铁皮石斛镁离子转运蛋白基因的克隆及表达分析

Cloning and expression analysis of a magnesium transporter gene in Dendrobium officinale

目的克隆珍稀濒危兰科药用植物铁皮石斛镁离子转运蛋白（magnesium transporter,MGT）基因（Do MGT1）并进行生物信息学和表达分析。方法采用RT-PCR和RACE技术,获得基因c DNA全长;利用生物信息学软件预测蛋白的理化性质、结构域及亚细胞定位等分子特征;用DNASTAR 6.0和MEGA 4.0分别进行氨基酸多序列比对和进化关系分析;借助实时定量PCR（q PCR）检测基因表达模式。结果克隆到Do MGT1（Gen Bank注册号KJ995532）,c DNA全长1 625 bp,编码一条由425个氨基酸组成的肽链,相对分子质量47 400,等电点4.79;Do MGT1蛋白含有Cor A家族镁离子转运蛋白保守结构域（313~420）和镁离子转运蛋白MRS2/LPE10保守域（27~420）;Do MGT1与多种植物MGTs基因一致性为46%~63%,编码蛋白与水稻Os MGTD、Os MGTE、Os MGTF等聚在一起,与At MGT4共同构成植物MGTs基因的一大类群;Do MGT1基因转录本在石斛根、茎、叶器官中为组成型表达,叶中相对表达量较高,为根中的3.46倍;茎次之,为根的1.54倍。结论成功克隆到一个镁离子转运蛋白基因Do MGT1,其转录本在叶中高的表达特征暗示该基因可能在铁皮石斛叶的生长发育中发挥重要作用。

Objective This study was carried out to clone a magnesium transporter gene Do MGT1 from a medicinally important endangered orchid species Dendrobium officinale, followed by bioinformatics and expression analysis. Methods RT-PCR and RACE technologies were used to isolate the full-length gene. The characteristics of physicochemical properties, conserved domains, and subcellular localization of Do MGT1 protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 4.0 softwares, respectively. Real time quantitative PCR was employed for gene expression analysis. Results The full-length c DNA of Do MGT1 was 1 625 bp in size, and encoded a 425-aa peptide chain with a molecular weight of 47 400 and an isoelectric point（p I） of 4.79; The deduced Do MGT1 protein contained the magnesiumion transporter Cor A-like（313—420） and MRS2/LEP10（27—420） conserved domain. Do MGT1 was highly similar（46%—63%） to MGTs genes from various plants. The deduced Do MGT1 protein was closely related to rice Os MGTD, Os MGTE, and Os MGTF proteins, and belonged to one clade of plant MGTs gene together with At MGT4. Do MGT1 transcripts were constitutively expressed in leaves, stems, and roots of D. officinale. The transcription level of Do MGT1 in leaves was the highest（3.46 fold higher than that of roots）, followed by that of stems（1.54 fold）. Conclusion The full-length Do MGT1 gene has been successfully cloned. The high expression level of Do MGT1 in D. officinale leaves suggests that the gene might play a vital regulatory role in the leaves.