Godio R P, Fouces R, Gudiña E J, Martín J F
Institute of Biotechnology of León, INBIOTEC, Science Park, Av. Real 1, 24006 Leon, Spain.
Curr Genet. 2004 Nov;46(5):287-94. doi: 10.1007/s00294-004-0533-5.
The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (P gpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.
担子菌亚侧耳（Hypholoma sublateritium）可产生抗肿瘤类异戊二烯化合物棒酸。该真菌的节孢子通过根癌农杆菌介导的接合作用进行转化。测试了携带不同调控序列以驱动潮霉素磷酸转移酶（hph）基因表达的五种质粒。为了在亚侧耳中表达异源基因，所使用的启动子至关重要。携带双孢蘑菇甘油醛-3-磷酸脱氢酶启动子（P gpd）的构建体显示出良好的转化效率，而带有子囊菌gpd启动子的构建体则无效。转化体克隆显示出质粒DNA的随机整合模式。大多数转化体显示出转化质粒的单拷贝整合，但约1.5%显示出双拷贝或多拷贝整合。所有分析的转化体在有丝分裂过程中都是稳定的，并且在没有抗生素的情况下保持整合的外源DNA。如逆转录聚合酶链反应（RT-PCR）研究所示，绿色荧光蛋白基因由双孢蘑菇gpd启动子表达，但未观察到明显荧光。亚侧耳的转化为该真菌中棒酸生物合成的基因操作开辟了道路。
