酿酒葡萄铁调节转运蛋白基因VvIRT1的克隆、表达与功能

doi:10.13560/j.cnki.biotech.bull.1985.2022-1552

生物技术通报 ›› 2023, Vol. 39 ›› Issue (8): 234-240.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1552

酿酒葡萄铁调节转运蛋白基因VvIRT1的克隆、表达与功能

宋志忠¹^,²^,⁴(), 徐维华², 肖慧琳², 唐美玲²^,³, 陈景辉³, 管雪强³, 刘万好¹^,²^,³()

1.鲁东大学农林工程研究院山东省高校作物高产抗逆分子模块育种重点实验室，烟台 264025
2.山东省烟台市农业科学研究院，烟台 265500
3.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，烟台 264000
4.剑桥大学植物系，英国剑桥 CB2 3EA

收稿日期:2022-12-23 出版日期:2023-08-26 发布日期:2023-09-05
通讯作者: 刘万好，男，硕士，高级农艺师，研究方向：葡萄栽培与分子生物学；E-mail: sliuwanhao1978@sina.com
作者简介:宋志忠，男，博士，副教授，研究方向：果树生理营养与分子生物学；E-mail: szhzh2000@163.com
基金资助:
国家现代农业产业技术体系建设专项资金(CARS-29-17);山东省重点研发计划（重大科技创新工程）(2022CXGC010605);国家留学基金(202208370080);烟台市科技计划项目(2020XCZX026);国家重点研发计划(2019YFD1000500)

Cloning, Expression and Function of Iron Regulated Transporter VvIRT1 in Wine Grape（Vitis vinifera L.）

SONG Zhi-zhong¹^,²^,⁴(), XU Wei-hua², XIAO Hui-lin², TANG Mei-ling²^,³, CHEN Jing-hui³, GUAN Xue-qiang³, LIU Wan-hao¹^,²^,³()

1. The Engineering Research Institute of Agriculture and Forestry, Ludong University, Key Laboratory of Molecular Module-Based Breeding of High Yield and Abiotic Resistant Plants in Universities of Shandong, Yantai 264025
2. Yantai Academy of Agricultural Sciences, Yantai 265500
3. Shandong Technology Innovation Center of Wine Grape and Wine, COFCO Great Wall Wine（Penglai）Co., Ltd, Yantai 264000
4. Department of Plant Science, University of Cambridge, Cambridge CB2 3EA, UK

Received:2022-12-23 Published:2023-08-26 Online:2023-09-05

摘要/Abstract

摘要：

克隆酿酒葡萄铁调节转运蛋白基因VvIRT，分析其表达模式及其潜在的生物学功能，为研究果树铁素吸收与高效利用机制提供理论依据。利用同源克隆法克隆酿酒葡萄VvIRT1，运用实时荧光定量PCR分析组织特异性表达模式及其在转录水平对不同铁素供应水平的响应特征，借助酵母异源表达系统分析其转运Fe²⁺的功能。从二倍体酿酒葡萄‘马瑟兰’中分离和鉴定了1个铁调节转运蛋白VvIRT1，系统发育树表明VvIRT1与芸香科柑橘CsIRT1和锦葵科陆地棉GhIRT1的遗传距离较近。VvIRT1在5年生成年树体新生根和组培幼苗根中特异表达；缺铁处理显著诱导了VvIRT1在组培幼苗全部组织（根、茎和叶）中表达，而高铁毒害显著抑制了VvIRT1在组培幼苗根中的表达。此外，酵母功能互补试验表明，VvIRT1能够恢复酵母突变菌株DEY1453的正常生长，VvIRT1具有转运外界Fe²⁺的能力。

关键词: 葡萄, 铁吸收与转运, 铁调节转运蛋白, 酵母功能互补

Abstract:

The genes VvIRT of iron regulated transporters（IRT）family from wine grape was cloned, its expression pattern and potential biological function were analyzed, which may provide theoretical foundation for Fe absorption and efficient utilization mechanisms of fruit trees. Homology cloning technology was used to clone the VvIRT1 gene from wine grape, real-time quantitative PCR was applied to analyze tissue-specific expression characteristics and their responses to different Fe supplies, and yeast heterologous expression system was to analyze the functional verification. The iron regulated transporter VvIRT1 from diploid wine grape ‘Marselan’ was isolated and determined. Phylogenetic tree analysis showed that VvIRT1 was closely clustered with citrus CsIRT1（Rutaceae）and upland cotton GhIRT1（Malvaceae）that the genetic distance among them was relatively close. VvIRT1 was specifically expressed in the roots of both 5-year-old mature trees and tissue-cultured seedlings. Fe depletion significantly induced the expressions of VvIRT1 in the whole tested seedlings, while high Fe toxicity significantly reduced the expressions of VvIRT1 in all tested seedlings in the roots of tissue-cultured seedlings. In addition, VvIRT1 recovered the normal growth of yeast mutant DEY1453 and had ability of transporting external Fe²⁺.

Key words: grape, Fe uptake and transport, iron regulated transporter, yeast functional complementation

宋志忠, 徐维华, 肖慧琳, 唐美玲, 陈景辉, 管雪强, 刘万好. 酿酒葡萄铁调节转运蛋白基因VvIRT1的克隆、表达与功能[J]. 生物技术通报, 2023, 39(8): 234-240.

SONG Zhi-zhong, XU Wei-hua, XIAO Hui-lin, TANG Mei-ling, CHEN Jing-hui, GUAN Xue-qiang, LIU Wan-hao. Cloning, Expression and Function of Iron Regulated Transporter VvIRT1 in Wine Grape（Vitis vinifera L.）[J]. Biotechnology Bulletin, 2023, 39(8): 234-240.

图/表 5

图1 不同植物IRT同源蛋白氨基酸序列的一致性分析 Ah：落花生；At：拟南芥；Cs：柑橘；Gh：陆地棉；Mx：小金海棠；Os：水稻；Pb：杜梨；Pm：梅；Pp：桃；Pt：白杨；Rs：萝卜；Sl：番茄；St：马铃薯；Vv：葡萄；Zm：玉米。下同

Fig. 1 Amino acid sequence identity analysis of IRT homologs from different plant species Ah: Arachis hypgaea; At: Arabidopsis thaliana; Cs: Citrus sinensis; Gh: Gossypium hirsutum; Mx: Malus xiaojinensis; Os: Oryza sativa; Pb: Prunus betulaefolia;Pm: Prunus mume; Pp: Prunus persica; Pt: Populus trichocarpa; Sl: Solanum lycopersicum; Vv: Vitis vinifera; Zm: Zea mays. The same below

图2 不同植物IRT同源蛋白系统发育树

Fig. 2 Phylogenetic tree of IRT homologs from different plant species

图3 葡萄VvIRT1的组织特异性表达分析

Fig. 3 Tissue-specific expression analysis of VvIRT1 in grape

图4 VvIRT1对不同铁素供应水平的响应在铁素处理与对照2个独立样品间进行t-检验（**P<0.01）

Fig. 4 Responses of VvIRT1 under different Fe supplies t-test analysis between iron treatment and control conditions（**P<0.01）

图5 VvIRT1的酵母功能互补验证

Fig. 5 Verification on the functional complementation of VvIRT1 in yeast

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酿酒葡萄铁调节转运蛋白基因VvIRT1的克隆、表达与功能

Cloning, Expression and Function of Iron Regulated Transporter VvIRT1 in Wine Grape（Vitis vinifera L.）

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