从细胞质到质外体：拟南芥中的潮霉素磷酸转移酶（HYG(R)）模型

From Cytosol to the Apoplast: The Hygromycin Phosphotransferase (HYG(R)) Model in Arabidopsis.

作者信息

Zhang Haiyan, Li Jinjin

机构信息

Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China.

出版信息

Methods Mol Biol. 2016;1459:81-90. doi: 10.1007/978-1-4939-3804-9_5.

DOI:10.1007/978-1-4939-3804-9_5

PMID:27665552

Abstract

The process by which proteins are secreted via endoplasmic reticulum (ER)/Golgi-independent mechanism is conveniently called unconventional protein secretion. Recent studies have revealed that unconventional protein secretion operates in plants, but little is known about its underlying mechanism and function. This chapter provides methods we have used to analyze unconventional character of hygromycin phosphotransferase (HYG(R)) secretion in plant cells. Following isolation of protoplasts from HYG (R) -GFP-transgenic plants and incubation with brefeldin A (BFA), an inhibitor of conventional secretory pathway, we easily obtain protein extracts from protoplasts and culture medium separately. These proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot analysis with anti-GFP antibodies.

摘要

蛋白质通过不依赖内质网（ER）/高尔基体的机制进行分泌的过程，被便捷地称为非常规蛋白质分泌。最近的研究表明，非常规蛋白质分泌在植物中发挥作用，但其潜在机制和功能却鲜为人知。本章提供了我们用于分析植物细胞中潮霉素磷酸转移酶（HYG(R)）分泌的非常规特性的方法。从转HYG(R)-GFP基因植物中分离原生质体，并与常规分泌途径的抑制剂布雷菲德菌素A（BFA）一起孵育后，我们可以轻松地分别从原生质体和培养基中获得蛋白质提取物。这些蛋白质通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分离，随后用抗GFP抗体进行蛋白质印迹分析。