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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 the sucrose transporter (DoSUT1) gene of rare and endangered medicinal plant Dendrobium candidum and analyze its bioinformatics and expression pattern. Methods RT-PCR and RACE were used to obtain the full-length genes. The bioinformatics software was used to predict the molecular properties of proteins such as physicochemical properties, domains and subcellular localization. DNA sequence alignment and evolutionary analysis were performed using DNASTAR 6.0 and MEGA4.0, respectively Relationship analysis; detection of gene expression patterns by real-time quantitative PCR. Results DoSUT1 (GenBank accession number KF876842) was isolated and its full-length cDNA was 1920bp encoding a polypeptide of 538 amino acids with a relative molecular mass of 57 400 and an isoelectric point of 9.61. DoSUT1 protein contained 12 transmembrane domains with higher plant sucrose / H ~ + cotransport families and the conserved domains (60-524,63-531) that help to diffuse the superfamily. DoSUT1 has high identity with the SUT gene of many plants, and has high homology with monocotyledonous plants such as maize and barley SUT4 in the SUT subfamily clustered together; DoSUT1 gene has tissue-specific expression, and its transcripts are highly expressed in the leaves and stems of Dendrobium, which are 2.783 and 2.150 times of the roots, respectively. Conclusion The molecular characterization of DoSUT1 gene lays a foundation for further study on the role of DoSUT1 gene in the regulation of glucose metabolism in Dendrobium candidum.