铁皮石斛类受体激酶DoRLK的基因克隆和分子特征

Molecular cloning and characterization of a novel DoRLK gene from Dendrobium officinale

目的克隆铁皮石斛Dendrobium officinale类受体激酶(receptor-like kinase,RLK)基因,命名为DoRLK(GenBank注册号ANC68272.1)。方法采用实时荧光定量PCR(qRT-PCR)和RACE技术获取基因全长;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;用软件DNASTAR 6.0和MEGA 7.0分别进行氨基酸多序列比对和进化关系分析;借助定量PCR检测基因表达模式。结果 DoR LK基因cD NA全长1 715 bp,编码1条由423个氨基酸组成的多肽,相对分子质量47 800.88,等电点为9.47。DoR LK蛋白包含RLK蛋白家族的1个蛋白激酶结构域(85~370)和一个跨膜基序(250~270)。DoR LK基因与多种植物RLK基因相似性很高(43.62%~63.35%),与小兰屿蝴蝶兰、海枣、芦笋等植物亲缘关系较近。DoR LK基因具有组织表达特异性,其转录本在石斛根中表达量较高,分别为叶和茎中的2.22、2.75倍。结论 DoR LK基因的分子鉴定为进一步揭示RLK在铁皮石斛与环境互作中的功能奠定基础。

Objective To identifiy a receptor-like kinase(RLK)encoding gene named as DoR LK(GenBank accession No.ANC68272.1)from Dendrobium officinale.Methods RT-PCR and RACE technologies were used to isolate the full length cDNA of DoR LK.Characteristics of physiochemical properties,conserved domains,and subcellular localization of the deduced DoRLK protein were determined by a series of bioinformatics tools.The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 7.0 softwares,respectively.Quantitative PCR was used for gene expression analysis.Results The results showed that the full length cDNA of DoRLK was 1715 bp in length and encoded a 423 aa protein with a molecular weight of 47800.88 and an isoelectric point(pI)of 9.47.The deduced DoR LK protein,like other RLK proteins,constituted one conserved domains(85—370)and one transmembrane motifs(250—270).Multiple sequence alignment and phylogenetic analyses demonstrated that DoR LK had high identity(43.62%—63.35%)to a number of RLK genes from various plants and was closely related to Phalaenopsis equestris,Phoenix dactylifera and Asparagus officinalis.Real time quantitative PCR(qPCR)analysis revealed that DoR LK was expressed in the three included organs.The transcripts were the most abundant in the roots with 2.22 fold over that in the l eaves,followed by that in the stems with 2.75 fold.Conclusion Molecular characterization of DoRLK will be useful for further functional elucidation of the gene involving in D.officinale responses to environmental factors.