| Takifugu obscurus is one of the most important freshwater aquaculture species in southern China.The testis of T.obscurus has high nutritional value,large-scale cultivation of all male T.obscurus can significantly improve the economic benefits of T.obscurus cultivation in China.Unfortunately,there are few studies on the sex regulation mechanism and sex control technology of T.obscurus.Using transcriptome,bioinformatics and molecular biology methods,this study preliminarily explored the molecular regulation mechanism of sex-related genes in the process of early development of T.obscurus.The main results are as follows:1.Identification of genetic sex of juvenile in T.obscurus.The genetic sex of T.obscurus was rapidly detected by ARMS-PCR.According to a missense mutation SNP locus linked to sex in amhr2 kinase domain of T.obscurus,specific allele primers were designed,that is,different kinds of mismatched bases were designed at different base sites at the 3'end of downstream PCR primers,and specific primers were screened.By optimizing the reaction system and conditions,an ARMS-PCR method was established for identifying the genetic sex differences of juvenile in T.obscurus.In addition,in order to determine the feasibility of using ARMS-PCR to identify the genetic sex of juvenile in T.obscurus,we also used direct sequencing and tissue section methods to identify the genetic sex and phenotypic sex of the samples.The results showed that the genetic sex of T.obscurus could be distinguished by ARMS-PCR,and the results of identification by ARMS-PCR were the same as those by direct sequencing,and also consistent with the phenotypic sex identified by tissue section.2.Transcriptome analysis of gonadal tissues in the early development stage of T.obscurusSequencing and bioinformatics analysis of gonadal differential transcriptome during 60~150 dph in T.obscurus were carried out.The results showed that the clean reads of ovary and testis were 54229278 and 52089726 respectively,of which 4358138 and 4387845 clean data were located in the reference genome respectively.33247 unigenes,including 9890 new genes,were obtained after splicing and assembling.The results of DEGs analysis showed that the number of DEG in ovaries was generally higher than that in testis during adjacent developmental stages,and the number of DEGs between sexes was much higher than that within sexes.The Pathway with significant enrichment was further screened.24 metabolic pathways involved in sex differentiation and gonadal development were obtained,especially in the metabolic pathways such as neuroactive ligand-receptor interaction,MAPK signaling pathway,and cell cycle,there are more differentially expressed genes.Acco rding to the DEGs in these metabolic pathways,8 genes with high expression in ovary,such as cyp19 a,cyp19b and hsd17b1,and 15 genes with high expression in testis,such as dmrt1,cyp11a1 and hsd3 b,were screened from the differentially expressed genes in metabolic pathways.These candidate genes provide important information for the study of the molecular regulation mechanism of sex determination and differentiation and the development of sex marker genes in T.obscurus.3.Molecular cloning and structural analysis of sex-related genes in T.obscurusBased on the the transcriptome data and known conservative sequences of puffer fish,degenerate primers of amhr2,cyp19 a,dmrt1,sox9 a and sox9 b genes were designed respectively.The full-length sequences of the five genes were cloned and their sequence structure and related bioinformatics characteristics were analyzed at the molecular level.The full-length of amhr2 gene cDNA sequence of T.obscurus was 1868bp(NCBI login number: MH218814),encoding 513 amino acids.The full-length of cyp19 a cDNA sequence was 1786bp(NCBI login number: MH218815),encoding 514 amino acids.The full-length of dmrt1 gene cDNA sequence was 1620bp(NCBI login number: MH218816),encoding 294 amino acids.The full-length of sox9 a cDNA sequence was 1274bp(NCBI login number: MH218818),encoding 227 amino acids,and the full-length of sox9 b cDNA sequence was 1943bp(NCBI login number: MH218819),encoding 489 amino acids.The results of basic physicochemical properties showed that Amhr2,Dmrt1,Sox9 a and Sox9 b all belonged to unstable hydrophilic proteins,while Cyp19 a belonged to stable hydrophilic proteins.The results of homology and phylogenetic analysis showed that Cyp19 a and Dmrt1 had the highest homology and the closest relationship with Takifugu rubripes and Takifugu niphobles,while Amhr2 and two Sox9 had the highest homology and the closest relationship with T.rubripes.4.Expression study of sex-related genes in T.obscurusThe expression of amhr2,cyp19 a,dmrt1,sox9 a and sox9 b in gonadal tissues of juveniles during 60~150 dph and different tissues of 1-year-old fish were quantitatively analyzed.The results of tissue expression showed that amhr2 was only expressed in gonads,and the expression of amhr2 in ovaries was significantly higher than that in testes.The expression of cyp19 a gene is mainly in muscle and ovary,followed by a small amount in other tissues.The expression of dmrt1 gene is mainly expressed in male fish,and its expression pattern is testicular-specific.The expression of sox9 a and sox9 b were the highest in the hypothalamus of female fish,and also high levels of expression in other tissues such as brain,pituitary,liver and eye.The results of gonadal tissue expression at the early stage of development showed that the expression of sox9 a and sox9 b in the testis at 90 dph male fish was significantly increased.The expression of cyp19 a in the ovary at 120 dph female fish was significantly increased,while the expression of dmrt1 in the testis at 120 dph male fish was significantly increased;and the expression of amhr2 in the ovary and testis at 150 dph was significantly increased.The above quantitative results indicate that these five genes are involved in gonadal development of T.obscurus,among which cyp19 a is related to ovarian development,dmrt1 is related to testicular development,and sox9 a and sox9 b are related to nerve regulation. |
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