《植物生理学报》 2017, 53(4): 729-735

棉花保卫细胞原生质体的有效分离与活性鉴定

赵翔, 朱金东, 孔培涛, 王雪娇, 张骁^*

河南大学棉花生物学国家重点实验室/植物逆境生物学教育部重点实验室/生命科学学院, 河南开封475004

通信作者：张；E-mail: xzhang@henu.edu.cn

摘　要：

模式植物拟南芥保卫细胞原生质体的分离技术已得到应用, 然而如何有效分离和纯化棉花保卫细胞原生质体, 已成为棉花气孔运动调控机理研究的限制因素。本文探索了一种棉花保卫细胞原生质体分离和活性鉴定的方法。我们选取生长2~3周的棉花幼叶下表皮, 利用1.3%纤维素酶和0.03%果胶酶进行一步酶解约1.5 h, 先低速15×g离心5 min, 再高速105×g离心10 min分离, 所获得保卫细胞原生质体的数量和纯度较好。利用膜片钳技术检测所分离得保卫细胞原生质体活性, 发现记录的全细胞K⁺电流稳定性好, 光处理明显增加K⁺的内流, 说明所检测原生质体的细胞质膜及其生理活性保持较好。该棉花保卫细胞原生质体分离和鉴定方法的建立, 可为研究棉花保卫细胞膜通透性、离子转运以及气孔运动机理提供基础。

关键词：棉花; 保卫细胞; 原生质体; K⁺电流

收稿：2016-11-07   修定：2017-03-02

资助：国家自然科学基金(31670289和31570294)、国家转基因生物新品种培育重大专项(2016ZX08005-004)和河南省基础与前沿技术研究计划(142300413225)。

Efficient isolation and identification of protoplasts from cotton guard cells

ZHAO Xiang, ZHU Jin-Dong, KONG Pei-Tao, WANG Xue-Jiao, ZHANG Xiao^*

State Key Laboratory of Cotton Biology, Key laboratory of Plant Stress Biology, College of Life Sciences, Henan University, Kaifeng, Henan 475004, China

Corresponding author: ZHANG Xiao; E-mail: xzhang@henu.edu.cn

Abstract:

Isolation of guard cell protoplast is a common approach in model plant Arabidopsis thaliana. However, how to isolate and purify the protoplasts of the guard cell has become a limiting factor for researching the mechanism of stomatal movement in cotton. In this study, a method for isolation and identification of the cotton guard cell protoplasts was investigated. Lower epidermis of cotton leaves was digested with 1.3% cellulase RS and 0.03% pectinase Y-23 for 1.5 hours. Its enzymatic hydrolysates was centrifugated at a low speed of 15×g for 5 min, and then at a higher speed of 105×g for 10 min, by which the higher quality and purity of guard cell protoplasts was obtained from the cotton leaves. We detected the physiological activity of protoplast by patch clamp technique, and found that the whole cell K⁺ current recorded stably and increased significantly induced by light, which proved that the physiological activity of protoplast was better. The isolation and identification of the protoplasts of cotton guard cells, effectively solved the difficult problem of cotton guard cell protoplast separation and provided a basis for studying the membrane permeability, ion transport and stomatal movement mechanism of cotton.

Key words: cotton; guard cell; protoplast; K⁺ current