Organelle specific expression of bamboo mitochondrial-encoded complex I subunits NAD1 and NAD4 in yeast Saccharomyces cerevisiae
| Autor: | Cheng-Hung Hsieh, 謝鎮鴻 |
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| Rok vydání: | 2012 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 100 NADH:ubiquinone oxidoreductase (complex I) is the first complex of the mitochondrial respiratory chain. It catalyzes electron transfer from NADH to ubiquinone (UQ) and pumps protons from matrix into inter-membrane space. The NAD1 and NAD4, which are the core subunits of complex I and encoded by mitochondrial nad1 and nad4 genes, reside at inner-membrane and function possibly to bind with ubiquinone and to translocate proton. To investigate the individual functional role of bamboo Phyllostachys edulis NAD1 and NAD4 subunits, they were heterologously expressed in the yeast Saccharomyces cerevisiae mitochondria. The cDNAs of bamboo mitochondrial nad1 and nad4 were cloned into a galactose inducible vector pYES2. The mitochondrial targeting (MT, encoded by mt) peptide gene and egfp gene were fused at 5’-terminal and 3’-terminal of nad1 or nad4, respectively. The constructed plasmids were confirmed by restriction enzyme digestion, PCR, and DNA sequencing, and then transformed into S. cerevisiae. The yeast transformants were selected. The mt-nad1-egfp and mt-nad4-egfp transcripts and MT-NAD1-EGFP and MT-NAD4-EGFP fusion proteins were observed in the yeast transformants. The localization of MT-NAD1-EGFP and MT-NAD4-EGFP fusion proteins at mitochondria of S. cerevisiae were confirmed by fluorescence microscopy. The oxygen consumption rates of yeast transformant mitochondria were measured and the rates of transformant mitochondria were found to be lower than the control. The results of this study may pave the way for further functional studies of NAD1 and NAD4 subunits. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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