Cloning And Function Prediction Of The Full-Length CDNA Of Porcine Sox9 Gene

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.