Expression and Localization of Arachis Hypogaea 9-Cis Epoxycarotenoid Dioxygenase 1 (AhNCED1) of Peanut Under Water Stress

摘要

The oxidative cleavage of cis-epoxycarotenoids catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) is considered to be the rate-limiting step in abscisic acid (ABA) biosynthesis. The optimal induction conditions for expressing the AhNCED1 (Arachis hypogaea 9-cis epoxycarotenoid dioxygenase 1) protein in E. coli BL21 (DE3) were established. The purified recombination AhNCEDl protein was used to generate a polyclonal antibody in rabbit. This purified antibody was used for western blotting analysis of protein samples extracted from different organs of peanut. Under normal growth conditions, water contents of leaves and stems in peanut were higher than that of roots, and AhNCED1 mRNA and protein were low in stems and leaves, but was not detectable in roots. After treatment with 20% PEG6000for 7 h, endogenous ABA predominantly accumulated in leaves and stems, and water contents of leaves and roots reduced more than that of stems. At the transcriptional level, AhNCED1 mRNA was strongly induced in roots and leaves. At expression of protein level, AhNCED1 were enhanced in both leaves and roots, but no change in stems. In transgenic Arabidopsis, AhNCED1-GFP fluorescence was detected in the root tips and cotyledons. Intense GFP-fluorescence signals were localized in chloroplasts of cotyledons in transgenic Arabidopsis treated with 20% PEG6000for 3h, and AhNCED1 targeted into chloroplasts was also indicated by western blotting in peanut. The results suggest that different spatio-temporal expression of AhNCED1 in leaves and roots related to water stress and these areas may be the main places that synthesize ABA in response to water stress.