PROKARYOTIC EXPRESSION OF Arachis hypogaea Nine- Cis- Epoxycarotenoid Dioxygenase1 RECOMBINANT PROTEIN

Abscisic acid (ABA) is an important hormone that mediates plant responses to abiotic stresses, including drought, salinity, and low temperature. The oxidative cleavage of cis-epoxycarotenoids catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) is considered to be the rate limiting step in ABA biosynthesis. We constructed a prokaryotic expression plasmid pProEX-HT-AhNCED1 and expressed a soluble fusion protein of about 66 kD in E. coli BL21 (DE3) cells induced by 0.2 mmol?L-1 IPTG at 28℃ for 4 h. The recombinant protein was purified with Ni/NTA affinity chromatography. Western blot assays revealed that there was a protein band, with a relative molecular mass of 66.0 kD, indicating that antiserum could react to the native protein expressed in peanut specifically. The CD spectrum of AhNCED1 showed that it is a typical random coil protein. The proportional content of -helixes of AhNCED1 protein increased from 20.9% to 82.5% under 30% PEG treated for 3h and 5% SDS made the proportional content of -helixs increase to 41.2%. Our research give some new insights for better understanding the function of AhNCED1 protein and the regulation mechanism of ABA biosynthesis.