小麦胁迫应答基因TaDSU的功能研究

发布时间：2018-05-10 06:40

本文选题：小麦 + SUMO蛋白酶　；参考：《山东大学》2017年硕士论文

【摘要】：小麦是一种重要的粮食作物。盐旱等逆境严重影响小麦产量,因而鉴定耐逆相关基因,并应用于耐逆小麦分子育种非常必要。本实验室利用不对称体细胞杂交技术,以普通小麦济南177(JN177)为亲本,培育了小麦渐渗系耐盐抗旱新品种山融3号(SR3)。通过转录组分析,鉴定了一个SR3中盐胁迫响应更显著SUMO蛋白酶基因TaDSU。SUMO化是重要的可逆性蛋白质修饰化,在生长发育和逆境响应中发挥重要作用。SUMO化的蛋白质可通过SUMO蛋白酶去除。但是,SUMO蛋白酶在非生物逆境响应的作用机制还知之甚少。为了认识SUMO蛋白酶在非生物胁迫应答中的功能,实验室前期构建了 TaDSU异源表达拟南芥转基因株系,并初步分析了 TaDSU在非生物胁迫应答中的功能。本论文在前期研究的基础上,利用TaDSU异源表达拟南芥转基因株系,开展了一下研究:1、进一步分析了TaDSU的耐逆功能及其生理基础与野生型相比,TaDSU过表达拟南芥具有以下特点:在萌发和幼苗生长阶段的耐盐能力均明显增强,盐胁迫下钠离子含量更低、钾离子含量和钾钠比更低;抗旱性提高,失水率降低;对甘露醇模拟的渗透胁迫耐受性增强,可溶性糖和脯氨酸等渗透调节物含量增加;对外源H2O2的抗性及过氧化氢酶活性明显提高,胁迫下叶片中H202水平降低;对ABA的敏感性降低。结果显示,TaDSU过表达通过对离子平衡维持、渗透平衡调节、抗氧化等抗逆生理基础的综合影响,提高了广谱性的耐逆能力。为了验证TaDSU在小麦是否提高广谱性的耐逆能力,构建了 TaDSU转基因小麦,共获得了 19株T2代阳性转基因株系。2、鉴定了TaDSU互作蛋白结合体外酶活实验和western bloting分析,初步证实了 TaDSU具有体外和体内酶活性。结合体外pull-down实验、质谱鉴定和生物信息学分析,筛选了 6个候选的TaDSU互作蛋白DIP1-DIP6,并通过酵母双杂交初步验证了 3个互作蛋白。TaDSU定位于细胞核,并且利用双分子荧光互补实验发现,TaDSU和DIP2在细胞核中相互作用。盐胁迫下,TaDSU过表达增强了 DIP2的富集及DIP2下游靶基因的表达。结果暗示,TaDSU通过和DIP2互作,影响DIP2的SUMO化和富集水平,调控DIP2下游基因的表达。3、初步鉴定了调控TaDSU表达的调控因子克隆了 1.1 kb的TaDSU启动子。构建了 TaDSU启动子-GUS转基因拟南芥,GUS染色分析显示,TaDSU在不同时期的多个组织中表达。TaDSU启动子含有DIP2结合元件,凝胶迁移实验显示,DIP2可以结合TaDSU启动子的DIP2结合元件区。综合以上结果推测,非生物胁迫诱导DIP2大量合成,后者促进TaDSU表达,TaDSU和DIP2结合,调控DIP2的SUMO化水平,提高DIP2下游基因表达,增强耐逆能力。
[Abstract]:Wheat is an important food crop. Salt drought and other stresses seriously affect the yield of wheat, so it is necessary to identify the stress tolerance related genes and to apply them to the molecular breeding of stress tolerant wheat. Using asymmetrical somatic cell hybridization technique and common wheat Jinan 177 (JN177) as parents, a new salt-tolerant and drought-resistant wheat variety, Shanrong No. 3, was developed in our laboratory. By transcriptome analysis, it was identified that the SUMO protease gene TaDSU.SUMO was an important reversible protein modification in a SR3. The proteins that play an important role in growth, development and stress response can be removed by SUMO protease. However, little is known about the mechanism of Sumo protease in response to abiotic stress. In order to understand the function of SUMO protease in abiotic stress response, TaDSU heterologous expression Arabidopsis transgenic lines were constructed in early laboratory, and the function of TaDSU in abiotic stress response was preliminarily analyzed. On the basis of previous studies, TaDSU was used to express Arabidopsis transgenic lines. The stress tolerance function and physiological basis of TaDSU were further analyzed. Compared with wild type, the overexpressed Arabidopsis thaliana with TaDSU had the following characteristics: salt tolerance of Arabidopsis thaliana during germination and seedling growth was significantly enhanced. Under salt stress, the content of sodium ion was lower, the content of potassium ion and the ratio of potassium and sodium were lower, the drought resistance was increased, the rate of water loss was decreased, the tolerance to osmotic stress simulated by mannitol was enhanced, and the content of osmotic regulators such as soluble sugar and proline increased. The resistance to exogenous H2O2 and the activity of catalase increased significantly, and the level of H202 in leaves decreased and the sensitivity to ABA decreased under stress. The results showed that the over-expression of TaDSU enhanced the broad-spectrum stress tolerance through the comprehensive effects on the physiological basis of stress resistance, such as ion balance maintenance, osmotic balance regulation and oxidation resistance. In order to verify the ability of TaDSU to improve broad-spectrum stress tolerance in wheat, TaDSU transgenic wheat was constructed, 19 T2 generation positive transgenic lines were obtained, and TaDSU interaction protein binding enzyme activity and western bloting analysis in vitro were identified. It was preliminarily confirmed that TaDSU had enzyme activity in vitro and in vivo. In combination with in vitro pull-down assay, mass spectrometry and bioinformatics analysis, six candidate TaDSU interaction proteins DIP1-DIP6 were screened, and three interacting proteins. TaDSU were preliminarily confirmed to be located in the nucleus by yeast two-hybrid. Furthermore, the interaction between TaDSU and DIP2 in the nucleus was found by bimolecular fluorescence complementary experiment. Overexpression of TaDSU enhanced the enrichment of DIP2 and the expression of target genes downstream of DIP2 under salt stress. The results suggested that TaDSU affected the level of SUMO and enrichment of DIP2 by interacting with DIP2, and regulated the expression of DIP2 downstream gene. The TaDSU promoter of 1.1 kb was identified as the regulator of TaDSU expression. Gus staining analysis of TaDSU promoter Gus showed that TaDSU expressed DIP2 binding elements in several tissues at different stages. Gel migration experiments showed that DIP2 could bind to the DIP2 binding element region of TaDSU promoter. These results suggest that abiotic stress induces a large amount of DIP2 synthesis, which promotes the binding of TaDSU to DIP2, regulates the SUMO level of DIP2, enhances the downstream gene expression of DIP2 and enhances the ability of stress tolerance.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S512.1

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