线纹尖塘鳢性别相关的2个dmrt基因结构特征及表达规律

Structure and expression of two sex-related dmrt genes in Oxyeleotris lineolata

关键词:  线纹尖塘鳢  Oxldmrt1  Oxldmrt3  基因结构  基因表达  荧光原位杂交

Key words:  Oxyeleotris lineolata  Oxldmrt1  Oxldmrt3  Gene structure  Gene expression  Fluorescence in situ hybridization

摘要:

线纹尖塘鳢(Oxyeleotris lineolata)具有典型性别生长二态性，雄鱼生长优势显著，doublesex and mab-3 related transcription factor (DMRT)家族是一个与性别决定相关的转录因子家族。基于线纹尖塘鳢性腺转录组数据，共获得2个dmrt基因的cDNA序列，分别命名为Oxldmrt1和Oxldmrt3，并采用PCR技术扩增验证2个基因的cDNA序列。运用生物信息学方法分析2个基因序列结构特征，结果显示，Oxldmrt1和Oxldmrt3开放阅读框分别为903 bp和1 363 bp，分别编码300个氨基酸和453个氨基酸；OxlDMRT1属于碱性蛋白，而OxlDMRT3属于酸性蛋白；2个基因均含有高度保守的DM结构域，OxlDMRT3还存在DMA结构域。氨基酸聚类分析显示，脊椎动物不同DMRT家族都是独立聚类，OxlDMRT1属于DMRT1家族，OxlDMRT3属于DMRT3家族，DMRT1家族最先聚类，再和DMRT3家族聚类。利用实时荧光定量PCR技术(real-time quantitative PCR, RT-qPCR)分析2个dmrt基因在雌鱼和雄鱼8个组织的表达水平，结果显示，2个基因在精巢中的表达量均显著高于其他组织(P<0.01)，Oxldmrt3在脑中也有少量表达；利用RT-qPCR分析2个dmrt基因在早期不同发育时期的表达谱，显示2个基因在受精卵中的表达量均最高，Oxldmrt1在眼囊期的表达量最低，而Oxldmrt3在出膜7 d时的表达量最低。利用荧光原位杂交(fluorescence in situ hybridization, FISH)对2个基因在精巢中的表达进行定位，显示2个基因在精巢中表达部位一致，均在精原细胞中有较强的表达信号。综上所述，Oxldmrt1和Oxldmrt3均在线纹尖塘鳢性腺胚胎发育阶段和精巢发育过程中起调节作用，而Oxldmrt1还可能参与胚胎后期性别决定和性别分化调控过程，Oxldmrt3还可能参与神经系统发育。本研究为线纹尖塘鳢性别决定与性别分化相关的分子机制研究提供了参考。

Abstract:

Oxyeleotris lineolata is a valuable economic fish cultured in Australia and countries of Southern Asia. It exhibits sex-related growth dimorphism; the males grow faster and have larger body size than females. Therefore, the efficient development of sex control breeding will be helpful to increase the yield and output value. However, the molecular mechanism regarding sex determination and gonadal differentiation of O. lineolata is poorly understood. Doublesex and mab-3 related transcription (DMRT) factors are known for their contributions in sex determination and differentiation. In this study, the Oxldmrt1 and Oxldmrt3 cDNA sequence was obtained from gonad transcriptome of O. lineolata, identified using PCR. The open reading frame (ORF) of Oxldmrt1 and Oxldmrt3 were 903 bp and 1 363 bp, encoding 300 and 453 amino acids, respectively. OxlDMRT1 belongs to basic proteins, whereas OxlDMRT3 belongs to acidic proteins. The two genes contain a highly conserved DM domain, and OxlDMRT3 also has a DMA domain. Phylogenetic analysis showed that OxlDMRT1 belonged to the DMRT1 family and OxlDMRT3 belonged to the DMRT3 family. The different DMRT families in vertebrates were clustered independently, with the DMRT1 family clustering first followed by the DMRT3 family. The expression levels of two genes in eight tissues of male and female fish were analyzed using RT-qPCR. The results showed that the expression levels of two dmrt genes were most highly expressed in the testis. The expression profiles of two dmrt genes at different developmental stages were analyzed using RT-qPCR. The results showed that the expression levels of two genes were highest in fertilized eggs, the expression level of Oxldmrt1 was the lowest in the optic capsule stage, and Oxldmrt3 was the lowest in the seven days post hatching. The expression and localize of the two genes in the testis were analyzed using fluorescence in situ hybridization (FISH). The results showed that the expression location of the two genes in the testis were identical, and both had strong expression signals in the spermatogonia. In conclusion, Oxldmrt1 and Oxldmrt3 are important in the early embryonic stage and testis development. Oxldmrt1 may also participate in regulating sex determination and differentiation in the late embryonic stage. Oxldmrt3 may also be involved in nervous system development. This study lays a foundation for the molecular mechanism of sex determination and sex differentiation in O. lineolata.