利用PCR-RFLP方法快速鉴别中国牛蒡属药用植物  

Rapid Identification of Chinese Medicinal Plants of Arctium by PCR-RFLP

作　　者：黄彦昌 宋跃岳 许亮[1] 郑汉[2] 董玉玮[3] 张娜 胡传银 窦德强[1] 康廷国[1] HUANG Yanchang;SONG Yueyue;XU Liang;ZHENG Han;DONG Yuwei;ZHANG Na;HU Chuanyin;DOU Deqiang;KANG Tingguo(College of Pharmacy,Liaoning University of Traditional Chinese Medicine,Dalian 116600,Liaoning,China;State Key Laboratory and Breeding Base of Daodi Herbs,Institute of Chinese Materia Medica.China Academy of Chinese Medical Sciences,Beijing 100700,China;Xuzhou University of Technology,Xuzhou 221018,Jiangsu,China;Xuzhou Tianma Jingan Food Co.,ld.,Xuzhou 221747,Jiangsu,China)

机构地区：[1]辽宁中医药大学药学院,辽宁大连116600 [2]中国中医科学院,中药资源中心,道地药材国家重点实验室,北京100700 [3]徐州工程学院,江苏徐州221018 [4]徐州天马敬安食品有限公司,江苏徐州221747

出　　处：《中华中医药学刊》2024年第12期80-83,I0021,共5页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(81874338);中央本级重大增减支项目“名贵中药资源可持续利用能力建设项目”(2060302);辽宁省“百千万人才工程”项目(2021921039)。

摘　　要：目的随着近十几年来国内学者对牛蒡属药用植物研究的深入,中国牛蒡属植物鉴定需要一种快速、准确的分子鉴别手段。因此研究拟通过利用聚合酶链式反应-限制性酶切长度多态性(polymerase chain reaction restriction frag-ment length polymorphism,PCR-RFLP)建立一种快速鉴定中国牛蒡属药用植物的方法。方法通过对两种中国牛蒡属药用植物牛蒡与毛头牛蒡常用鉴定DNA条形码进行限制性核酸内切酶图谱分析,找到牛蒡在内源转录间隔区1(internal transcribed spacer-1,ITS1)片段中有一个单核苷酸多态性(Single-nucleotide polymorphism,SNP)位点,正好为BsaAⅠ酶(YAC/GTR)的酶切位点,根据该酶切位点,设计引物对目标片段进行扩增。另外建立PCR体系:95℃预变性5min,循环反应40次(90℃20s,60℃20s,72℃20s),72℃延伸5min,4℃保温。建立限制性内切酶BsaAⅠ酶切体系,制作琼脂糖凝胶电泳观察结果。结果电泳结果表明在经过BsaAⅠ酶切后牛蒡将会产生长度分别为101bp与125bp的短条带,而毛头牛蒡未被切开仍为226bp的长条带,成功将两种药用植物区分开。该方法简单、快速、准确,满足日常对中国牛蒡属植物的鉴定。结论该实验通过从牛蒡与毛头牛蒡的ITS1序列入手,利用PCR-RFLP技术首次完成了对两种植物的鉴别研究,建立了中国牛蒡属植物的PCR-RFLP鉴别方法。Objective With the deepening of domestic scholars'research on medicinal plants of Arctium in the past decade,a rapid and accurate molecular identification method is needed for the identification of Arctium in China.Therefore,this study aims to establish a rapid identification method for medicinal plants of Arctium in China using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Method By conducting restriction endonuclease map analysis on commonly used i-dentification DNA barcodes of two medicinal plants of Arctium in China,Zhiwu Niubang(A.Lappa L.)and Maotou Niubang(A.tomentosum M.),it is found that Zhiwu Niubang(A.Lappa L.)has a single nucleotide polymorphism(SNP)site in the internal transcribed spacer 1(ITS1)fragment,which happens to be the cleavage site of BsaAⅠenzyme(YAC/GTR),and design primers to amplify the target fragment based on the enzyme cleavage site.In addition,it established the PCR system:pre denaturation at 95℃for 5 min,cyclic reactions for 40 times(90℃for 20 s,60℃for 20 s,72℃for 20 s),extension at 72℃for 5 min,and in-sulation at 4℃.The restriction endonuclease BsaAⅠdigestion system was established,and agarose gel electrophoresis was made to observe the results.Result The electrophoresis results showed that after BsaAⅠenzyme digestion,Zhiwu Niubang(A.Lappa L.)would produce short bands with lengths of 101 bp and 125 bp,respectively,while Maotou Niubang(A.tomentosum M.)will still have a long band of 226 bp without being digested,successfully distinguished two medicinal plants.This method is simple,fast and accurate,and meets the needs of daily identification of Arctium plants in China.Conclusion This experiment started with the ITS1 sequence of Zhiwu Niubang(A.Lappa L.)and Maotou Niubang(A.tomentosum M.),and used PCR-RFLP technolo-gy to complete the identification study of the two plants for the first time,establishing a PCR-RFLP identification method for Arc-tium plants in China.

关 键 词：牛蒡 毛头牛蒡 鉴别 PCR-RFLP 

分 类 号：R282.71[医药卫生—中药学]