茜草及其混伪品DNA条形码鉴定

Identification of Traditional Chinese Medicine Rubiae Radix et and Its Adulterants Using DNA Barcodes

目的正确鉴别中药材茜草及其混伪品。方法本实验对茜草及其混伪品共13个物种的psbA-trnH、matK和rbcL共317条序列进行分析，建立DNA条形码鉴定体系。采用MEGA5．1进行种内种间变异及K2P（Kimura-2-Parameter）遗传距离分析，并构建NJ系统聚类树，评价psbA-trnH、marK和rbcL序列及其组合对茜草与混伪品的鉴定能力。结果76份基原植物样品的DNA均能成功提取。psbA-trnH、matK和r6cJ已序列扩增成功率分别为100％、96％和99％。psbA-trnH、matK和r6吐单个序列及其两两组合，能将茜草与不同属的混伪品成功区分，但对同属其他物种鉴定效果不理想。psbA-trnH＋matK＋rbcL组合序列鉴定效果最好，种内K2P遗传距离为0～0．0014，种间K2P遗传距离0．0007～0．6303；NJ树显示茜草能与其混伪品茜草属其他8个物种以及紫金牛、丹参、川赤芍、蓬子菜明显区分。市场购买的30份未鉴定的茜草药材中，22份样品能成功扩增3个基因片段，采用NJ树分析，其中11份样品与茜草聚为一支，剩余样品聚为单独3支，为茜草属其他物种。结论psbA—trnH＋marK＋rbcJ已组合序列能作为DNA条形码对茜草及其混伪品进行鉴定。

OBJECTIVE To identify traditional Chinese medicine Rubiae Radix et Rhizoma and its adulterants using DNA bar- codes. METHODS A total of 317 sequences ofpsbA-trnH, matK and rbcL from 13 species were analized. The intra- and inter-specif- ic K2P genetic distances and neighbor-joining tree were calculated using MEGA5.1 program. RESULTS The DNAs were successfully extracted from 76 original plant samples. The amplification rates of psbA-trnH, matK and rbcL were 100% , 96% , and 99%, respec- tively. The adulterants could be identified from R. cordifolia by the single marker and the two combinations except for those adulterants in the Rubia genus. Fortunately, R. cordifolia could be identified from all the adulterants by psbA-trnH ＋ matK ＋ rbcL combination marker. The intra- and inter-specific K2P genetic distances of psbA-trnH ＋ marK ＋ rbcL were 0 - 0. 001 4 and 0. 000 7 - 0. 630 3. R. cordifolia could be identified from all＇the adulterants by the psbA-trnH ＋ matK ＋ rbcL combination using NJ tree method. Among the 30 unidentified samples of Rubiae Radix et Rhizoma which were collected from medicinal herb markets, all three markers, including psbA- trnH, matK and rbcL, could be amplified from 22 samples. The 11 samples of Rubiae Radix et Rhizoma clustered with R. cordifolia and the others were divided into three clusters by the psbA-trnH ＋ matK ＋ rbcL combination using NJ tree method. CONCLUSION psbA-trnH ＋ matK ＋ rbcL combination can be used as DNA barcode to identify R. cordifolia from adulterants.