Mechanism Analysis Of RING E3 Ligases TaDIS1 In Wheat(Triticum Aestivum L.)Tolerant To Drought Stress

In order to deal with the situations caused by abiotic stress,various mechanisms have been developed in plants.Among them,ubiquitin/26 S proteasome system is the one of the most important mechanisms.Drought is the most common abiotic stress and seriously jeopardizes wheat yield.Based on the preliminary analysis of the drought stress gene TaDIS1 in our laboratory,we screened the interaction protein of TaDIS1 by yeast two-hybrid screening assay,verified the interaction relationship in vitro and vivo,and analyzed the preliminary function of the interaction protein.Finally,the mechanism of TaDIS1 was analyzed by ubiquitination experiments and degradation experiments.The main results are as follows:1.Eight potential interaction proteins of TaDIS1 were screened by yeast double-hybrid screening assay.TaSTP is one of its interaction proteins.According to sequence alignment and structural analysis,TaSTP is a kind of stress protein,which may be related to ABA regulation pathway.TaSTP is a full-length 887 bp,89 bp 5' UTR region,462 bp ORF and The 336 bp gene in the 3'UTR region.2.Because yeast two-hybrid experiments may have false positives,we further verified the interaction between TaDIS1 and TaSTP through BiFC,Pull-Down,and CoIP experiments.In the BiFC experiment,fluoresce was found in intracellular endosomes of tobacco leaf cells,which indicates that TaDIS1 can interact with TaSTP in tobacco leaf cells;in the Pull-Down experiment,TaDIS1 could be pulled down by TaSTP in vitro;indicating that the two can interact.The results of CoIP experiment showed that there was an interaction between TaDIS1 and TaSTP in the tobacco cells.According to the experiments above,we can get that TaSTP can interact with TaDIS1 in vivo and vitro.3.In vitro ubiquitination experiments showed that TaDIS1 has E3 ubiquitin ligase activity and through the mutation analysis of its RING conserved domain,it was found that the RING conserved domain of TaDIS1 is required for its E3 ubiquitin ligase activity..4.The results of 26 S proteasome degradation assay showed that TaSTP is a substrate protein of TaDIS1,and TaSTP can be degraded via the 26 S proteasome pathway.5.According to the expression pattern analysis of TaSTP under simulated drought,NaCl and ABA stress,it was found that under 20% PEG6000 treatment,the mRNA expression level of TaSTP increased first and then decreased;Under 200 mM NaCl treatment,the expression level of TaSTP increased significantly;under100 ?M ABA treatment,the expression level of TaSTP tended to decrease rapidly initially,before remaining at a very low level with no obvious changes.These results demonstrate that TaSTP can respond to various stressful conditions.6.The subcellular localization results of TaDIS1 and TaSTP revealed that a green fluorescent signal was found in intracellular endosomes of tobacco leaf cells.It is speculated that the intracellular endosomes may be a Golgi apparatus,so the regulatory effect of TaDIS1 and TaSTP on the plant drought response may be related to Golgi apparatus.7.Based on the results above,we can conclude that the RING E3 ubiquitin ligase TaDIS1 in wheat can negatively regulate drought stress by degrading TaSTP via the 26 S proteasome pathway.This study provides a theoretical basis for wheat drought resistance breeding.