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| 军曹鱼性别决定与分化相关基因的鉴别及表达模式分析 |
| Identification and expression pattern analysis of genes related to sex determination and sex differentiation in cobia (Rachycentron canadum) |
| 投稿时间：2024-03-21 修订日期：2024-04-29 |
| DOI： |
| 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:国家重点研发计划项目 |
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| 中文摘要: |
| 为探讨军曹鱼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、Cyp19a2、Dmrt1、Gdf6a/6b、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)技术分析各基因在军曹鱼成鱼的组织表达分布情况及在性腺成熟过程中的表达量变化模式。染色体定位结果表明,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。共线性分析结果表明仅有军曹鱼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。系统进化分析结果显示,军曹鱼Amh、Dmrt1、Cyp19a2和Gdf6a与?鱼的亲缘关系最近、Gdf6b与日本花鲈的亲缘关系最近、而Sox9a和Sox9b与高体鰤的亲缘关系最近。qRT-PCR结果显示,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；Cyp19a2、Sox9a和Sox9b在脑、皮肤和心脏等组织中均有不同程度的表达；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),且在性腺成熟过程中除Sox9b显著下降之外均呈显著升高后下降的变化趋势。上述结果表明这些基因可能在军曹鱼精巢发育过程发挥重要作用,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, sex differentiation is 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), and a combination of both. Among them, GSD can be subdivided into two types: chromosomal sex determination and polygenic sex determination. Chromosomal sex determination is mainly the main sex-determining (SD) gene located on the sex chromosomes controlling the sex of the offspring; 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.
Rachycentron canadum is one of the most important seawater net-pen cultured fishes in the south of China. It has been shown that no heteromorphic sex chromosomes are found in the chromosomal karyotype of Rachycentron canadum, and there are no associated sex markers to distinguish between male and female sexes. Currently, there are limited reports on the sex determination and differentiation of Rachycentron canadum, and it is difficult to rely on the external morphology of Rachycentron canadum to differentiate the sexes of male and female Rachycentron canadum parents during the breeding process. In this study, based on the whole genome information of Rachycentron canadum obtained in the previous period, some reported fish sex determination and sex differentiation related genes (Amh、Cyp19a2、Dmrt1、Gdf6a/6b、Sox9a/9b) were identified from them; the genes were firstly subjected to chromosomal localisation, 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 carried out, and the amino acid sequences were compared using Protein BLAST on the NCBI database, and the amino acid sequences of other scleractinian fishes and higher vertebrates were downloaded separately, and the phylogenetic tree was constructed by 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 Rachycentron canadum 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 Rachycentron canadum, as well as providing a reference for the screening of sex markers in Rachycentron canadum. The main results are presented as follows.
Chromosomal localisation results showed that the five genes were located on seven different chromosomes of Rachycentron canadum without clustering. The results of covariance analyses showed that three of the five genes in Rachycentron canadum were covariant with XX/XY, ZZ/ZW and polygenic sex-determining fish, of which only the covariant gene for Amh was localised on the Y chromosome of XX/XY fish, all other genes are localised to autosomes. The results of interchromosomal covariance analyses showed homology between Chr7, where Amh is located, and the Y chromosome of XX/XY type fishes. 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, Cyp19a2 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 Rachycentron canadum, which may be male-specific genes of Rachycentron canadum; Gdf6a and Gdf6b were both highly expressed in the testis and skin, which may be male-biased genes; Cyp19a2, Sox9a and Sox9b are expressed to varying degrees in tissues such as brain, skin and heart. In the gonads of Rachycentron canadum 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 of them showed a trend of significant increase 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 the development of spermathecae in Rachycentron canadum, and the results may lay a foundation for revealing the regulatory mechanism of gonadal development in Rachycentron canadum. |
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