Construction of Vectors Specific for Virus Resistance and Transient Expression Analysis in Orchids
碩士 === 國立臺灣大學 === 園藝學研究所 === 92 === The application of RNA interference (RNAi) to disrupt gene expression has become a powerful tool in plants. To construct specific RNAi vectors for virus resistance in orchids, the third intron of Oncidium ethylene receptor gene Og-ERS1 was synthesized using polyme...
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| Format: | Others |
| Language: | zh-TW |
| Published: |
2004
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| Online Access: | http://ndltd.ncl.edu.tw/handle/76813062486765698709 |
| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 92 === The application of RNA interference (RNAi) to disrupt gene expression has become a powerful tool in plants. To construct specific RNAi vectors for virus resistance in orchids, the third intron of Oncidium ethylene receptor gene Og-ERS1 was synthesized using polymerase chain reaction (PCR). The resulting expression vector containing 2X Cauliflower mosaic virus (CaMV) 35S promoter driving RNAi cassette, was used for double-stranded RNA expression and comprised the full-length coding region sequences of Cymbidium mosaic virus (CyMV) or Odontoglossum ringspot virus (ORSV) coat protein gene in the sense and antisense configuration on either side of the intron fragment. Furthermore, four highly homologus regions of amino acid sequences of CyMV and ORSV coat protein isolated from various genera of orchids were used to construct double-stranded 25 bp small interference RNA (siRNA) expression plasmid.
Reporter plasmid encoding CyMV coat protein was co-transformed with antisense-sense RNAi effector plasmid via transient expression analysis in protoplasts from Phalaenopsis petals. The 1:1 molecule ratios between CyMV coat protein reporter plasmid and RNAi effector plasmid had 95% silencing effect using indirect enzyme linked immunosorbent assay for analysis. The proper time point to analyze the silencing effect of CyMV coat protein RNAi effector plasmid was 48 hr after co-transformation for 94% silencing. The silencing effect of full length CyMV coat protein RNAi construct was 100%. The production of coat protein was reduced by 54% for siRNA expression construct containing the third homologous region detected 48 hr after co-transformation. Immunoblot assays showed that CyMV coat protein expressed in transformed protoplasts from Phalaenopsis petals with the expected molecular mass of 27 kDa. No detectable CyMV coat protein in protoplasts co-transformed with reporter plasmid and RNAi effector plasmid was found. Decreased levels of coat protein were accumulated in protoplasts co-transformed with reporter plasmid and the siRNA expression construct containing third homologous region
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