Identification And Function Of Three Sex-related Genes In Hyriopsis Cumingii

Hyriopsis cumingii is an important freshwater pearl mussel in China,with high economicvalue.Studies have shown that H.cumingii is gonochorism,and adult males mussels have better pearling performance.There are many studies on genes related to gender regulation in high vertebrata animals,but the lack of systematicresearch on the sex determination mechanism of invertebrata,especially H.cumingii,has brought some difficulties to shellfish breeding.In this experiment,the full length of the genes HcWNT4,HcDax1,and HcSox7 of H.cumingii was cloned by the Race method(Rapid-amplification of cDNA ends).The sequence structure of these three genes,the expression of male and female tissues,and the expression of early gonad development were analyzed.Preliminary studies on gender determination and gender differentiation.The role of WNT signaling pathway and preliminary investigation through inhibitor injection.The specificresearch results are as follows:1.Full-length sequence analysisThe full-length cDNA of HcWNT4 gene is 1560 bp,of which 5'-UTR 396 bp,3'-UTR 24 bp,with open reading frame(ORF)of 1140 bp,can encode 379 amino acids,and the sequence contains the unique structure of the Wnt family area.The homology analysis of the amino acid sequence of HcWNT4 revealed that the evolutionary relationship and structural function of the gene between species were conserved.The full-length cDNA sequence of HcDax1 gene is 2719 bp,contains 188 bp and1295 bp of 5' and 3'-UTR,and the ORF region is 1236 bp,encoding 411 amino acids.The protein molecular weight is predicted to be 46.22 KDa and the theoretical isoelectricpoint is 5.96.It is a hydrophilicprotein with a conserved NR superfamily domain and contains the structural features of the nuclear receptor superfamily DBD and LBD.The full length of the HcSox7 gene sequence is 2315 bp,of which the HcSox7 gene has an ORF of 1668 bp,encoding a total of 555 amino acids,a 5'-UTR of 30 bp,and a 3'-UTR of 617 bp.The HcSox7 amino acid sequence analysis showed that the similarity with other species is low,and the protein molecular weight is predicted to be 62.00 KDa,the theoretical isoelectricpoint is 7.37,and the amino acid residue is hydrophilic.2.Analysis of spatiotemporal expression characteristics of three genesHcWNT4,Hcdax1,and HcSox7 genes are expressed in multiple tissues of male and female of H.cumingii(the mantle,the adductor muscle,the gill,the gonad,the foot and the liver),indicating that they are involved in diverse biological processes.The expression of HcWNT4 gene is higher in gills,foot,and liver,and there are significant differences between males and females in gonads,the adductor muscle,the foot.The expression level is high in January to March,and it is speculated that HcWNT4 is involved in the early development and gender differentiation process.The expression of HcDax1 gene is most different in gonads.The relative expression of female gonads and foot is significantly higher than that of males,and the expression level of 4 months of age is significantly increased.It is speculated that Hcdax1 gene plays a leading role in females.HcSox7 gene is highly expressed in the liver and gills,and the expression of females in gonads and foot is significantly higher than that of males,with the highest expression at 8 months of age,followed by 4 months.In situ hybridization results showed that HcWNT4,Hcdax1,and HcSox7 genes had blue hybridization signals on the oocytes of female H.cumingii,but the signals were not obvious in the testis,which may be involved in the gonad development process.3.IWR-1-endo inhibits Wnt signaling pathwayAt present,the Wnt signaling pathway has a greater effect on gender determination and gender differentiation in high vertebrates.In this experiment,H.cumingii at the age of 6 months was selected as the experimental sample,and different concentrations of IWR-1-endo inhibitors were injected.HcWNT4,HcSox7,and Key genes of WNT signaling pathway ?-catenin are down-regulated to varying degrees.It is speculated that there may be a Wnt signaling pathway in H.cumingii,and inhibition of this signaling pathway has a certain effect on key genes of sex.4.?-catenin immune response of key genes in WNT signaling pathway?-catenin is an important transcription factor of classical Wnt signaling pathway,and plays a key role in female gonad development and immune regulation.After injecting Aeromonas hydrophila and lipopolysaccharide in this experiment,it was found that the expression of Hc-?-catenin gene in blood cells and gills increased,which has been confirmed that the Hc-?-catenin gene can help the body to resist infection by A.hydrophila and LPS,and play an important role in the immune response.