pCAMBIA1300-AFH16-GFP重组质粒的构建及纯合转基因拟南芥植株的筛选
发布时间：2018-11-03 12:51
【摘要】：臭氧层变薄是地表紫外辐射(UV-B)强度增加的主要原因。研究表明地球UV-B辐射剂量都呈现逐年上升趋势,严重威胁地表生物的生命活动。对无法自主移动的植物来说,高能UV-B在形态建成、生理代谢等生命活动都具有较显著的影响。因此,研究植物如何响应UV-B辐射有着深远的科学意义与应用前景。植物细胞骨架一直处于动态的解聚与聚合过程中。是细胞中能够调整自身的结构响应细胞的生长发育以及外界刺激的一类重要的结构组成,在许多细胞代谢过程中起着关键的作用,比如细胞生长、胞质流动、细胞器的运动以及细胞分裂。这些功能的实现需要一些特定的辅助蛋白来调节细胞骨架动态行为。Formin蛋白(成蛋白)被认为是一种多结构域的肌动蛋白结合蛋白,但近来发现Formin还可以与微管结合,作为一个双功能蛋白涉及很多细胞过程的发生。与哺乳动物和酵母相比人们对植物中Formin蛋白的功能还知之甚少。拟南芥中AtFH16属于Ⅱ类Formin,已有的研究表明,其可能参与了细胞分裂的过程。本课题组长期致力于植物响应增强UV-B辐射的研究,并且前期发现微丝骨架的动态变化参与了UV-B辐射所导致的“分束分裂”现象的发生。综合文献分析,我们判断微丝响应这一过程的变化可能是依赖于Formin家族蛋白的。为了验证这一假设,(1)本研究用GFP作为融合报告基因,构建pCAMBIA1300-AFH16-GFP融合表达载体,测序正确后转入农杆菌,重组质粒通过农杆菌介导法导入拟南芥,筛选获得纯合转基因植株。(2)与此同时,将重组质粒导入烟草表皮细胞中使其瞬时表达,共聚焦检测可见绿色荧光,进一步证明构建成功。(3)对野生型植株与转基因植株做了表型对比后发现,转基因植株表现出主根伸长、叶片增大。进一步分析原因,表明AtFH16可能参与并促进细胞分裂。(4)最后对拟南芥中AtFH16的亚细胞定位的检测过程中发现,AtFH16定位在细胞质。载体的构建为后续研究At FH16与细胞骨架、细胞核以及探究“分束分裂”的机制提供了基础的实验材料。该载体的构建及转基因植物的获得,为课题组进一步验证AtFH16与“分束分裂”现象的发生提供了重要的实验材料。同时为课题组深入研究UV-B辐射后微丝骨架的动态调整的变化提供一定的依据。
[Abstract]:The thinning of the ozone layer is the main reason for the increase in the intensity of surface ultraviolet radiation (UV-B). The results show that the radiation dose of earth UV-B is increasing year by year, which seriously threatens the life activity of surface organisms. For plants unable to move independently, high energy UV-B has significant effects on morphogenesis, physiological metabolism and other life activities. Therefore, the study of how plants respond to UV-B radiation has profound scientific significance and application prospects. Plant cytoskeleton has been in the dynamic process of depolymerization and polymerization. It is a kind of important structural composition that can adjust its structure and respond to the growth and development of cells and external stimuli. It plays a key role in many cell metabolic processes, such as cell growth, cytoplasmic flow. Organelle movement and cell division. The realization of these functions requires specific auxiliaries to regulate cytoskeletal dynamic behavior. Formin protein is considered to be a multi-domain actin binding protein, but recently it has been found that Formin can also bind to microtubules. As a bifunctional protein, many cellular processes are involved. Little is known about the function of Formin proteins in plants compared with mammals and yeast. In Arabidopsis thaliana, AtFH16 belongs to class 鈪，
本文编号：2307844
[Abstract]:The thinning of the ozone layer is the main reason for the increase in the intensity of surface ultraviolet radiation (UV-B). The results show that the radiation dose of earth UV-B is increasing year by year, which seriously threatens the life activity of surface organisms. For plants unable to move independently, high energy UV-B has significant effects on morphogenesis, physiological metabolism and other life activities. Therefore, the study of how plants respond to UV-B radiation has profound scientific significance and application prospects. Plant cytoskeleton has been in the dynamic process of depolymerization and polymerization. It is a kind of important structural composition that can adjust its structure and respond to the growth and development of cells and external stimuli. It plays a key role in many cell metabolic processes, such as cell growth, cytoplasmic flow. Organelle movement and cell division. The realization of these functions requires specific auxiliaries to regulate cytoskeletal dynamic behavior. Formin protein is considered to be a multi-domain actin binding protein, but recently it has been found that Formin can also bind to microtubules. As a bifunctional protein, many cellular processes are involved. Little is known about the function of Formin proteins in plants compared with mammals and yeast. In Arabidopsis thaliana, AtFH16 belongs to class 鈪，
本文编号：2307844
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