基于ITS2序列的丹參及其混偽品分子鑒定研究

張萍　黎先軍　杜微波

摘要[目的]采用DNA條形碼及特異性引物PCR技術(shù)對(duì)中藥材丹參及其混偽品進(jìn)行分子鑒定研究。[方法]以核基因ITS2序列作為DNA 條形碼，對(duì)研究材料進(jìn)行 PCR 擴(kuò)增并雙向測(cè)序，將所得序列構(gòu)建NJ系統(tǒng)發(fā)育樹。利用Koetschan等建立的ITS2數(shù)據(jù)庫及其網(wǎng)站預(yù)測(cè)ITS2二級(jí)結(jié)構(gòu)，同時(shí)采用自設(shè)引物進(jìn)行特異性引物PCR鑒別研究。[結(jié)果]ITS2序列長度為470 bp左右；從系統(tǒng)聚類樹圖可以看出，丹參及其偽品分別聚在不同支，表現(xiàn)出單系性；比較二級(jí)結(jié)構(gòu)發(fā)現(xiàn)，丹參與甘西鼠尾差異甚微，與牛蒡在螺旋莖環(huán)的數(shù)目、大小、位置以及螺旋臂由中心環(huán)伸出時(shí)的轉(zhuǎn)角等方面具有明顯區(qū)別；通過特異性引物PCR技術(shù)可將丹參及其偽品進(jìn)行區(qū)分。[結(jié)論]DNA條形碼技術(shù)和特異性引物PCR技術(shù)均能夠有效地區(qū)別丹參及其混偽品，在中藥材的鑒定中具有重要的應(yīng)用前景。

關(guān)鍵詞丹參；DNA條形碼；二級(jí)結(jié)構(gòu)；PCR技術(shù)

中圖分類號(hào)R282.5文獻(xiàn)標(biāo)識(shí)碼A文章編號(hào)0517-6611（2017）30-0122-03

Abstract[Objective]The research aimed to study the molecular identification of Salvia miltiorrhiza and its adulterants by DNA barcode and specific primers PCR. [Method]The ITS2 sequence was used as DNA barcoding， and the materials were amplified by PCR and sequenced， and the NJ phylogenetic tree was constructed. The secondary structure of ITS2 was predicted by database established and its website by Koetschan et al.，and the selfdesigned primers were used to carry out specific primer PCR identification. [Result]ITS2 sequence length was around 470 bp. The results of cluster analysis showed that Salvia miltiorrhiza and its adulterants were clustered in different branches and showed single character. Compared with secondary structure， Salvia miltiorrhiza and Ganxi rat tail had little difference， and there were significant difference on the number， size， location of burdock in the spiral stem， and the rotation angle of the spiral arm from the central ring with burdock. The specific primers could distinguish the Salvia miltiorrhiza and its counterfeit by PCR technique. [Conclusion]DNA barcoding and specific primers PCR are effective in distinguishing Salviae miltiorrhiza and its adulterants， and it has an important application foreground in the identification of Chinese herbal medicines.

Key wordsSalvia Miltiorrhiza Bge.；DNA barcoding；Secondary structure；PCR technique

丹參在2015年版《中國藥典》一部中記載為唇形科植物丹參（Salvia miltiorrhiza Bge.）的干燥根和根莖，是我國傳統(tǒng)中藥，具有活血祛瘀、通經(jīng)止痛、涼血消癰等功效[1]。目前丹參的主產(chǎn)區(qū)主要集中于山東日照、陜西商洛、四川中江、河南內(nèi)鄉(xiāng)等地，多數(shù)為栽培品種，采用野生馴化、種子繁殖、根段無性繁殖等方式保存種質(zhì)資源[2]。丹參作為我國十大常用大宗藥材之一，在臨床應(yīng)用上已有2 000多年的歷史，由于其對(duì)心血管疾病有顯著療效，且具有抗凝、促纖溶、擴(kuò)血管、改善微循環(huán)、清除自由基、保護(hù)線粒體等廣泛的藥理作用，其需求量正在逐年增大[3]。……