Clone, Expression and Functional Analysis of Dihydroflavonol 4-Reductase Gene of Tea Plant (Camellia sinensis)
Dihydroflavonol 4-reductase (DFR) is a key enzyme in the biosynthesis of catechins in tea plant. However, the functions and the zymologic properties of DFR were not deeply identified in recent researches. The open reading frame of DFR gene, which encoding a 347 amino acids protein, was cloned from tea plant (Camellia sinensis) by RT-PCR. The deduced protein molecular weight was 38.69 kD and its theoretical isoelectric point was 6.02. The gene was cloned into the expression vector SUMO for expression in prokaryotic cells. The SDS-PAGE results showed that the dihydroflavonol 4-reductase peoteins was expressed in Escherichia coli BL21. The optimal inducing conditions including time, temperature and IPTG concentration were studied. The deduced protein was purified and its activity was detected by HPLC-MS method. The results indicated that purified protein showed the DFR activity, catalyzed the reduction reaction of DHQ and DHM. The research provides a valuable foundation for better understanding the substrate specificity and enzymatic properties of CsDFR.
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