Ⅰ Study of Tobacco mosaic virus : expression, purification and activity of methyltransferase.Ⅱ The effect of overexpressing Saccharomyces cerevisiae GPD1p on mitochondrial morphology.
| Autor: | Yu-Hung Lu, 呂毓鴻 |
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| Rok vydání: | 2013 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 PartⅠ Tobacco mosaic virus is the most common virus in the plant. The genome of this virus can be translated to protein which contains methyltransferase activity. In the past, a lot of researches focused on the viral nucleic acid methylation of the host. The host protein methylation has not yet been studied. In this study, we used recombinant E. coli to express heterologous protein. At first, E. coli. expressed the his-tag protein with inclusion body. We tried a number of conditions, such as cooling or adding surfactants, but it still expressed the protein with inclusion body. Some studies have found that GST tag could help protein fold with correct form. Therefore, we used pGEX4T-1 as a carrier vector to construct pGEX4T-1-MT and finally expressed the protein present in the supernatant. We purified the GST fusion protein with GSH column and reacted with Hot-SAM and tobacco crude protein. Experimental results showed that it had more substrate signal in the infected plants than uninfected plants. Therefore, E. coli can express heterologous proteins with activity and may allow host protein methylation. PartⅡ Previous studies have found that ADH3 protein in Saccharomyces cerevisiae affects morphology of mitochondria. The putative reason is that ADH3 protein can influence NADH/NAD+ ratio. It has been shown that GPD1 protein could synthesize glycerol and oxidize NADH to NAD+. Therefore, we constructed pYes2-GPD1-GFP and transformed it to S. cerevisiae. Experimental results show that after we induced the construct gene, pYes2-GPD1-GFP, the mitochondrial morphology of S. cerevisiae appeared fragmented for 83 percent. With non-induced genes, pYes2-GPD1-GFP and pYes2, the mitochondrial morphology of S. cerevisiae appeared tubular. When we replaced the medium glucose to glycerol, we found that both constructs, pYes2-GPD1-GFP and pYes2, made mitochondrial morphology to patterns appear fragmented. The result showed that the morphology of mitochondria may be influenced by NADH/NAD+ ratio. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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