| Cynoglossus semilaevis is an important aquaculture species that has a ZW/ZZ sex-determination system(females,ZW;males,ZZ).Some genotypic females may develop phenotypic male characteristics,and are known as pseudomales.In order to test the method of culling male and pseudomale seedings of C.semilaevis at 70 dph according to the morphological differences between different sex to improve the female percentage in the population.In order to test the scientific nature of the selection and selection,this study accurately identified the sex of C.semilaevis seedlings,measured morphological traits,and used the methods of data distribution test variance analysis,principal component analysis and discriminant analysis to test.And these pseudomales can develop mature testes and produce sperm.However,previous studies have shown that pseudomales cannot produce sperms bearing W chromosome(W sperm).For probing into the cause of the pseudomale do not produce W sperm,the age of 2.5 adult pseudomales and normal male gonads adopt the transcriptome study of proteomics and ubiquitin proteomic analysis,compare C.semilaevis pseudomales and males gonad after transcription and translation level differences,explores why pseudomales can’t produce sperm with a W chromosome.Results are follows:1)The results showed that the morphological traits of each gender group were normally distributed,and homogeneous in variance(P > 0.05).Variance analysis and principal component analysis of morphological traits showed that the differences between females and males were mainly concentrated on length and body height,and the mean length and body height of females were greater than those of males and pseudomales.The discriminant accuracy of females and males was 37% and 85%,respectively by using various standardized indicators.It was very difficult for pseudomales to discriminate,because they were close to males.The results show that the accuracy of male identification is relatively high,which can meet the requirements of early gender observation and identification of seedlings to improve the female percentage of C.semilaevis.2)Transcriptomic analysis and quantitative proteomic analysis.A total of 33,935 genes were identified and quantitated after calculating the expression level of each mapped gene.The intersection of DESeq2,Edge R,and NOISeq,identified 11,646 DEGs between the gonads of pseudomales and males.Of these,658 were upregulated and 10,988 were downregulated in pseudomales compared to males.Quantitative proteomic analysis,a TMT-labeled quantitative proteomics strategy was applied to investigate the characteristic proteins in the gonads of C.semilaevis pseudomales and males.A total of 5,846 proteins were quantified in this study.And 1,164 proteins were defined as DEPs between pseudomales and males,of which 424 were upregulated and740 were downregulated in pseudomales.3)Ubiquitin-proteome analysis.A label-free ubiquitin-modified quantitative proteomics strategy was used to study global ubiquitylation in the gonads of C.semilaevis pseudomales and males.Using this approach,we identified 410 proteins,721 peptides,and 795 ubiquitin-modified sites,of which 368 proteins,655 peptides,and 712 sites were quantified.And 471 peptides were defined as differentially expressed ubiquitin-modified proteins(DEUPs)between pseudomales and males,among which388 were upregulated and 83 were downregulated in pseudomales.Therefore,C.semilaevis pseudomales showed an overall higher ubiquitination level compared to males.4)Comparison of transcriptome and proteome data.Among the 5,759 quantified proteins that were searched against the NCBI nr database,3,794 were detected in the transcriptome datasets.The expression levels of all quantified proteins and their corresponding m RNAs showed a moderate correlation(r = 0.36,n = 3,794).The intersection of DEGs and DEPs included 254 genes/proteins,and a higher correlation was observed between the significantly differentially expressed proteins and m RNAs.It is intriguing that 225 of the total DEPs obtained showed the same direction of change at the transcriptional and translational levels,implying that the expression changes of these proteins are mainly driven by transcriptional changes.However,29 genes/proteins showed different directions of change between pseudomales and males,suggesting the existence of post-transcriptional regulation.5)Bioinformatics Analyses of the DEGs and DEPs.Functional enrichment of upregulated genes in pseudomales showed that the top-ranked biological processes GO terms(ranked by q-value)were non-coding RNA(nc RNA)processes and the negative regulation of m RNA processes,such as “nc RNA processing”,“ribosome biogenesis”,“mitotic cell cycle phase transition”,“r RNA processing”,and “ribonucleoprotein complex biogenesis”.In contrast to the pseudomale-biased gene enrichment results,the functional enrichment of male-biased genes showed that the top-ranked biological process GO terms were mainly the regulation of ion transport,signal transduction,and immune response.These terms included “regulation of signaling receptor activity”,“regulation of ERK1 and ERK2 cascade”,“regulation of cytosolic calcium ion concentration”,“regulation of acute inflammatory response”,“positive regulation of endocytosis”,and“positive regulation of cytosolic calcium ion concentration”.KEGG pathway enrichment analysis revealed that both DEGs and DEPs upregulated in the gonads of pseudomales were significantly enriched in “ribosome biogenesis in eukaryotes”.KEGG pathway enrichment analysis of the proteins corresponding to the DEUPs between pseudomales and males showed that “RNA degradation” and“spliceosome” were significantly enriched.6)Functional enrichment analysis of the differentially expressed genes/proteins showed that non-coding RNA metabolic processes and RNA degradation were more active in the gonads of pseudomales,and they showed higher levels of ubiquitination compared to males.What is interesting about the data is that,genes/proteins involved in meiosis did not show significant differences between pseudomales and males;however,there was an overall decrease in the expression levels of genes/proteins related to sperm morphogenesis in pseudomales.These genes included genes located exclusively on the Z chromosome,such as sperm flagellar protein 2(Spef2),doublesex and mab-3 related transcription factor 1(Dmrt1),doublesex and mab-3 related transcription factor 3(Dmrt3),and cilia-and flagella-associated protein 157(Cfap157).These findings characterized the transcriptional and proteomic profiles of the gonads of pseudomales and males,and suggested that W sperm were absent in pseudomales,possibly due to the disruption of sperm morphogenesis,resulting from round W sperm that lack the genes essential for sperm maturation.In word,the accuracy of male identification is relatively high,which can meet the requirements of early gender observation and identification of seedlings to improve the female percentage of Chinese tongue sole.These findings characterized the transcriptional and proteomic profiles of the gonads of pseudomales and males,and suggested that W sperm were absent in pseudomales,possibly due to the disruption of sperm morphogenesis,resulting from round W sperm that lack the genes essential for sperm maturation. |
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