泰国斗鱼Dmrt1基因的部分cDNA克隆及表达分析

Molecular Cloning,Partial Sequence and Tissue Expression of Dmrt1 Gene in Siamese Fighting Fish Betta splendens

Doublesex和Mab-3相关转录因子1(Dmrt1)隶属于DMRT家族,在脊椎动物性别决定、性别分化和性腺发育中具有重要作用。利用RACE克隆技术从泰国斗鱼脑中克隆Dmrt1基因的部分cDNA序列,分别采用半定量PCR和实时荧光定量PCR检测Dmrt1基因的组织分布和胚胎发育过程的表达变化。试验结果显示,克隆的泰国斗鱼Dmrt1基因的部分cDNA序列长为1037 bp,其中开放阅读框长为798 bp,编码265个氨基酸残基;进一步构建Dmrt1系统进化树,结果显示,泰国斗鱼与龟壳攀鲈亲缘关系最近;半定量PCR和实时荧光定量PCR结果显示,Dmrt1基因的表达具有明显的组织特异性及性别差异,在雄性个体中,Dmrt1基因在精巢和脾脏中特异表达,而在雌性个体中,Dmrt1基因在心脏中高表达,其次在脑、垂体、脾脏、肌肉、肾脏和肠道中也有少量表达,但未在性腺和肝脏中表达。在胚胎发育过程中,Dmrt1基因在受精卵的表达水平最高,而在原肠胚至心跳期表达量较低。Dmrt1可能在调控泰国斗鱼的雄性个体的生殖发育过程中发挥着重要作用。

Doublesex and Mab-3 related transcription factor 1(Dmrt1),a member of DMRT family,plays critical roles in sex determination,sex differentiation and gonadal development.The molecular cloning of a partial sequence of Dmrt1 was performed in the brain tissue of Siamese fighting fish Betta splendens by RACE,and Dmrt1 gene expression pattern was detected in all tissues and embryonic development of Siamese fighting fish by semi-quantitative RT-PCR and real-time quantitative RT-PCR.Siamese fighting fish had Dmrt1 consisting of 1037 nucleotides,with a 798 bp length of open reading frame(ORF),and coding 265 amino-acids.Phylogenetic analysis showed that Dmrt1 in Siamese fighting fish was more closely related to climbing perch Anabas testudineus.In addition,tissue-specific and sexual dimorphism expression profile of Dmrt1 was detected in Siamese fighting fish.In males,Dmrt1 was expressed in the testis and spleen exclusively.In females,however,Dmrt1 was highly expressed in the heart,weakly expressed in the brain,pituitary,spleen,muscle,kidney and intestine,without expression in ovary and liver.Moreover,during the period of embryonic development,Dmrt1 was predominately expressed in the fertilized eggs,and weakly from the gastrula stage to heart beating stage.The findings indicate that Dmrt1 may play an important role in male germ cell development,and reproductive regulation in spermatocyte maturation in Siamese fighting fish.