Integrative Omics Analysis Of Sex Reversal In Cynoglossus Semilaevis

Sex reversal is a complex biological phenomenon that exists widely in insects,amphibians,reptiles,and fish.Sex reversal can change the sex ratio of a population and increase breeding difficulty.The Cynoglossus semilaevis is one of the important marine aquaculture species of the Yellow Sea and the Bohai Sea of China.It is favored by consumers and farmers because of its delicious and fast growth rate.Cynoglossus semilaevis is of ZW-type sex determination,with females being heterogametic(ZW?)and males being homogametic(ZZ?),and the difference between the sexes is obvious.The growth rate of females is about 2 to 4 times that of males.When they reach 50-120 days of age,some females can be reversed into males,that is,pseudomales.This leads to a reduction in female rates in the population,increasing fry costs and management costs.This study performed a detailed functional analysis of gene and protein expression in female and pseudomale gonad combining transcriptome and proteomics analysis to reveal genes and their regulatory networks related to sexual reversal.The main research and results are as follows:1)Transcriptome analysis: Illumina Hi Seq Xten sequencing method was used to sequence the transcriptome of female and pseudomale gonads.A total of 46,317 transcripts and 25,761 genes were obtained from the sequencing results.Log2 FC ? 2and pvalue <0.05 were used as the criteria for differentially expressed genes.A total of1893 differentially expressed genes between female and pseudomale.Among the 1893 differentially expressed genes,sperm-binding proteins(LOC103393374 and LOC103396071)and Foxl2 were overexpressed in female gonads.The expression of Foxl2 in females was 44.21 times that of pseudomales,indicating the expression of Foxl2 It is highly related to gonad differentiation;in addition,the two genes Dmrt1 and Dmrt3 are highly expressed in pseudomale gonads.Functional enrichment analysis and pathway enrichment analysis indicate that initiation of DNA replication,wnt signaling pathways,cell development and oocyte maturation are four terms mainly enriched in female,and cell cycle,progesterone-mediated oocyte maturation and oocyte meiosis are three pathways enriched.While concentrated and cytoplasmic are two terms enriched in pseudomale,as well as adipose cytokine signaling is the significant pathways enriched.2)Proteomics analysis: Quantitative proteomics can be used to screen and finddifferentially expressed proteins between samples caused by any factor,qualify and quantify certain key proteins with bioinformatics analysis.In this study,TMT(Tandem Mass Tags)quantitative labeling technology was used to realize the qualitative and quantitative research of female and pseudomale gonadal proteomes.Using FC > 1.5 or FC < 2/3 and p-value < 0.05 as the criteria,a total of 324 differentially expressed proteins were found,of which 184 differentially expressed proteins were highly expressed in pseudomale and 140 differentially expressed proteins were in female.Female highly expressed proteins are enriched in cytoplasmic m RNA processing body assembly,RNA processing,and protein aggregation;enrichment in RNA degradation,spliceosome,and metabolic pathways.However,the most important term for the enrichment of pseudomale highly expressed proteins is to regulate the transcription and extension process using DNA as a template.Protein processing in the endoplasmic reticulum,PPAR signaling pathway and fatty acid biosynthesis are the three main pathways for the enrichment of pseudo-and-highly expressed proteins.3)Integrative analysis: Through the integrative analysis of transcriptome and proteomics,the size of the post-transcriptional regulation of different transcripts can be found.This part of the study compared the amino acid sequence of m RNA and protein.A total of 52 genes were selected from the comparison results,and their m RNA sequences and amino acid sequences of proteins were uniform.In addition,the Spearman correlation coefficients were calculated for the average fold changes of m RNA and protein expressions,indicating that the expression levels of these genes at the transcription level and protein level have a moderate degree of correlation(correlation coefficient ? = 0.59 and p <0.05).Among these 52 related genes,the m RNA expression levels and protein abundances of 46 differentially expressed genes are consistent(including 33 genes up-regulated in pseudomale individuals and 13 genes down-regulated in pseudomale individuals).This data indicates that 88% of the differential genes show consistent expression at both the transcriptional and protein levels.The genes whose transcription and protein levels are upregulated include YOD1deubiquitinase(Yod1),ubiquitin recognition factor(Ufd1)in ER degradation and MINDY lysine deubiquitinase 3(Mindy3).The down-regulated genes \ are Arhgdia,Zar1 and so on.The expression of six genes at the transcription and protein levels is not consistent.These results indicate that there is a complex post-transcriptional regulatory mechanism between m RNA and protein.