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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Codon Code 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。
Objective To study the intraspecific molecular identification of Rhododendron species in Wudang Mountain, solve the problem of re-formation due to small intraspecific difference in DNA barcode technology, standardize the authentic origin of Rhododendron, and ensure the quality of local medicinal herbs and clinical medication Effectiveness. Methods The sequence of ITS (internal transcribed spacers) was cloned and sequenced. ITS2 fragment was used as DNA barcode. The PCR products were amplified by polymerase chain reaction (PCR) and sequenced. The sequences were spliced by Codon Code Aligner and analyzed by software MEGA6 (Molecular Evolutionary Genetics Analysis 6.0), and constructed NJ-neighbor tree. ITS2 database and its website were established by Keller et al. To predict the secondary structure of ITS2. Results The sequence of ITS2 sequence in Wudang Mountain was identified by BLAST. The length of ITS2 sequence in the Wudang Mountains was 232 bp. Compared with the gene sequence of the flower of Aescin, 16 different positions There was only one variation site in HuaRongLou and K2R (Kimura-2-parameter) in the ITS2 sequence of Wudang Mountains, and the average K2P distance in the species was 0.0272. The constructed system Cluster tree can be seen in the region re-floor is divided into seven flower and ball medicine ball floor, two categories, Huazhong House is a variant of the seven-leaf flower, wide leaf re-building, Compared with ITS2 secondary structure, the number, size and position of stem rings in the spiral zone of Reproduction plants in Wudang Mountains were significantly different. Conclusion DNA barcoding has opened up a new way for the identification of medicinal plants of Rhizoma Paridis. Both the similarity search method, the nearest distance method and the NJ phylogenetic tree, etc. The results of the analysis fully show that ITS2 sequences can differentiate different species of Rhizoma The results of MEGA 6.0 also showed that the ITS2 gene of A. splendens, L. splendens L. and L. splendens was identical. The results of MEGA 6.0 also showed that the broadleaf, Flowers belong to one, does not support the broad-leaved floor, narrow-leaved floor to become an independent variant, it is proposed as a variant of aescinate flower; ITS2 DNA barcode sequence can effectively distinguish Rehanaia plants in the identification of Chinese herbal medicines Has an important application prospect in the implementation of standardized planting procedures can be used for the germplasm provenance of the correct identification.