Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (7): 1392-1404.doi: 10.3864/j.issn.0578-1752.2015.07.14

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Expression Analyses of R2R3-MYB Genes Related to Anthocyanin Biosynthesis in Rose

ZHAO Jia, LIU Rong, YANG Fan, LI Xin, LIU Hou-sheng, YAN Qian, XIAO Yue-hua   

  1. Biotechnology Research Center, Southwest University/Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops, Chongqing 400716
  • Received:2014-08-26 Online:2015-04-01 Published:2015-04-01

RichHTML

3

PDF

1075

Abstract: 【Objective】Anthocyanin is a major factor conferring pink or red to rose flower. R2R3-MYB proteins are key transcription factors controlling anthocyanin biosynthesis. This work aimed to clone homologous genes of anthocyanin-related R2R3-MYB protein from rose flowers, and further to analyze their relatedness to anthocyanin biosynthesis and coloration in rose petals, which may lay a foundation for gene engineering improvement of flower.【Method】With a degenerate primer designed according to the conserved sequence of anthocyanin-related R2R3-MYB proteins from various plants, complete coding sequences of corresponding homologous genes were amplified and cloned by using 3′-RACE and Y-RACE methods. Multiple sequence alignment and phylogenetic analysis were performed using typical sequences from the 4th and 6th subfamily of plant R2R3-MYB proteins (Sg4 and Sg6) and Arabidopsis R2R3-MYB proteins related to secondary metabolism with deduced proteins of the cloned genes. To elucidate the relatedness of the cloned genes to anthocyanin biosynthesis and coloration in rose petals, anthocyanin contents and expression levels of the two cloned R2R3-MYB protein genes were determined in rose petals of different colors. 【Result】Two R2R3-MYB protein genes (RhMYBs4-1 and RhMYBs6-1, GenBank accession Nos, KJ664810 and KJ664811, respectively) were cloned from red petals of rose ‘Red Success’. Sequence analyses indicated that RhMYBs4-1 and RhMYBs6-1, both conserved in the R2R3-MYB domain, were homologous to Sg4 and Sg6 R2R3-MYB proteins, respectively. RhMYBs4-1 contained the C1, C2 repressors and zinc finger domains typical of Sg4 R2R3-MYB proteins, whereas RhMYBs6-1 had the signature motifs ((A/S/G)NDV and KPRPR(T/S)) of Sg6 R2R3-MYB subfamily. Expression analyses showed that both RhMYBs4-1 and RhMYBs6-1 were expressed at a high level in the red petals of rose ‘Red Success’, but at low levels in leaves and stamens. Among rose petals of different colors, red petals had the highest anthocyanin contents, and the highest expression levels of both RhMYBs4-1 and RhMYBs6-1 genes. In pink rose petals, the anthocyanin content was less than 10% of that in red petals and the RhMYBs4-1 expression level was very low, whereas the expression level of RhMYBs6-1 in pink petals was comparable to that in red petals. 【Conclusion】Results of this study indicated that the RhMYBs4-1 and RhMYBs6-1 genes encoded Sg4 and Sg6 R2R3-MYB proteins, respectively. Both RhMYBs4-1 and RhMYBs6-1 were highly expressed in red petals and might be important regulators of anthocyanin biosynthesis and coloration in rose petals.

Key words: Rosa hybrida, petal color, anthocyanin, R2R3-MYB protein, gene expression

[1]    Buer C S, Imin N, Djordjevic M A. Flavonoids: new roles for old molecules. Journal of Integrative Plant Biology, 2010, 52: 98-111.
[2]    Katsumoto Y, Fukuchi-Mizutani M, Fukui Y, Brugliera F, Holton T A, Karan M, Nakamura N, Yonekura-Sakakibara K, Togami J, Pigeaire A, Tao G Q, Nehra N S, Lu C Y, Dyson B K, Tsuda S, Ashikari T, Kusumi T, Mason J G, Tanaka Y. Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. Plant and Cell Physiology, 2007, 48: 1589-1600.
[3]    Ogata J, Kanno Y, Itoh Y, Tsugawa H, Suzuki M. Plant biochemistry: anthocyanin biosynthesis in roses. Nature, 2005, 435: 757-758.
[4]    Koes R, Verweij W, Quattrocchio F. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends in Plant Science, 2005, 10: 236-242.
[5]    Bendahmane M, Dubois A, Raymond O, Bris M L. Genetics and genomics of flower initiation and development in roses. Journal of Experimental Botany, 2013, 64: 847-857.
[6]    Hichri I, Barrieu F, Bogs J, Kappel C, Delrot S, Lauvergeat V. Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. Journal of Experimental Botany, 2011, 62: 2465-2483.
[7]    Vimolmangkang S, Han Y, Wei G, Korban S. An apple MYB transcription factor, MdMYB3, is involved in regulation of anthocyanin biosynthesis and flower development. BMC Plant Biology, 2013, 13: 176.
[8]    Ben Zvi M M, Shklarman E, Masci T, Kalev H, Debener T, Shafir S, Ovadis M, Vainstein A. PAP1 transcription factor enhances production of phenylpropanoid and terpenoid scent compounds in rose flowers. The New Phytologist, 2012, 195: 335-345.
[9]    Mano H, Ogasawara F, Sato K, Higo H, Minobe Y. Isolation of a regulatory gene of anthocyanin biosynthesis in tuberous roots of purple-fleshed sweet potato. Plant Physiology, 2007, 143(3): 1252-1268.
[10]   Ravaglia D, Espley R, Henry-Kirk R, Andreotti C, Ziosi V, Hellens R, Costa G, Allan A. Transcriptional regulation of flavonoid biosynthesis in nectarine (Prunus persica) by a set of R2R3 MYB transcription factors. BMC Plant Biology, 2013, 13: 68.
[11]   Li L, Ban Z J, Li X H, Wu M Y, Wang A L, Jiang Y Q, Jiang Y H. Differential expression of anthocyanin biosynthetic genes and transcription factor PcMYB10 in pears (Pyrus communis L.). Plos One, 2012, 7: e46070.
[12]   Butelli E, Licciardello C, Zhang Y, Liu J, Mackay S, Bailey P, Reforgiato-Recupero G, Martin C. Retrotransposons control fruit- specific, cold-dependent accumulation of anthocyanins in blood oranges. The Plant Cell, 2012, 24: 1242-1255.
[13]   Wei Y Z, Hu F C, Hu G B, Li X J, Huang X M, Wang H C. Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi Chinensis Sonn. Plos One, 2011, 6: e19455.
[14]   Yamagishi M, Shimoyamada Y, Nakatsuka T, Masuda K. Two R2R3-MYB genes, homologs of petunia AN2, regulate anthocyanin biosyntheses in flower tepals, tepal spots and leaves of asiatic hybrid lily. Plant and Cell Physiology, 2010, 51: 463-474.
[15]   Lin-Wang K, Bolitho K, Grafton K, Kortstee A, Karunairetnam S, McGhie T K, Espley R V, Hellens R P, Allan A C. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in rosaceae. BMC Plant Biology, 2010, 10: 50.
[16]   Wang L, Han W, Xie C, Hou J, Fang Q, Gu J, Wang P G, Cheng J. Comparing the acceptor promiscuity of a Rosa hybrida glucosyltransferase RhGT1 and an engineered microbial glucosyltransferase OleD (PSA) toward a small flavonoid library. Carbohydrate Research, 2013, 368: 73-77.
[17]   Tanaka Y, Fukui Y, Fukuchi-Mizutani M, Holton T A, Higgins E, Kusumi T. Molecular cloning and characterization of Rosa hybrida dihydroflavonol 4-reductase gene. Plant and Cell Physiology, 1995, 36: 1023-1031.
[18]   谢吉容, 熊运海, 程在全, 黄兴奇. 月季MYB 基因cDNA 全长克隆和表达分析. 中国农业科学, 2008, 41(12): 4173-4179.
Xie J R, Xiong Y H, Cheng Z Q, Huang X Q. The full length cDNA cloning of rose MYB gene and its expression analysis. Scientia Agricultura Sinica, 2008, 41(12): 4173-4179. (in Chinese)
[19]   Lu P, Zhang C, Liu J, Liu X, Jiang G, Jiang X, Khan M A, Wang L, Hong B, Gao J. RhHB1 mediates the antagonism of gibberellins to ABA and ethylene during rose (Rosa hybrida) petal senescence. The Plant Journal, 2014, 78(4): 578-590.
[20]   肖月华, 罗明, 方卫国, 罗克明, 侯磊, 罗小英, 裴炎. 利用Y-RACE法进行棉花胚珠cDNA末端快速扩增. 中国生物化学与分子生物学报, 2002, 18(6): 688-692.
Xiao Y H, Luo M, Fang W G, Luo K M, Hou L, Luo X Y, Pei Y. Rapid amplification of cDNA ends from cotton ovules by Y-RACE method. Chinese Journal of Biochemistry and Molecular Biology, 2002, 18(6): 688-692. (in Chinese)
[21]   Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution. 2011, 28: 2731-2739.
[22]   Sanderson M J. Confidence limits on phylogenies: the bootstrap revisited. Cladistics, 1989, 5: 113-129.
[23]   Albert N W, Davies K M, Lewis D H, Zhang H, Montefiori M, Brendolise C, Boase M R, Ngo H, Jameson P E, Schwinn K E. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots.The Plant Cell, 2014, 26(3): 962-980.
[24]   Fornalé S, Shi X, Chai C, Encina A, Irar S, Capellades M, Fuguet E, Torres J L, Rovira P, Puigdomènech P, Rigau J, Grotewold E, Gray J, Caparrós-Ruiz D. ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux. The Plant Journal, 2010, 64: 633-644.
[25]   Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C, Weisshaar B, Martin C. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. The EMBO Journal, 2000, 19: 6150-6161.
[26]   Aharoni A, De Vos C H, Wein M, Sun Z, Greco R, Kroon A, Mol J N, O'Connell A P. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. The Plant Journal, 2001, 28: 319-332.
[27]   Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors in Arabidopsis. Trends in Plant Science, 2010, 15: 573-581.
[28] Koning-Boucoiran C F, Gitonga V W, Yan Z, Dolstra O, van der Linden C G, van der Schoot J, Uenk G E, Verlinden K, Smulders M J, Krens F A, Maliepaard C. The mode of inheritance in tetraploid cut roses. Theoretical and Applied Genetics, 2012, 125: 591-607.
[1] ZHANG KeKun,CHEN KeQin,LI WanPing,QIAO HaoRong,ZHANG JunXia,LIU FengZhi,FANG YuLin,WANG HaiBo. Effects of Irrigation Amount on Berry Development and Aroma Components Accumulation of Shine Muscat Grape in Root-Restricted Cultivation [J]. Scientia Agricultura Sinica, 2023, 56(1): 129-143.
[2] GU LiDan,LIU Yang,LI FangXiang,CHENG WeiNing. Cloning of Small Heat Shock Protein Gene Hsp21.9 in Sitodiplosis mosellana and Its Expression Characteristics During Diapause and Under Temperature Stresses [J]. Scientia Agricultura Sinica, 2023, 56(1): 79-89.
[3] LAI ChunWang, ZHOU XiaoJuan, CHEN Yan, LIU MengYu, XUE XiaoDong, XIAO XueChen, LIN WenZhong, LAI ZhongXiong, LIN YuLing. Identification of Ethylene Synthesis Pathway Genes in Longan and Its Response to ACC Treatment [J]. Scientia Agricultura Sinica, 2022, 55(3): 558-574.
[4] SHU JingTing,SHAN YanJu,JI GaiGe,ZHANG Ming,TU YunJie,LIU YiFan,JU XiaoJun,SHENG ZhongWei,TANG YanFei,LI Hua,ZOU JianMin. Relationship Between Expression Levels of Guangxi Partridge Chicken m6A Methyltransferase Genes, Myofiber Types and Myogenic Differentiation [J]. Scientia Agricultura Sinica, 2022, 55(3): 589-601.
[5] CHEN TingTing, FU WeiMeng, YU Jing, FENG BaoHua, LI GuangYan, FU GuanFu, TAO LongXing. The Photosynthesis Characteristics of Colored Rice Leaves and Its Relation with Antioxidant Capacity and Anthocyanin Content [J]. Scientia Agricultura Sinica, 2022, 55(3): 467-478.
[6] GUO ShaoLei,XU JianLan,WANG XiaoJun,SU ZiWen,ZHANG BinBin,MA RuiJuan,YU MingLiang. Genome-Wide Identification and Expression Analysis of XTH Gene Family in Peach Fruit During Storage [J]. Scientia Agricultura Sinica, 2022, 55(23): 4702-4716.
[7] SUN BaoJuan,WANG Rui,SUN GuangWen,WANG YiKui,LI Tao,GONG Chao,HENG Zhou,YOU Qian,LI ZhiLiang. Transcriptome and Metabolome Integrated Analysis of Epistatic Genetics Effects on Eggplant Peel Color [J]. Scientia Agricultura Sinica, 2022, 55(20): 3997-4010.
[8] KANG Chen,ZHAO XueFang,LI YaDong,TIAN ZheJuan,WANG Peng,WU ZhiMing. Genome-Wide Identification and Analysis of CC-NBS-LRR Family in Response to Downy Mildew and Powdery Mildew in Cucumis sativus [J]. Scientia Agricultura Sinica, 2022, 55(19): 3751-3766.
[9] YuXia WEN,Jian ZHANG,Qin WANG,Jing WANG,YueHong PEI,ShaoRui TIAN,GuangJin FAN,XiaoZhou MA,XianChao SUN. Cloning, Expression and Anti-TMV Function Analysis of Nicotiana benthamiana NbMBF1c [J]. Scientia Agricultura Sinica, 2022, 55(18): 3543-3555.
[10] JIN MengJiao,LIU Bo,WANG KangKang,ZHANG GuangZhong,QIAN WanQiang,WAN FangHao. Light Energy Utilization and Response of Chlorophyll Synthesis Under Different Light Intensities in Mikania micrantha [J]. Scientia Agricultura Sinica, 2022, 55(12): 2347-2359.
[11] XU XianBin,GENG XiaoYue,LI Hui,SUN LiJuan,ZHENG Huan,TAO JianMin. Transcriptome Analysis of Genes Involved in ABA-Induced Anthocyanin Accumulation in Grape [J]. Scientia Agricultura Sinica, 2022, 55(1): 134-151.
[12] YUAN JingLi,ZHENG HongLi,LIANG XianLi,MEI Jun,YU DongLiang,SUN YuQiang,KE LiPing. Influence of Anthocyanin Biosynthesis on Leaf and Fiber Color of Gossypium hirsutum L. [J]. Scientia Agricultura Sinica, 2021, 54(9): 1846-1855.
[13] SHU JingTing,JI GaiGe,SHAN YanJu,ZHANG Ming,JU XiaoJun,LIU YiFan,TU YunJie,SHENG ZhongWei,TANG YanFei,JIANG HuaLian,ZOU JianMin. Expression Analysis of IGF1-PI3K-Akt-Dependent Pathway Genes in Skeletal Muscle and Liver Tissue of Yellow Feather Broilers [J]. Scientia Agricultura Sinica, 2021, 54(9): 2027-2038.
[14] ZHAO Ke,ZHENG Lin,DU MeiXia,LONG JunHong,HE YongRui,CHEN ShanChun,ZOU XiuPing. Response Characteristics of Plant SAR and Its Signaling Gene CsSABP2 to Huanglongbing Infection in Citrus [J]. Scientia Agricultura Sinica, 2021, 54(8): 1638-1652.
[15] ZHAO Le,YANG HaiLi,LI JiaLu,YANG YongHeng,ZHANG Rong,CHENG WenQiang,CHENG Lei,ZHAO YongJu. Expression Patterns of TETs and Programmed Cell Death Related Genes in Oviduct and Uterus of Early Pregnancy Goats [J]. Scientia Agricultura Sinica, 2021, 54(4): 845-854.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd