光唇鱼MSTN基因克隆、组织表达及SNP位点筛查

摘要

肌肉生长抑制素(myostatin,MSTN)是骨骼肌生长的负调控因子,MSTN基因在遗传育种上有很大的潜在应用价值.光唇鱼(Acrossocheilus fasciatus)是一种具有较大开发前景的经济鱼类,但其生长速度较慢.为了解光唇鱼MSTN基因的特征并为生长性状标记辅助选择提供基础资料,本研究克隆了光唇鱼MSTN cDNA和基因组DNA序列、检测了组织表达情况并进行了单核苷酸多态性(single nucleotidepolymorphism,SNP)位点筛查.结果表明,光唇鱼MST cDNA序列全长2 163 bp,含有一个1 128 bp的开放阅读框(open reading frame,ORF)、编码375个氨基酸,预测的编码蛋白具有脊椎动物MSTN的序列特征(如含有1个蛋白酶水解位点RIRR、C端有9个保守的半胱氨酸残基).基于MSTN氨基酸序列的比对显示不同鱼类之间MSTN序列同源性较高(＞80％),而鱼类与其他脊椎动物同源性较低(65％左右);光唇鱼与其他鲤科鱼类的MSTN基因高度同源(＞96.5％).系统进化树中鱼类聚为一枝、其他脊椎动物聚为另一枝,光唇鱼MSTN位于鲤科鱼类的小簇中.半定量RT-PCR检测显示,MSTN mRNA在光唇鱼脑和肌肉中高表达、肠和肝脏中有微弱表达、其他组织中均未见表达.光唇鱼MSTN基因的DNA序列全长5 828bp,含有3个外显子和2个内含子,在第二内含子中检测到10个SNP位点,但与生长性状(体重、体长)不存在显著相关性(P＞0.05).研究结果为探究光唇鱼MSTN基因的功能和筛选生长性状相关辅助育种标记提供了参考资料.%Myostatin (MSTN) is the negative regulator in the muscular development.MSTN gene has great potential application values in genetic breeding.Acrossocheilus fasciatus is a kind of economic fish which has great prospects for the development,but its growth is slow.In order to clarify the gene characteristic and seek molecular markers associated with growth traits,the MSTN gene of A.fasciatus was cloned,detected its expression level in different tissues and screened single nucleotide poly morphism (SNP) sites.The full length of MSTN cDNA sequence was 2 163 bp,including a 1 128 bp open reading frame (ORF) that encoded 375 amino acids.The putative MSTN protein ofA.fasciatus had the essential characteristic of vertebrate MSTN,such as the proteolytic site RIRR and nine conserved cysteines in the C-terminal.Sequence alignment showed that homology of MSTNs were greater than 80％ among different teleost,and fish had low homology (approximately 65％) with other vertebrates.A.fasciatus MSTN was highly homologous with other Cyprinidae fish (＞96.5％).The phylogenetic tree based on MSTN amino acids showed all the fish clustered together,which was seperated from other vertebrates.A.fasciatus was located in the cluster of Cyprinidae.Semiquantitative RT-PCR showed that there were high level ofMSTN mRNA expression in brain and muscle,weak expression in liver and intestine,and no expression was detected in spleen,kidney,gill,fin and heart.The full-length of MSTN gene DNA sequence was 5 828 bp,which contained 3 exons and 2 introns.Ten SNPs sites were detected in the second intron,but there was no significant correlation (P＞0.05) between these SNP and growth traits (body weight and body length).These results provide reference for the function research of MSTN gene and seeking molecular markers associated with growth traits in A.fasciatus.