【作者】 张丽；

【导师】 李学军；

【作者基本信息】 西北农林科技大学 ， 作物学， 2019， 硕士

【摘要】 植物为了抵御非生物逆境胁迫造成的伤害,体内会产生各种应对的作用机制,其中,泛素/26S蛋白酶体系统是最重要的机制之一。干旱是最常见的非生物逆境,严重危害小麦的产量。本研究在前期克隆小麦干旱胁迫响应基因TaDIS1及功能初步分析的基础上,通过酵母双杂交技术筛选TaDIS1的互作蛋白及体内、外验证,并对其互作蛋白进行初步功能分析,最后通过体外泛素化实验和降解实验解析其作用机理。取得以下研究结果:1.通过酵母双杂实验筛选出TaDIS1的8个潜在互作蛋白,其中TaSTP是其互作蛋白之一,根据序列比对及结构分析结果表明该蛋白是一种逆境蛋白,可能与ABA调控途径有关。TaSTP基因全长为887bp,包含89bp的5’UTR区、462bp的ORF和336bp的3’UTR区。2.为了排除酵母双杂交实验可能出现的假阳性,我们通过BiFC,Pull-Down,CoIP实验对TaDIS1与TaSTP的互作关系进行验证。在BiFC实验中,发现TaDIS1和TaSTP在烟草叶片细胞的某一内含体发出荧光,表明TaDIS1和TaSTP在细胞内部互作;Pull-Down实验结果表明,TaDIS1在体外可以被TaSTP拉下来,说明二者可以相互作用;CoIP实验结果表明,在烟草细胞内,TaDIS1和TaSTP之间存在互作。上述结果表明无论在体内还是体外,TaDIS1与TaSTP之间都存在相互作用。3.体外泛素化实验结果表明TaDIS1具有E3泛素连接酶活性;通过对其RING保守结构域的突变分析,发现TaDIS1的RING保守结构域对其行使E3泛素连接酶活性是必需的。4.26S蛋白酶体降解实验结果表明,TaSTP是TaDIS1的底物蛋白,TaSTP可以通过26S蛋白酶体途径被降解。5.根据对在模拟干旱,NaCl和ABA的胁迫下的表达模式分析发现,在20%PEG6000干旱处理后,TaSTP在24h内呈现先增加后降低的趋势;在200mM的NaCL的处理下,TaSTP的表达趋势呈现明显增加;在100μM的ABA的处理下,TaSTP在最开始表现突然下降,之后变化幅度很小,一直维持在一个较低的水平。这个结果表明TaSTP对三种胁迫均有响应。6.TaDIS1和TaSTP亚细胞定位结果发现,在烟草叶片细胞的一种内含体中发现了绿色荧光信号,推测该内含体可能为高尔基体,因此TaDIS1与TaSTP在植物调控逆境中可能参与到了与高尔基体有关的跨膜运输等过程。7.综合上述结果得出,小麦中RING E3泛素连接酶TaDIS1可以通过26S蛋白酶体途径降解TaSTP来负调控干旱胁迫。该研究为小麦抗旱育种提供了理论依据。更多还原

【Abstract】 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 μM 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.更多还原