| 黄玉琴,徐佳乐,杨云生,张健东,黄建盛,马骞,谢瑞涛,陈刚.军曹鱼性别决定与分化相关基因的鉴别及表达模式分析.渔业科学进展,2025,46(3):1-11 |
| 军曹鱼性别决定与分化相关基因的鉴别及表达模式分析 |
| Sex determination and differentiation-related gene identification and expression pattern analysis in the cobia (Rachycentron canadum) |
| 投稿时间：2024-03-21 修订日期：2024-04-29 |
| DOI：10.19663/j.issn2095-9869.20240321003 |
| 中文关键词: 军曹鱼 性别 性腺发育 基因表达 系统进化 |
| 英文关键词: Rachycentron canadum Genders Gonadal growth Gene expression Phylogenetic |
| 基金项目:国家重点研发计划(2022YFD2401203)、广东省现代化海洋牧场适养品种核心技术攻关项目(2024-MRB-00-001)共同资助 |
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| 中文摘要: |
| 为探讨军曹鱼(Rachycentron canadum)性腺发育的调控机制，本研究从军曹鱼全基因组数据中筛选出5个与鱼类性别决定与分化相关的基因(Amh、Cyp19a1b、Dmrt1、Gdf6a/6b和Sox9a/9b)，利用生物信息学方法对其进行染色体定位、共线性分析和系统进化分析，并利用实时荧光定量PCR (qRT-PCR)分析各基因在军曹鱼成鱼各组织中的表达分布情况及在性腺成熟过程中的表达量变化模式。染色体定位结果显示，5个基因分别位于军曹鱼7条不同染色体上，不存在集簇现象。共线性分析结果显示，仅有军曹鱼Amh的共线性基因定位于XX/XY型鱼类的Y染色体上，其他基因均定位于常染色体上。系统进化分析结果显示，军曹鱼Amh、Dmrt1、Cyp19a1b和Gdf6a与䲟鱼(Echeneis naucrates)的亲缘关系最近，Gdf6b与日本花鲈(Lateolabrax japonicus)的亲缘关系最近，而Sox9a和Sox9b与高体 (Seriola dumerili)的亲缘关系最近。qRT-PCR结果显示，在军曹鱼Ⅲ~Ⅴ期性腺中，5 个基因在精巢中的表达量均极显著高于同时期卵巢(P<0.01)；此外，除Sox9b在性腺成熟过程中表达量显著降低，其余4个基因的表达水平均呈先显著升高而后显著降低的变化趋势。5个基因在不同组织的表达情况显示，Amh和Dmrt1仅在军曹鱼精巢中特异性表达，可能为军曹鱼雄性特异基因；Gdf6a和Gdf6b均在精巢和皮肤中高表达，可能为雄性偏向基因；Cyp19a1b、Sox9a和Sox9b在脑、皮肤和心脏等组织中均有不同程度的表达。上述结果表明，这些基因可能在军曹鱼精巢发育过程中发挥重要作用，相关研究结果可为揭示军曹鱼性腺发育的调控机制奠定基础。 |
| 英文摘要: |
| While sex determination in animals refers to the mechanism by which individuals develop into females or males, it presents the process by which undifferentiated gonads develop into mature testes or ovaries, with sex determination as a prerequisite. Among vertebrates, fish are at a primitive stage in the process of sex evolution, and their sex-determining mechanisms are primitive, diverse, and variable, and share the sex-determining modalities of all vertebrates. The mechanisms of sex determination in fish are complex and diverse, including genetic sex determination (GSD), environmental sex determination (ESD), or a combination of both. Among them, GSD can be subdivided into two types: chromosomal sex determination and polygenic sex determination. Chromosomal sex determination is the main sex-determining (SD) gene located on the sex chromosomes that determines the sex of the offspring; whereas polygenic sex determination is the combination of multiple genes on the genome or the cumulative effect of multiple alleles on a pair of chromosomes determining the sex of the offspring; virtually all are the result of the direct or indirect action of genes.
The cobia (Rachycentron canadum) is an important seawater net-pen cultured fish species in southern China. No heteromorphic sex chromosomes exist in the chromosomal karyotype of cobia, and there are no associated sex markers to distinguish between males and females. Furthermore, the sex determination and differentiation of cobia is limited, and it is difficult to rely on the external morphology of cobia to differentiate the sexes of male and female cobia parents during the breeding process. In this study, based on the whole genome information of cobia obtained in the previous period, some reported fish sex determination and sex differentiation related genes (Amh, Cyp19a1b, Dmrt1, Gdf6a/6b, and Sox9a/9b) were identified. The genes were subjected to chromosomal localization, whole genome and gene annotation files of XX/XY-type Oryzias latipes and Oreochromis niloticus, ZZ/ZW-type Cynoglossus semilaevis and Oreochromis aureus, and polygenic sex-determined zebrafish were then downloaded from the NCBI database for inter-species covariance analysis. Subsequently, phylogenetic analyses were performed, and the amino acid sequences were compared using Protein BLAST on the NCBI database. The amino acid sequences of other Osteichthyes and higher vertebrates were downloaded separately, and the phylogenetic tree was constructed using the neighbour-joining (NJ) method; followed by real-time fluorescence quantitative PCR (qRT-PCR) to detect the distribution of the expression of these genes in the tissues of adult cobia and their expression levels in gonadal tissues of different developmental stages, with the aim of screening for sex-specific genes, which will provide research materials for further exploring the mechanism of sex determination and differentiation of cobia, as well as providing a reference for the screening of sex markers in cobia.
Chromosomal localization revealed that the five genes were located on seven different chromosomes of cobia without clustering. The results of covariance analyses showed that three of the five genes in cobia were covariant with XX/XY, ZZ/ZW and polygenic sex-determining fish, of which only the covariant gene for Amh was localized on the Y chromosome of XX/XY fish, all other genes are localized to autosomes. The results of interchromosomal covariance analyses showed homology between Chr7, where Amh is located, and the Y chromosome of XX/XY type fish. Phylogenetic analyses showed that each of the five genes clustered together (Sox9a and Sox9b clustered together, Gdf6a and Gdf6b clustered together), and that the scleractinians formed a separate branch in each of the gene clusters, independent of other higher vertebrates; Amh, Dmrt1, Cyp19a1b and Gdf6a were the closest relatives to Echeneis naucrates, Gdf6b was the closest relative to Lateolabrax japonicus, and Sox9a and Sox9b were the closest relatives to Seriola dumerili.
The qRT-PCR results showed that Amh and Dmrt1 were specifically expressed only in the testis of cobia, which represent the male-specific genes of cobia. Gdf6a and Gdf6b were both highly expressed in the testis and skin, which may be male-biased genes. Finally, Cyp19a1b, Sox9a and Sox9b were expressed to varying degrees in tissues such as brain, skin, and heart. In the gonads of cobia at stages Ⅲ to Ⅴ, the expression of all five genes in the testis was extremely significantly higher than that in the ovary at the same period (P<0.01), and all were significant increased followed by a decrease in gonadal maturation, except for a significant decrease in Sox9b. The above results suggest that these genes may play important roles in spermathecae development in cobia, and the results may lay a foundation for revealing the regulatory mechanism of gonadal development in cobia. |
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