植物学报 ›› 2015, Vol. 50 ›› Issue (3): 394-404.doi: 10.3724/SP.J.1259.2015.00394

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pH敏感型荧光蛋白及其在植物细胞生物学中的应用

薛轶群1, 宋凯1, 范路生1, 万迎朗2, 林金星1,2,*()   

  1. 1中国科学院植物研究所, 北京 100093
    2北京林业大学生物科学与技术学院, 北京 100083
  • 收稿日期:2014-06-04 接受日期:2014-09-09 出版日期:2015-05-01 发布日期:2015-04-08
  • 通讯作者: 林金星 E-mail:linjx@ibcas.ac.cn
  • 作者简介:

    ? 共同第一作者

  • 基金资助:
    国家重点基础研究发展规划(No.2011CB809103)、国家自然科学基金创新研究群体(No.31121065)、北京林业大学杰出青年人才培育计划(No.JC2013-2)、新世纪优秀人才计划(No.NECT-12-0785)和国家自然科学基金(No.31271433)

pH-sensitive Fluorescent Proteins and Their Applications in Plant Cell Biology

Yiqun Xue1, Kai Song1, Lusheng Fan1, Yinglang Wan2, Jinxing Lin1, 2, *   

  1. 1Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
    2College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China
  • Received:2014-06-04 Accepted:2014-09-09 Online:2015-05-01 Published:2015-04-08
  • Contact: Lin Jinxing E-mail:linjx@ibcas.ac.cn
  • About author:

    ? These authors contributed equally to this paper

摘要:

pH敏感型荧光蛋白, 即pHluorin, 是荧光强度及光谱特征随环境pH值的变化而改变的一类荧光蛋白。人们通过对密码子使用偏好和特定剪切位点的修饰, 已使pHluorin及其衍生物成功地在动物、植物和真菌细胞中正常表达, 为测量细胞内微环境pH值的变化, 并研究活细胞内依赖或导致pH变化的生理过程提供了有力工具。该文总结了目前已报道的pH敏感型荧光蛋白的种类及特性, 并对其在细胞生物学, 特别是植物细胞生物学中的应用进行了详细介绍。随着报告基因技术及检测方法的不断改进, pHluorin将在植物科学领域发挥更大的作用。

Abstract:

pHluorin is a pH-sensitive variant of green fluorescent protein; its fluorescence signals display a strong pH dependence. Since the codon usage was optimized and the site of aberrant splicing was removed, pHluorin and its derivatives have been successfully expressed and used in animal, plant and fungi cells. These pH sensors have helped shed light on measure of the difference in pH of cellular microenvironment and many cell functions for which intracellular pH is an important modulator. Here, we review the characteristics of known pHluorins and their applications in cell biology, particularly plant cell biology. With the improvements in pHluorins by genetic engineering and the advancing in detection technology, the future for pHluorins in plant science is exciting.

图1

GFP及pHluorin的荧光激发光谱(Miesenböck et al., 1998) (A) 野生型GFP; (B) 比率pHluorin; (C) 盈缺pHluorin"

表1

部分pH敏感型荧光蛋白的光谱特性及pH测量范围(改编自Martinière et al., 2013a)"

荧光蛋白名称 类型 激发光(nm) 发射光(nm) pH范围 pKa 目前应用范畴 参考文献
pHluorin 比率 395/475 510 5.4-8.4 6.9 测量动物细胞内涵体和TGN中pH值 Miesenböck et al., 1998
pHluorin2 比率 395/475 510 5.4-8.4 6.9 检测动物细胞中激素受体内吞 Mahon, 2011
RaVC 比率 395/475 510 5.4-8.4 6.9 测量曲霉菌中胞质pH值 Bagar et al., 2009
pHGFP 比率 395/475 510 5.4-8.4 6.9 测量拟南芥根中胞质pH值 Moseyko and Feldman, 2001
Ratiometric
AtpHluorin
比率 395/475 510 5.4-8.4 6.9 测量拟南芥根质外体和细胞质中pH值 Gao et al., 2004
PRpHluorin 比率 395/475 510 5.4-8.4 6.9 测量拟南芥原生质体中细胞器pH值 Shen et al., 2013
Ecliptic pHluorin 盈缺 395/477 510 6.5-8.0 7.2 检测动物细胞突触囊泡胞吐 Miesenböck et al., 1998
Ecliptic AtpHluorin 盈缺 395/477 510 6.5-8.0 7.2 测量拟南芥根质外体和细胞质中pH值 Gao et al., 2004
PEpHluorin 盈缺 395/477 510 6.5-8.0 7.2 测量拟南芥原生质体中细胞器pH值 Shen et al., 2013
Superecliptic pHluorin 盈缺 477 510 5.5-8.5 7.2 检测动物细胞突触末端胞吞胞吐 Sankaranarayanan and Ryan, 2001
deGFP 比率 396 460/515 6-9.0 7.3 测量动物细胞中pH值 Hanson et al., 2002
E2GFP 比率 458/488 500/560 5-8.5 6.9/7.5 测量动物细胞中pH值 Bizzarri et al., 2006; Arosio et al., 2007
ptGFP 比率 390/475 540 3.8-8.2 7.3 测量拟南芥根质外体和细胞质中pH值 Schulte et al., 2006
pHlameleon 6 比率 458 481/533 5-7.5 5.6 基于FRET原理测量动物细胞中pH值 Esposito et al., 2008
pHusion 比率 488/458 510/600 4.5-8 5.8 基于FRET原理测量拟南芥叶肉和根
的固定细胞中质外体和胞质pH值
Gjetting et al., 2012
pHlash 比率 不适用 475/525 5.4-9 不适用 基于BRET原理测量动物细胞中胞质
pH值
Zhang et al., 2012
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