蝴蝶兰一个C3型PEPC基因的克隆及其低温胁迫下的表达分析

为研究蝴蝶兰对低温胁迫响应的分子调控机理,本研究采用RT-PCR及RACE的方法从蝴蝶兰叶片中克隆了一个C3型PEPC基因PhPEPC (登录号为:MK482383),该基因cDNA全长序列为3 448 bp,开放阅读框长度为2 898 bp,编码965个氨基酸;该蛋白包含一个PEPC酶结构域,具有2个PEPCase活性位点,86个磷酸化位点和7种motifs,为一酸性亲水性蛋白。系统进化分析显示,蝴蝶兰PhPEPC蛋白与兰科植物小兰屿蝴蝶兰、铁皮石斛、深圳拟兰等的PEPC蛋白亲缘关系最近。分析表明,PhPEPC基因在蝴蝶兰根中表达水平最高,在花器官中的表达水平次之,在叶和花梗中的表达水平最低;在13℃/8℃的昼/夜温度条件下,PhPEPC基因的表达水平先升高后降低,当恢复培养3 d时,该基因的表达水平又趋于升高。结果表明,蝴蝶兰PhPEPC基因参与了对低温胁迫的调控。本研究结果将有助于理解蝴蝶兰对低温胁迫的适应机制,并为蝴蝶兰新品种的遗传改良提供依据。

Cloning of a C3 Type PEPC Gene from Phalaenopsis and Its Expression under Low Temperature Stress

To study the molecular regulation mechanism Phalaenopsis in response to cold stress, a C3 type phosphoenolpyruvate carboxylase gene PhPEPC(GenBank accession number: MK482383) was cloned from the leaf of Phalaenopsis through RT-PCR and RACE method. The full-length cDNA sequence was 3 448 bp with an open reading frame(ORF) of 2 898 bp encoding 965 amino acids. The PhPEPC gene encoding protein contained a phosphoenolpyruvate carboxylase domain with two PEPCase active sites, 86 phosphorylation sites and 7 types motifs, it belonged to a acidic and hydrophilic protein. The phylogenetic tree showed that PhPEPC protein was closely related to the PEPC proteins from Phalaenopsis equestris, Dendrobium catenatum and Apostasia shenzhenica in Orchidaceae. The expression analysis showed that the PhPEPC gene was expressed with the highest level in root,the higher level in flower tissues, and the lowest level in leaf and pedicel. Under the 13℃/8℃ day/night temperature, the expression level of PhPEPC was increased firstly and then reduced gradually, the expression level of PhPEPC was increased after recovery for 3 d. The results suggested that PhPEPC involved in the molecular regulation of response to the cold stress. This study would be beneficial to understand the mechanism of low temperature tolerance in Phalaenopsis, and provide the basis for genetic improvement of new species in Phalaenopsis.