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An efficient PEG-mediated transient gene expression system in grape protoplasts and its application in subcellular localization studies of flavonoids biosynthesis enzymes

文献类型： 外文期刊

作者： Wang, Huiling 1 ; Wang, Wei 4 ; Zhan, Jicheng 2 ; Huang, Weidong 2 ; Xu, Haiying 1 ;

作者机构： 1.Beijing Acad Agr & Forestry Sci, Inst Forestry & Pomol, Beijing 100097, Peoples R China

2.China Agr Univ, Coll Food Sci & Nutr Engn, Beijing 100083, Peoples R China

3.Minist Agr, Key Lab Biol & Genet Improvement Hort Crops North, Beijing, Peoples R China

4.Chinese Acad Forestry, Res Inst Forestry, State Key Lab Tree Genet & Breeding, Beijing 100091, Peoples R China

关键词： Transient expression;Protoplast;Grape;Subcellular localization;Flavonoid

期刊名称：SCIENTIA HORTICULTURAE （ 影响因子：3.463； 五年影响因子：3.672 ）

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收录情况： SCI

摘要： Transient gene expression utilizing plant protoplasts has emerged as a refined method to identify proteins function in vivo. In this study, an improved protocol for preparation of protoplasts from grape berry (cabernet sauvignon) suspension cells and an optimized system for polyethylene glycol (PEG)-mediated transient transformation of the protoplasts with the application of the green fluorescent protein (GFP) as a reporter gene was presented. Step by step optimization showed that the quality of protoplasts as well as PEG-mediated transformation conditions including amount of plasmid DNA, the PEG concentration, transfection duration and the status of suspension cells used for protoplasts preparation were the major factors which influenced successful transient gene expression assays. Following the optimized protocol, up to half of the transfected protoplasts displayed green fluorescence in the total observed cells. To demonstrate the applicability of the system, the subcellular localization of VvCHS, VvCHI, VvUEGT and VvANR were investigated, revealing the cytoplasm and nucleus localization of flavonoids biosynthesis enzymes in grape. This improved transient gene expression system in grape protoplasts gives a rapid and efficient alternative for future research in grapevine molecular biology. (c) 2015 Elsevier B.V. All rights reserved.

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