Molecular Cloning,Analysis Of Sox17 And Cyp26A1 And The Influence Of Exogenous Hormone On Expression Of Sox17 And Cyp26A1 In The Pelodiscus Sinensis

Trionyx sinensis is one of the important economically aquatic animals in China.The growth difference between male and female individuals of T.sinensis can be observed.The male individuals have a faster growth rate with higher economic value.In recent years,a lot of research has been conducted on the sex regulation of the T.sinensis,but the molecular mechanisms of sex-determining of the T.sinensis are still unclearly.Lately,the RNA-Seq technology was used to screen out genes that related to sex differentiation in T.sinensis.In this study,the full-length cDNA sequences of T.sinensis Sox17 and Cyp26A1 were obtained by RACE technology,and the expression characteristics of Sox17 and Cyp26A1 in different tissues and different stages of embryo development were analyzed by qRT-PCR.Besides,the mRNA expression of Sox17 and Cyp26A1 in embryo of T.sinensis after17β-estradiol（E₂）and Letrozole（AI）treatment were detected.The full length of the Sox17 cDNA of T.sinensis was 3340 bp,including a 228bp5’-UTR,a 1894 bp 3’-UTR,and a 1218 bp open reading frame that encoded 405 amino acid residues.The Sox17 amino acid sequence showed as high as 94.07% similarity with Chrysemys picta bellii.Phylogenetic analysis showed that the T.sinensis is closely related to tortoises and birds,but farther from fish.qRT-PCR result revealed that Sox17 mRNA expression was the highest in ovary of adult T.sinensis,which was significantly higher than that of heart,liver,spleen,kidney,testis,intestine,muscle and brain tissue.The time of Sox17 mRNA expressed in embryo was consistent with the time of gonadal differentiation of T.sinensis.More,the expression level of Sox17 gradually increased from the gonadal differentiation,and then gradually returns to the initial level after reaching the maximum expression level at the 20 th stage.In addition,the study found that the expression of Sox17 in the gonads of pseudo-females（ZZ+E₂）was significantly up-regulated（p<0.05）compared with the control males（ZZ）,while the expression of Sox17 in pseudo-male（ZW+AI）gonads was significantly down-regulated compared with control female（ZW）individuals（p<0.05）.The studies indicated that the Sox17 may play an important role in the ovarian differentiation and development of T.sinensis.The full length of the Cyp26A1 cDNA of T.sinensis was 3340 bp,including a 66 bp5’-UTR,a 471 bp 3’-UTR containing a polyA structure,and a 1485 bp open reading frame,which encodes 494 amino acids.The Cyp26A1 amino acid sequence showed as high as 94.07%similarity with Chelonia mydas.The phylogenetic analysis showed that the T.sinensis is closely related to tortoises and birds,but relatively distant to fish.The q-PCR analysis showed that Cyp26A1 mRNA is highly specifically expressed in adult female of T.sinensis ovarian tissue,which is extremely significantly higher than that of heart,liver,spleen,kidney,intestine,muscle,brain tissue and testis tissue.The Cyp26A1 mRNA is mainly expressed in the 16 th to 21 st stages of embryonic development,and the expression of Cyp26A1 in the male（ZZ）gonads is extremely significantly higher than that of the female（ZW）gonads at the same period（p<0.01）.In addition,our study found that the expression of Cyp26A1 in pseudo-female（ZZ+E₂）induced by E₂ was significantly lower than the male（ZZ）individuals of control group.The expression of Cyp26A1 in pseudo-male（ZW+AI）induced by AI was significantly higher than that female（ZW）individuals of control group.It is suggested that the Cyp26A1 gene plays an important role in the reproductive activities of the female adult T.sinensis,and participates in the male sex determination process of the T.sinensis under the regulation of sex hormones during the embryonic development period.