The Korea Journal of Herbology (대한본초학회지)

The Korea Association of Herbology (대한본초학회)
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Development of SCAR Markers for the Discrimination of Rhei Radix et Rhizoma and Rhei Undulatai Rhizoma based on the RAPD

RAPD 분석을 통한 대황(大黃)과 종대황(種大黃) 감별용 SCAR 유전자 마커 개발

  • Moon, Byeong-Cheol (Center of Herbal Resources Research, Korea Institute of Oriental Medicine) ;
  • Lee, Young-Mi (Center of Herbal Resources Research, Korea Institute of Oriental Medicine) ;
  • Chun, Jin-Mi (Center of Herbal Resources Research, Korea Institute of Oriental Medicine) ;
  • Lee, A-Young (Center of Herbal Resources Research, Korea Institute of Oriental Medicine) ;
  • Yoon, Tae-Sook (Center of Herbal Resources Research, Korea Institute of Oriental Medicine) ;
  • Cheon, Myeong-Sook (Center of Herbal Resources Research, Korea Institute of Oriental Medicine) ;
  • Choo, Byung-Kil (Department of Crop Agriculture and Life Science, Jeonbuk National University) ;
  • Kim, Ho-Kyoung (Center of Herbal Resources Research, Korea Institute of Oriental Medicine)
  • 문병철 (한국한의학연구원 한약자원연구센터) ;
  • 이영미 (한국한의학연구원 한약자원연구센터) ;
  • 천진미 (한국한의학연구원 한약자원연구센터) ;
  • 이아영 (한국한의학연구원 한약자원연구센터) ;
  • 윤태숙 (한국한의학연구원 한약자원연구센터) ;
  • 전명숙 (한국한의학연구원 한약자원연구센터) ;
  • 추병길 (전북대학교 작물생명과학과) ;
  • 김호경 (한국한의학연구원 한약자원연구센터)
  • Published : 2009.12.30
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Abstract

Objectives : Due to the morphological similarity and frequent occurrence of intermediate forms as well as morphological variations of aerial part, the correct identification between Rhei Radix et Rhizoma and Rhei Undulatai Rhizoma is very difficult. To develop a reliable method for correct identification and improving the quality standards of Rhei Radix et Rhizoma and Rhei Undulatai Rhizoma, we analyzed RAPD and developed SCAR marker. Methods : To amplify target DNA at the genomic level, 32 Operon 10-mer random primers were applied with four Rheum species, R. officinale, R. palmatum, R. tanguticum and R. undulatum. The nucleotide sequences were determined and species-specific primers were prepared depending on the species-specific RAPD amplicons after subcloned into the pGEM-Teasy vector. To develop the SCAR markers, species-specific PCR amplification and multiplex-PCR were carried out using the single species-specific primer pairs and combinations of them, respectively. Results : We used RAPD analysis of four Rheum plant species to obtain several species-specific RAPD amplicons. From nucleotide sequences of these RAPD amplicons, we developed two SCAR markers that amplified 314 bp and 390 bp DNA fragments in only R. undulatum but not in R. officinale, R. palmatum, R. tanguticum and R. undulatum, for distinguishing Rhei Undulatai Rhizoma and Rhei Radix et Rhizoma. Furthermore, we established SCAR markers for the simultaneous discrimination of the three species within a single reaction by using multiplex-PCR. Conclusions : These genetic markers can be used for the efficient discrimination of plants species and commercial herbal medicines between Rhei Undulatai Rhizoma and Rhei Radix et Rhizoma, to ultimately prevent indiscriminate distribution and prescription of these herbal medicines.

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References

  1. 顧觀光 輯, 楊鵬擧 校主. 新農本草經 校主. 北京 : 學園出版社. 1998 : 93-4.
  2. 전명숙, 윤태숙, 최고야, 김승주, 이아영, 문병철, 추병길, 김호경. 마우스 Raw264.7 세포에 대한 4종 대황류 추출물의 항염증 활성 비교. 한국약용작물학회지. 2009 ; 17 : 109-114.
  3. 식품의약품안전청. 대한약전. 서울 : 식품의약품안전청. 2007 : 919.
  4. 식품의약품안전청. 대한약전외한약(생약)규격집. 서울 : 식품의약품안전청. 2004 : 319.
  5. 김선제, 한신희, 이영종. 대황의 형태 감별에 관한 연구. 대한본초학회지. 2006 ; 21 : 171-6.
  6. Joshi K, Minami M, Kawahara K, Nakamura I, Shibata T. Molecular merkers in herbal drug technology. Curr Sci. 2004 ; 87 : 159-165.
  7. Zhang YB, Shaw PC, Sze CW, Wang ZT, Tong Y. Molecuar authentication of chinese herbal materials. Food Drug Anal. 2007 ; 15 : 1-9.
  8. Sucher NJ, Carles MC. Genome-based approaches to the authentication of medicinal plants. Planta Med. 2008 ; 74 : 603-623. https://doi.org/10.1055/s-2008-1074517
  9. Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphi는 amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990 ; 18 : 6531-5. https://doi.org/10.1093/nar/18.22.6531
  10. Welsh J, Mcclelland M. Genomic fingerprints produced by PCR with consensus tRNA gene primers. Nucleic Acids Res. 1991 ; 19 : 861-6. https://doi.org/10.1093/nar/19.4.861
  11. Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Marc Z. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 1995 ; 23 : 4407-14. https://doi.org/10.1093/nar/23.21.4407
  12. Desmarais E, Lanneluc I, Lagnel J. Direct amplification of length polymorphism. (DALP), or how to get and characterize new genetic markers in many species. Nucleic Acids Res. 1998 ; 26 : 1458-65. https://doi.org/10.1093/nar/26.6.1458
  13. Paran I, Michlmore RW. Development of reliable PCR based markers linked to downy mildew resistnace genes in lettuce. Theor Appl Genet. 1993 ; 85 : 985-93.
  14. 방경환, 성정숙, 박충헌, 김동순, 박춘근, 유홍섭, 박희운, 성낙술. 해부형태적 특징과 SCAR marker를 이용한 백출의 기원식물 판별. 한국약용작물학회지. 2004 ; 12 : 53-9.
  15. 임정현, 김동순, 성정숙, 방경환, 김옥태, 차선우, 박희운. SCAR 마커를 이용한 황기와 몽고황기 판별. 한국약용작물학회. 2007 ; 15 : 51-5.
  16. Choo BK, Moon BK, Ji Y, Kim BB, Choi G, Yoon T, Kim HK. Development of SCAR markers for the discrimination of three species of medicinal plants, Angelica dicursiva(Peucedanum decursivum), Peucedanum praeruptorum and Anthricus sylvestris, based on the internal transcribed spacer(ITS) sequence and random amplified polymorphic DNA (RAPD). Biol Pharm Bull. 2009 ; 32 : 24-30. https://doi.org/10.1248/bpb.32.24
  17. 김형환, 박수연, 안덕균, 박성규. 장엽대황과 종대황이 흰쥐의 흉부대동맥 혈관이완에 미치는 영향. 대한본초학회지. 2004 ; 19 : 99-105.
  18. 김미화, 장스엽, 이제현, 고성권, 육창수. 대황 속의 Sennoside A, B의 HPLC에 의한 정량. 경희약대논문집. 2000 ; 28 : 115-20.
  19. 강종성, 박기주, 오은기, 이은실, 황귀서, 이현선, 김영호. 패턴분석법에 의한 대황의 비교 연구. 생약학회지. 2008 ; 39 : 179-85.
  20. Yang DY, Fushimi H, Cai SQ, Komatsu K. Molecular analysis of Rheum species used as Rhei Rhizoma based on the chloroplast matK gene sequence and its application for identification. Biol Pharm Bull. 2004 ; 27 : 375-83. https://doi.org/10.1248/bpb.27.375
  21. Yang DY, Fushimi H, Cai SQ, Komatsu K. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and amplification refractory mutation system(ARMS) analysis of medicinally used Rheum species and their application for identification of Rhei Rhizoma. Biol Pharm Bull. 2004 ; 27 :661-9. https://doi.org/10.1248/bpb.27.661
  22. Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH. Multiplex PCR: critical parameters and step-by-step protocol. Biotechnique. 1997 ; 23 : 504-511.