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Mechanistic Study of Electrofusion of Plant Protoplasts

植物原生质体电融合机理研究

基本信息

批准号：
63560062
负责人：
KAKUTANI Tadaaki
金额：
$ 1.22万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

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.

1. 开发了一种利用介电泳聚焦的新型电融合方法。使用这种方法，研究了大麦叶肉原生质体的电融合作为所施加的脉冲和交流场的函数。在通过脉冲电场诱导融合后施加交流场导致融合的原生质体对立即向两个电极瞬时延伸。延伸随着脉冲或交流场强度的增加而增加，但是当从熔化感应到施加交流场所花费的时间长于约10秒时，没有观察到这种现象。这些结果表明，处于可熔或融合状态的质膜的性质与融合诱导之前的质膜有很大不同，并且可熔膜状态的寿命约为10秒。对这种现象的定量分析将为融合原生质体质膜的研究提供一种有用的方法。 2．采用细胞电泳法研究了大麦叶肉和萝芙木培养细胞原生质体电融合产生的异源融合原生质体的表面电荷密度。结果表明，融合原生质体的表面电荷密度本质上是由亲本原生质体的表面电荷密度和尺寸决定的。 3．该方法被改进，用于使用荧光探针 diS-C_3-(5) 评估植物原生质体的膜电位。导出了荧光猝灭与膜电位之间的定量关系，并通过紫草培养细胞（MA系）分离的原生质体进行了实验验证。该方法用于研究 Ca 、 La 离子钙调蛋白拮抗剂（据报道这些化学物质影响电融合过程）对原生质体膜电位的影响。