| Summary: | 碩士 === 國立中興大學 === 農業生物科技學研究所 === 90 === Bamboo mosaic potexirus (BaMV) and potato virus X (PVX) belong to potexvirus group whose genome consists of a single-stranded, positive-sense RNA molecule. In BaMV RNA, the sequence in the 3´ UTR has been demonstrated to interact with the viral RNA-dependent RNA polymerase. In PVX RNA, an eight-nucleotide sequence in the 3´UTR is required for host protein binding and viral RNA multiplication. The 3´ UTR of BaMV RNA can be divided into 3 domains, ABC, D and E and form a tertiary structure comprising four stem-loops and a pseudoknot structure. The structure of the 3´ UTR of PVX RNA has only been predicted by mfold program and shows a simply three-stem-loop structure. In previous study, the 3´ UTR of PVX RNA could be recognized by BaMV replicase, and used as a template to synthesize the minus strand in vitro. In this study, we would like to know whether the 3´ UTR of BaMV RNA could be replaced with the 3´ UTR of PVX RNA in vivo as that was shown in vitro. We have designed eight different mutations with the replacement of the 3´ UTR of PVX RNA at different region of the 3´ UTR of BaMV RNA. Results showed that no mutant could replicate in protoplasts and plants of Nicotiana benthamiana except BaMV/PABCDE. It suggested that the 3´ UTR of BaMV RNA could not be replaced with the 3´ UTR of PVX RNA in vivo. However, in an in vitro replication assay rABCP could reach to 80% that of wild type. It suggested that the 3´ UTR of PVX RNA could functionally replace D and E domains of the 3´ UTR of BaMV RNA in vitro. Results of kinetic study of the RNAs with BaMV replicase showed that the KM and Vmax of r138/40A, rABCP, rP, and rDE is 302, 296, 533, and 210 nM, and 63, 21, 25, and17 min-1, respectively. These results indicated that the replication failure of mutant BaMV/ABCP in protoplasts and plants could be due to the less efficient of replication rate in minus strand RNA synthesis.
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