茄萼花色苷合成相关基因DFR和MYB克隆及表达分析

doi:10.3864/j.issn.0578-1752.2017.14.014

摘要/Abstract

摘要： 【目的】花色苷是一类通过类黄酮途径合成的水溶性次生代谢产物，既能使植物的不同器官呈现红、紫、蓝等颜色，还有利于人体健康。紫茄富含花色苷，但是有关茄萼花色苷生物合成的分子机制还不是很清楚。本研究旨在通过克隆茄萼花色苷合成相关基因DFR和MYB，测定其在不同发育时期不同颜色茄萼中的表达量，探究DFR和MYB在茄萼花色苷合成中的作用。【方法】选用绿萼和紫萼长茄（Solanum melongena L.）果萼为试材，测定不同pH条件下茄萼花色苷含量；通过RACE方法分离克隆DFR和MYB cDNA全长序列，分析DFR和MYB的保守结构域及序列特征；分别对DFR和MYB及其同源蛋白序列进行系统进化分析，构建系统进化树来进一步分析鉴定基因；使用ExPASy网站提供的在线分析软件SOPMA预测蛋白质二级结构；利用实时荧光定量PCR方法检测目的基因在不同发育阶段果萼中的表达情况。【结果】从绿萼和紫萼长茄果萼中克隆了DFR和MYB片段，分别命名为ouSmDFR、dongSmDFR和ouSmMYB、dongSmMYB，GenBank登录号分别为：KX224250、KX224251和KX224253、KX224254。ouSmDFR和dongSmDFR全长分别为1 285 bp和1 249 bp，开放阅读框为858 bp和864 bp，分别编码285个和287个氨基酸；ouSmMYB和dongSmMYB全长分别为969 bp和959 bp，开放阅读框均为462 bp，编码153个氨基酸。蛋白质二级结构分析表明α-螺旋和无规则卷曲均为两个DFR蛋白和两个MYB蛋白的主要二级结构元件。序列比对表明DFR蛋白具有NADPH结构域（NADPH binding domain）和底物特异性结合结构域（Substrate specific binding domain），属于NADB-Rossmann超基因家族；MYB蛋白属于R2R3-MYB转录因子，具有R2、R3两个MYB结构域和bHLH结合域。ouSmDFR和dongSmDFR与StDFR和SlDFR具有相对较高的同源性；ouSmMYB和dongSmMYB与EsMYB同源性较高。花色苷含量测定显示，紫萼果茄萼花色苷含量较高且随着果实的发育成熟而逐渐增加；而绿萼茄萼几乎检测不到花色苷。荧光实时定量PCR分析表明，DFR和MYB在紫萼长茄果萼中表达量均远高于绿萼长茄；从初蕾期到盛花期，紫萼长茄果萼中DFR和MYB表达量逐渐升高，而绿萼长茄则几乎没有变化，与两个品种茄萼颜色变化相一致。【结论】ouSmDFR和dongSmDFR属于NADB-Rossmann超基因家族，ouSmMYB和dongSmMYB为典型R2R3-MYB转录因子，DFR和MYB在紫萼长茄果萼中表达明显高于绿萼长茄。推测DFR和MYB在茄萼呈色中发挥作用，并且参与花色苷生物合成。

关键词: 萼片, 花色苷, DFR, MYB, 基因表达, 茄

Abstract: 【Objective】Anthocyanins are the water-soluble secondary metabolites synthesized via the flavonoid pathway responsible for red, violet, and blue in different kinds of plant organs. These pigments are of benefit to human health. Eggplant is rich in anthocyanins. The molecular mechanism of anthocyanin biosynthesis in eggplant calyx is still unclear. The primary objectives of this experiment are to clone DFR and MYB genes in the eggplant, detect their expression at different developmental stages and in calyx with different colors, explore the role of DFR and MYB in anthocyanin synthesis in calyx of eggplant. This work expanded our knowledge about the molecular mechanism of anthocyanin biosynthesis in eggplant peel and calyx. 【Method】Green calyx and purple calyx of eggplants were used as materials. The content of anthocyanins in the calyx was measured by spectrophotometry at different pH. cDNA sequence of the DFR and MYB were isolated and cloned by using RACE techniques. Analyze the conserved domains and characteristics of DFR protein were analyzed. Phylogenetic analysis of homologous protein sequences of other plants and DFR was conducted, the phylogenetic tree was constructed to further analyze and identify genes. To predict its secondary structure, SPOMA was used on-line which provided by the ExPAsy website. The bioinformatics analysis of MYB was the same as DFR. Expression of DFR and MYB genes in calyx with different colors were investigated through quantitative real-time PCR.【Result】In this study, two full-length cDNA sequences of DFR were successfully cloned and denoted as ouSmDFR and dongSmDFR (GenBank accession Nos KX224250 and KX224251). ouSmDFR is 1 285 bp in cDNA length with an open reading frame of 858 bp corresponding to 285 amino acid residues. dongSmDFR is 1 249 bp in cDNA length with an open reading frame of 864 bp, which encoding 287 amino acids. Two full-length cDNA sequences of MYB gene were cloned and denoted as ouSmMYB and dongSmMYB (GenBank accession Nos KX224253 and KX224254). The corresponding cDNAs are 969 bp and 959 bp in length and the deduced proteins contained 153 amino acids, respectively. Results of secondary structure analysis exhibited that α-Helix and random coil were primary secondary structural components of the two DFR genes and two MYB genes. Sequence analysis showed that ouSmDFR and dongSmDFR have a NADPH-binding domain and substrate specific binding domain. ouSmMYB and dongSmMYB belonged to the R2R3-MYB transcription factors, which have R2 and R3 conserved domains and a bHLH binding domain. Phylogenetic tree analysis indicated that ouSmDFR and dongSmDFR have a close relation with StDFR and SlDFR, ouSmMYB and dongSmMYB have the closest genetic relationship with EsMYB. Results of total anthocyanins measurement showed that high contents of anthocyanins were detected in purple calyx and increased gradually, while no anthocyanins were detected in green calyx. The qRT-PCR analysis indicated that the expression of DFR and MYB genes in the purple calyx were much higher than that in the green calyx. Moreover, from early budding to full flowering,the expression of dongSmDFR and dongSmMYB increased gradually. However, ouSmDFR and ouSmMYB almost had no change, which were consistented with the color variation of calyx.【Conclusion】ouSmDFR and dongSmDFR belong to the NADB-Rossmann superfamily, ouSmMYB and dongSmMYB are typical R2R3-MYB transcription factors. The expression of DFR and MYB in the purple calyx is significantly higher than that in the green calyx. Therefore, it is speculated that the DFR and MYB genes play a role in the color of eggplants’ calyx. Furthermore, DFR and MYB are involved in the process of anthocyanin biosynthesis.

Key words: calyx, anthocyanins, DFR, MYB, gene expression; Solanum melongena L.

王海竹，曲红云，周婷婷，徐启江. 茄萼花色苷合成相关基因DFR和MYB克隆及表达分析[J]. 中国农业科学, 2017, 50(14): 2781-2792.

WANG HaiZhu, QU HongYun, ZHOU TingTing, XU QiJiang. Cloning and Expression Analysis of Anthocyanin Biosynthesis-Associated DFR and MYB Genes in Calyx of Eggplant (Solanum melongena L.)[J]. Scientia Agricultura Sinica, 2017, 50(14): 2781-2792.

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参考文献

[1] Hirakawa H, Shirasawa K, Miyatake K, Nunome T, Negoro S, Ohyama A, Yamaguchi H, Sato S, Isobe S, Tabata S, Fukuoka H. Draft genome sequence of eggplant (Solanum melongena L.): the representative solanum species indigenous to the old world. DNA Research, 2014, 21(6): 649-660.

[2] Wu X, Beecher G R, Holden J M, Haytowitz D B, Gebhardt S E, Prior R L. Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption.Journal of Agricultural and Food Chemistry, 2006, 54(11): 4069-4075.

[3] MARSZALEK K, WOZNIAK ?, KRUSZEWSKI B, SKAPAKA S. The effect of high pressure techniques on the stability of anthocyanins in fruit and vegetables. International Journal of Molecular Sciences, 2017, 18(2): 277.

[4] YOUSUF B, GUL K, WANI A A, SINGH P. Health benefits of anthocyanins and their encapsulation for potential use in food systems: A review.Critical Reviews in Food Science and Nutrition, 2016, 56(13): 2223-2230.

[5] SCHWINN K E, BOASE M R, BRADLEY J M, LEWIS D H, DEROLES S C, MARTIN C R, DAVIES K M. MYB and bHLH transcription factor transgenes increase anthocyanin pigmentation in petunia and lisianthus plants, and the petunia phenotypes are strongly enhanced under field conditions. Frontiers in Plant Science, 2014, 5: 603.

[6] IBRAHEEM F, GAFFOOR I, TAN Q, SHYU C R, CHOPRA S. A sorghum MYB transcription factor induces 3-deoxyanthocyanidins and enhances resistance against leaf blights in maize. Molecules, 2015, 20(2): 2388-2404.

[7] SCHWINN K, VENAIL J, SHANG Y, MACKAY S, ALM V, BUTELLI E, OYAMA R, BAILEY P, DAVIES K, MARTIN C. A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. The Plant Cell, 2006, 18(4): 831-851.

[8] 赵启明, 李范, 李萍. 花青素生物合成关键酶的研究进展. 生物技术通报, 2012, 28(12): 25-32.

ZHAO Q M, LI F, LI P. Research advances on core enzymes of anthocyanidin biosynthesis. Biotechnology Bulletin, 2012, 28(12): 25-32. (in Chinese)

[9] 焦淑珍, 刘雅莉, 娄倩, 姜玲. 葡萄风信子二氢黄酮醇4-还原酶基因(DFR)的克隆与表达分析. 农业生物技术学报, 2014, 22(5): 529-540.

JIAO S Z, LIU Y L, LOU Q, JIANG L. Cloning and expression analysis of dihydroflavonol 4-reductase gene (DFR) from Grape hyacinth (Muscari armeniacum). Journal of Agricultural Biotechnology, 2014, 22(5): 529-540. (in Chinese)

[10] 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(8): 2465-2483.

[11] BROUN P. Transcriptional control of flavonoid biosynthesis: a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis. Current Opinion in Plant Biology, 2005, 8(3): 272-279.

[12] HUANG W, SUN W, LV H, LUO M, ZENG S, PATTANAIK S, YUAN L, WANG Y. A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway. PLoS One, 2013, 8(8): e70778.

[13] FENG S, WANG Y, YANG S, XU Y, CHEN X. Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10. Planta, 2010, 232(1): 245-255.

[14] STARKEVIC P, PAUKSTYTE J, KAZANAVICIUTE V, DENKOVSKIEN? E, STANYS V, BENDOKAS V, ŠIKŠNIANAS T, RA?ANSKIEN? A, RA?ANSKAS R. Expression and anthocyanin biosynthesis-modulating potential of Sweet cherry (Prunus avium L.) MYB10 and bHLH genes. PLoS One, 2015, 10(5): e0126991.

[15] ESPLEY R V, HELLENS R P, PUTTERILL J, STEVENSON D E, KUTTY-AMMA S, ALLAN A C. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. The Plant Journal, 2007, 49(3): 414-427.

[16] 满玉萍, 李刚, 刘虹, 王彦昌, 覃瑞. ‘红阳’猕猴桃MYB基因的克隆与表达. 华中农业大学学报, 2012, 31(6): 679-685.

MAN Y P, LI G, LIU H, WANG Y C, QIN R. Cloning and expression analysis of MYB in Actinidia chinesis ‘Hong yang’. Journal of Huazhong Agricultural University, 2012, 31(6): 679-685. (in Chinese)

[17] MATHEWS H, CLENDENNEN S K, CALDWELL C G, LIU X L, CONNORS K, MATHEIS N, SCHUSTER D K, MENASCO D J, WAGONER W, LIGHTNER J, WAGNER D R. Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. The Plant Cell, 2003, 15(8): 1689-1703.

[18] ZHANG Y, HU Z, CHU G, HUANG C, TIAN S, ZHAO Z, CHEN G. Anthocyanin accumulation and molecular analysis of anthocyanin biosynthesis-associated genes in eggplant (Solanum melongena L.). Journal of Agricultural and Food Chemistry, 2014, 62(13): 2906-2912.

[19] JIANG M M, REN L, LIAN H L, LIU Y, CHEN H Y. Novel insight into the mechanism underlying light-controlled anthocyanin accumulation in eggplant (Solanum melongena L.). Plant Science, 2016, 249: 46-58.

[20] WROLSTAD R E, CULBERTSON J D, CORNWELL C J, MATTICK L R. Detection of adulteration in blackberry juice concentrates and wines Journal–Association of Official Analytical Chemists, 1982, 65 (6): 1417-1423.

[21] 朱奇朗, 李晓波, 肖向文, 李雪源, 黄先忠, 郑巨云, 艾先涛. 不同色彩矮牵牛DFR基因的克隆与生物信息学分析. 生物技术通报, 2013, 29(9): 68-76.

ZHU Q L, LI X B, XIAO X W, LI X Y, HUANG X Z, ZHENG J Y, AI X T. Cloning and bioinformatical analysis of dihydroflavonol 4-reductase gene（DFR）from Petunia hybrida with different Color. Biotechnology Bulletin, 2013, 29(9): 68-76. (in Chinese)

[22] 周惠, 文锦芬, 邓明华,朱海山. 植物花青素生物合成相关基因研究进展. 辣椒杂志, 2011(4): 1-7.

ZHOU H,WEN J F,DENG M H, ZHU H S. Research progress in plant anthocyanidin biosynthesis genes. Journal of China Capsicum, 2011(4): 1-7. (in Chinese)

[23] MIOSIC S, THILL J, MILOSEVIC M, GOSCH C, POBER S, MOLITOR C, EJAZ S, ROMPEL A, STICH K, HALBWIRTH H. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species. PLoS One, 2014, 9(11): e112707.

[24] WANG L, ZHU Y, WANG P, FAN Q, WU Y, PENG Q Z, XIA G X, WU J H. Functional characterization of a dihydroflavanol 4-reductase from the fiber of upland cotton (Gossypium hirsutum). Molecules, 2016, 21(2): 32.

[25] LUO P, NING G, WANG Z, SHEN Y, JIN H, LI P, HUANG S, ZHAO J, BAO M. Disequilibrium of flavonol synthase and dihydroflavonol- 4-reductase expression associated tightly to white vs. red color flower formation in plants. Frontiers in Plant Science, 2015, 6: 1257.

[26] 焦淑珍. 葡萄风信子DFR基因克隆及表达分析[D]. 杨凌: 西北农林科技大学, 2014.

JIAO S Z. Cloning and expression analysis of DFR genes from grape hyacinths [D]. Yangling: Northwest Agriculture and Forestry University, 2014. (in Chinese)

[27] GROTEWOLD E,SAINZ M B,TAGLIANI L,HERNANDEZ J M,BOWEN B,CHANDLER V L. Identification of the residues in the Myb domain of maize C1 that specify the interaction with the bHLH cofactor R. Proceedings of the National Academy of Sciences, 2000, 97: 13579-13584.

[28] STRACKE R, WERBER M, WEISSHAAR B. The R2R3-MYB gene family in Arabidopsis thaliana. Current Opinion in Plant Biology, 2001, 4(5): 447-456

[29] Albert N W, Griffiths A G, Cousins G R, Verry I M, Williams W M. Anthocyanin leaf markings are regulated by a family

of R2R3-MYB genes in the genus Trifolium. New Phytologist, 2015, 205(2): 882-893.

[30] FELLER A, MACHEMER K, BRAUN E L, GROTEWOLD E. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant Journal, 2011, 66(1): 94-116.

[31] 赵佳, 刘荣, 杨帆, 李鑫, 刘厚生, 严倩, 肖月华. 月季花青素苷相关R2R3-MYB蛋白基因的克隆和表达分析. 中国农业科学, 2015, 48(7): 1392-1404.

ZHAO J, LIU R, YANG F, LI XIN, LIU H S, YAN Q, XIAO Y H. Cloning and expression analyses of R2R3-MYB genes related to anthocyanin biosynthesis in rose. Scientia Agricultura Sinica, 2015, 48(7): 1392-1404. (in Chinese)

[32] 许玲, 卫培培, 张大勇, 徐照龙, 何晓兰, 黄益洪, 马鸿翔, 邵宏波. 大豆转录因子基因GmMYB111的克隆及功能分析. 中国农业科学, 2015, 48(15): 3079-3089.

XU L, WEI P P, ZHANG D Y, XU Z L, HE X L, HUANG Y H,MA H X, SHAO H B. Expression and function analysis of the transcription factor GmMYB111 in soybean. Scientia Agricultura Sinica, 2015, 48(15): 3079-3089. (in Chinese)

[33] WANG Z, MENG D, WANG A, LI T, JIANG S, CONG P, LI T. The methylation of the PcMYB10 promoter is associated with green- skinned sport in Max Red Bartlett pear. Plant Physiology, 2013, 162(2): 885-896.

[34] VIMOLMANGKANG S, HAN Y, WEI G, KORBAN S S. An apple MYB transcription factor, MdMYB3, is involved in regulation of anthocyanin biosynthesis and flower development. BMC Plant Biology, 2013, 13: 176-189.

[35] BOGS J, JAFFE F W, TAKOS A M, WALKER A R, ROBINSON S P. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiology, 2007, 143(3): 1347-1361.

[36] TERRIER N, TORREGROSA L, AGEORGES A, VIALET S, VERRIÈS C, CHEYNIER V, ROMIEU C. Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway. Plant Physiology, 2009, 149(2): 1028-1041.

[37] LIM S H, SONG J H, KIM D H, KIM J K, LEE J Y, KIM Y M, HA S H. Activation of anthocyanin biosynthesis by expression of the radish R2R3-MYB transcription factor gene RsMYB1. Plant Cell Reports, 2016, 35(3): 641-653.