铁皮石斛过氧化氢酶基因DoCAT1的鉴定与表达分析

Identification and Expression Analysis of A Catalase Gene DoCAT1 in Dendrobium officinale

目的分离珍稀濒危药用铁皮石斛过氧化氢酶基因Do CAT1cDNA全长,并进行生物信息和表达分析。方法采用RACE技术获基因全长;利用生物信息软件预测基因编码蛋白的理化性质、结构域和亚细胞定位等分子特性;用DNASTAR 6. 0和MEGA 6. 0分别进行氨基酸多序列比对和进化树构建分析;借助实时荧光定量PCR检测基因的表达模式。结果克隆到Do CAT1(Gen Bank获得注册号KF876840),c DNA全长1 796 bp,ORF长1 479 bp,编码蛋白含492个氨基酸,相对分子质量56. 90×103,等电点6. 7。Do CAT1蛋白不含跨膜域和信号肽,包含过氧化氢酶核心结构域(18~401)、活性位点(54~70)、血红素结合位点(344~352)和多个保守基序; Do CAT1与植物CATs蛋白一致性为83. 3%~88. 3%,所在分支隶属于CATs分子进化的Ⅲ分支,与单子叶植物CATs亲缘关系近; Do CAT1基因的表达呈器官特异性,根中相对表达量较高,为叶中的4. 27倍,叶中次之,茎中表达量很低。结论 Do CAT1基因鉴定与表达特征为石斛生长发育和抗逆生理提供分子基础。

OBJECTIVE To isolate and characterize a catalase gene DoCAT1, the full length cDNA was cloned, in a rare endangered medicinal orchid species Dendrobium officinale, followed by bioinformatics analysis and expression pattern detection. METH- ODS RACE Technology was used for gene clone. Characteristics of physiochemical properties, conserved domains and subcellular localization of the protein were determined using a series of bioinformatics tools. The analyses of muhiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 6. 0 softwares, respectively. Real time quantitative PCR was used for gene expression analysis. RESULTS The full length cDNA of DoCAT1 was 1 796 bp in length, encoding a 492 amino acid protein with a molecular weight of 56. 90 × 10 and an isoelectric point of 6. 7. The deduced DoCAT1 protein, without transmembrane or signal peptide residues, contains catalase core domain （18 -401 ） , active site （54 -70） , heme binding site （344 -352） and multiple conserved motifs. DoCAT1 had high identities （83.3% - 88. 3% ） with CATs proteins from various plants ; DoCAT1 belonged to class m of the CATs evolutionary tree. DoCAT1 gene was differentially and organs specifically expressed. The transcripts were more abundant in the roots, with 4. 27 fold, then in the leaves and the lowest in the stems. CONCLUSION Molecular characterization of DoCAT1 provides important basis for the growth and development and stress adaptation in D. officinale.