月季中R2R3-MYB基因RhMYB113c调控花青素苷合成

doi:10.16420/j.issn.0513-353x.2021-0630

园艺学报 ›› 2022, Vol. 49 ›› Issue (9): 1957-1966.doi: 10.16420/j.issn.0513-353x.2021-0630

月季中R2R3-MYB基因RhMYB113c调控花青素苷合成

李茂福¹^,²^,³, 杨媛¹^,²^,⁴, 王华¹^,²^,³, 范又维¹^,²^,³, 孙佩¹^,²^,³, 金万梅¹^,²^,³^,^*()

¹北京市农林科学院林业果树研究所,北京100093
²农业农村部华北地区园艺作物生物学与种质创制重点实验室,北京100093
³北京市功能花卉工程技术研究中心,北京100093
⁴北京市落叶果树工程技术研究中心,北京100093

收稿日期:2021-07-02 修回日期:2021-09-10 出版日期:2022-09-25 发布日期:2022-10-08
通讯作者: 金万梅 E-mail:jwm0809@163.com
基金资助:
北京市农林科学院科技创新能力专项(KJCX20200205);北京市农林科学院科技创新能力专项(KJCX20200425);北京市农林科学院科技创新能力专项(KJCX20200204);北京市农林科学院林业果树研究所青年科研基金项目(LGYJJ202003)

Analysis the Function of R2R3 MYB Transcription Factor RhMYB113c on Regulating Anthocyanin Synthesis in Rosa hybrida

LI Maofu¹^,²^,³, YANG Yuan¹^,²^,⁴, WANG Hua¹^,²^,³, FAN Youwei¹^,²^,³, SUN Pei¹^,²^,³, JIN Wanmei¹^,²^,³^,^*()

¹Institute of Forestry and Pomology,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100093,China
²Key Laboratory of Biology and Genetic Improvement of Horticultural Crops（North China）,Ministry of Agriculture and Rural Affairs,Beijing 100093,China
³Beijing Engineering Research Center of Functional Floriculture,Beijing 100093,China
⁴Beijing Engineering Research Center for Deciduous Fruit Trees,Beijing 100093,China

Received:2021-07-02 Revised:2021-09-10 Online:2022-09-25 Published:2022-10-08
Contact: JIN Wanmei E-mail:jwm0809@163.com

摘要/Abstract

摘要：

以月季红色花品种‘卡罗拉’（Rosa hybrida‘Carola’）为材料,从其花瓣cDNA 中克隆到1个R2R3-MYB 基因,命名为RhMYB113c。其开放阅读框为699 bp,编码233个氨基酸。蛋白多重序列比对分析发现RhMYB113c含有R2和R3结构域。系统进化树分析表明,RhMYB113c与花青素苷合成激活因子AtMYB114、AtMYB113、AtMYB75、AtMYB90亲缘关系最近。组织特异性表达检测发现,RhMYB113c主要在‘卡罗拉’花瓣和花柱中表达,在白色花品种‘白缎’所有组织中均不表达。不同花发育时期的基因表达显示,RhMYB113c在‘卡罗拉’未着色花瓣中不表达,在盛花期花瓣中高表达,而在白色品种的每个时期均不表达。酵母双杂交和烟草瞬时表达试验证实RhMYB113c调控花青素苷的合成,从而影响月季红色花瓣着色。

关键词: 月季, 花瓣, RhMYB113c基因, 花青素苷合成

Abstract:

An R2R3 MYB gene,named RhMYB113c,was cloned from cDNA of the petal in Rosa hybrida‘Carola’. The open reading frame of RhMYB113c was 699 base pair,encoding 233 amino acids. The multiple sequence alignment analysis showed that RhMYB113c contained R2 and R3 domains. Phylogenetic tree results indicated that RhMYB113c was closely related to anthocyanin synthesis activator AtMYB114,AtMYB113,AtMYB75,and AtMYB90. Tissue specific expression showed that RhMYB113c was mainly expressed in petals and styles in‘Carola’,but not in all tissues of white flower variety ‘White Satin’. Gene expression at different flower development stages indicated that RhMYB113c was not expressed in‘Carola’uncolored petals,highly expressed in full flowering stage,but not in all stage of white flower varieties‘White Satin’. Yeast two hybrid and tobacco transient expression experiments suggested that RhMYB113c may regulate anthocyanin synthesis and determine the color of rose petals.

Key words: Rosa, petal, RhMYB113c, anthocyanin synthesis

中图分类号:

S685.12

李茂福, 杨媛, 王华, 范又维, 孙佩, 金万梅. 月季中R2R3-MYB基因RhMYB113c调控花青素苷合成[J]. 园艺学报, 2022, 49(9): 1957-1966.

LI Maofu, YANG Yuan, WANG Hua, FAN Youwei, SUN Pei, JIN Wanmei. Analysis the Function of R2R3 MYB Transcription Factor RhMYB113c on Regulating Anthocyanin Synthesis in Rosa hybrida[J]. Acta Horticulturae Sinica, 2022, 49(9): 1957-1966.

图/表 7

表1 本研究中所用引物序列

Table 1 Primers sequence used in this study

目的 Purpose	引物名称 Primer name	引物序列（5′-3′） Primer sequences
基因克隆 Gene clone	RhMYB113c	F：CAACGTCATTAACTGTGGGATC R：GCCGTGAGCAGTGGCTTTC
瞬时转化 Transient expression	RhMYB113c	F：GAAATTtctagaATGGAGGACCAGTCGGGTTTGAG R：GAAATTgagctcTCATTATCGATCTAAGAATGTCATCC
瞬时转化 Transient expression	RcbHLH	F：GAAATTtcgcgaATGGCTACACCGCCACCGAGTAGTAGC R：GAAATTgagctcTCATTATCGATCTAAGAATGTCATCC
酵母双杂交 Yeast two-hybrid assay	RhMYB113c:AD,RhMYB113c:BD	F：GAAATTcatatgATGGAGGACCAGTCGGGTTTGAG R：GAAATTgaattcTCATTATCGATCTAAGAATGTCATCC
实时荧光定量PCR RT-qPCR	RhMYB113c	F：ACCAAGCGGCGTCGGGACAAAC R：CCCGTCAAACAGAGTGAACTGGTCG
实时荧光定量PCR RT-qPCR	Actin	F：GGCTGTTCTCTCTCTGTATGC R：TTCTGGGCACCTGAATCTC

图1 月季‘卡罗拉’和‘白缎’盛花期花瓣花青素含量

Fig. 1 Anthocyanin content in petals of‘Carola’and‘White Satin’at full bloom

图2 RhMYB113c在叶片、茎、花柱和花瓣中的组织表达

Fig. 2 The RhMYB113c gene expression pattern analysis in leaf,stem,style,and petal

图3 RhMYB113c 在月季‘卡罗拉’和‘白缎’蕾期未着色花瓣和盛开花瓣的表达分析

Fig. 3 The RhMYB113c gene expression in uncolored petal of‘Carola’and‘White Satin’at bud stage and petals at full bloom

图4 月季RhMYB113c与其他MYB蛋白序列系统进化树

Fig. 4 Phylogenetic tree of amino acid sequence of RhMYB113c and MYB of other plants

图5 RhMYB113c与RcbHLH、RcWD40之间的互作分析

Fig. 5 The yeast two-hybrid tests assay for the interaction of RhMYB113c with RcWD40 and RcbHLH

图6 过表达RhMYB113c烟草叶片表型及花青素苷含量（HT-V为空载体）

Fig. 6 The phenotype and anthocyanin content of overexpression of RhMYB113c in tobacco leaves（HT-V was empty vector）

参考文献 34

[1]	An J P, Wang X F, Zhang X W, Xu H F, Bi S Q, You C X, Hao Y J. 2020. An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnol Journal, 18 (2):337-353. doi: 10.1111/pbi.13201 URL
[2]	Cutanda-Perez M C, Ageorges A, Gomez C, Vialet S, Terrier N, Romieu C, Torregrosa L. 2009. Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport. Plant Molecular Biology, 69 (6):633-648. doi: 10.1007/s11103-008-9446-x pmid: 19096760
[3]	Espley R V, Hellens R P, Putterill J, Stevenson D E, Kutty-Amma S, Allan A C. 2007. Red colouration in apple fruit is due to the activity of the MYB transcription factor,MdMYB10. Plant Journal, 49 (3):414-427.
[4]	Gil-Muñoz F, Sánchez-Navarro J A, Besada C, Salvador A, Badenes M L, Naval M D M, Ríos G. 2020. MBW complexes impinge on anthocyanidin reductase gene regulation for proanthocyanidin biosynthesis in persimmon fruit. Science Reports, 10 (1):3543.
[5]	Gonzalez A, Zhao M, Leavitt J M, Lloyd A M. 2008. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant Journal, 53 (5):814-827. doi: 10.1111/j.1365-313X.2007.03373.x URL
[6]	Gu Z, Zhu J, Hao Q, Yuan Y W, Duan Y W, Men S, Wang Q, Hou Q, Liu Z A, Shu Q, Wang L. 2019. A novel R2R3-MYB transcription factor contributes to petal bblotch formation by regulating organ-specificexpression of PsCHS in tree peony (Paeonia suffruticosa). Plant Cell Physiology, 60 (3):599-611. doi: 10.1093/pcp/pcy232 URL
[7]	Han Y, Yu J, Zhao T, Cheng T, Wang J, Yang W, Pan H, Zhang Q. 2019. Dissecting the genome-wide evolution and function of R2R3-MYB transcription factor family in Rosa chinensis. Genes (Basel),doi:10.3390/genes10100823.
[8]	Hsu C C, Chen Y Y, Tsai W C, Chen W H, Chen H H. 2015. Three R2R3-MYB transcription factors regulate distinct floral pigmentation patterning in Phalaenopsis spp. Plant Physiology, 168 (1):175-191. doi: 10.1104/pp.114.254599 URL
[9]	Jiang S, Sun Q, Zhang T, Liu W, Wang N, Chen X. 2021. MdMYB114regulates anthocyanin biosynthesis and functions downstream of MdbZIP4-like in apple fruit. Plant Journal, 257:153353.
[10]	Jin W M, Wang H, Li M F, Wang J, Yang Y, Zhang X, Yan G, Zhang H, Liu J S, Zhang K C. 2016. The R2R3 MYB transcription factor PavMYB10.1 involves in anthocyanin biosynthesis and determines fruit skin colour in sweet cherry(Prunus avium L.). Plant Biotechnol Journal, 14 (11):2120-2133. doi: 10.1111/pbi.12568 URL
[11]	Jiu S, Guan L, Leng X, Zhang K, Haider M S, Yu X, Zhu X, Zheng T, Ge M, Wang C, Jia H, Shangguan L, Zhang C, Tang X, Abdullah M, Javed H U, Han J, Dong Z, Fang J. 2021. The role of VvMYBA2r and VvMYBA2w alleles of the MYBA 2 locus in the regulation of anthocyanin biosynthesis for molecular breeding of grape(Vitis spp.)skin coloration. Plant Biotechnol Journal, 19 (6):1216-1239. doi: 10.1111/pbi.13543 URL
[12]	Li Maofu, Yang Yuan, Wang Hua, Liu Jiashen, Jin Wanmei. 2017. Cloning and expression analysis on RcbHLH gene from Rosa chinensis and interaction analysis between MYB and WD 40 transcription factors. Acta Horticulturae Sinica, 44 (10):1949-1958. (in Chinese)
	李茂福, 杨媛, 王华, 刘佳棽, 金万梅. 2017. 月季bHLH基因的克隆、表达及其与MYB和WD40的互作分析. 园艺学报, 44 (10):1949-1958.
[13]	Li S. 2014. Transcriptional control of flavonoid biosynthesis:fine-tuning of the MYB-bHLH-WD40(MBW)complex. Plant Signal Behavior, 9 (1):e27522. doi: 10.4161/psb.27522 URL
[14]	Li Y M, Liang J, Zeng X Z, Guo H, Luo Y W, Kear P, Zhang S M, Zhu G T. 2021. Genome-wide analysis of MYB gene family in potato provides insights into tissue-specific regulation of anthocyanin biosynthesis. Horticultural Plant Journal, 7 (2):129-141. doi: 10.1016/j.hpj.2020.12.001 URL
[15]	Lin-Wang K, Bolitho K, Grafton K, Kortstee A, Karunairetnam S, McGhie T K, Espley R V, Hellens R P, Allan A C. 2010. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biology, 10:50. doi: 10.1186/1471-2229-10-50 pmid: 20302676
[16]	Mazalovska M, Kouokam J C. 2020. Transiently expressed mistletoe lectin II in Nicotiana benthamiana demonstrates anticancer activity in vitro. Molecules, 25 (11):2562. doi: 10.3390/molecules25112562 URL
[17]	Meng Y, Wang Z. 2019. The MYB activator WHITE PETAL1 associates with MtTT8 and MtWD40-1 to regulate carotenoid-derived flower pigmentation in Medicago truncatula. Frontiers Plant Science, 31 (11):2751-2767.
[18]	Saigo T, Wang T, Watanabe M, Tohge T. 2020. Diversity of anthocyanin and proanthocyanin biosynthesis in land plants. Current Opinion in Plant Biology, 55:93-99. doi: S1369-5266(20)30041-8 pmid: 32387820
[19]	Sainsbury F, Thuenemann E C, Lomonossoff G P. 2009. pEAQ:versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol Journal, 7 (7):682-693. doi: 10.1111/j.1467-7652.2009.00434.x URL
[20]	Schaart J G, Dubos C, Romero De La Fuente I, van Houwelingen A M, de Vos R C, Jonker H H, Xu W, Routaboul J M, Lepiniec L, Bovy A G. 2013. Identification and characterization of MYB-bHLH-WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry(Fragaria × ananassa)fruits. New Phytologist, 197 (2):454-467.
[21]	Schwinn K, Venail J, Shang Y, Mackay S, Alm V, Butelli E, Oyama R, Bailey P, Davies K, Martin C. 2006. A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. Plant Cell, 18 (4):831-851. pmid: 16531495
[22]	Shen Y, Sun T, Pan Q, Anupol N, Chen H, Shi J, Liu F, Deqiang D, Wang C, Zhao J, Yang S, Wang C, Liu J, Bao M, Ning G. 2019. RrMYB5- and RrMYB10-regulated flavonoid biosynthesis plays a pivotal role in feedback loop responding to wounding and oxidation in Rosa rugosa. Plant Biotechnol Journal, 17 (11):2078-2095. doi: 10.1111/pbi.13123 URL
[23]	Stracke R, Werber M, Weisshaar B. 2001. The R2R3-MYB gene family in Arabidopsis thaliana. Current Opinion in Plant Biology, 4 (5):447-456. pmid: 11597504
[24]	Swarnkar M K, Kumar P. 2021. Prickle morphogenesis in rose is coupled with secondary metabolite accumulation and governed by canonical MBW transcriptional complex. Journal Plant Physiology, 5 (6):e00325.
[25]	Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods. Molecular Biology and Evolution, 28 (10):2731-2739. doi: 10.1093/molbev/msr121 URL
[26]	Tanaka Y, Sasaki N, Ohmiya A. 2008. Biosynthesis of plant pigments:anthocyanins,betalains and carotenoids. Plant Journal, 54 (4):733-749. doi: 10.1111/j.1365-313X.2008.03447.x URL
[27]	Teng S, Keurentjes J, Bentsink L, Koornneef M, Smeekens S. 2005. Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene. Plant Physiology, 139 (4):1840-1852. doi: 10.1104/pp.105.066688 URL
[28]	Walker A R, Lee E, Bogs J, McDavid D A, Thomas M R, Robinson S P. 2007. White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant Journal, 49 (5):772-785. pmid: 17316172
[29]	Wang H, Zhang H, Yang Y, Li M F, Zhang Y T, Liu J, Dong J, Li J, Butelli E, Xue Z, Wang A, Wang G Z, Martin C, Jin W M. 2020. The control of red colour by a family of MYB transcription factors in octoploid strawberry(Fragaria × ananassa)fruits. Plant Biotechnol Journal, 18:1169-1184.
[30]	Xi H C, He Y J, Chen H Y. 2021. Functional characterization of SmbHLH13in anthocyanin biosynthesis and flowering in eggplant. Horticultural Plant Journal, 7 (1):73-80. doi: 10.1016/j.hpj.2020.08.006 URL
[31]	Yamagishi M, Shimoyamada Y, Nakatsuka T, Masuda K. 2010. Two R2R3-MYB genes,homologs of Petunia AN2,regulate anthocyanin biosyntheses in flower tepals,tepal spots and leaves of asiatic hybrid lily. Plant Cell Physiology, 51 (3):463-474. doi: 10.1093/pcp/pcq011 URL
[32]	Yuan Y W, Sagawa J M, Frost L, Vela J P, Bradshaw H D. 2014. Transcriptional control of floral anthocyanin pigmentation in monkeyflowers (Mimulus). New Phytologist, 204 (4):1013-1027. doi: 10.1111/nph.12968 URL
[33]	Zhang Y, Xu S, Ma H, Duan X, Gao S, Zhou X, Cheng Y. 2021. The R2R3-MYB gene PsMYB58 positively regulates anthocyanin biosynthesis in tree peony flowers. Plant Physiology Biochemistry, 164:279-288. doi: 10.1016/j.plaphy.2021.04.034 URL
[34]	Zhu Ziguo, Zhang Qingtian, Han Zhen, Li Bo, Li Guodong, Li Xiujie. 2021. VvMYB6,an R2R3-MYB transcription factor,is involved in anthocyanin biosynthesis of grapevine. Acta Horticulturae Sinica, 48 (3):465-476. (in Chinese)
	朱自果, 张庆田, 韩真, 李勃, 李国栋, 李秀杰. 2021. 欧洲葡萄VvMYB6正向调控花青素合成. 园艺学报, 48 (3):465-476.

月季中R2R3-MYB基因RhMYB113c调控花青素苷合成

Analysis the Function of R2R3 MYB Transcription Factor RhMYB113c on Regulating Anthocyanin Synthesis in Rosa hybrida

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 34

相关文章 15

编辑推荐

Metrics

本文评价

访问总数:　当日访问总数:　当前在线人数:

[1]	欧阳丽莹, 黄艳飞, 陈君梅, 阳淑, 练华山. 微型月季新品种‘九儿’[J]. 园艺学报, 2022, 49(S2): 223-224.
[2]	练华山, 欧阳丽莹, 吴珊, 陈君梅, 舒彬. 微型月季新品种‘夜华’[J]. 园艺学报, 2022, 49(S2): 225-226.
[3]	陈君梅, 练华山, 黄艳飞, 禹婷, 蒋天仪. 微型月季新品种‘流星’[J]. 园艺学报, 2022, 49(S2): 227-228.
[4]	贾鑫, 曾臻, 陈月, 冯慧, 吕英民, 赵世伟. 月季‘月月粉’RcDREB2A的克隆与表达分析[J]. 园艺学报, 2022, 49(9): 1945-1956.
[5]	邓娇, 苏梦月, 刘雪莲, 欧克芳, 户正荣, 杨平仿. 基于转录组分析揭示双色花莲‘大洒锦’花色形成机理[J]. 园艺学报, 2022, 49(2): 365-377.
[6]	李茂福, 杨媛, 王华, 范又维, 孙佩, 金万梅. 月季自交不亲和性S-RNase的鉴定与分析[J]. 园艺学报, 2022, 49(1): 157-165.
[7]	洪燕红, 叶清华, 李泽坤, 王威, 谢倩, 陈清西, 陈建清. 红花草莓‘莓红’花瓣花色苷积累及其MYB基因的表达分析[J]. 园艺学报, 2021, 48(8): 1470-1484.
[8]	杨婷, 薛珍珍, 李娜, 郎校安, 李凌飞, 钟春梅. 铁十字秋海棠斑叶发育过程内参基因筛选及验证[J]. 园艺学报, 2021, 48(11): 2251-2261.
[9]	庄玥莹, 周利君, 程璧瑄, 于超, 罗乐, 潘会堂, 张启翔. 基于转录组测序的香水月季花香代谢基因研究[J]. 园艺学报, 2021, 48(11): 2262-2274.
[10]	温佳辛, 王超林, 冯慧, 李珊珊, 王亮生, 武荣花, 赵世伟. 月季花色研究进展[J]. 园艺学报, 2021, 48(10): 2044-2056.
[11]	吴洪娥, 金晶, 吴楠, 董万鹏, 朱立, 周洪英. 丰花月季新品种‘花好月圆’[J]. 园艺学报, 2020, 47(S2): 3077-3078.
[12]	丁爱琴1,2，韩坤桀1，杨立晨1，张竹君1，耿子雯1，付鲁峰1，刘庆华1,，姜新强1,. 月季RhNAC31互作蛋白的筛选及分析[J]. 园艺学报, 2020, 47(7): 1335-1344.
[13]	王焕，郑日如，曹声海，张通，史若明，王彩云，罗靖**. 月季花瓣特异表达启动子的筛选和鉴定[J]. 园艺学报, 2020, 47(4): 686-698.
[14]	高述民 1，周燕 1,2,*，范莉娟 1,2，巢阳 2. 微型月季新品种‘桔月’[J]. 园艺学报, 2020, 47(2): 401-402.
[15]	王丽花1，段金辉2,*，李清云2，段云晟2，薛祖旺2，范荣旭2. 月季新品种‘金焰’[J]. 园艺学报, 2020, 47(1): 199-200.

月季中R2R3-MYB基因RhMYB113c调控花青素苷合成

Analysis the Function of R2R3 MYB Transcription Factor RhMYB113c on Regulating Anthocyanin Synthesis in Rosa hybrida

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 34

相关文章 15

编辑推荐

Metrics

本文评价

访问总数: 当日访问总数: 当前在线人数:

访问总数:　当日访问总数:　当前在线人数: