Molecular Characterization and Expression Profiles of Myrosinase Gene (RsMyr2) in Radish (Raphanus sativus L.)
PAN Yan1, XU Yuan-yuan1, ZHU Xian-wen2, LIU Zhe1, GONG Yi-qin1, XU Liang1, GONG Mao-yong1, and LIU Li-wang1
1、National Key Laboratory of Crop Genetics and Germplasm Enhancement/Engineering Research Center of Horticultural Crop Germplasm
Enhancement and Utilization, Ministry of Education/Key Laboratory of Horticultural Crop Biology and Genetic Improvement (East China),
Ministry of Agriculture/College of Horticulture, Nanjing Agricultural University, Nanjing 210095, P.R.China
2、Department of Plant Sciences, North Dakota State University, Fargo, ND 58105, USA
摘要 Myrosinase is a defense-related enzyme and is capable of hydrolyzing glucosinolates into a variety of compounds, some of which are toxic to pathogens and herbivores. Many studies revealed that a number of important vegetables or oil crops contain the myrosinase-glucosinolate system. However, the related promoter and genomic DNA sequences as well as expression profiles of myrosinase gene remain largely unexplored in radish (Raphanus sativus). In this study, the 2 798 bp genomic DNA sequence, designated as RsMyr2, was isolated and analyzed in radish. The RsMyr2 consisting of 12 exons and 11 introns reflected the common gene structure of myrosinases. Using the genomic DNA walking approach, the 5´-flanking region upstream of RsMyr2 with length of 1 711 bp was successfully isolated. PLACE and PlantCARE analyses revealed that this upstream region could be the promoter of RsMyr2, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and regulatory motifs responsive to defense and stresses. Furthermore, recombinant pET-RsMyr2 protein separated by SDS-PAGE was identified as myrosinase with mass spectrometry. Real-time PCR analysis showed differential expression profiles of RsMyr2 in leaf, stem and root at different developmental stages (e.g., higher expression in leaf at cotyledon stage and lower in flesh root at mature stage). Additionally, the RsMyr2 gene exhibited up-regulated expression when treated with abscisic acid (ABA), methyl jasmonate (MeJA) and hydrogen peroxide (H2O2), whereas it was down-regulated by wounding (WO) treatment. The findings indicated that the expression of RsMyr2 gene was differentially regulated by these stress treatments. These results could provide new insight into elucidating the molecular characterization and biological function of myrosinase in radish.
Abstract Myrosinase is a defense-related enzyme and is capable of hydrolyzing glucosinolates into a variety of compounds, some of which are toxic to pathogens and herbivores. Many studies revealed that a number of important vegetables or oil crops contain the myrosinase-glucosinolate system. However, the related promoter and genomic DNA sequences as well as expression profiles of myrosinase gene remain largely unexplored in radish (Raphanus sativus). In this study, the 2 798 bp genomic DNA sequence, designated as RsMyr2, was isolated and analyzed in radish. The RsMyr2 consisting of 12 exons and 11 introns reflected the common gene structure of myrosinases. Using the genomic DNA walking approach, the 5´-flanking region upstream of RsMyr2 with length of 1 711 bp was successfully isolated. PLACE and PlantCARE analyses revealed that this upstream region could be the promoter of RsMyr2, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and regulatory motifs responsive to defense and stresses. Furthermore, recombinant pET-RsMyr2 protein separated by SDS-PAGE was identified as myrosinase with mass spectrometry. Real-time PCR analysis showed differential expression profiles of RsMyr2 in leaf, stem and root at different developmental stages (e.g., higher expression in leaf at cotyledon stage and lower in flesh root at mature stage). Additionally, the RsMyr2 gene exhibited up-regulated expression when treated with abscisic acid (ABA), methyl jasmonate (MeJA) and hydrogen peroxide (H2O2), whereas it was down-regulated by wounding (WO) treatment. The findings indicated that the expression of RsMyr2 gene was differentially regulated by these stress treatments. These results could provide new insight into elucidating the molecular characterization and biological function of myrosinase in radish.
This work was partially supported by grants from the National Natural Science Foundation of China (31171956, 31372064) the National Key Technologies R&D Program of China (2012BAD02B01), the Key Technologies R&D Program of Jiangsu Province, China (BE2013429), the PAPD and JASTI [CX(12)2006].
PAN Yan1, XU Yuan-yuan1, ZHU Xian-wen2, LIU Zhe1, GONG Yi-qin1, XU Liang1, GONG Mao-yong1, and LIU Li-wang1.
2014.
Molecular Characterization and Expression Profiles of Myrosinase Gene (RsMyr2) in Radish (Raphanus sativus L.). Journal of Integrative Agriculture, 13(9): 1877-1888.
Alvarez S, He Y, Chen S. 2008. Comparative investigationsof the glucosinolate-myrosinase system in Arabidopsissuspension cells and hypocotyls. Plant and Cell Physiology,49, 324-333
Andersson D, Chakrabartyb R, Bejaia S, Zhangc J M, RaskdL, Meijer J. 2009. Myrosinases from root and leaves ofArabidopsis thaliana have different catalytic properties.Phytochemistry, 70, 1345-1354
Andréasson E, Jørgensen L B. 2003. Chapter four Localizationof plant myrosinases and glucosinolates. Phytochemistry,37, 79-99
Barth C, Jander G. 2006. Arabidopsis myrosinases TGG1 andTGG2 have redundant function in glucosinolate breakdownand insect defense. The Plant Journal, 46, 549-562
Chu Y F, Sun J, Wu X, Liu R H. 2002. Antioxidant andantiproliferative activities of common vegetables. Journalof Agricultural and Food Chemistry, 50, 6910-6916
Ediage E N, di Mavungu J D, Scippo M L, Schneider Y J,Larondelle Y, Callebaut A, Robbens J, van PeteghemC, de Saeger S. 2011. Screening, identification andquantification of glucosinolates in black radish (Raphanussativus L. niger) based dietary supplements using liquidchromatography coupled with a photodiode array andliquid chromatography-mass spectrometry. Journal ofChromatography (A), 1218, 4395-4405
Eriksson S, Ek B, Xue J, Rask L, Meijer. 2001. Identificationand characterization of soluble and insoluble myrosinaseisoenzymes in different organs of Sinapis alba. PhysiologiaPlantarum, 111, 353-364
Eulgem T, Somssich I E. 2007. Networks of WRKYtranscription factors in defense signaling. Current Opinionin Plant Biology, 10, 366-371
Eulgem T. 2005. Regulation of the Arabidopsis defencetranscriptome. Trends in Plant Science, 10, 71-78
Hara M, Fujii Y, Sasada Y, Kuboi T. 2000. cDNA cloning ofradish (Raphanus sativus) myrosinase and tissue-specificexpression in root. Plant and Cell Physiology, 41, 1102-1109
Hashem F A, Motawea H, El-Shabrawy A E, Shaker K, El-Sherbini S. 2012. Myrosinase hydrolysates of Brassicaoleraceae L. var. italica reduces the risk of colon cancer.Phytotherapy Research, 26, 743-747
Hopkins R J, van Dam N M, van Loon J A. 2009. Role of glucosinolates in insect-plant relationships andmultitrophic interactions. Annual Review Entomology,54, 57-83
Husebye H, Chadchawan S, Winge P, Thangstad O P, BonesA M. 2002. Guard cell and phloem idioblast-specificexpression of thioglucoside glucohydrolase 1 (myrosinase)in Arabidopsis. Journal of Plant Physiology, 128, 1180-1188
Jiang L N, Wang L J, Liu L W, Zhu X W, Zhai L L, Gong Y Q.2012. Development and characterization of cDNA librarybased novel EST-SSR marker in radish (Raphanus sativusL.). Scientia Horticulturae, 140, 164-172
Jost R, Altschmied L, Bloem E, Bogs J, Gershenzon J, HähnelU, Hänsch R, Hartmann T, Kopriva S, Kruse C, MendelR R, Papenbrock J, Reichelt M, Rennenberg H, SchnuqE, Schmidt A, Textor S, Tokuhisa J, Wachter A, WirtzM, et al. 2005. Expression profiling of metabolic genesin response to methyl jasmonate reveals regulation ofgenes of primary and secondary sulfur-related pathwaysin Arabidopsis thaliana. Photosynthesis Research, 86,491-508
Jun B K, Seo S G, Kim J S, Lee Y, Shin M R, Choi H S, YiB Y, Kim S H. 2011. Molecular cloning and expressionanalysis of Bro-GS-elong and Bro-myro from Brassicaoleracea. Genes & Genomics, 33, 299-305
Kim S T, Kim S G, Hwang D H, Kang S Y, Kim H J, Lee B H,Lee J J, Kang K Y. 2004. Proteomic analysis of pathogenresponsiveproteins from rice leaves induced by rice blastfungus, Magnaporthe grisea. Journal of Proteomics, 4,3569-3578
Kissen R, Rossiter J T, Bones A M. 2009. The ‘mustard oilbomb’: not so easy to assemble?! Localization, expressionand distribution of the components of the myrosinaseenzyme system. Phytochemistry Reviews, 8, 69-86
Koroleva O A, Davies A, Deeken R, Thorpe M R, Tomos A D,Hedrich R. 2000. Identification of a new glucosinolate-richcell type in Arabidopsis flower stalk. Plant Physiology,124, 599-608
Kuchernig J C, Backenköhler A, Lübbecke M, BurowM, Wittstock U. 2011. A thiocyanate-forming proteingenerates multiple products upon allylglucosinolatebreakdown in Thlaspi arvense. Phytochemistry, 72, 1699-1709
Kusnierczyk A, Winge P, Midelfart H, Armbruster WS, Rossiter J T, Bones A M. 2007. Transcriptionalresponses of Arabidopsis thaliana ecotypes with differentglucosinolate profiles after attack by polyphagous Myzuspersicae and oligophagous Brevicoryne brassicae. Journalof Experimental Botany, 58, 2537-2552
Li Q, Eigenbrode S D, Stringham G R, Thiagarajah M R. 2000.Feeding and growth of Plutella xylostella and Spodopteraeridania on Brassica juncea with varying glucosinolateconcentrations and myrosinase activities. Journal ofChemical Ecology, 26, 2401-2419
Li X, Kushad M M. 2005. Purification and characterization ofmyrosinase from horseradish (Armoracia rusticana) roots.Plant Physiology and Biochemistry, 43, 503-511
Li X, Wen M Z, Bohnert H J, Schuler M A, Kushad M M.2007. Myrosinase in horseradish (Armoracia rusticana)root: Isolation of a full-length cDNA and its heterologousexpression in Spodoptera frugiperda insect cells. PlantScience, 172, 1095-1102
Liu L, Guo W, Zhu X, Zhang T. 2003. Inheritance and finemapping of fertility-restoration for cytoplasmic malesterility in Gossypium hirsutum L. Theoretical and AppliedGenetics, 106, 461-469
Livak K J, Schmittgen T D. 2001. Analysis of relative geneexpression data using real-time quantitative PCR and the2-ΔΔCT method. Methods, 25, 402-408
Malik M S, Riley M B, Norsworthy J K, Bridges W J R. 2010.Variation of glucosinolates in wild radish (Raphanusraphanistrum) accessions. Journal of Agricultural andFood Chemistry, 58, 11626-11632
Marchler-Bauer A, Anderson J B, Chitsaz F, Derbyshire M K,deWeese-Scott C, Fong J H, Geer L Y, Geer R C, GonzalesN R. 2009. CDD: Specific functional annotation with theconserved domain database. Nucleic Acids Research, 37,D205-D210.Martin N, Müller C. 2007. Induction of plant responsesby a sequestering insect: Relationship of glucosinolateconcentration and myrosinase activity. Basic and AppliedEcology, 8, 13-25
McCully M E, Miller C, Sprague S J, Huang C H, KirkegaardJ A. 2008. Distribution of glucosinolates and sulphur-richcells in roots of field-grown canola (Brassica napus). NewPhytologist, 180, 193-205
Müller C, Sieling N. 2006. Effects of glucosinolate andmyrosinase levels in Brassica juncea on a glucosinolatesequestering herbivore and vice versa. Chemoecology,16, 191-201
Nitz I, Berkefeld H, Puzio P S, Grundler F M. 2001. Pyk10,a seedling and root specific gene and promoter fromArabidopsis thaliana. Plant Science, 161, 337-346
Plesch G, Ehrhardt T, Mueller-Roeber B. 2001. Involvementof TAAAG elements suggests a role for Dof transcriptionfactors in guard cell-specific gene expression. NuclearPlant Journal, 28, 455-464
Pontoppidan B, Hopkins R, Rask L, Meijer J. 2003. Infestationby cabbage aphid (Brevicoryne brassicae) on oilseedrape (Brassica napus) causes a long lasting induction ofthe myrosinase system. Entomologia Experimetalis EtApplicata, 109, 55-62
Pontoppidan B, Hopkins R, Rask L, Meijer J. 2005. Differentialwound induction of the myrosinase system in oilseedrape (Brassica napus): contrasting insect damage withmechanical damage. Plant Science, 168, 715-722
Rask L, Andréasson E, Ekbom B, Eriksson S, PontoppidanB, Meijer J. 2000. Myrosinase: Gene family evolutionand herbivore defense in Brassicaceae. Plant MolecularBiology, 42, 93-113
Reymond P, Weber H, Damond M, Farmer E E. 2000.Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell,12, 707-719
Sarosh B, Meijer J. 2007. Transcriptional profiling by cDNAAFLPreveals novel insights between methyl jasmonate,wounding and insect attack in Brassica napus. PlantMolecular Biology, 64, 425-438
Sooyeon L, Joonhee L, Jong-Kee K. 2009. Analysis ofisothiocyanates in newly generated vegetables, Baemuchae(×Brassicoraphanus) as affected by growth. InternationalJournal of Food Science and Technology, 44, 1401-1407
Stotz H U, Sawada Y, Shimada Y, Hirai M Y, Sasaki E,Krischke M, Brown P D, Saito K, Kamiya Y. 2011.Role of camalexin, indole glucosinolates, and side chainmodification of glucosinolate-derived isothiocyanates indefense of Arabidopsis against Sclerotinia sclerotiorum.The Plant Journal, 67, 81-93
Stranger B E, Mitchell-Olds T. 2005. Nucleotide variation atthe myrosinase-encoding locus, TGG1, and quantitativemyrosinase enzyme activity variation in Arabidopsisthaliana. Molecular Ecology, 14, 295-309
Textor S, Gershenzon J. 2009. Herbivore induction of theglucosinolate-myrosinase defense system: Major trends,biochemical bases and ecological significance. PhytochemReviews, 8, 149-170
Thangstad O P, Gilde B, Chadchawan S, Seem M, Husebye H,Bradley D, Bones A M. 2004. Cell specific, cross-speciesexpression of myrosinases in Brassica napus, Arabidopsisthaliana and Nicotiana tabacum. Plant Molecular Biology,54, 597-611
Toufighi K, Brady S M, Austin R, Ly E, Provart N J. 2005. TheBotany Array Resource: e-Northerns, Expression Angling,and promoter analyses. The Plant Journal, 43, 153-163
Travers-Martin N, Müller C. 2007. Specificity of inductionresponses in Sinapis alba L. and their effects on a specialistherbivore. Journal of Chemical Ecology, 33, 1582-1597
Tsujimoto-Inui Y, Naito Y, Sakurai N, Suzuki H, Sasaki R,Takahashi H, Ohtsuki N, Nakano T, Yanagisawa S, ShibataD, Uchimiya H, Shinshi H, Suzuki K. 2009. Functionalgenomics of the Dof transcription factor family genes insuspension-cultured cells of Arabidopsis thaliana. PlantBiotechnology Journal, 26, 15-28
Ueda H, Nishiyama C, Shimada T, Koumoto Y, HayashiY, Kondo M, Takahashi T, Ohtomo I, Nishimura M,Hara-Nishimura I. 2006. AtVAM3 is required for normalspecification of idioblasts, myrosin cells. Plant CellPhysiology, 47, 164-175
Wang H, Wu J, Sun S L, Liu B, Cheng F, Sun R F, Wang XW. 2011. Glucosinolate biosynthetic genes in Brassicarapa. Gene, 487, 135-142
Wang M, Li D, Sun X, Zhu Y J, Nong H, Zhang J. 2009a.Characterization of a root-specific β-thioglucosideglucohydrolase gene in Carica papaya and its recombinantprotein expressed in Pichia pastoris. Plant Science, 177,716-723
Wang S, Li D, Tan D, Gong S, Sun X, Meijer J, Zhang J.2009b. The two non-functional myrosinase genes TGG3and TGG6 in Arabidopsis are expressed predominantly inpollen. Plant Science, 177, 371-375
Wielanek M, Urbanek H. 2006. Ehanced glucotropaeolinproduction in hairy root cultures of Tropaeolum majus L.by combining elicitation and precursor feeding. Plant CellTissue and Organ Culture, 57, 39-45
Winde I, Wittstock U. 2011. Insect herbivore countersadaptations to the plant glucosinolate-myrosinase system.Phytochemistry, 72, 1566-1575
Xu L, Wang L G, Gong Y Q, Dai W H, Wang Y, Zhu XW, Wen T C, Liu L W. 2012a. Genetic linkage mapconstruction and QTL mapping of cadmium accumulationin radish (Raphanus sativus L.). Theoretical and AppliedGenetics, 125, 659-670
Xu Y Y, Zhu X W, Gong Y Q, Xu L, W Y, Liu L W. 2012b.Evaluation of reference genes for gene expressionstudies in radish (Raphanus stativs L.) using quantitativereal-time PCR. Biochemical and Biophysical ResearchCommunications, 424, 398-403
Yang G, Wang D F, Dong Z Q, Wang Q L, Pruski G W, YouM S. 2012. Characterization of a myrosinase cDNA fromBrassica parachinensis and its defense against Plutellaxylostella after suppression. Insect Science, 19, 461-471
Zhang J, Pontoppidan B, Xue J, Rask L, Meijer J. 2002. Thethird myrosinase gene TGG3 in Arabidopsis thaliana is apseudo-gene specifically expressed in stamen and petal.Physiologia Plantarum, 115, 25-34
Zhang Y Y, Xu L, Zhu X W, Gong Y Q, Xiang F, Sun X C,Liu L W. 2013. Proteomic analysis of heat stress responsein leaves of radish (Raphanus sativus L.). Plant MolecularBiology Repopter, 31, 195-203
Zhao Z, Zhang W, Stanley B A, Assmann S M. 2008.Functional proteomics of Arabidopsis thaliana guard cellsuncovers new stomatal signaling pathways. Plant Cell,20, 3210-3226
Zimmermann P, Hirsch-Hoffmann M, Hennig L, GruissemW. 2004. GENEVESTIGATOR. Arabidopsis microarraydatabase and analysis toolbox. Plant Physiology, 136,2621-2632
RONG Rui-juan, WU Peng-cheng, LAN Jin-ping, WEI Han-fu, WEI Jian, CHEN Hao, SHI Jia-nan, HAO Yu-jie, LIU Li-juan, DOU Shi-juan, LI Li-yun, WU Lin, LIU Si-qi, YIN Chang-cheng, LIU Guo-zhen. Western blot detection of PMI protein in transgenic rice[J]. >Journal of Integrative Agriculture, 2016, 15(4): 726-734.