牙鲆肌肉生长抑制素(MSTN)基因克隆  

Cloning and expression analysis of the myostatin (MSTN) gene in Paralichthys olivaceus

作　　者：徐建勇[1] 陈松林[1] 

机构地区：[1]中国水产科学研究院黄海水产研究所农业部海洋渔业资源可持续利用重点开放实验室

出　　处：《水产学报》2008年第4期497-506,共10页Journal of Fisheries of China

基　　金：国家“八六三”高技术研究发展计划(2006AA10A402);山东省泰山学者工程专项资助

摘　　要：采用同源克隆及基因组步移的方法,分离克隆了牙鲆肌肉生长抑制素(MSTN)基因。经过序列分析及cDNA验证,牙鲆MSTN基因具有3个外显子和2个内含子,编码377个氨基酸。5′侧翼区含有8个TATA框,一个CAAT框,6个E框;3′侧翼区含有加尾信号。通过同源分析,牙鲆MSTNC末端含有9个保守半胱氨酸残基和一个RVRR蛋白酶酶切位点;通过进化树分析,牙鲆MSTN与鱼类MSTN基因聚为一支。RT-PCR分析表明,牙鲆MSTN在胚胎发育中不表达或表达量较低,说明MSTN在牙鲆胚胎发育中并不起重要作用;其在各组织中的表达,随个体和环境的不同而有差异,暗示MSTN的表达受外界因素调控。Myostatin （MSTN） gene was isolated from Japanese flounder （Paralichthys olivaceus） by homology cloning strategy and genome walking. Two pairs of degenerate primers were designed according to the conserved region of MSTN genes from different fish. Full length DNA sequence of MSTN gene was obtained after one step of 3′ genome walking and two steps of 5′ genome walking. By sequence analysis and verified in cDNA level, Japanese flounder MSTN gene included three exons and two introns, encoding a 374-amino-acid protein. Eight TATA boxes, one CAAT box and six E boxes were located in 5′ flanking region. A polyA signal existed in 3′ flanking region. Two single nucleotide polymorphism （SNP） sites were discovered within MSTN cDNA, one located in coding region with synonymous mutation and the other located in 3′ untranslated region. Nine conserved cysteine residues and an RXXR proteolytic cleavage domain were detected in the C terminal of protein. MSTN in Japanese flounder was highly homologous with MSTN genes in fish via phylogeny analysis, which had64.5%, 63.5%, 64.5%, 65.5%, 80.1%, 83.5%, 84.5%, 92.6%, 90.5%, 77.5% identity with human, house mouse, chicken, zebrafish-2, zebrafish-1, rainbow trout-2, rainbow trout-1, striped bass, red seabream and channel catfish, respectively. The MSTN gene of Japanese flounder might belong to type 1 MSTN, according to identity analysis. The RT-PCR analysis demonstrated that MSTN was undetectable during embryo development, indicating low expression or no expression during embryo development. MSTN had high expression in brain of Japanese flounder, indicating the important role in neural development. The expression of MSTN in kidney indicated the potential role in immunity. However, the expression of MSTN of Japanese flounder in muscle was low. Starvation could induce reduction of MSTN expression in muscle. Unspliced transcript was discovered in normal individual, which was verified by sequencing. The spliced transcript was about 1 270 bp and the unspliced transcript w

关 键 词：牙鲆 肌肉生长抑制素 克隆 

分 类 号：Q785[生物学—分子生物学] S917[农业科学—水产科学]