葡萄转录因子VvERF2耐盐功能鉴定

doi:10.3864/j.issn.0578-1752.2024.02.009

Abstract

Abstract:

【Objective】 In order to give references for future study on the mechanism of the AP2/ERF superfamily on grapes, the protein bioinformatics analysis of grape transcription factor VvERF2 was performed. Additionally, the procedures of gene cloning and homologous genetic transformation were employed for exploring the function of VvERF2 under salt stress in grape callus. 【Method】 For the bioinformatics analysis of the VvERF2 protein, the NCBI Blast database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and additional online resources were utilized. The Thompson seedless (Vitis vinifera L.) callus was used as the material, and the grape homologous genetic transformation system of VvERF2 were constructed. The transgenic callus phenotype was determined by growth volume, total sugar, total acid, and other factors. Free proline, antioxidant enzyme activity, and other indices were used to assess the salt tolerance of transgenic callus. 【Result】 Based on the bioinformatical analysis of VvERF2 and the 7 most homologous orthologous protein sequences, the VvERF2 gene encoded 240 amino acids, which were quite similar to those of tomatoes and figs, with protein homology percentages of 78% and 67%, respectively. The amino acid residues in eight species varied from 240 to 348, their molecular weights from 26.43 to 38.60 kDa, their theoretical isoelectric points from 5.54 to 8.68, and their index of fatty amino acids were all belonged to unstable proteins, which was higher than 66%. The physicochemical properties of amino acid sequences in different species were quite different. In addition, the promoter of VvERF2 gene had a variety of cis-acting element related to abscisic acid and other transcription factors, such as MYB. Particularly, VvERF2 expressed specificity in different tissues, with callus exhibiting the lowest level of expression. Following salt stress, however, VvERF2 gene expression increased to three times that of the control group. Transgenic results showed that after overexpression of VvERF2 gene in grape callus, growth amount, total acid, total phenol content and antioxidant activity of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) were significantly increased. The content of total protein and free proline in transgenic callus were almost higher than those in wild-type callus treated with different concentrations of NaCl. 【Conclusion】 The overexpression of VvERF2 promoted callus growth and accumulation of secondary metabolites, such as phenolic substances, and improved salt tolerance of grape.

Key words: grape, VvERF2, salt stress, functional identification

DAI YingZi, GUO HongYang, YANG ZhiFeng, WANG XianPu, XU LiLi. Identification of Salt Resistance Functional of Grape Transcription Factor VvERF2[J].Scientia Agricultura Sinica, 2024, 57(2): 336-348.

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名称 Name	上游引物 Forward primer (5′-3′)	下游引物 Reverse primer (5′-3′)
VvERF2	AACATCCTCATCACCCCTCCAT	AACGGTGTCCATCCGACATCA
β-Actin	CTTGCATCCCTCAGCACCTT	TCCTGTGGACAATGGATGGA

物种 Species	ID号 ID number	氨基酸残基数 Number of amino acid residue	分子量 Molecular weight (kDa)	理论等电点 Theoretical isoelectric point	不稳定系数 Instability index (%)	脂肪指数Aliphatic index	平均亲水性 Average hydrophilicity
葡萄 Vitis vinifera	XP_002279585.2	240	26.43	6.94	62.32	68.67	-0.555
苹果 Malus domestica	XP_008352414	276	29.62	8.45	57.23	70.98	-0.457
草莓 Fragaria ananassa	AZL19475	295	31.60	6.76	56.57	71.80	-0.363
中国白梨 Pyrus bretschneideri	XP_009369170.2	269	28.82	8.47	52.47	68.85	-0.455
桃 Prunus persica	XP_007209449	281	30.16	9.09	59.26	76.65	-0.411
无花果 Ficus carica	QID57935.1	348	38.60	5.54	55.88	66.75	-0.570
拟南芥 Arabidopsis thaliana	AB008103.1	266	28.97	8.68	56.72	67.86	-0.499
番茄 Solanum lycopersicum	NP_001316388.2	242	26.76	7.68	71.59	69.34	-0.433

元件 Motifs	ABRE	ARE	MYB	MYC	Box4	CGTCA-motif	MRE
数量 Quantity	2	2	4	6	7	4	2
功能 Function	参与脱落酸信号响应 Participate in abscisic acid signal response	厌氧诱导调控元件 Anaerobic induction regulatory element			参与光信号响应 Participate in optical signal response	茉莉酸甲酯响应信号调控元件 Methyl jasmonate responsive signal regulatory element	参与光信号响应的MYB结合位点 MYB binding sites involved in optical signal response

愈伤组织 Callus	生长量 Growth (g·dish^-1 FW)	总糖 Total sugar (mg·g^-1 FW)	总酸 Total acid (mg·g^-1 FW)	总酚 Total phenol (mg·g^-1 FW)	DPPH抗氧化活性 DPPH antioxidant activity (mg·g^-1 FW)	FRAP抗氧化活性 FRAP antioxidant activity (mg·g^-1 FW)	ABTS抗氧化活性 ABTS antioxidant activity (mg·g^-1 FW)
wt	0.33±0.017	26.04±1.02	0.14±0.0088	0.13±0.0012	0.86±0.010	1.60±0.45	1.38±0.32
erf2-1	0.41±0.065*	27.48±1.98	0.11±0.0069**	0.17±0.0032**	1.22±0.012*	2.88±0.27*	1.75±0.12*
erf2-2	0.40±0.027*	26.99±1.34	0.08±0.0060*	0.18±0.0021*	1.33±0.021*	3.51±0.073**	2.38±0.23*
erf2-3	0.39±0.051*	28.02±2.18	0.10±0.0021*	0.18±0.0032**	1.40±0.030*	3.51±0.10**	2.45±0.10*

Identification of Salt Resistance Functional of Grape Transcription Factor VvERF2

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