利用简并引物克隆黑莓、树莓肌动蛋白基因片段及表达分析  

Cloning and Expression Analysis of Actin Gene Fragment from Blackberry and Raspberry Using Degenerate Primers

作　　者：张春红[1] 王小敏[1] 王鲁北[1] 吴文龙[1] 李维林[1] 

机构地区：[1]江苏省中国科学院植物研究所,南京210014

出　　处：《生物技术通报》2011年第8期123-128,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31000753);江苏省农业科技自主创新基金项目(CX[10]109);江苏省科技基础设施建设计划项目(BM2009041;BM2010458)

摘　　要：采用同源克隆法,利用设计的一对肌动蛋白(actin)基因简并引物,从黑莓、树莓和悬钩子杂种3个悬钩子植物品种均得到一条743 bp的actin cDNA片段,推测编码247个氨基酸。序列比对发现,克隆的actin基因推导的氨基酸序列和其他植物的肌动蛋白氨基酸序列具有高度保守性,经数据库搜索后将来自黑莓和树莓品种的actin cDNA片段在GenBank中登录,登录号分别为HQ439558和HQ439559。不同来源的植物actin蛋白系统进化树的聚类结果表明,树莓和悬钩子杂种亲缘关系较为密切。actin基因在3个悬钩子植物品种不同组织中的RT-PCR分析结果表明,其在检测的不同组织中均有一定程度的表达量,可能都属于组成型表达的肌动蛋白基因。研究结果为利用actin基因作为内参进一步研究黑莓和树莓其他基因的丰度奠定基础。In this report,the cDNA fragment of 743 bp encoding 247 amino acid residues was similarly cloned from blackberry,raspberry and rubus hybrid berry cultivars by homologous cloning using a pair of degenerate primers.Multiple sequence alignment and database search results showed that the three amino acid sequences deduced from the cloned actin cDNA sequences exhibited high conservation with those from other plants.Consequently,the actin cDNA sequences from blackberry and raspberry cultivars were submitted into GenBank and the accession numbers were HQ439558 and HQ439559 respectively.Phylogeny analysis by using different plant actin protein sources showed that the actin protein from raspberry and rubus hybrid berry cultivars had close genetic relationship.RT-PCR analysis of three actin genes from different rubus cultivars showed that they uniformly had expression level in different tissues detected and thus may belong to constitutive expression.The results laid basis for identification of gene transcription level in rubus using actin as internal control.

关 键 词：悬钩子 肌动蛋白基因 简并引物 序列分析 半定量RT-PCR 

分 类 号：S663.2[农业科学—果树学]