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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.
Item Metadata
Title |
Characterization of scytalone dehydratase and reductase genes and expression of melanin biosynthesis genes in Ophiostoma floccosum
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2002
|
Description |
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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Extent |
17641576 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-10-05
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0090694
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2002-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.