类整合素蛋白与微管蛋白在植物响应渗透胁迫中的作用
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摘要
整合素蛋白是动物细胞粘附分子的重要成员,属于一类跨膜蛋白的超家族,由α-亚基和β-亚基通过非共价键连接而成。它的胞外域能识别并结合各种含有RGD短肽序列的胞外基质分子,而在胞内域连接细胞骨架和信号分子,从而构成了胞外基质-质膜-细胞骨架连续体,成为了动物细胞内外信号转导过程中的关键分子之一,在细胞的运动、增殖、分化和凋亡等方面起着重要的作用。近年来的研究表明:胞外基质-质膜-细胞骨架连续体是所有真核细胞所共有的一种结构,这种结构中的联结分子在植物、真菌和动物细胞中可能是相同或类似的。但对植物细胞内是否存在连接细胞壁组分与细胞骨架的分子,如存在,该类分子的特性又是怎样的还知之甚少。有证据表明在植物细胞的质膜上存在类似动物细胞整合素的蛋白。
干旱等逆境引起的渗透胁迫是影响植物生长发育的主要因素之一。阐明植物对渗透胁迫的响应机制,增强植物的抗性具有重要的意义。渗透胁迫导致植物细胞产生质壁分离,而细胞壁与质膜间的粘附结构对于维持植物细胞正常的生理功能至关重要,特别是在对渗透胁迫的反应过程中。作为细胞壁-质膜-细胞骨架连续体的重要分子,质膜上类似于动物细胞整合素蛋白分子以及与细胞骨架间的相互作用很可能参与了渗透胁迫所诱导的许多生理生化响应过程。
本试验以拟南芥(Arabidopsis thaliana,Columbia生态型)叶片悬浮培养细胞和玉米(Zea maize L.)幼苗为试验材料,综合利用分子生物学、植物生理学、生物化学和细胞生物学等技术,研究了植物细胞细胞壁与质膜间的相互作用、类整合素和微管蛋白的功能、细胞学定位以及编码类整合素蛋白和微管蛋白基因的克隆与转化,同时研究了类整合素蛋白、细胞骨架及两者的相互作用在渗透胁迫诱导细胞合成ABA中的作用,为进一步深入研究植物细胞壁-质膜-细胞骨架连续体在渗透胁迫信号传导过程中的作用提供一些理论依据。主要结果如下:
1、应用差速离心和密度梯度离心分离纯化细胞不同部分(膜部分和可溶性部分),结合Western Blotting技术分析了相关蛋白的表达。结果表明,在拟南芥悬浮细胞和玉米幼苗根尖细胞的不溶性部分存在与人整合素β1亚基抗体特异反应的蛋白质带,其分子量分别在110kDa和80kDa。用人整合素β1亚基抗体作探针,采用间接免疫荧光定位的方法,在拟南芥悬浮细胞的细胞膜和玉米幼苗根尖细胞
Adverse environmental factors, such drought, salinity, extreme temperature, etc, have deleterious effects on plant growth and development, thus the final plant productivity. It is understandable that, when plants are imposed to drought stress, the cell wall and/or plasma membrane are the most possible candidates for the primary sensing site(s). In plants, much progress has been made in understanding the complexity of the cell wall chemistry and cell wall extension, but there is still little information concerning its dynamic nature and interaction between it and the plasma membrane. Over the past two decades, evidence has been accumulated that the plant cell wall may interact with many proteins of the plasma membrane, like the extracellular matrix (ECM) in animals, and these in turn may interact directly or indirectly with the cytoskeleton. Integrin is one of the most important plasma membrane proteins in animals, which interacts with ECM and cytoskeleton and plays important roles in many physiological processes. In present study, several techniques, including immunological, biochemical and molecular biological techniques, have been used to explore whether there existed a similar molecule in plasma membrane, like integrin in animals, and if so, its possible interaction with cell wall and cytoskeleton. In addition, what are the roles of cell wall-plasma membrane-cytoskeleton continnum in the responses to osmotic stress? The results indicated that
1. Western blotting and immuno-fluorescent localization studies, using a monoclonal antibody againstβ1 subunit of human integrin, showed that the integrin-like protein localized in the plasma membrane fraction in Arabidopsis suspension cells and both cell membrane and nucleus of maize root cells with a molecular weight of ca.110 kDa and 80 kDa for Arabidopsis suspension cells and maize roots, respectively.
2. Under normal conditions (non-osmotic stress), the cell wall and plasma membrane was adhered together around the cells. However, the cell wall and plasma membrane was separated (plasmolyzed) partially when cells were exposed to osmotic stress. When cells were treated with RGD, a polypeptide that blocks the link between
干旱等逆境引起的渗透胁迫是影响植物生长发育的主要因素之一。阐明植物对渗透胁迫的响应机制,增强植物的抗性具有重要的意义。渗透胁迫导致植物细胞产生质壁分离,而细胞壁与质膜间的粘附结构对于维持植物细胞正常的生理功能至关重要,特别是在对渗透胁迫的反应过程中。作为细胞壁-质膜-细胞骨架连续体的重要分子,质膜上类似于动物细胞整合素蛋白分子以及与细胞骨架间的相互作用很可能参与了渗透胁迫所诱导的许多生理生化响应过程。
本试验以拟南芥(Arabidopsis thaliana,Columbia生态型)叶片悬浮培养细胞和玉米(Zea maize L.)幼苗为试验材料,综合利用分子生物学、植物生理学、生物化学和细胞生物学等技术,研究了植物细胞细胞壁与质膜间的相互作用、类整合素和微管蛋白的功能、细胞学定位以及编码类整合素蛋白和微管蛋白基因的克隆与转化,同时研究了类整合素蛋白、细胞骨架及两者的相互作用在渗透胁迫诱导细胞合成ABA中的作用,为进一步深入研究植物细胞壁-质膜-细胞骨架连续体在渗透胁迫信号传导过程中的作用提供一些理论依据。主要结果如下:
1、应用差速离心和密度梯度离心分离纯化细胞不同部分(膜部分和可溶性部分),结合Western Blotting技术分析了相关蛋白的表达。结果表明,在拟南芥悬浮细胞和玉米幼苗根尖细胞的不溶性部分存在与人整合素β1亚基抗体特异反应的蛋白质带,其分子量分别在110kDa和80kDa。用人整合素β1亚基抗体作探针,采用间接免疫荧光定位的方法,在拟南芥悬浮细胞的细胞膜和玉米幼苗根尖细胞
Adverse environmental factors, such drought, salinity, extreme temperature, etc, have deleterious effects on plant growth and development, thus the final plant productivity. It is understandable that, when plants are imposed to drought stress, the cell wall and/or plasma membrane are the most possible candidates for the primary sensing site(s). In plants, much progress has been made in understanding the complexity of the cell wall chemistry and cell wall extension, but there is still little information concerning its dynamic nature and interaction between it and the plasma membrane. Over the past two decades, evidence has been accumulated that the plant cell wall may interact with many proteins of the plasma membrane, like the extracellular matrix (ECM) in animals, and these in turn may interact directly or indirectly with the cytoskeleton. Integrin is one of the most important plasma membrane proteins in animals, which interacts with ECM and cytoskeleton and plays important roles in many physiological processes. In present study, several techniques, including immunological, biochemical and molecular biological techniques, have been used to explore whether there existed a similar molecule in plasma membrane, like integrin in animals, and if so, its possible interaction with cell wall and cytoskeleton. In addition, what are the roles of cell wall-plasma membrane-cytoskeleton continnum in the responses to osmotic stress? The results indicated that
1. Western blotting and immuno-fluorescent localization studies, using a monoclonal antibody againstβ1 subunit of human integrin, showed that the integrin-like protein localized in the plasma membrane fraction in Arabidopsis suspension cells and both cell membrane and nucleus of maize root cells with a molecular weight of ca.110 kDa and 80 kDa for Arabidopsis suspension cells and maize roots, respectively.
2. Under normal conditions (non-osmotic stress), the cell wall and plasma membrane was adhered together around the cells. However, the cell wall and plasma membrane was separated (plasmolyzed) partially when cells were exposed to osmotic stress. When cells were treated with RGD, a polypeptide that blocks the link between
引文
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