The Cloning And Expression Analyzing Of Dazl And Boule In Germ Cells In The Chinese Soft-shell Turtle

Gene dazl and boule belong to the DAZ(Deleted in Azoospermia)gene family.The DAZ family is one of the germ cell specific genes across the animal phyla,consist of daz,dazl,and boule encoding RNA-binding proteins that regulate germ cell development and differentiation.In mammal,the deletion of gene daz leads to azoospermia in male.While the DAZ gene family present dynamic expression pattern in stages of germ cells during gametogenesis,which provide excellent markers for investigating the germ cells' development,differentiation and maturation in fish and mammal.Chinese soft-shell turtle(Pelodiscus sinensis)is a kind of common species in China and has high economic value of edible and medicinal function.However,as an important model for evolutionary and developmental biology,it is still largely uncertain that the mechanisms by which the germ cells develop and differentiate in turtles and that is not conducive to the conservation of germplasm and the breeding of improved species of turtles.In this study,we used the Chinese soft-shell turtle as a model,cloned turtle dazl?boule and observed their expression in germ cells to study the germ cells differentiation in turtle species.Specific research content:1.The cloning of gene dazl and boule of Chinese soft-shell turtle:We cloned a turtle dazl cDNA fragment of 1007 bp,containing a 197 bp 5' untranslated region(UTR),a 33 bp 3' UTR and a open reading frame(ORF)for 258 amino acid residues.The predicted turtle Dazl is maximally 96% identical to western painted turtle Dazl protein and 75% identical to mouse Dazl protein.A turtle boule cDNA fragment of 1005 bp,containing a 57 bp 3' UTR and a open reading frame(ORF)for 315 amino acid residues.The predicted turtle Boule is maximally 92% identical to sea turtle Boule protein and 88% identical to mouse Boule protein.2.Analysing of turtle dazl mRNA and boule mRNA expression in different tissues by RT-PCR:The RT-PCR result showed that the turtle dazl and boule transcript were absent in all somatic tissues but abundant in adult ovary and testis.3.Chemical and fluorescence in situ hybridization of turtle dazl mRNA and boule mRNA during Spermatogenesis:In 7 month testis,the dazl mRNA signals focused in spermatogonia.In 2 year old adult testis,the dazl mRNA signals were strongly dispersed in primary spermatocyte and secondary spermatocyte,weakly in spermatogonia,barely detected in spermatids and sperm.In 3 year old adult testis,the signals were detected in few spermatogonia,strongly dispersed in primary spermatocyte and weakly in secondary spermatocyte.In 7 month testis,the boule mRNA signals focused in spermatogonia.In 2 year old adult testis,the boule mRNA signals were strongly in primary spermatocyte and secondary spermatocyte,weakly in spermatogonia,and barely detected in spermatids and sperm.In 3 year old adult testis,the signals were still strongly dispersed in primary spermatocyte.4.Chemical and fluorescence in situ hybridization of turtle dazl mRNA and boule mRNA during Oogenesis:In ovary,the dazl mRNA and boule mRNA signals were strong in early primary stages of oocytes and displayed a uniform distribution;as oocytes grown into larger size,the signals concentrated in the perinuclear cytoplasm became weak as vitellogenesis further developed.In a conclusion,the findings in this study suggested that gene dazl and gene boule would play important roles in germ cells differentiation during gametogenesis in Chinese soft-shell turtle.The characterization of gene dazl and gene boule in the Chinese soft-shell turtle would provide the basis for further invstigations on reproductive development in other turtles.