两种瞬时表达体系研究拟南芥Profilin-1的亚细胞定位

Subcellular Localization of Profilin-1 from Arabidopsis Utilizing Two Transient Expression Systems

使用两种瞬时表达方法研究Profilin-1(PRF1)的亚细胞定位,并比较了2种瞬时表达体系在亚细胞定位研究中的优缺点。利用拟南芥幼叶作为材料,提取叶片的RNA,采用特异性引物RT-PCR的方法克隆PRF1基因,连接到p CAMBIA1300-GFP的改造载体上,成功的构建p CAMBIA1300-GFP-PRF1的表达载体。然后分别利用PEG转化拟南芥原生质体、农杆菌浸染烟草叶片两种技术进行了瞬时表达,并在激光共聚焦显微镜下观察绿色荧光蛋白(GFP)融合蛋白的表达。研究结果表明,将PRF1基因导入拟南芥的原生质体和烟草表皮细胞后,融合蛋白绿色荧光均能被观察到,PRF1基因与GFP融合蛋白的产物在烟草表皮细胞中主要定位在细胞质和外周细胞器中,在拟南芥的原生质体中的细胞核和细胞质中都有定位。两种不同的瞬时表达体系中PRF1蛋白的定位出现了不同,这可能与同源或异源表达的植物的特性相关。

Here we study the subcellular localization of profilin-1 （ PRF1 ） using two transient expression methods and compare the advantages and disadvantages of two transient expression methods in the subcellular localization. Using the leaves of Arabidopsis thaliana as material, the total RNA from the leaves was extracted, and PRF1 gene was cloned using specific primers by RT-PCR and then ligated to pCAMBIA13OO-GFP vectors, finally one plant expression vector pCAMBIA1300- GFP-PRF1 was constructed successfully. Two transient expression systems, agroinfiltration of tobacco leaves, and PEG transformation of protoplasts isolated from the leaves of Arabidopsis thaliana were adopted. The expression of the fusion proteins was observed by a confocal laser scanning microscopy. The results showed that the green fluorescence of the fusion protein was observed when the PRF1 gene was introduced into the protoplasts of A. thaliana and tobacco epidermal cells, the product of PRF1 gene and GFP fusion protein was mainly localized in the cytoplasm of tobacco epidermal cells, and there was also an expression in the nuclear cell organelle. PRF1 was localized in the nucleus and cytoplasm ofArabidopsis thaliana. The localization of PRF1 protein was different in two different transient expression systems, which may be related to the characteristics of the plants that are homologous or heterologous expression.