Effects Of Sox 9α Gene Expression Patterns And OP On Expression Of Sox 9α In Testis

In order to explore the effects of Octylphenol on Sox9a which involved in larvae gonads differentiation and maintenance the function of adults sexual gland of Rana chensinensis. Firstly, we cloned the Sox9a in R.chensinensis adult testis, splicing all sequencing results with SeqMan; Using Blast, ORF, SignalP, ProParam, SMART, NPS@, SWISS-MODEL software to analyze the homology of spliced sequence and predict open reading frame, signal peptide, protein molecular weight and isoelectric point, functional domain, protein secondary and tertiary structures. Then, in order to analyze the evolutionary status of R.chensinensis Sox9a,12species of amphibians and reptiles were selected used for ClustalW2analysis, and constructing evolutionary trees with MEGA5.1. Secondly, The expression of Sox9a mRNA was examined in a wide range of tissues and organs, including adult testis, ovary, kidey, liver and brain of R.chensinensis, and the stage32-36tadpoles of R.chensinensis using Semi-quantity RT-PCR. Finally, the male adult of R.chensinensis were exposed to10-5,10"6,10-7,10-8,0mol/L OP, and the testicles were sampled at10d、20d and30d. The expression levels of Sox9a mRNA was examined in the testicles using Real-time Quantity PCR. The experimental and analytical results are as follows:1. Sox9a cDNA was first isolated from testicles, spanning1580bp and containing a1449bp open reading frame (ORF). The initatioin codon position of ORF was consisted with the initiation codon of Rana rugosa Sox9a. The deduced amino acid sequence encodes of482amino acids without frameshift reading phenomenon, sharing97%amino acids sequence identity with Rana rugosa Sox9a, showing85%with Alligator mississippiensis, and85%with Trachemys scripta. The sequence reported in this paper had been deposited in the GenBank data base (Accession No. KC442293). Analysing the structure and function of the deduced protein sequence, it had no N-terminal signal sequence, indicating that it’s a non-secreted protein. The molecular weight was54kDa, while the isoelectric point was6.32. The sequence contained a HMG-box domain (105-175), a BEN domain (75-147), a DnaJ domain (107-152), a GIT domain (120-150), a CG-1domain (237-276), a HSF domain (135-291), a PAH domain (376-414) and a DUF1518(304-354) domain. The secondary structure of the deduced protein sequence comprised17.39%α-helix,5.38%extended strand, and 77.23%free curled.2.The multiple sequences alignment and phylogenetic analysis by ClustalW2and MEGA5.1revealed that the amino acids sequence of R.chensinensis Sox9a were conserved in HMG-box domain. The12selected species were divided into3groups on the evolutionary relationships, Caudata, Anuran and reptiles. R.chensinensis and Rana rugosa belonged to one group which indicated that they are more relative on evolutionary relationships.3. The results of semi-quantity RT-PCR showed that Sox9a were expressed in adult testis, ovary, kidney, liver, and brain of R.chensinensis, significantly in testis and ovary; Sox9a also expressed in larvae of stage32-38, male and female of stage43-45and female of stage46of R.chensinensis. These results suggest that Sox9a likely to be involved in larvae gonads differentiation and maintenance the function of adult sexual gland of R.chensinensis.4. Comparing with the control group, the relative expression of Sox9a mRNA in each exposure groups was significantly down-regulated, which was examined in the testicles using Real-time Quantity PCR. In10d exposure group, OP induced an initial significantly down-regulated of Sox9a mRNA at10-8mol/L and thereafter clear dose-dependent decreases of Sox9a mRNA levels were observed. In20d exposure group, the result showed clear dose-dependent increases of Sox9a mRNA levels except10-8mol/L group. In30d exposure group, with the increase of concentration of OP, the relationship of Sox9a mRNA expression with the concentration of OP was irregular. Overall, OP exposure can down-regulated the Sox9a mRNA expression, but the regularity of Sox9a mRNA expression with the OP concentration and exposure time was not strong.5.In situ hybridization showed that the Sox9a mRNA positive expression signals exists in the spermatogenic cells of R.chensinensis.