264 2011 7 4 4 Global Traditional Chinese Medicine July 2011 Vol. 4 No. 4 ITS2 ITS2 DNA NJ Schultz ITS2 ITS2 ITS2 220 bp 230 bp NJ 83% ITS2 DNA DNA ITS2 R282. 5 A doi 10. 3969 /j. issn. 1674-1749. 2011. 04. 007 Molecular identification of radix et rhizoma clematis and its adulterants based on ITS2 DNA barcode ZENG Xu LI Li YE Ning et al. Department of Resources Science of Traditional Chinese Medicines China Pharmaceutical University Nanjing 210009 China Corresponding author LIU Zhi-hua E-mail zhliu@ implad. ac. cn QIN Min-jian E-mail minjianqin@ 163. com Abstract Objective To discriminate radix et rhizoma clematis and its adulterants and also assure the quality and clinicalsafety of this traditional Chinese medicinal material. Methods In the study ITS2 sequence of the clematis and its adulterants were evaluated according to sequence length GC content intra - specific variation as well as the inter - specific divergence. NJ tree of radix et rhizoma clematis its adulterants had been constructed using MEGA4 software. The ITS2 secondary structure was predicted using the ITS2 database and website constructed by Schultz et al. Results The length of ITS2 sequence of the origin plants of radix et rhizoma clematis were 220 bp and 230 bp respectively. In the phylogenetic tree the three origin plants could be clustered together and the favorability rating was 85 %. From the branches of the plants the distribution center of the clematis genera in southwesten China conformed with the theory of center spread periphery. The difference between radix et rhizoma clematis and other species could be found in the number size position of loop and the angle of helix exsertion based on comparing the ITS2 secondary structure of the origin plants of radix et rhizoma clematis and its adulterants. Conclusion can be used to correctly identify radix et rhizoma clematis and its adulterants and it plays an important role in the identification of traditional Chinese medicine. Key words Radix et rhizoma clematis DNA barcoding ITS2 Identification 5113033 D08080203640901 1986-2009 E-mail xzeng. liulab@ gmail. com 1979- E-mail zhliu@ implad. ac. cn 1962- E-mail minjianqin@ 163. com ITS2 转载
2011 7 4 4 Global Traditional Chinese Medicine July 2011 Vol. 4 No. 4 265 2010 Clematis chinensis Osbeck Clematis hexapetala Pall. Clematis manshurica Rupr. 1 1. 1 1 Genbank Smilax Clematis 2 3 Inula neruosa Wall. Sarandra glabra Thunb. NaRai 1 Material 4 Clematis hexapetala GQ434606 Clematis manshurica GU732608 Clematis lasiandra GU732600 Paeonia lactiflora Pall. Clematis pogonandra GU732618 Clematis uncinata GU732637 Sarcandra glabra GQ434455 Sarcandra glabra GQ434454 Sarcandra glabra AF280408 Sarcandra glabra AF203635 DNA DNA barcoding Podophyllum hexandrum Podophyllum hexandrum GQ434794 AJ717334 DNA Podophyllum hexandrum AJ717332 Paeonia lactiflora GQ434601 Smilax lasioneura AY775251 7 2003 Smilax herbacea AY775249 PaulHebert C COI 1. 2 1. 2. 1 DNA 9 70% DNA 15 mg 30 mg DNA Tiangen Biotech Co. China DNA rdna 5. 8S 26S rrna 1. 2. 2 PCR ITS2 ITS2 5'-GCGATACTTGGTGTGAAT- DNA 3' 5'-GACGCTTCTCCAGACTACAAT-3' DNA PCR MgCl 2 2 μl 25 mm dntp 2 μl 2. 5 mm PCR buffer 2. 5 μl 10 1. 0 μl 2. 5 ITS2 μm Sangon Co. China 1. 0 U Biocolor DNA BioScience & Technology Co. China DNA 1 Clematis chinensis Clematis hexapetala Clematis hexapetala Genbank Latin Name Genbank No. GU732584 GU732597 AY055385
266 2011 7 4 4 Global Traditional Chinese Medicine July 2011 Vol. 4 No. 4 1 μl 25 μl 94 5 94 30 56 30 72 45 40 72 10 PCR PolyC PolyG GC Tiangen Biotech Co. China 69. 5% Clematis manshurica ABI 3730XL Applied Biosystems Co. USA 1. 2. 3 CodonCode Aligner 3. 71 ITS2 0. 006 HMMer 0. 017 5. 8S 26S ITS2 MEGA 4. 0 1. 083 1. 077 1. 146 2. 2 Kimura 222 Parameter K2P NJ neighbour-2-joining method NJ Genbank ITS2 1000 bootstrap 3 230 Schul- bp151 bp C-G tz ITS2 Schultz 16 1 bp 5 bp 209 ITS2 ITS2 19 2 2. 1 PolyC PolyG GC 69. 9% Clematis hexapetala 220 bp 230 bp PolyC PolyG GC 69. 9% Kimura-2 bp 222 bp 230 bp K2P 0. 003 K2P 0. 005 2. 3 ITS2 ITS2 K2P 0. 708 Clematis chinensis 230 bp 1
2011 7 4 4 Global Traditional Chinese Medicine July 2011 Vol. 4 No. 4 267 1 1 ITS2
268 2011 7 4 4 Global Traditional Chinese Medicine July 2011 Vol. 4 No. 4 2. 4 ITS2 DNA ITS2 ITS 7 8 6 100% DNA 92. 7% ITS2 DNA 6 3 83% 2 2. 5 ITS2 3 3 Ⅳ Ⅳ 2 1 64% 2 Ⅳ I Ⅲ ITS2 20 3 Helleboroideae 21 ITS2 DNA 2 ITS2 NJ bootstrap1000 50%
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