Mapping The Interaction Domain Of Maize Stress-Tolerance Transcription Factor ABP9and Its Interact Protein NCP1

ABP9is a bZIP transcription factor which was idendified by yeast one hybrid using ABRE2of CAT1promoter as bait. It had been proven that ABP9can modulate ABA and ROS signaling to improve plantstress tolerance in transgenic plants of ABP9overexpression. NCP1(ABP Nine Complex Protein1) wasscreened as an interact protein of ABP9by yeast two hybrid assay. Sequence alignment found thatNCP1belongs to NINJA（Novel Interactor of JAZ）family protein and shares21%similarity witharabidopsis AFP1（ABI five binding protein1）. AFPs is an interaction protein of arabidopsis bZIPtranscription factor ABI5and can repress the function of ABI5. However, unlike to AFP1and otherNINJA family proteins, NCP1lacks the EAR motif which function as a repression domain. Interestingly,despite lacks the EAR motif, NCP1can still repress the ABP9response to ABA and ROS and otherstresses signaling. For the interaction domain of ABP9and NCP1is unkown, we can’t make sure howABP9was regulated by NCP1. In this study, we attempt to map the interaction domain of ABP9andNCP1and to get more clue of the mechanism of this regulation.The main results obtained are as followed:1) Aseries of truncated transient expression vectors ofABP9and NCP1were gained.2) We found that the light intensity of the maize material growth condition is the key factor that affectthe protoplast transformation efficiency. In addition, we optimized the maize protoplast transientsystem and brought the transform efficiency up to60%. By this system, we expressed the truncatedand full length of ABP9and NCP1, and then proved that NCP1can’t affect the accumulation ofABP9protein.3) The interaction domain of ABP9and NCP1was mapped to the middle part of the ABP9and Cterminal of the NCP1by CoIP assay.4) By sequence alginment, we speculated that ABP9151-169AA and NCP1213-261AA were thedirect interaction sites and the prediction was confirmed by CoIP assay.5) Amodel of how NCP1regulates ABP9was raised.In summary,the maize protoplast transfection system was optimized. The interation sites of ABP9andNCP1were determined.A model of how NCP1regulates ABP9has also been raised. This study offeressome clues for how NCP1regultes ABP9and a few theoretical foundation of plant gene regulationmechanism in ABA signaling pathway.