本文關(guān)鍵詞：AT14A在擬南芥響應(yīng)PEG模擬干旱脅迫中的功能分析　出處：《揚(yáng)州大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： AT14A 類整合素蛋白 擬南芥 干旱脅迫 信號(hào)轉(zhuǎn)導(dǎo) 氧化損傷 脫落酸

【摘要】：干旱是影響作物生長(zhǎng)發(fā)育,限制產(chǎn)量的重要環(huán)境脅迫因子之一。闡明植物對(duì)干旱脅迫的響應(yīng)機(jī)理是植物抗性機(jī)制研究中重要的途徑之一。當(dāng)植物受到干旱脅迫時(shí),植物細(xì)胞感知干旱信號(hào),以啟動(dòng)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo),從而得以生存。干旱脅迫可以影響植物細(xì)胞質(zhì)膜和細(xì)胞壁之間的相互作用。細(xì)胞壁和/或細(xì)胞壁—細(xì)胞膜的相互作用部位是感知干旱脅迫的重要部位。已有的研究表明,植物細(xì)胞中存在細(xì)胞壁-質(zhì)膜-細(xì)胞骨架連續(xù)體,其在植物響應(yīng)干旱脅迫過(guò)程中具有重要調(diào)控作用。AT14A介導(dǎo)了細(xì)胞壁-質(zhì)膜-細(xì)胞骨架連續(xù)體,它是連續(xù)體的中間組分,具有調(diào)節(jié)細(xì)胞壁和細(xì)胞骨架組織的重要作用。通過(guò)擬南芥AT14A的序列分析,發(fā)現(xiàn)其與動(dòng)物整合素存在序列相似性,因此推斷AT14A可能為一種類整合素蛋白。為了深入研究AT14A的功能,尤其在擬南芥響應(yīng)干旱脅迫中的作用機(jī)理,本論文以AT14A相關(guān)基因型(野生、缺失、過(guò)表達(dá))擬南芥懸浮細(xì)胞和植株為實(shí)驗(yàn)材料,用PEG-6000模擬干旱脅迫處理,綜合利用基因表達(dá)分析、生理生化分析、轉(zhuǎn)錄組學(xué)、定量蛋白組學(xué)和生物信息學(xué)等多種生物學(xué)方法分析AT14A的功能。主要結(jié)果如下：1. RT-PCR試驗(yàn)結(jié)果表明PEG模擬干旱脅迫能誘導(dǎo)懸浮細(xì)胞AT14A的表達(dá)。相對(duì)于野生型懸浮細(xì)胞,AT14A過(guò)表達(dá)(AT14A-OE)懸浮細(xì)胞具有更高的生物量積累和存活率,因此它更能適應(yīng)干旱脅迫。而且,AT14A-OE懸浮細(xì)胞具有較低的過(guò)氧化氫積累、膜脂過(guò)氧化程度和電解質(zhì)滲漏；較高的可溶性蛋白含量和抗氧化酶活性(如POD和APX),因此它表現(xiàn)出更好的防護(hù)氧化損傷能力�？傊�,這些研究結(jié)果表明,AT14A在擬南芥懸浮細(xì)胞響應(yīng)PEG模擬干旱脅迫中具有重要的調(diào)控作用。AT14A的過(guò)表達(dá)提高了細(xì)胞對(duì)干旱脅迫的適應(yīng)性。它通過(guò)調(diào)控抗氧化防護(hù)系統(tǒng)應(yīng)答,抑制ROS產(chǎn)生、膜脂過(guò)氧化和離子滲透水平來(lái)緩解干旱脅迫誘導(dǎo)的氧化損傷。2.AT14A在擬南芥植株根、莖、葉和花中均有表達(dá),尤其葉片中的表達(dá)較高。PEG模擬干旱脅迫能誘導(dǎo)葉片AT14A的表達(dá)。與野生型(WT)植株相比,缺失突變體(at14a)植株對(duì)土壤干旱更敏感。通過(guò)轉(zhuǎn)錄組學(xué)的方法,分析兩基因型(WT和at14a)植株響應(yīng)干旱脅迫后的差異基因。野生型植株的葉片差異基因中681個(gè)表達(dá)上調(diào),613個(gè)表達(dá)下調(diào)；缺失突變體植株的葉片差異基因中1018個(gè)表達(dá)上調(diào),1413個(gè)表達(dá)下調(diào)。定量PCR驗(yàn)證結(jié)果顯示,兩基因型植株ABA合成相關(guān)基因(AA03, NCED5)和依賴于ABA的干旱相關(guān)基因(RAB18, RD22)的表達(dá)量均升高了,但相比較于野生型(WT)植株,缺失突變體(atl4a)植株中,AAO3, NCED5, RAB18, RD22的表達(dá)均較低。隨著干旱時(shí)間的延長(zhǎng),兩基因型(WT和at14a)植株葉片ABA的含量均升高,復(fù)水后ABA含量均下降,但相比較于野生型(WT)植株,缺失突變體(at14a)植株ABA含量均較低。這些研究結(jié)果表明AT14A在干旱脅迫誘導(dǎo)ABA合成中發(fā)揮重要作用。它通過(guò)調(diào)控ABA合成關(guān)鍵基因(AAO3,NCED5)的表達(dá),從而調(diào)控ABA的合成,最終調(diào)控干旱脅迫誘導(dǎo)ABA的合成過(guò)程。同時(shí),AT14A也通過(guò)調(diào)控依賴于ABA途徑的干旱相關(guān)基因(RAB18, RD22)的表達(dá)來(lái)適應(yīng)脅迫。3.通過(guò)定量蛋白組學(xué)的方法,分析兩基因型(WT和at14a)植株響應(yīng)干旱脅迫后的差異蛋白。干旱脅迫前,兩基因型植株的差異蛋白共123個(gè),表達(dá)上調(diào)的71個(gè),表達(dá)下調(diào)52個(gè)；干旱處理后,兩基因型植株的差異蛋白共625個(gè),表達(dá)上調(diào)的339個(gè),表達(dá)下調(diào)286個(gè)。干旱脅迫后,兩基因型植株的抗氧化防護(hù)酶APX1和V-H+ATPase E1的表達(dá)均升高,但相比較于野生型(WT)植株,缺失突變體(at14a)植株中這些分子的表達(dá)量更低。這些研究結(jié)果進(jìn)一步驗(yàn)證了AT14A參與細(xì)胞防護(hù)氧化損傷。這些結(jié)果也表明AT14A通過(guò)調(diào)控V-H+ATPase E1的表達(dá)來(lái)響應(yīng)干旱脅迫。總之,AT14A在擬南芥應(yīng)答干旱脅迫中有重要作用。這些結(jié)果不僅有利于我們對(duì)AT14A蛋白在干旱脅迫的感知和信號(hào)轉(zhuǎn)導(dǎo)調(diào)控機(jī)理方面的理解,而且對(duì)利用現(xiàn)代生物技術(shù)提高植物的抗旱性具有重要的指導(dǎo)意義。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q945.78

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