Isolation of a gene from the melanin pathway of the sapstaining fungi Ophiostoma piceae using PCR

IRG/WP 97-10219

R Eagen, J Kronstad, C Breuil

To prevent sapstaining fungi from discoloring wood, it is necessary to determine what factors affect the biosynthesis and characteristics of the pigment(s) and to identify the genes involved in the pathway. Using inhibitors and heterologous DNA probes from Alternaria alternata, we suggest that melanin, the pigment of Ophiostoma piceae, is produced by the dihydroxynaphthalene (DHN) pathway. Recently, sequences were published for one of the enzymes in the DHN pathway of Colletotrichtum lagenarium, a cucumber pathogen, and Magnaporthe grisea, a pathogen of rice. From this information we synthesized degenerate oligonucleotides to the conserved regions of the trihydroxynaphthalene (THR1) and tetrahydroxynaphthalene reductase (ThnR) genes. Using these primers and genomic O. piceae as template DNA, we obtained a 365 nucleotide PCR product. The deduced amino acid sequence of the product had 85% homology to the Thr1 of C. lagenarium and 80% homology to the ThnR of M. grisea. This PCR product will be used to screen a genomic library of O. piceae in order to isolate the entire gene sequence in the melanin pathway. Complete characterization of the genes involved should facilitate more direct development of anti-stain strategies.


Keywords: SAPSTAINING FUNGI; OPHIOSTOMATACEAE; PCR; PIGMENT; MELANIN; DHN

Conference: 97-05-25/30 Whistler, British Columbia, Canada


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