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目的:建立基于DNA条形码技术的中药材苍术种苗鉴定方法。方法:收集中药材苍术及其易混品原植物样品28份,构建苍术种苗鉴定参考核糖体内部转录间隔区2(ITS2)条形码数据库。从河北、山东、山西、内蒙古、辽宁及湖北等苍术主产地收集苍术种苗52份。通过提取基因组DNA,聚合酶链式反应(PCR)扩增,双向测序获得其ITS2条形码序列。应用PUAP 4.0软件计算种内、种间遗传距离,利用MEGA 7.0软件构建邻接树,对中药材苍术种苗进行DNA条形码鉴定。结果:中药材苍术及其易混品原植物ITS2条形码序列具有稳定的序列差异,邻接树呈现单系性,可以明显区分苍术及其易混品;基于标准参考数据库,42份苍术种苗为朝鲜苍术(80.8%),7份苍术种苗为苍术(13.5%),3份苍术种苗为白术(5.7%)。结论:基于ITS2序列可以准确区分中药材苍术及其混伪品种苗。苍术种苗基原物种鉴定结果以朝鲜苍术为主,提示苍术临床用药存在潜在安全风险。
OBJECTIVE: To establish a method for the identification of Cangzhu seedlings based on DNA barcoding technology. Methods: Twenty-eight samples of Chinese herb medicinal plants and their mixed samples were collected to establish the barley seedling identification reference ribosomal internal transcribed spacer 2 (ITS2) barcode database. From the main producing areas of Hebei, Shandong, Shanxi, Inner Mongolia, Liaoning and Hubei, 52 cultivars of herb were collected. ITS2 barcode sequence was obtained by extracting genomic DNA, polymerase chain reaction (PCR) amplification and bidirectional sequencing. PUAP 4.0 software was used to calculate the intraspecific and interspecific genetic distance. The MEGA 7.0 software was used to construct the contiguous tree and the DNA was identified by DNA barcoding. Results: The ITS2 barcode sequence of Chinese herbal medicine and its mixture had stable sequence differences. The adjacent trees showed monophyleticity, which could clearly distinguish between herbaceous stems and their mixed products. Based on the standard reference database, 42 herbaceous seedlings were North Korea Atractylodes (80.8%), seven herbaceous seedlings were herbaceous (13.5%), and three herbaceous seedlings were Atractylodes (5.7%). Conclusion: Based on the ITS2 sequence, it is possible to accurately distinguish Chinese herbaceous plants and their adulterated seedlings. The results of the identification of the original species of Atractylodes seedling seedlings were mainly from Korean herb, suggesting that there is a potential safety risk in the clinical use of Atractylodes.