小麥穎果篩分子與導(dǎo)管發(fā)育中細(xì)胞骨架動態(tài)變化及ACPase定位的比較研究
發(fā)布時間：2019-04-18 07:49
【摘要】：小麥(Triticum aestivum L.)穎果腹部韌皮部篩分子(sieve element,SEs)的主要功能是運(yùn)輸有機(jī)營養(yǎng)物質(zhì),木質(zhì)部導(dǎo)管(tracheary elements,TEs)的主要功能是運(yùn)輸水分和無機(jī)鹽。前期研究表明,小麥穎果腹部韌皮部篩分子的發(fā)育經(jīng)歷了特殊的PCD過程,發(fā)育成熟的SEs仍具有細(xì)胞活性,能將營養(yǎng)物質(zhì)運(yùn)輸并儲存于小麥胚乳中。本實(shí)驗(yàn)采用生物電鏡、超微細(xì)胞化學(xué)與免疫熒光等技術(shù)手段,比較觀察了篩分子和導(dǎo)管發(fā)育過程中超微結(jié)構(gòu)的特征,并對細(xì)胞骨架的動態(tài)變化及ACPase分布的時空變化進(jìn)行觀察,進(jìn)而探討它們與PCD的關(guān)系。主要結(jié)果如下:1.顯微與超微結(jié)構(gòu)觀察表明,篩分子呈對稱性半圓形分布于導(dǎo)管的兩側(cè),其細(xì)胞質(zhì)主要通過液泡膜內(nèi)陷包裹細(xì)胞器,形成自噬體進(jìn)行降解,最終仍保留了部分細(xì)胞器碎片,篩分子仍然存活;而在導(dǎo)管分化中,主要是液泡破裂后,胞內(nèi)形成自噬泡發(fā)揮自噬功能,最后細(xì)胞質(zhì)被完全降解,導(dǎo)管最終死亡,形成管狀分子,且在花后6-7 d時,導(dǎo)管細(xì)胞壁多處發(fā)生溶解,形成了端壁孔。2.通過吖啶橙/碘化丙啶熒光染色結(jié)果顯示,篩分子和導(dǎo)管細(xì)胞壁均被染成黃綠色,細(xì)胞核被染成紅色。多數(shù)篩分子細(xì)胞核降解主要發(fā)生在花后3-5 d,而導(dǎo)管發(fā)育過程中細(xì)胞核的降解主要發(fā)生在花后2 d,在花后3 d時,細(xì)胞核已被完全降解。3.間接免疫熒光標(biāo)記微管結(jié)合透射電子顯微鏡觀察發(fā)現(xiàn),隨著分化的進(jìn)行,篩分子和導(dǎo)管內(nèi)微管主要靠近細(xì)胞壁周圍分布,尤以細(xì)胞壁加厚的位置分布較多。亞細(xì)胞水平觀察發(fā)現(xiàn)微管周圍有很多小囊泡。篩分子內(nèi)靠近細(xì)胞壁周圍的微管從花后5 d開始減少直至降解,而導(dǎo)管中微管在花后4 d出現(xiàn)橫向微管,但在花后6-7 d時,有的導(dǎo)管分子細(xì)胞壁上的微管增多。微絲蛋白的免疫熒光結(jié)果顯示,花后2 d,篩分子與導(dǎo)管內(nèi)微絲均呈網(wǎng)絡(luò)狀。隨著分化進(jìn)行,微絲均在細(xì)胞壁加厚的位置分布較多。篩分子內(nèi)靠近細(xì)胞壁周圍的微絲從花后5 d開始減少直至降解。而導(dǎo)管中微絲在花后4 d出現(xiàn)橫向微絲,花后6-7 d時,導(dǎo)管細(xì)胞內(nèi)橫向微絲減少。4.ACPase定位結(jié)果表明,在導(dǎo)管液泡膜破裂后,ACPase活性多位于線粒體內(nèi)自噬空泡內(nèi)、內(nèi)質(zhì)網(wǎng)等發(fā)生降解的細(xì)胞器上。在篩分子發(fā)育初期,僅在液泡內(nèi)檢測到ACPase活性,且液泡內(nèi)包裹少量細(xì)胞質(zhì)。隨著發(fā)育的進(jìn)行,不僅在發(fā)生降解的線粒體等細(xì)胞器上檢測到了ACPase活性,在成熟篩分子胞間連絲上也檢測到了ACPase活性。綜上所述,小麥篩分子和導(dǎo)管分化過程中均出現(xiàn)了細(xì)胞核降解,內(nèi)含物缺失等典型的細(xì)胞程序性死亡現(xiàn)象,但二者降解細(xì)胞質(zhì)的方式不同,篩分子中主要是微自噬的過程,而導(dǎo)管中則是形成自噬泡來降解細(xì)胞質(zhì),且導(dǎo)管內(nèi)含物的降解要比篩分子快。從形態(tài)變化來看,導(dǎo)管細(xì)胞壁發(fā)生了溶解,形成了端壁孔。更重要的是,分化成熟的篩分子是活細(xì)胞,而導(dǎo)管是死細(xì)胞。篩分子和導(dǎo)管中細(xì)胞骨架的動態(tài)變化可能對細(xì)胞壁的發(fā)生具有調(diào)控作用。ACPase不僅與胞內(nèi)物質(zhì)的降解有一定相關(guān)性,自噬泡內(nèi)ACPase活性也介導(dǎo)了細(xì)胞器的消亡過程,同時,分布于胞間連絲上的ACPase可能還與細(xì)胞間的物質(zhì)運(yùn)輸和信息傳遞有關(guān)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S512.1
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S512.1
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