J Plant Biotechnol (2015) 42:245 256 DOI:http://dx.doi.org/10.5010/JPB.2015.42.3.245 ISSN 1229-2818 (Print) ISSN 2384-1397 (Online) Research Article 배추유묘의글루코시놀레이트합성기작에미치는 LED 혼합광의효과 문정현 정미정 이수인 이준구 황현승 유재웅 김용록 박세원 김진아 Effect of LED mixed light conditions on the glucosinolate pathway in brassica rapa Junghyun Moon Mi Jeong Jeong Soo In Lee Jun Gu Lee Hyunseung Hwang Jaewoong Yu Yong-Rok Kim Se Won Park Jin A Kim Received: 9 June 2015 / Revised: 15 July 2015 / Accepted: 5 August 2015 c Korean Society for Plant Biotechnology Abstract In the agricultural industries, LEDs are used as supplementary, as well as main lighting sources in closed cultivation systems. In cultivation using artificial light sources, various light qualities have been tried to supplement fluorescent lamps to promote plant growth and metabolism. Microarray analysis of Brassica rapa seedlings under blue and fluorescent mixed with blue light conditions identified changes in three genes of the glucosinolate pathway. This attracted attention as functional materials highly expressed 3.6-4.6 fold under latter condition. We selected four more J. Moon M. J. Jeong S. I. Lee J. A. Kim ( ) 농촌진흥청국립농업과학원생물소재공학과 (Functional Biomaterial Division, National Academy of Agricultural Science, Rural Development Administration, Suwon, 441-707, Republic of Korea) e-mail: jakim72@korea.kr J. G. Lee 전북대학교농업생명과학대학 (Department of Horticulture, Chonbuk National University, Jeonju 561-756, Korea) H. Hwang 서울대학교식물생산과학부 (Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul, 151-744 Korea) J. Yu S. W. Park ( ) 건국대학교분자생명공학과 (Department of Molecular Biotechnology, Konkuk University, Seoul 143-701, Korea) e-mail: swpark@konkuk.ac.kr Y. R. Kim 연세대학교이과대학화학과 (Department of Chemistry, Yonsei University, Shinchon-Dong 134, Seodaemun-Gu, Seoul 120 749, Republic of Korea) genes of the glucosinolate pathway from the Brassica database and tested their expression changes under fluorescent light mixed with red, green, and blue, respectively. Some genes increased expression under red and blue mixed conditions. The Bra026058, Bra015379, and Bra021429; the orthologous genes of CYP79F1, ST5a, and FMOGS-OX1 in Arabidopsis, are highly expressed in Brassica rapa under fluorescent mixed with blue light conditions. Further, Bra029355, Bra034180, Bra024634, and Bra022448; the orthologous genes of MAM1, AOP3, UGT74B1, and BCAT4 in Arabidopsis, are highly expressed in Brassica rapa under fluorescent mixed with red light conditions. The various light conditions had unique effects on the varieties of Brassica, resulting in differences in glucosinolate synthesis. However, in some varieties, glucosinolate synthesis increased under mixed blue light conditions. These results will help to construct artificial light facilities, which increase functional crops production. Keywords Blue light, Brassica rapa, Glucosinolate, LED, Microarray 서론 광은식물에게있어일차적인에너지원으로대부분의식물발달단계를조절하고광합성효율을유지하는역할을한다 (Hangarter 1997). 태양광은식물재배에있어서최적의조건이지만일일중또는일년중끊임없이변하고다양한파장을가지고있기때문에식물의광합성작용에사용되는파장은많지않다 (Lund et al. 2007). 조사된
246 J Plant Biotechnol (2015) 42:245 256 광은광질별로특이수용체의광화학적반응에의해조절되며식물의발육에서로다른역할을한다 (Butler et al. 1964) 는것이밝혀진이후식물의생육에특정파장의광이어떤영향을미치는지에대한연구가계속되어왔다. 광파장과작물의생육과의관계에대한오랜연구결과적색광은식물체의광합성에영향을미치고청색광은형태학적으로식물체의건전한생장에필수요소임이밝혀졌다 (Okamoto et al. 1996). 또한애기장대에청색광을조사하였을때하배축의신장을억제하고자엽확장을유발하며, 적색광은하배축신장과자엽확장을촉진한다는보고가있었다 (Whitelam and Halliday 2007). 자연의기후에영향을받지않고작물을안정적으로재배하기위해이용되는다양한시설내에서의식물의생리는피복재를통해투과되는광량, 광질및광조사시간등에의해영향을받는다 (Klein 1979). 식물이필요로하는광을인공조명장치로대체하기도하는데태양광을대신하여식물에유용한광질과광도를조사할수있도록고압나트륨등, 메탈헬라이드등, 형광등, LED 등등다양한인공광원을개발하여왔다 (Kim 2010). 특히 LED 등은무수은으로환경친화적이고경량이며, 전력절감이탁월하고수명이길면서도구동회로가간단하다는것과함께특정광질을쉽게만들수있는장점이있다 (Hwang et al. 2004). 또한 LED 전등의발광파장을식물엽록소의흡수피크와거의일치시킬수있어광합성에유리하고, 낮은전압으로작동할수있다 (Kim 2010). 재배목적에따라맞춤식광질로특정한광질을이용할수있다는장점을이용하여엽채류재배에 LED 광원을이용하는사례가늘어나고있지만식물의발아와생장그리고기능성물질의함유량과의관계을규명하고이를조절해채소의품질을향상시키는조사기술에관한연구는아직미비하다 (An et al. 2011; Cho et al. 2008; Choi et al. 2014). 배추 (Chinese cabbage) 는국내에서소비량이가장많은채소이며, 제놈서열분석이완료 (Wang et al. 2011) 되어생리실험뿐아니라이를분자수준으로검정할수있는매우유용한작물이며모델식물인애기장대와의비교제놈연구를통해다양한관련유전자를동정할수있다 (Kim et al. 2012). 또한유채, 브로콜리, 갓, 등새싹채소로각광받고있는작물 (Lee et al. 2007) 과같은속 (Kim et al. 2012; Nagaharu 1935) 에속하여제놈의일부를공유하고있으므로배추연구를다른십자화과에적용하는것이가능하다. 또한배추는민간과한방에서화상및감기의치료, 갈증해소, 소화촉진등의효능을가지는것으로전해지고있으며, 근래에들어서는생리활성효과연구에대한관심이증가하고있다 (Cha et al. 2000). 특히십자화과채소 (Cruciferous vegatable) 의생리활성성분중하나인글루코시놀레이트 (glucosinolate) 는 2 차대사산물들중하나로서병충해나질병등에대한식물의생체방어반응 에관여한다 (Redovniković et al. 2008). 또한글루코시놀레이트는강력한항암작용을하며종자와새싹에다량함유되어있는것으로보고되어있다 (Lund et al. 2001; Faulkner et al. 1998). 본연구에서는 LED 의광질에반응하는배추초기유묘의글루코시놀레이트합성기작과연관된유전자발현변화를추적하였다. 청색광을배추유묘에조사하였을때 2 차대사산물생산에관여하는유전자들의발현이변화하였다는이전의연구 (Kim et al. 2013; Mun et al. 2014) 결과를적용하여청색광을보광함으로써배추유묘의품질을변화시킬수있는지알아보기위한실험을수행하였다. 유묘의재배에이용되는광환경은단일광보다는자연광이나형광등에다양한광질을보광 (Kim 2010) 하는경우가많다. 본실험에서는엽록소 (chlorophyll) 의두가지흡수파장인청색광과적색광을혼합한조건에서반응하는글루코시놀레이트합성유전자의발현변화를관찰하였다. 이러한연구는특정파장영역의선택이가능한 LED 를적절히이용할수있는조건을규명함으로써작물의수량증대와품질향상에광원을효과적으로이용할수있는기초자료가될수있으며, 다양한배추속작물의재배에도적용가능할것으로사료된다. 재료및방법 식물재료 배추추지부 (B.rapa ssp. pekinensis cv. Chiifu) hi 종자를 18 시간동안물에침지시킨후원예용상토에파종하고유묘의생장은형광등 ( 오슬람삼파장전구, FPL36EX-D, 오슬람코리아, 한국 ) 과 LED 전구가장착된생장상을이용하였다. 생장상의온도는 23 C 로유지하였고 16 시간동안은광을조사하고 8 시간동안은암상태가되도록시간을설정하여광을조사하였다. 글루코시놀레이트분석에이용된 Tsao Huang Pa (IT no. 221747) 와 BP79 (IT no. 221766) 아종은국립농업과학원유전자원센터에서분양받은종자를이용하였으며파종및생육은지부와같은방법으로수행하였다. 광처리 청색광에의해발현변화하는유전자의전사체분석을위한실험의광조사조건은형광등 (FL: Fluorescent) 과청색단독광 (B: Blue) 그리고형광등에청색광 (FLB: Fluorescent + Blue) 을혼합한세가지조건을이용하였다. 16 시간일장의형광등조건에서 4 일간자란유묘는 2 일간의암배양후각광조사조건에 24 시간동안둔후자엽을비롯한
J Plant Biotechnol (2015) 42:245 256 247 지상부를수확하였다. 각각의광도는 150 μmol m- 2 s- 1 로하였는데, 형광등과청색광혼합조건은각각의광도를 75 μmol m- 2 s- 1 로맞추어총광도가 150 μmol m- 2 s- 1 가되도록조절하였다. 다양한혼합광조건에의해변화하는배추유묘의글루코시놀레이트관련유전자발현분석실험에는형광등 (FL) 과형광등에적색광 (FLR: Fluorescent + Red), 녹색광 (FLG: Fluorescent + Green), 청색광 (FLB) 을섞은혼합광을이용하였으며각각의광도는 250 μmol m- 2 s- 1 로하였다. 광원이장착된생육상은광도와일장및온도를제어할수있는장비 (HB-302S, 한백과학, Korea) 를이용하였으며흡수및형광스펙트럼은각각 U-2900 와 F-4500 (Hitachi, Japan) 을이용하여측정하였다 (Fig. 1). 유묘는생육단계를달리하여광을조사하였는데 7 일묘와 14 일묘는각각처음 4 일, 11 일간 16 시간일장의형광등조건에서생육시키고 2 일간의암배양후 24 일간동안각각의광조사조건에두고유묘를수확하였다. 글루코시놀레이트분석을위한실험에이용된광조건은각각 FL, FLR, 그리고 FLB 로혼합광조건실험과동일하게키운 7 일유묘를수확하여분석하였다. 마이크로어레이분석 형광등 (FL: Fluorescent) 과청색단독광 (B: Blue) 그리고형광등에청색광 (FLB: Fluorescent + Blue) 을혼합한세가지조건에 24 시간노출된 지부 (B.rapa ssp. pekinensis cv. Chiifu)h 유묘는지상부를채취하였다. 각각의식물체를 2 개씩채취하여 2 반복실험을수행할수있도록액체질소에얼려막자사발을이용해마쇄하였고, RNeasy Plant Mini Kit (Qiagen, Germany) 를사용하여 RNA 를추출하였다. 추출한총 RNA 샘플은 NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., USA) 를이용하여 260 nm 와 280 nm 에서정량하였다. 총 RNA 로부터 cdna 를합성하기위해 RevertAid iderrma, ienti Strand cdna Synthesis Kit (Fermentas, Lithuania) 를사용하였다. MinElute Reaction Cleanup Kit (Qiagen, Germany) 를사용하여 cdna 를정제하고 1 μg 의 cdna 에 30 μl 의 Cy3-9mer primers (Sigma-Aldrich, USA) 를넣고 98 C 에서 10 분간열처리하여 Cy3 표지된 DNA 를합성하였다. 10 μl 의 50X dntp mix ( 각 10 mm) 와 2 μl 의 Klenow fragment (50U primers) (Sigma-Aldrich, USA) 를넣고 37 C 에서 2 시간동안반응시킨후, 표지된 DNA 를 isopropanol 로침전시켜 13 μl 의물로녹였다. Spectrophotometer 로농도를측정하여 10 mg 의 DNA 만을마이크로어레이혼성화에이용하였다. 각샘플에혼성화완충액 (NimbleGen, USA) 을넣고마이크로어레이와함께 42 C 에서 16-18 시간동안 MAUI chamber (Biomicro, USA) 내에서혼성화반응을진행하였다. Wash Ⅰ, Ⅱ, Ⅲ (NimbleGen, USA) 로세척하여남아있는 Cy3 를제거하고 건조시킨후, GenePix scanner 4000B (Axon, USA) 로 Cy3 신호를스캔하였다. 분석에사용된마이크로어레이는 NimbleGen Inc (http://www.nimblegen.com/) 에서제작되었으며혼성화반응과데이터분석은 GGBIO (http://www.ggbio. com) 에서수행되었다. 혼성화반응수행후 Nimblescan (NimbleGen, USA) 프로그램으로이미지신호를분석하였다. 데이터의정규화및분석은 cubic spline normalization 와 Robust Multi-Chip Analysis (RMA) 를이용하였다 (Irizarry et al. 2003; Workman et al. 2002). 다중분석 (Smyth 2004) 수행후 P Value 가 0.05 이하인값을취하고다른광조건에서의데이터와비교하여 2 배이상발현증가혹은 2 배이하발현감소한유전자만을선별하여 Acuity 3.1 (Axon Instruments) 로통계분석수행하였다. Arabidopsis thaliana TAIR9 (http://www.arabidopsis.org/) 를기반으로하여배추유전자와 match 된유전자수는총 18,725 개이며, GOMINER (Ashburner et al. 2000; Zeeberg et al. 2003) (http://www. geneontology.org, http://discover.nci.nih.gov/gominer/) 로유의한 GO (Gene ontology) term 을분석하였는데 GO term 과 expression mode (Under, Over, Change) 에따라각유전자를분류하였다. One-sided Fisher 의유의성검정방법으로 P Value 를계산하였고, 100 개를무작위로취해서 False discovery rate (FDR) 값이 0.05 미만인 GO term 을추출하였고, 이를생물학적과정 (biological processes), 분자기능 (molecular function), 그리고세포요소 (cellular component) 세가지로분류하였다. FL 조건과비교하여 FLB 에서발현이증가또는감소하는유전자에대해서는 AgriGO (Zhou et al. 2003; http://bioinfo.cau.edu.cn/agrigo/index.php) 를이용하여추가분석을수행하였다. 유전자발현분석 추출한총 RNA 5 μg 으로 cdna EcoDry Premix (Oligo dt) (Clontech Laboratories, Inc., Takara Bio company, USA) 를사용하여 cdna 를합성하였다. cdna 합성을위해서 42 C, 60 분반응후 70 C 에서 10 분간반응을정지시켰다. 일차합성된 cdna 를주형으로하여 primer 를이용하여 Prime Taq DNA pol. (GeNet Bio, Korea) 로 PCR 반응을수행하였다 (BIOER thermal cycler: Bioer Technology Co., Ltd., China). PCR 조건은 pre-denaturation 는 95 C 에서 5 분, 95 C 에서 30 초 denaturation, 프라이머의 GC content 에따라 58 C 또는 60 C 에서 30 초 annealing, 72 C 1 분의 extension 과정을 25 ~ 36 회반복하여진행한후 72 C 10 분간 final extension 후에종료하였다. 반응이끝난후 1% agarose gel (SeaKem GTG Agarose, Lonza, USA) 에전기영동하고 ethidium bromide (EtBr) 로염색하였다.
248 J Plant Biotechnol (2015) 42:245 256 프라이머제작 글루코시놀레이트합성유전자를특이적으로증폭하기위한프라이머는배추제놈 EST 데이터베이스와 NCBI 유전자정보를통해얻은염기서열을 primer3 program (http: //bioinfo.ut.ee/primer3-0.4.0) 을이용하여제작하였다 (Table 4). 유전자발현정량분석 전기영동을통해발현변화를확인한유전자의정량분석 (Quantitative PCR analysis) 을수행하였다. 3 반복으로진행하였으며 Forward primer/reverse primer 는각각 3 pmol, 50X ROX dye 는 0.4 μl, DEPC-distilled water 3.6 μl, 10 μl AccuPower 2X GreenStar qpcr Master Mix (Bioneer, Korea) 를첨가하여총반응부피를 20 μl 로하고, BIO-RAD Realtime thermal cycler (CFX) 를사용하여 PCR 반응을수행하였다. PCR 조건은 95 C 에서 10 분 pre-denaturation 시킨후, 95 C 10 초, 55 C 10 초, 72 C 30 초반응을 39 회반복하고 72 C 에서 10 초간반응시킨후종료하였다. 글루코시놀레이트함량분석 마쇄한시료 0.1 g 을 15 ml conical tube 에취하여 80 C 로 끓인 75% methanol 2 ml 을가한다음약하게혼합하여항온수조 (80 C) 에서 60 분추출후 2,000 rpm, 10 C, 10 분간원심분리하였다. 원심분리후상층액을회수하여 2 ml e-tube 에옮기고, 추출액전체를 DEAE column 에흘린후, sulfatase 200 μl 를처리한후밀봉하여상온조건에서하룻밤동안방치한후, column 에 3 차증류수 0.5 ml 로 3 회녹여 2 ml e-tube 에담은뒤분석전에 0.2 µm syringe filter 로여과하여분석하였다. Sephadex-A-25 powder 를적당량시약병에덜어 1 차증류수로 2 번세척및침전시키고약 10 분후에증류수를따라내고남은 slurry 에 0.1 M sodium acetate (ph 4.0) 100 ml 을혼합하였다. 24 시간경과후 ACQUITY Ultra performance liquid chromatography (UPLC) H-Class System (Waters, USA) 를이용하여 UPLC 분석을수행하였다. 결과및고찰 청색광혼합광조건에반응하는유전자발현변화 실험을수행한생장상의광조건은적, 청, 녹색광의균일한파장을조사할수있으며 (Fig. 1A), 적색광은 625 nm, 녹색광은 524 nm, 청색광은 455 nm 의파장을안정적으로 Fig. 1 Spectral distribution and intensity of LED chambers. LED bulbs in chambers irradiated sharp peaks of Blue, Green, and Red light (A). Each peak point at 455, 524, and 625nm. Fluorescent light (FL) was mixed half and half with each blue (B), green (C), and red (D) LED, respectively. Each color line reflected the light source bulb, blue, green, and red. Black line indicates fluorescent light. Sky-blue, light green, and pink indicate spectral distributions under mixed conditions with blue, green, and red, respectively
J Plant Biotechnol (2015) 42:245 256 249 구현할수있고, 형광등을조사하면서적, 청, 녹색광을동시에조사할수있는장비를이용하였다 (Fig. 1B, C, and D). 4 일간동일한형광등조건에서자란유묘는광조건에서유도되었던물질변화및유전자발현효과를배재하고자 (Huseby et al. 2013) 2 일간암배양한후형광등조건 (FL: Fluorescent lamp) 과형광등에청색광을혼합한조건 (FLB: Fluorescent lamp +Blue), 그리고청색단일광 (B: Blue) 조건에 24 시간동안노출시켜총 7 일된유묘의마이크로어레이분석을수행하였다. 형광등조건을기준으로형광등과청색광을혼합하여처리한 ( 청색혼합광 ) 조건과청색단일광조건에서의유전자발현분석데이터를계층군집화하여분석한결과, 청색단일광을조사한경우형광등조건과비교해유전자발현의변화가매우크게나타났으나청색혼합광조건에서는발현변화한유전자의수가크게줄었다 (Fig. 2). 청색단일광조건에서발현이 2 배이상증가한유전자수는배추제놈유전자 4,553 개중 1,513 개이고발현이억제된유전자의수는 1,789 개였으나형광등에청색광을혼합하여조사한유묘내에서발현이증가한유전자의수는 12 개, 억제된유전자는 10 개로줄었다 (Table 1). GO 분석을수행한결과, 청색단일광조건에서는 1,295 개와 1,598 개의유전자들이각각발현증가및감소하였고혼합광조건에서는각각 11 개와 8 개의유전자가각각발현증가및감소하였다 (Table 2). 형광등과청색광혼합조건에서발현변화를보인유전자들의 GO term 을분석한후 FDR 값이 0.05 이하인 GO term 을추출한결과십자화과에다량함유되어있다고알려진 (Kim 2004) 글루코시놀레이트합성기작에관여하는유전자가 19 개유전자중 3 개있음을확인하였다 (Table 3). 마이크로어레이데이터베이스의염기서열번호 (SEC ID) 로 Bra026058, Bra029355, Bra034180 유전자는각각글루코시놀레이트합성에관여하는유전자 AT1G16410 (CYP79F1), AT5G23010 (MAM1), AT4G03050 (AOP3) 유전자로확인되었다 (Table 4). 이들유전자는청색단일광에서는발현이감소되었으나 ( 각각 -3.3, -5.3, -6.4 배 ) 청색과형광등혼합조건에서는각각 4.4, 4.6, 3.6 배발현이증가하였다 ( 그림 3A). 식물은기질의산화를저해하거나지연시켜노화를방지하는항산화제 (Halliwell 1997) 나암의발생을억제하는항암물질의천연자원이다 (Osawa et al. 1992). 이들식물화학물질들 (phytochemicals) Table 1 Light regulated Gene expression changes: Summary showed up-regulated and down-regulated gene expression, beyond twofold, by each light condition (P Value < 0.05). Expressed genes in 7 day old seedlings of B. rapa, after 24 hours of light condition treatment, (continuous fluorescent light (FL), blue (B), and fluorescent + blue light (FLB)) for 24 hours were compared to fluorescent light (FL) Sample set Up regulation Down regulation Total significant genes B/FL z 1,513 1,789 3,302 FLB/FL y 12 10 22 z blue light versus fluorescent light(fl). y blue light + fluorescent light versus fluorescent light(fl). Fig. 2 Overview of light-regulated genome expression by cluster display: Hierarchical clustering analysis was performed with genes up or down regulated twofold above by light condition in each light quality. Genes with a P Value below 0.05 were collected. For hierarchical clustering analysis, we used average log2 ratios. The color scales are shown below. Positive numbers represent gene up-regulation (red color) and negative numbers represent gene down-regulation (green color). Expressed genes in 7 day old seedlings of B. rapa, after 24 hour treatment with light conditions of: fluorescent light (FL), fluorescent + blue light (FLB) and blue light alone (B) were compared to fluorescent light (FL). FL/FL, continuous fluorescent light versus fluorescent light; FLB/FL, continuous fluorescent + blue light versus fluorescent light; B/FL, continuous blue light versus fluorescent light Table 2 GO analysis of genes significantly regulated by light conditions. The number of B. rapa genes which have orthologs in Arabidopsis TAIR9 were calculated. With a False Discovery Rate < 0.05, Gominer categorized each gene according to mode of gene expression, either up- or down- regulated Sample set Up regulation Down regulation Total significant genes B/FL z 1,295 1,598 2,893 FLB/FL y 11 8 19 z blue light versus fluorescent light (FL). y blue light + fluorescent light versus fluorescent light (FL).
250 J Plant Biotechnol (2015) 42:245 256 Fig. 3 Gene expression in B. rapa seedlings under blue light mixed conditions. We screened the gene change under Fluorescent + blue light (FLB) conditions compared with Fluorescent light (FL) conditions. Genes changes in response to FLB/FL are shown in the graph with those in response to B/FL (blue light (B) compared with fluorescent light (FL)) (A). White and black indicate FLB/FL and B/FL, respectively. Error bars were calculated by two replicates in microarray data. Hierarchical clustering analysis was performed with genes up or down regulated by both light conditions, FLB and B (B). Genes with a P Value below 0.05 were collected. For hierarchical clustering analysis, we used average log2 ratios. The color scales are shown at the top. Positive numbers represent up-regulation (red color) and negative numbers represent down-regulation (green color). We clustered the data using KMC analysis of MeV4.8 (MultiExperiment Viewer : http://www.tm4.org/mev.html) program Table 3 GO term enrichment analysis of genes regulated by light conditions in each light combination. GO term enrichments were calculated with AgriGO ( Zhou et al., 2003; http://bioinfo.cau.edu.cn/agrigo/index.php). The 37,767 genes were used as total gene set in the AgriGO web-site for Arabidopsis. Analysis was performed to compare FL to FLB. AgriGO categorized each gene according to their GO terms, biological processes, cellular components, and molecular functions. We submitted 19 genes that were up-or down-regulated by FLB conditions compared to FL and collected GO terms with FDR less than 0.05 GO term Ontology Description Number in input list P Value FDR GO:0019758 P glycosinolate biosynthetic process 3 1.40E-06 5.40E-05 GO:0016144 P S-glycoside biosynthetic process 3 1.40E-06 5.40E-05 GO:0019761 P glucosinolate biosynthetic process 3 1.40E-06 5.40E-05 GO:0019757 P glycosinolate metabolic process 3 4.60E-06 8.80E-05 GO:0019760 P glucosinolate metabolic process 3 4.60E-06 8.80E-05 GO:0016143 P S-glycoside metabolic process 3 4.60E-06 8.80E-05 GO:0016138 P glycoside biosynthetic process 3 9.30E-06 0.00015 GO:0016137 P glycoside metabolic process 3 2.10E-05 0.0003 GO:0044272 P sulfur compound biosynthetic process 3 2.80E-05 0.00035 GO:0034637 P cellular carbohydrate biosynthetic process 3 9.70E-05 0.0011 GO:0006790 P sulfur metabolic process 3 0.00018 0.0019 GO:0016051 P carbohydrate biosynthetic process 4 0.00036 0.0034 GO:0005975 P carbohydrate metabolic process 3 0.00082 0.0073 GO:0044262 P cellular carbohydrate metabolic process 3 0.0012 0.0095 GO:0019748 P secondary metabolic process 3 0.0018 0.014
J Plant Biotechnol (2015) 42:245 256 251 Table 4 List of genes used in RT-PCR analysis. Putative genes related to the glucosinolate pathway were screened in the B. rapa gene chip database of GGBio (http://www.ggbio.com/) SEQ_ID Bra026058 Bra029355 A.thaliana homologue AT1G16410 AT5G23010 Annotation CYP79F1, BUS1, SPS1; CYP79F1 (CYTOCHROME P450 79F1); oxidoreductase, acting on paired donors, with incorporation or reduction of molecular oxygen, NADH or NADPH as one donor, and incorporation of one atom of oxygen MAM1, IMS3; MAM1 (METHYLTHIOALKYLMALATE SYNTHASE 1); 2-isopropylmalate synthase/ methylthioalkylmalate synthase Bra022448 AT3G19710 BCAT4; branched-chain aminotransferase4 Bra024634 AT1G24100 UGT74B1; UDP-glucosyl transferase 74B1 Bra015379 AT1G05000 STSa; Phosphotyrosine protein phosphatases superfamily Bra021429 AT3G02580 FMGSOX1/ STE1, DWF7, BUL1; sterol 1 Bra034180 AT4G03050 AOP3; 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein Forward primer 5-3 Reverse primer 5-3 5 -TCGTCAAGATACCACCCTCG-3 5 -ACGACACGAAACGCAATTCA-3 5 -CGAACAAGCTCCCAGACAAG-3 5 -GGCGATGGTTTGGATGGTTT-3 5 -CCGGGGAGAGGAACATTGTA-3 5 -CACTTGGAAAAGCACGACGA-3 5 -CGAATCCTTCAAGCTCCACG-3 5 -GAAGGACAGAGCAAACGGTG-3 5 -CTGACAAGTTCACCACCACG-3 5 -TTAGCCGGATCAGGGAATCC-3 5 -CCGCATTTCCTCCAGACATG-3 5 -AGCAGAGTGTACCAAGGCAT-3 5 -AACTGCTACGCCCTGATTGT-3 5 -TCATCAGTCGCAAACGGTAA-3 의조성은식물의품종과재배동안의다양한환경요인에따라달라지는데그중광은매우중요한요인중하나이다 (Kopsell and Kopsell 2008). 적상추의이식후초기유묘의활착동안청색광을조사하게되면생장이좋아지고카로티노이드와같은항산화물질이증가한다 (Johkan et al. 2010). 또한 Lee et al. (2010) 은보리잎추출물의 ABTS (diammonium salts) radical-scavenging 활성이청색광조사조건에서가장높았음을확인하여항산화제의합성에청색광이유리한조건임을증명하였다. 본실험에서청색광단독조건은글루코시놀레이트합성유전자의발현을억제하였으나식물의재배에널리이용되고있는형광등에청색광을혼합한조건은합성기작에관여하는유전자의발현을유도할수있음을확인하였다. 다양한혼합광조건과글루코시놀레이트합성유전자의발현변화 마이크로어레이데이터를분석하여청색광혼합조건에서발현이현저히증가하는것으로확인된 Bra026058, Bra029355, Bra034180 외에서도글루코시놀레이트합성유전자의배추제놈내이종상동유전자를마이이크로어레이데이터베이스에서추출하였다 (GGBio). Bra024634 (At1g24100; UGT74B1), Bra022448 (At3g19710; BCAT4), Bra015379 (At1g74100; ST5a) 및 Bra021429 (AT1G65860; FMO GS-OX1) 4 개유전자를더선발하여다양한혼합광조건에서의발현변화를함께살펴보았다. 광조건은형광등과형광등에 LED 전구로적색 (FLR), 녹색 (FLG), 청색 (FLB) 을각각혼합하여처리하였고, 또한광질의효과가유묘의생장단계에따라달라지는지의여부를조사하기위해각각 4 일과 11 일동안동일한형광등조건에서자란유묘를 2 일간암배양하여 4 가지광조건에 24 시간노출시킨후유묘의지상부내유전자발현량을분석하였다. 마이크로어레이분석을통해형광등과청색혼합광조건에서발현이 4.4 배증가한것으로확인되었던 Bra026058 는 7 일유묘에서는 FL 조건과비교하여발현량이 38 배나증가함을보였는데이는 FLR 에서 4.8 및 FLG 에서 7.8 배발현이증가한것과비교하여현저히큰차이를보였다. 그러나 14 일된유묘에서는광조건간에유전자발현차이를보이지않았다 (Fig. 4 and Fig. 5A). 또한 Bra029355 와 Bra034180 두유전자는 7 일유묘에서 FLB 조건에서의발현량보다 FLR 조건에서발현량이더많았다 (Fig. 4, Figs. 5B and 5C). 특히 Bra029355 는 FL 조건보다 6 배, FLB 조건과비교하여 4 배의발현증가를보였다. 데이터베이스에서따로선발하였던 Bra022448 과 Bra024634 는각각 7 일과 14 일유묘에서 6.5 배, 1.2 배 FLR 조건에반응하여발현량이증가하였으며 (Fig. 4, Fig. 5D and 5E), Bra015379 와
252 J Plant Biotechnol (2015) 42:245 256 Fig. 4 RT-PCR analysis of genes expressed in Brassica rapa under different light conditions: Genes (listed in Table 4) were analyzed by reverse-transcript (RT) PCR. RT-PCR products for up-regulated genes in Brassica rapa under four light conditions: fluorescent light (FL), fluorescent + red light (FLR), fluorescent + green light (FLG), fluorescent + blue light (FLB) were loaded in 1% agarose gel and electrophoresis carried out Bra021429 는각각 7 일유묘에서 1.3 배과 14 일유묘에서 1.4 배 FLB 조건에반응하여발현량이더많았다 (Fig. 4, Figs. 5F and 5G). 애기장대 CYP79F 와 MAM1 유전자는암상태에서는발현이줄었다가광에노출이되면직접적으로발현이증가하는유전자이다 (Huseby et al. 2013). CYP79F1 와 MAM1 유전자의이종상동서열인 Bra026058 와 Bra029355 역시 2 일간의암배양후광에노출되면서발현이증가하였으며각각 FLB 와 FLR 조건에서발현이증가한것으로보아각각의유전자에영향을주는광질이서로다를수있음을보여주었다. 알리패틱글루코시놀레이트의초기단계에서사슬연장 (chain elongation) 의촉매효소인 MAM1 과산화를통해핵심구조 (core structure) 를형성하는 P450 효소중하나인 CYP79F1 유전자 (Zang et al. 2009) 의상동유전자가각각다른광조건에의해발현이유도되었다는본연구의결과는청색광과적색광을적절히보광하여배추유묘내글루코시놀레이트합성을촉진시킬수있음을보여주었다. 녹색광을혼합한조건인 FLG 에서는다른광조건과비교하여특이적인변화를보이지않았다. 이전의결과를보면고압나트륨램프 (HPS) 에 LED 광을보광하여비타민 C, 페놀화합물, 카로티노이드와같은항산화물질의합성을촉진함으로써새 Fig. 5 Quantitative PCR analysis of Brassica rapa genes expressed by different light conditions: Genes listed in Table 4 were quantitatively analyzed by real-time PCR. Quantitative RT-PCR of mrna regulated by four light conditions: fluorescent light (FL), fluorescent + red light (FLR), fluorescent + green light (FLG), and fluorescent + blue light (FLB). qrt-pcr was performed on: Bra026058 (A), Bra029355 (B), Bra034180 (C), Bra022448 (D), Bra024634 (E), Bra015379 (F), and Bra021429 (G) with three technical replications using the Bio-Rad system and the SYBR Green I master mix in a 20ul volume. White and black indicate 7 and 14 day old B. rapa seedlings, respectively. Error bars were calculated from three replicates of experiments
J Plant Biotechnol (2015) 42:245 256 253 싹의영양성분을향상시켰다 (Urbonavičiūtė et al. 2009). 또한적색과청색광을단독으로사용하는것보다혼합하여조사함으로써인도고무나무의체내전분함량을증가시켰으며, 청색광을단독으로사용하는것보다는적색광과의혼합을통해항산화물질생산을더촉진시킬수있었다 (Heo et al. 2010; Johkan et al. 2010). 광질은식물의생장과체내물질의조성을조절하는가장중요한환경신호이다 (Shiga et al. 2009). 특히청색광은식물체내플라보노이드와안토시아닌의축적을유도하며 (Ebisawa et al. 2008; Kojima et al. 2010) 비타민 C 와 E 의함량을증가시키는역할을하는것으로알려져있다 (Awad et al. 2001). 본연구에서는형광등에청색광과적색광을보광함으로써글루코시놀레이트합성에관여하는유전자들의발현이다양하게변화하는것을관찰하였다. 이러한결과는실제재배에청색광과적색광을적절히혼용해보광하거나생육시기별로광질을재배함으로써배추유묘내글루코시놀레이트를비롯한유용한영양성분을효율적으로축적시킬수있는데응용할수있을것이다. 또한식물의생육시기나대사물의합성기작에따라보광의효과를높일수있는광질이다를수있음을시사하였으며글루코시놀레이트의합성단계및생육시기에맞춰물질의생산효율을더높일수있는광질조합을구체적으로구현할수있는실험이필요할것으로사료된다. 글루코시놀레이트함량분석 광질의효과가배추유묘의글루코시놀레이트함량에도실제로영향을주지는알아보기위해형광등, 형광등과 적색광혼합, 형광등과청색광혼합조건에서생육한 7 일유묘를수확하여 UPLC 분석을수행하였다. 분석에사용한스탠다드글루코시놀레이는프로고이트린 (progoitrin:pro), 글루코라파닌 (glucoraphanin: rapha), 글루코나핀 (gluconapin: napin), 글루코브라시카나핀 (glucobrassicanapin: canapin) 글루코브라시신 (glucobrassicin: brass), 글루코나스투르틴 (gluconasturtin: nastur) 총 6 가지이고이중글루코브라시신은인돌글루토시놀레이트 (indolic glucosinolate) 이고, 다른 5 종은모두알리패틱글루코시놀레이트 (aliphatic glucosinolate) 에속한다. 글루코시놀레이트합성기작에관여하는유전자발현실험에이용되었던지부품종은글루코시놀레이트함량이비교적적은품종으로, 본실험의결과에서프로고이트린의대사량이청색혼합광조건에서증가하였고글루코나핀의경우적색혼합광에서증가한듯보이나큰변화를관찰할수없었다 (Fig. 6A). 또한글루코시놀레이트함량이적은품종으로알려진 Tsao Huang Pa 역시글루코나핀은적색혼합광에서약간증가하였지만다른종류의글루코시놀레이트는광질과무관하게생산되었다 (Fig. 6B). 상대적으로글루코시놀레이트함량이많은것으로알려진 BP79 배추의경우프로고이트린을비롯하여분석에사용된 6 종의글루코시놀레이트가모두청색혼합광에서증가하였다. 특히발암물질을억제하는아이소싸이오사이아네이트 (isothiocyanate) 의전구체로알려진글루코나핀 (Padilla et al. 2007) 의경우적색혼합광조건과비교하여청색혼합광조건에서함량이 1.2 배증가하였다. 또한광범위한암억제효과를가지며식물의방어기작에도관여 (Brew et al. 2009; Pedras et al. 2006) 하는것으로알려진글루코브라시신은 0.019 μm/ Fig. 6 Glucosinolate contents in three Chinese cabbage varieties: Tsao Huang Pa, Chiifu, and BP79. The seedlings were grown for 7 days. After they were cultured for four days under fluorescent light and 2 days in the dark, they were treated for 24 hrs under three light conditions. The white, gray, and black bars indicate fluorescent light (FL), fluorescent + red light (FLR), and fluorescent + blue light (FLB), respectively. The names of glucosinolate trivial were written by abbreviation: pro, progoitrin; rapha, glucoraphanin; napin, gluconapin; canapin, glucobrassicanapin; brass, glucobrassicin; nastur, gluconasturtin
254 J Plant Biotechnol (2015) 42:245 256 gdw 였던형광등조건에서보다적색혼합광과청색혼합광조건에서함량이각각 0.03 과 0.055 μm/gdw 로 2 배와 3 배증가하였다 (Fig. 6) 본실험은배추유묘의초기생육동안 2 차대사산물합성에영향을주는것으로알려진청색광과적생광을형광등에혼합한조건이배추의유용성분인글루코시놀레이트대사에영향을줄수있음을유전자발현과물질을분석을통해증명하였다. 온실이나식물공장에서주, 조보광원으로이용이늘고있는 LED 광원은다양한파장의광을선택적으로이용할수있다. 이러한 LED 의특성을이용하여식물의생육단계와목적하는대사물의생산시기에맞춰관련유전자들의발현을향상시킬수있는광질과조사시기및조사량에대한실험은광원을효율적으로이용하고작물의기능성을향상시키는시설조건을마련하는데기초자료를제공할수있다. 또한김치의원료로만이용되었던배추유묘의글루코시놀레이트의함량을조절하여기능성식품소재로서이용범위를넓힐수있는가능성을제시하였다. 글루코시놀레이트생합성은광에의해조절되는황산동화 (sulphate assimilation) 작용의영향을받으므로밤보다는낮시간에대사산물의량이많으며 (Huseby et al. 2013), 황산동화에관여하는유전자와함께글루코시놀레이트합성유전자들이광에의해조절을받는다는보고가있다. 광질과함께일일단위의광조사주기와각대사단계별영향및대사산물의량에관한상관관계에대한연구역시필요하며이러한연구는인공광을이용하여유묘를생육하는시설에서광원을효율적으로이용하면서기능적으로우수한작물을생산하는조건을조성하는데유용한기초자료가될것으로사료된다. 사사 본연구는농촌진흥청국립농업과학원연구과제 ( 과제번호 : PJ01002502) 와차세대바이오그린 21 사업 (PJ01106902) 의지원에의해수행되었음. 적요 농업에서의 LED 는단지광을보충해주는역할로만이용되는것뿐아니라, 시설재배시스템에있어매우중요한주광원이다. 인공광을이용한시설재배에서주광인형광등에다양한파장의광질을혼합하여식물의생육및대사를증진시키는시도가있어왔다. 주광인형광등에청색광을혼합한조건과청색단독광조건을배추유묘에조사한후마이크로어레이분석을수행한결과배추속 작물의기능성물질로주목을받고있는글루코시놀레이트합성에관여하는일부유전자들중청색혼합광조건에서 3.6-4.6 배는증가하는유전자 3 개를찾을수있었다. 글루코시놀레이트합성유전자를배추데이터베이스에서더선발하여적, 녹, 청색광을형광등에각각혼합한조건에반응하여발현이변하하는지관찰하였는데청색광혼합조건뿐아니라적색광혼합조건에서도발현이증가하는유전자들이있었다. 각각애기장대 CYP79F1, ST5a, FMOGS-OX1 와이종상동유전자인 Bra026058, Bra015379 와 Bra021429 는청색광조건에서발현량이증가하였으나 MAM1, AOP3, UGT74B1, BCAT4 의이종상동유전자인 Bra029355, Bra034180, Bra024634, Bra022448 은청색광보다적색혼합광조건에서발현이증가하였다. 혼합광처리에의한글루코시놀레이트합성의효과는배추품종에따라차이를보였으나몇가지글루코시놀레이트가청색광을혼합한조건에서합성이증가하였다. 이러한결과는유묘의생육시설에서기능적으로우수한작물을생산하는인공광조건을조성하는데유용한기초자료가될것으로사료된다. References An CG, Hwang YH, An JU, Yoon HS, Chang YH, Shon GM, Hwang SJ (2011) Effect of LEDs (Light Emitting Diodes) irradiation on growth of paprika (Capsicum annuum Cupra ). J Bio-Env Cont 20:253-257 Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25:25-29 Awad MA, Wagenmakers PS, Jager AD (2001) Effects of light on flavonoid and chlorogenic acid levels in the skin of Jonagold apples. Sci Hort 88:289-298 Brew CT, Aronchik I, Kosco K, McCammon J, Bjeldanes LF, Firestone GL (2009) Indole-3-carbinol inhibits MDA-MB- 231 breast cancer cell motility and induces stress fibers and focal adhesion formation by activation of Rho kinase activity. Int J Cancer 124:2294-2302 Butler WL, Siegelman HW, Miller CO (1964) Denaturation of phytochrome. Biochem 3:851-857 Cha YS, Oh SH (2000) Investigation of gamma-aminobutyric acid in Chinese cabbage and effects of the cabbage diets on lipid metabolism and liver function of rats administered with ethanol. J Korean Soc Food Sci Nutr 29:500-505 Cho JA, Son DM, Kim JM, Seo BS, Yan SY, Kim BW, Heo BG (2008) Effects of LEDs on the germination, growth and physiological activities of amaranth sprouts. Kor J Hort Sci Technol 26:106-112
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