| Pelodiscus sinensis belongs to the Reptilia and Testudines.Male individuals are larger than the female,its growth speed is faster,its skirt is wide and thick,its nutrition is rich and its market price is high,so it is more popular with the industry and consumers.Therefore,the breeding of full males is of great significance to the breeding industry.The key to achieve this goal is to explore its sex determination and differentiation.However,Rspo1,Wnt4 and Beta-catenin1 were important regulatory factors of female sex determination and differentiation.In this study,the full-length c DNA of Wnt4 gene of Pelodiscus sinensis was cloned,and analyzed the expression characteristics of Rspo1,Wnt4 and Beta-catenin1 genes in the adult tissues of Pelodiscus sinensis and different developmental stages of embryos,and the effect of Wnt agonist and Wnt/β-catenin inhibitor on gene expression.The main research is as follows:1.In this study,the full-length c DNA sequence of Wnt4 gene of Pelodiscus sinensis was cloned,with a full length of 2432 bp,an open reading frame of 1095 bp,encoding 364 amino acids,48 bp 5’-Untranslated Region and 1289 bp 3’-Untranslated Region.The molecular weight of Wnt4 protein is 40.36 k D,the theoretical isoelectric point is 9.04,which has 24 conserved cysteine sequences of Wnt protein family.Wnt4 gene of Pelodiscus sinensis has the highest homology with Chelonia mydas and Terrapene carolina triunguis,up to 91.7%.The phylogeny showed that Wnt4 protein was clustered into three branches: mammal and reptile,bony fish and shellfish.Wnt4 of Pelodiscus sinensis gathers with reptiles,such as Chelonia mydas,Terrapene carolina triunguis and American alligators.It has the closest relationship with Chelonia mdas and Terrapene carolina triunguis and the farthest relationship with Azumapecten farreri.2.qRT-PCR was used to detect the expression of Rspo1,Wnt4 and Beta-catenin1.The expression characteristics of Rspo1,Wnt4 and Beta-catenin1 were analyzed in adult tissues,embryos of different developmental stages,Wnt agonist 1 and Wnt/β-Catenin inhibitors.(1).Expression characteristics in adult Pelodiscus sinensisWnt4 gene was expressed in the brain,gonadal,heart,liver,spleen,lung,kidney,intestine and muscle tissues of adult Pelodiscus sinensis,showing tissue differences.The highest expression was found in the gonadal gland,followed by the brain and lung tissues,and the lowest expression was found in the intestinal tissues.The expression of Wnt4 in the female ovary was significantly higher than that in the male testis(P<0.05).The expression of Beta-catenin1 gene in tissues was also extensive and showed some tissue differences,and the expression in the brain and gonads was significantly higher than that in other tissues(P<0.05).Wnt4 and Beta-catenin1 genes may be involved in various biological processes,especially in gonadal development or function maintenance.(2).Expression characteristics in embryos at different developmental stagesRspo1,Wnt4 and Beta-catenin1 genes were expressed in the 14 th to 21 st stages of embryonic development.The expression of Rspo1 gene increased gradually in female embryos,but decreased gradually in male embryos.It was expressed in the 14 th stage,and gradually increased in the 15 th to 21 st stages,and the expression in the female embryo was significantly higher than that in the male(P<0.05).The expression in the female embryo at the 19 th,20th and 21 st stages was highly significantly higher than that in the male(P<0.01).The expression of Wnt4 gene increased gradually in female embryos,and maintained a low level in male embryos.It had been expressed in the 14 th stage,the expression in the female embryo was significantly higher than male(P<0.05).In the 15 th to 21 st stages,the expression in the female embryo always maintained high expression level.The expression in the female embryo at the 15 th to 21 st was highly significantly higher than that in the male(P<0.01).The expression of Beta-catenin1 gene in embryo increased first and then decreased.It had been expressed in the 14 th stage,increased gradually from the 14 th stage to the 16 th stage,and reached the maximum in the 16 th stage.The expression in the 18 th and 21 st stages of male embryo was significantly higher than that in the females(P < 0.05).It is suggested that Rspo1,Wnt4 and Beta-catenin1 genes may play an important role in the process of embryonic development,especially in the gonadal differentiation of Pelodiscus sinensis.(3).Effect of Wnt agonist 1 on gene expressionEffects of Wnt agonist 1 on Rspo1 gene expression: On the 15 th day of hatching,the embryos of Pelodiscus sinensis.were treated with Wnt agonist 1.In female embryos,after treatment of 1 mg/mL,the expression of Rspo1 gene was up-regulated at 36 h ～ 168 h,and reached the peak at 72 h.After treatment of 2.5 mg/mL,the expression was up-regulated at 24 h ～ 168 h,peaked at 72 h.But after treatment of 5 mg/mL,the expression was significantly up-regulated at 6 h ～ 168 h(P<0.05).In male embryos,after treatment of 1 mg/mL,the expression was up regulated expression at 48 h ～ 168 h and reached the peak at 72 h.After treatment of 2.5 mg/mL,the expression was up-regulated at 36 h ～ 168 h and reached the peak at 72 h.While after treatment of 5 mg/mL,the expression was up-regulated at 6 h ～ 168 h and reached the peak at 72 h.With the concentration increased,the gene began to up-regulated earlier,and the up-regulated level also increased.Effects of Wnt agonist 1 on Wnt4 gene expression: in female embryos,after treatment of 1 mg/mL,the expression of Wnt4 gene was up-regulated at 36 h ～ 96 h,peaked at 72 h,and returned to normal at 120 h to 168 h.After treatment of 2.5 mg/mL and 5 mg/mL,the expression was up regulated expression at 24 h ～ 168 h,peaked at 72 h.In male embryos,after treatment of 1 mg/mL,the expression was significantly up-regulated at 6 h ～36 h(P<0.05),peaked at 36 h,and gradually decreased at 48 h ～ 168 h.After treatment of 2.5 mg/mL,the expression was significantly up-regulated at 6 h ～ 96 h(P<0.05),peaked at 96 h,and returned to normal level at 120 h ～ 168 h.But after treatment of 5 mg/mL,the expression was significantly up-regulated at 6 h ～ 168 h(P<0.05),peaked at 96 h.Effects of Wnt agonist 1 on Beta-catenin1 gene expression: in female embryos,after treatment of 1 mg/mL,the expression of Beta-catenin1 gene was up-regulated at 36 h ～ 168 h,and reached the peak at 72 h.While after treatment of 2.5 mg/mL and 5 mg/mL,the expression was up-regulated at 6h ～168 h.In male embryos,after treatment of 1 mg/mL,the expression was up-regulated at 24 h ～ 168 h and reached the peak at 48 h.After treatment of 2.5 mg/mL,the expression was up-regulated at 6 h ～ 168 h and reached the peak at 48 h.While after treatment of 5 mg/mL,the expression was significantly up-regulated at 6 h ～ 168 h(P<0.05)and reached the peak at 72 h.(4).Effects of Wnt/β-catenin inhibitor on gene expressionEffect of Wnt/β-catenin inhibitor on Rspo1 gene expression: in female embryos,after treatment of 1 mg/mL,the expression of Rspo1 gene was significantly down regulated at 6 h ～48 h(P<0.05),and maintained at the normal expression level after 72 h.After treatment of 2.5 mg/mL,the expression was significantly down regulated at 6h and maintained at the normal expression level after 12 h.After treatment of 5 mg/mL,the expression was up regulated at 6 h ～ 168 h.In male embryos,after treatment of 1 mg/mL and 2.5 mg/mL,the expression was significantly down regulated at 12 h ～ 36 h(P<0.05).While after treatment of 5 mg/mL,the expression was up regulated at 6 h ～ 168 h,peaked at 72 h.Effect of Wnt/β-catenin inhibitor on Wnt4 gene expression: in female embryos,after treatment of 1 mg/mL,the expression of Wnt4 gene was down-regulated at 6 h ～168 h.After treatment of 2.5 mg/mL,the expression was down regulated at 6 h ～ 120 h,and returned to normal level at 144 h ～ 168 h.However,after treatment of 5 mg/mL,the expression was up-regulated at 6 h～12 h and up regulated 24 h ～ 144 h,peaked at 96 h,returned to normal level at 168 h.In male embryos,after treatment of 1 mg/mL,the expression was down-regulated slightly at 6 h ～ 12 h,and decreased significantly at 24 h ～ 168 h(P<0.05).After treatment of 2.5 mg/mL,the expression was increased slightly at 6 h ～ 24 h and decreased significantly at 36 h ～ 168 h(P<0.05).After treatment of 5 mg/mL,the expression was up-regulated at 6 h ～ 168 h,and reached the peak at 96 h.The effect of Wnt/β-catenin inhibitor on Beta-catenin1 gene expression: in female embryos,after treatment of 1 mg/mL,the expression of Beta-catenin1 gene was down regulated at 6 h ～ 36 h,up regulated at 72 h ～ 96 h,and returned to normal expression level at 120 h ～ 168 h.After treatment of 2.5 mg/mL,the expression was normal at 6 h ～ 48 h,up regulated significantly at 72 h ～ 168 h(P<0.05).After treatment of 5 mg/mL,the expression was up regulated significantly at 6 h ～ 168 h(P<0.05),and reached the peak at 36 h.In male embryos,after treatment of 1 mg/mL,the expression of Beta-catenin1 gene was down regulated at 6 h ～ 120 h and returned to the normal level at 144 h ～ 168 h.After treatment of 2.5 mg/mL,the expression was down regulated at 6 h ～ 120 h and up regulated at 144 h ～ 168 h.While after treatment of 5 mg/mL,the expression was up-regulated at 6 h ～ 168 h and reached the peak at 36 h. |
PDF Full Text Request