Journal of Radiation Industry 6 (2) : 47~52 (22) Review Paper 감마선변이체스프레이국화 ARTI-purple 의화색관련유전자발현분석 성상엽,2 이유미 김상훈 하보근 강시용 김진백 김홍기 2 김동섭, * 한국원자력연구원방사선과학연구소, 2 충남대학교응용생물학전공 Expression Analysis of Flower Color Related Genes in Spray-type ARTI-purple Developed by Gamma-ray Mutagenesis Sang Yeop Sung,2, Yu-Mi Lee, Sang Hoon Kim, Bo-Keun Ha, Si-Yong Kang, Jin-Baek Kim, Hong Gi Kim 2 and Dong Sub Kim, * Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 58-85, Korea 2 Department of Agricultural Biology, Graduate School, Chungnam National University, Daejeon, Korea Abstract - Anthocyanins are major plant pigment and produced through phenylpropanoid pathway. In this study, anthocyanin biosynthesis mechanisms of chrysanthemum flowers were studied using Argus and flower color mutant ARTI-purple which were induced by 4 Gy gamma irradiation (Co 6 ). And, three chrysanthemums, Ford, Yeonja and Orando were additionally used as the check varieties to understand the relationship between flower color and expression patterns of genes. The expression patterns of the anthocyanin biosynthetic genes were matched with the flower color of the check varieties. High anthocyanin concentration of Orando showed the high expression of anthocyanin biosynthetic genes. In the white flower of Ford, expressions of CHI, DFR and ANS were not identified. Despite different flower color, Argus and ARTI-purple showed different expression patterns compared with the check varieties. From the dot blot analysis, we screened the seven genes showing the different expressions between Argus and ARTI-purple. Key words : Chrysanthemum, Authocyanin, Flower, Gamma irradiation, Expression 서 론 국화 (Chrysanthemum morifolium) 는장미등과함께 * Corresponding author: Dong Sub Kim, Tel. +82-63-57-33, Fax. +82-63-57-339, E-mail. bioplant@kaeri.re.kr 화훼시장에서높은시장가치를가지는화훼류로서, 다양한화형과화색으로분류되고, 화색은안토시아닌및카로티노이드등의색소의구성및함량에따라결정된다. 화려한화색과화형은소비자의선호도에가장큰영향을미치는요소로현재까지일부연구가이루어졌다 (Schwinn et al. 993; Hayashi and Todoriki 996; Ohmiya 47
48 성상엽 이유미 김상훈 하보근 강시용 김진백 김홍기 김동섭 et al. 26). 여러색소중안토사이닌은페닐프로파노이드 (phenylpropanoid) 경로에서유도되는제일크고아주다양한식물색소그룹이다. 수산기와메틸기의수와위치에따라또한 glycosylation과방향족또는지방족 acylation에따라색과안정성이다른다양한형태의안토시아닌분자가되며, 붉은색, 노란색, 보라색등이대표적으로확인된다 (Springob et al. 23). 안토시아닌은국화의화색을결정하는주요색소임에도불구하고, 각성분의생합성과정에대한연구는국화에서활발하게이루어지지못했다. 그동안국화의안토시아닌합성유전자는 chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid-3-hydroxylase (F3H), flavonoid-3 -hydroxylase (F3 H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), anthocyanidin 3-O-glucoside-6 -O-malonyltransferase (3MaT), anthocyanidin 3-O-glucoside-3, 6 -O-dimalonyltransferase (3MaT2) 등, 구조합성효소가확인되었다 (Suzuki et al. 24; Seo et al. 27). 하지만국화에서화색에따른이러한유전자의발현양상에대한연구는미비하다. 한편, 국화화훼품종의개발은주로전통육종방법인교배육종을비롯하여방사선을이용한돌연변이육종을통해이뤄지고있으며, 최근에는형질전환기술을통해서도시도되고있다. 특히, 자연돌연변이빈도는지극히낮은확률로발생되며, 돌연변이의빈도를증가시키기위하여감마선, 이온빔, 전자선등을이용한방사선돌연변이육종법은다른화학적돌연변이유기보다돌연변이처리과정이안전하고용이하다. 국화의다양한화형, 화색품종의개량및품질향상을위해방사선돌연변이육종기술이이용되고있으며, 전세계화훼시장에서돌연변이육종에의해등록된화훼류는약 6 여품종으로확인되며, 그가운데 254개가국화품종이며, X선과 γ선을이용하여 5% 이상이네덜란드와인도에서개발되었다 (FAO-IAEA; http://www.mvgs.iaea.org/ MVD/Default.htm) (Yamaguchi et al. 28). 최근일본에서도 Imajin, Arajin, Emi-akari, Princess kagawa, Dreaming, Ray Sunrise 등 ion beam과 X선조사를통한품종개발이보고된바있으며국내에서도조직배양기술과감마선돌연변이육종기술을접목하여국화신품종개발을위한육종연구가보고되고있다 (Park 25; Park et al. 27; Lee et al. 28). 본연구에서는 Argus 국화와이로부터유기된돌연변이품종 ARTI-purple 을활용한국화화색관련유전자연구를 Ford, Orando, Yeonja 품종과비교하여실시했으며, 국화화색을결정하는안토시아닌생합성에관여하는주요유전자의발현양상을확인하고, 추가로 안토시아닌함량에영향을미치는것으로추정되는잠재적유전자를추가로분리하고발현양상을확인하였다. 재료및방법. 실험재료국화 Argus 품종을 25년에줄기의기내배양을통해대량증식한후, 유식물체에한국원자력연구원저준위감마선조사시설 ( 6 Co) 에서 4 Gy를조사하여 3주간의발근후방사선육종시험장온실에정식하였다. 그리고 26년에서 27년사이에삽목증식을통해고정된 ARTI-purple 품종을육성하였다 (Park et al. 27). 그리고대조군으로 Ford Orando, Yeonja 를사용하였다. 2. Total RNA 분리국화는개화후약 2일이경과하면국화화형및화색이완성된다. 그리고이후로시간이진행되면꽃잎에서안토시아닌침적이나타나며, 노화가진행된다. 국화샘플링은개화후 2일째만개한꽃잎 (Ray floret) 을채취하였으며, -8 C 에보관하였다. 채취한꽃잎 (Ray floret) 2g을막자사발로곱게갈은후, ml의 Trizol regeant 를이용하여제안된 protocol을따라 RNA를추출하였다 (Invitrogen, USA). 이후 DNase 처리및 NanoDrop ND- spectrophotometer (NanoDrop Technologies, USA) 을이용하여정량을하였다. 3. Dot blot 분석 ) Preparation of membrane 분리된 cdna clones로부터유전자발현차이의확인을위해, reverse dot blot hybridization을수행하였다. 먼저국화꽃잎으로부터수집된 cdna library clone을 5 μl volume으로 saturation PCR을하였고, 이를 Hybond N+ membranes (Amersham, Germany) 에 vacuum blotter (Gibco BRL) 을이용하여 blotting 하였다. 그리고 CL- Ultraviolet Crosslinker (UVP Inc, England) 을이용하여고정하고 4 C 에서 digoxigenin-labeled cdna를 hybridization 하기전까지보관하였다. 2) Probe synthesis and hybridization of membranes Argus 와 ARTI-purple 의 total RNAs을분리하고 DIG DNA labeling kit (Roche, Switzerland) 을이용하여 digoxigenin-labeled cdnas을합성하였다. 그리고 4 C 에서보관중인 membrane을 2시간동안 62 C 에서 pre-hybridiza-
국화변이체 ARTI-purple 의화색유전자분석 49 tion한후digoxigenin-labeled cdnas μg을 6시간동안 62 C 에서 hybridization 하였다. DIG Nucleic acid detection kit (Roche, Switzerland) 을이용하여 membranes을세척하고, X-ray films에 3분간노출하였다. 그리고최종적으로 Argus 와 ARTI-purple 으로부터얻어진두장의 membrane에각각clone을비교하였다. 4. 프라이머제작및 relative RT-PCR 분석실험에이용한프라이머는 cdna library를통해확인된시퀀스를이용하여 gene specific primer를 Primer 3 Software (http://www.genome.wi.mit.edu/cgi-bin/primer/ primer3.cgi) 를통해제작하였다. Relative RT-PCR은 Pfaffl (2) 의실험을참조하여진행하였다. 분리된 RNA를 ng μl - 로정량하고 α-tubulin을 reference 유전자로이용하였다. 그리고 Eco TM Real-Time PCR system을통해 SYBR green II fluorescence dye (Takara, Japan) 을이용하여발현을검정하였다. 결과및논의. 안토시아닌생합성유전자의분리및발현분석안토시아닌생합성유전자의연구는아라비돕시스, 페튜니아, 옥수수, 포도등여러식물체에서오랫동안연구 되어왔으며, 지금까지상당한연구의결과가누적되었다 (Yamaguchi et al. 999; Kobayashi et al. 2; Park et al. 28). 안토시아닌합성은크게 Phenylpropanoid biosynthesis 경로에서안토시아닌전구체인 Cinnamoyl-CoA가합성되며, Flavonoid biosynthesis 경로를통해 naringenin 에서안토시아닌전구체 flavonoid류와이들의복합적구조체인안토시아닌생합성이이루어진다 (Casta neda- ~ Ovando et al. 29). 국화 Argus 와이로부터유기된보라색화색변이체 ARTI-purple 의안토시아닌합성유전자의발현양상을확인하기위해추가적인대조군으로흰색꽃잎, 분홍색꽃잎, 자홍색꽃잎의색상을가지는 Ford, Yeonja, Orando 세품종의발현양상을함께비교분석하였다 (Fig. ). 안토시아닌합성유전자의발현양상을확인하기위해, Argus 와 ARTI-purple 그리고이미분리된유전자를활용하여유사도가가장높은 conserved region에대한프라이머를 8~2 bp 사이로제작하였다 (Table ). 분석결과, 대조군으로확인한 Ford, Yeonja, Orando 세품종에서 CHI, F3H, DFR, ANS의발현양상이국화꽃잎에서나타나는안토시아닌함량과유사하게증가하는패턴으로확인이되었으며, 흰색꽃잎의 Ford 는 F3H, DFR, ANS의유전자발현이확인되지않았다 (Fig. 2). 하지만 Argus 와 ARTI-purple 에서유전자의발현양상은큰차이가없이나타나거나 CHI, F3H의발현은보라색꽃잎을가지는 ARTI-purple 에서오히려감소 Argus ARTI-purple Ford Yeonja Orando Fig.. Used plant materials. Table. Summary of differential expression in qpcr of the five chrysanthemum varieties. Clone number was arbitrarily annotated in clones of cdna library. All of genes description is best matched with NCBI blastx databases Clone No. Accession No. Annotation [Species] E-value EA55 AAD393. LIM domain protein WLIM- [Helianthus annuus] 2E-99 EF22 ADD7996. 4-3-3 family protein [Dimocarpus longan] E-27 EG55 ACK37352. S-adenosyl-L-methionine synthetase [Chrysanthemum coronarium]. ED28 ACW8366.2 mevalonate diphosphate decarboxylase [Panax ginseng] E-32 EF28 XP_25738. phosphofructokinase, putative [Ricinus communis] 2E-55 EA5 ACF7637. putative arogenate dehydratase [Capsicum annuum] E-8 EB87 ACN3827. glutathione S-transferase [Vitis amurensis] E-8
5 성상엽 이유미 김상훈 하보근 강시용 김진백 김홍기 김동섭 (A) 2.5 2 Argus ARTI-purple (A) 3 2.5 Argus ARTI-purple Relative expression Relative expression.5.5 (B).8.5.2.9.6.3 CHS CHI F3H DFR ANS Ford Yeonja Orando Relative expression 2.5.5 (B).7.6 Relative expression.5.4.3.2 EA55 Ford Yeonja Orando EF22 EG55 ED28 EF28 EA5 EB87 CHS CHI F3H DFR ANS Fig. 2. Relative expressions of the anthocyanin biosynthetic genes in five chrysanthemums. A: qpcr of Argus and ARTIpurple, B: qpcr of check varieties. 하는것으로확인이되었다. 이러한사실로부터 Argus 품종은 colorless의안토시아닌류를가지거나, 꽃잎에서안토시아닌의합성이다른이화작용과함께일어나꽃잎에서안토시아닌이축적이되지못하는것으로추정되었다. 즉 Argus 와 ARTI-purple 에서나타나는화색의차이는대조군에서확인된안토시아닌합성유전자의발현양상에따른안토시아닌함량의차이가아니라다른요인이추가적으로작용할가능성이있는것으로확인되었다. 2. Argus 와 ARTI-purple 에서다르게발현되는유전자분석 추가적으로 Argus 와 ARTI-purple 사이에화색의차이, 즉안토시아닌의함량의차이가나타나는원인을추적하기위해서 Argus 와 ARTI-purple 간에발현적차이를보이는유전자를 dot blot hybridization을통해발현을스크린하였으며, 그결과로부터발현에서차이를보인 7개유전자를대조군국화품종과함께 real-time. EA55 EF22 EG55 ED28 EF28 EA5 EB87 Fig. 3. Relative expressions of differentially expressed seven genes by dot blot analysis between Argus and ARTI-purple, in five chrysanthemums. A: qpcr of Argus and ARTI-purple, B: qpcr of check varieties. PCR분석을수행하였다 (Table ). LIM domain protein WLIM-, 4-3-3 family protein, S-adenosyl-L-methionine synthetase, mevalonate diphosphate decarboxylase, phosphofructokinase, arogenate dehydratase, glutathione S- transferase는모두 ARTI-purple 에서발현이증가한것으로확인되었다. 특히 glutathione S-transferase는페튜니아의 AN9과높은 homology를가지고있었으며 (positive identities; 8%), 대조군세품종에서발현양상은안토시아닌함량과매우유사하게변화하는것을확인할수있었으나, Argus 와 ARTI-purple 사이에서는발현차이가크게나타나지않았다. 또한 S-adenosyl-L-methionine synthetase는대조군품종에서매우낮은발현양상 ( Argus 대비 %, ARTI-purple 대비 5% 수준 ) 을띄고, ARTI-purple 에서발현이가장큰폭으로증가한것을확인할수있었다 ( 약 2배 ). 그리고 Argus 와 ARTI-pur-
국화변이체 ARTI-purple 의화색유전자분석 5 ple 사이에서 Mevalonate diphosphate decarboxylase의발현이가장큰폭의증가를보였다 (Fig. 3). 결론안토시아닌은가끔어린식물조직에서나타나며, 조직이성숙함에따라사라지기도한다. 일반적으로안토시아닌의기능은가시광선과자외선을흡수하기때문에조직을보호하기위해차광역할을하는것으로추정되며, 또한산화방지능력이있어산화적인피해로부터세포를보호하는것으로추정된다 (Mol et al. 998; Winkel- Shirley 22; Zhang et al. 24). 가시적으로나타나는안토시아닌함량의손실은엽록소의증가, 안토시아닌생합성유전자의발현감소, 광보호제가필요하지않을때의식물의안토시아닌분해능력증가등의복합요인에기인된것으로추정되며, 반대로낙엽과같이조직의노후와함께확인되는안토시아닌의증가는여전히확실한해답을찾지못하고있는실정이다. 최근안토시아닌의연구는안토시아닌생합성유전자에의존적으로이루어졌으나, 브룬펠시아꽃잎에서안토시아닌의분해가능성을처음으로제시및확인하였다 (Vaknin et al. 25; Oren-Shamir 29; Bar-Akiva et al. 2). 본연구에서우리는국화로부터안토시아닌의생합성에관여하는주요유전자를분리하고, 발현양상을확인하였으며, 국화꽃잎에서나타나는안토시아닌함량과비교하여유전자발현양상을비교한결과, 대조군에서는안토시아닌함량과유사하게합성유전자의발현이증가되는것을확인할수있었으나, Argus 품종과감마선돌연변이체 ARTI-purple 에서는이러한특성을확인할수없었다. 이러한결과는국화꽃잎에서나타나는안토시아닌함량은추가적으로안토시아닌함량제어에관여된메커니즘의존재나안토시아닌의분해, 또는 colorless anthocyanin의존재가능성을시사한다. 그리고추가적으로확인한유전자의발현양상확인을통해확인된 glutathione S-transferase는안토시아닌의 channel protein으로알려진페튜니아의 AN9 유전자와 ortholog의가능성이있을것으로판단되며, 그외확인된유전자에대해추가적인연구를통해서국화에서안토시아닌함량에영향을미치는추가적인요소또는메커니즘연구를진행할수있을것으로기대된다. 참고문헌 Bar-Akiva A, Ovadia R, Rogachev I, Bar-Or C, Bar E, Freiman Z, Nissim-Levi A, Gollop N, Lewinsohn E, Aharoni A, Weiss D, Koltai H and Oren-Shamir M. 2. Metabolic networking in brunfelsia calycina petals after flower opening. Journal of Experimental Botany 6(5):393-43. Casta ~ neda-ovando A, Pacheco-Hernández MdL, Páez-Hernández ME, Rodríguez JA and Galán-Vidal CA. 29. Chemical studies of anthocyanins: A review. Food Chemistry 3(4):859-87. Hayashi T. and Todoriki S. 996. Sugars prevent the detrimental effects of gamma irradiation on cut chrysanthemums. HortScience 3():7-9. Kobayashi S, Ishimaru M, Ding CK, Yakushiji H and Goto N. 2. Comparison of udp-glucose:flavonoid 3-o-glucosyltransferase (ufgt) gene sequences between white grapes (vitis vinifera) and their sports with red skin. Plant Science 6(3): 543-55. Lee GJ, Chung SJ, Park IS, Lee JS, Kim JB, Kim DS and Kang SY. 28. Variation in the phenotypic features and transcripts of color mutants of chrysanthemum (Dendranthema grandiflorum) Derived from Gamma ray Mutagenesis. J Plant Biol. 5(6):48-423. Mol J, Grotewold E and Koes R. 998. How genes paint flowers and seeds. Trends in Plant Science 3(6):22-27. Ohmiya A, Kishimoto S, Aida R, Yoshioka S and Sumitomo K. 26. Carotenoid cleavage dioxygenase (cmccd4a) contributes to white color formation in chrysanthemum petals. Plant Physiology 42(3):93-2. Oren-Shamir M. 29. Does anthocyanin degradation play a significant role in determining pigment concentration in plants? Plant Science 77(4):3-36. Park IS and Song HS. 25. Current trends of mutation breeding by radiation technology at domestic and foreign ornamentals. J. Kor. Flower Res. Soc. 3(3):84-2. Park IS, Lee GJ, Kim DS, Chung SJ, Kim JB, Song HS, Goo DH and Kang SY. 27. Mutation breeding of a spray chrysanthemum Argus by gamma-ray irradiation and tissue culture. Flower Res. J. 5():52-57. Park J-S, Kim J-B, Cho K-J, Cheon C-I, Sung M-K, Choung M-G and Roh K-H. 28. Arabidopsis r2r3-myb transcription factor atmyb6 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (lactuca sativa) Plant Cell Reports 27(6):985-994. Pfaffl MW. 2. A new mathematical model for relative quantification in real-time rt-pcr. Nucleic Acids Research 29(9): e45. Schwinn KE, Markham KR and Giveno NK. 993. Floral flavonoids and the potential for pelargonidin biosynthesis in commercial chrysanthemum cultivars. Phytochemistry 35():45-5. Seo J, Kim S, Kim J, Cha H and Liu J. 27. Co-expression of
52 성상엽 이유미 김상훈 하보근 강시용 김진백 김홍기 김동섭 flavonoid 3 5 -hydroxylase andflavonoid 3 -hydroxylase accelerates decolorization in transgenic chrysanthemum petals. Journal of Plant Biology 5(6):626-63. Springob K, Nakajima J-i, Yamazaki M and Saito K. 23. Recent advances in the biosynthesis and accumulation of anthocyanins. Natural Product Reports 2(3):288-33. Suzuki H, Nakayama T, Yamaguchi M-a and Nishino T. 24. Cdna cloning and characterization of two dendranthema morifolium anthocyanin malonyltransferases with different functional activities. Plant Science 66():89-96. Vaknin H, Bar-Akiva A, Ovadia R, Nissim-Levi A, Forer I, Weiss D and Oren-Shamir M. 25. Active anthocyanin degradation in brunfelsia calycina (yesterday-today-tomorrow) flowers. Planta 222():9-26. Winkel-Shirley B. 22. Biosynthesis of flavonoids and effects of stress. Current Opinion in Plant Biology 5(3):28-223. Yamaguchi H, Shimizu A, Degi K and Morishita T. 28. Effects of dose and dose rate of gamma ray irradiation on mutation induction and nuclear DNA content in chrysanthemum. Breeding Science 58(3):33-335. Yamaguchi M-A, Oshida N, Nakayama M, Koshioka M, Yamaguchi Y and Ino I. 999. Anthocyanidin 3-glucoside malonyltransferase from dahlia variabilis. Phytochemistry 52(): 5-8. Zhang Z, Pang X, Xuewu D, Ji Z and Jiang Y. 24. Role of peroxidase in anthocyanin degradation in litchi fruit pericarp. Food Chemistry 9(-2):47-52. Manuscript Received: March 26, 22 Revised: April 2, 22 Revision Accepted: May 22, 22