Cloning And Differential Expression Analysis Of Ovarian Developmental Genes Rspo1 And ?-catenin Gene In Chinese Giant Salamander(Andrias Davidianus)

The Rspo1/Wnt/?-catenin signaling pathway involved of Rspo1(R-spondin1)and ?-catenin is one of the Wnt-mediated classical signaling pathways and plays a crucial part in female sex determination and differentiation.In recent years,the role of the Rspo1 and ?-catenin genes in the genital system has received increasing attention,especially in early sex determination and differentiation of vertebrates.In this study,Rspo1 and ?-catenin genes c DNA complete sequence of Chinese giant salamander are cloned by reverse transcription polymerase chain reaction(RT-PCR)and rapid amplification of c DNA ends(RACE),and their biological characteristics are also analyzed.Analysis of Real-time PCR is performed to examine the expression in various tissues and development stage of the Chinese giant salamander.The expressions of other gonadal development-related genes Fox L2,Cyp19 a,Dmrt1 and Sox9 in the ovaries at different stages of development were comprehensively analyzed to explore the role of Rspo1 and ?-catenin genes in ovarian development.We the following results have been obtained:1.Chinese giant salamander Rspo1 gene c DNA sequence contains 902 bp and encodes 261 aa.Its encoded protein has an N-terminal signal peptide,two furin-like domains(FU1 and FU2),and a thrombin-sensitive protein type 1 domain(TSP1).There are also a signal peptides and a nuclear localization signal regions(NLS),suggest that signal peptide is present in the endoplasmic reticulum and Golgi,in the classical secretory pathway.A conserved N-glycosylation site exists in Rspo1 of Chinese giant salamander,indicating that it is involved in the regulation of the Wnt/?-catenin signaling pathway.2.Chinese giant salamander ?-catenin gene c DNA sequence contains 2343 bp and encodes 780 aa.Its encoded protein sequence contains 12 Armadillo(arm)repeat region domains.,the 12 arm domain of the Chinese giant salamander ?-catenin protein was highly conserved compared to other species,with only a few amino acid differences.Among the important phosphorylation sites in mammalian ?-catenin,except for the remaining phosphorylation sites of Y3,which are present in Chinese giant salamander,it is speculated that the functions of these phosphorylation sites are also present in the Chinese giant ?-catenin protein.3.Through the comparative analysis of gene evolution,it is found that:the Rspo1 CDS of the Chinese giant salamander forms a branch with the amphibians and is at the outermost part of its branch,while the protein sequence with the reptiles and birds cluster into a large group and are at the outermost periphery;the Chinese giant salamander ?-catenin CDS doesn't form a cluster with the amphibians and forms a branch with the reptiles and birds and is located at the outermost periphery of the branches.The protein sequence and the amphibians cluster into a cluster and are at the outermost periphery.4.The expression of Rspo1 and ?-catenin in the ovaries of Chinese giant salamander was significantly higher than the testis in the early stage,indicating that Rspo1 and ?-catenin mainly regulate the development and differentiation of ovaries in the early stage of gonadal development of Chinese giant salamander.Both Rspo1 and ?-catenin of Chinese giant salamander were expressed in other tissues,and showed significant specificity of spatial,temporal and gender.5.We combined the expression levels of Rspo1 and ?-catenin with Sox9,Dmrt1,Fox L2 and Cyp19 a in the ovary of different developmental stages of Chinese giant salamander.The genetic model of Rspo1 and ?-catenin involved in the development of gonads in Chinese giant salamander is proposed,which provides a theoretical basis for revealing the molecular mechanism of amphibians,especially the differentiation and development of ovary and the interaction between somatic and germ cells.