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

• 园艺 • 上一篇    下一篇

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. 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:
    上官凌飞,E-mail:
  • 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. 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:

【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AtCSD1AtCCS1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