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|標題:||利用RNA干擾降低小球藻Chlorella PsbO 蛋白轉譯以誘導產氫|
Using RNA interference to knockdown PsbO protein to induce hydrogenase expression in Chlorella
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The PsbO protein is one of the oxygen-evolving complex (OEC) subunits of Photosystem II (PSII), the water splitting enzyme of photosynthesis. It concerned with the stability of PSII for O2 evolution. O2 is a powerful suppressor of hydrogenase (HydA) gene expression and a potent inhibitor of HydA enzymatic activity. While cultivated in sulfur-deprived medium, the PSII activity of green algae would be inhibited and the O2 evolution would be decreased. Therefore, the circumstance became anaerobic, leading to the induction of HydA to produce H2. However, the cells would be gradually dead because of lacking sulfur. We attempted to knockdown the psbO gene expression of Chlorella sp. DT (DT) by short interference RNA (siRNA) as PsbO suppression would lead to the inhibition of O2 evolution and induction of HydA. The DT cells were transformed with siRNA vectors of pHm3A/psbO-a and pHm3A/psbO-b which contained the antisense gene fragments of antisense-psbO-a and antisense-psbO-b, respectively. The presence of transformed plasmids was detected by PCR amplification of the hpt, merA, siRNA-psbO-a and siRNA-psbO-b gene fragments from genomic DNA isolated from DT transformants. The results suggested that the siRNA plasmids had been successfully transformed into DT. PsbO protein was detected by western blotting. The amounts of PsbO in DT transformants were decreased to 42-66% as compared to that in DT wild type. Under sulfur-supplied condition, the transcription of hydA and the translation of HydA in transformants could be induced in these transformants. H2 concentrations of DT transformants were about 98.5-352.3 ml l-1 and higher than that of DT wild type about 30.3 ml l-1. These results indicate that the decrease of the PsbO protein can induce HydA expression to increase H2 production.
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