Sato T, Yaegashi K, Ishii S, Hirano T, Kajiwara S, Shishido K, Enei H
Iwate Biotechnology Research Center, Japan.
Biosci Biotechnol Biochem. 1998 Dec;62(12):2346-50. doi: 10.1271/bbb.62.2346.
We have used the restriction enzyme-mediated DNA integration (REMI) method to establish a transformation system in Lentinus edodes using the recombinant plasmid pLC1-hph, which contains the L. edodes transcriptional signals and an Escherichia coli hygromycin B phosphotransferase gene. Protoplasts of L. edodes were treated by the PEG transformation mixture containing 50 units of SalI, which cleaves pLC1-hph at a single site, yielding about 15 transformants per 2.5 micrograms of DNA. The conventional PEG transformation without SalI, however, yielded only 1.5 transformants per 25 micrograms of DNA. The optimal amount of SalI for increased transformation was 50 units. In the case of transformation with SphI, which cleaves the plasmid at one site, the optimal amount of the enzyme was 2.5 units. Southern blot analysis of the SphI-derived transformants suggested that 50% of the plasmid integrations were REMI events.
我们使用了限制酶介导的DNA整合（REMI）方法，利用重组质粒pLC1-hph在香菇中建立了转化系统，该质粒包含香菇转录信号和大肠杆菌潮霉素B磷酸转移酶基因。香菇原生质体用含有50单位SalI的PEG转化混合物处理，SalI在单个位点切割pLC1-hph，每2.5微克DNA产生约15个转化体。然而，没有SalI的传统PEG转化，每25微克DNA仅产生1.5个转化体。增加转化的SalI最佳用量为50单位。在用SphI（在一个位点切割质粒）进行转化的情况下，该酶的最佳用量为2.5单位。对SphI衍生的转化体进行的Southern印迹分析表明，50%的质粒整合是REMI事件。
