Screening And Functional Study Of Sex Determining Genes In Portunus Trituberculatus

Portunus trituberculatus is the main caught and mariculture crab in China,which has important economic value.Generally male crabs grow faster before sexual maturity,but female crabs grow faster after sexual maturity.The market value of female crabs with well-developed ovaries is often several times as that of male crabs.Therefore,the research on sex determination of P.trituberculatus and the establishment of sex control technology will significantly improve the efficiency of monasexual culture.Due to the complexity of crustacean genomes,studies on the mechanism of sex determination are relatively backward.In recent years,studies on sex QTL mapping,identification of sex determination types and development of sex identification techniques have been reported,which laid a foundation for the interpretation of sex determination mechanism in P.trituberculatus.However,the lack of research progress on sex determination genes in P.trituberculatus restricts the comprehensive analysis of sex determination mechanism of this species and greatly restricts the research and development of sex control technology.On the basis of previous work,this study intends to carry out screening and functional studies of sex-determination related genes in P.trituberculatus.Through comparative transcriptome study and combined with SD region location information,sex-determination related genes are accurately screened.Further,RNAi and other molecular biological techniques were used to study the mechanism of key candidate genes in sex determination.The main results are as follows:1.Sex identification and transcriptome sequencing of Portunus trituberculatus larvae during developmentDNA and RNA materials were prepared from P.trituberculatus at 6developmental stages from zoea to juvenile phase 2 by co-extraction method.The DNA template was used to identify individual sex in combination with the sex identification technology established in the early stage of the laboratory.Then,the male and female RNA of each period were mixed separately to prepare the male and female RNA mixing pool.Transcriptome sequencing was performed on the RNA mixing pool based on Illumina sequencing.The sequenced sequences were compared to the reference genome and the prediction of new transcripts was performed,and then gene expression levels were quantified.A total of 86.84 G clean bases were obtained in the transcriptome.The mean values of Q20 and Q30 were96.355 and 91.6175,respectively,and the average percentage of genome mapping was 84.21%.2.Screening and expression analysis of genes related to sex determination in Portunus trituberculatusA total of 1915 male and female differential genes(DEG)were screened by comparative transcriptome analysis.GO enrichment analysis results showed that DEG was mainly enriched in the "biological process" of chitin metabolism and glucose-containing compound metabolism.KEGG enrichment analysis showed that DEG was mainly enriched in DNA replication,retinol metabolism and neuroactive ligand-receptor interactions.A total of 8 candidate genes were screened by combined analysis of DEG and SD regions,and the expression levels of 3 genes were higher in males than in females.It is particularly noteworthy that Contig475.13 is not expressed at all stages in females but only expressed in males.Named PtDMY,we believed that this gene is a key candidate gene for sex determination in P.trituberculatus.3.Functional study of PtDMY gene in sex determination in Portunus trituberculatusPrimers were designed for different regions of PtDMY gene,and conventional PCR amplification was performed using male and female genomic DNA templates.It was found that PtDMY gene was only amplified in males,and almost no amplification was found in females,which was closely linked to sex.Rt-q PCR and WB were used to detect the expression of the gene at different developmental stages at transcriptional level and translation level,respectively.The results showed that the gene was mainly expressed in males and the highest expression was found in zoea stage,suggesting that this stage was the key stage for sex determination.The results of tissue expression distribution also showed that the gene was only expressed in males and showed a pan-tissue expression pattern,and the highest expression level was found in the androgenic gland.Further fluorescence in situ hybridization and immunofluorescence showed that it was mainly located in the testis(spermatocytes)and androgenic glands.The expression of PtDMY gene was successfully knocked down by RNAi technology,and it was found that the known sex-related genes IAG,Dmrt and SPATA showed a significantly down-regulated expression trend after PtDMY knockdown.It was speculated that PtDMY gene was a key gene upstream of the sex-determined pathway.