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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。
However, how to effectively isolate and purify cottonseed protoplasts has become a limiting factor in the regulation of stomatal movement in cotton. This article explored a method of protoplast isolation and activity identification of cotton guard cells. We selected two to three weeks after the growth of young cotton leaf epidermis, using 1.3% cellulase and 0.03% pectinase for one-step enzymatic hydrolysis for about 1.5 h, first low-speed 15 × g centrifugation 5 min, and then high-speed 105 × g 10 min Isolated, the number and purity of the protoplasts of guard cells obtained are better. Patch clamp technique was used to detect the activity of protoplasts isolated from guard cells. It was found that the stability of K + current recorded in whole cells was good, and the influx of K + was significantly increased by light treatment, indicating that the plasma membrane and its physiological activity of the tested protoplasts remained relatively good . The establishment of the method of isolation and identification of protoplasts of cotton guard cells may provide a basis for studying membrane permeability, ion transport and stomatal movement mechanism of cotton guard cells.