【作者】 梁峰；

【导师】 杨清；

【作者基本信息】 南京农业大学 ， 细胞生物学， 2007， 博士

【摘要】 本文运用光学显微镜、电子显微镜、X-射线能谱和免疫荧光等技术,以慈姑叶柄通气组织发育过程为主要研究对象,研究了细胞周质微管(corticalmicrotubules,CMTs)的组织形式与细胞形态发生的关系,并运用基因工程手段对微管和微管相关蛋白的活体探针技术进行了初步的研究。主要研究结果如下:慈姑叶柄的通气组织是一种裂生式通气组织具有复杂的整齐结构,这种类似于蜂巢结构的通气组织由构成圆柱体气室壁的细胞和横隔膜细胞构成,成熟的横隔膜上形成复杂的维管束网络。在叶柄发育早期,通气组织气体空间增长的主要因素是细胞分裂;而在叶柄发育后期,通气组织的气体空间的增长主要因素是细胞伸展。在横隔膜细胞形成细胞间气体空间的过程中,CMTs定位在那些将要形成通气空间或者正在形成通气空间的区域,然后,这些区域细胞壁加厚,表明CMTs参与细胞间缝隙的形成是通过调节细胞壁的沉积,继而调控细胞形态来实现的。在横隔膜细胞伸展时期,CMTs分布在细胞的周边,包括通气缝隙边缘和相邻细胞的边界,说明周质微观参与细胞的伸展并导致细胞间通气缝隙的扩展。这些结果显示CMTs的重组与细胞间通气缝隙的定位与形成有关,是通过调节细胞壁的局部沉积改变细胞的形状。CMTs参与调节横隔膜上维管束(vascular bundle,VB)形成过程的细胞形态变化。结合细胞形态和CMTs的组织形式,将横隔膜上的维管束划分为初生和次生两种类型。维管束的前身是已经分化的横隔膜组织,提示水分疏导组织和通气组织的横隔膜组织是同源的,同时也表明横隔膜除了机械支持作用外,很可能还具有气体交换的功能;形态上明显分化的横隔膜细胞能够恢复分生能力并发育成维管束,显示横隔膜细胞较旺盛的居间分生能力;横隔膜上维管束的形成过程有明显的位置效应,这种位置效应的产生应该是细胞内外信号总和的输出,是一种多细胞调控的机制。运用冰冻扫描电镜X-射线能谱分析(CSEM-EDX)定点测量慈姑叶柄通气组织不同发育时期细胞壁的矿质元素组成。发现在不同发育阶段,细胞壁的元素含量呈现动态变化,表明细胞壁(质外体物质)的元素构成有很大的变动范围。通气组织横隔膜细胞和圆柱体气腔壁细胞的元素构成变化有相似的趋势,表明这种变化与组织的发育阶段关系密切。早期的通气组织细胞壁大量积累钾和氯,暗示早期的气体空间充满液体(组织液):镁在细胞快速伸展前出现一个峰值,可能参与细胞伸展的调控;伸展中细胞的细胞壁积累高浓度的铜和锌,并不影响细胞的正常功能;而钙的出现使细胞壁硬度增加,终止细胞伸展。细胞壁的一些元素含量之间呈现较高的相关性,其中K与Cl及Cu与Zn之间成较高的正相关。Cu和Zn在细胞壁中的积累呈高度的直线关系,回归分析显示,二者呈现定量关系,推测它们可能有共同的或者类似的转运和吸收机制。运用基因工程手段构建了三个融合蛋白植物表达质粒:pTUA6-GFP、pGFP-TUA6和pMAP65-6-GFP。分别转入拟南芥获得一批能够表达相应融合蛋白的株系,经过进一步的筛选工作,可能获得用于活体研究的探针材料。更多还原

【Abstract】 The shape of plant cell has long been the cornerstone of diverse areas of plant research but it is only recently that molecular-genetic and cell-biological tools were been effectively combined for dissecting plant cell morphogenesis.Increased understanding of the polar growth characteristics of model cell types,the availability of many morphological mutants and significant advances in fluorescent-protein-aided live-cell visualization have provided the major impetus for these analyses.The cytoskeleton and its regulators have emerged as essential components of the scaffold involved in fabricating plant cell shape.In this thesis Sagittaria trifolia Lwere used to study the cortical microtubule(CMT) organization and their relationship with cell morphogenesis.The development of aerenchyma in the petiole of S.trifolia was studied by means of light-microscope,scanning electron microscope,transmission electron microscope and immunofluorescence,focusing on the formation of intercellular spaces in diaphragms and its relationship with the organization of CMT arrays.A complex and organized honeycomb-like schizogenous aerenchyma formed by cylinders and vascular diaphragms was observed in the petiole of S.trifolia at different developmental stages.Cell division was the primary factor contributing to the increased volume of air spaces at early stages,while cell enlargement became the primary factor at later stages.The CMTs localized at the sites where intercellular spaces and the secondary cell walls would be formed or deposited during the formation of intercellular spaces by the separation of diaphragm cells.CMTs were observed at the boundary of diaphragm cells and the fringes of intercellular spaces at later developmental stages,where cell expansion occurred rapidly.These observations supported the hypothesis that reorganization of CMT arrays might be related to the formation of air spaces in diaphragms and are involved in the deposition of secondary cell walls.The vascular bundles appeared in diaphragm of the aerenchyma in petioles of S. trifolia being classified to primeray and secondary vascular bundle.All vascular bundles were come of diaphragm cells by changing their shapes.In this process,the diaphragm cells differentiated into tracheary elements with the CMTs reorganization. This result suggested that the diaphragm cells and the tracheary elements might share similar developmental characteristics.Essential mineral elements like Potassium(K),Mg,Cu,Zn,Ca and P in the cell wall of aerenchyma cells in petioles of S.trifolia at five different developmental stages were analysed by CSEM-EDX.At early stage,K and Cl concentrations in cell wall were high up to 36%and 4.3%of dry weight,respectively.It supported the hypotheses that aerenchyma spaces were filled with liquid at early developmental stages of aerenchyma in S.trifolia petiole.Magnesium concentration was high at stage 2,up to 0.86%of dry weight.Zinc and Cu were detected only at rapid expansion stages,during which the concentrations were up to 1.5 and 2.5%, respectively.Calcium was detected in the cell wall only at mature stages,the concentration was high up to 1.3%of dry weight at stage 5.These results confirmed that the element concentration of aerenchyma cell wall undergwent dynamic changes during the different developmental stages,and a low Ca with high Zn and Cu concentration were needed for cell expansion.Cu and Zn deposition in the cell wall showed a significant positive linear correlation,suggesting that these two elements shared same or similar uptake and transport mechanism in plants.Three plasmids,named pTUA-6-GFP,pGFP-TUA6 and pMAP65-6-GFP were constructed and transferred into in Arabidopsis.The expression of these fusion genes in Arabidopsis will further be analysed and used to analyze the function of CMT or/and MAP65-6 with fluorescent protein aided live-cell visualizations.更多还原