Establishment and Application of Two-dimensional Gel Electrophoresis Technology System for Total Protein of Camellia sinensis Leaf

doi:10.3969/j.issn.1009-7791.2019.03.003

Subtropical Plant Science ›› 2019, Vol. 48 ›› Issue (03): 220-226.DOI: 10.3969/j.issn.1009-7791.2019.03.003

• Plant physiology and biochemistry • Previous Articles Next Articles

Establishment and Application of Two-dimensional Gel Electrophoresis Technology System for Total Protein of Camellia sinensis Leaf

ZHOU Lin, SHEN Jia-zhi, DUAN Yu, WANG Ting, WANG Yu-hua, ZHU Xu-jun, MA Yuan-chun, FANG Wan-ping

(1.Tea Research Institute, Nanjing Agricultural University, Nanjing 210095, Jiangsu China; 2.Forestry and Pomology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China)

Received:2019-07-21 Revised:2019-08-30 Online:2019-09-30 Published:2019-09-30
Contact: FANG Wan-ping

茶树叶片蛋白双向电泳体系建立与应用

周琳，申加枝，段玉，王婷，王玉花，朱旭君，马媛春，房婉萍

(1.南京农业大学茶叶科学研究所, 江苏南京 210095；2.上海市农业科学院林木果树研究所, 上海 201403)

通讯作者: 房婉萍
作者简介:周琳，博士，从事茶树生理生化和种质资源研究。
基金资助:
江苏省农业(茶叶)产业技术体系(JATS[2018]280)；国家现代农业产业技术体系建设专项资金资助(CARS-19)；江苏省重点研发计划项目(BE2016417)；南京市科技计划项目(201805064)

Abstract

Abstract: In order to carry out the isolation and identification of the differential proteins under the cold and drought stress of tea plants, using the strong stress-resistant tea tree variety ‘Yingshuang’ as experimental material. The two-dimensional gel electrophoresis technology system suitable for leaf was screened by extraction method, IPG strip pH range, protein content, separation gel concentration and staining method. The results showed that total protein was extracted by TCA-acetone or Tris-HCl method, using 17 cm pH 4—7 IPG strip and 1.6—2.2 mg protein for isoelectric focusing, using 13.5% polyacrylamide gel for electrophoresis, subsequently staining by high sensitivity Coomassie Brilliant Blue R-250 method. Finally, the protein of each molecular weight of the leaf was sufficiently separated, and a 2-DE pattern with high resolution, clear background and good reproducibility was obtained. The system was suitable for the analysis of differential proteins in leaves of ‘Yingshuang’ under cold and drought stress.

Key words: Camellia sinensis, leaf, two-dimensional gel electrophoresis, system establishment, application

摘要： 为开展茶树Camellia sinensis低温和干旱胁迫下差异蛋白的分离和鉴定，以抗逆性较强的茶树品种‘迎霜’为试材，通过对提取方法、IPG胶条pH范围、上样量、分离胶浓度、染色方法的比较，筛选适用于茶树叶片的蛋白质双向电泳体系。结果表明，采用TCA-丙酮法或Tris-HCl法提取叶片总蛋白，选用17 cm pH 4～7 IPG胶条用于等电聚焦，选择1.6～2.2 mg上样量、13.5%聚丙烯酰胺凝胶进行分离，随后通过高敏考马斯亮蓝R-250法染色；最终，叶片各分子量的蛋白充分分离，获得的双向电泳图谱分辨率高、背景清晰、重复性好，适用于‘迎霜’低温和干旱胁迫下叶片差异蛋白分析。

关键词: 茶树, 叶片, 蛋白双向电泳, 体系建立, 应用

CLC Number:

S571.1

ZHOU Lin, SHEN Jia-zhi, DUAN Yu, WANG Ting, WANG Yu-hua, ZHU Xu-jun, MA Yuan-chun, FANG Wan-ping. Establishment and Application of Two-dimensional Gel Electrophoresis Technology System for Total Protein of Camellia sinensis Leaf[J]. Subtropical Plant Science, 2019, 48(03): 220-226.

周琳，申加枝，段玉，王婷，王玉花，朱旭君，马媛春，房婉萍. 茶树叶片蛋白双向电泳体系建立与应用[J]. 亚热带植物科学, 2019, 48(03): 220-226.

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Establishment and Application of Two-dimensional Gel Electrophoresis Technology System for Total Protein of Camellia sinensis Leaf

茶树叶片蛋白双向电泳体系建立与应用

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