which coding a 509-amino acid(aa) residues.We find it could had 10 promoters and there is a big CpG island which from 187bp to 1541bp about 1355bp length containing 151 CpG sites.It contains no signal peptide,typical hydrophobic regions or transmembrane region,but the nucleoprotein residing in nucleolus.There is a HMG box which as main functional region contained of 71-amino acid(aa) residues from 104aa to 174aa of proteide sequence.A conserved domain database alignment reveals that the protein contains several conserved domains,including HMG,DnaJ,GIT,HSF and Prim-Zn-Ribbon domain.We speculate Sox9 encoded proteins are capable of binding to DNA in a sequence-specific manner,participating in chromatin modification and transcriptional regulation.3 Real-time PCR analysis of Sox9 mRNA tissue-specific expression profilesAccording to the double standard curve method,real-time fluorescence quantitative PCR was performed to analyze the relative expression levels in various tissues.The results showed that Sox9 gene was expressed broadly in various tissues,but at very different levels. The strongest expression was observed in pituitary,cartilage and gonad,whereas the lowest was seen in spleen,thymus and lymph,suggesting that Sox9 protein plays important roles in porcine gonad,brain and chondrogenesis development function.更多还原