Cloning,Expression Analysis Of Aqp1aa,Lipocalin In Half-smooth Tongue Sole(Cynoglossus Semilaevis)

Half-smooth tongue sole(Cynoglossus semilaevis)is an important marine aquaculture fish in China.Due to the serious male and female differences,the growth of male individuals is slow and the of female individuals faster.Therefore,it is very important to cultivate the whole female seed for breeding of C.semilaevis.In addition,C.semilaevis employs the female heterogamety sex determination system ZZ/ZW with a heteromorphic W chromosome.and there is a natural reversal phenomenon.Since the sex-determining mechanism and the sex-determining gene of C.semilaeus are not clear,the study of the sex-related genes of C.semilaevis is vital importance for the selection of the C.semilaevis breeding.In this paper,two sex-related genes of C.semilaevis were cloned and analyzed,and the results were as follows:Aquaporins(AQPs)are transmembrane water channel proteins that allow water and some small molecules to pass through the plasma membrane of cells in animals.Aquaporin 1(aqp1)is the first isoform identified on the cell membrane of erythrocytes whose functions include cell migration,fat metabolism,body immunity,and neuro-signal transduction.In addition,some studies have found that the gene is also involved in male spermatogenesis.In this work,we cloned the full length of aqp1 aa gene of C.semilaevis and the related bioinformatics analysis was carried out to explore the evolutionary relationship between C.semilaevis and other teleosts.The aqp1 aa gene was 1411 bp in length and 786 bp in ORF,encoding 261 amino acids.Bioinformatics analysis revealed that the aqp1 aa gene contained a hydrophobic channel which consist by six transmembrane alpha helices and a highly conserved aspartate-proline-alanine motif,which is the aquaporin superfamily Specific label.Through sequence alignment and evolutionary analysis,it was found that the aqp1 aa was highly conserved in teleosts,especially the ?-helix and family label were the highest.The results of qPCR showed that the expression level of the gene was the highest in the testis,followed by the brain.In the different developmental periods,the expression level was significantly higher at 105 dph and constantly increased until 2 years of age in the testis of male.In different phenotypes,the expression level of aqp1 aa in the testis of male and pseudo-male was significantly larger than that of the overy,the expression level in DMRT1–knock out male and triploid male was the lowest.The results of in situ hybridization showed that the aqp1 aa hybrid signals was mainly found in spermatozoa and spermatids.The results of in situ hybridization of gonad in different periods were consistent with the results of qPCR.Therefore,we predicted that aqp1 aa was important in the process of spermatogenesis.The lipocalin superfamily is a class of extracellular secreted proteins of small molecules.These proteins are different in sequence,but they are highly conserved in crystal structure and have four hydrogen bonds.Lipid carrier protein is mainly through the use of small molecules to exercise the function: it can be combined with a series of hydrophobic small molecules or cell surface receptors to form soluble macromolecular complex to complete its function.In this study,the full-length cDNA of lipocalin gene was obtained and the bioinformatics analysis was carried out.The transcription factor binding site of the gene and its evolutionary relationship between species were studied.The gene mRNA was different in different tissues The expression of lipocalin gene was the highest in the ovary of Cynoglossus semilaevis,and the expression level of the lipocalin gene was higher in the ovary,and the expression level of the lipocalin gene was higher in the ovary of 105 days.In situ hybridization results showed that the lipocalin gene had significant hybridization signals in the ovaries of the III and IV oocytes.These results indicate that the lipocalin gene is very important to the development of the egg,which provide reference for the study of the sex determination mechanism of C.semilaevis.