Weltring K M, Turgeon B G, Yoder O C, VanEtten H D
Department of Plant Pathology, Cornell University, Ithaca, NY 14853.
Gene. 1988 Sep 7;68(2):335-44. doi: 10.1016/0378-1119(88)90036-4.
Detoxification of the pea phytoalexin pisatin via demethylation, mediated by a cytochrome P-450 monooxygenase, is thought to be important for pathogenicity of the fungus Nectria haematococca on pea. To isolate a fungal gene encoding pisatin demethylating activity (pda), we transformed Aspergillus nidulans with a genomic library of N. haematococca DNA constructed in a cosmid which carried the A. nidulans trpC gene. Transformants were selected for Trp+ and then screened for pda. One transformant among 1250 tested was Pda+ and was less sensitive to pisatin in culture than Pda- A. nidulans. The cosmid containing the gene (PDA) conferring this activity was recovered by phage lambda packaging of transformant genomic DNA. When A. nidulans was transformed with the cloned cosmid, 98% of the Trp+ transformants were Pda+. RNA blots probed with a 3.35 kb subclone carrying PDA indicated that the gene is expressed constitutively in A. nidulans but is inducible by pisatin in N. haematococca.
由细胞色素P - 450单加氧酶介导的豌豆植保素豌豆素通过去甲基化作用进行解毒，这一过程被认为对血红色丛赤壳菌在豌豆上的致病性很重要。为了分离编码豌豆素去甲基化活性（pda）的真菌基因，我们用构建在携带构巢曲霉trpC基因的黏粒中的血红色丛赤壳菌DNA基因组文库转化构巢曲霉。选择色氨酸营养型（Trp+）转化子，然后筛选pda。在1250个测试转化子中，有一个转化子是Pda+，并且在培养中比Pda-构巢曲霉对豌豆素的敏感性更低。通过对转化子基因组DNA进行λ噬菌体包装回收了含有赋予这种活性的基因（PDA）的黏粒。当用克隆的黏粒转化构巢曲霉时，98%的色氨酸营养型（Trp+）转化子是Pda+。用携带PDA的3.35 kb亚克隆进行RNA印迹杂交表明，该基因在构巢曲霉中组成型表达，但在血红色丛赤壳菌中可被豌豆素诱导表达。
