红鳍东方鲀gsdf基因的克隆及表达模式分析

Cloning and expression profiling of the gsdf gene in redfin puffer Takifugu rubripes

为研究红鳍东方纯Takifugu rubripes的性别决定因子（gonadal soma derivedfactor，GSDF），利用cD-NA末端快速扩增技术（RACE）首次克隆了红鳍东方纯舻妒基因（Trgsdf）的cDNA全长序列（GenBank登陆号：KR914667）。结果表明：TrgsdfcDNA序列全长为1734bp，其中5’端非编码区144bp，开放阅读框648bp，3’端非编码区942bp，共编码215个氨基酸；预测的氨基酸序列中存在1个长度为19个氨基酸的信号肽和相同长度的跨膜区，1个N-糖基化位点NST，1个TGF-β家族成员特有的保守结构域；BLAST同源性分析结果显示，红鳍东方纯GSDF氨基酸序列与其他鱼类的相似性为26％～58％；系统发育分析结果显示，鱼类GSDF单独聚为一支，与TGF-β超家族内的其他成员分开，红鳍东方纯与青鳐Oryzias latipes GSDF的亲缘关系最近，先聚为一支，后与三斑海猪鱼Halichoeres trimaculatus聚在一起，与矛尾鱼Latimeria menadoensi5的GSDF亲缘关系最远：应用RT-PCR技术检测TrgsdfmRNA在雌性和雄性红鳍东方纯成鱼不同组织中的表达，结果显示，TrgsdfmRNA在卵巢和精巢中高表达，在皮肤和肌肉组织中微量表达，在其他组织中无表达：采用相对实时荧光定量PCR方法比较了成鱼卵巢和精巢中TrgsdfmRNA的表达量，结果显示，TrgsdfmRNA在精巢中的表达量显著高于卵巢（P〈0．05），约为卵巢表达量的6倍。研究表明，鲈妒基因可能在红鳍东方纯的性腺尤其是精巢的分化和发育过程中起着重要的作用。

The full length cDNA of gsdf gene (Trgsdf, GenBank accession No. KR914667)was first cloned in red-fin puffer Takifugu rubripeshis using rapid amplification of cDNA ends ( RACE) . The results showed that the Trgsdf gene had 1734 bp in length, with 144 bp of 5'-untranslated region ( UTR) , 942 bp of 3'-UTR and 648 bp of the coding region encoding a 215 amino acids predicted protein. The predicted protein contained a signal peptide (19 amino acid long) , a transmembrane region (19 amino acid long) , an N-glycosylation site( NST) and a conserverd domain of the TGF-βsuperfamily. A BLAST search against the NCBI protein database showed that the Trgsdf-en-coding protein shared 26%-58% identity with the GSDF proteins found in other fish species. Phylogenetic analysis revealed that the fish GSDF proteins had a clade separated from the other members of the TGF-βsuperfamily. Mo-reover, the Trgsdf-encoding protein showed a higher similarity with the medaka Oryzias latipes GSDF and the prot-ogynous wrasse Halichoeres trimaculatus gsdf as compared with the GSDF in other species. The expression pattern of Trgsdf in various tissues by RT-PCR ( reverse transcription polymerase chain reaction) showed that there was Trgs-df transcripts in both female and male redfin puffer, with a high expression level in ovary and testis, low expression level in skin and muscle and without expression in other tissues. Relative real-time PCR showed that there was sig-nificantly higher expression level of Trgsdf in testis than that in ovary ( P<0 . 05 ) . The findings suggest that GSDF might be involved in the development of gonad ( especially in testis differention and development) in redfin puffer.