Mechanistic Study of Electrofusion of Plant Protoplasts

1. 提出了一种利用介电泳聚焦的新型电熔方法。利用该方法研究了大麦叶肉原生质体电融合对脉冲电场和交流电场的影响。脉冲电场诱导融合后施加交流电场，使原生质体的融合对立即向两个电极瞬时延伸。随着脉冲或交流电场强度的增加，延伸量增加，但当从聚变感应到施加交流电场的时间超过10 s时，这种现象就没有出现。这些结果表明，处于易熔或熔合状态的质膜的性能与熔合诱导前有很大的不同，可熔膜状态的寿命约为10 s。这一现象的定量分析将为研究融合原生质体的质膜提供有用的方法。用细胞电泳法研究了大麦叶肉原生质体和粗尾狼原生质体电融合后的表面电荷密度。结果表明，融合原生质体的表面电荷密度主要由各亲本原生质体的表面电荷密度和大小决定。提出了利用荧光探针diS-C_3-(5)评价植物原生质体膜电位的方法。建立了红紫草培养细胞（MA系）原生质体荧光猝灭与膜电位之间的定量关系，并进行了实验验证。这种方法被应用于研究钙离子、钙调素拮抗剂（这些化学物质被报道影响电融合过程）对原生质体膜电位的影响。

1. A new electrofusion method using dielectrophoretic focusing was developed. Using this method electrofusion of barley mesophyll protoplasts was studied as function of applied pulse and ac fields. Application of ac field after induction of fusion by pulse electric field caused immediately transient extension of the fusing pair of protoplasts toward both electrodes. The extension increased with increasing strength of pulse or ac field but this phenomenon was not observed when the time taken from the fusion induction to application of the ac field was longer than about 10 s. These results suggest that the properties of the plasma membrane being in the fusible or fusing state are very different from those before the fusion induction and that the life time of the fusible membrane state is about 10 s. Quantitative analysis of this phenomenon will provide a useful approach for the study of the plasma membrane of the fusing protoplasts.2. The surface charge density of the hetro-fused protoplast, which was produced by electrofusion from protoplasts of barley mesophyll and Rauwolflia sperpentina cultured cells, was studied by cell electrophoresis. The results indicated that the surface charge density of the fused protoplasts is essentially determined by the surface charge density and size of each parent protoplast.3. The methodology was advanced for the evaluation of membrane potentials of plant protoplasts using a fluorescent probe, diS-C_3-(5). The quantitative relationship between the fluorescent quenching and membrane potential was derived and was verified experimentally for protoplast isolated from Lithospermum erythrorhizon cultured cell (MA line). This method is being applied to study the effects of Ca , La ions calmoduline antagonists (these chemicals were reported to influence the electrofusion processes) on the membrane potential of the protoplasts.