苗药八爪金龙基原植物DNA条型码的筛选与鉴定  

Screening and Identification on ITS Sequences of Original Plants from Ardisia crispa

作　　者：潘婕 刘畅 俸婷婷 刘雄伟[1] 周永强 石慧 周英 Pan Jie;Liu Chang;Feng Tingting;Liu Xiongwei;Zhou Yongqiang;Shi Hui;Zhou Ying(Research Center for Development of Traditional Chinese Medicine,Research Center for Application and Development of Medicine and Food Dual­use Resources,School of Pharmacy,Guizhou University of Traditional Chinese Medicine,Guiyang,550025;Guizhou Engineering Center for Innova­tive Traditional Chinese Medicine and Ethnic Medicine,Guizhou University,Guiyang,550025)

机构地区：[1]贵州中医药大学药学院,药食两用资源应用与开发研究中心,中药材开发技术研究中心,贵阳550025 [2]贵州大学,贵州省药食同源植物资源开发工程技术研究中心,贵阳550025

出　　处：《分子植物育种》2020年第24期8187-8195,共9页Molecular Plant Breeding

基　　金：国家重点研发计划项目(2018YFC1708100);贵州省高层次创新型人才培养项目(黔科合人才(2015)4032号);贵州中医药大学2018年度学术新苗培养及创新探索专项([2018]5766号-9);贵州中医药大学博士启动基金(贵中医博士启动[2019]04号)共同资助。

摘　　要：八爪金龙属紫金牛科(Myrsinaceae)紫金牛属(Ardisia)植物,是常用民族药,其抗病毒、抗心律失常、抗肿瘤的效果显著。八爪金龙的基原植物表型极度相似,导致其应用混乱。本研究筛选适用于八爪金龙基原植物鉴定的DNA条形码序列,以探索高效准确鉴定八爪金龙基原植物的新方法。提取了百两金、朱砂根和红凉伞的植物基因组DNA,利用DNA条形码序列(ITS, ITS2, RbcL, PsbA-trnH)对其进行PCR扩增和测序,比较各序列的扩增率和测序率,应用Blast方法来评估各序列的鉴定效率。同时与9种紫金牛属植物的相应序列进行比对,构建NJ树。结果表明:ITS、ITS2、RbcL序列的扩增成功率和测序成功率均为100%,PsbA-trnH序列的扩增成功率和测序成功率分别为97.7%和96.7%。4个候选序列的BLAST成功率结果:ITS=ITS2 (100%)>PsbA-trnH (99.2%)>RbcL (33.3%)。ITS序列的平均种间遗传距离大于平均种内遗传距离。系统发育树构建结果显示,ITS2序列的NJ树可将百两金、朱砂根和红凉伞单聚成一支,与同属植物区别开;PsbA-trnH片段将朱砂根、红凉伞单聚为一支,区别于其他同属植物;ITS序列能将不同基原聚成不同的分支,区别于同属植物,但朱砂根和红凉伞需进一步研究。各结果显示ITS序列较其他序列有明显优势,本研究建立了基于ITS序列的八爪金龙基原的DNA条形码鉴定技术方法,为临床准确用药提供依据。Radix Ardisia, a genus of Myrsinaceae of Ardisia is commonly used as national medicine for antiviral,antiarrhythmic and antitumor treatment. However, phenotypes of these species are quite similar, which often leads to confusing applications. In the present study, to screen suitable DNA barcoding and explore an effectively and efficient identification method for three original species from Radix ardisia. The total DNA of Ardisia crispa,Ardisia crenata Sims, and Ardisia crenata Sims var. bicolor was extracted. The DNA barcoding sequences(ITS, ITS2,RbcL, PSBA-TRNH) were used for PCR amplification and sequencing, and the amplification and sequencing rates of each sequence were compared. Then BLAST was used to evaluate the identification efficiency of each sequence.. At the same time, the NJ tree was constructed by comparing with the corresponding sequences of nine species of Ardisia. The results showed that the rate of amplification and sequencing of ITS, ITS2 and RbcL was100%, and the PsbA-trnH were 97.7% and 96.7%. The Blast success rate result is as follows: ITS=ITS2(100%)>PsbA-trnH(99.2%)>RbcL(33.3%). The average interspecific genetic distance is more than the average intraspecific genetic distance of ITS sequence. According to the phylogenetic tree of ITS2, Ardisia crispa, Ardisia crenata Sims,and Ardisia crenata Sims var. bicolor were grouped into a single branch, distinguished from the same genus. The Ardisia crenata Sims and Ardisia crenata Sims var. bicolor were clustered into a single branch, which was distinguished from other plants of the same genus by PsbA-trnH. All primordia could cluster into different branches, which is different from the same genus based on ITS. However, Ardisia crenata Sims and Ardisia crenata Sims var. bicolor need to be further studied. The results showed that ITS sequence was superior to other sequences. The DNA barcoding technique for identification of Ardisia crispa was established on the basis of ITS sequences. This study provided the basis for accurate clinical medication.

关 键 词：八爪金龙(Raxid Ardisia) 近缘植物 DNA条形码 ITS序列 

分 类 号：S567.19[农业科学—中草药栽培]