Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (21): 4178-4185.doi: 10.3864/j.issn.0578-1752.2017.21.012

• HORTICULTURE • Previous Articles     Next Articles

Molecular Cloning and Expression Analysis of Cytokinins Responsive Gene MdMYB308 in Red Flesh Apple

WANG YiCheng, WANG Nan, XU HaiFeng, ZHANG ZongYing, JIANG ShengHui, ZHANG Jing, QU ChangZhi, CHEN XueSen   

  1. College of Horticultural Science and Engineering, Shandong Agricultural University/State Key Laboratory of Crop Biology, Tai’an 271018, Shandong
  • Received:2017-05-07 Online:2017-11-01 Published:2017-11-01

RichHTML

6

PDF

473

Abstract: 【Objective】Cytokinin is an important hormone in the regulation of anthocyanin synthesis in plants. To develop the theory and technology for red flesh apple breeding, the function in the cytokinin regulating anthocyanin metabolism of MYB transcription factor gene MdMYB308 in ‘Zihong No.3’ from Malus sieversii f. neidzwetzkyana F1 population was studied. 【Method】The callus induced from the leaves of ‘Zihong No.3’ apple was used as materials. The MdMYB308 in ‘Zihong No.3’ was cloned and its bioinformation was analyzed. The expression level of MdMYB308 and anthocyanin biosynthesis related genes in callus which grown on different concentrations of 6-BA was studied by the qRT-PCR. Meanwhile, the interaction between MdbHLH3 and MdMYB308 was verified by yeast two-hybrid system and bimolecular fluorescence complementation assay. 【Result】 The full length of MdMYB308 in ‘Zihong No.3’ was cloned, and the gene was 768 bp which encoded 255 amino acids. It was predicted that the molecular mass of this protein was 28.37 kD, and pI was 8.94. A phylogenetic tree indicated that MdMYB308, AtMYBL2, FaMYB1 and AtMYB4 are located in the same evolutionary branch. The aligned protein sequences revealed that MdMYB308 contain the EAR motif. Furthermore, the content of anthocyanin rose as 6-BA concentration increased as well. The transcript levels of anthocyanin structural genes (MdCHS, MdDFR, MdUFGT) and transcription factors (MdMYB10, MdbHLH3) were significantly higher in callus grown on 1 mg?L-1 6-BA compared with 6-BA-deprived callus. In contrast, the expression of MdMYB308 was inhibited. The results of yeast two hybrid experiments and bimolecular fluorescence complementation assays showed that the MdMYB308 could interact with MdbHLH3.【Conclusion】Cytokinin (6-BA) could promote anthocyanin accumulation by down-regulating the expression of MdMYB308 which may destroy the combination of MdMYB308 and MdbHLH3.

Key words: apple, cytokinin, MdMYB308, yeast two hybrid experiments, bimolecular fluorescence complementation assays

[1]    王延玲, 张艳敏, 冯守千, 宋杨, 徐玉亭, 张友朋, 陈学森. 新疆红肉苹果果皮果肉呈色差异机理. 中国农业科学, 2012, 45(13): 2771-2778.
WANG Y L, ZHANG Y M, FENG S Q, SONG Y, XU Y T, ZHANG Y P, CHEN X S. The mechanism of red coloring difference between skin and cortex in Malus sieversii f. neidzwetzkyana (Dieck) Langenf. Scientia Agricultura Sinica, 2012, 45(13): 2771-2778. (in Chinese)
[2]    ZHANG C Y, CHEN X S, HE T M, LIU X L, FENG T, YUAN Z H. Genetic structure of Malus sieversii population from Xinjiang, China, revealed by SSR markers. Journal of Genetics and Genomics, 2007, 34(10): 947-955.
[3]    张艳敏, 冯涛, 张春雨, 何天明, 张小燕, 吴传金, 刘遵春, 王艳玲, 束怀瑞, 陈学森. 新疆野苹果研究进展. 园艺学报, 2009, 36(3): 447-452.
ZHANG Y M, FENG T, ZHANG C Y, HE T M, ZHANG X Y, WU C J, LIU Z C, WANG Y L, SHU H R, CHEN X S. Advances in research of the Malus sieversii (Lebed.) Roem. Acta Horticulturae Sinica, 2009, 36(3): 447-452. (in Chinese)
[4]    HU B, ZHAO J, LAI B, QIN Y, WANG H, HU G. Lcgst4 is an anthocyanin-related glutathione s-transferase gene in litchi chinensis sonn. Plant Cell Reports, 2016, 35(4): 831-843.
[5]    LI W, LIU Y, ZENG S, XIAO G, WANG G, WANG Y, PENG M, HUANG H W. Gene expression profiling of development and anthocyanin accumulation in kiwifruit (actinidia chinensis) based on transcriptome sequencing. PLoS ONE, 2015, 10(8): e0136439.
[6]    TOHGE T, ZHANG Y, PETEREK S, MATROS A, RALLAPALLI G, TANDRON Y A, BUTELLI E, KALLAM K, HERTKORN N, MOCK H P, MARTIN C, FERNIE A R. Ectopic expression of snapdragon transcription factors facilitates the identification of genes encoding enzymes of anthocyanin decoration in tomato. Plant Journal for Cell & Molecular Biology, 2015, 83(4): 686-704.
[7]    ZHAO X, YUAN Z, FENG L, FANG Y. Cloning and expression of anthocyanin biosynthetic genes in red and white pomegranate. Journal of Plant Research, 2015, 128(4): 687-696.
[8]    GUAN L, DAI Z, WU B H, WU, J, MERLIN I, HILBERT G, RENAUD C, GOMES E, EDWARDS E, LI S H, DELROT S. Anthocyanin biosynthesis is differentially regulated by light in the skin and flesh of white-fleshed and teinturier grape berries. Planta, 2016, 243(1): 23-41.
[9]    LU Y, ZHANG M, MENG X, WAN H, ZHANG J, TIAN J, HAO S, JIN K, YAO Y. Photoperiod and shading regulate coloration and anthocyanin accumulation in the leaves of malus crabapples. Plant Cell Tissue & Organ Culture, 2015, 121(3): 619-632.
[10]   MORO L, HASSIMOTTO N M A, PURGATTO E. Postharvest auxin and methyl jasmonate effect on anthocyanin biosynthesis in red raspberry (Rubus idaeus L.). Journal of Plant Growth Regulation, 2017, 4(1): 1-10.
[11]   MOVAHED N, PASTORE C, CELLINI A, ALLEGRO G, VALENTINI G, ZENONI S, CAVALLINI E, DINCA E, TORNIELLI G B, FILIPPETTI I. The grapevine vviprx31 peroxidase as a candidate gene involved in anthocyanin degradation in ripening berries under high temperature. Journal of Plant Research, 2016, 129(3): 1-14.
[12]   ZHANG H N, LI W C, WANG H C, SHI S Y, BO S, LIU L Q, WEI Y Z, XIE J H. Transcriptome profiling of light-regulated anthocyanin biosynthesis in the pericarp of litchi. Frontiers in Plant Science, 2016, 7(225): 963-976.
[13]   OZEKI Y, KOMAMINE A. Induction of anthocyanin synthesis in relation to embryogenesis in a carrot suspension culture: correlation of metabolic differentiation with morphological differentiation. Physiologia Plantarum, 1981, 53(4): 570-577.
[14]   PECKET R C, BASSIM T A H. The effect of kinetin in relation to photocontrol of anthocyanin biosynthesis in Brassica oleracea. Phytochemistry, 1974, 13(8): 1395-1399.
[15]   DEIKMAN J, HAMMER P E. Induction of anthocyanin accumulation by cytokinins in Arabidopsis thaliana. Plant Physiology, 1995, 108(1): 47-57.
[16]   DAS P K, DONG H S, CHOI S B, YOO S D, CHOI G, PARK Y I. Cytokinins enhance sugar-induced anthocyanin biosynthesis in Arabidopsis. Molecules and Cells, 2012, 34(1): 93-101.
[17]   DUBOS C, LE G J, BAUDRY A, HUEP G, LANET E, DEBEAUJON I, ROUTABOUL J M, ALBORESI A, WEISSHAAR B, LEPINIEC L. MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. Plant Journal, 2008, 55(6): 940-953.
[18]   CHEN X S, FENG T, ZHANG Y M, HE T M, FENG J R, ZHANG C Y. Genetic diversity of volatile components in Xinjiang wild apple (Malus sieversii). Journal of Genetics and Genomics, 2007, 34(2): 171-179.
[19]   JI X H , WANG Y T , ZHANG R, WU S J , AN M M, LI M, WANG C Z , CHEN X L , ZHANG Y M , CHEN X S. Effect of auxin, cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell, Tissue and Organ Culture, 2015, 120(1): 325-337.
[20]   刘静轩, 许海峰, 王得云, 张宗营, 王意程, 左卫芳, 王楠, 姜生辉, 毛志泉, 陈学森. 两个耐贮性不同的红肉苹果株系果实硬度与香气成分及相关酶活性与基因表达差异分析. 园艺学报, 2017, 44(2): 330-342.
LIU J X, XU H F, WANG D Y, ZHANG Z Y, WANG Y C, ZUO W F, WANG N, JIANG S H, MAO Z Q, CHEN X S. Changes of firmness, aroma, cell wall-modifying enzyme activities and analysis of related-gene expression in 2 red flesh apple strains during fruit storage. Acta Horticulturae Sinica, 2017, 44(2): 330-342. (in Chinese)
[21]   许海峰, 王楠, 姜生辉, 王意程, 刘静轩, 曲常志, 王得云, 左卫 芳, 张晶, 冀晓昊, 张宗营, 毛志泉, 陈学森. 新疆红肉苹果杂种一代4个株系类黄酮含量比较及其合成相关基因表达分析. 中国农业科学, 2016, 49(16): 3174-3187.
XU H F, WANG N, JIANG S H, WANG Y C, LIU J X, QU C Z, WANG D Y, ZUO W F, ZHANG J, JI X H, ZHANG Z Y, MAO Z Q, CHEN X S. Comparison of content and analysis of biosynthesis- related genes in flavonoid among four strains of Malus sieversii f. neidzwetzkyana F1 population. Scientia Agricultura Sinica, 2016, 49(16): 3174-3187. (in Chinese)
[22]   ZHANG Z Y, JIANG S H, WANG N, LI M, JI X H, SUN S S, LIU J X, WANG D Y, XU H F, QI S M, WU S J, FEI Z J, FENG S Q, CHEN X S. Identification of differentially expressed genes associated with apple fruit ripening and softening by suppression subtractive hybridization. PLoS ONE, 2015, 10(12): e0146061.
[23]   AN X H, TIAN Y, CHEN K Q, LIU X J, LIU D D, XIE X B. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant Cell Physiology, 2015, 56(4): 650-662.
[24]   SUN J J, WANG Y C, CHEN X S, GONG X J, WANG N, MA L, QIU Y F,WANG Y L, FENG S Q. Effects of methyl jasmonate and abscisic acid on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell Tissue Organ Culture, 2017, 130:227-237.
[25]   CIFTCI-YILMAZ S, MORSY M R, SONG L H, COUTU A, KRIZEK B A, LEWIS M W, WARREN D, CUSHMAN J, CONNOLLY E L, MITTLER R. The EAR-motif of the Cys2/His2-type zinc finger protein Zat7 plays a key role in the defense response of Arabidopsis to salinity stress. Journalof Biological Chemistry, 2007, 282(21): 9260-9268.
[26]   HIRATSU K, OHTA M, MATSUI K, OHME-TAKAGI M. The SUPERMAN protein is an active repressor whose carboxy-terminal repression domain is required for the development of normal flowers. Febsletters, 2002, 514(2): 351-354.
[27]   KAZAN K. Negative regulation of defence and stress genes by EAR-motif-containing repressors. Trends in Plant Science, 2006, 11(3): 109-112.
[28]   HEMM M R, HERRMANN K M, CHAPPLE C. AtMYB4:A transcription factor general in the battle against UV. Trends in Plant Science, 2001, 6(4): 135-136.
[29]   JIN H L, COMINELLI E, BAILEY P, PARR A, MEHRTENS F, JONES J, TONELLI C. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO Journal, 2000, 19(22): 6150-6161.
[30]   AHARONI A, De VOS C, 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. Plant Journal for Cell & Molecular Biology, 2001, 28(3): 319-332.
[31]   PAOLOCCI F, ROBBINS M P, PASSERI V, HAUCK B, MORRIS P, RUBINI A ARCIONI S, DAMIANI F. The strawberry transcription factor FaMYB1 inhibits the biosynthesis of proanthocyanidins in Lotus corniculatus leaves. Journal of Experimental Botany, 2011, 62(3): 1189-1200.
[1] SU YiFan, YANG ZhanXu, WANG Di, MAO JunCheng, WEI MengMeng, CHEN Ze, BAI XinRan, CHU TianGe, MA ChangNing, QIAO MingFei, SUN Quan, HU DaGang. Effects of 2, 4-Epibrassinolide on Postharvest Storage Quality and Physiological Performance of Apple [J]. Scientia Agricultura Sinica, 2026, 59(7): 1536-1551.
[2] CONG QiQi, ZHANG JingYi, MENG XiangLong, DAI PengBo, LI Bo, HU TongLe, WANG ShuTong, CAO KeQiang, WANG YaNan. Identification of Hypovirus in Apple Ring Rot Fungus Botryosphaeria dothidea and Detection of Virus-Carrying Status in China [J]. Scientia Agricultura Sinica, 2025, 58(3): 478-492.
[3] PAN Yuan, WANG De, LIU Nan, MENG XiangLong, DAI PengBo, LI Bo, HU TongLe, WANG ShuTong, CAO KeQiang, WANG YaNan. Evaluation of the Effectiveness of Two High-Throughput Sequencing Techniques in Identifying Apple Viruses and Identification of Two Novel Viruses [J]. Scientia Agricultura Sinica, 2025, 58(2): 266-280.
[4] ZHANG Yi, LIU Ying, CHENG CunGang, LI YanQing, LI Zhuang. Effects of Combined Application Proportion of Cow Manure and Chemical Fertilizer on Soil Organic Carbon Pool and Enzyme Activity in Apple Orchard [J]. Scientia Agricultura Sinica, 2024, 57(20): 4107-4118.
[5] ZHOU HanMi, MA LinShuang, SUN QiLi, CHEN JiaGeng, LI JiChen, SU YuMin, CHEN Cheng, WU Qi. Optimization of Integrated Water and Nitrogen Regulation System in Apple Based on Multi-Objective Comprehensive Evaluation [J]. Scientia Agricultura Sinica, 2024, 57(18): 3654-3670.
[6] ZENG YanXin, GONG HaoNan, YOU ChunXiang, LU JingSheng, GAO WenSheng, WANG XiaoFei. Effects of Different Rootstocks on Growth and Fruit Quality of Young Ruixianghong Apple Trees with Multi-Stem Shape [J]. Scientia Agricultura Sinica, 2024, 57(14): 2847-2861.
[7] ZHANG HaiQing, ZHANG HengTao, GAO QiMing, YAO JiaLong, WANG YaRong, LIU ZhenZhen, MENG XiangPeng, ZHOU Zhe, YAN ZhenLi. Transcriptome Analysis for Screening Key Genes Related to Regulating Branching Ability in Apple [J]. Scientia Agricultura Sinica, 2024, 57(10): 1995-2009.
[8] SUN Zheng, LAI ZhongXiao, ZHAO XiaoMin, JIANG ZhiLi, CHEN GuangYou, MA ZhiQing. Application Evaluation of the Whole-Process Biological Management Scheme for Apple Pests in the Weibei Dry Highland [J]. Scientia Agricultura Sinica, 2023, 56(6): 1102-1112.
[9] ZHENG WenYan, CHANG YuanSheng, HE Ping, HE XiaoWen, WANG Sen, GAO WenSheng, LI LinGuang, WANG HaiBo. Development and Validation of KASP Markers Based on a Whole- Genome Resequencing Approach in a Hybrid Population of Luli × Red No. 1 [J]. Scientia Agricultura Sinica, 2023, 56(5): 935-950.
[10] WANG ZiDun, WANG Hui, FENG YuChen, ZHANG XueLiang, YAN LeiYu, LIU XiaoJie, ZHAO ZhengYang. Effects of Different Color Fruit Bags on Quality of Ruixue Apple Fruits [J]. Scientia Agricultura Sinica, 2023, 56(4): 729-740.
[11] LI XingXing, ZHOU GuoFu, LUO GuanYu, CHEN SiRong, ZHANG JinLong, CHEN GuoHua, ZHANG XiaoMing. Selection Preference and Adaptability of Bactrocera dorsalis to Different Varieties of Malus pumila [J]. Scientia Agricultura Sinica, 2023, 56(17): 3358-3371.
[12] LI MinJi, LI XingLiang, ZHANG Qiang, ZHOU Jia, YANG YuZhang, ZHOU BeiBei, ZHANG JunKe, WEI QinPing. Effects of Different Distances from Original Planting Row on Tree Growth and Fruit Yield of Young Trees of G935 Dwarf Rootstock Miyato Fuji Under Continuous Cropping [J]. Scientia Agricultura Sinica, 2023, 56(17): 3412-3419.
[13] FU Shan, LIANG Ye, XU JiuLiang, RUAN YunZe, LUO Jian, LI TingYu. Comprehensive Evaluation of Fruit Texture and Taste Quality of Pineapple Based on Multiple Methods [J]. Scientia Agricultura Sinica, 2023, 56(15): 3006-3019.
[14] LI JiaQi, XUN Mi, SHI JunYuan, SONG JianFei, SHI YuJia, ZHANG WeiWei, YANG HongQiang. Response Characteristics of Rhizosphere and Root Endosphere Bacteria and Rhizosphere Enzyme Activities to Soil Compaction Stress in Young Apple Tree [J]. Scientia Agricultura Sinica, 2023, 56(13): 2563-2573.
[15] DONG YongXin,WEI QiWei,HONG Hao,HUANG Ying,ZHAO YanXiao,FENG MingFeng,DOU DaoLong,XU Yi,TAO XiaoRong. Establishment of ALSV-Induced Gene Silencing in Chinese Soybean Cultivars [J]. Scientia Agricultura Sinica, 2022, 55(9): 1710-1722.
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