鹅myf6基因的克隆，胚胎期以及填饲后表达特征分析  

Molecular Cloning of Goose(Anser anser) myf6 Gene and Its Expression During Embryonic Stage and After Overfeeding

作　　者：邵芳[1] 张燕萍[1] 王星果[1,2] 唐瑞璟 龚道清[2] 顾志良[1] 

机构地区：[1]常熟理工学院生物与食品工程学院,常熟215500 [2]扬州大学动物科技学院扬州225009

出　　处：《农业生物技术学报》2014年第2期158-167,共10页Journal of Agricultural Biotechnology

基　　金：江苏省六大人才高峰项目(第六批);苏州市科技项目(农业支撑)(No.SNG201245)

摘　　要：myf6基因属于MyoD家族成员之一,为成肌细胞分化成肌管的重要调控因子。本研究旨在克隆鹅(Anser anser)myf6基因,并进一步探讨该基因的结构与功能,揭示其在胚胎期和填饲后的表达特征。以鹅肌肉总RNA为模板,采用RACE的方法,克隆该基因全长cDNA序列,并进行生物信息学分析。运用实时荧光定量PCR检测myf6基因在鹅不同胚胎期mRNA的表达规律,以及填饲对其表达的影响。鹅myf6全长cDNA为1 196 bp,包括90 bp的5'UTR,383 bp的3'UTR和723 bp的开放阅读框(ORF),共编码240个氨基酸(GenBank登录号:JQ905627)。经预测鹅myf6编码蛋白的分子量为26 214.4,等电点为5.68,平均亲水性为-0.595,为不稳定亲水蛋白。预测鹅myf6蛋白具有Basic和HLH这两个明显的结构域,bHLH蛋白结构域形成剪刀状α-螺旋二聚体,且不同物种间HLH氨基酸序列高度同源。鹅myf6基因CDS区序列与鸭(Anas platyrhynchos)同源性最高,为98.62%,与哺乳类同源性在81%左右,与鱼类同源性较低,仅62%～66%。构建的系统进化树显示,所有哺乳类、鸟类、硬骨鱼各形成一个大的分支,两栖类形成独立分支,鹅首先与绿头鸭(Anas platyrhynchos)聚成一支,分子进化地位上关系最近,其次聚类顺序较近的物种为红原鸡(Gallus gallus),该结果与这些物种在生物学上的分类是较一致的。荧光定量PCR结果显示,鹅myf6 mRNA在胚胎早期第7天就有少量的表达,7天以后表达量逐渐上升,随后在胚胎中期表达量明显升高;其在E18肌肉组织中表达量达到高峰后下降,胚胎发育后期E21 myf6 mRNA的表达量是E25的近4倍,E25后逐渐趋于平稳;E18与E14、E15、E25和E28的表达差异极显著(P<0.01),E21与E25、E28的表达也差异极显著(P<0.01),与E14、E15的表达差异显著(P<0.05)。在鹅的胸肌和腿肌组织中,填饲组myf6 mRNA表达量高于对照组中该基因的表达量,且胸肌组织达到显著水平(P<0.05)。鹅myf6基因的表达具有组织特异性,该结The transcription factor myf6 is a member of the MyoD family that plays a key role in myogenic lineage determination. This study was carried out to clone and analyze sequence of goose （Anser anser） myf6 gene, and to reveal its expression during embryonic stage and after overfeeding. In order to obtain full-length of myf6 cDNA, RT-PCR and rapid-amplification cDNA ends （RACE） were used. The structure and function of myf6 gene was analyzed by bioinformatics methods. Real-time fluorescence quantitative RT-PCR was performed to detect myf6 mRNA expression levels in various embryonic stages and after overfeeding status. A full-length of 1 196 bp myf6 cDNA contains an open reading frame, the 90 bp 5＇UTR and the 383 bp 3＇UTR （GenBank Accession： JQ905627）. The open reading frame was 723 bp, which encodes polypeptides of 240 amino acids, with a calculated relative molecular mass of 26.214 4 kD and pI of 5.68. The myf6 was a kind of hydrophilic and unstable protein, with average of hydropathicity -0.595. The myf6 protein was predicted to distinctly had Basic and HLH protein domains, and the bHLH domain of goose myf6 was forming α-helix dimer that was like a scissors, and HLH sequence was highly homologous among different species. Multiple sequence comparisons showed Anser anser myf6 had high homology with Anas platyrhynchoss at 98.62%, about 81% with mammals, and the lowest with fish at only 62%~66%. Phylogenetic relationship analysis indicated that all mammals, birds, bony fish each was formed a large branch, and amphibians was formed an independent branch. Anser anser was firstly clustered with Anas platyrhynchoss and also closely related with Gallus gallus, which was similar to the results of the biological classification. Real-time PCR was performed to analyze the expression profiling of myf6, results showed that goose myf6 was expressed in early embryos at E7, then the expression level gradually increased, the expression level of myf6 in muscle reached the peak at E18, then decreased and stabilized be

关 键 词：鹅 myf6 胚胎期 填饲 组织表达分析 

分 类 号：S882.6[农业科学—特种经济动物饲养]