【作者】 房克凤；

【导师】 周嫦； 孙蒙祥；

【作者基本信息】 武汉大学 ， 发育生物学， 2003， 博士

【摘要】 本文以异硫氰酸荧光素标记的凝集素(主要为伴刀豆凝集素、麦芽凝集素和大豆凝集素)为探针，对蓝猪耳和烟草等的雌、雄细胞以及胚胎细胞表面凝集素受体进行了定位，并利用共聚焦激光扫描显微术(CLSM)、冷电荷耦合仪(CCD)和动态录象CCD获得图象，以MetaMorph和Pro-Plus软件进行图象和数据分析，以探讨凝集素受体的基本分布模式及与被子植物双受精的可能关系。主要结果如下： 1．利用蓝猪耳的半裸露胚囊这一特性，建立了四种不同的实验系统，即对酶解法得到的雌性细胞、解剖法得到的雌性细胞、原位酶解的雌性细胞、不经任何酶解的原位雌性细胞分别进行标记，观察、比较荧光的分布形式及强度。从而证实：在本实验中用的几种酶组合对凝集素受体的影响并不大，即由酶解及解剖得到的雌性细胞的荧光在分布形式及强度上没有明显的差异；对比原位酶解与离体酶解得到的雌性细胞的荧光，证实机械操作对荧光的分布形式及强度也没有明显的影响。从而建立了探讨性细胞表面凝集素受体的可靠技术。 2．在烟草活体的中央细胞及卵细胞表面发现了伴刀豆凝集素受体的极性分布或者称之为“镶嵌性分布”。在大部分的中央细胞膜表面，该凝集素受体主要分布在靠近核的部位，而在相对侧的荧光要弱或缺失。在相同的条件下，不均匀分布表现多样，即荧光弱或无的部分占整个细胞表面的大小有差异。在卵细胞膜表面，凝集素受体在靠近核的部位较另一端要少。这一现象仅在小鼠的卵细胞表面发现过。而在高等植物中这是首次发现。受精后这种极性分布现象消失。我们利用下述方法对其进行了反复验证:l)胚囊或性细胞分离出来后，直接进行标记;2)胚囊或性细胞分离出来后先用多聚甲醛进行固定，清洗后标记;3)F队固定胚珠，酶解法分离出胚囊，进行标记;4)标记完毕后延长保温时间。从而排除了酶解的影响、机械操作的影响及膜的流动性的影响。证实了结果的可靠性。这一现象的发现表明动、植物受精过程中可能存在相似的调控机制。麦芽凝集素受体在受精前的中央细胞和卵细胞膜表面均匀分布，而在受精后的合子和初生胚乳细胞膜表面缺失。 3.用凝集素作为荧光探针对蚕豆、莺尾和朱顶红生殖细胞质膜表面的凝集素受体进行标记。结果显示，在不同植物中均有部分生殖细胞不能被凝集素探针标记。这可能是导致部分精子表面不能被同种凝集素标记的重要原因。同一种凝集素受体在不同物种的生殖细胞上分布不一致，不同的凝集素受体在同一种植物生殖细胞上的分布模式亦有不同。在蚕豆和莺尾的生殖细胞表面均有这三种凝集素的受体。在朱顶红生殖细胞表面有前两种凝集素的受体，分布比较均一，但是没有大豆凝集素的受体。此外，发现在具尾生殖细胞表面有凝集素受体极性分布的现象，为探讨精细胞功能和其表面糖蛋白分布的可能差异提供了重要启示。 4.为进一步探讨由生殖细胞到精子的发育过程中细胞质膜表面凝集素受体的可能变化及两个精子的可能差异。以同为二细胞花粉的烟草为材料，将其生殖细胞及成熟的雄性生殖单位分离出来，用凝集素作为荧光探针，对生殖细胞与精细胞表面荧光的分布形式及强度进行比较，同时对来自同一个花粉管中的一对精细胞表面的荧光的分布形式及强度进行比较，发现靠近营养核的精细胞表面的荧光略强于另一个精细胞，但差异并不显著。在荧光的分布形式上没有很大的差别。荧光均匀分布在精细胞表面膜上。连接两个精细胞的横壁也发出很亮的荧光。生殖细胞表面的荧光强度要梢强于精细胞，但差异不明显。我们的实验没有证实前人提出的精细胞表面糖蛋白分布的二型性。认为两类精子的存在可能意味着生活精细胞也有授精能力的差异。 5.发现了蓝猪耳胚囊表面伴刀豆凝集素受体的分布形式与密度随胚囊的成熟、授粉及受精过程同步变化。在开花前两天，凝集素受体在胚囊表面均匀分布，而授粉后凝集素受体在胚囊的珠孔端聚集，形成一个冠状的荧光带，随着花粉管逐渐接近胚囊，胚囊表面的凝集素受体出现在丝状器部位。而花粉管穿入胚囊后，除珠孔端和丝状器部位有很强的荧光外，退化助细胞处也出现了较强的荧光。受精完毕后，珠孔端的荧光逐渐减弱，荧光在整个胚囊的表面又呈均匀分布。这一规律表明凝集素受体的分布与授粉、受精作用的正常进行有密切的关系。 6.以实验手段观察凝集素对蓝猪耳花粉萌发、花粉管生长、烟草性细胞离体融合的影响。用凝集素处理柱头后授新鲜花粉，发现凝集素对花粉的正常萌发没有影响。但是对花粉管的正常生长有影响，即花粉管的生长速率较对照组慢，受精率也稍低。同时，凝集素也用来诱导性细胞原生质体的离体融合。多种浓度的凝集素(>50魄/ml)可以成功诱导不同来源的原生质体的粘连，包括中央细胞原生质体与体细胞原生质体、中央细胞原生质体与助细胞原生质体、中央细胞原生质体和卵细胞原生质体、中央细胞与中央细胞原生质体及中央细胞原生质体与精细胞等不同组合，并且不同浓度的凝集素诱导粘连的速率也不同。浓度越高，粘连的速度也越快，反之亦然。但是这些浓度的凝集素未能诱导原更多还原

【Abstract】 The interaction between lectins and their specific receptors is believed to play a critical role during fertilization in animals and some lower plants. However, for higher plants there is almost no information on lectins or their receptors related to male and female gametes and fertilization. Therefore, fluorescein isothiocyanate (FITC) conjugated lectins, namely concanavalin agglutinin (Con A), wheat germ agglutinin (WGA) and soybean agglutinin (SBA), were used to localize lectin receptors on plasma membrane of male and female gametes and other relevant sexual cells, as well as embryos of various developmental stages of Nicotiana tabacum and Torenia fournieri by cooled charge coupled device (CCD) and Confocal Laser Scanning Microscope (CLSM). Main results are described as follows:1. As the first attempt, it was designed to reveal the general pattern of lectin receptor distribution on the surface of female cells, namely egg cells, central cells and synergids of Torenia fournieri and, especially, toensure the possible effects of cell isolation procedure on the distribution of lectin receptors by four different systems. Con A and WGA receptors on the surface of both in vitro and in situ living female cells were localized by FITC conjugated Con A and WGA as probes. We demonstrated that enzymatic treatment and isolation procedure did not notably modify the surface characteristic of the female cells and the distributional patterns of Con A and WGA receptors. It was also found that Con A receptors were distributed differently on the surface of the female cells, with the strongest fluorescent signal on central cells and the weakest on egg cells. Calcium could greatly enhance the binding of Con A to cell surface. A polar distribution pattern of Con A receptors in embryo sacs was observed. The receptors were obviously densest at the filiform apparatus of the synergids. The basic pattern of WGA receptor distribution was similar to that of Con A’s. However, the fluorescent signal of WGA was much weaker than that of Con A. Fluorescent patches of FITC-WGA were usually found on the cells surface.2. FITC conjugated Con A and WGA were used as probes to localize their receptors on the surface of unfertilized and fertilized female cells of tobacco. Polar distribution of Con A receptors was observed on the plasma membrane of unfertilized egg cells and central cells. But after fertilization, Con A receptors were distributed uniformly on the surface of female cells. Relation between localization of nucleus and polar distribution of Con A receptors on surface of central cell and egg cell was also studied. Results indicated that Con A receptors were usually denser on membrane surface near nucleus than other part of central cells, while in egg cells it was contrary to that of central cell. Factors influencing distribution of Con A receptors were also investigated. WGA receptors were also present on plasma membrane of central cell, egg cell and synergid. But after fertilization, no WGA receptors weredetected on plasma membrane of zygote and primary endosperm cell. Clearly, fertilization induced redistribution of lectin receptors on female cell surface.3. FITC-lectins were used to localize their specific receptors on the plasma membrane of generative cells (GCs) isolated from Vicia faba Lind., Iris tectorium Maxim, and Hippeastrum vittatum Herb.. The investigation on possible distributive dynamic of lectin receptors during developmental process from generative cells to sperm cells was performed. All the three lectin receptors were found on the surface of generative cells of V. faba and /. tectorium. However, on generative cells of H. vittatum only Con A and WGA receptors were observed. The same lectin receptors on the generative cells from different species showed various distributive patterns. The distribution of various lectin receptors on the same generative cells also showed different characteristics; Lectin receptors were totally absent on some generative cells of all three investigated species;更多还原