基于ITS序列构建系统发育树鉴定市售黄芪饮片基源的研究  

Original identification of Commercial Slices of Astragali Radix by Construction of Phylogenetic Trees Based on ITS Sequences

作　　者：刘建军[1] 黄世锋[1] 代文婷[1] 翟丹丹 宗凯 LIU Jianjun;HUANG Shifeng;DAI Wenting;ZHAI Dandan;ZONG Kai(Dept.of Pharmacy,the Second People’s Hospital of Hefei,Anhui Hefei 230011,China;Technical Center,Hefei Customs District P.R.China,Anhui Hefei 230022,China)

机构地区：[1]合肥市第二人民医院药学部,安徽合肥230011 [2]中华人民共和国合肥海关技术中心,安徽合肥230022

出　　处：《中国医院用药评价与分析》2020年第2期154-157,共4页Evaluation and Analysis of Drug-use in Hospitals of China

基　　金：国家质检总局科技计划项目(No.2015IK001).

摘　　要：目的:建立一种基于内转录间隔区(internal transcribed spacer,ITS)序列的系统发育树方法,快速、准确地鉴别市售黄芪饮片的基源。方法:提取经鉴别的不同来源的14份蒙古黄芪药材和8份膜荚黄芪药材的DNA,设计并合成ITS通用引物,经扩增和测序后采用MEGA 6.0软件将测序结果用邻接法构建系统发育树;收集市场上销售的16份黄芪饮片,采用设计的引物进行扩增后电泳并测序,按照邻接法构建系统发育树对其种类进行区分鉴定。结果:蒙古黄芪和膜荚黄芪ITS序列同源性极高,在整个ITS全序列区域仅第128位碱基上有G/A突变、第619位碱基上有G/-/T/C突变、第620位碱基上有T/C突变、第621位碱基上有C/A突变、第622位碱基上有A/-/C突变以及第623位碱基上有C/A/-突变;通过基因测序结果所建立的系统发育树,可以区分22份黄芪样品中膜荚黄芪和蒙古黄芪的遗传进化关系;对16份市售黄芪饮片的基源进行鉴别,其中12份为蒙古黄芪,4份为膜荚黄芪。结论:基于ITS序列构建的系统发育树,可有助于蒙古黄芪和膜荚黄芪药材或饮片的基源鉴别。OBJECTIVE:To establish the phylogenetic trees based on internal transcribed spacer(ITS)gene sequence,so as to quickly and accurately identify the origin of commercial slices of Astragali Radix.METHODS:The DNA of 14 samples of Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao and 8 samples of Astragalus membranaceus(Fisch.)Bge.from different sources were extracted.ITS universal primers were designed and synthesized.After the amplification and sequencing,the phylogeny trees for all species were constructed by Neighbor-Joining(NJ)tools with MEGA 6.0 software.Totally 16 kinds of Astragali Radix pieces were collected from the market and then amplified by PCR with designed primers.The PCR products were directly sequenced and its species were differentiated and identified by constructing phylogenetic trees according to NJ method.RESULTS:ITS sequence had high homology for Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao and Astragalus membranaceus(Fisch.)Bge..There were only a few mutation types in the whole ITS sequence region,such as G/A on base 128 and G/-/T/C on base 619,T/C on base 620,C/A on base 621,A/-/C on base 622,and C/A/-on base 623.With the help of the phylogenetic trees established by gene sequencing,the genetic and evolutionary relationship between Astragalus membranaceus(Fisch.)Bge.and Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao in 22 samples can be distinguished,which can also provide the origin identification for 16 kinds of Astragali Radix in the market,including 12 for Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao and 4 for Astragalus membranaceus(Fisch.)Bge..CONCLUSIONS:The phylogenetic trees based on ITS sequence can be helpful for the original identification of Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao and Astragalus membranaceus(Fisch.)Bge..

关 键 词：ITS序列 系统发育树 黄芪饮片 基源鉴别 

分 类 号：R284.1[医药卫生—中药学]