| In this study,the prokaryotic expression vectors pET-28a(+)-MaDFR2 b and pMAL-c5x-MaDFR2 b were constructed according to the sequence of MaDFR2 b cloned from grape hyacinth ’Armenia’ in the early stage of the study group.The conditions of different induction temperature,IPTG concentration and induction time were studied.Under the induction of fusion protein expression,the expression of the recombinant fusion protein MaDFR2 b was detected by SDS-PAGE,and the optimal induction conditions of the soluble fusion protein were selected.The obtained soluble recombinant fusion protein was purified by affinity chromatography and subjected to in vitro enzymatic reaction to identify its activity.The results are as follows:1.The expression of MaDFR2 b protein in grape hyacinth was induced by prokaryotic expression in vitro.The results showed that the soluble MaDFR2 b fusion protein could be efficiently induced in the E.coli expression system at 28°C and 0.5mM IPTG concentration at the induction of 6 hours.The target protein was purified by affinity chromatography.The purity of the target protein was 90% after SDS-PAGE analysis and Quantity-one analysis.The purified MaDFR2 b fusion protein achieved the purity required for subsequent experiments.2.The obtained target protein was in vitro enzymatic reaction experiment with dihydroflavonol as substrate.The results of high performance liquid chromatography showed that grape hyacinth MaDFR2 b could catalyze dihydroquercetin and dihydromyricetin,but could not catalyze dihydrokaewol..3.Agrobacterium-mediated transient transformation experiments: Construction of MaDFR2 b overexpression vector,transient transformation of the rose ’Awakening’ by Agrobacterium injection.The results showed that the rose petals infected with the overexpression vector carrying functional genes showed blue plaques.. |
