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1 Volume 42 Number 1 March 31, 2015 Vol. 42 No. 1 March

2 Volume 42 Number 1 March 31, 2015 Vol. 42 No. 1 March

3 한국식물생명공학회지 Journal of Plant Biotechnology ISSN Volume 42, Number 1 March 31, 2015 편집위원회 Editorial Board 편집위원장강권규 ( 한경대학교 ) kykang@hknu.ac.kr 편집간사박영훈 ( 부산대학교 ) ypark@pusan.ac.kr < 오믹스및분자마커 > 책임편집위원 조용구 ( 충북대학교 ) ygcho@cbnu.ac.kr 편집위원 김선태 ( 부산대학교 ) stkim5505@gmail.com 김성길 ( 전남대학교 ) dronion@jnu.ac.kr 류호진 ( 충북대학교 ) hjryu96@chungbuk.ac.kr 염인화 ( 안동대학교 ) iyeam@andong.ac.kr 유수철 ( 한경대학교 ) scyoo@hknu.ac.kr < 형질전환및대사공학 > 책임편집위원 정우식 ( 경상대학교 ) chungws@gnu.ac.kr 편집위원 이병현 ( 경상대학교 ) hyun@gnu.ac.kr 이행순 ( 한국생명공학연구원 ) hslee@kribb.re.kr 정재철 ( 한국생명공학연구원 ) jcjeong@kribb.re.kr 최동욱 ( 전남대학교 ) dwchoi63@jnu.ac.kr 최영림 ( 국립산림과학원 ) yichoi99@forest.go.kr < 조직배양및 2 차대사 > 책임편집위원 김창길 ( 경북대학교 ) ckkim@knu.ac.kr 편집위원 김종보 ( 건국대학교 ) jbhee@hanmail.net 박소영 ( 충북대학교 ) soypark7@chungbuk.ac.kr 안영옥 ( 동부한농 ) yoahn@dongbu.com 정유진 ( 한경대학교 ) yuyu1216@hknu.ac.kr Editor-in-Chief Kang, Kwon-Kyoo HanKyong National University Associate Editor Park, Young-Hun Pusan National University Managing Eiditors Cho, Yong-Gu Chungbuk National University Chung, Woo-Sik Gyeongsang National University Kim, Chang-Kil Kyungpook National University Editors Ahn, Young-Ock DongBu Farm Hannong Choi, Dong-Woog Chonnam National University Choi, Young-Lim Korea Forest Research Institute Jeong, Jae-Cheol Korea Research Institute of Bioscience and Biotechnology Jung, Yu-Jin HanKyong National University Kim, Jong-Bo Konkuk University Kim, Seong-Gil Chonnam National University Kim, Sun-Tae Pusan National University Lee, Bung-Hyun Gyeongsang National University Lee, Haeng-Soon Korea Research Institute of Bioscience and Biotechnology Park, So-Young Chungbuk National University Ryu, Ho-Jin Chungbuk National University Yeam, In-Hwa Andong National University Yoo, Soo-Cheul HanKyong National University Aims and Scope Journal of Plant Biotechnology (JPB) is an international open access journal published four issues of a yearly volume on March 31, June 30, September 30, and December 31 by The Korean Society for Plant Biotechnology (KSPBT) founded in JPB publishes original, peer-reviewed articles dealing with advanced scientific aspects of plant biotechnology, which includes molecular biology, genetics, genomics, proteomics, and metabolomics. JPB does not exclude studies on lower plants including algae and cyanobacteria if studies are carried out within the aspects described above. Electronic Edition Journal of Plant Biotechnology allows open access to all published articles. Membership of The Korean Society for Plant Biotechnology (KSPBT) is required to access the electronic free full-text PDF format, available at and Subscription Information Abbreviated title is J. Plant Biotechnol. ISSN print edition: Digital objet identifier(doi): Editorial Office The Korean Society of Plant Biotechnology, 2017 Hongin Tower Officetel, 28 Daehak-ro, Yuseong-gu, Daejeon , Korea. Tel: , Fax: , kspbt@kspbt.or.kr Society Website: Printing Company CIR / Yejang-dong, Jung-gu, Seoul , Republic of Korea Tel: , Fax: , circom@chol.com Copyright c 2015 by the Korean Society for Plant Biotechnology This journal was supported by the Korean Federation of Science and Technology Societies Grant funded by the Korean Government. Printed on Mar 26, 2015 and published on Mar 31, 2015 한국식물생명공학회 The Korean Society for Plant Biotechnology

4 Journal of Plant Biotechnology Vol. 42 No. 1 March 31, 2015 CONTENTS Review 식물의생장및발달과정에서 Glycogen synthase kinase 3 (GSK3) 유전자의역할류호진 1 The functional roles of plant glycogen synthase kinase 3 (GSK3) in plant growth and development Hojin Ryu 약용작물의기원판별에관한분자생물학적기술개발현황한은희 김윤희 이신우 6 Development of molecular biological techniques for the differentiation of medicinal plant species Eun-Heui Han Yun-Hee Kim Shin-Woo Lee Research Articles Absence of AVP1 transcripts in wild type watermelon scions grafted onto transgenic bottle gourd rootstocks Byung Oh Kim Jeung-Sul Han Kyung il Park Su Min Jeon Chang Kil Kim 13 CodA 고발현형질전환고구마의산화및건조스트레스내성증가박성철 김명덕 김선하 김윤희 정재철 이행순 곽상수 19 Enhanced drought and oxidative stress tolerance in transgenic sweetpotato expressing a coda gene Sung-Chul Park Myoung Duck Kim Sun Ha Kim Yun-Hee Kim Jae Cheol Jeong Haeng-Soon Lee Sang-Soo Kwak 배검은별무늬병 (Venturia nashicola) 고도저항성 유래 PR-10 유전자의특성천재안 김세희 조강희 김대현 최인명 신일섭 25 Characterization of PR-10 gene derived from highly resistant pear inoculated with scab (Venturia nashicola) Jae An Chun Se Hee Kim Kang Hee Cho Dae Hyun Kim In Myong Choi Il Sheob Shin 생물반응기를이용한적하수오부정근의바이오매스와생리활성물질대량생산이경주 박영기 김자영 정택규 윤경섭 백기엽 박소영 34 Production of biomass and bioactive compounds from adventitious root cultures of Polygonum multiflorum using air-lift bioreactors Kyung-Ju Lee Youngki Park Ja-Young Kim Taek-Kyu Jeong Kyung-Seop Yun Kee-Yoeup Paek So-Young Park 전라남도지역감바이로이드의감염상황및무병화효율연구김대현 김인수 이건섭 조인숙 조강희 신일섭 김세희 천재안 최인명 43 Current occurrence of persimmon viroid and citrus viroid in persimmon in JellaNam-do and testing for viroid inactivation methods Dae Hyun Kim In-Soo Kim Gunsup Lee In-Sook Cho Kang Hee Cho Il Sheob Shin Se Hee Kim Jae An Chun In-Myung Choi ON THE COVER: Adventitious root culture of P. multiflorum. A. Mother plants in greenhouse, B. In vitro grown plantlets. C. Adventitious root culture on petri-dish, D. Adventitious root culture in 300 ml flask, E. Harvested root from liquid culture, F. Adventitious root culture in 3 L air-lift bioreactor after 4 weeks of culture (p. 41)

5 양앵두왜성대목 Gisela 5 의기내번식을위한정단배양조건의최적화쉬쥔핑 강인규 김창길 한증술 최철 49 Optimization of apical tip culture condition for In Vitro propagation of Gisela 5 dwarf cherry rootstock Junping Xu In-Kyu Kang Chang Kil Kim Jeung-Sul Han Cheol Choi 울릉도자생식물삼나물 (Aruncusdioicus) 의항산화활성검증김동희 문용선 손준호 55 Verification of anti-oxidative activity of Aruncus dioicus, a native plant of Ulleungdo Dong-Hee Kim Yong-Sun Moon Jun-Ho Son 적외선분광스펙트럼및기체크로마토그라피분석데이터의다변량통계분석을이용한대두종자지방산함량예측안명숙 지은이 송승엽 안준우 정원중 민성란 김석원 60 Simultaneous estimation of fatty acids contents from soybean seeds using fourier transform infrared spectroscopy and gas chromatography by multivariate analysis Myung Suk Ahn Eun Yee Ji Seung Yeob Song Joon Woo Ahn Won Joong Jeong Sung Ran Min Suk Weon Kim

6 J Plant Biotechnol (2015) 42:1 5 DOI: ISSN Review 식물의생장및발달과정에서 Glycogen synthase kinase 3 (GSK3) 유전자의역할 류호진 The functional roles of plant glycogen synthase kinase 3 (GSK3) in plant growth and development Hojin Ryu Received: 15 March 2015 / Revised: 24 March 2015 / Accepted: 24 March 2015 c Korean Society for Plant Biotechnology Abstract The biological roles of glycogen synthase kinase 3 (GSK3) proteins have long been extensively explored in eukaryotic organisms including fungi, animals and plants. This gene family has evolutionary well conserved kinase domain and shares similar phosphorylation properties to their substrate proteins. However, their specific biological roles are surprisingly distinct in different organisms. GSK3s play key role in key regulating the cytoskeleton and metabolic processes in animal systems, but plant GSKs are involved in quite different processes, such as flower development, brassinosteroid signaling, abiotic stresses, and organogenesis. In particular, recent studies have reported the critical multiple functions of BIN2 and its related paralogues plant GSK3s during organogenesis via connecting hormonal or developmental programs. In this review, we outline the recent understanding in the versatile functions related in physiological and biochemical relevance, which are mediated by plant GSK3s in various cellular signaling. 서언 진핵생물에서발견되는 Glycogen synthase kinase 3 (GSK3) 단백질은동물의인슐린에의해조절되는 Glycogen 합성의최종단계에관여하는중요단백질에인산화를유도하는특성을지니는인산화효소를코딩하는유전자로처음발견되었다 (Saidi et al. 2012). 현재까지많은연구그룹에 H. J. Ryu ( ) 충북대학교자연과학대학생물학과 (Department of Biology, Chungbuk National University, Cheongju, , Korea) hjryu96@chungbuk.ac.kr 의해 GSK3 유전자들의기능이연구되어오고있으며, 그중요성이최근부각되고있는유전자중에하나이다. 특히세포의운명결정인자, 세포사멸, wnt 신호전달에서의주요조절인자로서그중요성이동물에서많이알려져왔다 (Saidi et al. 2012). 동물의 wnt signaling 은매우다양한동물의발달및발생에관여하는것으로알려져있으며, GSK3β 에의해전사인자 β-catenin 의인산화를통한활성도조절기작이잘알려져있다. 이러한중요성과함께 GSK3 은알츠하이머, 암과같은주요질병과도매우밀접하게연결되어있다는사실들이최근지속적으로보고되고있다. 현재까지두개의유전자 (GSK3α/β) 에의해동물의 GSK3 단백질이코딩되어지고있으며, 동물에서는약 80 여종류의주요단백질들이 GSK3 인산화효소에의해인산화가일어난다는사실이잘알려져왔다 (Sutherland 2011). 모델식물애기장대의게놈정보가알려진이후동물의 GSK3 단백질과유사성이존재하는 10 개의 GSK3-like 유전자들이애기장대의게놈에존재하고있음이알려져왔다 (Jonak and Hirt 2002; Saidi et al. 2012). 식물에서 GSK3 유전자에대한연구가본격적으로이뤄진것은식물호르몬브라시노스테로이드에대한반응성이나타나지않는난쟁이표현형을보이는 brassinosteroid-insensitive 2 (bin2) /dwarf12 돌연변이가발견되고, 그원인이되는유전자 BIN2 의기능학적연구가진행되면서부터이다 (Choe et al. 2002; He et al. 2002; Li and Nam 2002). 브라시노스테로이드신호전달체계에서 BIN2 는주요전사인자 BES1/BZR1 의인산화를통해브라시노스테로이드신호전달을억제하게되는기작이잘알려져있다 (Ryu et al. 2007). 또한 Tang, Ryu 등은식물 GSK3 인산화효소 BIN2 에의해인산화가일어나는 BES1 (BRI1-EMS-SUPPRESSOR 1), BZR1 (BRASSINAZOLE-RESISTANT 1) 의 Ser/Thr 인산화좌를동

7 2 J Plant Biotechnol (2015) 42:1 5 정하였다 (Ryu et al. 2010; Ryu et al. 2007; Tang et al. 2008). 애기장대에서발견된 10 개의 GSK3 유전자들은단백질을구성하는아미노산서열을중심으로총 4 개의그룹으로구분되어지며 (Jonak and Hirt 2002; Saidi et al. 2012), 최근까지 BIN2 가포함되어져있는그룹 Ⅱ (BIN2, BIL1 (BIN2-LIKE 1), BIL2 (BIN2-LIKE 2)) 유전자들의기능학적연구가브라시노스테로이드신호전달을중심으로이뤄져왔다 (Yin et al. 2005). 하지만최근연구들은 BIN2/GSK3 유전자가브라시노스테로이드이외에도다양한호르몬신호전달뿐만아니라다양한식물의생장및발달에관여한다는증거가보고되고있다. 이러한증거들은동물에서와마찬가지로식물에서도 GSK3 에의해유도되는인산화과정들이발달과생장에매우중요하게작용하고있음을예상하게해준다. 본리뷰에서는최근에발견된 GSK3 유전자의새로운기능들이어떻게식물의발달과생장에관여하고있는지를서술하고자한다. 또한향후이들중요성이부각되고있는 GSK3 유전자의기능연구를통해어떠한생명공학적발전을이룰수있는지를제시하고자한다. Brassinosteroids ( 브라시노스테로이드 ) 식물호르몬은식물의발달및생장에매우중요한역할을담당하고있는생리물질이다. 식물의 GSK3 가식물호르몬신호전달에직접적으로관여되는사실이알려진것은 BIN2 유전자에의해브라시노스테로이드신호전달이억제되는것이알려지면서부터이다 (Kutschera and Wang 2012). 식물에서브라시노스테로이드신호전달은모델식물애기장대를통해많은연구가진행되어졌으며, 대부분의주요신호전달인자들이동정되어왔다. 브라시노스테로이드는생체막에존재하는수용체 BRI1 (BRASSIN- OSTEROID-INSENSITIVE 1) 에의해인식이되어신호개시가이뤄지게된다. 이들신호는최종적으로두전사인자 BES1 과 BZR1 의활성화를통해다양한브라시스토스테로이드효과를나타내게된다. 두전사인자 BES1 과 BZR1 의활성은인산화상태에따라달라지게되는데, Ryu 등에의해발견된기작은 BIN2 에의한인산화가전사인자들의핵으로부터세포질로의이동을통해전사인자들의비활성화를유도함을보여주고있다 (Ryu et al. 2010; Ryu et al. 2007). BES1/BZR1 전사인자에 BIN2 에의해인산화가일어나는아미노산의위치는 LC/MS/MS 기법과전기영동을통한 gel-shift 기법을통해어느정도알려져있으며, 특히 단백질과의결합을통해핵과세포질의위치가결정되는데매우중요한역할을하는것으로알려졌다 (Ryu et al. 2010; Ryu et al. 2007). BIN2 이외에가장유사한아미노산서열을보유하고있는그룹 Ⅱ (BIN2, BIL1 (BIN2-LIKE), BIL2) 유전자들의기능학적연구가브라시노스테로이드신호전달에서매 우유사한기능을하고있음이보고되었다 (Charrier et al. 2002). BIN2 유전자의 T-DNA knock-out 돌연변이체가야생형식물체와큰차이를보이지않는것에비해, bin2 bil1 bil2 triple knock-out 돌연변이체는브라시노스테로이드신호전달이강하게활성화되는표현형이나타났다 (Vert and Chory 2006). 이러한결과는그룹 Ⅱ GSK3 유전자의기능이브라시노스테로이드신호전달에상호협력적으로작용하고있음을보여주고있다. 이외에도최근연구에의하면그룹 Ⅲ 에속하는 AtGSK31 유전자또한브라시노스테로이드신호전달에음성적으로작용함이알려졌다 (Zhang et al. 2013). AtGSK31 유전자는 BES1/BZR1 단백질에인산화를유도하였으며, 이들유전자가과발현된애기장대에서브라시노스테로이드가약해졌을때나타나는난쟁이표현형이보고되었다 (Zhang et al. 2013). 이러한결과는그룹 Ⅱ 에서특이적으로브라시노스테로이드신호전달에관여될것으로여겨졌으나, 다른식물의 GSK3 도식물의발달과정중에다양하게브라시노스테로이드신호전달에관여할수있는가능성을보여주고있다. Auxin ( 옥신 ) 과기관형성 식물의발달과정에서 morphogen 으로알려진옥신은식물의전생애과정에서필요로하는다양한발달과생장프로그램에매우밀접하게관여하고있는호르몬이다. 옥신은수용체 TIR1 (TRANSPORT INHIBITOR RESPONSE 1) 에의해인식이되면서신호전달의개시가된다 (Lau et al. 2008). 옥신에의해활성화되는 TIR1 은옥신신호의음성적조절자 AUX/IAA 단백질의분해과정을촉진하여전사인자 ARF 들의활성을유도하게된다 (Lau et al. 2008). 애기장대의게놈에는 23 개의 ARF 유전자들이존재하는것으로알려져있으며이들의기능은흥미롭게도일부는옥신신호의활성인자, 일부 ARF (AUXIN RESPONSE FACTOR) 유전자들은억제인자로서의역할을수행하는것으로알려져있다 (Guilfoyle 2007; Guilfoyle and Hagen 2007). Vert et al. (2008) 은옥신신호의억제인자로작용하는 ARF2 ( 가 BIN2 에의해인산화를직접적으로일으키며, ARF2 (AUXIN RESPONSE FACTOR 2) 의 DNA 결합력과전사인자능력을상쇄시킴으로써기능을억제한다는사실을규명하였다 (Vert et al. 2008). 이러한결과를바탕으로 BIN2 에의한 ARF2 의억제효과가최종적으로는브라시노스테로이드와옥신신호전달과의상호보완적인연관성에대한의문이어느정도풀리게되었다. 하지만브라시노스테로이드신호전달에서 BIN2 의음성적조절효과와옥신신호전달에서의양성적조절인자효과에대한상보적인결과는논란으로이어지고있다. Cho et al. (2014) 은식물의곁뿌리형성과정에서 BIN2 에의한 ARF7 과 ARF19 의활성화기작을보고하였다 (Cho

8 J Plant Biotechnol (2015) 42:1 5 3 et al. 2014). BIN2 에의한 ARF7 의 S698/S707 아미노산의직접적인인산화를확인하였고, 인산화는 AUX/IAA 와의결합력을현저하게낮춰주면서옥신신호전달의전사인자로서의기능을높이는기작을보고하였다. 또한 BIN2 의조절기작이브라시노스테로이드신호전달과는별개로 TDIF/TDR (TDIF RECEPTOR) 경로를통해이뤄짐이확인되었다 (Cho et al. 2014). 현재까지 BIN2 의인산화효소기능이조절되는기작은브라시노스테로이드에의한조절이유일했지만, TDIF/TDR 경로가곁뿌리의발달에긴밀하게작용하고있음이확인되었다. ABA 와스트레스반응 초기식물에서의 GSK3 의기능연구들은스트레스내성과긴밀한관계가존재하고있을가능성을제기하고있다 (Jonak et al. 1995; Jonak and Hirt 2002). 특히염스트레스에의해식물의 GSK3 유전자들 (AtSK13, AtSK31, AtSK42) 의발현이강하게증가됨이보고되었다 (Charrier et al. 2002). 최근연구는 AtSK11 에의한 G6PD 단백질의인산화가직접적으로염스트레스에의해조절되는세포내산화과정을조절하는주요기작이라는연구결과가발표되었다 (Dal Santo et al. 2012). 식물의 GSK3 가스트레스반응성및 ABA 신호전달에관련되어있다는사실은 BIN2 의 gain-of-function 돌연변이 bin2-1 에서더욱분명하게발견되었다. bin2-1 에서 ABA 에대한반응성이매우강하게나타난다는사실이보고되었으며 (Li and Nam 2002), bin2-3 bil1 bil2 triple knock-out 돌연변이체에서는 ABA 에대한반응성이매우낮게나타남이보고되었다 (Cai et al. 2014; Ryu et al. 2014). 즉 BIN2 인산화효소의기능이 ABA 신호전달에양성적으로관여하고있음을의미하고있다. 이러한원인에대한분자기전이최근연구에의해보고되었다. Cai et al. (2014) 은 BIN2 에의해 ABA 신호전달에관여하는인산화효소 SnRK2.2 (SNF1-RELATED PROTEIN KINASE 2.2) 와 SnRK2.3 (SNF1- RELATED PROTEIN KINASE 2.3) 이인산화가이뤄지며, 이러한인산화는 ABA 신호전달의활성화에직접적인작용을하고있음이보고되었다 (Cai et al. 2014). 특히 SnRK2.3 의인산화좌는 S172, S176, S177 과 T180 으로규명되었고, 이러한인산화좌는다른 SnRK2 의유전자에도진화적으로보존되어져있다는사실이알려졌다. 이러한결과는 BIN2 또는다른식물 GSK3 에의해 SnRK 단백질의인산화가더이뤄질수있음을의미하며, 다양한신호전달과매우밀접하게관련되어져있을가능성을보여주고있다. 물기관이다. 생체막에존재하는수용체단백질 ERECTA 의활성화에의해개시되는기공발달은 YODA 에의해활성화되는 MAPK (MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE) 신호전달이매우중요하게관여하고있는것으로잘알려져있다 (Bergmann and Sack 2007; Lampard and Bergmann 2007). 최근연구들은식물의 GSK3 에의해유도되는인산화과정이기공발달에매우긴밀하게작용하고있음을보여주고있다. Kim et al. (2012) 과 Khan et al. (2013) 은 BIN2 에의한직접적인 YODA 와하위 MAPKK4/5 의인산화를규명하였다 (Khan et al. 2013; Kim et al. 2012). 이러한주요기공발달신호전달단백질들의인산화는기공발달을촉진시키는것으로규명되었다 (Kim et al. 2012). 이러한 BIN2 의기공발달활성화기작은브라시노스테로이드에의해식물의잎에서음성적으로조절된다는것이보고되었다. 하지만 Gudesblat et al. (2012) 이러한결과와는상반된연구결과를발표하였다 (Gudesblat et al. 2012). 브라시노스테로이드합성돌연변이의하배축에서기공의발달이현저히감소되어있다는것이발견되고, 이러한원인은 BIN2 에의한기공발달신호전달의주요전사인자 SPCH 의인산화임이알려졌다 (Gudesblat et al. 2012). 이러한상반된결과는식물의발달과정에서서로다른신호전달경로를통해매우중요한조절이이뤄질수있음을의미한다. 향후좀더자세한연구를통해다양한식물의발달프로그램과 GSK3 가어떠한상호작용을일으키는지에대한해답을제시해야할것이다. 결론 본논문을통해식물의발달및호르몬신호전달과정에서 GSK3 에의해유도되는다양한유전자들의인산화를조사하였다. 주요발달프로그램및호르몬신호전달에서다양한단백질이 GSK3 의타겟이되고있으며, 인산화에의한유전자들의기능이매우견고하게조절되고있음을확인할수있었다 (Fig. 1). 하지만현재까지의연구 GSK3 와기공발달 식물의기공은광합성을의해필요로하는 CO 2 의흡수뿐만아니라수분조절등에매우중요하게작용하고있는식 Fig. 1 Plant GSK3 mediated signaling network reviewed in this work

9 4 J Plant Biotechnol (2015) 42:1 5 는대부분이 BIN2 및그룹 Ⅱ 에속하는 BIL1, BIL2 에의한인산화과정이주대상이되어왔다. 하지만모델식물애기장대에는총 10 개의 GSK3 유전자가존재하며, 대부분의식물에서도많은수의 GSK3 유전자를지니고있음이최근알려지고있다. 즉현재까지알려진 BIN2 에의해조절되는다양한신호전달경로이외에도다양한 GSK3 에의해정교하게조절되는발달프로그램이매우다양하게존재하고있음을의미한다. 본논문을통해확인할수있듯이주요유전자들의인산화는식물의생장및발달에매우중요한역할을담당하고있다. 특히동물에서는 2 개의 GSK3 가암, 발달, 생식, 세포분열등매우중요한역할을담당하고있다는사실은매우고무적인것이다. 식물의경우에는좀더많은수의 GSK3 들이존재하면서, 아마도식물발달및생장을체계적으로조절할것으로예상된다. 좀더심층적이고체계적인연구를통한 GSK3 유전자의기능연구는식물을통한생명현상의이해뿐만아니라다양한생명공학적인접근을통한작물개량에매우중요한정보를제공해줄수있으리라판단된다. 사사 본연구는 2014 학년도충북대학교학술연구지원사업의연구비지원과정부 ( 미래창조과학부 ) 신진연구지원사업 (NRF-2013R1A1A ) 지원에의해연구되었음. References Bergmann DC, Sack FD (2007) Stomatal development. Annu Rev Plant Biol 58 : Cai Z, Liu J, Wang H, Yang C, Chen Y, Li Y, Pan S, Dong R, Tang G, Barajas-Lopez Jde D, Fujii H, Wang X (2014) GSK3-like kinases positively modulate abscisic acid signaling through phosphorylating subgroup III SnRK2s in Arabidopsis. Proc Natl Acad Sci USA 111 : Charrier B, Champion A, Henry Y, Kreis M (2002) Expression profiling of the whole Arabidopsis shaggy-like kinase multigene family by real-time reverse transcriptase-polymerase chain reaction. Plant Physiol 130 : Cho H, Ryu H, Rho S, Hill K, Smith S, Audenaert D, Park J, Han S, Beeckman T, Bennett MJ, Hwang D, De Smet I, Hwang I (2014) A secreted peptide acts on BIN2-mediated phosphorylation of ARFs to potentiate auxin response during lateral root development. Nat Cell Biol 16 : Choe S, Schmitz RJ, Fujioka S, Takatsuto S, Lee MO, Yoshida S, Feldmann KA, Tax FE (2002) Arabidopsis brassinosteroidinsensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase. Plant Physiol 130 : Dal Santo S, Stampfl H, Krasensky J, Kempa S, Gibon Y, Petutschnig E, Rozhon W, Heuck A, Clausen T, Jonak C (2012) Stress-induced GSK3 regulates the redox stress response by phosphorylating glucose-6-phosphate dehydrogenase in Arabidopsis. Plant Cell 24 : Gudesblat GE, Betti C, Russinova E (2012) Brassinosteroids tailor stomatal production to different environments. Trends Plant Sci 17 : Gudesblat GE, Schneider-Pizon J, Betti C, Mayerhofer J, Vanhoutte I, van Dongen W, Boeren S, Zhiponova M, de Vries S, Jonak C, Russinova E (2012) SPEECHLESS integrates brassinosteroid and stomata signalling pathways. Nat Cell Biol 14 : Guilfoyle T (2007) Plant biology: sticking with auxin. Nature 446 : Guilfoyle TJ, Hagen G (2007) Auxin response factors. Curr Opi Plant Biol 10 : He JX, Gendron JM, Yang Y, Li J, Wang ZY (2002) The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proc Natl Acad Sci USA 99 : Jonak C, Heberle-Bors E, Hirt H (1995) Inflorescence-specific expression of AtK-1, a novel Arabidopsis thaliana homologue of shaggy/glycogen synthase kinase-3. Plant Mol Biol 27 : Jonak C, Hirt H (2002) Glycogen synthase kinase 3/SHAGGY-like kinases in plants: an emerging family with novel functions. Trends Plant Sci 7 : Khan M, Rozhon W, Bigeard J, Pflieger D, Husar S, Pitzschke A, Teige M, Jonak C, Hirt H, Poppenberger B (2013) Brassinosteroid-regulated GSK3/Shaggy-like kinases phosphorylate mitogen-activated protein (MAP) kinase kinases, which control stomata development in Arabidopsis thaliana. J Biol Chem 288 : Kim TW, Michniewicz M, Bergmann DC, Wang ZY (2012) Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway. Nature 482 : Kutschera U, Wang ZY (2012) Brassinosteroid action in flowering plants: a Darwinian perspective. J Exp Bot 63 : Lampard GR, Bergmann DC (2007) A Shout-Out to Stomatal Development: How the bhlh Proteins SPEECHLESS, MUTE and FAMA Regulate Cell Division and Cell Fate. Plant signaling & behavior 2 : Lau S, Jurgens G, De Smet I (2008) The evolving complexity of the auxin pathway. The Plant cell 20 : Li J, Nam KH (2002) Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase. Science 295 : Ryu H, Cho H, Bae W, Hwang I (2014) Control of early seedling development by BES1/TPL/HDA19-mediated epigenetic regulation of ABI3. Nature Commun 5 : 4138 Ryu H, Cho H, Kim K, Hwang I (2010) Phosphorylation dependent nucleocytoplasmic shuttling of BES1 is a key regulatory event in brassinosteroid signaling. Mol Cells 29 : Ryu H, Kim K, Cho H, Park J, Choe S, Hwang I (2007) Nucleocytoplasmic shuttling of BZR1 mediated by phospho-

10 J Plant Biotechnol (2015) 42:1 5 5 rylation is essential in Arabidopsis brassinosteroid signaling. Plant Cell 19 : Saidi Y, Hearn TJ, Coates JC (2012) Function and evolution of 'green' GSK3/Shaggy-like kinases. Trends Plant Sci 17 : Sutherland C (2011) What Are the bona fide GSK3 Substrates? Inter J Alzheimer's Disease 2011 : Tang W, Deng Z, Oses-Prieto JA, Suzuki N, Zhu S, Zhang X, Burlingame AL, Wang ZY (2008) Proteomics studies of brassinosteroid signal transduction using prefractionation and two-dimensional DIGE. Mol Cell Proteom : MCP 7 : Vert G, Walcher CL, Chory J, Nemhauser JL (2008) Integration of auxin and brassinosteroid pathways by Auxin Response Factor 2. Proc Natl Acad Sci USA 105 : Yin Y, Vafeados D, Tao Y, Yoshida S, Asami T, Chory J (2005) A new class of transcription factors mediates brassinosteroidregulated gene expression in Arabidopsis. Cell 120 : Zhang D, Chen ZG, Liu SH, Dong ZQ, Dalin M, Bao SS, Hu YW, Wei FC (2013) Galectin-3 gene silencing inhibits migration and invasion of human tongue cancer cells in vitro via downregulating beta-catenin. Acta Pharm Sinica 34 :

11 J Plant Biotechnol (2015) 42:6 12 DOI: ISSN Review 약용작물의기원판별에관한분자생물학적기술개발현황 한은희 김윤희 이신우 Development of molecular biological techniques for the differentiation of medicinal plant species Eun-Heui Han Yun-Hee Kim Shin-Woo Lee Received: 5 March 2015 / Revised: 15 March 2015 / Accepted: 15 March 2015 c Korean Society for Plant Biotechnology Abstract Medicinal plants resources are becoming important assets since their usages have been expanded to the development of functional foods for human health, more attractive cosmetics, and pharmaceutical industries. However, their phylogenetic origins and names are different from each country and quite often they are mixed each other resulting in the confusion for consumers. In particular, when they are very similar based on their morphological characteristics and distributed as dried roots, it is extremely difficult to differentiate their origins even by specialists. Recently, DNA barcodes have been extensively applied to identify their origin of medicinal plant species. In this review, we tried to overview the current research achievements for the development of suitable DNA barcodes regarding to the differentiation of medicinal plant species. Furthermore, more advanced techniques including amplification refractory mutation system (ARMS)-PCR, multiplex single base extension (MSBE), high-resolution melting (HRM) curve analyses are also discussed for their practical applications in the authentification of particular medicinal plant species. E.-H. Han S.-W. Lee ( ) 경남과학기술대학교, 생명과학대학, 농학ㆍ한약자원학부 (Dept. of Agronomy & Medicinal Plant Resources, Gyeongnam National University of Science & Technology, JinJu, Korea) shinwlee@gntech.ac.kr Y.-H. Kim 경상대학교, 사범대학, 생물교육과 ( 농업생명과학연구원 ) (Dept. of Biology Education, College of Education, IALS, Gyeongsang National University, Jinju, Korea) 서론 최근약용작물은기존에이용되던한약재뿐만아니라기능성식품, 화장품, 의약품등의원료로그용도가점차확대됨으로서수요가기하급수적으로늘어나고있는실정이며가격또한상승하고있는실정이다. 그러나우리나라는수입에의존도가높아이러한한약재의수급불균형이발생할가능성이높다. 또한한약재의경우유통과정에서그기원이부정확하거나명칭이유사하여혼 오용되는사례가많다. 특히국가별로한약재를약으로사용하면서약용식물의기원을다르게규정하고있어우리나라는한약재의정확한기원을모르고위품이시장에유통되는경우가많아유통질서가제대로확립되지않아향후중국과일본등 3 국간에분쟁우려가많이발생할것으로우려되고있다. 기존의관능검사, 형태학적, 이화학적, 세포생화학적검사법등에의존하기보다는보다과학적인진보된기술의도입이시급한실정이다. 이러한문제점을해결하기위하여 Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Sequence Characterized Amplified regions (SCARs), Cleaved Amplified Polymorphic Sequence (CAPS), Amplificon Length Polymorphism (ALP), Sequence Tagged Site-Restriction fragment Length Polymorphism (STS-RFLP) 등의분자생물학적기술을적용하기위하여지난수년간많은연구자들에의하여수행되어왔으나아직까지반복성과신뢰도등의문제점으로인하여실제한약재의위품을구분하기에유용한기술의개발은아직미진한실정이다. 따라서최근에는단일염기다형성 (single nucleotide polymorphism, SNP) 을보이는부분을이용하는 DNA barcodes 의개발에관한연구에많은관심을갖게되었다. DNA barcodes

12 J Plant Biotechnol (2015) 42: 라는용어는 Arnot et al. (1993) 에의하여처음사용되었으며, 그후 Hebert et al. (2003) 에의하여 cytochrome C oxidase 유전자단편을이용한동물의 barcoding 에관한논문의발표와함께전성기를맞이하였다. 이후 Consortium for the Barcode of Life (CBOL, 가조직되어국제적인 DNA barcoding 연구가활성화되기시작하였다. 그러나아직까지도동물의 barcode 는미토콘드리아 cytochrome C oxidase I (COI) 유전자에해당하는 658bp 가가장유효한것으로결정될수있었으나식물의경우에는논란이많고 COI 처럼대표할수있는 barcode 가없는실정이다. 최근에는 Next Generation DNA Sequencing (NGS) 기술의급속한발전과함께생물다양성의유지보존과관련된자연생태계에서채취한샘플중에서단일종의판별, 다양한가공기술에의하여개발된가공식품에포함된단일종의판별, GMO 의혼재여부판별등다양한분야에응용될수있는기술의개발에관한연구가활발하게보고되고있다 (Valentini et al. 2008). 따라서본리뷰논문에서는최근까지보고된주요약용작물특히향후중국, 일본등과국가간분쟁의우려가높은것으로사료되는하수오, 당귀등의주요약용작물을중심으로그기원을판별하기위하여사용된분자생물학적기술 (DNA barcoding 등 ) 에관하여기술의원리, 성과, 실용화가능성그리고향후문제점등을정리하여보았다. 식물의분류에이용되는 Barcodes 개발현황 DNA barcoding 에있어서 3 가지주요원칙은표준화 (standardization), 최소화 (minimalism), 응용범위 (scalability) 이라고기술된바있다 (Hollingsworth et al. 2011). 동물의경우에는 COI DNA barcode 가이원칙에아주적절한것으로확인되었다 (Hebert et al, 2003). 그러나식물의경우에는엽록체의게놈에존재하는 rpoc1, rpob, matk, trnh-psba, rbcl, atpf-h, psbki 유전자등에관한많은연구결과가발표되었으나, 현재까지의연구결과에의하면한가지만으로는충분하지않으며이들중여러개를복합적으로비교분석한결과어느정도만족할만한결과를얻었다고하였다. 예를들면 rpoc1+rpob+matk 또는 rpoc1+matk+ trnh-psba (Chase et al, 2007); rbcl+trnh-psba (Kress and Erickson, 2007) 등의조합에의하여어느정도분류가가능하였다고하였다. 또한 CBOL 의 Plant working group 의검토결과에의하면 rbcl 과 matk 를조합한경우에가장좋은결과를얻을수있었으며이조합을 core barcode 로정한바있다 (CBOL, 2009). matk 와 rbcl barcode 는모두애기장대 (Arabidopsis thaliana) 의엽록체게놈의염기서열로부터프라이머를디자인하여전장의유전자단편을 polymerase chain reaction (PCR) 로증폭하여얻은단편을염기서열로확인한것이다. 식물 체의 matk 유전자는진화속도가가장빠른것으로알려져있어동물의 COI barcode 와가장유사한것이다. 그러나불행하게도애기장대에서디자인한프라이머가모든식물의 matk 유전자를증폭시킬수있는광역프라이머로사용될수없다는게단점이다. 따라서 clade-specific primer 또는 phylogenic tree 상에서보다가까운그룹에서디자인한프라이머세트를다시개발하여사용하여야한다 (Wickie et al. 2009; Dunning et al. 2010; Kuo et al. 2011). 반면에 rbcl 프라이머는비교적넓은범위의식물종을대상으로 PCR 증폭이가능하나그만큼상동성이높아다형성 (polymorphism) 을많이나타내지않는것이단점이라고알려져있다. 한편 rbcl+matk core barcode 에이어그다음으로많이연구되어온엽록체 barcodes 는 trnh-psba 의유전자간 DNA (IGS, intergenic spacer) 영역이다. 이 barcodes 를사용하여 Ficus (Roy et al. 2010), Alnus (Ren et al. 2010), Quercus (Piredda, 2010) and Salix (von Cräutlein et al. 2011) 속의종간구분에는오히려 rbcl+matk core barcode 보다좋은결과를보였다고하였다. 또한 trnl intron 지역그리고 trnl 과 trnf 사이의 IGS 영역역시많은연구대상이되어왔다. 특히 trnl intron 에는작은 stem-loop 구조를하고있어이지역주변의잘보존되어있는양쪽에서디자인한프라이머세트로증폭하여이영역을 minibarcode 화하여아주유용하게사용될수있을것이라고주장된바도있다 (Valentini et al. 2009). 약용작물의분류및위품판별을위한 barcodes 개발현황 이미전술한바와같이약용작물의경우에는국가별로명칭이다르고서로혼재하여사용되는경우가많으며, 모양이나형태가유사한것을위품으로유통되는경우가많다. 특히건조된뿌리나약간의 1 차가공과정만하여도전문가가아닌일반인은구분을할수없어서시중에유통되는이들위품을판별하기위한 barcodes 를개발한다면그응용성은무한할것으로사료된다. 따라서약용작물의구분을위한 barcodes 의개발은핵내라이보좀구성 RNA 를암호하는 (nuclear ribosomal) DNA 의 internal transcribed spacer region (ITS1 과 ITS2) 과염록체게놈의여러가지 DNA 단편을이용하여연구되어왔다. 먼저 ITS 를이용한연구현황을 Table 1 에요약하였다. 감초등이속하여있는콩과 (Fabaceae) 에해당하는약용작물들의 ITS2 영역을 PCR 로증폭하여염기서열분석결과를비교하여종내에는평균 1.7% 의변이를보이고전체적으로는 0 에서 14.4% 까지변이를보였다고하였다. 종간에는 0 에서 63% 까지의변이를보여평균 8.6% 의변이를보였다고하였다. 따라서시중에유통되는위품들의판별에도유용하게사용될수있었다고보고된바있다

13 8 J Plant Biotechnol (2015) 42:6 12 Table 1 List of medicinal plant species that have been differentiated by using internal transcribed spacer (ITS) of nuclear ribosomal DNA as a barcode Family/genus/species References Comments Fabaceae (Leguminosae) 753 genera 4,800 species Gao et al Chen et al (Licorice) Glycyrrhiza uralensis polymorphism (92.7%) 50,790 plants 12,221 animals Yao et al Angelica species He et al Breeae & Cirsii herba Moon et al Angelica species Kim et al Schizonepta spike Baigalmaa et al Hyung-Gae Fallopia multiflora Sun et al Cnidii Rhizoma Song et al Fallopia multiflora Zheng et al Ligusticum chuanxiong hort vs Cnidium officinale Makino Liu et al primer design for differentiation by PCR Chinese Chuanxiong vs Japan Chuanxiong (Gao et al. 2010). Chen 등 (2010) 은 753 속 4,800 종에해당하는방대한시료에대하여 ITS2 영역의염기서열을분석한결과 92.7% 에해당하는종의구분이가능하여약용식물의종간구분을위한범용바코드로지정하자고제의를하였다. 또한 Yao et al. (2010) 도 50,790 종의식물과 12,221 종의동물에대하여 ITS2 영역의염기서열을비교분석한결과 67 ~ 91% 까지종의구분이가능하였으며여기에다가 ITS2 영역의 secondary structure 를이용하면범용으로사용될수있는식물용 barcode 로의사용가능성을제시하였다. 또한 He 등 (2011) 도당귀속에속하는다양한종에대하여 ITS 지역의염기서열을비교하여 phylogenic tree 를제작하여유연관계를분석할수있었다고하였다. 특히중국의시중에서말린뿌리상태로유통되어구분이어려운당귀시료들을시중에서수집하여조사한결과일부위품의판별이가능하였다고하였다. Kim 등 (2012) 도 ITS 지역을이용하여분석한결과토당귀와일당귀가중국당귀보다유연관계가가까운것으로조사되었으며, 한 중 일 3 국에서각기다른기원종으로규정하고있는당귀 ( 當歸 ) 류의구별을위해시중에서유통중인당귀 ( 當歸 ) 를종별로 3 점씩, 총 9 점을구입하여미각패턴과 ITS DNA 의염기서열을비교한결과일치하는결과를얻었다고보고하였다 (Kim et al. 2012). 천궁역시 ITS1 과 ITS2 유전자단편의염기서열을비교 Table 2 Chloroplast barcodes for the differentiation of medicinal plant species Family/Genus/Species Chloroplast barcodes References Polygonaceae rbcl, trnh-psba, ndhj, rpob, rpoc, accd Song et al Cnidium officinale, trnk Ligusticum chuanxiong Zhu et al Breeae, Cirsii herba matk, rbcl Moon et al 2013 Pinellia species matk, rbcl Lee et al Fallopia multiflora (Thumb) Harald matk, rbcl, psba-trnh Sun et al Fagopyrum species trnk, matk Ohsako & Ohnishi,2001 Cnidium officinale, matk Ligusticum chuanxiong Liu et al Fallopia multiflora (Thumb) Harald matk Yan et al Liriope platyphylla Wang et Tang, Phiopogon japonicus Ker-Gwaler rpoc1 Park et al 분석한결과토천궁과일천궁, 중국천궁은모두천궁속 (Cnidium) 보다는고본속 (Ligusticum) 과같은분계조를형성하고있는것으로나타났으며, 또한한국산천궁과중국산천궁의두집단간에식별이가능한 SNPs (Single nucleotide polymorphisms) 를확보하여서로간에구분이가능하였다고함 (Song et al. 2009). Baigalmaa et al. (2009) 은 26 계통의형개 (Schizonepeta spike) 로알려진한약재시료를채취하여 ITS 영역의염기서열을분석하여비교한결과 Schizonepeta tenuifolia plants 로표기된 9 종은모두진품으로판명할수있었다고보고하였다. 또한 Song et al. (2009) 은일천궁, 토천궁, 중국천궁을수집하여 ITS 영역의염기서열을분석한결과이들을구분할수있는 SNP 들을확인할수있었다고하였다. 이외에도 6 계통의 Fallopia multiflora 에속하는시료를대상으로 ITS 영역의 SNP 를구분할수있는 primer 를디자인하여위품으로부터종을판별하는데성공하였다고보고한바있다 (Zheng et al. 2009). 또한중국의 Liu 등도이미 2002 년도에일본천궁 (Cnidium officinale Makino) 과중국천궁 (Ligusticum chuanxiong Hort) 의 ITS 영역의염기서열을조사한결과단한개의염기치환이있었다고보고하였다 (Liu et al. 2002). 다음으로엽록체 barcodes 의개발을위한연구를 Table 2 에요약하였다. 이들을검토하여보면적하수오등이속한붉은조롱과 (Polygonaceae) 약용작물을위품으로부터판별하기위하여 18 종에속하는 38 계통에대하여 6 가지의엽록체 barcodes 와핵내존재하는 ITS 등을 PCR 로증폭한다음염기서열을분석하여조사한결과 trnh-psba 가가장

14 J Plant Biotechnol (2015) 42: 높은다양성을보였고 (20.05%), 위품에해당되는유사약용작물들을쉽게판별할수가있었다고보고하였다 (Song et al. 2009). 대계 ( 大薊 ) 와소계 ( 小薊 ) 의기원종을구분하기위하여 rdna-its, matk 및 rbcl 부위 DNA 바코드분석을수행한결과 ITS 가가장많은다형성을보여이들의구분은물론위품인 Carduus 속의지느러미엉겅퀴를종단위에서감별할수있었다고보고하였다 (Moon et al. 2013). 반하 ( 半夏 ) 의기원식물을구분하기위하여 Pinellia ternata, Pinellia pedatisecta, Pinellia tripartita, Typhonium flagelliforme 에속하는 19 계통을채취하여 matk 와 rbcl 두유전자영역의염기서열을비교한결과, matk 유전자영역에서 rbcl 유전자보다반하류의종감별에유용한 SNP 를다수포함하고있어 matk 유전자가 rbcl 유전자보다반하류의종감별에더유용한 DNA 바코드로이용될수있을것으로사료된다고보고하였다 (Lee et al. 2013) 또한 Sun et al. (2013) 은 Fallopia multiflora 종에속하는계통들을중국의 17 성에서서식또는재배되고있는야생종과재배종으로부터 105 계통을수집하여 matk, rbcl, psba-trnh 그리고 ITS barcodes 들을사용하여분석한결과 psba-trnh 가가장양호한결과를보였으며이들 4 가지 barcodes 를조합하여보다판별효율을증진시킬수있었다고하였다. 또한 trnk 와 matk 영역을이용하여 Polygonaceae 과의메밀속 (Fagopyrum) 에속하는 F. leptopodum 종으로알려진 10 계통과과 F. statice 종으로알려진 5 계통을대상으로분석한결과상호간에 9 개의염기치환이있었음을확인하고구분이가능하였다고하였다 (Ohsako & Ohnishi 2001). 이외에도중국천궁과일본천궁의 matk 영역을비교분석한결과단하나의염기치환을확인하였다고하였으며 (Liu et al. 2002), 중국에서만서식하는것으로알려진중국토종인 Fallopia multiflora (Thumb) Harald 의 matk 영역의염기서열을이용하여위품을구분할수있는 SNP 를확인할수있었다고하였다 (Yan et al. 2008). 뿐만아니라국내외에서수집된 26 계통의맥문동에대하여 rpoc1 유전자의염기서열을비교분석하여 SNP 를확인하고이들을판별할수있는프라이머까지개발하여국내특허를획득한것으로알려졌다 (Park et al. 2014). DNA barcodes 를이용한진보된기술의개발현황 전술한바와같이 DNA barcodes 는 SNPs 를찾아내어상호간에구분을하는기술로고도로전문화된전문가가필요하다. 따라서보다단순화되고효율적으로반복이가능하고저렴한가격에수행할수있는기술들을개발하여현장에서실용화가가능하여야한다. 이러한관점에서가장쉬운방법은 SNP 를포함하는프라이머를제작하여 PCR 에의하여증폭되는단편을전기영동하여밴드의유무로판단하는것일것이다. 그러나대부분의경우 Table 3 Advanced techniques for the identification of medicinal plant species Techniques Target regions Family/Genus /Species ARMS-PCR Chs Panax ginseng MSBE HRM trnk ITS, trnl-f, rpl36-ps8 matk ITS2 Chloroplast IGS(62) Cnidium officinale Ligusticum chuanxiong Berry species Hellevorous niger Veratrum niger Sideritis species References Yang et al KR Patent Zhu et al Jaakola et al Mader et al Kalivao et al Panax species Kim et al 2013 에단하나또는두개의염기서열의차이가나는프라이머를제작하여 PCR 을수행하였을경우에도동일한 PCR 산물을생산하기때문에보다정밀한기술이필요하다. 현재까지이러한진보된기술을이용하여약용작물에적용한사례들을요약하여보면 Table 3 과같다. Amplification refractory mutation system (ARMS) 일반적으로 PCR 은 3 - 말단의염기가주형과완전히일치할때에가장확실하게이용될수있는기술이다. 그러나실제로는 3 - 말단의염기가하나혹은두개틀려도 PCR 로증폭이가능하기때문에판별이어려운경우가대부분이다. 따라서 ARMS-PCR 은이러한문제점을극복하기위하여 SNP 에가장가깝게인접한 2, 3 번째염기서열을변경하여 SNP 프라이머의특이성을높이고자하는기술이다. 실제로인삼의경우금품종과청선종에만유일하게나타나는 SNP 를포함하는프라이머를제작하여 ARMS-PCR 을수행한결과다른인삼품종과구분이가능한특정밴드를생산할수있었다. 이때프라이머는 SNP 가 3 - 말단에위치하게하고그로부터 5 - 쪽으로 3 번째의염기서열을변이가일어나도록디자인하여금품종과청선종은한개의염기서열이차이가있으나다른품종들은두개의염기서열이차이가나게하여 PCR 을수행하였을경우보다확실하게밴드의유무를확인할수있는결과를얻어이기술을특허로등록하였다 (Yang et al. 2012). ARMS 기술은 Newton et al.(1989) 에의하여처음보고된기술로서, 기본적으로는 allele-specific (AS)-PCR 기술이다. 그러나예를들어 G 가 C 로점돌연변이 (point mutation) 가일어난경우에 ARMS primer 의 3 - 말단은 G 가되어야치

15 10 J Plant Biotechnol (2015) 42:6 12 Table 4 The strength of mismatch pairings (Little, 1994) Strength Maximum Strong Medium Weak None Mismatch types G-A, C-T, T-T C-C A-A, G-G, C-A, G-T A-T, G-C 환된 C 와결합이될것이다. 하지만이프라이머는정상적인유전자단편과도 G-G mismatch 결합이가능하며이는약한 mismatch 이어서 primer 의 extension 을효율적으로저해하지못하여 PCR 반응에의하여증폭된동일한크기의밴드를형성한다. 다만강한 mismatch 에해당하는 C-C, G-A, A-A 의경우에만 100% 또는 95% 까지염기서열의합성을저해할수있다. 따라서 3'- 말단의 SNP 위치에서 2, 3, 4 번째에 C-C, G-A, A-A mismatch 염기를도입하여제작한프라이머를사용하여 PCR 증폭을 100% 저해할수있도록고안한기술이다. 일반적으로 3'- 말단의 SNP 가약하면두번째 mismatch 는강한것으로디자인한다. 그리고두번째것을디자인하여결과를보고세번째염기를변경하도록디자인것을시도한다. Litte(1994) 는 mismatch 결합의강약을 Table 4 와같이보고하였으며이들조합을적당하게사용하여프라이머를제작하여몇번의시도를거쳐가장적당한조건을설정하는것이중요하다고하였다. Multiplex Single base Extension (MSBE) 분석기술 이기술은일종의 mini-sequencing 기술로분석하고자하는 SNP 를포함하는부위를증폭한다음 dntp 와프라이머를제거한후 SNP 바로앞부분까지를포함하는프라이머 (extension primer) 를넣고형광색소로표지된 ddntp 를이용하여하나의염기만신장시킨다. 이렇게하면서로다른형광색을나타내는 ddntp 중하나만신장하고반응은종결되며이를자동염기서열분석장치를이용하여분석하는기술이다. 따라서이기술은여러종의 SNP 를포함하는부위를이용하여길이가서로다른여러개의 extension primer 를사용하면 2 종이상의시료가혼재되어있을경우에이들을구분하기에용이한기술이다. 약용작물에이용된사례로는천궁즉 Cnidium officinale ( 일본에서사용되는천궁학명 ) 과 Ligusticum chuanxiong ( 중국에서사용되는천궁학명 ) 을확실하게구분이가능하였다고하였다. 보다자세하게살펴보면이들의속간구분을위하여엽록체유전자인 trnk 유전자의염기서열을비교분석하여상류 (upstream) 에서각각 767, 924, 964 번째염기서열에해당하는위치에서나타나는 SNP 를확인하 고, 이위치에서각각 14mer, 23mer, 30mer 길이로디자인된프라이머를사용하여 multiplex single base extension (MSBE) 을수행하여두종을쉽게구분할수있었다고하였다 (Zhu et al. 2007). 또한이기술로 Dongxiong 이라고불리는중국의또다른지역에서재배되고있는천궁은그기원이 Cnidium officinale 인것으로판명할수있었다. 중국에서는천궁을 Chuanxiong 이라고부르며일본식발음으로는 Senkyu, 한국에서는천궁이라고부른다. 이들의외부형태학적인모양은유사하나그식물기원은서로다르다. 그러나 rbcl 유전자와 18S rrna 유전자의염기서열을조사한결과상호간에 100% 일치하여구분이불가능하였다고하였다 (Fushimi et al. 1997). High-resolution melting (HRM) curve 분석기술 post-pcr melting curve 분석기술은 SNP 를포함하는유전자단편을증폭시킨다음초당 0.5 ~ 1.0 C 씩온도를증가시키면서 melting curve 를조사하여그차이를이용하여종을판별하는기술이다. 주로 SYBR Green 이라는형광염색화합물을사용하는데이는이중나선 (dsdna) 을한 DNA 의 minor groove 에삽입되면서강한발색을나타낸다. PCR 로이중나선 DNA 를많이증폭하게되면그만큼상대적으로발색이강하여진다. 따라서 SYBR Green dye 를포함하는반응용액에서 PCR 로증폭시킨이중나선 DNA 를초당 0.5 ~ 1.0 C 씩온도를올리면서 melting curve 를보면초기에많은양의이중나선 DNA 가존재하기때문에강한발색에서온도를서서히상승하면서단일가닥으로 melting 되면서발색정도가서서히약하여진다. 이러한 melting curve 의굴곡의차이를갖고 data 를분석하거나또는시간별온도에대한음의형광값 (-df/dt) 으로계산하여얻은 melting peak 를갖고분석을한다. 그러나이기술로는아주적은수 ( 하나또는두개 ) 의 SNP 를구분할수없다는단점이있다. 따라서보다정밀하고효율이띄어난것으로알려진 High Resolution Melting (HRM) Curve analysis 기술이보고되었다. 이기술의기본원리는 post-pcr melting curve analysis 기술과동일하다. 그러나고농도에서도 DNA 중합효소의기능을저해하지않는형광색소인 LC Green PLUS, Eva Green, SYT09, ResoLight 등을사용하여모든증폭된 DNA 의전체길이에형광색소가삽입이될수있도록하고, 아주미세한단위즉초당 0.01 ~ 0.2 C 로온도상승조절이가능한특수기계 (High Resolution Melting Master, Roche Diagnostics GmbH, Mannheim, Germany 또는 SsofatTM EvaGreen supermix, BioRad Laboratories Inc., Hercules, CA, USA, Light Cycler R 480) 를사용한다. 이렇게하면하나의 SNP 차이도구분이가능하다. 따라서이기술은극도로민감하여해바라기, 땅콩, 옥수수, 참깨, 쌀등에서추출한기름에적

16 J Plant Biotechnol (2015) 42: 용하여상호간에구분이가능하다고보고하였다 (Vietina et al. 2013). 이러한연구결과는향후 HRM 기술의응용범위는각종한약재의가공품뿐만아니라한약재를이용한탕재등에적용하는등그응용범위가크게늘어갈것으로전망된다. ITS 영역또는엽록체 DNA 에해당하는 trnl-f, rpl36-ps8 영역을증폭하여얻은단편을대상으로 HRM 분석기술을적용한결과시중에유통되는야생형은물론다양한베리 (berry) 종들의판별이가능하였다고하였다. 특히불과 4 시간내에 DNA 시료를채취하여 HRM 분석까지마칠수있었다고하였다. 또한가공제품의원료로사용되는혼합시료로부터손쉽게특정종의존재여부를판별할수있었다고보고하며, 향후유통과정에서위품들을판별할수있는기술로크게이용될것이라고예견하였다 (Jaakola et al. 2010). 또한크리스마스장미라고불리는 Helleborus niger 와영국에서 Black Hellebore 로불리는 Veratrum niger 와시중에쉽게혼재하여유통되므로이들을구분하기위한 barcode 로 matk 영역을선택하여 HRM 기술을적용한결과 1 : 1,000 비율로모르는시료에혼재되어있거나 1 : 200,000 비율로 Veratrum niger 가혼재한경우에도판별이가능할정도로민감하였다고보고하였다 (Mader et al. 2011). 또한최근에는그리스의고산지대에서군락을이루고있는 Sidertis 종은약용식물로항균, 소염, 진정제로효과가띄어나차로애용되고있는식물인데다양한종류의종이자라며일부는약효가아주띄어나고일부는독성도있는것으로알려져이들을정확하게판별할수있는기술이필요하였다. 따라서 ITS2 영역에대한 HRM 분석기술을적용하여특별한처리과정이필요없고짧은시간에아주효율적으로서로간에구분이가능하였다고보고된바있다 (Kalivas et al. 2014). 또한인삼의엽록체게놈의모든유전자간 DNA 의염기서열을분석하고이들에대한 HRM 분석기술을적용하여본결과 62 개의 IGS 영역에서다형성을확인하였으며이중에서특히 trne-trnt, trnt-psbd, ndhfrpl32, rpl14-rpl16 spaces 가인삼종간에가장다양성이높게나타났으며, 분자시계 (molecular clocks) 를계산한결과 130 만년전에 P. notoginseng 이다른 Panax 종으로부터분지되었을것이라고추정하였다 (Kim et al. 2013). 사사 본연구는농림축산식품부농생명산업기술개발사업 ( 과제번호 : SB050) 에의해이루어진결과입니다. References Arnot DE, Roper C, Bayoumi RA (1993) Digital codes from hypervariable tandemly repeated DNA sequences in the Plasmodium falciparum circumsporozoite gene can genetically barcode isolates. Mol Biochem Parasitol 61:15-24 Baigalmaa J, Kim MK, Noh JH, Hua S, Yang DC (2009) Phylogenetic analysis of Schizonepeta Spike on the basis of DNA sequences. K J Med Crop Sci 17:46-53 CBOL Plant Working Group (2009) A DNA barcode for land plants. Proc Natl Acad Sci 106: Chase MW, Cowan RS, Hollingsworth PM, van den Berg C, Madriñán S, Petersen G, Seberg O, Jørgsensen T, Cameron KM, Carine M, Pedersen N, Hedderson TAJ, Conrad F, Salazar GA, Richardson JE, Hollingsworth ML, Barraclough TG, Kelly L, Wilkinson M (2007) A proposal for a standardised protocol to barcode all land plants. Taxon 56: Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, Luo K, Li Y, Li X, Jia X, Lin Y, Leon C (2010) Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One 5:1-8, e8613 Dunning LT, Savolainen V (2010) Broad-scale amplification of matk for DNA barcoding plants, a technical note. Botanical J of the Linnean Society 164:1-9 Fushimi H, Komatsu K, Isobe M, Namba T (1997) A new approach for the identification of a Chinese traditional medicine, Chuanxiong by 18S ribosomal RNA gene sequences. Phytomedicin 3: Gao T, Yao H, Song J, Liu C, Zhu Y, Ma X, Pang X, Xu H, Chen S (2010) Identification of medicinal plants in the family Fabaceae using a potential DNA barcode ITS 2. J Ethnopharmacology 130: He Y, Hou P, Fan G, Song Z, Liu H, Li Y, Zhang Y (2011) Internal transcribed spacers (ITS) identification of Angelica anomala Lallem Chuanbaizhi (in Chinese) cultivars collected in Sichuan and their molecular phylogenetic analysis with other Angelica L. species. J Med Plants Res 5: Hebert PDN, Cywinska A, Ball SL, de Waard JR (2003) Biological identifications through DNA barcodes. Proc Royal Soc London, series B 270: Hebert PDN, Ratnasingham S, de Waard JR. (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Biol Sci 270:S96-99 Hollingsworth PM, Graham SW, Little DP (2011) Choosing and using a plant DNA barcode. PLoS One 6:e19254 Jaakola L, Suokas M, Haggman (2010) Novel approaches based on DNA barcoding and high-resolution melting of amplicons for authenticity analyses of berry species. Food Chem 123: Kalivas A, Ganopoulos I, Xanthopoulou A, Chatzopoulou P, Tsaftaris A, Madesis P (2014) DNA barcode ITS2 coupled with high resolution melting (HRM) analysis for taxonomic identification of Sideritis species growing in Greece. Mol Biol Rep 41: Kim JH, Jung JY, Choi HI, Kim NH, Park JY, Lee Y, Yang TJ (2013) Diversity and evolution of major Panax species revealed

17 12 J Plant Biotechnol (2015) 42:6 12 by scanning the entire chloroplast intergenic spacer sequences. Genet Resour Crop Evol 60: Kim YH, Choi G, Lee HW, Lee GH, Chae SW, Kim YH, Lee MY (2012) Comparison of Angelica species roots using taste sensor and DNA sequencing analysis. Kor J Herbology 27:37-42 Kress WJ, Erickson DL (2007) A two-locus global DNA barcode for land plants: The coding rbcl gene complements the non-coding trnh-psba spacer region. PLoS ONE 2: e508. Kuo LY, Li FW, Chiou WL, Wang CN (2011) First insights into fern matk phylogeny. Mol Phylo Evol 59: Lee YM, Moon BC, Ji Y, Kim WJ, Kim HK (2013) Molecular Authentication of Pinelliae Tuber from its adulterants by the analysis of DNA barcodes, matk and rbcl genes. Kor. J. Herbology 2013;28(6):53-58 Little S (1994) Amplification-refractory mutation system (ARMS) analysis of point mutations. In. current protocols in human molecular genetics. (Eds. Nicholas C Dracopoli et al.) Jhon Wiley & Sons, IOOnc. New York, pp Liu YP, Cao H, Han GR, Fushimi H, Komatsu K (2002) MatK ITS nucleotide sequencing of crude drug chuanxiong and phylogenetic relationship between their species from China and Japan. Yao Xue Xue Bao 37:63-68 Mader E, Ruzicka J, Schmiderer C, Novak J (2011) Quantitative high-resolution melting analysis for detecting adulterations. Anal Biochem 409: Moon BC, Lee YM, Ji Y, Choi G, Chun JM, Kim HK (2013) Molecular authentication and phylogenetic analysis of plant species for Breeae and Cirsii Herba based on DNA barcodes. Kor J Herbology 28:75-84 Newton CR, Graham A, Heptinstall LE (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system(arms). Nucl Acids Res 17: Ohsako T, Ohnishi O (2001) Nucleotide sequence variation of the chloroplast trnk/matk region in two wild Fagopyrum (Polygonaceae) species, F. leptopodum and F. statice. Genes Genet Syst 76:39-46 Park YJ, Kwon SO, Park JW (2014) Identification method of Liriope species and identification probe thereof. Korea patent Piredda R, Simeone MC, Attimonelli M, Bellarosa R, Schirone B (2010) Prospects of barcoding the Italian wild dendroflora: oaks reveal severe limitations to tracking species identity. Mol Eco Ress 11:72-73 Ren BQ, Xiang XG, Chen ZD (2010) Species identification of Alnus (Betulaceae) using nrdna and cpdna genetic markers. Mol Eco Res 10: Roy S, Tyagi A, Shukla V, Kumar A, Singh UM, et al. (2010) Universal plant DNA barcode loci may not work in complex groups: A case study with Indian Berberis species. PLoS ONE 5: e13674 Song IG, An BR, Seo BI, Park SJ (2009) Molecular marker to identify and origin of Cnidii Rhizoma from Korea and China. Kor J Herbology 24:1-8 Sun XQ, Bai MM, Yao H, Gao JI, Li MM, Hang YY (2013) DNA barcoding of populations of Fallopia multiflora, an indigenous herb in China. Genet Mol Res 12: Valentini A, Pompanon F, Taberlet P (2008) DNA barcoding for ecologists. Trends Ecol Evol 24: Valentini A, Miquel C, Nawaz MA, Bellemain EVA, Coissac E, Pompanon F, Gielly L, Ruaud C, Nascetti G, Wincker P, Swenson JE, Taberlet P (2009) New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trnl approach. Mol Ecol Res 9:51-60 Vietina M, Agrimonti C, Marmiroli N (2013) Detection of plant oil DNA using high resolution melting (HRM) post PCR analysis: A tool for disclosure of olive oil adulteration. Food Chem 141: von Cräutlein M, Korpelainen H, Pietiläinen M, Rikkinen J (2011) DNA barcoding: a tool for improved taxon identification and detection of species diversity. Biodiversity and Conservation 20: Wicke S, Quandt D (2009) Universal primers for amplification of the trnk/matk region in land plants. Anales del Jardín Botánico de Madrid 66: Yan P, Pang QH, Jiao XW, Zhao X, Shen YJ, Zhao SJ (2008) Genetic variation and identification of cultivated Fallopia multiflora and its wild relatives by using chloroplast matk and 18S rrna gene sequences. Planta Med 74: Yang DC, Wang HD (2012) SNP primers for Gumpoong and Chungsun selection of Korean ginseng (Panax ginseng C.A. Meyer) and method for Gumpoong and Chungsun distinciton using the same. KR patent Yao H, Song J, Liu C, Luo K, Han J, Li Y, Pang X, Xu H, Zhu Y, Xiao P, Chen S (2010) Use of ITS2 region as the universal DNA barcode for plants and animals. PLoS ONE 5:e13102 Zheng CJ, Zhao SJ, Zhao ZH, Guo J (2009) Molecular authentification of the traditional medicinal plant Fallopia multiflora. Planta Med 75: Zhu S, Fushimi H, Han G, Tsuchida T, Uno T, Takano A, Komatsu K, (2007) Molecular identification of Chuanxiong by nucleotide sequence and multiplex single base extension analysis on chloroplast trnk gene. Biol Pharm Bull 30:

18 J Plant Biotechnol (2015) 42:13 18 DOI: ISSN Research Article Absence of AVP1 transcripts in wild type watermelon scions grafted onto transgenic bottle gourd rootstocks Byung Oh Kim Jeung-Sul Han Kyung il Park Su Min Jeon Chang Kil Kim Received: 6 March 2015 / Revised: 15 March 2015 / Accepted: 15 March 2015 c Korean Society for Plant Biotechnology Abstract In this study we confirmed the stable integration of Arabidopsis AVP1 in the genomes of bottle gourd T 3 homozygous lines and its transcription, and additionally evaluated possibility of translocation of the AVP1 mrna from transgenic bottle gourd rootstocks to wild type watermelon scions. Each AVP1 gene in two bottle gourd T3 lines is abundantly expressed under a field condition. Given the grafting between wild type watermelon scions and AVP1- expressing bottle gourd rootstocks, no translocation of the AVP1 mrna was detected in leaves, both sexual flowers, and fruits of the scions. Keywords Bottle ground, Gene modified, Graft, RT-PCR, Watermelon Introduction Grafting is now a popular technique for the cultivation of the horticultural crops including cucurbitaceae fruit vegetables, which has been developed not only to control growth and development of the scion but also to enhance tolerance against soil-borne diseases and/or abiotic stresses, such as salinity, low temperature and drought (Jang et al. 2012; Kubota et al. 2008; Lee 1994). In some special regions, where land utility is B. O. Kim School of Food science & Biotechnology, Kyungpook National University, Daegu , Korea J.-S. Han Department of Ecological Environment, Kyungpook National University, Sangju , Korea K. I. Park Department of Horticulture & Life Science, Yeungnam University, Gyeongsan , Korea S. M. Jeon C. K. Kim ( ) Department of Horticultural Science, Kyungpook National University, Daegu , Korea ckkim@knu.ac.kr extremely limited, the allied crops are repeatedly cultivated all the year round (Kubota et al. 2008; Lee 1994), which increases specific pathogens and salinity of the rhizosphere. To overcome the disadvantages of the intensive cultivation, improvement of rootstocks by using genetic engineering is being attempted as a solution (Han et al. 2009; Smolka et al. 2010; Wang et al. 2012). Control of abiotic stresses is an important element to increase total yields in modern agriculture. Plants respond to various abiotic stresses by altering their turgor pressure in vacuoles in order to accomplish selective permeation of solutes through proton pumps (Gaxiola et al. 2001; McNeil et al. 1999). A vacuolar H + -pyrophosphatase encoded by the AVP1 gene is one of the proton pumps in Arabidopsis (Sarafian, et al. 1992) and generates an H + electrochemical gradient across the tonoplast (Zhen et al. 1997). Several transgenic plants overexpressing AVP1 have been shown to be more tolerant to salt- and drought-stress than their counterparts (Gaxiola et al. 2001; Jeong et al. 2013; Park et al. 2012; Pasapula et al. 2011). Bottle gourd (Lagenaria siceraria Standl.) that gives host-specific resistance against a Fusarium spp. is one of the most popular rootstocks for watermelon, although acute disorders such as sudden wilt caused by complicated abiotic stresses appear occasionally under cultivation conditions in year round production systems (Edelstein et al. 1999; Kubota et al. 2008; Lee 1994; Park et al. 2005b). Therefore, transgenic bottle gourd with modified abiotic stress-related traits is being considered as a rootstock of watermelon production (Han et al. 2009). However, the utilization of a transgenic rootstock might bring both sides of the coin owing to feasible long-distance transport of molecules derived from the transgene: to indirectly improve horticultural traits of the scion and to provoke controversy about social acceptance or rejection (Harada 2010; Kim et al. 2001). Here we focused on ascertaining transgene RNA transmission from rootstock to scion, which is one of the most important factors together with protein movement evaluating

19 14 J Plant Biotechnol (2015) 42:13 18 the substantial equivalence (Millstone et al. 1999). Two bottle gourd lines expressing Arabidopsis AVP1 were used as rootstocks for watermelon. Molecular characteristics of the AVP1 and its transcript were analyzed in the two transgenic lines, and the translocation of the transcripts of AVP1 and Bar (a selection marker gene) was tested in each part of watermelon scion. Materials and Methods Plant materials and transformation Bottle gourd (Lagenaria siceraria G5 ) transformation was performed by means of the Agrobacterium-mediated transformation method using cotyledon explants as described (Han et al. 2004; 2005; 2009). A. tumefaciens strain LBA4404, with the prg521 plasmid that was generated by replacing the selectable marker cassette of prg395 plasmid (Park et al. 2005a) with the Nos-pro/Bar/Nos-ter of pcb302 plamid (Xiang et al. 1999), was used for this study. Collectively, the T-DNA region of prg521 plasmid was consisted of LB/tandem 35S-pro/AVP1/Poly A/Nos-pro/Bar/Nos-ter/RB. The T 3 lines, BGAVP05, was developed through phosphinothricin (Duchefa Biochemie, the Netherlands) at 2 mg/l supplementation for selecting T 0 plants in vitro, and herbicide Basta TM (Kyungnoog, Korea) at 0.3%(v/v) treatment and polymerase chain reaction (PCR) analysis for succeeding generations. Final T 3 generation of the BGAVP05 lines and wild type bottle gourd were sown in plastic trays filled with commercial organic soil. After 3 weeks, young plants were transplanted into plastic pots (30 35 cm) and then further grown in a greenhouse at Kyungpook National University located in Daegu, Korea. These plants were then subjected to nucleic acids analyses. Meanwhile, 1 week delayed seedlings of two commercial watermelons (Citrullus vulgaris prince and speed ) were grafted onto the two transgenic and wild type bottle gourd lines. After graft unions were stabilized, grafted plants were also transplanted and grown under the same conditions indicated above for non-grafted bottle gourd lines (Fig. 1). PCR and Reverse transcription polymerase chain reaction (RT-PCR) analyses DNAs for PCR analysis and total RNAs for RT-PCR were extracted by using the HiYield TM Genomic DNA Mini Kit (Real Biotech Corporation, Taiwan) and the RNeasy R Plant Mini Kit (Qiagen, Germany), respectively. Up to 0.1 g of young leaves of bottle gourd plants for DNA extraction and up to 0.3 g each of samples from bottle gourds (young leaves and stems, and male and female flowers) and watermelons (young leaves, male and female flowers, and flesh of fruits) for RNA Fig. 1 Growth and development of watermelon plants grafted onto bottle gourd rootstocks expressing AVP1. (A) Acclimatization of grafted watermelon young plants. (B) Formation of graft union between wild type watermelon scion (arrow head) and AVP1-expressing bottle gourd rootstock (arrow). (C) The potted plants of grafted watermelon plants growing in a glasshouse. Female flowers of watermelon, cultivars Speed (D1) and Prince (D2). Fruits of watermelon cultivars Speed (E1) and Prince (E2)