山葡萄VaSR基因家族的鉴定及VaSR1抗寒功能验证与互作蛋白筛选

doi:10.16420/j.issn.0513-353x.2024-0109

摘要/Abstract

摘要：

为探究葡萄中丝氨酸/精氨酸富集剪接因子（SR）的抗寒功能，对山葡萄（Vitis amurensis Rupr.）低温休眠期不同温度时段处理下的枝条韧皮部转录组中筛选出的VaSR1进行克隆、抗寒功能鉴定与互作蛋白筛选。从山葡萄基因组数据库中鉴定到了13个VaSR家族成员，其保守功能结构域为RRM_SF superfamily。对其进行系统进化和共线性分析，结果显示VaSR的多数成员被聚类到SC亚族，且与苹果SR蛋白亲缘关系较近。对VaSR1进行抗寒功能验证，转基因拟南芥植株的冰冻存活率显著低于野生型；4 ℃低温胁迫后转基因植株叶片中可溶性糖含量和抗氧化酶及淀粉酶活性显著低于野生型，而淀粉含量显著增加；酵母双杂交显示VaU1SNRNP和VaCYP63是VaSR1的互作蛋白，具有促进前体mRNA的剪接和参与调节细胞中相关基因的聚腺苷酸化作用。综上，VaSR1过表达降低了转基因拟南芥中的淀粉酶活性，导致叶片中可溶性糖含量下降，减弱了植株的低温耐受性。此外，山葡萄中存在VaU1SNRNP和VaCYP63，能够与VaSR1发生互作。

关键词: 山葡萄, 转录因子, VaSR1, 低温胁迫, 酵母双杂交

Abstract:

To investigate the cold-resistance function of serine/arginine-rich splicing factor（SR）in grapevine，the present study selected VaSR1 from the transcriptome data of the phloem of branches treated with various temperature periods during the low-temperature dormancy period of Vitis amurensis Rupr.，and performed cloning，identification of cold-resistant functions，and screening of interacting proteins. Thirteen VaSR family members with the conserved functional domain RRM_SF superfamily were identified from the genome database of V. amurensis. Phylogenetic and collinearity analysis revealed that the majority of the members of the VaSRs were clustered into the SC subfamily and were closely related to the SR proteins of apple. The cold tolerance function of VaSR1 was verified，and the freezing survival rate of transgenic Arabidopsis plants was significantly lower than that of the wild type；the soluble sugar content and the activities of antioxidant enzymes and amylase in the leaf of transgenic plants were significantly lower than those of the wild type after 4 ℃ cold stress，while the starch content was significantly increased；the yeast two-hybridization elucidated that VaU1SNRNP and VaCYP63 were the intercalating proteins of VaSR1 with the ability to promote precursor mRNA splicing and involved in regulating polyadenylation of related genes in cells. Collectively，VaSR1 overexpression reduced amylase activity in transgenic plants，leading to a reduction in soluble sugar content in the leaves and attenuating the low-temperature tolerance of the plants. Furthermore，the presence of VaU1SNRNP and VaCYP63 in mountain grapes was capable of interacting with VaSR1.

Key words: Vitis amurensis, transcription factor, VaSR1, cold stress, yeast two-hybrid

梁国平, 曾宝珍, 刘铭, 边志远, 陈佰鸿, 毛娟. 山葡萄VaSR基因家族的鉴定及VaSR1抗寒功能验证与互作蛋白筛选[J]. 园艺学报, 2025, 52(1): 37-50.

LIANG Guoping, ZENG Baozhen, LIU Ming, BIAN Zhiyuan, CHEN Baihong, MAO Juan. Identification of VaSR Gene Family in Vitis amurensis，Verification of Cold Resistance Function of VaSR1 and Screening of Interacting Proteins[J]. Acta Horticulturae Sinica, 2025, 52(1): 37-50.

图/表 11

表1 山葡萄VaSR基因家族成员基本理化性质

Table 1 Physicochemical properties of members of the VaSR gene family in Vitis amurensis

基因 Gene	基因ID Gene ID	基因全长/ bp Gene length	CDS全长/ bp CDS length	氨基酸残基数 Amino acid residue number	相对分子量 Molecular weight	等电点 pI	不稳定指数 Instability index	亲水性 Hydropathicity index
VaSR1	VIT_209s0002g06810	1 599	921	307	32 952.0	4.70	55.38	-0.61
VaSR45A	VIT_200s0264g00120	3 478	783	261	30 353.1	11.50	87.59	49.50
VaSR45A-like	VIT_202s0025g00090	3 345	732	244	27 996.2	11.70	101.00	-1.23
VaSR45B	VIT_206s0004g06010	2 145	993	331	38 616.9	11.80	133.80	-1.42
VaRS31	VIT_215s0046g00050	2 580	726	242	28 559.7	9.80	74.15	-1.15
VaRS41	VIT_216s0100g00450	3 441	1 437	479	55 565.7	10.50	90.20	-1.65
VaRS41-like	VIT_215s0048g01870	3 980	924	308	35 855.2	9.90	63.80	-0.96
VaRSZ2-like	VIT_204s0069g00800	4 085	549	183	20 726.9	11.40	104.10	-1.34
VaRSZ22A	VIT_201s0026g00250	8 907	540	180	19 863.1	11.30	93.50	-1.13
VaRSZ32	VIT_208s0040g02860	2 897	801	267	30 402.1	10.52	110.00	-1.72
VaRSZ32-like	VIT_213s0067g03600	2 846	978	326	37 282.2	10.59	90.92	-1.57
VaSC35	VIT_218s0001g05550	2 089	774	258	29 815.9	11.26	94.78	-1.62
VaSC35-like	VIT_212s0142g00110	6 748	768	256	29 827.9	11.03	82.21	-1.40

图1 山葡萄VaSR基因家族成员的motif（A）、功能结构域（B）和基因结构（C）

Fig. 1 Motif（A），functional domain（B），and gene structure（C）of members of the VaSR gene family in Vitis amurensis

图2 拟南芥（At）、番茄（Sl）、苹果（Md）和山葡萄（Va）SR蛋白的系统进化树

Fig. 2 Phylogenetic tree of SR proteins in Arabidopsis thaliana（At），Solanum lycopersicum（Sl），Malus × domestica（Md）， and Vitis amurensis（Va）

图3 拟南芥（At）、番茄（Sl）、苹果（Md）和山葡萄（Va）SR基因家族成员之间的共线性关系绿色：两对基因之间存在 ≤ 50%的共线性；橙色：两对基因之间存在 ≤ 75%的共线性；红色：两对基因之间存在 ≤ 99%的共线性

Fig. 3 Co-linear relationships among SR gene family members of Arabidopsis thaliana（At），Solanum lycopersicum（Sl），Malus × domestica（Md），and Vitis amurensis（Va） Green：CO-linear relationships ≤ 50% between the two pairs of genes；Orange：Co-linear relationships ≤ 75% between the two pairs of genes；Red：Co-linear relationships ≤ 99% between the two pairs of genes

图4 VaSR的组织表达特异性

Fig. 4 Tissue-specific expression of members of the VaSRs

图5 不同温度阶段葡萄枝条韧皮部VaSR1基因的表达（A）及过表达VaSR1拟南芥冰冻后的表型（C）和存活率（B） * 表示不同处理与对照或转基因株系与野生型相比在0.05水平上具有显著性差异。下同

Fig. 5 Expression of VaSR1（A）in the phloem of branches at different temperature stages and survival rate（B）and phenotype（C）of overexpressing-VaSR1 Arabidopsis under frozen treatment * represents significant differences for different treatment with control or transgenic strain with wild type（Col）at P < 0.05. The same below

图6 4 ℃低温胁迫前后Col和过表达VaSR1拟南芥植株叶片的相对电导率和抗氧化酶活性变化

Fig. 6 Changes of relative electrolyte leakage and antioxidant enzyme activities in Col and overexpressing-VaSR1 Arabidopsis under 4 ℃ cold stress

图7 低温胁迫前后Col和过表达VaSR1拟南芥植株叶片中淀粉酶活性和淀粉含量的变化

Fig. 7 Changes of amylase activity and starch content in Col and overexpressing-VaSR1 Arabidopsis under cold stress

图8 低温胁迫前后Col和过表达VaSR1拟南芥植株叶片中可溶性糖含量的变化

Fig. 8 Changes of soluble sugar content in Col and overexpressing-VaSR1 Arabidopsis under cold stress

图9 酵母双杂交验证与VaSR1互作的蛋白

Fig. 9 Yeast two-hybridization to verify proteins interacting with VaSR1

表2 与VaSR1互作的蛋白信息

Table 2 Protein information interacting with VaSR1

互作蛋白 Interacting proteins	互作蛋白ID Interacting proteins ID	基因长度/bp Gene length	氨基酸数 Amino acid residue number	互作蛋白功能结构域 Interacting proteins function domain	互作蛋白功能描述 Interacting proteins character
U1SNRNP （尿苷酸小分子1核糖体蛋白-70K U1 small nuclear ribonucleoprotein 70 kD）	XP_010649159	9 406	514	RRM_snRNP70 U1snRNP70_N PRK12678 superfamily	促进前体mRNA的剪接，并参与调节细胞中基因的聚腺苷酸化位点数 Promotes splicing of precursor mRNA and involved in regulating the polyadenylation site number of genes in cells
CYP63 （肽基—丙酰基顺式转运异构酶CYP63 Peptidyl- prolyl cis-trans isomerase）	RVX20698	6 800	755	肽基丙酰异构酶结构域Peptidyl propionyl isomerase domain（PpiB）	催化寡肽中脯氨酸酰亚胺肽键的顺反异构化作用Catalyze the cis-trans isomerization of proline imide peptide bonds in oligo- peptides

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