绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻活体生殖细胞及精细胞的微丝骨架

摘要

利用绿色荧光蛋白(GFP)基因结合鼠Talin基因表达技术及水稻(Oryza sativa L.)转基因技术,筛选出表达稳定和具等位基因型的第三代转基因水稻.在其活体花粉的4个发育阶段(Ⅰ.小孢子晚期;Ⅱ.二细胞早期;Ⅲ.二细胞晚期;Ⅳ.三细胞阶段),观察了细胞内微丝骨架的分布和结构形态的变化.发现在这4个花粉发育阶段,花粉内的营养核、生殖核、生殖细胞和精细胞都在不同的发育阶段出现位移.而这些位移与微丝骨架的结构变化和运动有密切关系.在胞质中央的微丝网络以及细胞周质的网络不断变化和互动,导致营养核、生殖核或生殖细胞和精细胞的定向位移.在活体生殖细胞和精细胞内,存有一股与细胞纵轴平行排列的微丝骨架.这些微丝骨架对生殖细胞及精细胞可以提供移动的动力,这对生殖细胞或精细胞在花管内以及胚囊内的运动(包括独自游动)提供了依据.%Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels theactin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryzasativa L.) plant, A005-G-T1-2. Observations were made on pollen at four major developmental stages, viz.Ⅰ. uni-nucleate microspore stage; Ⅱ. early bi-cellular pollen stage; Ⅲ. late bi-cellular pollen stage; and Ⅳ.tri-cellular pollen stage. At each of these developmental stages vegetative nucleus, generative nucleus/cell, and sperm cells were seen undergoing continuous and coordinated motion and migration. Thesemovements seemed to be influenced by associated microfilament networks existing in the pollen. Basedon these observations we propose that it is the interaction between the microfilament networks (usuallyone existing in the central cytoplasm and another in the cortex) that controls the dynamic movement ofthe vegetative nucleus, generative nucleus/cell and sperm cells. Furthermore, we have also observed thatthere is an array of microfilaments (oriented mostly parallel to the long axis of the cell) existing in thegenerative and sperm cells. As far as we are aware, this is the first report showing the existence ofmicrofilaments in living generative and sperm cells of rice pollen. The implication and significance of theexistence of microfilaments in generative and sperm cells in rendering self-propelled motion of these cellsin relation to their passage and movement in the pollen tube and embryo sac for fertilization were discussed.