半滑舌鳎Gadd45g3基因的克隆与表达分析

Cloning and expression analysis of Gadd45g3 in half-smooth tongue sole(Cynoglossus semilaevis)

生长阻滞和DNA损伤诱生蛋白45 （growth arrest and DNA damage inducible protein 45,Gadd45）基因在胚胎发育和性别决定中起重要作用.本研究通过RACE方法在半滑舌鳎（Cynoglossus semilaevis）中克隆得到了1个Gadd45g同源基因,命名为Gadd45g3.该基因全长1 147 bp,编码155个氨基酸残基.氨基酸序列分析表明,半滑舌鳎Gadd45g3与其他鱼类Gadd45g-like的同源性为79％～88％,与硬骨鱼和高等脊椎动物的Gadd45g同源性为58％～67％.对多个物种的Gadd45g同源基因的CDS区进行Ka/Ks分析,发现Ka/Ks均小于0.3.FISH结果显示,半滑舌鳎Gadd45g3基因位于常染色体上.实时荧光定量PCR结果显示,Gadd45g3在雌雄成鱼的脑中都有很高的表达,说明其参与了神经发育;在成熟性腺中,Gadd45g3的表达具有精巢特异性,说明Gadd45g3是性别相关基因;而在不同发育时期的性腺中,Gadd45g3的表达量在孵化后20 d的雄性仔鱼中显著高于同龄雌鱼（P＜0.05）,但在孵化后95 d的雌雄仔鱼中均有高表达,说明在半滑舌鳎性别决定和性腺发育中起着重要作用.

The half-smooth tongue sole, Cynoglossus semilaevis, is an increasingly important marine flatfish of potentially significant aquaculture value in China. It exhibits prominent sexually dimorphic growth in which females grow two to three times faster and larger than males. The growth arrest and DNA-damage-inducible protein 45 gamma （Gadd45g） plays a major role in embryonic development and sex determination. We evaluated the physiological function of a homologous Gadd45g gene on sex determination in C. semilaevis. Using RACE, we obtained a Gadd45g homologous gene from half-smooth tongue sole （named Gadd45g3）. The full length Gadd45g3 was 1 147 bp, encoding 155 amino acid residues. A ribosomal_L7Ae domain was found in the sequence. Amino acid sequence analysis revealed that C. semilaevis Gadd45g3 shared 79%-88% identity with Gadd45g-like proteins in other fish and 58%--67% with Gadd45g in bony fish and higher vertebrates. The phylogenetic tree suggested that Gadd45g3 in C. semilaevis and Gadd45g-like in other fish were closely clustered in one branch because of their high similarity. Gadd45g-like genes were homologous with Gadd45g and typical for fish. They were produced from the additional fish-specific whole genome duplication. The ratio of Ka to Ks was used to diagnose the pattern of sequence evolution. The Ka/Ks for all comparisons of Gadd4593 with homologous genes in other species was 〈0.3 suggesting that Gadd45g homologous genes are highly conserved. However, the Ka/Ks ratio for Gadd4593 and Gadd45g homologous genes in mammals was higher than the former comparisons, suggesting that the function of Gadd4593 in C. semilaevis varies from that of Gadd45g in mice. FISH results revealed that the Gadd45g3 gene is located on the autosome. Gadd4593 expression was highest in the brain of both males and females, suggesting it is involved in the neurological development of C. semilaevis. Gadd45g3 expression was significantly higher in the testis of mature males and neo-males than in the ovary. In 20-day-old male larva, the expression of Gadd45g3 was markedly higher in the gonad than in same-aged females, whereas there was no difference in the level of gonadal expression between sexes in 95-day-old larva. Our results suggest that Gadd45g3 is a gender related gene that is necessary for testis maturation. Additionally, Gadd45g3 is involved in sex determination prior to gonadal differentiation. Moreover, Gadd45g3 also plays an important role in the early development of the ovary and testis.