Cloning And Expression Of Sox9 From Andrias Davidianus

The Sox9 (SRY-related high mobility group-box gene9) is an important gene related to the embryo growth in early period, with a conserved HMG-box. Sox 9, one of the important Sox gene family, plays a key role in controlling Mammal's sex determination. Amphibian is a critical link in Biological phylogenic evolution from aquatic animal to Domestic animal. Andrias davidianus is the biggest Amphibian among the existed Amphibian. Therefore, the study of Sox9 gene will not only expound the evolution of sex determination mechanism from lower invertebrates to vertebrates, but also can offer the proof for studying the Sox9 evolution and determining sex and analyzing the function of Andrias davidianus.Designing the special primer based on the Sox9 HMG-box, which has been obtained, to get the Full-Length cDNA of Sox9 with the template of the ovary of Andrias davidianus. Its Full-Length is 1335 bp, including the 192 bp of 5'-UTR sequence,732bp of open reading frame and 411bp of 3'-UTR sequence. The open reading frame is 732 bp totally, which encodes 243 amino acids, on which the N region, from aa 104 to 174, has 71-amino acid (aa) protein as highly conservative HMG-box. Phylogenetic analysis further proved that it was Sox9 that we had obtained. Comparing the deduced amino acid sequence of Andrias davidianus Sox9 to that of nine species showed that their similarity is over 86%, which showed that the Sox9 is more conservative; we also analyzed the construction, characteristic of Andrias davidianus Sox9 sequence and its protein function adopting bioinformatics.In order to confer the function of protein, we studied six tissues expression of Andrias davidianus using RT-PCR technique. The tissues include ovary, heart, liver, spleen, spermary and muscle. The result showed that the expression was observed in ovary, spermary, heart and liver, but did not find in the tissues of muscle and spleen, which showed that the expression of Sox9 has no characteristic of special expression in these tissues, while expresses in some certain tissues, meanwhile, pointed out that Sox9 has taken in the development of these tissues. Sox9, which obtained in this experimentation, is related to the gonad development and differentiation because of expression happened in ovary and spermary.