葡萄转录因子VvERF2耐盐功能鉴定

doi:10.3864/j.issn.0578-1752.2024.02.009

摘要/Abstract

摘要：

【目的】对葡萄转录因子VvERF2进行蛋白质生物信息学分析，利用基因克隆、同源遗传转化技术，探索该转录因子在葡萄愈伤组织盐胁迫下的功能，为进一步研究AP2/ERF超家族对葡萄的作用机理提供参考。【方法】借助NCBI-Blast（https://blast.ncbi.nlm.nih.gov/Blast.cgi）等在线数据库工具对VvERF2蛋白进行生物信息学分析；以‘无核白’葡萄（Vitis. vinifera L.）愈伤组织为材料，构建葡萄VvERF2同源遗传转化体系，结合生长量、总糖、总酸等理化指标鉴定转基因愈伤组织表型；设定不同盐浓度梯度，通过游离脯氨酸、抗氧化酶活性等生理指标鉴定转基因愈伤组织耐盐功能。【结果】对VvERF2及一致性最高的7个直系同源蛋白序列进行生物信息学分析，发现VvERF2编码240个氨基酸，与番茄、无花果氨基酸序列高度相似，蛋白同源性分别为78%和67%。8种不同物种的氨基酸残基数为240—348个，分子量为26.43—38.60 kDa，理论等电点在5.54—8.68，脂肪氨基酸指数均大于66%，均属于不稳定性蛋白；不同物种氨基酸序列理化性质存在差异较大。此外，VvERF2启动子存在多种与脱落酸等激素及MYB等转录因子相关的顺式作用元件。VvERF2具有组织表达特异性，在愈伤组织中表达水平最低，且受外源盐胁迫诱导显著上调（为对照组的3倍）。转基因结果表明，VvERF2在葡萄愈伤组织中过量表达后，生长量、总酸、总酚含量及DPPH（1,1-二苯基-2-三硝基苯肼，1,1-Diphenyl-2- trinitrophenylhydrazine）等抗氧化活性均显著升高，不同浓度外源NaCl处理后，转基因愈伤组织总蛋白、游离脯氨酸等含量均高于野生型愈伤组织。【结论】 VvERF2过量表达促进葡萄愈伤组织生长量和次生代谢产物相关物如酚类物质的含量积累，从而提高葡萄耐盐性。

关键词: 葡萄, VvERF2, 盐胁迫, 功能鉴定

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

代瑛姿, 郭宏扬, 杨志峰, 王宪璞, 许丽丽. 葡萄转录因子VvERF2耐盐功能鉴定[J]. 中国农业科学, 2024, 57(2): 336-348.

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*