野生铁皮石斛rDNA ITS序列分析

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。

Analysis of rDNA ITS Sequence of Wild Dendrobium Officinale

This study was aimed to determine the sequence and analyze rDNA ITS sequences of samples of wild Dendrobium officinale from different origins. The feasibility of using ITS sequence to identify the origin of Dendrobium officinale was discussed. Total DNA was extracted and PCR was applied by using primer ITS5F/ITS4R. Then the PCR products are electrophoresised, purified, sequenced and submitted to GenBank. Clustal W was used to compare the multiple alignment of all 33 ITS sequences, including 11 submitted sequences in this article. Dendrobium nobile and Bulbophyllum odoratissimum were used as outgroup to analyze phylogeny by building neighbor-joining tree. The results showed that 11 sequences were selected to be submitted to the GenBank from all sequences. The achieved accession number was JF803235-JF803245. A total of 645 homologous sites, 9 informative sites (3 sites of ITS1, 4 sites of 5.8S, and 2 sites of ITS2) were obtained. Phylogenetic analysis showed that Dendrobium nobile and Bulbophyllum odoratissimum were in the base of the tree, and the other 32 Dendrobium officinale together were formed on large branches. No correspondence between the origin and the topology of phylogenetic tree were founded. It was concluded that due to the current lack of comprehensive standard of Dendrobium officinale samples and ITS reference sequence, only using ITS sequences cannot get enough phylogenetic information sites. Therefore, it is not feasible to speculate the origin of Dendrobium officinale.