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Characterization of scytalone dehydratase and reductase genes and expression of melanin biosynthesis genes in Ophiostoma floccosum Wang, Honglong

Abstract

Wood sapstain is a significant economic problem for the lumber industry. The discoloration of sapwood is mainly caused by sapstain fungi, which grow on wood and produce dark or brown pigment. The aim of the thesis was to obtain some molecular information about the pigmentation of sapstain fungi by cloning and characterizing the major melanin genes. A transformation system is the prerequisite for conducting both gene disruption and genetic complementation of an organism. Transformation systems were set up for Ophiostoma floccosum 387N and other major sapstain fungal species such as Ophiostoma piceae using the transformation vectors pAN7-l and pCJ31004. This transformation system was applied to attempt to disrupt the cloned genes, THN1 encoding a melanin pathway reductase gene and OSD1 encoding a scytalone dehydrates gene in 387N. Unfortunately, no disruptant was identified by screening more than 2,000 transformants. We concluded that homologous DNA integration in O. floccosum 378 would be a rare event. We isolated and characterized a putative scytalone dehydratase gene (OSD1) from O. floccosum 387N encoding a predicted polypeptide sequence of 216 amino acids that shared high homology to other fungal melanin scytalone dehydratases. The function of OSD1 was determined by complementing a Colletotrichum lagenarium scytalone dehydratase deficient mutant. OSD1 was able to restore the melanization and pathogenicity of the mutants. A reductase gene (THN2) encoding a protein of 284 amino acids was isolated, and it shared a 44% amino acid identity to the O. floccosum THN1 genes' deduced protein sequence. We confirmed the function of the THN2 gene by complementing the DHN melanin deficient, non-pathogenic mutants of C. lagenarium and Magnaporthe grisea that lack the 1,3,8-trihydroxynaphthalene reductase gene. Sequence analysis of all available fungal melanin reductases showed that two groups of the reductases are present in fungal DHN melanin biosynthetic pathway. THN1 and THN2 belonged to different groups. We tried to complement a double mutant of M. grisea, where the 1,3,6,8-tetrahydroxynaphthalene reductase gene and the 1,3,8- trihydroxynaphthalene reductase gene have been knocked out, using THN1, THN2 and the combination of THN1 and THN2, respectively. The results indicated that both reductases can not function as the 1,3,6,8-tetrahydroxynaphthalene reductase. However, whether they function in a similar way in O. floccosum remains unknown. A partial melanin PKS gene (OPKS1) was cloned in O. floccosum. The expression of the melanin genes, OPKS1, THN1, OSD1 and THN2 was associated with the mycelial differentiation and affected by nutrients.

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