半滑舌鳎Shh基因的克隆与表达及甲基化分析

Cloning, expression and methylation analysis of the Shh gene in half-smooth tongue sole(Cynoglossus semilaevis)

通过RACE方法获得半滑舌鳎（Cynoglossus semilaevis）Hh基因家族的Shh基因,该基因全长1 922 bp,其中5′UTR 266 bp,3′UTR 360 bp,ORF1 296 bp,编码431个氨基酸,经预测该多肽的相对分子质量为47.28 k D,理论等电点6.95,具有Hh基因家族特有的保守结构域。氨基酸序列分析表明,半滑舌鳎Shh与牙鲆的同源性最高,为82%,与其他鱼类同源性为74%~81%,与非洲爪蟾的同源性为61%,与人和鼠的同源性为63%~64%。甲基化结果显示,Shh基因在1龄卵巢中的甲基化程度普遍低于雄鱼与伪雄鱼精巢。基因表达分析显示,Shh基因在胚胎期的囊胚期表达显著高于其它时期（P〈0.05）,在雌雄成鱼8个组织器官均表达,在雌性性腺分化的关键期孵化后50 d,雌性性腺中表达量较前期升高,且显著高于同时期雄性性腺中的表达（P〈0.05）,在雄性性腺分化的关键期80~95 d,Shh基因在雄性中的表达水平显著升高（P〈0.05）,在8月龄、1龄及2龄雌鱼性腺中表达显著高于雄鱼与伪雄鱼。本研究表明,半滑舌鳎Shh基因在进化中高度保守,与胚胎分化、组织器官形成、雌雄性腺分化及性腺发育密切相关。

The hedgehog（HH） signaling pathway plays an important role in embryonic development and adult tissue balance. To understand the function of the Shh（sonic hedgehog） gene in gonadal differentiation and development, we cloned Shh from the Cynoglossus semilaevis.The full-length Shh c DNA was 1922 bp, including a 1296-bp open reading frame（ORF）, a 266-bp 5′-untranslated region（UTR） and a 360-bp 3′-UTR. The c DNA encoded a predicted protein of 431 amino acid residues. Phylogenetic analysis showed that the Shh putative protein belonged to the HH family, with typical HH-N and HH-C domains. Amino acid sequence analysis revealed that C. semilaevis Shh shared 74%–82% identity with Shh proteins in other fish and 60%–70% with Shh proteins from higher vertebrates. The Cp G methylation levels of Shh were significantly lower in females than in males and neo-males. Real-time quantitative PCR showed that the relative expression of the Shh gene was significantly higher in the blastula stage than in other embryonic developmental stages（P〈0.05）. Shh was expressed in eight organs in males and females, and at the critical period of female gonad differentiation（50 d）, the relative expression of the Shh gene sharply increased compared with earlier stages and was significantly higher than at the same period in the male gonads（P〈0.05）. In male gonads, during the critical period of differentiation（80 to 95 d）, the relative expression of the Shh gene was significantly higher than in the female gonads. In 5-month, 8-month and 1-year-old gonads, the relative expression of the Shh gene was significantly higher in females than in males and pseudo males. These results suggest that the Shh gene is highly conserved in evolution and plays an important role in embryonic differentiation, tissue and organ formation, gonadal differentiation and the development of C. semilaevis.