Molecular cloning and expression analysis of Dmrt4 gene in Megalobrama amblycephala | 团头鲂Dmrt4基因的克隆与表达分析

chinois. 为研究团头鲂Dmrt4基因的可能作用,采用RACE-PCR技术克隆了团头鲂卵巢Dmrt4基因的cDNA全长序列,采用实时荧光定量PCR技术对其胚胎及出膜后30d内各期、成鱼不同组织、雌雄性腺各分期的表达进行研究。结果表明,团头鲂Dmrt4基因至少存在2种不同的剪接形式,分别命名为Dmrt4a和Dmrt4b。其中,Dmrt4acDNA全长1 265bp,编码352个氨基酸,含DM和DMA 2个结构域;Dmrt4b cDNA全长1 081bp,编码281个氨基酸,仅含DMA结构域。氨基酸序列同源性分析显示,团头鲂Dmrt4与牙鲆、奥利亚罗非鱼的同源性最高。实时荧光定量PCR结果表明,Dmrt4基因在团头鲂胚胎期至出膜后30d内均有表达,且在受精后32h的表达量最高;成鱼卵巢的表达量显著高于其他组织（P〈0.01）;Ⅱ期卵巢的表达量显著高于其他时期,也显著高于精巢各分期（P〈0.01）。上述结果表明,Dmrt4基因可能对团头鲂卵母细胞初期的生长起到重要作用,在雌鱼性腺发育过程中亦发挥一定的作用。

anglais. The Dmrt4 gene was cloned from the ovary of Megalobrama amblycephala via using rapid amplification of eDNA ends （RACE） PCR. The relative expression of Dmrt4 gene at different sta- ges of embryonic and post-embryonic development, in different adult tissues, and at different stages of gonad in M. amblycephala were analyzed by quantitative real-time PCR. The results showed that the Dmrt4 gene in M. amblycephala contains at least two isoforms named Dmrt4a and Dmrt4b,respective- ly. The eDNA of Drnrt4a was 1 265 bp encoding 352 amino acids containing DM and DMA domains,and the eDNA of Dmrt4b was 1 081 bp encoding 281 amino acids containing DMA domain. The amino acids sequences of Dmrt4 genes in M. amblycephala had high identities with that of Paralichthys olivaceus and Oreochromis aureus. The Dmrt4 gene was expressed from embryo to 30 dph （day post hatching） continuously and reached the peak at 32 hpf （hour post fertilization）,the gene expression in ovary was significantly （P〈O. 01） higher than in other adult tissues,and the gene expression at Stage II of the o- vary was significantly （P〈0.01） higher than that of other ovary stages and the testis stages, suggesting that the Dmrt4 gene may play a vital role in primary oocytes and female gonad development in M. am- blycephala.