铁皮石斛蔗糖转运蛋白基因的分离和表达分析

Isolation and expression analysis of a sucrose transporter gene in Dendrobium officinale

目的分离珍稀濒危药用植物铁皮石斛蔗糖转运蛋白(sucrose transporter,SUT)基因(DoSUT1)并进行生物信息学和表达模式分析。方法采用RT-PCR和RACE技术获基因全长;利用生物信息学软件预测蛋白的理化性质、结构域和亚细胞定位等分子特性;用DNASTAR 6.0和MEGA4.0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检测基因表达模式。结果分离到DoSUT1(GenBank注册号KF876842),cDNA全长1920bp,编码一条由538个氨基酸组成的多肽,相对分子质量57 400,等电点9.61;DoSUT1蛋白含12个跨膜域,具有高等植物蔗糖/H^+共转运家族和协助扩散超家族的保守结构域(60-524、63-531);DoSUT1与多种植物SUT基因编码蛋白有较高的一致性,与玉米、大麦等单子叶植物SUT亚家族中的SUT4聚类在一起;DoSUT1基因具有组织表达特异性,其转录本在石斛叶和茎中表达量较高,分别为根中的2.783和2.150倍。结论 DoSUT1基因的分子特征为进一步研究其在铁皮石斛糖代谢调控中的作用奠定基础。

Objective To isolate and characterize a sucrose transporter （SUT） gene DoSUT1 in a rare endangered medicinal orchid species Dendrobium officinale. Methods RT-PCR and RACE technologies were used to isolate the full length cDNA of DoSUT 1. Characteristics ofphysiochemical properties, conserved domains, and subcellular localization of the deduced DoSUT 1 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 used for gene expression analysis. Results The full length cDNA of DoSUT1 was 1 920 bp in length and encoded a 538-aa protein with molecular weight of 57 400 and isoelectric point of 9.61; The deduced DoSUT1 protein, with 12 transmembrane regions, contained the sucrose/H＋ symporter （SUC/SUT） and the major facilitator superfamily, （MFS） conserved domains （60-524 and 63-531）. DoSUT1 had high identities with SUTs proteins from various plants; DoSUT1 belonged to the l b subgroup of SUT evolutionary tree, and was closely related to the monocot maize and barley SUT4. DoSUT1 was differentially expressed in the three included organs. The transcripts were the most abundant in the leaves with 2.783 folds over that in the roots, followed by that in the stems with 2.150 fold. Conclusion Molecular characterization of DoSUT1 will be useful for further functional determination of the gene involving in the regulation of sugar metabolism ofD. officinale.