Cloning Of Sox2and Sox19Gene Full CDNA And Their Tissue Expressing Analysis In Andrias Davidianus

Chinese giant salamander is one of the largest amphibians on the earth. It is an endemic rare species in china which has been list in nationally protected animals at two class, and in appendix I of CITES convention. And it possesses a important value in science research, edible, medicine and ornamental. The Sox gene family is a family involving in individual development and sex differentiation. It has a highly conservation in evolution process. By studying of the Sox gene family, we not only can reveal the molecular mechanism of animal sex determination and differentiation, the regulation embryonic development and the regulatory mechanism in the process of a variety of tissues and organs development, but also can provide the molecular basis for the diagnosis and treatment of some diseases. In this paper, we clone the genes of giant salamander Sox2and Sox19, analyze their biological characteristics and study their organization and expression situation so as to set a foundation for the following further understanding of the structure and function of Sox2and Sox19genes in Chinese giant salamander; as well as lay the foundation for revealing the Chinese giant salamander system evolution, the molecular mechanism of sex determination and differentiation and the regulation of embryonic development.By using RACE technique, we amplify the full length cDNA of Chinese giant salamander Sox2gene, it is2233bp in length. And its opening reading frame is totally963bp, encods320amino acids. The online analytical results of full length cDNA of giant salamander Sox2gene on NCBI BEAST show that the similarity with Cynops pyrrhogaster and Notophthalmus viridescens is89%and88%respectively; that is between76%and82%among Xenopus laevis, Mus musculus, Homo sapiens, Equus caballus, Felis catus, Bubalus bubalis and Gallus gallus with Chinese giant salamander. By analyzing the protein coded by Sox2gene with the same method, it shows that the highest similarity with Cynops pyrrhogaster is92%; and that of it with Mus musculus, Xenopus laevis, Salmo salar, Trachinotus blochii, Homo sapiens, Equus caballus, Gallus gallus and some other species is between69%and80%. Based on the amino acids sequences of Sox gene, we construct the phylogenetic tree by Neighbor-joining method to repeat10,000times in MEGA5.0. The results show that they can be divided into three distinct clades. One clade consists of two species:Notophthalmus viridescens and Cynops pyrrhogaste. The fish are gathered together and formed a big clade. While Xenopus laevis and Xenopus tropicalis get together with mammals and birds and form another clade in different lower level branch. The results indicate that the evolutionary relationship of Notophthalmus viridescens and Cynops pyrrhogaster is closer which is line with the evolution of species.By using RACE technique, we amplify the full length cDNA of Chinese giant salamander Sox19gene which is1290bp in length. And its opening reading frame is totally858bp, encods285amino acids. The online analytical results of full length cDNA of giant salamander Sox19gene in NCBI BLAST show that the similarity with Takifugu rubripes, Hippoglossus and M.musculus is85%,87%and8%respectively. By using the same method to analyze the protein encoded by the salamander Sox19gene, the result shows that the similarity with Takifugu rubripes, Danio rerio, Dicentrarchus labrax and M.musculus is55%,58%,64%and12%respectively. Based on the amino acids sequences of Sox19gene, we construct the phylogenetic tree. The results show that they can be divided into three distinct clades. The salamander becomes a group alone; the fish get together become a big group and the mus musculus forms a group alone. The analysis result shows that the evolutionary relationship of salamander and fish is much closer than mammals, which is basically agree with the species evolution process.This paper also makes the expression profile analysis in the Chinese giant salamander’s heart, kidney, ovaries and intestinal tissue on the basis of the Chinese giant salamander Sox2and Sox19gene’s cDNA sequence. The results show that the Chinese giant salamander Sox2gene’s protein expression quantity is highest in ovarian, followed in the kidney, Almost no expressed in intestinal tract. However the Chinese giant salamander Sox19gene’s protein expression quantity is highest in heart, followed in ovarian, the protein expression quantity is similar between kidney and intestinal tract, the protein expression quantity in kidney is higher than in intestinal tract.