文献类型：期刊文献

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

作者：Wang, Huiling[1,2] Wang, Wei[4] Zhan, Jicheng[2] Huang, Weidong[2] Xu, Haiying[1,3]

第一作者：Wang, Huiling

通信作者：Huang, WD[1]

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

年份：2015

卷号：191

起止页码：82-89

外文期刊名：SCIENTIA HORTICULTURAE

收录：;Scopus(收录号：2-s2.0-84929459488);WOS:【SCI-EXPANDED(收录号:WOS:000357230100011)】；

基金：We thank Professor Dapeng Zhang (Tsinghua University, China) for the gift of the plasmid pEZS-NL. This research was supported by the National Key Technology R&D Program during the "12th Five-Year Plan" period (No. 2012BAD31B07-01) and the Special Fund for the Construction of Scientific and Technological Innovation Capability (No. KJCX20140110).

语种：英文

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

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.