Proteomic Analysis of Differential Protein Expression of Bamboo mosaic virus Infected Nicotiana benthamiana

• Main Authors: Li-Chin Wu, 吳麗琴
• Other Authors: Yau-Heiu Hsu
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/89582669429601137127

碩士 === 國立中興大學 === 生物科技學研究所 === 95 === Bamboo mosaic virus (BaMV), is a single-stranded positive-sense RNA virus with flexuous rod-shaped morphology. A number of studies have been devoted to analyze the replication of plus-stranded RNA viruses; several host proteins are known to be involved in assembling the viral RNA replication complex, activating the complex for RNA synthesis, and other steps. However, the host has the defense system to against viral replication and spreading which are also mediated through proteins. In this study, we have tried to identify the differentially expressed proteins between mock and BaMV inoculated N. benthamiana leaves by proteomics approach. Since positive-sense RNA virus replication is usually associated with rearrangements of membranous organelles, we first isolated the membrane fraction (P30) of the N. benthamiana leaves. Proteins associated with P30 fraction were resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE) and identified by MALDI-TOF MS. Comparison of protein patterns from P30 fractions of mock and BaMV-infected N. benthamiana in 2-D gels revealed several differential expressed proteins. The differential expression of ribosomal protein L25, RubisCO small chain, calmodulin-1 and glycoprotein endopeptidase-like protein. Among these proteins, ribosomal protein L25 was only found in P30 fractions sample from BaMV-infected plants. It had been reported that ribosomal protein L25 is homologous to general stress proteins CTC. We utilized TRV- based virus-induced gene silencing (VIGS) system to generate L25-knockdown plants and to investigate the effect of this protein on BaMV accumulation. Real-time PCR results showed that the levels of the L25 mRNA in the various L25 knock-down plant were reduced to about 25 and 40 % to those of the control plants. Western blots and northern blots were used to analyze the accumulation of viral coat protein and viral RNA at seven day post-inoculation of BaMV virions. Results showed that the accumulation of BaMV coat protein and viral RNA in L25-knockdown plant were reduced. But no interference on the accumulation of FoMV coat protein was observed in all L25-knockdown plants. Together, these data suggest that L25 likely plays an important role in the BaMV accumulation. Keywords：Bamboo mosaic virus / host factors / proteomic