半滑舌鳎促滤泡激素基因全长cDNA的克隆与组织表达分析

Full length cDNA cloning and tissue expression analysis of follicle-stimulating hormone (FSH) from half-smooth tongue sole Cynoglossus semilaevis Günther

中文关键词: 半滑舌鳎  促滤泡激素基因  cDNA克隆  组织表达  实时荧光定量PCR

英文关键词: Cynoglossus semilaevis Günther  FSH gene  cDNA cloning  Tissue expression  Quantitative real-time PCR

中文摘要:

      从半滑舌鳎Cynoglossus semilaevis Günther脑垂体中提取总RNA，利用RT-PCR和RACE技术首次克隆得到半滑舌鳎促滤泡激素（FSH）基因全长cDNA序列。半滑舌鳎FSH cDNA全长为541bp，开放阅读框为393bp，编码130个氨基酸（GenBank序列登录号为：JQ277933）。发现一个N-糖基化位点：24-27 NTT。与其他脊椎动物的FSH成熟肽氨基酸序列同源性比较表明：半滑舌鳎FSH与鲽形目和鲈形目鱼类FSH同源性为42%~49%，与鲤形目和高等脊椎动物FSH同源性为27%~31%。用MEGA 4.0软件构建了NJ树，获得的进化树表明半滑舌鳎FSH与其他鱼类FSH聚类，亲缘关系较近。实时荧光定量组织表达分析表明，FSH mRNA除在垂体中大量表达外，其他组织也有表达，尤以脑、性腺表达量较高。半滑舌鳎FSH mRNA在垂体以外组织中的广泛表达，暗示FSH可能具有广泛的生理功能。

英文摘要:

      Total RNA was extracted from the pituitary gland of half-smooth tongue sole Cynoglossus semilaevis Günther, and the follicle stimulating hormone (FSH) was cloned by using RT-PCR and RACE technology for the first time. The cDNA of FSH is 541bp, and the open reading frame was 393bp encoding peptides of 130 amino acids. This cDNA sequence was submitted to GenBank with Accession No. JQ277933. One N-glycosylation site was found in the deduced amino acid sequence. Multiple amino acid sequences alignments were performed among vertebrates including Osteichthyes, Amphibia, Reptilia, Aves and Mammalia. It showed that half-smooth tongue sole FSH mature peptide was more similar to their orthologs of Pleuronectiformes and Perciformes which was 42%-49% identity than to Cypriniformes and mammalian which was 27%-31% identity. The pairwise distances were computed and phylogenetic trees were constructed using NJ method based on the codon sequences of the chosen species by MEGA 4.0 software. The result indicated that the FSH of half-smooth tongue sole was clustered together with other fish FSH, and the half-smooth tongue sole gene shared high homology with teleost. Real-time PCR analysis showed that FSH mRNA was expressed at the highest level in pituitary. Also strong amplification signals were detected in all other tissues, especially brain and gonad. FSH mRNA of half-smooth tongue sole expressed widely in non-pituitary tissues may indicate that it has more physiological functions.