Cloning and sequencing ofphbA gene of poly-β-hydroxybuty rate synthesis and its expression analysis
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The gene of the first key enzyme of poly-β-hydroxybuty rate synthesis, 3-ketothiolase, has been amplified and cloned from chromosomal DNA ofAlcaligenes eutrophus H16 by PCR. DNA sequencing results show thatphbA cloned in pBluescriptSK+ has an identical sequence with that reported previously except for one base pair. The plant constitutive expression vector has been constructed and tobacco has been transformed in order to examine thephbA gene function and the efficiency ofctp gene product. SDS-polyacrylamide gel electrophoresis result shows that thectp gene product could direct foreign protein into plastid efficiently andphbA gene could be translated into corresponding protein with correct size. The enzyme activity analysis of 3-ketothiolase shows that the enzyme could catalyze acetyl-CoA to form acetoacetyl-CoA.
KeywordsphbA gene poly-β-hydroxybutyrate 3-ketothiolase
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