张 岗1, 2,胡本祥1,张大为2,陈 伟1,郭顺星2*.铁皮石斛咖啡酰辅酶A氧甲基转移酶基因的分离与表达分析[J].中草药,2014,45():
铁皮石斛咖啡酰辅酶A氧甲基转移酶基因的分离与表达分析
Isolation and expression analysis of caffeoyl CoA O-methyltransferase gene in Dendrobium officinale
  
DOI:
中文关键词:  铁皮石斛  甲基转移酶  基因克隆  序列分析  实时定量PCR
英文关键词:Dendrobium officinale  methyltransferase  gene cloning  sequence analysis  real time quantitative PCR
基金项目:国家自然科学基金资助(31070300, 31101608);陕西省青年科技新星项目(2012KJXX-44);陕西省教育厅专项科研计划项目(2013JK0829)
作者单位
张 岗1, 2,胡本祥1,张大为2,陈 伟1,郭顺星2* 1. 陕西中医学院药学院/陕西省中药基础与新药研究重点实验室陕西 西安 712046 2. 中国医学科学院-北京协和医学院药用植物研究所北京 100193 
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中文摘要:
      目的 分离珍稀濒危药用植物铁皮石斛咖啡酰辅酶A氧甲基转移酶(caffeoyl CoA O-methyltransferase,CCoAOMT)基因(DoOMT)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cDNA全长;利用生物信息学软件预测蛋白的理化性质、结构域和三维建模等分子特性;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检测基因表达模式。结果 分离到DoOMT(GenBank注册号KF876839),cDNA全长1 005 bp,编码一条由239个氨基酸组成的多肽,相对分子质量为2.708×104,等电点5.03;DoOMT蛋白不含跨膜域和信号肽,具有氧甲基转移酶family 3、甲基转移酶的保守结构域(13~238、31~238)和8个保守基序;DoOMT与植物CCoAOMTs蛋白一致性为49.4%~78.7%,所在分支隶属于CCoAOMTs分子进化的1b类群,与单子叶植物香草亲缘关系最近;DoOMT基因转录本在石斛根、茎、叶器官中为组成型表达,茎中相对表达量较高,为叶中的4.562倍,根和叶中显著差异。结论 铁皮石斛咖啡酰辅酶A氧甲基转移酶基因DoOMT的分子特征为进一步研究其在铁皮石斛次生代谢和生长发育过程中的作用奠定基础。
英文摘要:
      Objective To isolate the caffeoyl CoA O-methyltransferase (CCoAOMT) gene (DoOMT) in a rare endangered medicinal orchid species Dendrobium officinale, and to carry out the bioinformatics and expression mode analysis. Methods RT-PCR and RACE technologies were used to obtain the full length cDNA of DoOMT gene. The characteristics of physiochemical properties, conserved domains, and three dimensional structure of the deduced DoOMT protein were determined using a series of bioinformatic 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 DoOMT was 1 005 bp in length and encoded a 239-aa protein with a molecular weight of 27 080 and an isoelectric point of 5.03; The deduced DoOMT protein, without transmembrane or signal peptide residues, contained the oxygen methyltransferase family 3, methyltransferase conserved domains (13—238, 31—238), and eight conserved signature sequences. DoOMT had high identities (49.4%—78.7%) with CCoAOMTs proteins from various plants; DoOMT belonged to the 1b subgroup of the CCoAOMT evolutionary tree, and was closely related to the monocot Vanilla planifolia. DoOMT transcripts were constitutively expressed in the leaves, stems, and roots. The transcription level of DoOMT was markedly higher than that of leaves with 4.562 fold, whereas the transcript amount showed no significant difference in leaves and roots. Conclusion Molecular characterization of DoOMT will be useful for further functional determination of the gene involving in the secondary metabolism along with growth and development in D. officinale.
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