Introduction

We investigated the main factors affecting the efficacy of protoplast isolation, including leaf-obtaining period, cutting shapes of leaf material, enzyme concentration, enzymolysis time, and centrifugal speed.

Methods

Protoplast isolation was optimal on the condition of 20 days of leaf materials, 2-mm filament of leaves, 1.6% RS and 0.8% R-10, 80 min of enzymolysis, and 700 rpm of centrifugation, resulting in the best yield (1.19 X 106 protoplasts/g FW) and vitality (80.34%) of mesophyll protoplasts. The transient expression vector pGFPl with green fluorescent protein was transfected into the obtained protoplasts from castor by polyethylene glycol-mediated method with a transformation efficiency of 12.37%.

Results

Moreover, the applicability of the system for studying the subcellular localization of Re FATA (an acyl-ACP thioesterase) was validated via the protoplast isolation and transient expression protocol in this study.

Discussion

Collectively, the efficient mesophyll protoplast isolation and protoplast transient expression system facilitate to analyze the function of specific gene in castor (Ricinus communis L).

中文翻译：

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蓖麻瞬时表达系统的高效叶肉原生质体分离与开发

介绍

我们研究了影响原生质体分离效果的主要因素，包括叶片获得期、叶片材料的切割形状、酶浓度、酶解时间和离心速度。

方法

原生质体分离在叶材20天、叶丝2-mm、RS 1.6%、R-10 0.8%、酶解80 min、离心700 rpm条件下最佳，产量最佳(1.19 X 106 个原生质体/g FW) 和叶肉原生质体的活力 (80.34%)。用聚乙二醇介导的方法将带有绿色荧光蛋白的瞬时表达载体pGFP1转染到所得蓖麻原生质体中，转化率为12.37%。

结果

此外，该系统在研究 Re FATA（一种酰基 ACP 硫酯酶）的亚细胞定位方面的适用性通过本研究中的原生质体分离和瞬时表达协议得到验证。

讨论

总的来说，高效的叶肉原生质体分离和原生质体瞬时表达系统有助于分析蓖麻(Ricinus community L)中特定基因的功能。