Cloning,Expression And Function Analysis Of Wnt4 And Structure,Function Analysis Of Genes In Wnt Signaling Pathway In Yellow River Carp

Wnt4(wingless-type MMTV integration site family,member 4)belonged to the Wnt family,it was found in both invertebrates and vertebrates,and involved in many important processes.It has been proved that Wnt4 gene was play a major role in ovarian differential in mammals.In this study,using the forward and backward primers designed on the basis of the conserved region amino acid sequence of the Wnt4 genes registered in Genbank.We obtained the full length c DNA of Wnt4 in Yellow River carp,the complete cDNA sequence was 2489 nucleotides in length,consisting of a5'-untranslated region of 445 bp and a 3'-untranslated region(including poly A)of 988 bp.The open reading frame was 1056 bp in length and encoded a protein of 352 amino acids with a predicted molecular weight of 39.41 kDa,and theoretical isoelectric point was 8.61.It had 20 kinds of nitrogen amino acids in amino acid sequence,which 39 of negatively charged amino acid(Asp + Glu),48 of positively charged amino acids(Arg and Lys).It was a secreted protein with a signal peptide of 22 amino acids and a transmembrane region.In the Yellow River carp WNT4 protein secondary structure analysis of alpha helix is occupied 30.11%,beta Angle is occupied 9.94%,random coil accounted for 40.34%,extending chain accounted for 19.6%.Homology comparison data showed: the Yellow River carp with the homology is very high between teleost,the Yellow River carp Wnt4 and zebrafish Wnt4 is the similarity,and the highest was 98.3%.These data suggested that the Yellow River carp Wnt4 genes close with carp shape on the evolutionary purpose genetic relationship.Semi-quantitative RT-PCR,fluorescent real-time RT-PCR and In situ hybridization were conducted to determine the expression of Wnt4 gene in different stages during embryonic development and tissues of males and females Yellow River carp,the same results were teal and credible.All stages of embryonicdevelopment detected the expression of Wnt4,in the gastrula stage had the highest expression level.Whole mount in situ hybridization showed that the positive signal was detected from the endoderm at gastrula stage to the liver at larvae,and the expression had a high level in liver at the 3-mo and adult samples.The same results were found in Paralichthys olivaceus and Crassostrea gigas.Together,these results provided the evidence that Wnt4 had a potential function at liver development in teleosts.In the ovary,Wnt4 expression level was higher in adult ovary than stage ? ovary,and localization of Wnt4 transcript by ISH showed that Wnt4 was expressed in the cytoplasm of oocyte.The same results were reported and we could speculated that the Wnt4 involved ovary development and function maintain,not ovary differentiation.In the testis,the expression gradually increased during development and peaked in the mature testis,and more remarkable,the expression level in mature testis was higher than mature ovary.This expression pattern was not conservation with mammals,but same with some other teleosts,we speculated that it could be involved in the delay of steroidogenesis in males.To sum up,the Wnt4 gene might involve in male and female gonads development process.Wnt signaling plays major role in sex determination and sex differentiation in mammals.In order to explain its potential implication during these processes in Yellow River carp,we used a chemical inhibitor IWR-1-endo to inhibit Wnt signaling before gonads differentiation.After the treatment,the Wnt target genes and six female marker genes were down-regulated,however,four male marker genes were not unity.To sum up,because of female marker genes were down-regulated after inhibitor treatment,we speculated that Wnt signaling was implicated in the process of female gonadal development and the function in male gonadal development needs further research.