Grapevine VvARF18 Regulates Fruit Expansion and Screening of its Interacting Proteins

doi:10.16420/j.issn.0513-353x.2024-0594

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (4): 821-834.doi: 10.16420/j.issn.0513-353x.2024-0594

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Grapevine VvARF18 Regulates Fruit Expansion and Screening of its Interacting Proteins

LIANG Jing, ZENG Baozhen, LIANG Guoping, MAO Juan, CHEN Baihong^*()

College of Horticulture，Gansu Agricultural University，Lanzhou 730000

Received:2024-10-11 Revised:2025-03-10 Online:2025-05-08 Published:2025-04-25
Contact: CHEN Baihong

Abstract

Abstract:

In order to investigate the molecular mechanism of grape VvARF18 in fruit development，‘Red Globe’grapes（Vitis vinifera‘Red Globe’）were used as materials，the VvARF18 gene was cloned，and analyzed for its bioinformatics，and the gene was heterologously overexpressed in tomato to verify its effect on fruit development. The CDS of the VvARF18 gene was 22 109 bp in length，coding for 703 amino acids，with a pI of 6.50. Phylogenetic analysis showed that VvARF18 was the closest related to the VrARF18 protein of Vitis riparis；the gene was heterologously overexpressed in tomato. Expression，the longitudinal and transverse diameters of T₃ generation transgenic tomato fruits were significantly higher than those of the wild type，and its volume and single fruit mass were significantly increased，suggesting that VvARF18 has a positive regulatory effect on fruit expansion；transcriptional activation assay found that VvARF18 was not toxic but had self-activating activity，and screening of proteins interacting with its conserved functional structural domain B3-Auxin revealed a potential interplay relationship with the signaling complex VvCOP9.

Key words: grape, VrARF18, subcelluar localization, functional verification, yeast two-hybrid

LIANG Jing, ZENG Baozhen, LIANG Guoping, MAO Juan, CHEN Baihong. Grapevine VvARF18 Regulates Fruit Expansion and Screening of its Interacting Proteins[J]. Acta Horticulturae Sinica, 2025, 52(4): 821-834.

Figures/Tables 8

Table 1 Primers used in this study

用途 Application	引物名称 Primer name	引物序列（5′-3′）Primer sequence
构建过表达载体Construction of overexpression vectors	VvARF18	F：TTTGGAGAGGACACGCTCGAGATGGCGCATGGGAATAATATCA
构建过表达载体Construction of overexpression vectors		R：GCCCTTGCTCACCATGAATTCCTATGGTTCAGTTCTTAACTCTGAATCC
构建BD载体 Building the BD Carrier	VvARF18-BD	F：AGGCCGAATTCCCGGGGATCCTTATGGCGCATGGGAATAATATCA
构建BD载体 Building the BD Carrier		R：CCGCTGCAGGTCGACGGATCCCTATGGTTCAGTTCTTAACTCTGAATCC
	VvARF18-1	F：AGGCCGAATTCCCGGGGATCCTTATGGCGCATGGGAATAATATCA
		R：CCGCTGCAGGTCGACGGATCCCTAAAATGAATGAACGGTTTGTTTTG
	VvARF18-2	F：AGGCCGAATTCCCGGGGATCCTTATGTGCAAGATTTTAACGGCCT
		R：CCGCTGCAGGTCGACGGATCCCTACTTCAATGAACGCCATTTAGAA
	VvARF18-3	F：AGGCCGAATTCCCGGGGATCCTTATGATTCAATGGGATGAACCTGC
		R：CCGCTGCAGGTCGACGGATCCCTATGGTTCAGTTCTTAACTCTGAATCC
	VvCOP9	F：GTGGGCATCGATACGGGATCCTTATGGAACCCTACTCCTTCACATCC
		R：CAGCTCGAGCTCGATGGATCCTCAAGTTTCAATCATGGGTTCTGG
内参基因 Reference gene	Actin	F：GCCGACTACAACATCCAGAAGG
内参基因 Reference gene		R：TGCAACACAGCGAGCTTAACC

Fig. 1 Bioinformatics analysis of VvARF18 A：Gene structure analysis；B：Protein secondary structure prediction；C：Protein tertiary structure prediction；D：Protein molecule transmembrane structural domain prediction；E：Protein hydrophilicity prediction；F：Phylogenetic tree of close homologs

Fig. 2 Analysis of the 2 000 bp2 000 bp promoter before VvARF18

Fig. 3 PCR electrophoresis map of VvARF18 gene A：VvARF18 gene cloning（M：Trans2K DNA marker；1-3 indicates the target bands obtained from cloning）；B：VvARF18 gene expression vector Escherichia. coli bacterial liquid PCR results（M：Trans5K DNA marker；1-5 indicates VvARF18 gene expression vector Escherichia. coli bacterial liquid PCR band）；C：VvARF18 gene Agrobacterium bacterial liquid PCR results（M：Trans2K DNA marker；1-5 indicates VvARF18 gene expression vector Agrobacterium liquid PCR bands）

Fig. 4 Subcellular localization of VvARF18

Fig. 5 Validation of VvARF18 overexpressing tomato plants（A，B）and fruit morphology（C-F）and fruit cell observation（G，H） A：PCR identification of VvARF18 transgenic lines；B：Expression of VvARF18 in wild-type（WT）and VvARF18 overexpressing plants（OE-3，OE-5，OE-9）；C：Phenotypes of wild-type（WT）and overexpressing VvARF18 transgenic plants；D-F：Single fruit mass，longitudinal diameter and transverse diameter of overexpressed VvARF18 transgenic tomato；G，H：Cytological map of wild-type（WT）and overexpressing VvARF18；M：Trans5 000 bp000 bp DNA；P：Positive control；N：Negative control；1-10：PCR bands of 10 strains of transgenic tomato identified. * indicates significant difference at 0.05 level compared with wild-type

Fig. 6 Analysis of VvARF18 transcriptional activation A：Self-activating activity assay；B：Protein structural domain segmentation；C：Segmented assay of self-activating activity. 10-1，10-2，and 10-3 represent dilutions. pGBKT7 for BD

Fig. 7 Screening of VvARF18-2-pGBKT7 library（A）and identification of PCR amplification（B）and validation of COP9 interactions（C） M：Trans2K DNA Marker；1-17 represent PCR bands for selected genes after screening library. DDO：SD/-Trp-Leu，QDO：SD/-Ade-His-Leu-Trp，and X：X-α-gal. 10-1，10-2，10-3 represent dilutions

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Grapevine VvARF18 Regulates Fruit Expansion and Screening of its Interacting Proteins

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