小麦颖果筛分子与导管发育中细胞骨架动态变化及ACPase定位的比较研究

发布时间：2019-04-18 07:49

【摘要】：小麦(Triticum aestivum L.)颖果腹部韧皮部筛分子(sieve element,SEs)的主要功能是运输有机营养物质,木质部导管(tracheary elements,TEs)的主要功能是运输水分和无机盐。前期研究表明,小麦颖果腹部韧皮部筛分子的发育经历了特殊的PCD过程,发育成熟的SEs仍具有细胞活性,能将营养物质运输并储存于小麦胚乳中。本实验采用生物电镜、超微细胞化学与免疫荧光等技术手段,比较观察了筛分子和导管发育过程中超微结构的特征,并对细胞骨架的动态变化及ACPase分布的时空变化进行观察,进而探讨它们与PCD的关系。主要结果如下:1.显微与超微结构观察表明,筛分子呈对称性半圆形分布于导管的两侧,其细胞质主要通过液泡膜内陷包裹细胞器,形成自噬体进行降解,最终仍保留了部分细胞器碎片,筛分子仍然存活;而在导管分化中,主要是液泡破裂后,胞内形成自噬泡发挥自噬功能,最后细胞质被完全降解,导管最终死亡,形成管状分子,且在花后6-7 d时,导管细胞壁多处发生溶解,形成了端壁孔。2.通过吖啶橙/碘化丙啶荧光染色结果显示,筛分子和导管细胞壁均被染成黄绿色,细胞核被染成红色。多数筛分子细胞核降解主要发生在花后3-5 d,而导管发育过程中细胞核的降解主要发生在花后2 d,在花后3 d时,细胞核已被完全降解。3.间接免疫荧光标记微管结合透射电子显微镜观察发现,随着分化的进行,筛分子和导管内微管主要靠近细胞壁周围分布,尤以细胞壁加厚的位置分布较多。亚细胞水平观察发现微管周围有很多小囊泡。筛分子内靠近细胞壁周围的微管从花后5 d开始减少直至降解,而导管中微管在花后4 d出现横向微管,但在花后6-7 d时,有的导管分子细胞壁上的微管增多。微丝蛋白的免疫荧光结果显示,花后2 d,筛分子与导管内微丝均呈网络状。随着分化进行,微丝均在细胞壁加厚的位置分布较多。筛分子内靠近细胞壁周围的微丝从花后5 d开始减少直至降解。而导管中微丝在花后4 d出现横向微丝,花后6-7 d时,导管细胞内横向微丝减少。4.ACPase定位结果表明,在导管液泡膜破裂后,ACPase活性多位于线粒体内自噬空泡内、内质网等发生降解的细胞器上。在筛分子发育初期,仅在液泡内检测到ACPase活性,且液泡内包裹少量细胞质。随着发育的进行,不仅在发生降解的线粒体等细胞器上检测到了ACPase活性,在成熟筛分子胞间连丝上也检测到了ACPase活性。综上所述,小麦筛分子和导管分化过程中均出现了细胞核降解,内含物缺失等典型的细胞程序性死亡现象,但二者降解细胞质的方式不同,筛分子中主要是微自噬的过程,而导管中则是形成自噬泡来降解细胞质,且导管内含物的降解要比筛分子快。从形态变化来看,导管细胞壁发生了溶解,形成了端壁孔。更重要的是,分化成熟的筛分子是活细胞,而导管是死细胞。筛分子和导管中细胞骨架的动态变化可能对细胞壁的发生具有调控作用。ACPase不仅与胞内物质的降解有一定相关性,自噬泡内ACPase活性也介导了细胞器的消亡过程,同时,分布于胞间连丝上的ACPase可能还与细胞间的物质运输和信息传递有关。
[Abstract]:The main function of the sieve element (SEs) is to transport organic nutrients, and the main function of the xylem ducts (SEs) is to transport water and inorganic salts. The early studies have shown that the development of the phloem screen in the wheat caryopsis has experienced a special PCD process, and the mature SEs still has cell activity, which can transport and store nutrients in the endosperm of wheat. In this experiment, the characteristics of the ultrastructures in the development of the screen and the catheter were compared and the changes of the cytoskeleton and the spatial and temporal changes of the ACPase distribution were observed, and the relationship between them and the PCD was also discussed. The main results are as follows:1. The microscopic and ultrastructural observations show that the screen molecules are symmetrically and semi-circular distributed on both sides of the catheter, and the cytoplasm of the sieve is mainly through the vacuole membrane to be trapped in the organelles to form a self-phagemid to be degraded, part of the organelle fragments are still retained, the screen molecules are still alive, and in the differentiation of the catheter, After the vacuole is broken, the autophagy is formed in the cell to perform autophagy function, the last cytoplasm is completely degraded, the catheter is finally killed, the tubular molecule is formed, and when 6 to 7 days after the flower, the cell wall of the catheter is dissolved, and the end wall hole is formed. The results showed that the cell walls of both the screen and the catheter were stained with yellow green and the nuclei were stained with red color. The nuclear degradation of most screen molecules mainly occurred in 3-5 days after the flower, while the cell nuclei in the course of the development of the catheter mainly occurred 2 days after the flower, and at 3 d after the flower, the cell nucleus was completely degraded. The microtubule-binding transmission electron microscope with indirect immunofluorescence showed that, with the development of the differentiation, the microtubules in the screen and the catheter were mainly distributed around the cell wall, especially in the thickened location of the cell wall. The subcellular level observed that there were many vesicles around the microtubules. The microtubules near the cell wall in the screen molecule begin to decrease from the back 5d to degradation, while the microtubules in the catheter show transverse microtubules after the flower, but at 6-7 days of the flower, the microtubules on the cell wall of the catheter molecule are increased. The results of the immunofluorescence of microfilament protein showed that the microfilaments in the 2 d, the screen and the inside of the catheter were in the form of network. As the differentiation progresses, the microfilaments are distributed more in the thickened position of the cell wall. The microfilaments located near the cell wall in the screen molecule begin to decrease from the back 5d until it is degraded. The results showed that, after the rupture of the vacuole membrane of the catheter, the activity of the ACPase was more localized in the mitochondria, such as the autophagy, endoplasmic reticulum and the like. In that early stage of the molecular development of the screen, the ACPase activity was only detect in the vacuole, and a small amount of the cytoplasm was wrapped in the vacuole. ACPase activity was not only detected on the organelles such as the mitochondria, but also the activity of the ACPase was also detected on the intercell of the mature screen. In conclusion, there are some typical cell-programmed cell death in the process of molecular and catheter differentiation of wheat and the process of microautophagy in the screen molecule. In that catheter, the autophagy is formed to degrade the cytoplasm, and the degradation of the contents of the catheter is to be faster than that of the screen. In the form of morphological changes, the cell wall of the catheter was dissolved and the end wall hole was formed. More importantly, the differentiated mature screen molecule is a living cell, and the catheter is a dead cell. The dynamic changes of the cytoskeleton in the screen molecule and the catheter may have a regulatory effect on the occurrence of the cell wall. ACPase not only has a certain correlation with the degradation of the intracellular substance, but the activity of the ACPase in the autophagy also mediates the extinction process of the organelles, and the ACPase, which is distributed on the intercell, may also be related to the transport of substance and the transfer of information between the cells.
【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】：S512.1

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