| The Hyriopsis cumingii is an important freshwater pearl mussel in China.Related studies have shown that male have superiority in pearl production compared to females.There is a lack of systematic inquiry into the gender forming mechanisms of the Hyriopsis cumingii,bivalve,and entire mollusks.The diversity of gender forms is often the result of the interaction of environmental factors and sex chromosome or cytosolic sex-related genes.There are some proven signaling pathway genes in the sex determination and differentiation of higher vertebrates.This experiment selected three classical genes,Transformer-2?,?-catenin and SOX2,and studied their sequence,structure,expression of male and female tissues,expression during early development and cell localization,aiming to find out whether there is a certain correlation with the formation of gender.In addition,based on the previous work,a preliminary study was performed on the function of the 3'-specific sequence of the M-type COII gene to explore its role in sex differentiation.1.Cloning and expression analysis of Transformer-2? gene from H.cumingiiThe full-length cDNA sequence of Transformer-2? gene from H.cumingii was obtained and cloned.It contains a 92 bp 5? untranslated region and a 881 bp 3? untranslated region.The open reading frame is 894 bp and encodes 297 amino acids.The predicted relative molecular weight of the protein is 3,4661.2,the theoretical isoelectric point is 11.02,no signal peptide or transmembrane structure,includeing a RNA recognition motif(RRM)and a serine/arginine enrichment region(SR).Fluorescent quantitative results showed that the expression of Transformer-2? gene in male tissues was higher than that in corresponding female tissues.Transformer-2? gene was presumed to have different splicing complex in male and female,which led sex to different directions.Expression during the period found that there was an expression peak at about 3 days of embryonic stage indicating that it may play an important role in embryonic development.In situ hybridization showed that there were signals both in female and male gonads,indicating the bidirectional splicing function of the gene.2.Cloning and expression analysis of ?-catenin gene from H.cumingiiThe cDNA sequence of ?-catenin gene was cloned and the full length was 5544 bp.The open reading frame was 2463 bp.It encoded 820 amino acids,including 1355 bp 5'untranslated region,and 1726 bp 3'untranslated region.It had on signal peptides and transmembrane structure.In the center of the protein domain,there are 12 imperfect armadillo repeat domains(ARM),and this domain is highly conserved among species.Quantitative fluorescence results of male and female tissues showed that in addition to the mantle,the expression of female tissues was significantly higher than that of males(P<0.01).The ?-catenin gene itself was a pro-ovariety gene,indicating that it plays an important role in the process of female gonadal development.Expression during the period showed a peak on the third day of the embryonic period which was speculated to participate in the embryonic development process.The in situ hybridization signal is stronger in females than in males.In conclusion,the ?-catenin gene plays a decisive role in the formation of female sex,but it is not necessary in males.3.Cloning and expression analysis of SOX2 gene from H.cumingiiThe cDNA sequence of the SOX2 gene was cloned and its sequence length was 1840 bp,its 5' UTR was 163 bpand its 3' UTR was 1005 bp,including a 672 bp open reading frame which encodes 223 amino acids.There is an AATAAA box and a PolyA tail at the 3' end and 9 ATTTA units within the 3' UTR.The molecular weight is 25136.15 u,the theoretical isoelectric point is 10.19,the average hydrophilicity coefficient is-1.243,and glutamine accounts for the highest proportion in the amino acid composition,which is 10.3%.There is no signal peptide and transmembrane structure.The amino acid sequence contains an HMG-box composed of 72 amino acids and a SOXp domain composed of 48 amino acids,thus it can be concluded that it belongs to the SOX family genes.Fluorescent quantitative results showed that both male and female had the highest expression level in the foot,and almost no expression in the liver.Female gonads expressed significantly more than females,demonstrating the versatility of the SOX2 gene.The period expression also showed the highest in about 3 days which indicates it also participated in the development of the embryo.In situ hybridization showed that the female signal was stronger.4.Western blot analysis of COII geneOur research shows that there are two different sequences of M-type and F-type COII genes in the mitochondrial gene sequence of H.cumingii,of which the M-type COII gene 3' has 546 bp more sequences than F-type.We used this excess and guess that there may be a functional sequence to design specific antibodies to detect the expression of the male and female tissues.It was found that only the male gonads existed,the remaining tissues and females were basically absent.At the protein level,the gene was specifically expressed in males. |
PDF Full Text Request