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Transformation of basidiomycetes protoplasts by electroinjection of plasmid DNA

电注射质粒 DNA 转化担子菌原生质体

基本信息

批准号：
62560161
负责人：
MIURA Kiyoshi
金额：
$ 1.22万
依托单位：
Hokkaido 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：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

The aim of present study is to obatin basic information with regard to the basidiomycetes cell by electroinjection of plasmid DNA, through the experimental process of applying electrical stimulation to basidiomycetes protoplasts.With regard to protoplalst fusion, the following observations were made. Adhesion to the electrode occurred at a frequency of 2 MHz in an AC-field with a field strength of 50 V/cm or more; pearl chains were generated at a field strength of 750 V/cm. At AC field strengths over 1.25 KV/cm. It became more difficult for the protoplasts to separate from the electrode. Adhesion between fifferent species of protoplasts was accomplished by first placing on species of protpolast into a fusion chamber, adhering them to the electrode with an AC-field strength of 125 V/cm, and then adding a different species of protoplast to the chamber and applying an ACfield of 250 V/cm. Under these conditions, it was possible to cause fusion with a DC pulse voltage of 10 KV/cm and a duration of 6 sec. The maximum fusion ratio was 10% with this procedure. The rotation of protoplasts, which prevents fusion, halted at voltages less than or equal to 250 V/cm.The equipment used for the present study was of the author's own design, leaving room for improvement in its operability. In order to obtain the AC and pulse voltages required for fusion, the electrode in the fusion chamber must be extremely marrow (40-50 m), and this resulted in some difficulties with regard to the manufacture of the chamber as well as the fusion of rotoplasts. The greatest advantages of the completely closed fusion chamber are: (1) it allows the maintenance of sterile conditions in non-sterile atmosphere, and (2) adhesion between different species of protoplasts can easily be accomplished by varying the AC voltage.

本研究的目的是通过对担子菌原生质体进行电刺激的实验过程，通过电注射质粒DNA来获得有关担子菌细胞的基本信息。对于原生质体融合，进行了以下观察。在场强为 50 V/cm 或更高的交流场中，频率为 2 MHz 时发生电极粘附；珍珠链在 750 V/cm 的场强下生成。交流场强超过 1.25 KV/cm。原生质体与电极分离变得更加困难。不同种类的原生质体之间的粘附是通过以下方式实现的：首先将原生质体种类放入融合室中，将它们粘附到具有125V/cm的交流场强度的电极上，然后将不同种类的原生质体添加到室中并施加250V/cm的交流场。在这些条件下，可以用10KV/cm的DC脉冲电压和持续时间6秒来引起熔合。此过程的最大融合率为 10%。原生质体的旋转在电压低于或等于250V/cm时停止，从而阻止融合。本研究中使用的设备是作者自己设计的，其可操作性还有改进的空间。为了获得融合所需的交流和脉冲电压，融合室中的电极必须非常细（40-50 m），这给室的制造以及旋转体的融合带来了一些困难。完全封闭的融合室的最大优点是：（1）它允许在非无菌气氛中维持无菌条件，（2）通过改变交流电压可以轻松地实现不同种类的原生质体之间的粘附。