RNAi-mediated gene silencing of lanosterol 14α- demethylase (ERG11)-encoding gene in ganoderma boninense
Oil palm basal stem rot (BSR) disease is caused by several species of Ganoderma including Ganoderma boninense. Available molecular tools for G. boninense are essential for providing the knowledge on the fungal infection process in oil palm. The objectives of this study are to isolate the full-len...
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| Format: | Thesis |
| Language: | English |
| Published: |
2022
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/104437/1/LIM%20FOOK%20HWA%20-IR.pdf |
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| Summary: | Oil palm basal stem rot (BSR) disease is caused by several species of
Ganoderma including Ganoderma boninense. Available molecular tools for G.
boninense are essential for providing the knowledge on the fungal infection
process in oil palm. The objectives of this study are to isolate the full-length cDNA
encoding Lanosterol 14α-demethylase (GbERG11) from G. boninense, to
optimize a polyethylene glycol (PEG)-mediated protoplast transformation
protocol for G. boninense, to perform the GbERG11 gene functional study via
RNAi-mediated gene silencing approach and to transform the hpRNA-GbERG11
vectors into oil palm. A full-length 1980 bp cDNA encoding GbERG11 was
successfully isolated and the GbERG11 shared high similarity (91%) to ERG11
from other basidiomycete fungi. Southern blot and genome data analyses
indicated that there is only a single copy of GbERG11 gene in the G. boninense
genome. An average concentration of 107/ml viable protoplasts were
successfully isolated from G. boninense mycelium. The G. boninense PEGmediated
protoplast transformation using 1 μg of transformation vector, 25% of
PEG solution, 10 min of pre-transformation incubation and 30 min of posttransformation
incubation has improved the transformation efficiency by 33.5
folds on average. Three hpRNA vectors corresponding to different regions of
GbERG11 were prepared using the in vitro recombination between the entry
vectors (containing different target regions of GbERG11) and hpRNA vector,
pH7GWIWG2(I). The G. boninense transformed with the hpRNA vectors have
shown reduced growth, expression of ERG11 and ergosterol content as much
as 57.3%, 32.9% and 42.9%, respectively as compared to the PER71 (wild type).
Less severe infection symptoms were observed on oil palm plantlets inoculated
with G. boninense transformants as compared to the G. boninense PER71 (wild
type) in the initial stages of in vitro inoculation study. Besides that, particle
bombardment of oil palm calli with the hpRNA vectors (GbERG11) was
performed. The selection process has led to regeneration of 8 putative
transgenic greenish polyembryoids. As a conclusion, a gene function study for
G. boninense has been successfully developed by using the PEG-mediated
protoplast transformation and RNAi-mediated gene silencing approaches.
Reduced GbERG11 gene expression followed by reduced ergosterol content
was observed in the G. boninense transformants indicating the functional of this
molecular tool. The G. boninense transformants have showed reduced
pathogenicity towards oil palm at the initial stages of the in vitro inoculation study,
which could highlight the GbERG11 gene role at the initial stage of infection. The
developed molecular tools in this study can be applied for studying other genes
in G. boninense, as well as other Ganoderma species or basidiomycete fungi.
The putative transgenic oil palm plantlets can be further evaluated especially on
the resistance against G. boninense, which could indicate the potential
application of RNAi-mediated gene silencing in protecting oil palm against the
Ganoderma infection. |
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