[关键词]
[摘要]
目的 对武当山区重楼属植物进行种内分子鉴定研究,以DNA条形码技术解决重楼因种内形态差异小而难以鉴别的问题,规范重楼的正品来源,确保本地药材的质量以及临床用药的有效性。方法 对ITS(internal transcribed spacers)序列进行克隆测序,以核基因ITS2片段作为DNA条形码,对研究材料进行PCR(polymerase chain reaction)扩增并双向测序,所得序列经CodonCode Aligner拼接后,用软件MEGA6.0(Molecular Evolutionary Genetics Analysis 6.0)进行相关数据分析,并构建NJ邻接树(neighbor-joining tree),利用Keller等建立的ITS2数据库及其网站预测ITS2的二级结构。结果 经BLAST鉴定,采集的武当山区重楼样本来源准确;武当山区重楼属植物ITS2序列长度均为232 bp,与七叶一枝花的基因序列相比,球药隔重楼有16个变异位点,华重楼只有1个变异位点;武当山区重楼的ITS2序列种内K2P(Kimura-2-parameter)距离为0~0.073 6,种内平均K2P距离为0.027 2;由所构建的系统聚类树可以看出,本地区重楼主要分为七叶一枝花和球药隔重楼2大类,华重楼为七叶一枝花的变种,宽叶重楼、狭叶重楼与七叶一枝花为一支,不支持宽叶重楼、狭叶重楼成为独立的变种;比较ITS2二级结构发现,武当山区重楼属植物螺旋区的茎环数目、大小、位置有明显差异。结论 DNA条形码为重楼属药用植物鉴别开辟一条新途径,不论是相似性搜索法、最近距离法还是NJ系统发育树等方法,其分析结果均充分表明,ITS2序列可将不同品种重楼药材很好地区分开;七叶一枝花、狭叶重楼、宽叶重楼的ITS2基因232个碱基序列完全相同,MEGA 6.0的分析结果也表明宽叶重楼、狭叶重楼与七叶一枝花属于一支,不支持宽叶重楼、狭叶重楼成为独立的变种,建议作为七叶一枝花的变型处理;ITS2作为DNA条形码序列能够有效地区别重楼属植物,在中药材的鉴定中具有重要的应用前景,在实现规范化种植规程中可用于种质种源的正确鉴定。
[Key word]
[Abstract]
Objective DNA barcoding technology, a molecular identification method, is applied to Paris polyphylla collected from Wudang Mountain area in order to solve the differential problem caused by small morphology difference, make authentic origins canonical, and guarantee the quality and clinical curative effect of this medical material in our area. Methods In this study, the second internal transcribed spacers (ITS2) of ribosomal DNA were amplified and sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. To perform a phylogenetic study, the genetic distances were computed using molecular evolutionary genetics analysis 6.0 (MEGA 6.0) in accordance with the Kimura-2-parameter (K2P) model. The phylogenetic tree was constructed using the neighbor-joining (NJ) and Kimura-2-parameter methods. The ITS2 secondary structures were predicted using ITS2 database and website which was found by Keller. Results Identified by BLAST, P. polyphylla collected in Wudang mountain area was proved to be precise. The results showed that the ITS2 sequence length was 232 bp. It was indicated that their K2P distance were 0-0.073 6 and the average was 0.027 2. Compared with the gene sequence of P. polyphylla, there were 16 variation loci in P. fargesii and only one variation locus in P. polyphylla var. chinensis. As seen from the NJ tree, P. polyphylla in this area was mainly divided into two categories, P. polyphylla and P. fargesii. Obviously, P. polyphylla var. chinensis was a variation of P. polyphylla. Meanwhile, it was not tenable that P. polyphylla var. stenophylla and P. polyphylla var. latifolia were the variations of P. polyphylla because they belonged to a branch. To compare the ITS2 secondary structure of P. polyphylla, we noticed the differences in the amount, size, and position of loop of helix zone. Conclusion DNA barcoding technology opens up a new way for the identification of P. polyphylla. Regardless of what methods were used, such as similarity search, nearest distance, and NJ tree method, the analysis results have fully demonstrated that ITS2 sequences could be used to correctly identify the different varieties of P. polyphylla. It is identical in 232 base sequence of ITS2 to P. polyphylla var. stenophylla, P. polyphylla var. latifolia, and P. polyphylla. The results computed by Mega 6.0 also show that they belong to a branch. It is more appropriate to say P. polyphylla var. stenophylla and P. polyphylla var. latifolia are variants of P. polyphylla. Hence, as a DNA barcode sequence, ITS2 can be applied to intraspecific discrimination of P. polyphylla. Furthermore, the application of ITS2 in the identification of traditional Chinese medicine has an important prospect. And it can be used to correct identification of germplasm resources in GAP procedures.
[中图分类号]
[基金项目]
湖北省2011计划“武当山区重楼等特色中药资源调查及品质比较研究”;2014年湖北省中青年创新团队项目(T201414,武当山区道地中药材GAP关键技术研究);湖北省药用植物综合利用工程技术研究中心项目(GC2015204)