半滑舌鳎Dmrt4基因DNA全序列的克隆与表达分析

克隆了半滑舌鳎(Cynoglossus semilaevis)Dmrt4基因DNA全序列,得到的序列长度为2271 bp,包含1个内含子,2个外显子,与其他鱼类的同源性在90%以上.Dmrt4基因在雌性和雄性半滑舌鳎的7个组织中都有表达,有差异表达的组织是脑、垂体和性腺,雄性的表达显著高于雌性(P<0.05).高温处理组的伪雄鱼中Dmrt4基因的表达与对照组的雌鱼没有显著差异(P>0.05),但是甲基睾酮处理组的雄鱼和伪雄鱼的表达显著高于对照组的雌鱼和雄鱼(P<0.05).Dmrt4基因的表达在胚胎发育过程中呈现先升高后降低的趋势,在原肠期表达量最高,显著高于其他几个时期(P<0.05),从尾芽形成期开始恢复到多细胞期的表达水平,之后几个时期 Dmrt4基因的表达无显著变化(P>0.05),一直到出膜前保持恒定.研究表明, Dmrt4基因是雄性中优势表达的基因,并且受雄性基激素调节,但并不是性反转的必要因素.这一研究为半滑舌鳎全雌群体的建立提供了分子水平的研究基础.

Molecular cloning and expression analysis of Dmrt4 gene in half- smooth tongue sole (Cynoglossus semilaevis)

A complete Dmrt4 DNA sequence was cloned from Cynoglossus semilaevis. The DNA sequence was   2 271 bp long, containing one intron and two exons. The NJ-phylogenetic tree indicated that the Dmrt4 gene of C.semilaevis had high homology to other fishes. Real-time PCR revealed that Dmrt4 was expressed in all seven tissues we examined in both sexes. Expression was significantly higher in the male pituitary than in other tissues. The expression of Dmrt4 in the gonads of methyl testosterone-treated males and neo-males was higher than in normal males. However, there was no significant difference between normal females and the high temperature-treated neo-males. This indicates that Dmrt4 is not critical for sex reversal. Dmrt4 was expressed at significantly higher levels during the gastrula stage than during other stages. Dmrt4 was expressed at higher levels in males than in females. Our results provide a molecular basis for the formation of all female groups of C.semilaevis.