High-efficiency transformation of Chlamydomonas reinhardtii by electroporation.
High-efficiency transformation of Chlamydomonas reinhardtii by electroporation.
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发表时间：
1998-04
期刊：
影响因子：
3.3
通讯作者：
Kosuke Shimogawara;S. Fujiwara;A. Grossman;H. Usuda
中科院分区：
文献类型：
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作者：
Kosuke Shimogawara;S. Fujiwara;A. Grossman;H. Usuda
我们建立了一种高效的电转化单细胞、绿色衣藻的方法。在质粒pJD 67（其含有ARG 7）存在下，使用菌株CC 3395和CC 425（缺乏氨基琥珀酸裂解酶（由ARG 7编码）的无细胞壁突变体）的电穿孔来优化引入外源DNA的条件。改变的条件包括渗透压、温度、外源DNA浓度、电压和电容。优化后，获得的最大转化频率为每微克DNA 2 x 10（5）个转化子;该频率比使用玻璃珠引入外源DNA的当前标准方法获得的频率高两个数量级。本文所述的电穿孔方法具有普遍的实用性，并使得通过莱茵衣藻突变体的直接互补来分离基因成为可能。
We have established a high-efficiency method for transforming the unicellular, green alga Chlamydomonas reinhardtii by electroporation. Electroporation of strains CC3395 and CC425, cell wall-less mutants devoid of argininosuccinate lyase (encoded by ARG7), in the presence of the plasmid pJD67 (which contains ARG7) was used to optimize conditions for the introduction of exogenous DNA. The conditions that were varied included osmolarity, temperature, concentration of exogenous DNA, voltage and capacitance. Following optimization, the maximum transformation frequency obtained was 2 x 10(5) transformants per microg of DNA; this frequency is two orders of magnitude higher than obtained with the current standard method using glass beads to introduce exogenous DNA. The electroporation procedure described in this article is of general utility, and makes it feasible to isolate genes by direct complementation of Chlamydomonas reinhardtii mutants.