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

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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"

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