石榴花青苷合成相关基因PgMYB111的克隆与功能分析

doi:10.16420/j.issn.0513-353x.2020-0707

摘要/Abstract

摘要：

为研究R2R3-MYB转录因子调控石榴花青苷合成的机理,以石榴品系‘榴花红’（红花）和‘榴花白’（白花）为试材,利用同源克隆技术分离出1个调控花青苷生物合成的R2R3-MYB基因PgMYB111（Pg012177.1）。其CDS全长为1 101 bp,编码366个氨基酸。多重比对分析表明,PgMYB111蛋白的N端含有1个R2R3保守结构域,以及1个与bHLH蛋白互作的[D/E]Lx2[R/K]x3Lx6Lx3R结合位点。系统发育树分析表明,PgMYB111与苹果和扁桃具有较高的同源性,聚为一簇。亚细胞定位试验发现,PgMYB111定位于细胞核。PgMYB111在‘榴花红’中的表达量是‘榴花白’的4.3倍,且‘榴花红’花瓣花青苷含量也显著高于‘榴花白’。烟草瞬时表达结果表明转基因烟草叶片的花青苷含量和基因表达量呈先上升后下降的趋势,均在第5天达到最高,分别是未侵染叶片的3.8倍和8.9倍,pBI121空载叶片的3.17倍和5.57倍。结果表明PgMYB111参与了花青苷合成途径并促进其合成。

关键词: 石榴, 花青苷, 基因克隆, 亚细胞定位, 花色

Abstract:

To study the regulatory mechanism of the anthocyanin synthesis pathway in pomegranate,we isolated PgMYB111 gene from the petals of‘Liuhuahong’and‘Liuhuabai’. Results showed that the cDNA sequence contained a 1 101 bp open reading frame（ORF）encoding a 366 amino acid polypeptide. Multiple alignment analysis of amino acid sequences showed that the N-terminus of PgMYB111 protein contained a conserved R2R3 domain and a binding site of [D/E]Lx2[R/K]x3Lx6Lx3R interacted with bHLH protein. Furthermore,the amino acid sequence had the highest similarity with MYB transcription factors in Malus × domestica and Prunus dulcis. Subcellular localization assays suggested that PgMYB111 localized in the nucleus. qRT-PCR analysis indicated that the expression of PgMYB111 gene in‘Liuhuahong’ was 4.3 times higher than that in‘Liuhuabai’. In addition,the anthocyanin content in‘Liuhuahong’petal is significantly higher than that in‘Liuhuabai’petal. The results of tobacco transient expression showed that the expression level of the gene increased at first and then decreased. The anthocyanin content in transgenic tobacco leaves was consistent with the gene expression,and reached the highest level on the fifth day. They were 3.8 times and 8.9 times higher than those in uninfected leaves,and 3.17 times and 5.57 times higher than those in pBI121 leaves,respectively. In summary,PgMYB111 could involve in the anthocyanin synthesis pathway and facilitate its synthesis.

Key words: pomegranate, anthocyanin, gene cloning, subcellular localization, flower colour

中图分类号:

S665.4

王沙, 张心慧, 赵玉洁, 李变变, 招雪晴, 沈雨, 董建梅, 苑兆和. 石榴花青苷合成相关基因PgMYB111的克隆与功能分析[J]. 园艺学报, 2022, 49(9): 1883-1894.

WANG Sha, ZHANG Xinhui, ZHAO Yujie, LI Bianbian, ZHAO Xueqing, SHEN Yu, DONG Jianmei, YUAN Zhaohe. Cloning and Functional Analysis of PgMYB111 Related to Anthocyanin Synthesis in Pomegranate[J]. Acta Horticulturae Sinica, 2022, 49(9): 1883-1894.

图/表 9

表1 基因克隆、亚细胞定位以及特异性表达分析引物表

Table 1 Primers for the gene cloning,subcellular localization and qRT-PCR

注释 Annotation	引物 Primer	引物序列（5′-3′） Primer sequence
基因克隆 Gene clone	PgMYB111	F：ATGGGGAGGAAACCAT;R：TCAAGTTTTAGTGATCTTCTC F：gagaacacgggggactctagaATGGGGAGGAAACCAT R：gcccttgctcaccatggatccTCAAGTTTTAGTGATCTTCTC
亚细胞定位 Subcellular localization	GFP-PgMYB111
基因表达 Gene expression	PgMYB111	F：ATCTCTCCCCAAGAATGCC;R：AGACATGACGCTGTTACCCAA
	PgCHS	F：AGCGGACTACAAGCTCA;R：CACTCGCGCATCAGCA
	PgDFR	F：CCGGCATCGCAAAGCTC;R：CACAGCCCCGACGAAC
	PgUFGT	F：CGCCCATATCTACACTGACC;R：AGTCCAAATCGCCGAAG
	PgANS	F：CCTATGCAAACTCCCCGAA;R：TGCTTCCCGTACTCGTTG
	PgActin	F：AGTCCTCTTCCAGCCATCTC;R：CACTGAGCACAATGTTTCCA

表2 石榴花瓣的CIE L*,a*,b*值

Table 2 Date of CIE L*,a*,b* in petals of pomegranate

材料 Meterial	花色 Color	L^*	a^*	b^*	C^*	h
榴花红Liuhuahong	红色Red	54.35 ± 1.05	49.73 ± 1.18	31.42 ± 1.33	58.93 ± 0.97	32.42 ± 1.45
榴花白Liuhuabai	白色White	85.82 ± 0.92	-1.14 ± 0.20	7.07 ± 0.29	7.16 ± 0.29	-80.83 ± 1.45

图1 ‘榴花白’和‘榴花红’盛花期花朵表型（A）及花青苷含量（B）不同小写字母表示在0.05水平差异显著。

Fig. 1 Phenotype（A）and anthocyanin content（B）in full bloom of‘Liuhuabai’and‘Liuhuahong’flowers Different lowercase letters indicate significant differences at 0.05 level.

图2 PgMYB111基因克隆 M：DL ladder 2000 DNA marker;1：石榴cDNA模板。

Fig. 2 Gene cloning of PgMYB111 M：DL ladder 2000 DNA marker;1：Pomegranate cDNA template.

图3 蛋白多重序列比对 AtMYB111：拟南芥（NP_199744.1）;CsMYB111：柑橘（XP_006488875.1）;VvMYB111：葡萄（RVW39925.1）;MdMYB111：苹果（XP_028951203.1）;EgMYB111：油棕（XP_010921536.1）;AhMYB111：花生（XP_025692112.1）;PdMYB111：扁桃（XP_034227785.1）;PtMYB111：毛果杨（XP_002316017.2）;BrMYB111：芜菁（XP_009151728.1）;AtMYB11：拟南芥（NP_191820.1）;AtMYB12：拟南芥（ABB03913.1）;AtMYB90：拟南芥（NP_176813.1）;AtMYB75：拟南芥（ABB03879.1）;AtMYB113：拟南芥（OAP11934.1）;AtMYB114：拟南芥（AEE34502.1）;AN2：矮牵牛（BAP28593.1）。

Fig. 3 Multiple sequence alignment of the proteins AtMYB111：Arabidopsis thaliana（NP_199744.1）;CsMYB111：Citrus reticulata（XP_006488875.1）;VvMYB111：Vitis vinifera（RVW39925.1）;MdMYB111：Malus × domestica（XP_028951203.1）;EgMYB111：Elaeis guineensis（XP_010921536.1）;AhMYB111：Arachis hypogaea（XP_025692112.1）;PdMYB111：Amygdalus communis（XP_034227785.1）;PtMYB111：Populus trichocarpa（XP_002316017.2）;BrMYB111：Brassica rapa（XP_009151728.1）;AtMYB11：Arabidopsis thaliana（NP_191820.1）;AtMYB12：Arabidopsis thaliana（ABB03913.1）;AtMYB90：Arabidopsis thaliana（NP_176813.1）;AtMYB75：Arabidopsis thaliana（ABB03879.1）;AtMYB113：Arabidopsis thaliana（OAP11934.1）;AtMYB114：Arabidopsis thaliana（AEE34502.1）;AN2：Petunia hybrida（BAP28593.1）.

图4 系统发育树

Fig. 4 Phylogentic analysis

图5 PgMYB111蛋白亚细胞定位

Fig. 5 Subcellular localization of PgMYB111

图6 PgMYB111的组织表达（A）与PgMYB111（B）、PgCHS（C）、PgDFR（D）、PgUFGT（E）及PgANS（F）在‘榴花红’和‘榴花白’中的表达不同小写字母表示在0.05水平差异显著。下同。

Fig. 6 Expression of PgMYB111 in different tissues（A）and expression of PgMYB111（B）,PgCHS（C）,PgDFR（D）,PgUFGT（E）and PgANS（F）in ‘Liuhuahong’ and ‘Liuhuabai’ Different lowercase letters indicate significant differences at 0.05 level. The same below.

图7 烟草瞬时表达结果

Fig. 7 Tobacco transient expression results

参考文献 55

[1]	An X H, Tian Y, Chen K Q, Liu X J, Liu D, Xie X B, Cheng C G, Cong P H, Hao Y J. 2015. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant and Cell Physiology, 4 (56):650-662.
[2]	Arlotta C, Puglia G D, Genovese C, Toscano V, Karlova R, Beekwilder J, de Vos R C H, Raccuia S A. 2020. MYB5-like and bHLH influence flavonoid composition in pomegranate. Plant Science, 298:110563. doi: 10.1016/j.plantsci.2020.110563 URL
[3]	Bac-Molenaar J A, Fradin E F, Rienstra J A, Vreugdenhil D, Keurentjes J J. 2015. GWA mapping of anthocyanin accumulation reveals balancing selection of MYB90 in Arabidopsis thaliana. PLoS ONE, 10 (11):e0143212. doi: 10.1371/journal.pone.0143212 URL
[4]	Balakrishnan S, Gao S, Lercher M, Hu S, Chen W. 2019. Evolview v3:a webserver for visualization,annotation,and management of phylogenetic trees. Nucleic Acids Research, 47:W270-W275. doi: 10.1093/nar/gkz357
[5]	Ban Y, Honda C, Hatsuyama Y, Igarashi M, Bessho H, Moriguchi T. 2007. Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant and Cell Physiology, 48 (7):958-970. pmid: 17526919
[6]	Borevitz J O, Xia Y, Blount J, Dixon R A, Lamb C. 2000. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell, 12 (12):2383-2394. pmid: 11148285
[7]	Chaves-Silva S, Santos A L, Chalfun-Júnior A, Zhao J, Peres L, Benedito V A. 2018. Understanding the genetic regulation of anthocyanin biosynthesis in plants—tools for breeding purple varieties of fruits and vegetables. Phytochemistry, 153:11-27. doi: S0031-9422(18)30147-X pmid: 29803860
[8]	Chen Y H, Yang X Y, He K, Liu M H, Li J G, Gao Z F, Lin Z Q, Zhang Y F, Wang X X, Qiu X M, Shen Y P, Zhang L, Deng X H, Luo J C, Deng X W, ChenZ L, GuH Y, Qu L J. 2006. The MYB transcription factor superfamily of Arabidopsis:expression analysis and phylogenetic comparison with the rice MYB family. Plant Molecular Biology, 60:107-124. doi: 10.1007/s11103-005-2910-y URL
[9]	El-Gebali S, Mistry J, Bateman A, Eddy S, Luciani A, Potter S, Qureshi M, Richardson L, Salazar G, Smart A. 2019. The Pfam protein families database in 2019. Nucleic Acids Research, 47 (D1):d427-d432.
[10]	Falcone Ferreyra M L, Rius S P, Casati P. 2012. Flavonoids:biosynthesis,biological functions and biotechnological applications. Front Plant Science, 3 (222):222.
[11]	Gonzalez A, Zhao M, Leavitt J M, Lloyd A M. 2010. 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
[12]	Hichri I, Barrieu F, Bogs J, Kappel C, Delrot S, Lauvergeat V. 2011. Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. Journal of Experimental Botany, 62 (8):2465-2483. doi: 10.1093/jxb/erq442 pmid: 21278228
[13]	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.
[14]	Kumar S, Stecher G, Tamura K. 2016. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33 (7):1870-1874. doi: 10.1093/molbev/msw054 URL
[15]	Lefort V, Longueville J E, Gascuel O. 2017. SMS:Smart model selection in PhyML. Molecular Biology and Evolution, 4 (9):2422-2424.
[16]	Leon R, Lightbourn L A, Melina L, Amarillas L. 2020. Differential gene expression of anthocyanin biosynthetic genes under low temperature and ultraviolet-B radiation in bell pepper(Capsicum annuum). International Journal of Agriculture and Biology, 23 (3):531-538.
[17]	Li M, Cao Y T, Ye S R, Irshad M, Pan T F, Qiu D L. 2016. Isolation of CHS gene from Brunfelsia acuminata flowers and its regulation in Anthocyanin Biosysthesis. Molecules, 22 (1):44. doi: 10.3390/molecules22010044 URL
[18]	Li B, Fan R, Guo S, Wang P, Zhu X, Fan Y, Chen Y, He K, Kumar A, Shi J, Wang Y, Li L, Hu L, Song C. 2019. The Arabidopsis MYB transcription factor,MYB111 modulates salt responses by regulating flavonoid biosynthesis. Environmental and Experimental Botany, 166:103807. doi: 10.1016/j.envexpbot.2019.103807 URL
[19]	Li H, Duan S Y, Sun W L, Wang S F, Zhang J, Song T T, Tian J, Yao Y C. 2022. Identification,through transcriptome analysis,of transcription factors that regulate anthocyanin biosynthesis in different parts of red-fleshed apple‘May’fruit. Horticultural Plant Journal, 8 (1):11-21. doi: 10.1016/j.hpj.2021.07.001 URL
[20]	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
[21]	Lin Qifang, Liu Tingting, Liu Jieru, Cai Ming, Cheng Tangren, Wang Jia, Zhang Qixiang, Pan Huitang. 2021. Flavonoids composition and content in petals of Lagerstroemia and Heimia species and cultivars. Acta Horticulturae Sinica, 48 (10):1956-1968. (in Chinese)
	林启芳, 刘婷婷, 刘洁茹, 蔡明, 程堂仁, 王佳, 张启翔, 潘会堂. 2021. 紫薇属与黄薇属植物花瓣类黄酮组成及含量分析. 园艺学报, 48 (10):1956-1968.
[22]	Liu Y, Tikunov Y, Schouten R E, Marcelis L F M, Visser R G F, Bovy A. 2018. Anthocyanin biosynthesis and degradation mechanisms in solanaceous vegetables:a review. Frontiers in Chemistry, 6:52. doi: 10.3389/fchem.2018.00052 URL
[23]	Liu Xin, Chen Yunzhu, Kim P, Kim M J, Song H, Li Yuhua, Wang Yu. 2020. Progress on molecular mechanism and regulation of tomato fruit color formation. Acta Horticulturae Sinica, 47 (9):1689-1704. (in Chinese)
	刘昕, 陈韵竹, Kim Pyol, Kim Min-jun, Song Hyondok, 李玉花, 王宇. 2020. 番茄果实颜色形成的分子机制及调控研究进展. 园艺学报, 47 (9):1689-1704.
[24]	Livak K, Schmittgen T. 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2^-ΔΔCT method. Methods, 25 (4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[25]	Lou Q, Liu Y L, Qi Y Y, Jiao S Z, Tian F F, Jiang L, Wang Y J. 2014. Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth. Journal of Experimental Botany, 65 (12):3157-3164. doi: 10.1093/jxb/eru168 pmid: 24790110
[26]	Lu Suwen, Zheng Xuanang, Wang Jiayang, Fang Jinggui. 2021. Research progress on the metabolism of flavonoids in grape. Acta Horticulturae Sinica, 48 (12):2506-2524. (in Chinese)
	卢素文, 郑暄昂, 王佳洋, 房经贵. 2021. 葡萄类黄酮代谢研究进展. 园艺学报, 48 (12):2506-2524.
[27]	Ma D W, Constable C P. 2019. MYB repressors as regulators of phenylpropanoid metabolism in plants. Trends in Plant Science, 24 (3):275-289. doi: S1360-1385(18)30290-5 pmid: 30704824
[28]	Martin C, Paz-Ares J. 1997. MYB transcription factors in plants. Trends in Genetics, 13 (2):67-73. doi: 10.1016/s0168-9525(96)10049-4 pmid: 9055608
[29]	Matsui K, Oshima Y, Mitsuda N, Sakamoto S, Nishiba Y, Walker A R, Ohme-Takagi M, Robinson S P, Yasui Y, Mori M, Takami H. 2018. Buckwheat R2R3 MYB transcription factor FeMYBF1 regulates flavonol biosynthesis. Plant Science, 74:466-475.
[30]	Nakabayashi R, Yonekura-Sakakibara K, Urano K, Suzuki M, Yamada Y, Nishizawa T, Matsuda F, Kojima M, Sakakibara H, Shinozaki K, Michael A J, Tohge T, Yamazaki M, Saito K. 2014. Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids. Plant Journal, 77:367-379. doi: 10.1111/tpj.12388 URL
[31]	Petroni K, Tonelli C. 2011. Recent advances on the regulation of anthocyanin synthesis in reproductive organs. Plant Sci, 181 (3):219-229. doi: 10.1016/j.plantsci.2011.05.009 pmid: 21763532
[32]	Ramya M, Kwon O K, An H R, Park P M, Baek Y S, Park P H. 2017. Floral scent:regulation and role of MYB transcription factors. Phytochemistry Letters, 19:114-120. doi: 10.1016/j.phytol.2016.12.015 URL
[33]	Shen H, Chou K. 2010. Cell-PLoc 2.0:an improved package of web-servers for predicting subcellular localization of proteins in various organisms. Natural Science, 2 (10):1090-1103. doi: 10.4236/ns.2010.210136 URL
[34]	Stracke R, Ishihara H G, Barsch A, Mehrtens F, Niehaus K, Weisshaar B. 2010. Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant Journal, 50 (4):660-677. doi: 10.1111/j.1365-313X.2007.03078.x URL
[35]	Takos A M, Jaffé F W, Jacob S R, Bogs J, Robinson S P, Walker A R. 2006. Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiology, 142 (3):1216-1232. pmid: 17012405
[36]	Wang H, Wang X, Yu C, Wang C, Jin Y, Zhang H. 2020. MYB transcription factor PdMYB118 directly interacts with bHLH transcription factor PdTT8 to regulate wound-induced anthocyanin biosynthesis in poplar. BMC Plant Biology, 20:173. doi: 10.1186/s12870-020-02389-1 URL
[37]	Wang S, Chu Z, Jia R, Dan F, Shen X, Li Y, Ding X. 2018. SlMYB12 regulates flavonol synthesis in three different cherry tomato varieties. Scientific Reports, 8 (1):1582. doi: 10.1038/s41598-018-19214-3 URL
[38]	Winkelshirley B. 2001. Flavonoid biosynthesis. A colorful model for genetics,biochemistry,cell biology,and biotechnology. Plant Physiology, 126 (2):485-493. pmid: 11402179
[39]	Xi H C, He Y J, Chen H Y. 2021. Functional characterization of Smbhlh13 in anthocyanin biosynthesis and flowering in eggplant. Horticultural Plant Journal, 7 (1):73-80. doi: 10.1016/j.hpj.2020.08.006 URL
[40]	Xu W J, Dubos C, Lepiniec L. 2015. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Science, 20 (3):176-185. doi: 10.1016/j.tplants.2014.12.001 URL
[41]	Xu Y, Zheng H, Wang Q, Khalilurrehman M, Meng L S, Tao J M. 2019. Comparison among‘Benitaka’grape,ABA-treated‘Benitaka’,and its deeper-colored bud mutation revealed the coloring mechanisms on grapes. Journal of Plant Interactions, 14 (1):102-109. doi: 10.1080/17429145.2018.1547843 URL
[42]	Xu Y, Zhu X, Gong Y, Xu L, Wang Y, Liu L. 2012. Evaluation of reference genes for gene expression studies in radish(Raphanus sativus L.)using quantitative real-time PCR. Biochemical and Biophysical Research Communications. 424 (3):398-403.
[43]	Xu Z S, Yang Q Q, Feng K, Yu X, Xiong A S. 2020. DcMYB113,a root-specific R2R3-MYB,conditions anthocyanin biosynthesis and modification in carrot. Plant Biotechnol Journal, 18 (7):1585-1597. doi: 10.1111/pbi.13325 URL
[44]	Yang C Q, Sha G Y, Wei T, Ma B Q, Li C Y, Li P M, Zou Y J, Xu L F, Ma F W. 2021. Linkage map and QTL mapping of red flesh locus in apple using a R1R1 × R6R6 population. Horticultural Plant Journal, 7 (5):393-400. doi: 10.1016/j.hpj.2020.12.008 URL
[45]	Yang Guanxian, Xu Haifeng, Zhang Jing, Wang Nan, Fang Hongcheng, Jiang Shenghui, Wang Yicheng, Su Mengyu, Chen Xuesen. 2019. Functional identification of apple anthocyanin regulatory gene MdMYB111. Acta Horticulturae Sinica, 46 (5):832-840. (in Chinese)
	杨官显, 许海峰, 张静, 王楠, 房鸿成, 姜生辉, 王意程, 苏梦雨, 陈学森. 2019. 苹果花青苷调控基因MdMYB111的功能鉴定. 园艺学报, 46 (5):832-840.
[46]	Yuan Z, Fang Y, Zhang T, Fei Z, Han F, Liu C, Liu M, Xiao W, Zhang W, Wu S, Zhang M, Ju Y, Xu H, Dai H, Liu Y, Chen Y, Wang L, Zhou J, Guan D, Yan M, Xia Y, Huang X, Liu D, Wei H, Zheng H. 2018. The pomegranate(Punica granatum L.)genome provides insights into fruit quality and ovule developmental biology. Plant Biotechnology Journal, 16 (7):1363-1374. doi: 10.1111/pbi.12875 URL
[47]	Yuan Zhaohe. 2015. Chinese fruit tree science and practice pomegranate,Xi’an:Shaanxi Science and Technology Press:26-42. (in Chinese)
	苑兆和. 2015. 中国果树科学与实践·石榴. 西安:陕西科学技术出版社:26-42.
[48]	Zhai R, Zhao Y, Wu M, Yang J, Li X, Liu H, Wu T, Liang F, Yang C, Wang Z, Ma F, Xu L. 2019. The MYB transcription factor PbMYB12b positively regulates flavonol biosynthesis in pear fruit. BMC Plant Biology, 19 (1):85. doi: 10.1186/s12870-019-1687-0 pmid: 30791875
[49]	Zhang K, Logacheva M D, Meng Y, Hu J P, Wan D P, Li L, Janovska D, Wang Z Y, Georgiev M I, Yu Z, Yang F Y, Yan M L, Zhou M L. 2018. Jasmonate-responsive MYB factors spatially repress rutin biosynthesis in Fagopyrum tataricum. Journal of Experimental Botany, 69 (8):1955-1966. doi: 10.1093/jxb/ery032 URL
[50]	Zhang T, Xu H, Yang G, Wang N, Zhang J, Wang Y, Jiang S, Fang H, Zhang Z, Chen X. 2019. Molecular mechanism of MYB111 and WRKY40 involved in anthocyanin biosynthesis in red-fleshed apple callus. Plant Cell Tissue and Organ Culture, 139:467-478. doi: 10.1007/s11240-019-01637-z URL
[51]	Zhang Zhen, Li Chenzhiyu, Zhang Ya, Li Xi, Wang Shuo, Wang Xinyu, Chen Xuesen, Feng shouqian. 2021. Inhibition of pear anthocyanin synthesis by the PcHB12 gene in European pears. Acta Horticulturae Sinica, 48 (3):456-464. (in Chinese)
	张震, 李晨智宇, 张雅, 李玺, 王烁, 王鑫玉, 陈学森, 冯守千. 2021. 西洋梨PcHB12基因抑制果实花青苷的合成. 园艺学报, 48 (3):456-464.
[52]	Zhao Xueqing, Yuan Zhaohe. 2019. Expression profiles of fruit color related genes CHS and CHI in Punica granatum L. Genomics and Applied Biology, 38 (5):2175-2182. (in Chinese)
	招雪晴, 苑兆和. 2019. 石榴果色合成相关基因CHS和CHI的表达特性分析. 基因组学与应用生物学, 38 (5):2175-2182.
[53]	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.
[54]	Zhong C, Tang Y, Pang B, Li X, Yang Y, Deng J, Feng C, Li L, Ren G, Wang Y, Peng J, Sun S, Liang S, Wang X. 2020. The R2R3-MYB transcription factor GhMYB1a regulates flavonol and anthocyanin accumulation in Gerbera hybrida. Horticulture Research, 7:78. doi: 10.1038/s41438-020-0296-2 URL
[55]	Zimmermann I M, Heim M A, Weisshaar B, Uhrig J F. 2004. Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant Journal, 40 (1):22-34. pmid: 15361138