Structure,Expression Pattern And Its Relationship With MiRNA Targets Analysis Of Gsdfs Gene In Yellow River Carp(Cyprinus Carpio Var)

The TGF-?(transformation growth factor-?,TGF-?)superfamily plays an important role in the reproduction,development and growth of vertebrates.Gsdf is a new member of the TGF-? superfamily,which is only present in fish.And it is specifically expressed in the gonads in most teleost fish.It has become a strong candidate gene for fish sex determination gene research.Therefore,this study conducted a preliminary study on the structure and function of Gsdf gene in the Yellow River carp.In this study,we first screened the Gsdfs gene sequence from transcriptome sequencing data of Yellow River carp constructed by our laboratory.Two correct full-length sequences were then determined by DNAman alignment,Blast search,and PCR sequencing.The results showed that: The full length of the Gsdf1 gene is 1444 bp,encoding 154 amino acids,has a TGF-? domain and 8 conserved cysteine residues,contains 4 exons and 3 introns;the full length of the Gsdf2 gene is 1355 bp,encoding 191 amino acids,has a TGF-? domain and 8 conserved cysteine residues,containing 5 exons and 4 introns.The results of CLUSTALW showed that the amino acid sequences of Gsdf1 and Gsdf2 were highly consistent with the Gsdf amino acid sequences of zebrafish,and the identities were 49.3% and 57.0%,respectively.The similarities with Takifugu rubripes were very low,only 19.9% and 24.5%.Phylogenetic tree based on MEGA6 software showed that,Gsdf1 and Gsdf2 of the Yellow River carp were clustered with the Gsdf gene of zebrafish,which is consistent with the amino acid alignment results.It is speculated that the Gsdfs gene in the Yellow River carp is closely related to the Gsdf gene of zebrafish in evolution.Chromosome synteny analyses revealed that there were several conserved genes around the Gsdf of other teleost fish: NUP54,PPEF2,AFF1 and KLHL8,but the Gsdfs in the Yellow River carp did not find a conserved gene adjacent to it.It is speculated that it may have been rearranged during the evolution process.Numerous essential transcription factor binding sites(TFs)were predicted within the 2000 bp upstream of the 5' end of these genes.These TFs include SOX,FOXL,SF1 and SRY,which suggesting that the Gsdfs gene in the Yellow River carp may be involved in gonadal development and differentiation.Location and tissue distribution analyses of of Gsdf1 and Gsdf2 genes in different tissues of juvenile and adult fish and different stages of embryonic development by Quantitative real-time RT-PCR(qRT-PCR)and in situ hybridization(ISH).The results showed that Gsdf1 and Gsdf2 were mainly expressed in the gonads,and after sexual maturity,the expression level in the testis was much higher than that in the ovar.Gsdf1 and Gsdf2 are highly expressed in the early stage of embryonic development,and then the expression level is down-regulated;in the mature gonads,the positive signal of Gsdf1 and Gsdf2 in the testis is more intense,mainly concentrated in the supporting cells around the spermatogonial cells,and only weakly expressed in the granule cells of the ovary.This indicates that the Gsdfs gene may play an important role in the development and differentiation of the gonads of the Yellow River carp,especially in the development and differentiation of the testis.The miRNA plays a variety of roles in the regulation of cell growth and developmental processes.With the gradual deepening of the research on the expression of miRNA regulatory genes,the study of the interaction between miRNA and its target genes becomes more important.Therefore,this experiment combined with the miRNA library of the gonads of the Yellow River carp constructed by the laboratory and Targetscan software predicted that Gsdf1 is a potential target gene of miR-22 a and miR-210.In order to study the regulatory relationship between Gsdf1 and two miRNAs,the dual luciferase reporter gene detection system was used to verify the target relationship between the two miRNAs and Gsdf1.The results indicated that Gsdf1 is a direct target of miR-22 a and miR-210.Finally,this experiment successfully produced a polyclonal antibody to the Gsdf1 gene of the Yellow River carp.The target relationship between miR-22 a and miR-210 and Gsdf1 was double verified by Western Blot and Elisa.It was once again proved that Gsdf1 in the Yellow River carp was the target gene of miR-22 a and miR-210.It is speculated that miR-22 a and miR-210 may participate in the sex differentiation and development of the Yellow River carp through the regulation of Gsdf1,but the specific mechanism needs further research.In summary,we speculate that the Gsdfs gene plays an important role in the development and differentiation of the gonads of the Yellow River carp.This is very important for studying the gonad development,differentiation of the Yellow River carp and the molecular mechanism of the development of the testis of the Yellow River carp.Significance,while at the same time may provide some help in the exploration of fish sex determination mechanism.