AT14A在拟南芥响应PEG模拟干旱胁迫中的功能分析

发布时间：2017-12-28 19:25

本文关键词：AT14A在拟南芥响应PEG模拟干旱胁迫中的功能分析　出处：《扬州大学》2016年博士论文　论文类型：学位论文

【摘要】：干旱是影响作物生长发育,限制产量的重要环境胁迫因子之一。阐明植物对干旱胁迫的响应机理是植物抗性机制研究中重要的途径之一。当植物受到干旱胁迫时,植物细胞感知干旱信号,以启动细胞信号转导,从而得以生存。干旱胁迫可以影响植物细胞质膜和细胞壁之间的相互作用。细胞壁和/或细胞壁—细胞膜的相互作用部位是感知干旱胁迫的重要部位。已有的研究表明,植物细胞中存在细胞壁-质膜-细胞骨架连续体,其在植物响应干旱胁迫过程中具有重要调控作用。AT14A介导了细胞壁-质膜-细胞骨架连续体,它是连续体的中间组分,具有调节细胞壁和细胞骨架组织的重要作用。通过拟南芥AT14A的序列分析,发现其与动物整合素存在序列相似性,因此推断AT14A可能为一种类整合素蛋白。为了深入研究AT14A的功能,尤其在拟南芥响应干旱胁迫中的作用机理,本论文以AT14A相关基因型(野生、缺失、过表达)拟南芥悬浮细胞和植株为实验材料,用PEG-6000模拟干旱胁迫处理,综合利用基因表达分析、生理生化分析、转录组学、定量蛋白组学和生物信息学等多种生物学方法分析AT14A的功能。主要结果如下：1. RT-PCR试验结果表明PEG模拟干旱胁迫能诱导悬浮细胞AT14A的表达。相对于野生型悬浮细胞,AT14A过表达(AT14A-OE)悬浮细胞具有更高的生物量积累和存活率,因此它更能适应干旱胁迫。而且,AT14A-OE悬浮细胞具有较低的过氧化氢积累、膜脂过氧化程度和电解质渗漏；较高的可溶性蛋白含量和抗氧化酶活性(如POD和APX),因此它表现出更好的防护氧化损伤能力。总之,这些研究结果表明,AT14A在拟南芥悬浮细胞响应PEG模拟干旱胁迫中具有重要的调控作用。AT14A的过表达提高了细胞对干旱胁迫的适应性。它通过调控抗氧化防护系统应答,抑制ROS产生、膜脂过氧化和离子渗透水平来缓解干旱胁迫诱导的氧化损伤。2.AT14A在拟南芥植株根、茎、叶和花中均有表达,尤其叶片中的表达较高。PEG模拟干旱胁迫能诱导叶片AT14A的表达。与野生型(WT)植株相比,缺失突变体(at14a)植株对土壤干旱更敏感。通过转录组学的方法,分析两基因型(WT和at14a)植株响应干旱胁迫后的差异基因。野生型植株的叶片差异基因中681个表达上调,613个表达下调；缺失突变体植株的叶片差异基因中1018个表达上调,1413个表达下调。定量PCR验证结果显示,两基因型植株ABA合成相关基因(AA03, NCED5)和依赖于ABA的干旱相关基因(RAB18, RD22)的表达量均升高了,但相比较于野生型(WT)植株,缺失突变体(atl4a)植株中,AAO3, NCED5, RAB18, RD22的表达均较低。随着干旱时间的延长,两基因型(WT和at14a)植株叶片ABA的含量均升高,复水后ABA含量均下降,但相比较于野生型(WT)植株,缺失突变体(at14a)植株ABA含量均较低。这些研究结果表明AT14A在干旱胁迫诱导ABA合成中发挥重要作用。它通过调控ABA合成关键基因(AAO3,NCED5)的表达,从而调控ABA的合成,最终调控干旱胁迫诱导ABA的合成过程。同时,AT14A也通过调控依赖于ABA途径的干旱相关基因(RAB18, RD22)的表达来适应胁迫。3.通过定量蛋白组学的方法,分析两基因型(WT和at14a)植株响应干旱胁迫后的差异蛋白。干旱胁迫前,两基因型植株的差异蛋白共123个,表达上调的71个,表达下调52个；干旱处理后,两基因型植株的差异蛋白共625个,表达上调的339个,表达下调286个。干旱胁迫后,两基因型植株的抗氧化防护酶APX1和V-H+ATPase E1的表达均升高,但相比较于野生型(WT)植株,缺失突变体(at14a)植株中这些分子的表达量更低。这些研究结果进一步验证了AT14A参与细胞防护氧化损伤。这些结果也表明AT14A通过调控V-H+ATPase E1的表达来响应干旱胁迫。总之,AT14A在拟南芥应答干旱胁迫中有重要作用。这些结果不仅有利于我们对AT14A蛋白在干旱胁迫的感知和信号转导调控机理方面的理解,而且对利用现代生物技术提高植物的抗旱性具有重要的指导意义。
[Abstract]:Drought is one of the important environmental stress factors that affect the growth and development of crops and limit the yield. Clarifying the response mechanism of plants to drought stress is one of the important ways to study the mechanism of plant resistance. When plants are subjected to drought stress, plant cells perceive drought signals to activate cell signal transduction and thus survive. Drought stress can affect the interaction between plasma membrane and cell wall of plant cells. The interaction site of cell wall and / or cell wall - cell membrane is an important part of sensing drought stress. Previous studies have shown that the cell wall - plasmalemma - cytoskeleton exists in plant cells, which plays an important role in the response to drought stress in plants. AT14A mediates the cell wall plasmalemma cytoskeleton, which is the intermediate component of the continuum, and plays an important role in regulating the cell wall and cytoskeleton. According to the sequence analysis of Arabidopsis AT14A, it was found that it was similar to the sequence of animal integrin. Therefore, it was concluded that AT14A might be a kind of integrin protein. In order to study the function of AT14A, especially in Arabidopsis in response to drought stress in this thesis, AT14A (wild type gene, deletion and overexpression of Arabidopsis plants and suspension cells) as experimental materials, using PEG-6000 simulated drought stress, comprehensive utilization of gene expression analysis, physiological and biochemical analysis, transcriptomics, quantitative proteomics and bioinformatics analysis of AT14A and other biological methods. The main results are as follows: 1. the results of 1. RT-PCR test showed that PEG simulated drought stress could induce the expression of AT14A in the suspension cells. Compared with wild type suspension cells, AT14A overexpressed (AT14A-OE) suspension cells have higher biomass accumulation and survival rate, so it is more able to adapt to drought stress. Moreover, AT14A-OE suspension cells had lower accumulation of hydrogen peroxide, membrane lipid peroxidation and electrolyte leakage, higher soluble protein content and antioxidant enzyme activities (such as POD and APX), so it showed better protection against oxidative damage. In conclusion, these results suggest that AT14A plays an important role in regulating drought stress in Arabidopsis thaliana cells in response to PEG simulated drought stress. The overexpression of AT14A increased the adaptability of cells to drought stress. It inhibits the oxidative damage induced by drought stress by regulating the response of the antioxidant protection system and inhibiting the production of ROS, membrane lipid peroxidation and ion penetration. 2.AT14A was expressed in the roots, stems, leaves and flowers of Arabidopsis, especially in leaves. PEG simulated drought stress can induce the expression of AT14A in leaves. Compared with the wild type (WT) plants, the deletion mutant (at14a) plants are more sensitive to soil drought. Through the method of transcriptional analysis, the genes of two genotypes (WT and at14a) were analyzed in response to drought stress. The expression of 681 genes in leaf differentially expressed genes and 613 down regulated genes in wild type plants were increased. 1018 of the differentially expressed genes in the mutant plants were up-regulated and 1413 down regulated. Quantitative PCR results showed that two genotypes, ABA synthesis related genes (AA03, NCED5) and ABA dependent drought related genes (RAB18, RD22) expression were increased, but compared to the wild type (WT) plants, mutant (atl4a) plant, AAO3, NCED5, RAB18. The expression of RD22 was low. With the prolongation of time, because the two types (WT and at14a) in leaves of ABA were increased after rewatering, ABA content decreased, but compared to the wild type (WT) plants, mutant (at14a) ABA content in plant were lower. These results suggest that AT14A plays an important role in the induction of ABA synthesis by drought stress. It regulates the synthesis of ABA by regulating the expression of the key gene (AAO3, NCED5) of ABA synthesis, and ultimately regulates the synthesis process of ABA induced by drought stress. At the same time, AT14A also regulates the expression of drought related genes (RAB18, RD22) dependent on the ABA pathway to adapt to stress. 3. by quantitative proteomics, the differential proteins of the plants of the two genotypes (WT and at14a) were analyzed in response to drought stress. Before drought stress, there were 123 differentially expressed proteins in two genotypes, 71 up-regulated expression and 52 down regulated expression. After drought treatment, there were 625 differential expression proteins in two genotype plants, 339 expressed up-regulated and 286 down regulated. After drought stress, the expression of APX1 and V-H+ATPase E1 in two genotype plants increased, but compared with the wild type (WT) plants, the expression of these molecules in the deletion mutant (at14a) plants was lower. These findings further demonstrate that AT14A is involved in oxidative damage in cell protection. These results also suggest that AT14A responds to drought stress by regulating the expression of V-H+ATPase E1. In conclusion, AT14A plays an important role in response to drought stress in Arabidopsis. These results not only help us to understand AT14A protein's perception and signal transduction mechanism in drought stress, but also have important guiding significance for improving drought resistance of plants by modern biotechnology.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q945.78

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