Characteristic And Functional Analysis Of Rspondin1 And R-spondin3 Genes In Tongue Sole(Cynoglossus Semilaevis)

Tongue sole(Cynoglossus semilaevis) is one of the most important mariculture commercial fish with great differences between male and female ones. There are four members in R-spondin (Rspo) protein family including Rspol-4 which are all secretory proteins. All the members in Rspo protein family contain highly similar conserved domains including two furin-like cysteine rich domains (FU) and one thrombospondin-type 1 domain (TSP1). It had been proved that all Rspo proteins can interact with Wnt signaling pathways and play different significant roles in vertebrates’ development process. The Rspo1 gene, as a best candidate gene for ovary determinant and differentiate, has prominent effect on sex determination, skin differentiation and malignancy. As another important member of Rspo family, Rspo3 gene can determine the fate of hemopoietic stem cells, promote angiogenesis and control the growth of the mammals’ placenta. The purpose of this experiment is to get the complete cDNA of Rspol and RspoS genes of tongue sole through homology cloning and RACE PCR (Rapid amplification of cDNA ends PCR); make preliminary exploration on these two genes’expressions by in situ hybridization and quantitatine RT-PCR and combined these results and bioinformatics analysis to predict the function of the two genes.1. The cloning and expression analysis of Rspol gene of tongue soleDegenerate primers and RACE primers were secured by homologous comparison. After cloning and jointing them, we got the ORF of Rspol gene in tongue sole that ORF were 834 bp, encoding 277 amino acids, and its molecular weight was about 30.52 kDa. Protein structure prediction displayed that Rspol gene had three domains including FU1, FU2 and TSP1, which are located at the points of 31-86,93-137 and 154-216, respectively. The results of protein structure prediction showed that the amino acid sequences of Rspol gene of tongue sole enjoyed highly similarity with that of other species especially marine fish, which had been further proved by the construction of a phylogenetic tree. The tree was obviously divided into two branches:differentiation of this gene in fish was big that the similarity among different species of fish reached 40%-60%, while that of tetrapods were above 99%. The analysis and prediction of transcription factors in promoter region and found bingding sites of some gender related transcription factors like Sox-5, SRY, which provided help to study the gene function. The results of quantitative real-time PCR (qRT-PCR) showed that during the embryonic developmental stages the expressions of Rspol gene started to increase dramatically after the formation of kupffer’s vesicle in tongue sole. Considering its effect on sex differentiation, we compared the different expressions of the Rspol gene in female and male tongue sole tissues, and found that the expression existed in both sex. However, the magnitude of Rspol gene expressions in ovary was obviously higher than those in testis, and the former was twice of the latter, which was consist with the results of the previous reports, indicating the importance of Rspol gene in sex determination. Its expressions also existed in non-gonadal tissues such as brain and heart, but there was no big difference between male and female fish. The results of in situ hybridization tissue slices showed that Rspol gene was mainly located in oocyte nucleus, spermatogonium and spermatocyte, and there was no signal in spermatid, which was consisted with the research results of zebrafish Rspol gene. After the hybridization of the RNA probes with the same concentration, we found that the signal in ovary was much stronger than that in testis, which indirectly proved the magnitude of Rspol gene expressions in ovary were much higher than those in testis. With the help of zebrafish, the model organism, we explored what effect of Rspol gene of tongue sole might have on the development of zebrafish and Wnt signaling pathway when it was injected into the model organism. When the Rspol-GFP plasmid was injected, signals could be detected in most parts of the experimental subject, and embryonic malformation rate was rising accordingly. However, when the Rspol and Wnt3a mRNA of tongue sole was over-expression in embryos, the Wnt signaling pathway were blocked.2. The cloning and expression analysis of Rspo3 gene of tongue soleThrough homology cloning and RACE PCR technology, we got Rspo3 gene of tongue sole, whose ORF were 987 bp, encoding 328 amino acids and its molecular weight was about 38.07 kDa. Protein structure prediction showed that apart from three conserved domains including FU1 (36-88), FU2 (94-137) and TSP1 (200-257), Rspo3 gene had another domain, FU3 (141-185). According to the comparative analysis, these three FU domains exclusively belong to fish’s Rspo3 gene, and don’t exist in mammals’like human and mouse Rspo3 gene. The phylogenetic tree was also divided into two branches:the fish was separated into sea fish and freshwater fish, which more clearly shows the evolutionary relationships. Quantitative RT-PCR results showed that the expression of Rspo3 gene existed in almost every parts of the adult fish, especially in brain and gill tissues. The microinjection of Rspo3-GFP plasmid of tongue sole had significant influences on the zebrafish’s development. Normal embryo had less and more concentrated fluorescence signals, while malformed embryos might have curly or shortened tail, mal-developed head, generally atrophy and so on. Transfected brain cells, which were cultured in vitro, with Rspo3-GFP plasmid, we found signals appeared in nucleus not cytoplasm.