Structure, Expression Pattern And Function Analyses Of Three Genes Of Soxf Subfamily In Yellow River Carp (Cyprinus Carpio Var)

The SoxF(Sry-related high-mobility group box)gene subfamily includes three members,Sox7,Sox17 and Sox18.It is widespread in both vertebrates and invertebrates.It is involved in regulating the development and differentiation of blood vessels and lymphatics.In additionit,it plays a crucial role in the process of neurogenesis.In this study,the full-length of Sox7,Sox17 and Sox18 were obtained based on the sequencing results of carp ovary transcriptome in our laboratory and DNAMAN sequence alignment.The results of gene structure analysis showed that the full-length cDNA sequence of carp Sox7 was 2339 bp,including a 265 bp 5'-UTR and a 880 bp 3'-UTR;The ORF was 1194 bp.The predicted amino acid sequence was 397 residues long.The full-length cDNA sequence of carp Sox17 was 1653 bp;it contained an ORF of 999 bp,a 5'-UTR of 134 bp,and a 3'-UTR of 520 bp.The predicted amino acid sequence was 332 residues long.The full-length cDNA sequence of carp Soxl8 was 2458 bp;it contained a 5'-UTR of 243 bp,a 3'-UTR of 895 bp,and an ORF of 1320 bp that predicted 439 amino acids.Within the ORF there is a 72 amino acid highly conserved(HMG)DNA binding domain,the DNA binding site included 14(RMNFMAKRANKGWR)amino acids.The results of CLUSTALW showed that the amino acid sequences of Sox7,Sox17 and Sox18 were highly consistent with the Sox7,Sox17 and Sox18 amino acid sequences of zebrafish,and the identities were 96.4%,95.8%and 71.5%,respectively.The similarity to mammals and birds is low.Phylogenetic tree based on MEGA6 software showed that Sox7,Sox17 and Sox18 belonged to three different branches.Sox7,Sox17 and Soxl8 of carp were grouped with Sox7,Sox17 and Sox18 of zebrafish respectively,suggesting that carp Sox7,Sox18 and zebrafish Sox7,Sox18 were homologous,respectively,carp Sox17 and Sinocyclocheilus rhinocerous Sox17 was homologous.Chromosome homology analysis showed that there were two copies of Sox7 in the Yellow River common carp,one located on the scaffold 1136,the gene around the chromosome was relatively conservative,and the other was located on the scaffold 77 and the surrounding genes were changed.Among the genes around Sox17,only lyplal is conserved.The two copies of Sox18 in the Yellow River common carp are adjacent.The surrounding genes are conserved in fish but the gene arrangement and orientation are different.It is speculated that chromosomal rearrangements or gene insertions occur frequently in the process of fish genome replication,but the arrangement of conserved genes is very consistent.The 2000 bp 5'flanking sequences upstream of Sox7,Sox17 and Sox18 were analyzed with genomatix software.There was a high similarity with the zebrafish and tilapia Sox7,Sox17 and Sox18 in transcription factor binding site.Neurogenesis,angiogenesis,muscle and liver-specific transcription factor binding sites were found.Therefore speculated that Sox7,Sox17,Sox18 closely related to neurogenesis and blood vessels of the carp.In order to further study the function of Sox7,Sox17 and Sox18 in the carp,we analyzed the expression pattern of Sox7,Sox17 and Sox18 in different tissues of carp embryos and sexual maturity individuals by real-time qRT-PCR.Sox7 had the highest expression in nerve embryonic stage.Sox17 had the highest expression in gastrulation stage.But Sox 18 hardly expressed in all stages of embryo.Sox7,Sox17 and Sox18 were abundantly expressed in brain.Sox7 and Sox18 were the highest expressed in mesencephalon and Sox 17 was highest in epencephalon.The result corresponds to the analysis of transcription factor binding sites.The localization of Sox7,Sox17 and Sox18 in the gonads,brain and embryos at six stages were detected by in situ hybridization.The three genes were not expressed in the ovary,weak expression in the testicles.Sox7 were more expressed in the brain than Sox 17 and Sox18.In embryos,Sox7 had a strong positive signal in blastula stage,neuroblastoma stage and caudal period.Soxl 7 had strong expression signal in blastula stage and gastrula stage,and strongly expressed in gill and internal organs after three days of harvest.Sox18 was barely expressed during embryogenesis,and the expression in gills and offal was weaker than Sox 7 and Sox17 after three days of harvest.To further explore the relationship between Cyprinus carpio SoxF(CcSoxF)subfamily and neurogenesis,angiogenesis,the toxicological experiments were designed to treat yellow carp embryos and juveniles with different concentrations of silver ions(Ag+)and silver nanoparticles(AgNPs).After treatment,there were cases of spondylolisthesis,caudal flexion,pericardial edema,and overlap of the above deformities.Significant changes have also occurred in the expression of Sox7,Soxl 7 and Sox18.The expression had changed mainly in the embryonic blastocyst stage,gastrulation stage,neuroblastoma and juvenile heart,eye,brain,spleen.These results are consistent with the functional sites of the SoxF subfamily gene.Based on the above findings,the discovery and validation of Sox7,Sox17,Sox 18,simple bioinformatics analysis,expression pattern analysis and gene expression changes after drug treatment will reveal the potential functional of CcSoxF subfamily in neuronal and vascular differentiation,the specific regulatory mechanism remains to be further studied.