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

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技术获取基因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2.708×10-4,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,根和叶中无显著差异。结论铁皮石斛CCoAOMT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。

Objective To isolate the caffeoyl CoA O-methyltransferase （CCoAOMT） gene （DoOMT） in a rare endangered medicinal orchid species Dendrobium oJficinale, 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 ofDoOMTwas 1 005 bp in length and encoded a 239-amino acid protein with a molecular weight of 2.708 ×104 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 and 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 lb 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 DoOMTwas markedly higher than that in the leaves with 4.562 fold, whereas the transcript amount showed no significant difference in the leaves and roots. Conclusion Molecular characterization of DoOMT will be useful for the further ftmctional determination of the gene involving in the secondary metabolism along with growth and development in D. officinale.