植物肌动蛋白的纯化及荧光标记

Preparation of Actin and Fluorescent Actin Analogs from Plant Cells 

以植物花粉为材料,利用肌动蛋白可以与其单体结合蛋白———profilin特异性结合的特性及profilin的多聚脯氨酸亲和柱层析法,获得较大量、高纯度,具有活性的植物肌动蛋白,并用羧酸俄勒冈绿对所获纯化肌动蛋白进行了荧光标记。结果显示,从10g玉米(ZeamaysL.)花粉中可得到1.2mg具有绿色荧光的肌动蛋白,标记率为60%。体外实验结果表明,所得荧光肌动蛋白在适宜条件下可聚合成绿色荧光微丝。

The plant actin cytoskeleton provides a dynamic cytoplasmic framework for many fundamental cellular processes like cytoplasmic streaming, cytokinesis and morphogenesis. Understanding the actin organization and structure in plants requires the generation of new probes for measuring actin dynamics in living cells. Fluorescent analog cytochemistry presents an unrivaled opportunity to probe the actin cytoskeleton in living cells. Such method using in the study of plant actin cytoskeleton has not been reported. By using this method, based on the affinity chromatography of profilin with PLP_Sepharose (PLP: poly_L_proline) for actin purification, the author obtained 6 mg of >98% in purity, polymerizable actin from 10 g of maize (Zea mays L.) pollen, and this actin was successfully labeled with Oregon Green 488 carboxylic acid. From 10 g of maize pollen, 1.2 mg with 60% dye/protein ratio, polymerizable, fluorescent actin analog was obtained. The study yields an effective method for purifying plant actin and preparing fluorescent analog, which may provide facilities for the study of actin dynamics in plant cells.