松针的显微及分子鉴别研究

目的: 利用显微和分子鉴定技术对不同植物基源的松针药材进行鉴别，为松针的鉴别提供科学依据。方法: 采用基源、显微鉴定的方法研究马尾松（Pinus massoniana Lamb.）、黑松（Pinus thunbergii Parl.）和华山松（Pinus armandii Franch.）的特征；利用PCR扩增和双向测序分析植物DNA条形码ITS2和rbcL序列特征；采用分子进化遗传分析（MEGA）6.0软件进行种间、种内Kimura2-parameter（K2P）遗传距离计算，并构建邻接系统聚类树。结果: 三种松针数目及长度、维管束数、气孔线的分布、树脂道数目及分布等具有明显区别。马尾松、黑松和华山松的ITS2序列长度分别为470、469和470 bp，rbcL序列长度均为553 bp；ITS2序列在马尾松、黑松和华山松的种内变异率分别为0%、0.2%和2.8%，rbcL序列在这三种松针样品的种内变异率分别为0%、2.4%和1.1%。ITS2序列在松针的种内和种间遗传距离中不存在显著的条形码间隔；而rbcL序列的种内和种间距离存在一定的条形码间隔。邻接系统树结果表明，三种松针药材聚为不同的分支，rbcL条形码序列可鉴别马尾松、黑松、华山松及其近缘的植物。结论: 利用基源、显微及分子鉴定技术可准确、快速地鉴别马尾松、黑松和华山松，为松针的质量评价和分类鉴别提供了方法和依据。
Abstract: Objective: To identify pine needles from different plant origins by microscopic and molecular approaches. Methods: The characteristics of pine needles of Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were investigated via plant morphology and microscopic characteristics. ITS2 and rbcL were analyzed with PCR amplification and bi-directional sequencing. MEGA 6.0 was used to calculate the intra-and inter-specific Kimura-2-Parameter (K2P) distances, and the phylogenetic tree was constructed by using the neighbor-joining (NJ) method. Results: There were significant differences in the number and length of pine needles, number of vascular bundles, distribution of stomatal lines, number and distribution of resin channels among three kinds of pine needles. The lengths of ITS2 sequences of Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were 470, 469 and 470 bp, respectively. The lengths of rbcL sequences in three kinds of pine needles were 553 bp. The intraspecific variation rates of ITS2 sequences in Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were 0%, 0.2%, and 2.8%, respectively; and the intraspecific variation rates of rbcL sequences were 0%, 2.4%, and 1.1%, respectively. There was no significant barcoding gap in intraspecific and interspecific genetic distances of ITS2 sequences. The intraspecific and interspecific distances of rbcL sequences were clearly separated in the barcoding gap test. The NJ tree based on rbcL showed that the three pine needles clustered into three separate groups, indicating that rbcL DNA marker could distinguish the Pinus massoniana Lamb., Pinus thunbergii Parl., Pinus armandii Franch. and its close relative species. Conclusions: The three types of pine needles can be distinguished accurately and rapidly by microscopic and molecular identification. The study provides methodology and experimental basis for the quality evaluation and