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目的:测定和分析不同产地野生铁皮石斛样品的rDNA ITS序列,探讨利用ITS序列推测铁皮石斛样品产地来源的可行性。方法:提取总基因组DNA,使用通用引物ITS5F/ITS4R进行PCR扩增,产物经电泳检测、纯化和测序后用Sequin 11.0提交GenBank。用Bioedit 7.0中的Clustal W对GenBank中包括本研究提交的11条在内的所有33条铁皮石斛的ITS序列进行多重序列比对,随后以金钗石斛和密花石豆兰为外群,用邻接法构建NJ系统发育树。结果:从所有测定的ITS序列中选择11条提交GenBank,获得登录号JF803235-JF803245。所有铁皮石斛经比对后共发现645个同源位点,9个信息位点(ITS1区3个,5.8 S区4个,ITS2区2个)。系统发育分析发现,金钗石斛和密花石豆兰位于树的基部,另外32条铁皮石斛共同形成单个大的分支,未发现产地来源与系统树的拓扑结构之间存在对应关系。结论:由于缺乏完善的铁皮石斛标准品及其DNA参考序列的提交标准,同时单个ITS序列仅能提供有限的系统发育信息位点,因此通过ITS序列分析的方法推测铁皮石斛样品的产地来源目前尚不具有可行性。
Objective: To determine and analyze the rDNA ITS sequence of Dendrobium officinale in different areas, and to explore the feasibility of using ITS sequence to infer the origin of Dendrobium officinale samples. Methods: Total genomic DNA was extracted and amplified by universal primer ITS5F / ITS4R. The product was detected by electrophoresis, purified and sequenced and submitted to GenBank with Sequin 11.0. Multiple sequence alignment of ITS sequences of all 33 species of Dendrobium candidum, including 11 strains submitted in this study, was carried out in GenBank using Clustal W in Bioedit 7.0. Subsequently, the ITS sequences of Dendrobium nobile, Construction of NJ phylogenetic tree. Results: Eleven selected ITS sequences were submitted to GenBank for accession number JF803235-JF803245. A total of 645 homologous sites and 9 information sites (3 in ITS1, 4 in 5.8 S and 2 in ITS2) were found in all Dendrobium candidum. Phylogenetic analysis showed that Dendrobium nobile and Dactylis odoina were located at the base of the tree, while the other 32 Dendrobium species together formed a single large branch. There was no correspondence between the origin and the topological structure of the phylogenetic tree. CONCLUSIONS: The origin of D. candidum samples was inferred by means of ITS sequence analysis due to the lack of a complete set of standards for the submission of standards and reference sequences for D. officinale, and for a single ITS sequence to provide only limited information on phylogenetic information Not feasible.