水稻有机硅调控养分运输及细胞壁组分诱导硅沉积的研究

发布时间：2018-06-03 02:13

本文选题：硅 + 养分运输　；参考：《华中农业大学》2017年硕士论文

【摘要】：硅在土壤中的丰度位列第二,且有益于植物的生长。水稻(Oryza sativa)是一种典型的喜硅植物,在其体内积累了大量的硅,其中地上部分干重高达10%。这些硅主要以无定型的二氧化硅沉积在茎和叶的特化细胞内或细胞壁上,只有微量硅与细胞壁交联形成硅-细胞壁复合物(有机硅)。大量的研究表明硅营养能够帮助水稻缓解环境胁迫和调节养分运输,然而硅的生物学功能与其相对应的分子机制尚未完全清楚。为了排除组织、器官和植株水平上多因子作用的复杂性,我们选用遗传背景和发育阶段完全一致的水稻悬浮细胞作为研究对象,采用非损伤微测技术(NMT)、同位素标记相对和绝对定量的蛋白质组学技术(iTRAQ)以及其它化学与生物的手段,在细胞水平系统地研究硅与养分运输和蛋白表达的关系。同时利用原子力显微镜(AFM)模拟了不同细胞壁组分对二氧化硅沉积的影响。主要结果总结如下:1、硅-细胞壁有机复合物提高了细胞壁的机械性质,进而有利于细胞膜的稳定,从而促进了水稻悬浮细胞对铵态氮(NH_4~+)的吸收在正常状态和1%聚乙二醇6000(PEG 6000)处理的条件下,借助NMT测得加硅(+Si)细胞NH_4~+的平均内流速率大于缺硅(-Si)细胞的速率。总RNA和总蛋白的测定,发现两者的浓度均是+Si细胞高于-Si细胞,说明硅的添加提高了单细胞的氮代谢水平。XPS和AFM分析可知,硅是以有机硅的形式存在于细胞壁上,而且提高了细胞壁的致密程度和力学性质。细胞壁的机械性质与膜稳定性紧密相关,不同条件下,其它的离子流的监测、电解质渗漏的测定和荧光染料DiBAC4(3)标记的膜电势变化均表明+Si细胞的质膜要更加稳定。2、硅营养能够引起水稻悬浮细胞蛋白的差异性表达在无胁迫的条件下,iTRAQ结果表明,硅营养调控了74蛋白的表达,其中下调和上调的蛋白分别为51和23个。分析可知,硅营养的缺失使得细胞壁存在缺陷,因此-Si细胞遭受各种环境胁迫,引起蛋白的差异性表达。这些蛋白主要是与细胞壁合成相关的酶以及抗逆蛋白。3、不同细胞壁组分对植物体内二氧化硅(SiO_2)沉积的诱导能力不同,且半纤维素为主要的诱导因子运用原子力显微镜,以不同细胞壁组分修饰的云母为基底,在体外模拟了二氧化硅在植物体内的沉积,结果表明半纤维素的主要成分木聚糖比果胶更容易诱导SiO_2成核。
[Abstract]:Silicon is the second most abundant in the soil and beneficial to plant growth. Rice (Oryza sativa) is a typical silicon-loving plant, which accumulates a large amount of silicon in its body, in which the dry weight of the aboveground part is as high as 10%. These silicon is mainly deposited in the specialized cell or cell wall of stem and leaf by amorphous silica. Only a trace of silicon is cross-linked with the cell wall to form a silicon-cell wall complex (organosilicon). A large number of studies have shown that silicon nutrition can help rice alleviate environmental stress and regulate nutrient transport. However, the biological function of silicon and its corresponding molecular mechanism have not been fully understood. In order to eliminate the complexity of multifactorial action at the tissue, organ and plant levels, we chose rice suspended cells with identical genetic background and developmental stage as the research objects. The relationship between silicon and nutrient transport and protein expression at the cellular level was systematically studied at the cellular level by using noninvasive microassay, relative and absolute quantitative proteomics techniques with isotopic labeling and other chemical and biological methods. At the same time, AFM was used to simulate the effect of different cell wall components on the deposition of silica. The main results are summarized as follows: 1. The silicon-cell wall organic complex improves the mechanical properties of the cell wall and contributes to the stability of the cell membrane. Thus, the absorption of ammonium nitrogen by rice suspension cells under normal condition and 1% polyethylene glycol (6000(PEG) treatment was promoted. The average inflow rate of Si-added cells was higher than that of Si-deficient cells by means of NMT. The concentrations of total RNA and total protein were both higher in Si cells than in -Si cells, indicating that the addition of silicon increased the nitrogen metabolism level of single cells. XPS and AFM analysis showed that silicon existed in the form of organosilicon on the cell wall. Moreover, the density and mechanical properties of the cell wall were improved. The mechanical properties of the cell wall are closely related to the stability of the membrane, and other ion currents are monitored under different conditions. The measurement of electrolyte leakage and the change of membrane potential labeled by fluorescent dye DiBAC4C3 indicated that the plasma membrane of Si cells should be more stable, and silicon nutrition could induce the differential expression of protein in rice suspension cells under the condition of no stress. Silicon nutrition regulated the expression of 74 protein, of which the downregulation and upregulation were 51 and 23, respectively. The results showed that the cell wall was deficient due to the lack of silicon nutrition, so that the -Si cells were subjected to various environmental stresses, which led to the differential expression of protein. These proteins are mainly enzymes associated with cell wall synthesis and stress-resistant proteins. Different cell wall components have different inductive effects on the deposition of SiO-2) in plants, and hemicellulose is the main inducer using atomic force microscopy (AFM). The deposition of silicon dioxide in plant was simulated in vitro on the base of mica modified by different cell wall components. The results showed that xylan, the main component of hemicellulose, was easier to induce SiO_2 nucleation than pectin.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511

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