| Summary: | 博士 === 國立中興大學 === 植物病理學系所 === 96 === The genus Potyvirus is the largest and economically most important plant virus group.
The objective of this study was directed to generate transgenic plants with a high level of
resistance against different potyviruses using the artificial micro RNA (amiRNA) approach. In
order to create a broad-spectrum resistance to different viruses, highly conserved regions among
16 potyviral genomes were identified by sequence alignment. The 273-nt pre-miR159a from
Arabidopsis was chosen as the backbone for the construction and expression of amiRNAs. Using
oligonucleotide-directed mutagenesis, a 21 nucleotide region of miR159 was replaced by a
synthetic 21-nucleotide sequence targeting at the highly conserved regions of potyviral CI, NIb,
or CP genes. Six single, one double and one triple precursor amiRNAs were created. The
constructed sequences were then moved to the binary vector pBA-DC-HA or pB2T-DC-HA. To
check the expression of designed amiRNAs, the total RNA was isolated from agro-infiltrated
leaves and detected by northern blotting with a specific probe against the corresponding
amiRNA sequence (21nt). Our results revealed that all constructed amiRNAs in pre-amiRNA
constructs were efficiently expressed in agroinfiltrated leaves. Agrobacterium-mediated
transformation method was used to transform plants of Nicotiana benthamiana. Transgenic lines
were obtained and their resistance against potyvirus was evaluated by mechanical challenge with
Turnip mosaic virus under greenhouse conditions. Some transgenic lines showed symptom delay
in comparison to non-transgenic plants. One transgenic line expressing the constructed three
amiRNAs exhibited a high level of resistance, indicating that the amiRNA approach targeting at
highly conserved regions of potyviral genomes is a good strategy for generating transgenic
resistance against potyvirus. Whether the transgenic lines provide broad-spectrum resistance
against different potyviruses remains to be further tested.
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