| The razor clam,Sinonovacula constricta is one of the four major cultured mollusks in China,with the important economic value and artificial breeding benefits.Gonadal development is one of the important research fields in the developmental biology.The theories of gonadal development have been applied to the genetic breeding of aquatic economic animals by domestic and foreign scholars,which aim to obtain new varieties with larger bodies,faster growth speed,stronger fecundity and better meat quality.However,the mechanism of gonadal development in the bivalves is not clear,and the relevant information is limited,which is unfavorable to new variety breeding and sustainable development of mollusks.In the present study,sex-related genes,proteins and metabolites were identified by combining analysis of female and male gonadal transcriptome,proteome and metabolome.The full-length c DNA sequences of sex-related genes were cloned and sequences were analyzed,and the expression pattern and cell localization of sex-related genes/proteins in gonadal development were analyzed by q RT-PCR and immunofluorescence.Furthermore,si RNA interference and lentivirus overexpression were used to investigate the expression changes of sex-related genes,which is important to analyze the roles of sex-related genes in gonadal development.The regulation mechanisms of gonadal development of these genes in the razor clam were preliminarily analyzed,which will enrich the theory of gonadal development in mollusks.The major results of this study are presented as follows:1.In the gonadal transcriptome analysis of S.constricta,44 DEGs(differential genes)related to gonadal development were identified,including sex determination and gonadal development genes such as Dmrt4/5-1,Sox9,Fem-1b,Fem-1c and Beta-catenin,as well as genes Vg,CYP17A1 and TSSK involved in gametogenesis;The results of gonadal proteomic analysis showed 1462 DEPs(differential proteins)were identified,and 43 DEPs related to gonadal development were also identified.DEPs such as SPGT2,Tektin protein family,TSSK protein family and so on were significantly up-regulated in testis,and 17β-HSD12,Cytochrome P450,Beta-catenin,etc.were significantly up-regulated in ovary.A total of 409 DEMs(differential metabolites)were identified in gonadal metabolomes,and Uridine was significantly up-regulated in the ovary,which might be used as a potential sex differential metabolic marker.23 DEMs related to gonadal development were identified.16,16-Dimethyl prostaglandin A2,Progesterone,Leucylproline and DL-Arginine,etc.were significantly up-regulated in testis,and Pregnenolone,Methyltestosterone,etc.were significantly up-regulated in ovary.618 shared DEGs/DEPs were identified in the combining analysis of gonadal transcriptome and proteome,and 485 DEGs/DEPs showed the same expression trend.22 potential DEGs/DEPs related to gonadal development and KEGG pathways such as m TOR signal pathway and Notch signal pathway were obtained by correlation analysis of transcriptome and proteome.There are 31 KEGG pathways associated with transcriptome and metabolome,such as Steroid biosynthesis pathway related to gonadal development and so on.In the correlation analysis of gonadal proteome and metabolome,there are11 shared KEGG pathways.The results of this study provide basic information about the potential genetic mechanisms of gonadal development that will be useful for further studies of reproduction and breeding in S.constricta.2.There were 1458 bp in the Open Reading Frame(ORF)sequences of ScSox9 gene,encoding a protein of 485 amino acids.ScSox9 protein is highly conservative,containing the HMG frame of Sox protein family.ScSox9 gene was expressed in all tested tissues,and the expression levels in the testis were significantly higher than those in ovary(P<0.05);ScSox9 gene was all expressed in the four stages of gonadal development(proliferative stage,growing stage,mature stage and spawning stage),and the expression levels in testis were always higher than those in ovary at the same stages(P<0.05).ScSox9 gene was expressed the highest in the growing stages of testis.However,there was no significant change in the ovarian development cycle and the expression level remained low.The results of immunofluorescence showed that ScSox9 protein was mainly located in germ cells at different stages of gonadal development,such as oocytes,oocytes and mature oocytes of ovary,spermatogonia,spermatocytes and mature sperm of testis.After si RNA interference,the expression levels of ScSox9 gene in testis and ovary decreased significantly(P<0.01),indicating the effectiveness of si RNA interference.The expression of ScSox9 gene in testis and ovary all increased significantly(P<0.01)after lentivirus over-expression,indicating lentivirus over-expression vector ScSox9-OE was effective.The results presumed that ScSox9 gene might be involved in gonadal development,especially in the testis development and spermatogenesis.3.The ORF sequences of ScFem-1b and ScFem-1c genes were 1911 bp and 2035 bp respectively,encoding 636 and 622 amino acids.The amino acid sequence of ScFem-1b protein contained 6 Anyrin repeats(ANKs),and ScFem-1c protein contains 7 Ank repeats,which were the typical conservative domains of Fem family.ScFem-1b and ScFem-1c genes were all expressed in tested tissues,and the expression levels in testis was significantly higher than those in ovary(P<0.05).In testis,ScFem-1b gene was expressed highest in the mature stage,and the expression level of ScFem-1c gene was the highest in the growing stage.In ovary,the expression level of ScFem-1b and ScFem-1c genes remained low in the ovarian development cycle without significant difference.ScFem-1b and ScFem-1c proteins were both located in the cytoplasm of oogonium,oocytes and mature oocytes of ovary and spermatogoniums,spermatocytes and mature spermsof testis.The expression levels of ScFem-1b and ScFem-1c genes in testis and ovary decreased significantly after si RNA interference(P<0.01),and increased significantly after lentivirus over-expression(P<0.01).The results showed that ScFem-1b gene might play important roles in the testis functional maintenance and sperm spawning,while ScFem-1c gene might be involved in testis development and spermatogenesis.4.There were 1170 bp and 1125 bp in the ORF sequences of ScDmrt4/5-1 and ScDmrt4/5-2genes,respectively,encoding 389 and 374 amino acids,which contained the conserved domains DM and DMA.The results of amino acid homology sequence alignment and phylogenetic analysis showed that the two Dmrt genes could be identified as Dmrt4/5-1 and Dmrt4/5-2.ScDmrt4/5-1 and ScDmrt4/5-2 genes were both expressed in all tested tissues,and the expression levels in testis were all significantly higher than those in ovary(P<0.05).ScDmrt4/5-1 gene was expressed the highest in the mature stage of testis,and expressed the highest in spawning stage of ovary.In testis,ScDmrt4/5-2 gene was expressed highest in the mature stage,and the expression level was low in each ovarian development stage without significant difference.ScDmrt4/5-1 and ScDmrt4/5-2protein were both mainly located in germ cells at different stages of gonadal development,such as oocytes,oocytes and mature oocytes of ovary,spermatogonia,spermatocytes and mature sperm of testis.The results implied that ScDmrt4/5-1 and ScDmrt4/5-2 might be mainly involved in the testis development and functional maintenance. |
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