Cloning And Tissue Expression Analysis Of CDNA Encoding Sox3?Sox5 Gene In Chinese Giant Salamander (Andrias Davidianus)

The transcription factor of Sox(Sry-related HMG box)family is a large gene family that associated with the Sry(sex determination region of Ychromosome),and its members contain a highly conserved group of high-mobility group(HMG-box)Binding domain.Sox gene is widely distributed across the animal kingdom and has been subdivided into groups A to K based on their sequence similarity and gene structure.So far,more than 100 members have been found in humans,mammals,birds,fishes and fruit flies.The Sox3 gene belongs to the SoxBl subfamily and is a single exon gene located on the X chromosome and plays a role in the development of embryonic urogenital ridge and the nervous system.The Sox5 gene belongs to the SoxD subfamily,and its encoded proteins play an important role in embryonic development,cell fate decisions,cartilage formation processes and spermatogenesis.In this study,Sox3 cDNA sequence(No.KY745789)and Sox5(No.KY745790)gene sequences are cloned by reverse transcription polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE),polymerase chain reaction(PCR),and their biological characteristics are also analyzed.Real-time PCR of analysis is performed to examine the expression in various tissues of the Chinese giant salamander.The results we abtained are as follow:1.The full-length cDNA sequence of Sox3 gene of Chinese giant salamander was 1396bp,including 5'UTR of 151bp and 3'UTR of 348bp,and 897bp of cds,encoding 298 amino acids which are provided with a-71aa-HMG-box from 35 to 105.NCBI Accession number is KY745789.The Sox3 protein of the Chinese giant salamander was compared with other vertebrate Sox3 protein by DNAMAN software.The results showed that the Sox3 protein had a highly-conserved H-box domain,and the H-box domain contained 71 amino acids.The similarity analysis showed that Sox3 protein of the Chinese giant salamander was similar to that of other vertebrate Sox3 protein.Nucleotide and amino acid sequences are highly similar to amphibians,and are similar to mammals.Phylogenetic tree analysis showed that Sox3 gene of Chinese giant salamander was highly similar in non-fish vertebrates,and the Chinese giant salamander and Cynops pyrrhogaster were in a branch and were in parallel branch nodes with amphibians,birds,reptiles,mammals and fish groups.2.The full-length cDNA sequence of Chinese giant salamander Sox5 gene was 2930bp,including 5'UTR of 159bp and 3'UTR of 446bp,and 2325bp of CDS which encoded 774 amino acids with a 71 aa HMG-box from 566 to 636.NCBI Accession number is KY745790.The Sox5 protein of the Chinese giant salamander was compared with other vertebrate Sox5 protein by DNAMAN software.The results showed that the Sox5 protein also had a highly conserved H-box domain,the H-box domain contained 51 amino acids,The similarity analysis showed that the nucleotide and amino acid sequences of Sox5 were similar to amphibians and reptiles,and the similarity was lower to that of mammals.The results of phylogenetic tree analysis showed the evolution of Sox5 in different species of Chinese giant salamander,in which vertebrates and invertebrates were clustered into one group.Among the large groups of vertebrates,fish are clustered together as a large group;Amphibians,birds,reptiles and mammals gathered as a large group,Among them Chinese giant salamander and birds,reptiles and mammals clustered into a subgroup,while the Xenopus laevis,nanorana parkeri were a separate branch,The results showed that the evolution rate of Sox5 gene was faster to that of other amphibians.3.The expression of Sox3 and Sox5 genes in Chinese giant salamander were analyzed by Real-time PCR.The results shows that the expression of Sox3 gene is particularly highly and lowly expressed in the testis and the ovary,respectively,which is significantly different from the expression of Sox3 gene in other species that have been reported.The expression of Sox5 gene is highest in testis of immature male,but the expression is relatively low.In various tissues of mature males,the expression is highest in kidney and ovary of mature female.The expression have largest difference in the testis,compared mature male to immature male.Expression of Sox5 gene in the pancreas did not show any significant differences between male and female.