水稻小G蛋白OsRab5b的亚细胞定位研究

doi:10.13560/j.cnki.biotech.bull.1985.2015.11.017

生物技术通报 ›› 2015, Vol. 31 ›› Issue (11): 139-145.doi: 10.13560/j.cnki.biotech.bull.1985.2015.11.017

水稻小G蛋白OsRab5b的亚细胞定位研究

邵军丽^1,2, 龙跃生^2,3, 徐增富^2,4

1.广东医学院公共卫生学院，东莞 523808；
2.中山大学基因工程教育部重点实验室有害生物控制与资源利用国家重点实验室，广州 510275；
3.广州医科大学附属第二医院神经科学研究所，广州 510260；
4.中国科学院西双版纳热带植物园热带植物资源可持续利用重点实验室，昆明 650223

收稿日期:2015-03-03 出版日期:2015-11-26 发布日期:2015-11-26
作者简介:邵军丽，女，博士，研究方向: 基因功能和基因工程；E-mail: 327862795@qq.com
基金资助:
国家高技术研究发展计划(“863”计划)(2007AA02Z102)

Subcellular Localization of Rice Small G Protein OsRab5b in BY-2 Cells

Shao Junli^1，2
Long Yuesheng^2，3
Xu Zengfu^2，4

1. School of Public Health，Guangdong Medical College，Dongguan 523808；
2. Key Laboratory of Gene Engineering of the Ministry of Education，School of Life Sciences，Sun Yat-sen University，Guangzhou 510275；
3. Institute of Neuroscience，The Second Affiliated Hospital of Guangzhou Medical University，Guangzhou 510260；
4. Key Laboratory of Tropical Plant Resources and Sustainable Use，Xishuangbanna Tropical Botanical Garden，Chinese Academy of Sciences，Kunming 650223

Received:2015-03-03 Published:2015-11-26 Online:2015-11-26

摘要/Abstract

摘要： 旨在研究水稻OsRab5b亚细胞定位及第二位甘氨酸在蛋白定位中的作用。利用药物、细胞器标记物和示踪染料处理OsRab5b-GFP与OsRab5b(Gly2Ala)-GFP的转基因BY-2细胞，然后用激光共聚焦显微镜观察。结果表明，OsRab5b-GFP转基因细胞呈现出大量的分散点状结构和少量细胞质弥散信号；wortmannin处理可以使OsRab5b-GFP标记的点状结构膨胀成小的环状结构；采用100 μg/mL布雷菲德菌素A(BFA)处理可使OsRab5b-GFP标记的结构聚集。大部分的OsRab5b-GFP信号都可以与前液泡区标记蛋白VSR_At-1共定位。在内吞示踪染料FM4-64摄取的第60 min，OsRab5b-GFP标记的结构大部分被FM4-64标记。突变体OsRab5b(Gly2Ala)-GFP弥散分布于细胞核和细胞质内，而且不受wortmannin或BFA药物处理的影响。OsRab5b定位于BY-2细胞的前液泡区，Gly2在蛋白准确定位方面发挥重要作用。

关键词: OsRab5b, 亚细胞定位, 前液泡区, 内吞体

Abstract: To investigate the subcellular localization of rice OsRab5b and the Gly2 role in the protein subcellular localization. OsRab5b-GFP and OsRab5b(Gly2Ala)-GFP plasmids were transformed into BY-2 cells. The laser confocal microscope was employed to observe the transgenic cells treated with drugs, a marker protein VSR_At-1 or an endocytosis tracer dye FM4-64. Results showed Punctuate and diffuse signals were observed in the OsRab5b-GFP transgenic cells. Wortmannin at a concentration of 16.5 μmol/L led to the change of the OsRab5b-GFP marked organelles into small vacuoles. The signals of OsRab5b-GFP were aggregated after the treatment of Brefeldin A(BFA)at a concentration of 100 μg/mL, but not at 10 μg/mL. Most of the OsRab5b-GFP positive signals colocalized with the VSR_At-1 positive organelles in the cells with and without wortmannin or BFA treatment. Additionally, most of the OsRab5b-GFP marked organelles colocalized with the endosomal marker FM4-64 after 60-min incubation. The diffuse signals of the mutation protein OsRab5b(Gly2Ala)-GFP were observed in the nuclei and cytoplasm of the transgenic cells, which were not affected by the wortmannin or BFA treatment. OsRab5b is localized to the prevacuolar compartment of BY-2 cells and the Gly2 site is essential for the correct subcellular localization of OsRab5b.

Key words: OsRab5b, subcellular localization, prevacuolar compartment(PVC), endosome

邵军丽, 龙跃生, 徐增富. 水稻小G蛋白OsRab5b的亚细胞定位研究[J]. 生物技术通报, 2015, 31(11): 139-145.

Shao Junli,Long Yuesheng, Xu Zengfu. Subcellular Localization of Rice Small G Protein OsRab5b in BY-2 Cells[J]. Biotechnology Bulletin, 2015, 31(11): 139-145.

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水稻小G蛋白OsRab5b的亚细胞定位研究

Subcellular Localization of Rice Small G Protein OsRab5b in BY-2 Cells

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