目的 克隆当归Angelica sinensis咖啡酸-O-甲基转移酶(caffeic acid O-methyltransferases,COMT)编码基因全长cDNA,并对序列进行生物信息学分析。方法 提取当归叶片总RNA为cDNA合成模板,利用同源克隆结合cDNA末端快速扩增(RACE)技术克隆当归COMT全长cDNA,并利用NCBI、ExPASy网站上的Blast N、Blast P、ORF Finder、Compute PI/Mw、ProtScale、PROSITE、SWISS-MODEL等序列在线分析工具和MEGA、DNAMAN生物信息学软件对序列进行分析。结果 获得COMT全长cDNA序列,并在GenBank注册(登录号KP188587)。序列分析表明,克隆的cDNA全长为1436 bp,其中包括5'-UTR(76 bp)和3'-UTR(362 bp),含有1个1098 bp的完整开放阅读框(opening reading frame,ORF),编码365个氨基酸的多肽链;预测蛋白质相对分子质量为40230,理论等电点(pI)为5.43;无信号肽,具有典型的Ⅱ型氧甲基转移酶结构域SAM_OMT_Ⅱ。结论 首次克隆当归COMT基因全长cDNA,为当归COMT基因功能研究和当归阿魏酸生物合成与调控的机制研究奠定基础。

Objective To clone the full-length cDNA of caffeic acid O-methyltransferase(COMT) encoding gene from Angelica sinensis and to perform bioinformatic analysis for the cDNA sequence. Methods Extracting the total RNA from the leaves of A. sinensis as cDNA synthesis template, the full length COMT cDNA of A. sinensis was cloned through homology-based cloning and rapid amplification of cDNA ends(RACE) technique. The bioinformatics of the cDNA sequence was analyzed by Blast N, Blast P, ORF Finder, Compute PI/Mw, ProtScale, PROSITE, and SWISS-MODEL sequences online analysis tools on NCBI, ExPASy, DNAMAN, and MEGA softwares. Results The full-length of COMT cDNA(1436 bp) was obtained(GenBank accession number:KP188587). It included 5'-UTR(76 bp) and 3'-UTR(362 bp), with an open reading frame(ORF) of 1098 bp, encoding 365 amino acid polypeptides. The relative molecular mass of COMT calculated was 40230, theoretical isoelectric point(PI) was 5.43, and there was no signal peptide in COMT. The protein sequence contained typical class Ⅱ O-methyltransferases domain:SAM_OMT_Ⅱ. Conclusion A novol cDNA encoding COMT from A. sinensis is cloned. This work might establish an experimental basis for exploring COMT gene function and the biosynthetic and regulation of ferulic acid(FA) in A. sinensis.

国家自然科学基金资助项目(81260616,81060327)