葡萄VvARF18调控果实膨大及其互作蛋白筛选

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

摘要/Abstract

摘要：

为探究葡萄VvARF18在果实发育中的分子机制，以‘红地球’葡萄（Vitis vinifera‘Red Globe’）为材料，克隆了VvARF18基因，对其进行生物信息分析；将该基因在番茄中异源过表达以验证其对果实发育的影响。VvARF18基因CDS长度为2 109 bp2 109 bp，编码703个氨基酸，pI为6.50。系统进化分析表明，VvARF18与河岸葡萄（Vitis riparis）VrARF18蛋白的亲缘关系最近；在番茄中异源过表达，T₃代转基因番茄果实纵横径显著高于野生型，其体积与单果质量明显增加，表明VvARF18对果实膨大具有正调控的作用；转录激活试验发现VvARF18没有毒性但具有自激活活性，筛选与其保守功能结构域B3-Auxin存在互作的蛋白，发现其与信号复合体VvCOP9存在潜在互作关系。

关键词: 葡萄, VvARF18, 亚细胞定位, 功能验证, 酵母双杂

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

梁靖, 曾宝珍, 梁国平, 毛娟, 陈佰鸿. 葡萄VvARF18调控果实膨大及其互作蛋白筛选[J]. 园艺学报, 2025, 52(4): 821-834.

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.

图/表 8

表1 本研究中的引物序列

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

图1 VvARF18的生物信息分析 A：基因结构分析；B：蛋白二级结构预测；C：蛋白三级结构预测；D：蛋白分子跨膜结构域预测；E：蛋白亲水性预测；F：近缘同源系统发育树

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

图2 VvARF18上游22 000 bp启动子分析

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

图3 VvARF18基因PCR电泳图谱 A：VvARF18基因克隆（M：Trans2K DNA marker；11 ~ 3表示克隆所得目的条带）；B：VvARF18基因表达载体大肠杆菌菌液PCR结果（M：Trans5K DNA marker；1 ~1 ~ 5表示VvARF18基因表达载体大肠杆菌菌液PCR条带）；C：VvARF18基因农杆菌菌液PCR结果（M：Trans2K DNA marker；1 ~1 ~ 5表示VvARF18基因表达载体农杆菌菌液PCR条带）

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）

图 4 VvARF18的亚细胞定位

Fig. 4 Subcellular localization of VvARF18

图5 VvARF18过表达番茄植株的验证（A、B）及果实形态（CC ~ F）和果实细胞观察（G、H） A：PCR鉴定VvARF18转基因株系；B：野生型（WT）和VvARF18过表达植株（OE-3、OE-5、OE-9）中VvARF18的表达量。C：野生型（WT）和过表达VvARF18转基因植株的表型；DD ~ E：过表达VvARF18转基因番茄单果质量、果实纵径和横径；G、H：野生型（WT）和过表达VvARF18的细胞图。M：Trans 5 000 bp5 000 bp DNA marker；P：阳性对照；N：阴性对照；11 ~ 10：鉴定转基因番茄10个株系的PCR条带。* * 表示与野生型相比在0.05水平上差异显著；不同小写字母表示材料间差异显著（P P < 0.05）

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

图6 VvARF18转录激活分析 A：自激活活性检测；B：蛋白结构域分段；C：自激活活性分段检测。10-1、10-2、10-3代表稀释倍数。BD为pGBKT7

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

图7 VvARF18-2-pGBKT7文库筛选（A）及PCR扩增鉴定（B）和COP9互作验证（C） M：Trans2K DNA Marker；1 1 ~ 17代表筛库后部分基因的PCR条带。DDO：SD/-Trp-Leu，QDO：SD/-Ade-His-Leu-Trp，X：X-α-gal。10-1， 10-2，10-3代表稀释倍数

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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