葡萄自噬基因ATG8f的克隆及其功能分析

doi:10.3864/j.issn.0578-1752.2025.01.012

中国农业科学 ›› 2025, Vol. 58 ›› Issue (1): 156-169.doi: 10.3864/j.issn.0578-1752.2025.01.012

葡萄自噬基因ATG8f的克隆及其功能分析

葛艺¹^,²(), 郑秋玲³^,⁴, 陈梦霞¹^,², 夏家欣¹^,², 方项²^,⁵^,^*(), 唐美玲³^,⁴, 房经贵¹^,²^,⁶, 上官凌飞¹^,²^,⁶^,^*()

¹ 南京农业大学园艺学院，南京 210095
² 江苏省果树品种改良与种苗繁育工程研究中心，南京 210095
³ 山东省烟台市农业科学研究院，山东烟台 264003
⁴ 烟台市特色果品质量安全与营养重点实验室（筹），山东烟台 264003
⁵ 江苏农林职业技术学院农学园艺学院，江苏句容 212400
⁶ 宁夏贺兰山东麓葡萄酒产业技术协同创新中心，银川 750299

收稿日期:2024-06-04 接受日期:2024-07-30 出版日期:2025-01-01 发布日期:2025-01-07
通信作者:
方项，E-mail：fangxiang@jsafc.edu.cn。
上官凌飞，E-mail：shangguanlf@njau.edu.cn。
联系方式: 葛艺，E-mail：17509671667@163.com。
基金资助:
烟台市农业科学院北方种谷“揭榜挂帅”项目; 国家级大学生创新创业训练计划(202310307076Z); 江苏农林职业技术学院高层次人才科研启动项目(2024rc16); 江苏省自然科学基金青年基金(BK20240503); 宁夏回族自治区重点研发项目(2023BCF01001); 宁夏贺兰山东麓葡萄酒产业技术协同创新中心科技人才开放课题(CXZXKT2024005); 江苏省高等学校优势学科项目(PAPD)

Cloning and Functional Analysis of the Autophagy Gene ATG8f in the Grapevine

GE Yi¹^,²(), ZHENG QiuLing³^,⁴, CHEN MengXia¹^,², XIA JiaXin¹^,², FANG Xiang²^,⁵^,^*(), TANG MeiLing³^,⁴, FANG JingGui¹^,²^,⁶, SHANGGUAN LingFei¹^,²^,⁶^,^*()

¹ Department of Horticulture, Nanjing Agricultural University, Nanjing 210095
² Fruit Crop Genetic Improvement and Seedling Propagation Engineering Research Center of Jiangsu Province, Nanjing 210095
³ Yantai Academy of Agricultural Sciences in Shandong Province, Yantai 264003, Shandong
⁴ Yantai Key Laboratory of Quality Safety and Nutrition of Characteristic Fruits, Yantai 264003, Shandong
⁵ School of Agronomy and Horticulture, Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, Jiangsu
⁶ Collaborative Innovation Center for Wine Industry Technology of Ningxia Helan Mountain Eastern Foothills, Yinchuan 750299

Received:2024-06-04 Accepted:2024-07-30 Published:2025-01-01 Online:2025-01-07

摘要/Abstract

摘要：

【目的】通过对葡萄自噬基因ATG8f的克隆与功能分析，探讨其在植物铜胁迫耐受调控中的作用，为植物耐铜机制解析与调控网络构建奠定基础。【方法】基于本课题组铜胁迫下‘阳光玫瑰’葡萄多组学测序分析结果，筛选获得细胞自噬基因VlATG8f，并分析其序列特征，克隆CDS序列，构建VlATG8f过表达载体。通过烟草瞬时表达试验研究VlATG8f蛋白的亚细胞定位，利用花序浸染法使其在拟南芥中异源表达，观察和测定野生型和转基因株系的种子发芽率及根长，比较其在铜胁迫处理下的生长发育状况。利用实时荧光定量PCR技术分析相关抗氧化基因的表达水平，并采用酵母双杂交技术和双分子荧光互补试验初步筛选VlATG8f的互作蛋白。【结果】‘阳光玫瑰’ATG8f的CDS为393 bp，编码130个氨基酸。生物学信息分析发现，拟南芥、番茄、苹果、白梨、桃和葡萄ATG8f的蛋白序列较为保守，都含有一个Ubl_ATG8保守结构域，可能具有相似的功能。亚细胞定位发现，VlATG8f蛋白定位于细胞核和细胞膜。正常条件下，野生型和过表达VlATG8f株系生长状态良好，叶片青翠，但在铜胁迫下株系矮小，甚至出现了黄化、叶片卷曲、根毛细短和侧根增多的现象。但与野生型相比，过表达VlATG8f的拟南芥幼苗在铜处理后生长受限较小，种子发芽率和根长受抑制作用较轻，过氧化氢（H₂O₂）积累较低，表明VlATG8f的过表达增强了转基因株系对铜的耐受性。此外，VlATG8f过表达植株在铜胁迫下抗氧化基因AtCSD1和AtCCS1表达量增加，参与ROS合成的关键基因RbohB表达量降低，促进了对ROS的清除。通过酵母双杂交技术筛选到23个与VlATG8f可能互作的蛋白。BiFC进一步验证发现PYL8、MnSOD、AUX22D、bZIP17和COT44与VlATG8f之间存在较强的互作。【结论】过表达VlATG8f可能通过增强抗氧化系统能力、降低ROS合成和促进植物激素合成等途径，提高转基因株系的耐铜性，减轻铜毒性对植物的伤害。

关键词: 葡萄, 自噬, 铜胁迫, VlATG8f, 抗氧化基因

Abstract:

【Objective】This study aims to investigate the role of the grape autophagy gene ATG8f in regulating copper stress tolerance in plants, thereby laying the foundation for elucidating copper tolerance mechanisms and constructing regulatory networks. 【Method】Based on multi-omics sequencing analysis of Shine Muscat grapevine under copper stress, the autophagy gene VlATG8f was identified and its sequence characteristics were analyzed. The CDS sequence of VlATG8f was cloned and an overexpression vector was constructed. The subcellular localization of the VlATG8f protein was studied through transient expression in tobacco. Heterologous expression of VlATG8f in Arabidopsis was achieved using the floral dip method. Seed germination rate and root length were measured to compare the growth and development of wild-type and transgenic lines under copper stress. Real-time quantitative PCR was used to analyze the expression levels of related antioxidant genes, and yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays were used to preliminarily screen for VlATG8f- interacting proteins. 【Result】The CDS of VlATG8f was 393 bp, encoding 130 amino acids. Bioinformatics analysis revealed that the ATG8f protein sequences in Arabidopsis, tomato, apple, pear, peach, and grapevine were relatively conserved, all containing a Ubl_ATG8 conserved domain, indicating potential functional were conserved. Subcellular localization showed that the VlATG8f protein was located on both the nucleus and cell membrane. Under normal conditions, both wild-type and VlATG8f-overexpressing lines exhibited healthy growth with green leaves. Under copper stress, the transgenic lines were less stunted and exhibited milder symptoms of chlorosis, leaf curling, reduced root hair size, and increased lateral roots compared to wild-type plants. Overexpression of VlATG8f in Arabidopsis resulted in less growth inhibition under copper stress, higher seed germination rate, longer root, and lower accumulation of hydrogen peroxide (H₂O₂), indicating enhanced copper tolerance. Additionally, the expression of antioxidant genes AtCSD1 and AtCCS1 increased in VlATG8f-overexpressing plants under copper stress, while the expression of the key ROS synthesis gene RbohB decreased, promoting ROS scavenging. Yeast two-hybrid screening identified 23 potential interacting proteins with VlATG8f. BiFC further confirmed strong interactions between VlATG8f and proteins such as PYL8, MnSOD, AUX22D, bZIP17, and COT44. 【Conclusion】Overexpression of VlATG8f may enhance copper tolerance in transgenic lines by strengthening the antioxidant system, reducing ROS synthesis, and promoting the synthesis of plant hormones, thereby mitigating copper toxicity in plants.

Key words: grapevine, autophagy, copper stress, VlATG8f, antioxidant-related genes

葛艺, 郑秋玲, 陈梦霞, 夏家欣, 方项, 唐美玲, 房经贵, 上官凌飞. 葡萄自噬基因ATG8f的克隆及其功能分析[J]. 中国农业科学, 2025, 58(1): 156-169.

GE Yi, ZHENG QiuLing, CHEN MengXia, XIA JiaXin, FANG Xiang, TANG MeiLing, FANG JingGui, SHANGGUAN LingFei. Cloning and Functional Analysis of the Autophagy Gene ATG8f in the Grapevine[J]. Scientia Agricultura Sinica, 2025, 58(1): 156-169.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2025.01.012

https://www.chinaagrisci.com/CN/Y2025/V58/I1/156

图/表 8

表1

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表2

筛选到的VlATG8f候选互作蛋白"

编号 Code	基因 Gene	蛋白ID Protein ID	描述 Description
1	VvDRIP2	XP_010651942.1	E3泛素蛋白连接酶E3 ubiquitin protein ligase DRIP2
2	VvsGR1	XP_002282183.1	滞绿蛋白1，叶绿体Protein STAY-GREEN 1, chloroplastic
3	VvPsaD	XP_002281825.1	光系统Ⅰ反应中心亚基Ⅱ，叶绿体Photosystem Ⅰ reaction center subunit Ⅱ, chloroplastic
4	VvGST	XP_002262842.1	谷胱甘肽S-转移酶Glutathione S-transferase
5	VvRHF2a	XP_010653244.1	E3泛素蛋白连接酶RHF2A亚型X2 E3 ubiquitin-protein ligase RHF2A isoform X2
6	VvAUX22D	XP_002281696.1	生长素诱导蛋白22D Auxin-induced protein 22D
7	VvPYL8	XP_010659134.1	脱落酸受体PYL8 Abscisic acid receptor PYL8
8	VvHIPP39	XP_002274512.1	重金属相关异戊二烯化植物蛋白39 Heavy metal-associated isoprenylated plant protein 39
9	VvbZIP17	XP_010653644.1	bZIP转录因子17 bZIP transcription factor 17
10	VvGOLS1	XP_002265947.1	半乳糖醇合酶1 Galactinol synthase 1
11	VvCOT44	XP_002283263.1	半胱氨酸蛋白酶COT44 Cysteine proteinase COT44
12	VvsDH6	XP_002268190.1	琥珀酸脱氢酶亚基6，线粒体Succinate dehydrogenase subunit 6, mitochondrial
13	VvALS1	XP_002274073.1	乙酰乳酸合酶1，叶绿体Acetolactate synthase 1, chloroplastic
14	VvPGDH3	XP_002273552.1	D-3-磷酸甘油酸脱氢酶3，叶绿体D-3-phosphoglycerate dehydrogenase 3, chloroplastic
15	VvEXO	XP_002285762.1	磷酸诱导酶Phosphoinductase 1
16	VvPP6-ARS-C	XP_002268442.1	丝氨酸/苏氨酸蛋白磷酸酶6调节锚蛋白重复亚基C Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit C
17	VvDRM homolog 4	XP_003632757.1	休眠相关蛋白同源物4亚型X2 Dormancy-associated protein homolog 4 isoform X2
18	VvOEE1	XP_002274796.1	放氧增强蛋白1，叶绿体Oxygen-evolving enhancer protein 1, chloroplastic
19	VvFKBP13	XP_002272628.1	肽基脯氨酰顺反异构酶FKBP13，叶绿体Peptidyl-prolyl cis-trans isomerase FKBP13, chloroplastic
20	VvMnSOD	NP_001268135.1	锰超氧化物歧化酶Manganese superoxide dismutase
21	VvVAMP711	XP_002285207.1	囊泡相关膜蛋白711 Vesicle-associated membrane protein 711
22	VvPAT2	XP_019072271.1	蛋白驱动蛋白轻链相关2 Protein kinesin light chain-related 2
23	VvHCF136	XP_002278958.1	光系统Ⅱ稳定性/组装因子HCF136，叶绿体 Photosystem Ⅱ stability/assembly factor HCF136, chloroplastic

表2

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引物名称 Primers name	正向引物 Forward primers (5′-3′)	反向引物 Reverse primers (5′-3′)	用途 Application
VlATG8f-F/R	TTTGCTGATGAAGGAGGAATGGC	TCGACAATCACCGGAATCCTGT	拟南芥定量引物 Arabidopsis thaliana quantitative primers
AtRbohB-F/R	CCGGGGATGATTACCTCAGC	CGATGTCAATGCCGCTCTTG
AtGPX6-F/R	CAAAGCTGCCCCAGTCTACA	GGTGAGGTAGTTGGTGCGAA
AtCSD1-F/R	CCCTGAGGATGCTAATCGACAT	TGGCAATCAGTGATTGTGAAGG
AtTRX1-F/R	ACATGGAACGAGCAGCTTCA	GCCTGTATCGCCCAATCACT
AtCCS1-F/R	GCAAACTGGTCGAAAAGCTC	GTCAGGGCCTTTGAATTCTG
AtP5CS1-F/R	GCAGCTTTGCGGATCTTCAG	GATATGGGGCTCTTCGGGTG
AtMDAR4-F/R	GGAATGCGGTTGTTATCGGC	ACCAAGTCGGCTGGTAAGTG
AtPEX4-F/R	CTCCTTACGAAGGCGGTGTT	TTCTTAGGCATAGCGGCGAG	拟南芥定量引物（Actin） Arabidopsis thaliana quantitative primers
Hyg-F/R	GGTCGCGGAGGCTATGGATGC	GCTTCTGCGGGCGATTTGTGT	转基因拟南芥鉴定 Identification of transgenic Arabidopsis thaliana
1302-VlATG8f	GAAGATCTATGATTTTTGCTGATGAAGGAGGA	GGACTAGTATGTGGTATCTCATATCCAAACGT	构建植物过表达载体 Construction of plant overexpression vectors
1305-VlATG8f	AGGACAGCCCAGATCACTAGTATGATTTTTGCTGATGAAGGAGGA	GCTCACCATGGATCCCCCGGGATGTGGTATCTCATATCCAAACGT	构建亚细胞定位载体 Construction of subcellular localized vectors
BD-VlATG8f	ATGGCCATGGAGGCCGAATTCATGATTTTTGCTGATGAAGGAGGA	CCGCTGCAGGTCGACGGATCCATGTGGTATCTCATATCCAAACGT	构建BD载体 Construction of the BD vector

葡萄自噬基因ATG8f的克隆及其功能分析

Cloning and Functional Analysis of the Autophagy Gene ATG8f in the Grapevine

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