Cloning and homologous expression of hydrogenase in Chlorella sp. DT
| Autor: | Ting-Ting Kuo, 郭婷婷 |
|---|---|
| Rok vydání: | 2011 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 99 Green algae have a photosynthetic system similar to plants but can produce hydrogen by using sun light under anaerobic conditions. The hydrogenase (HydA) of green algae accepts electrons directly from ferredoxin and generate hydrogen. HydA is nuclear-encoded by hydA gene, and its transcription and activity are strictly inhibited by oxygen. Chlorella sp.DT (DT), a Taiwanese isolate, was able to produce hydrogen under anaerobic or sulfur-deprived (-S) conditions. In this study, we attempted to homologously overexpressed hydA to enhance hydrogen production in DT under normal condition. The nucleotide sequences of genomic DNA and cDNA of DT hydA were sequenced, and found to be highly conserved as compared with other green algae. The coding region of DT hydA was constructed into plasmids and driven by the promoters which are commonly used in plants. The DT cells were transformed with plasmids pHm3A-hydA, pHyg3-hydA or pRpsbOa-hydA. The observation of homologous DT hydA fragment in DT transformants by PCR indicated that the plasmids were successfully transformed into DT. The observations of hydA transcript by RT-PCR and HydA protein by western blotting in certain DT transformants suggested that hydA could be expressed in these transformants under aerobic and S-supplied (+S) condition. The hydrogen contents in DT transformants are 7 to 10-fold higher than that in DT wild type under anaerobic and +S condition. These results suggested that the HydA protein was successfully homologously expressed to enhance hydrogen production in DT. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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