通过电穿孔法对莱茵衣藻进行高效转化。
High-efficiency transformation of Chlamydomonas reinhardtii by electroporation.
作者信息
Shimogawara K, Fujiwara S, Grossman A, Usuda H
机构信息
Laboratory of Chemistry, Teikyo University School of Medicine, Hachioji, Tokyo, Japan.
出版信息
Genetics. 1998 Apr;148(4):1821-8. doi: 10.1093/genetics/148.4.1821.
Abstract
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.
摘要
我们已经建立了一种通过电穿孔转化单细胞绿藻莱茵衣藻的高效方法。在质粒pJD67（含有ARG7）存在的情况下，对CC3395和CC425菌株（缺乏精氨琥珀酸裂解酶（由ARG7编码）的无细胞壁突变体）进行电穿孔，以优化外源DNA导入的条件。变化的条件包括渗透压、温度、外源DNA浓度、电压和电容。优化后，每微克DNA获得的最大转化频率为2×10⁵个转化体；该频率比目前使用玻璃珠导入外源DNA的标准方法高出两个数量级。本文所述的电穿孔程序具有普遍实用性，使得通过直接互补莱茵衣藻突变体来分离基因成为可能。
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