基于ITS序列分析铁皮石斛试管苗的亲缘关系.doc

基于ITS序列分析铁皮石斛试管苗的亲缘关系 顾慧芬1，庄意丽1，梅其春2 （1. 上海市中药研究所 上海 201203；2. 复旦大学生命科学学院 上海 200433） 摘要：采用分子系统学方法分析铁皮石斛试管苗及其近缘种的遗传多样性，为准确进行基源鉴定、阐明属内的种间关系及发现新的药用资源提供分子证据。采集三十多种石斛样品分离提取DNA， PCR扩增ITS区及5.8S rDNA完整序列并测序，用Mega 3.1软件进行系统学分析。采用邻接法和最大简约法分析铁皮石斛试管苗亲缘关系，对三十多种石斛类植物的ITS区序列进行分析，两种方法得到的系统树基本一致。ITS序列在石斛种间存在极其显著的差异，铁皮石斛与其它各种石斛均有各自特异性的序列位点，树状图显示铁皮石斛试管苗与野生铁皮石斛源于同一物种，而石斛属其它种与这两种遗传关系较远。 关键词：铁皮石斛试管苗；ITS序列；系统发育；亲缘关系 所属专业：生化与生物技术药物 The Phylogenetic Relationships of Tissue Culture Seedlings of Dendrobium candidum Based on Evidences from ITS Sequences GU Hui-fen1 ，ZHUANG Yi-1i1 ，MEI Qi-chun2 (1．Shanghai Institute of Chinese Materia Medica，Shanghai 201203，China； 2．School of Life Sciences，Fudan University，Shanghai 200433，China) Abstract： Molecular systematic techniques were applied to reveal the genetic diversity of medicinal plant of Dendrobium candidum and related species in Dendrobium. The internal transcribed spacer (ITS) as well as 5.8S rDNA sequences of more than 30 samples of Dendrobium were amplified using PCR method and sequenced．Mega 3.1 was used to analyze the genetic diversity within genus. Phylogenetic relationships among species in Dendrobium and tissue culture seedlings of Dendrobium candidum as outgroup were estimated by maximum parsimony and neighbor—joining methods with Mega 3.1 software，and similar ITS trees were obtained with the two methods The results of phylogenetic analysis also indicate that the tissue culture seedlings of Dendrobium candidum has a closed relationship with Dendrobium candidum, while both Dendrobium candidum and tissue culture seedlings of Dendrobium candidum are the most distant to the other species of Dendrobium. Key words：tissue culture seedlings of Dendrobium candidum；ITS sequence；phylogeny；relationship Dendrobium candidum Wall.ex Lindl），为兰科（Orchidaceae）石斛属（Dendrobium Sw.）多年附生草本，是我国传统名贵中药之一，为石斛之上品，具有润喉消毒、生津益胃、清热养阴等功效。药理研究显示铁皮石斛是一类很有价值的中药类免疫增强剂[1]-[2]，临床上也广泛的用于肿瘤病人手术及化疗后的辅助治疗。随着铁皮石斛研究的不断深入，市场对铁皮石斛的需求也不断增大。近年来通过无性繁殖即组织培养培植试管苗，获得了成功，实现了资源的可持续开发和利用。药