Cloning And Expression Analysis Of Aromatase? Sox9 And Sf1 Genes Of Gekko Japonicus

Temperature-dependent sex determination(TSD)in reptiles is one of the most fascinating phenomena that has received a great deal of attentions.Temperature can regulate the level of estrogen and the expression of gender specific genes in TSD.We chose Gekko japonicus,a lizard with TSD,as our research object.Aromatase?Sox9 and Sf1 were chosen as sex determination candidate genes in this study.At first,we cloned full-length cDNA sequence of Aromatase?Sox9 and Sf1 genes.Subsequently,we detected the mRNA expression of Aromatase?Sox9 and Sf1 genes.Through qRT-PCR,we compared the expression level of the three genes in different tissues and in different developmental stages between sexes,which supplies the basis for the research of the molecular mechanisms of sex determination and sex differentiation in G.japonicus.The main results are as follow:1.RACE-PCR was used to obtain the full lenth cDNA sequences of Aromatase?Sox9 and Sf1 genes.The length of three genes are 1908 bp,1895bp,and 2699 bp,encoding these as amino acids 506,494 and 509.Their molecular weight are 57.84kDa?55.29 kDa and 30.94 kDa.They all contains 20 kinds of essential amino acids,with a considerable difference on the composition of amino acid.The proteins encoded by Aromatase,Sox9 and Sf1 genes were found to be hydrophilic proteins with no signal peptides.We found two conserved structure domains of Sox9 and Sf1.Aromatase gene exists in a cytochrome P450 structure domain,with a long 461 amino acid sequence.Integrated results of multiple comparison,amino acid homology analysis and phylogenetic tree,We found Aromatase,Sox9 and Sf1 genes in the process of vertebrates evolution is relatively conservative.2.Three genes mRNA expression pattern were detected in different tissues and in different developmental stages of both gonads with qRT-PCR.The experimental results showed that genes expression of Aromatase,Sox9 and Sf1 are tissue specificity.(1)Expression ofAromatase gene in ovary was significantly higher than that in testis and other tissues;Expression of non-reproduction period was higher than reproduction period in ovary,with no difference in testis;Expression in the adults and the immatures were higher than than the jureniles in ovary.Expression in the adults period was higher than other periods in testis.(2)Sox9 gene expression in testis of G.japonicus was significantly higher than that in ovary;Expression of non-reproduction period was higher than reproduction period in both gonads;It presented higher expression significantly in the adults and the immatures than the jureniles in testis,and higher expression in the jureniles and the immatures than the adults in ovary.(3)Sf1 gene has a certain amount of expression in different tissues and Sf1 gene expression in testis was significantly higher than that in ovary;Expression of non-reproduction period is higher than reproduction period in both gonads;It presents sustainable growth from the jureniles to the adults in testis,with the highest expression in the adults.We speculated that Aromatase gene may be involved in the ovarian development and maturation process of G.japonicus,while Sox9 and Sf1 gene may participate in the testicular development and maturation process.