Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (3): 503-511.doi: 10.3864/j.issn.0578-1752.2019.03.010

• HORTICULTURE • Previous Articles     Next Articles

Molecular Cloning and Functional Characterization of VcNAC072 Reveals Its Involvement in Anthocyanin Accumulation in Blueberry

SONG Yang,LIU HongDi,WANG HaiBo,ZHANG HongJun,LIU FengZhi()   

  1. Research Institute of Pomology, Chinese Academy of Agricultural Sciences/Key Laboratory of Fruit Germplasm Resources Utilization, Ministry of Agriculture/Laboratory of Mineral Nutrition and Efficient Fertilization for Deciduous Fruits, Liaoning Province, Xingcheng 125100, Liaoning
  • Received:2018-08-27 Accepted:2018-09-28 Online:2019-02-01 Published:2019-02-14
  • Supported by:
    2015059

RichHTML

23

PDF

386

Abstract:

【Objective】 The objective of this study was to isolate a transcription factor VcNAC072 (NAM, ATAF1/2, CUC2) from blueberry by PCR technology and studying its expression, and to identify its role in anthocyanin biosynthesis. This study laid the foundation for further study of the molecular mechanism of VcNAC072 affecting anthocyanin biosynthesis in blueberry. 【Method】 The blueberry VcNAC072 gene was cloned by PCR technology from the fruits of blueberry (Vaccinium corymbosum ‘Duke’). The transgenic Arabidopsis thaliana were generated via Agrobacterium-mediated transformation. The differences in the anthocyanin accumulation were compared between transgenic and wild-type Arabidopsis. The yeast one-hybrid (Y1H) and transient expression assays were carried out to test the transcriptional regulation of MYB transcription factor AtPAP1 by VcNAC072. 【Result】 A blueberry VcNAC072 was cloned from blueberry. Sequence analysis showed that the coding domain sequence (CDS) of VcNAC072 was 1 032 bp, which encoded 343 amino acids. Protein structure analysis showed that VcNAC072 contained a NAC domain. Expression analysis showed that VcNAC072 was expressed at different developmental stages of the blueberry fruits. However, the expression levels varied, with the highest expression level in pink fruits and blue fruits and the relatively low transcript levels in green fruits. The content of anthocyanin in fruits was increased with the elevation of relative expression of VcNAC072. The sequence of AtPAP1 promoter was analyzed and a NAC binding motif was found. The VcNAC072 protein could interact with the promoter of AtPAP1. Moreover, the VcNAC072 could induce the expression of AtPAP1 and favorably contributed to anthocyanin accumulation in seeds of transgenic Arabidopsis. 【Conclusion】 It was speculated that VcNAC072 up-regulated anthocyanin accumulation in fruits of blueberry.

Key words: blueberry, NAC transcription factor, anthocyanin, expression analysis, functional identification

Table 1

Primers used in this study"

用途 Use 引物名称Primer name 序列（5′-3′）Primer sequence (5′-3′)
构建pRI101-VcNAC072表达载体
The construction of pRI101-VcNAC072 expression vector
构建pGADT7-VcNAC072表达载体
The construction of pGADT7-VcNAC072 expression vector
构建pAbAi-AtPAP1pro表达载体
The construction of pAbAi-AtPAP1pro expression vector
构建AtPAP1pro-GUS表达载体
The construction of AtPAP1pro-GUS expression vector
实时荧光定量qRT-PCR
Real time fluorescent quantitative PCR		 VcNAC072(pRI101)-F
VcNAC072(pRI101)-R
VcNAC072( pGADT7)-F
VcNAC072( pGADT7)-R
AtPAP1pro(pAbAi)-F
AtPAP1pro(pAbAi)-R
AtPAP1pro(GUS)-F
AtPAP1pro(GUS)-R
VcNAC072-(RT-PCR)-F
VcNAC072-(RT-PCR)-R
VcGAPDH-(RT-PCR)-F
VcGAPDH-(RT-PCR)-R
AtUBQ10-(RT-PCR)-F
AtUBQ10-(RT-PCR)-R
AtPAP1-(RT-PCR)-F
AtPAP1-(RT-PCR)-R
AtDFR-(RT-PCR)-F
AtDFR-(RT-PCR)-R
AtANS-(RT-PCR)-F
AtANS-(RT-PCR)-R		 GGAATTCCATATGATGGGAGTTCAGGAGTCCG
CGCGGATCCTCACTGCCGAAACCCGAATCC
GGAATTCCATATGATGGGAGTTCAGGAGTCCG
CGCGGATCCTCACTGCCGAAACCCGAATCC
CGAGCTCCTAATACATAAAATGTGGATATC
ACGCGTCGACGGAACAAAGATAGATACGTAA
CGGGGTACCCTAATACATAAAATGTGGATATC
CATGCCATGGGGAACAAAGATAGATACGTAA
GATGAGGTACAATGACATG
GAGTTCTGCTGAAACAAC
GGTTATCAATGATAGGTTTGGCA
CAGTCCTTGCTTGATGGACC
CGTTAAGACGTTGACTGGGAAAACT
GCTTTCACGTTATCAATGGTGTCA
GCTCTGATGAAGTCGATCTTC
CTACCTCTTGGCTTTCCTCT
GTCGGTCCATTCATCACAAC
TGAGCGTTGCATAAGTCGTC
TCAAGAAAGCCGGAGAAGAG
TTGTCCACTCGCGTTGTTAG

Fig. 1

Multiple sequence alignment of VcNAC072 and NAC protein from other plants"

Fig. 2

Phylogenetic relationships of VcNAC072 of blueberry and NAC protein of Arabidopsis"

Fig. 3

The change of relative expression of VcNAC072 and anthocyanidin content during fruit development"

Fig. 4

Effects of VcNAC072 over-expression on anthocyanin accumulation in Arabidopsis A: Phenotypes of transgenic VcNAC072 and wild-type Arabidopsis on the anthocyanin accumulation; B: Detection of the anthocyanin content; C: qRT-PCR analysis of the expression of anthocyanin biosynthesis genes, the value for WT was set to 1. Different lowercase letters indicate significant differences (P<0.05). The same as below"

Fig. 5

Y1H assay showed the interactions of VcNAC072 with promoter of AtPAP1"

Fig. 6

The effect of VcNAC072 on the gene expression of AtPAP1 by transient expression assays A: Quantitative analysis of relative GUS activity. B: Transient expression assay. a: pRI101 empty vector and AtPAP1pro-GUS. b: pRI101-VcNAC072 and AtPAP1pro-GUS"

[1] 李亚东, 孙海悦, 陈丽 . 我国蓝莓产业发展报告.中国果树, 2016(5):1-10.
LI Y D, SUN H Y, CHEN L . Report on the development of blueberry industry in China. China Fruits, 2016(5):1-10. (in Chinese)
[2] 吴林 . 中国蓝莓35年—科学研究与产业发展. 吉林农业大学学报, 2016,38(1):1-11.
WU L . Thirty-five years of research and industry development of blueberry in China. Journal of Jilin Agricultural University, 2016,38(1):1-11. (in Chinese)
[3] GORDILLO G, FANG H Q, KHANNA S, HARPER J, PHILIPS G, SEN C K . Oral administration of blueberry inhibits angiogenic tumor growth and enhances survival of mice with endothelial cell neoplasm. Antioxid Redox Signal, 2009,11(1):47-58.
doi: 10.1089/ars.2008.2150 pmid: 2933151
[4] BASU A, RHONE M, LYONS T J . Berries: Emerging impact on cardiovascular health. Nutrition Reviews, 2010,68(3):168-177.
doi: 10.1111/j.1753-4887.2010.00273.x pmid: 20384847
[5] SHAHNEJAT-BUSHEHRI S, TARKOWSKA D, SAKURABA Y, BALAZADEH S . Arabidopsis NAC transcription factor JUB1 regulates GA/BR metabolism and signalling. Nature Plants, 2016,2:16013.
[6] KIM H S, PARK B O, YOO J H, JUNG M S, LEE S M, HAN H J, KIM K E, KIM S H, LIM C O, YUN D J, LEE S Y, CHUNG W S . Identification of a calmodulin-binding NAC protein as a transcriptional repressor in Arabidopsis. Journal of Biological Chemistry, 2007,282(50):36292-36302.
[7] HONG Y B, ZHANG H J, HUANG L, LI D Y, SONG F M . Overexpression of a stress-responsive NAC transcription factor gene ONAC022 improves drought and salt tolerance in rice. Frontiers in Plant Science, 2016,7(e0116646):4.
doi: 10.3389/fpls.2016.00004 pmid: 26834774
[8] YU X W, LIU Y M, WANG S, TAO Y, WANG Z K, MIJITI A, WANG Z, ZHANG H, MA H . A chickpea stress-responsive NAC transcription factor,CarNAC5, confers enhanced tolerance to drought stress in transgenic. Plant Growth Regulation, 2016,79(2):187-197.
doi: 10.1007/s10725-015-0124-0
[9] KO J H, YANG S H, PARK A H, LEROUXEL O, HAN K H . ANAC012, a member of the plant-specific NAC transcription factor family, negatively regulates xylary fiber development in Arabidopsis thaliana. The Plant Journal, 2007,50(6):1035-1048.
doi: 10.1111/j.1365-313X.2007.03109.x pmid: 17565617
[10] ZHONG R, DEMURA T, YE Z H . SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers ofArabidopsis. The Plant Cell, 2006,18(11):3158-3170.
doi: 10.1105/tpc.106.047399 pmid: 17114348
[11] YAMAGUCHI M, KUBO M, FUKUDA H, DEMURA T . Vascular-related NAC-domain7 is involved in the differentiation of all types of xylem vessels in Arabidopsis roots and shoots. The Plant Journal, 2008,55(4):652-664.
doi: 10.1111/j.1365-313X.2008.03533.x pmid: 18445131
[12] ODA-YAMAMIZO C, MITSUDA N, SAKAMOTO S, OGAWA D, OHME-TAKAGI M, OHMIYA A . ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves. Scientific Reports, 2016,6:23609.
[13] TAKASAKI H, MARUYAMA K, TAKAHASHI F, FUJITA M, YOSHIDA T, NAKASHIMA K, MYOUGA F, TOYOOKA K, YAMAGUCHI-SHINOZAKI K, SHINOZAKI K . SNAC-As, stress-responsive NAC transcription factors, mediate ABA-inducible leaf senescence. The Plant Journal, 2015,84(6):1114-1123.
[14] ZHU Y, YAN J W, LIU W J, LIU L, SHENG Y, SUN Y, LI Y Y, SCHELLER H, JIANG M Y, HOU X L, NI L, ZHANG A Y . Phosphorylation of a NAC transcription factor by a calcium/ calmodulin-dependent protein kinase regulates abscisic acid-induced antioxidant defense in maize. Plant Physiology, 2016,171(3):1651-1664.
[15] ZHOU H, WANG K L, WANG H L, GU C, DARE A, ESPLEY R, HE H P, ALLAN A, HAN Y P . Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. The Plant Journal, 2015,82(1):105-121.
doi: 10.1111/tpj.12792 pmid: 25688923
[16] 安建平, 宋来庆, 赵玲玲, 由春香, 王小非, 郝玉金 . 苹果愈伤组织超表达MdNAC029促进花青苷积累. 园艺学报, 2018,45(5):845-854.
AN J P, SONG L Q, ZHAO L L, YOU C X, WANG X F, HAO Y J . Overexpression of MdNAC029 promotes anthocyanin accumulation in apple calli. Acta Horticulturae Sinica, 2018,45(5):845-854. (in Chinese)
[17] NURUZZAMAN M, MANIMEKALAI R, SHRRONI A M . Genome-wide analysis of NAC transcription factor family in rice. Gene, 2010,465(1/2):30-44.
doi: 10.1016/j.gene.2010.06.008 pmid: 20600702
[18] KIM S G, LEE S, SEO P J, KIM S K, KIM J K, PARK C M . Genome-scale screening and molecular characterization of membrane- bound transcription factors in Arabidopsis and rice. Genomics, 2010,95(1):56-65.
doi: 10.1016/j.ygeno.2009.09.003 pmid: 19766710
[19] SONG Y, LIU H D, ZHOU Q, ZHANG H J, ZHANG Z D, LI Y D, WANG H B, LIU F Z . High-throughput sequencing of highbush blueberry transcriptome and analysis of basic helix-loop-helix transcription factors. Journal of Integrative Agriculture, 2017,16(3):591-604.
doi: 10.1016/S2095-3119(16)61461-2
[20] YIN X R, ALLAN A C, CHEN K S, FERGUSON I B . Kiwifruit EIL and ERF genes involved in regulating fruit ripening. Plant Physiology, 2010,153(3):1280-1292.
[21] JEFFERSON R A, KAVANAGH T A, BEVAN M W . GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal, 1987,6(13), 3901-3907.
doi: 10.1089/dna.1987.6.583 pmid: 3327686
[22] PERTUZATTI P B, BARCIA M T , REBELLO L P G, GÓMEZ- ALONSO S, DUARTE R M T, DUARTE M C T, GODOY H T, HERMOSÍN-GUTIÉRREZ I .Antimicrobial activity and differentiation of anthocyanin profiles of rabbiteye and highbush blueberry using HPLC-DAD-ESI-MS and multivariate analysis. Journal of Functional Foods, 2016,26:506-516.
[23] ZHU X Y, CHEN J Y, XIE Z K, GAO J, REN G D, GAO S, ZHOU X, KUAI B K . Jasmonic acid promotes degreening via MYB2/3/4- and ANAC019/055/072-mediated regulation of major chlorophyll catabolic genes. The Plant Journal, 2015,84(3):597-610.
doi: 10.1111/tpj.13030 pmid: 26407000
[24] LI S, GAO J, YAO L Y, REN G D, ZHU X Y, GAO S, QIU K, ZHOU X, KUAI B K . The role of ANAC072 in the regulation of chlorophyll degradation during age- and dark- induced leaf senescence. Plant Cell Reports, 2016,35(8):1729-1741.
doi: 10.1007/s00299-016-1991-1 pmid: 27154758
[25] HUANG J C, PIATER L A, DUBERY I A . The NAC transcription factor gene ANAC072 is differentially expressed in Arabidopsis thaliana in response to microbe-associated molecular pattern (MAMP) molecules. Physiological and Molecular Plant Pathology, 2012,80(80):19-27.
doi: 10.1016/j.pmpp.2012.07.002
[26] TRAN L S, NAKASHIMA K, SAKUMA Y, SIMPSON S D, FUJITA Y, MARUYAMA K, FUJITA M, SEKI M, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K . Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. The Plant Cell, 2004,16(9):2481-2498.
doi: 10.1105/tpc.104.022699 pmid: 15319476
[27] LI X Y, SUN H Y, PEI J B, DONG Y Y, WANG F W, CHEN H, SUN Y P, WANG N, LI H Y, LI Y D . De novo sequencing and comparative analysis of the blueberry transcriptome to discover putative genes related to antioxidant. Gene, 2012,511(1):54-61.
doi: 10.1016/j.gene.2012.09.021 pmid: 22995346
[28] SUN H Y, LIU Y S, GAI Y Z, GENG J M, CHEN L, LIU H D, KANG L M, TIAN Y W, LI Y D . De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis, transport and regulation. BMC Genomics, 2015,16(1):652.
doi: 10.1186/s12864-015-1842-4 pmid: 4556307
[29] BOREVITZ J O, XIA Y J, BLOUNT J, DIXON R, LAMB C . Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. The Plant Cell, 2000,12(12):2383-2393.
doi: 10.2307/3871236 pmid: 11148285
[30] TAKOS A, JAFFÉ F, JACOB S, BOGS J, ROBINSON S, WALKER A . Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiology, 2006,142(3):1216-1232.
doi: 10.1104/pp.106.088104 pmid: 17012405
[31] YAO G F, MING M L, ALLAN A, GU C, LI L T, WU X, WANG R Z, CHANG Y J, QI K J, ZHANG S L, WU J . Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis. The Plant Journal, 2017,92:437-451.
[32] JAAKOLA L, POOLE M O, JONES M ,KÄMÄRÄINEN- KARPPINEN T,KOSKIMÄKI J,HOHTOLA A,HÄGGMAN H D,FRASER P,MANNING K J,KING G. A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits. Plant Physiology, 2010,153(4):1619-1629.
[33] SAKURABA Y, KIM Y S, HAN S H, LEE B D, PAEK N C . The Arabidopsis transcription factor NAC016 promotes drought stress responses by repressing AREB1 transcription through a trifurcate feed-forward regulatory loop involving NAP. The Plant Cell, 2015,27(6):1771-1787.
[34] QU Y T, DUAN M, ZHANG Z Q, DONG J L, WANG T . Overexpression of the Medicago falcata NAC transcription factor MfNAC3 enhances cold tolerance in Medicago truncatula. Environmental and Experimental Botany, 2016,129:67-76.
[1] LI ShiJia,LÜ ZiJing,ZHAO Jin. Identification of R2R3-MYB Subfamily in Chinese Jujube and Their Expression Pattern During the Fruit Development [J]. Scientia Agricultura Sinica, 2022, 55(6): 1199-1212.
[2] 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.
[3] YOU YuWan,ZHANG Yu,SUN JiaYi,ZHANG Wei. Genome-Wide Identification of NAC Family and Screening of Its Members Related to Prickle Development in Rosa chinensis Old Blush [J]. Scientia Agricultura Sinica, 2022, 55(24): 4895-4911.
[4] 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.
[5] 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.
[6] 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.
[7] LIU Chuang,GAO Zhen,YAO YuXin,DU YuanPeng. Functional Identification of Grape Potassium Ion Transporter VviHKT1;7 Under Salt Stress [J]. Scientia Agricultura Sinica, 2021, 54(9): 1952-1963.
[8] LÜ ShiKai, MA XiaoLong, ZHANG Min, DENG PingChuan, CHEN ChunHuan, ZHANG Hong, LIU XinLun, JI WanQuan. Post-transcriptional Regulation of TaNAC Genes by Alternative Splicing and MicroRNA in Common Wheat (Triticum aestivum L.) [J]. Scientia Agricultura Sinica, 2021, 54(22): 4709-4727.
[9] LIU YouChun,LIU WeiSheng,WANG XingDong,SUN Bin,LIU XiuLi,YANG YanMin,WEI Xin,YANG YuChun,ZHANG Duo,LIU Cheng,LI TianZhong. Identification of F1 Hybrids in Blueberry (Vaccinium corymbosum L.) Based on Specific-Locus Amplified Fragment Sequencing (SLAF-seq) [J]. Scientia Agricultura Sinica, 2021, 54(2): 370-378.
[10] HUANG JinFeng,LÜ TianXing,WANG Xu,WANG YingDa,WANG DongMei,YAN ZhongYe,LIU Zhi. Genome-Wide Identification and Expression Pattern Analysis of LRR-RLK Gene Family in Apple [J]. Scientia Agricultura Sinica, 2021, 54(14): 3097-3112.
[11] CUI HuLiang,HE Xia,ZHANG Qian. Anthocyanins and Flavonoids Accumulation Forms of Five Different Color Tree Peony Cultivars at Blooming Stages [J]. Scientia Agricultura Sinica, 2021, 54(13): 2858-2869.
[12] LIN Bing,CHEN YiQuan,ZHONG HuaiQin,YE XiuXian,FAN RongHui. Analysis of Key Genes About Flower Color Variation in Iris hollandica [J]. Scientia Agricultura Sinica, 2021, 54(12): 2644-2652.
[13] XU Ming,LIN ShiQiang,NI DongXin,YI HenJie,LIU JiangHong,YANG ZhiJian,ZHENG JinGui. Cloning and Function Characterization of Chalcone Synthase Gene AgCHS1 in Ampelopsis grossedentata [J]. Scientia Agricultura Sinica, 2020, 53(24): 5091-5103.
[14] WANG Feng,WANG XiuJie,ZHAO ShengNan,YAN JiaRong,BU Xin,ZHANG Ying,LIU YuFeng,XU Tao,QI MingFang,QI HongYan,LI TianLai. Light Regulation of Anthocyanin Biosynthesis in Horticultural Crops [J]. Scientia Agricultura Sinica, 2020, 53(23): 4904-4917.
[15] LU BaoShun,ZHU YongJing,ZHANG ShuTing,LÜ YuMeng,LI XiaoFei,SONG YuYang,LAI ZhongXiong,LIN YuLing. Whole-Genome Identification and Expression Analysis of SPL Gene Family in Dimocarpus Longan [J]. Scientia Agricultura Sinica, 2020, 53(20): 4259-4270.
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