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中国农学通报

中国农学通报 ›› 2023, Vol. 39 ›› Issue (34): 32-40.doi: 10.11924/j.issn.1000-6850.casb2023-0005

• 生物科学 • 上一篇    下一篇

葡萄铁还原酶FRO基因的克隆、鉴定与表达模式分析

王建萍1(), 刘万好1, 隋永超2,3, 唐美玲1,2,4, 李进4, 徐维华1, 宋志忠1,2,5()   

  1. 1 山东省烟台市农业科学研究院，山东烟台 264000
    2 鲁东大学农林工程研究院，山东烟台 264025
    3 龙口市农业技术推广中心果树站，山东烟台 265700
    4 山东省酿酒葡萄与葡萄酒技术创新中心/中粮长城葡萄酒（蓬莱）有限公司，山东烟台 264000
    5 剑桥大学植物系，英国剑桥 CB2 3EA
  • 收稿日期:2023-01-02 修回日期:2023-08-15 出版日期:2023-12-05 发布日期:2023-11-30
  • 通讯作者:
    宋志忠，男，1983年出生，山东菏泽人，副教授，博士，研究方向为果树生理营养与分子生物学。通信地址：264025 山东烟台市芝罘区红旗中路186号 鲁东大学农林工程研究院，Tel：0535-6664669，E-mail：。
  • 基金资助:
    国家现代农业产业技术体系“国家现代农业产业技术体系烟台葡萄实验站”(CARS-29-17); 山东省重点研发计划（重大科技创新工程）“酿酒葡萄与葡萄酒全产业链关键技术研发与应用”(2022CXGC010605); 国家留学基金委项目“葡萄香气关键物质形成的分子机制”(202208370080); 山东省良种工程项目“优质特色果树突破性新品种选育”(2020LZGC008)

Analysis of Cloning, Identification and Expression Pattern of Ferric Reduction Oxidase Genes in Grape

WANG Jianping1(), LIU Wanhao1, SUI Yongchao2,3, TANG Meiling1,2,4, LI Jin4, XU Weihua1, SONG Zhizhong1,2,5()   

  1. 1 Yantai Academy of Agricultural Science of Shandong, Yantai, Shandong 264000
    2 The Engineering Research Institute of Agriculture and Forestry, Ludong University, Yantai, Shandong 264025
    3 Fruit Tree Station of Longkou Agricultural Technology Promotion Center, Yantai, Shandong 265700
    4 Shandong Technology Innovation Center of Wine Grape and Wine/COFCO Great Wall Wine (Penglai) Co., Ltd., Yantai, Shandong 264000
    5 Department of Plant Science, University of Cambridge, Cambridge, CB2 3EA
  • Received:2023-01-02 Revised:2023-08-15 Published-:2023-12-05 Online:2023-11-30

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摘要：

以自主创新品种‘烟酿1号’葡萄为材料，筛选并克隆葡萄FRO家族基因，对其进行生物信息学鉴定和表达特征分析，为研究果树铁素营养与吸收利用机制提供理论依据。通过同源克隆法从‘烟酿1号’中克隆和鉴定了6个FRO家族基因，命名为VvFRO1~VvFRO6，属于典型的植物铁还原酶编码基因；10种不同科、属植物FRO蛋白的氨基酸水平一致性约为43.66%，分为2个亚族（Group I和II），其中，VvFRO1~VvFRO3属于Group I，VvFRO4~VvFRO6属于Group II；系统进化树表明VvFRO倾向于与资阳香橙和小金海棠的同源蛋白紧密聚在一起；VvFRO蛋白主要定位于细胞质膜，均含有10~11个跨膜区；实时荧光定量PCR分析表明，VvFRO3在‘烟酿1号’葡萄不同组织中的整体表达水平最高，VvFRO3和VvFRO2在成年树体叶片和幼苗叶片中高量表达，VvFRO5在硬核期和膨大期果实中高量表达，而VvFRO1、VvFRO4和VvFRO6的整体表达水平相对较低。此外，葡萄FRO家族基因在根部易受缺铁、200 mmol/L NaCl和10% PEG6000(w/v)处理的诱导而显著增加，但对低温(4℃)和热(45℃)胁迫变化不敏感，VvFRO3在根部的表达量受ABA处理显著诱导而上调，但受50 μmol/L高铁毒害显著抑制而降低。本研究可为解析葡萄铁的吸收与转运机制奠定分子基础。

关键词: 葡萄, 铁吸收, 铁还原酶, 非生物胁迫, 表达模式

Abstract:

Using the independently innovative grape variety ‘Yanniang No.1’ as the material, the FRO (ferric reduction oxidase) family genes were screened and cloned, and their bioinformatics identification and expression characteristics were analyzed. This study provides a theoretical basis for studying iron nutrition and the mechanism of absorption and utilization in fruit trees. In total, 6 FRO family genes were isolated from ‘Yanniang No.1’ by homologous cloning method, entitled by VvFRO1-VvFRO6, which belonged to the classic plant ferric reduction oxidase. The amino acid sequences of FRO proteins from 10 plants shared an overall identity of 43.66%, which were classified into 2 major groups (Groups I and II). VvFRO1-VvFRO3 belonged to Group I, while VvFRO4-VvFRO6 belonged to Group II. Phylogenetic tree analysis showed that VvFRO proteins were prone to be closely clustered with homologs form Citrus junos ‘Ziyang xiangcheng’ and Malus xiaojinensis, respectively. All VvFRO proteins were majorly localized in plasma membrane and contained 10-11 transmembrane domains (TMs). Quantitative real-time PCR (qRT-PCR) analysis showed that VvFRO3 was the most abundant expressed gene during different parts of ‘Yanniang No.1’ grape on the whole. VvFRO3 and VvFRO2 were highly expressed in leaves of both mature trees and seedlings, and VvFRO5 was highly expressed in fruits of hard core stage and veraison stage, while genes of VvFRO1, VvFRO4 and VvFRO6 were relatively lowly expressed. In addition, expression of FRO family genes in grape roots were easily induced by iron depletion, 200 mmol/L NaCl stress and 10% PEG6000 (w/v) stress, respectively, but changed little under low temperature (4℃) and heat (45℃) stresses. In particular, VvFRO3 in roots was upregulated under 200 μmol/L ABA treatment but down-regulated under 500 μmol/L iron toxicity stress. This study laid a molecular foundation for revealing iron absorption and transport mechanisms in grape.

Key words: grape, Fe uptake, ferric reduction oxidase, abiotic stress, expression pattern