基于ITS2序列广陈皮基原植物及药材的DNA分子鉴定

Identification of Citrus reticulate 'Chachi' and its original plant based on ITS2 sequence

目的选取ITS2序列对广陈皮基原植物及药材进行鉴定,为DNA条形码技术应用于广陈皮药材的鉴定研究提供理论依据。方法提取样品DNA,利用PCR技术对样品进行核基因ITS2片段扩增并双向测序。所得序列经Codon Code Aligner拼接后,用MEGA6.0软件进行数据分析;计算种内、种间Kimura-2-Parameter(K2P)遗传距离并采用邻接法构建NJ系统树。结果广陈皮种内最大K2P遗传距离远小于种间最小K2P遗传距离;由构建的系统聚类树可以看出,广陈皮基原植物及药材聚成一支,并与其他近缘种植物明显分开。广陈皮药材的测序成功率明显低于原植物的测序成功率,年份越高的广陈皮测序成功率越低。结论 ITS2序列作为DNA条形码可以有效地鉴别广陈皮基原植物及药材与其他近缘种植物,为广陈皮药材基原鉴定及临床安全用药提供了分子依据。

Objective To identify Citrus reticulate ＇Chachi＇ and its original plant with ITS2 sequence and provide theoretical basis for the application of DNA barcoding in the identification of Citrus reticulate ＇Chachi＇. Methods Total genomic DNA was isolated as template and the nuclear gene ITS2 region was amplified by PCR technology and sequenced in bidirection. The sequences was analyzed using the software Codon Code Aligner and MEGA 6.0. The intra-and inter-specific Kimura-2-Parameter（ K2P） distances were calculated and the phylogenetic tree was constructed by using the Neighbor-joining method. Results The maximum K2P genetic distance of inter-specific of Citrus reticulate ＇Chachi＇ and its original plant was lower than the mini-mum K2P genetic distance of its related species. In the cluster dendrogram, Citrus reticulate ＇Chachi＇ and its original plant could be obviously distinguished from the closely relative species. However,the success rate of sequencing of Citrus reticulate ＇Chachi＇ is obviously lower than that of its original plant,and the longer the year of Citrus reticulate ＇Chachi＇, the lower the success rate. Conclusion ITS2 barcoding could be used to distinguish the original plant of Citrus reticulate ＇Chachi＇ and its related species effectively,and provide important molecular evidence for the identification of Citrus reticulate ＇Chachi＇ and clinic safety in medicinal use.