Molecular Cloning,Expression Of Foxl2 And The Influence Of Temperature And Exogenous Hormone On Expression Of Foxl2 In The Pelodiscus Sinensis

Inheritance of gender is highly conserved in the long-term evolution.However, environmental sex determination(ESD) arises after organism genetically determined(GSD),which gender is relatively perfect.Their relationship is of great significance for understanding life evolution in the life course.Especially, the effect of environment and steroids on sex determination of animals belonging to ESD is reviewed,The phenomenon that temperature determines the sex ratio of offspring is widespread in reptiles,sex-related genes are more likely to be disturbed by exogenous hormones and temperature, response changes of Foxl2 in P.sinensis on this aspect are not clear.Pelodiscus sinensis is a kind of widespread breeding famous species,belonging Reptilia,Testudines,turtle family.Male turtles grow a third faster than female turtles, have high gum-content and are more welcomed by the market.Therefore,hatching a higher proportion of males has very important influence on aquaculture industry of P.sinensis in production,the research on molecular mechanism of sex determination in P.sinensis can lay a solid theoretical foundation for it.Foxl2(winged helix/forkhead transcription factor gene 2),belonging the winged helix or forkhead box transcription factor family members,had been detected in a variety of species,was involved in ovarian determination and/or differentiation as well as functional maintenance,and belonged to female-related gene in invertebrate. P.sinensis had not yet corresponding reports.To provide biological data to the molecular mechanism of sex determination in P.sinensis,in this study,the sequence characteristics,spatio-temporal expression profile at different development stages of Foxl2 gene and the influence of temperature and hormone on the gene expression had been analyzed by clone,expression technique and RT-PCR.The main findings were as follows. 1.Bioinformatics Anylysis of Foxl2 Gene.We cloned the middle length of Foxl2 cDNA(No.KP734210) from the ovary by RT-PCR.The middle-length cDNA of Foxl2 was 903 bp,comprised a 903 bp open reading frame(ORF)encoding 300 amino acids peptide and contained a typical DNA binding domain(FH);Phylogenetic analysis with other species revealed that the FH-domain of Foxl2 in P.sinensis were highly conserved;According to the BLASTP in NCBI,we carried on the analysis on amino acid sequence,P.sinensis had the nearest homologies with turtles,especially with the Trachemys scripta elegans;phylogenetic tree analysis showed that P.sinensis had the closest genetic distance with Chrysemys picta bellii.In addition,similar to the Foxl2 of other non-mammals,Foxl2 in P.sinensis contained neither the 14-polyalanine tract nor the glycine-raline repeats. 2.Cloning and Tissue Expression Pattern of Foxl2 Gene in P.sinensis.RT-PCR was employed to reveal the special expression pattern of Foxl2 in P.sinensis.Foxl2 expression had a high level in the ovary tissue and somewhat expression in brain,spleen,eye,kidney,muscle,liver and heart tissues.In addition, Foxl2 had weak expression in testis and was significantly lower than that in the ovary.Foxl2 in P.sinensis presented significant sexually dimorphic expression pattern.The results suggested that Foxl2 closely related to the development of female gonad in P.sinensis. 3.Effect of Foxl2 Expression in Pelodiscus Sinensis Treated with 17β-estradiol(E2) and 17α-Methyltestosterone(MT).In order to study the relationship between exogenous hormone and Foxl2 gene, P.Sinensis were treated with MT and E2 and the levels of Foxl2 expression were examined in the both sex individuals gonads after injection treatment for 6 h,12 h,24 h,48 h,7 d and 14 d,respectively.Results show that,E2 up-regulated the level of Foxl2;Under MT,the Foxl2 expression had no significant difference.Results indicated Foxl2 expression was hormone dependent. 4.Study of Foxl2 Expression Type during Development of Embryos in P.Sinensis Treated with 17β-estradiol(E2),17α-Methyltestosterone(MT)and Temperature.In this study,Foxl2 expression pattern in the whole embryo under the incubation temperature of 26.5℃ and 32℃ was investigated.Research showed:Foxl2 expression was the highest before 12 h,gradually decreased and sustained until 34 d,then reached its highest expression,and was higher at 32℃ than at 26.5℃.Foxl2 expression had a similar changing trend under 26.5℃ and 32℃, 34 d appeared to be a key time during the development process of gonad in P.sinensis.Foxl2 expression was controlled by parent form in early development of embryos in P.Sinensis.To investigate the effect of exogenous hormone on the sex determination and gonad differentiation in P.sinensis.Embryos were treated after 24 h incubation with E2 and MT.Results showed that,Both E2 and MT promoted Foxl2 expression, which expression peak by E2 appeared earlier than that by MT.