Cloning And Gonadal Expression Analysis Of Figla Gene In Chinese Soft-shell Turtle(Pelodiscus Sinensis)

figla(factor in the germline alpha)is a member of the bHLH(basic helix-loop-helix)family and plays a pivotal role in reproduction and gonadal development,especially in follicular formation and ovary development in most of vertebrates.figla posessing a highly conserved bHLH domain,has been identified and characterized in several vertebrates.Chinese soft-shell turtle(Pelodiscus sinensis)is one of the most common species of reptiles in aquaculture and has high value in medicine and nutrition.The overharvesting of turtles and the ruining of environment lead to serious germplasm degradation of species.Therefore,it needs developing new technologies of elite breeding through combining the traditional selective breeding and cell engineer breeding.Moreover,the germ cells are the basis of new breeding techniques,and the studies on germ cell diferentiation would be the prelude for further investigating the molecular mechanisms behind the gonadal develoment and gametogenesis in turtles.In this thesis,we analyzed the figla expression profiles in stages of germ cells in the Chinese soft-shell turtle.The major jobs are as follows:1.Cloning and characterizing the figla gene in Chinese soft-shell turtleHere,we cloned the cDNA sequence of figla gene from the gonads of Chinese soft-shell turtle.The isolated fragment of cDNA is 714 bp,containing a 3?-UTR of 72 bp and and a full open reading frame(ORF)of 642 bp.The deduced protein contains213 amino acids with a highly conserved bHLH domain and was designed as PSFigla.The aa alignment showed that PSFigla presents a highest identity to Figla of western painted turtle,up to 93%.Moreover,the analysis of NJ phylogenetic tree showed that the PSFigla was clustered into the same branch with Figla of the mammals and birds.2.Tissues specific expression of figla mRNASemi-quantitative PCR and qRT-PCR result showed that figla mRNA was exclusively expressed in the gonads,strongly in the ovary and weakly in the testis,and hardly detected expression in other tissues.3.The expression and celluar distribution of figla mRNA in ovaryIn situ hybridization analysis on ovarian cryosections showed that figla mRNA was expressed in almost all stages of follicles during oogenesis: the signals were strong in the primary oocyte(?-?),and distributed mainly in the cytoplasm of oocytes.As the oocytes growing(?-?),figla mRNA became concentrated into the follicular cells surrounding oocytes.The signal was gradually deduced in the cytoplasm,while became strong in the surrounding follicular cells.The findings of this study showed that figlagene may play a crucial role in follicle formation and ovary development in Chinese soft-shell turtle.4.The expression and distribution of figla mRNA in testisfigla mRNA showed dynamic expression in germ cells during spermatogenesis.figla mRNA was strongly expressed in spermatocytes and spermatids,weakly in spermatogonia,barely in somatic cells.The results showed that figla gene may also play a role in spermatogenesis in Chinese soft-shell turtle.In conclusion,figla gene could be a key factor regulating oogenesis and ovary development,also it maybe function in spermatogenesis in Chinese soft-shell turtle.This study would provide basic knowledge for further investigations on gametogenesis,gonadal development and determination in turtles or other reptiles.