西番莲PeERG基因克隆及其表达模式分析

Cloning and expression pattern analysis of PeERG gene from Passiflora edulis

关键词:  ERA蛋白, 西番莲, RACE, 表达模式, G蛋白, 低温胁迫

Key words:  Eecherichia coli Ras-like protein, Passiflora edulis, RACE, expression pattern, G protein, low temperature stress

摘要:

ERA(Eecherichia coli Ras-like protein)蛋白是与已知异三聚体G蛋白和小分子G蛋白不同的一种新的GTP结合蛋白。为了在木本植物中开展其同源基因ERG(ERA-like GTPase)克隆和功能验证的相关研究,该文首次在西番莲新品种‘平塘1号'中采用cDNA末端快速克隆(RACE)技术克隆鉴定1个ERG基因。结果表明:西番莲PeERG基因cDNA全长为1 518 bp,包括1 260 bp的开放阅读框、38 bp的5'-端非翻译区和220 bp的3'-端非翻译区,该基因编码蛋白由420个氨基酸残基组成,其二级结构含有丰富的α-螺旋和延伸链。PeERG蛋白不含跨膜区域,也不存在信号肽酶切位点,既在其N端有典型的GTPase保守结构域(GTPase domain)又在其C端有独特的RNA结合结构域(KH domain)。系统进化树分析表明,西番莲PeERG蛋白和水稻OsERG1、拟南芥AtERG1、大肠杆菌ERA位于同一进化分枝。实时定量PCR检测揭示PeERG基因在西番莲根、茎、叶、花、果中均有表达,叶中表达最高; 同时该基因响应低温胁迫信号,其表达呈动态变化模式。该研究首次鉴定和描述了木本植物西番莲的ERG基因,为深入挖掘西番莲特异基因资源提供参考,也有助于进一步探究ERG基因在植物中的生物学功能及其作用机制。

Abstract:

Different from the known trimetical G protein and small GTPase, ERA(Eocherichia coli Ras-like protein)is a new GTP-binding protein. In order to study cloning and functional verification of ERG(ERA-like GTPase)homologous gene in woody plants, one ERG gene was firstly isolated from a cold-tolerant variety of Passiflora edulis(‘Pingtang 1')through the rapid amplification of cDNA ends(RACE). The resutls were as follows: The full-length sequence of PeERG cDNA was 1 518 nucleotides, including a 1 260 bp open reading frame(ORF), flanked by a 38 bp 5'-untranslated region(UTR)and a 220 bp 3'-UTR. PeERG may encode a protein of 420 amino acids, and its secondary structure was rich in alpha helix and extended strand. PeERG protein did not contain both transmembrane region and signal peptidase cleavage site, and had two conserved domains: a GTPase domain and a KH domain. Phylogenetic tree revealed that PeERG was clustered into the same clade as OsERG1, AtERG1 and ERA.(4)Using real-time RT-PCR, the transcripts of PeERG were detectable in various tissues(roots, stems, leaves, flowers and fruits), and its dynamic expression pattern under low temperature stress was analyzed. Altogether, one ERG gene was isolated from woody plants for the first time. These results will be valuable for further study of the biological function of ERG in plants and enrich the excellent genetic resources of Passiflora edulis.