| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 103 === Abstract
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to genus Potexvirus of family Flexiviridae. To identify the differentially expressed host genes possibly involved in the infection cycle of BaMV, cDNA-AFLP technique was taken to identify those cDNA fragments after infection in Nicotiana benthamiana leaves. One of the downregulated genes ACAG1 is further characterized. To investigate whether ACAG1 is involved in regulating the infection cycle of BaMV, the ACAG1 cDNA fragment is cloned into Tobacco rattle virus (TRV)-based gene silencing vector and transformed to Agrobacterium tumefaciens. When the gene is knocked down by the silencing vector, the accumulation of BaMV coat protein is increase to approximately 2 folds of that of the control. Furthermore, the accumulation of BaMV coat protein in ACAG1-knockdown protoplasts is approximately 1.4 folds of that in the control protoplasts. These results suggest that the host gene ACAG1 could be involved in the replication step of BaMV infection cycle. In addition, the full-length cDNA of ACAG1 is cloned using rapid amplification of cDNA ends (RACE) technique. The identity of the ACAG1 gene is revealed as the non-specific ferredoxin NADP+ reductase (designated as NbFNR) when the sequence of the full-length cDNA is compared to the transcriptome database. Furthermore, NbFNR is transiently expressed in plants and localized in the chloroplasts, the accumulation of BaMV coat protein is reduced to approximately 76% of that of the control. The results suggest that NbFNR could play an inhibiting role in the infection cycle of BaMV. FNR is an oxidoreductase and functioning in the photosynthetic electron transfer chain that catalyzes the NADP+ and H2O to form NADPH and OH-. Since the plants in the light and dark phase were shown to have different affinity betweenFNR and Tic62/Trol and resulting in alkaline and acidic situation in the stroma, respectively. Thus, as BaMV infected in plants and growth in dark, the relative accumulation of BaMV RNA is reduced to 30% of that of the control. The results indicate that the plants in dark might have effect on BaMV RNA replication. Furthermore, I will construct mutants that remove the transit peptide or diminish the NAD binding site to examine the mechanism of the inhibitory role of FNR in BaMV replication.
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