| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 98 === Bamboo mosaic virus (BaMV), belonging to genus Potexvirus of the family Flexiviridae, is a plus-strand RNA virus. Previously, we have used the cDNA-AFLP technique to identify and isolate some differentially expressed cDNA fragments after BaMV inoculation on Nicotiana benthamiana leaves. These genes may play important roles in positively or negatively regulating the infection cycles of BaMV. Three differentially expressed cDNA fragments ACGT2，ACGT4 and ACGT11 were further characterized. ACGT2, ACGT4, and ACGT11 are orthologs with one of the proteins in photosystem II oxygen evolving complex, thioredoxin h-like protein, and plasma membrane intrinsic polypeptide, respectively, after blasting the sequence to the databased on workbench website. First, I cloned these three fragments into Tobacco rattle virus-based gene-silenced vector, and transformed to Agrobacterium tumefaciens to knock down the expression levels of the cDNA-associated genes. Through agro-infiltration on N. benthamiana leaves, virus was applied on the fourth leaf above the infiltrated leaf after 10 days of infiltration. Total proteins were extracted from the virus inoculated leaves after 5 days of inoculation for a western analysis to inspect the accumulation levels of coat protein. Results showed that the coat protein accumulation levels were increased about 150 to 180% in ACGT2，ACGT4 and ACGT11 knockdown plants to that in control plant. These results suggested that ACGT2, ACGT4 and ACGT11 may have the potential roles as innate immunity genes. The reduction of the expression of these genes could enhance the viral accumulation in plants. Further, I have inoculated BaMV RNA into protoplasts derived from the knockdown and control plants and showed no significant difference of the accumulation of BaMV coat protein. Over all of these results suggest that the expression of these genes did not have the interference on the replication of BaMV viral RNA but may have the inhibition on cell-to-cell movement. The BaMV CP accumulation detected on the 7dpi from ACGT11 knockdown plants was much more than the control plants. The average area of fluorescent viral lesions detected by fluorescence microscope is larger on ACGT11 knockdown plants than the control plants. These data suggest that decreasing ACGT11 expression do unchain the inhibition of BaMV. The fluorescence of GFP fused ACGT11 proteins were translocated from cell periphery to cytoplasm was detected by confocal microscope. These data indicate ACGT11 affect BaMV movement in an unknown mechanism.
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