基于ITS2序列的羌活及其混伪品的DNA条形码鉴定

Identification of Notopterygii Rhizoma et Radix and its adulterants using DNA barcoding method based on ITS2 sequence

目的对羌活及其混伪品进行分子鉴定,以确保该药材的质量以及临床疗效。方法利用PCR测序法,对样品进行核基因ITS2片段扩增并双向测序,所得序列经CodonCode Aligner拼接后,用软件MEGA4.0进行相关数据分析,并构建邻接(NJ)树。利用已建立的ITS2数据库及其网站预测ITS2二级结构。结果羌活与宽叶羌活ITS2序列长度均为228 bp,二者种内平均K2P(Kimura-2-parameter)遗传距离均远远小于其与混伪品的种间平均K2P遗传距离;由所构建的系统聚类树图可以看出,羌活与宽叶羌活均表现出了单系性,而同时又与其他混伪品明显分开;比较ITS2二级结构发现,羌活基原植物与其混伪品在4个螺旋区的茎环数目、大小、位置以及螺旋发出时的角度均有明显差异。结论 ITS2序列作为DNA条形码可以方便快捷地鉴别羌活及其混伪品,为其种质资源鉴定及临床安全用药提供了重要分子依据。

Objective To establish and optimize the MSAP reaction system for analysis on epigenetic diversity in Akebia trifoliata.Methods The leaves of A.trifoliata were as materials,using orthogonal L16（45） method to establish pre-amplification and selective amplification optimum reaction system of the MSAP.Results The optimal MSAP reaction systems include： 10 U HpaII/MspI and EcoR I in the enzyme digestion;in the 20 μL pre-amplication mixture contained 2 μL of ligation products,125 ng of each pre-selective primers,E00 and HM0;0.312 5 mmol/L of dNTPs,1.875 mmol/L of Mg2＋,2 U of Taq DNA polymerase;in the 20 μL selective amplification mixture contained 5 μL of 1∶200 diluted pre-amplification products,50 ng of each selective primer,0.250 mmol/L of dNTPs,1.250 mmol/L of Mg2＋,and 1 U of Taq DNA polymerase.Using the optimal MSAP reaction system to screen for six pairs of effective primers from 80 pairs of primers of MSAP.Conclusion Those results provide fundamental reference for further epigenetic studies on A.trifoliata DNA methylation.