RUI: Antibodies to Flowering Plant Sperm: Development and Specificity of Epitopes

在开花植物中，受精过程是种子和果实产生的基础。 虽然双受精的结构事件已被描述，但精子和卵子相互识别、粘附和融合的分子机制尚不清楚。 植物精子嵌入花粉粒中，并沿着生长的花粉管向下移动。 在卵附近，花粉管释放出两个精子：一个与卵融合形成胚胎，另一个与卵附近的中央细胞融合形成胚乳，支持发育中的胚胎的生长。 植物繁殖中一个主要的未解问题是精子如何识别卵子和中央细胞，粘附和融合。 该提案的目标是表征精子细胞膜上的分子。 在索斯沃思博士的实验室里，已经鉴定出三种与芸苔属精子细胞结合的抗体。 本研究的具体目的是（1）从（a）花药中表达的时间，（B）组织特异性表达，（c）抗体结合的种特异性，（d）分子量，（e）细胞膜抗原决定簇，（f）细胞膜抗原和（e）在细胞中的位置和（2）让大学生参与研究。 将从花药、小孢子、花粉或花粉管中分离精子和导致其发育的细胞，并通过免疫细胞化学和免疫印迹法测定抗体结合。 这些结果将进一步了解植物繁殖中的基本发育机制，并将导致对育性和不育性的理解：从不相容的杂交中产生杂交种，并在需要雄性不育以防止自交的情况下防止受精。 精子特异性膜分子的鉴定将有助于未来的遗传学研究。 本科生将获得积极的研究有价值的见解。 通过解决问题和协助实现目标，他们将在了解研究方法和发展技术技能方面获得经验，这将有助于他们作为科学家的发展。

In flowering plants, the process of fertilization underlies production of seeds and fruits. Although structural events of double fertilization have been described, the molecular mechanisms by which sperm and egg recognize each other, adhere and fuse are unknown. Plant sperm are embedded in pollen grains and move down growing pollen tubes. Near the egg, a pollen tube releases two sperm: one fuses with the egg to form the embryo, the other fuses with the central cell, adjacent to the egg, to form endosperm that supports growth of the developing embryo. A major unanswered question in plant reproduction is how sperm recognize eggs and central cells, adhere, and fuse. The goal of this proposal is to characterize molecules on sperm cell membranes. In Dr. Southworth's laboratory, three antibodies that bind to Brassica sperm cells have been identified. One anti-sperm antibody, BRSP1, has been partially characterized with regard to species specificity, presence of carbohydrate in the epitope, and molecular weight.The specific objectives of this proposal are (1) to characterize Brassica sperm membrane epitopes with regard to (a) timing of expression in the anther, (b) tissue-specific expression, (c) species-specificity of antibody binding, (d) molecular weight, and (e) location in the cell and (2) to involve undergraduates in research. Sperm and the cells leading to their development will be isolated from anthers, microspores, pollen, or pollen tubes and antibody binding will be determined by immunocytochemistry and by immunoblotting. The results will further knowledge of basic developmental mechanisms that function in plant reproduction and will lead to understanding of fertility and sterility: producing hybrids from incompatible crosses and preventing fertilization where male sterility is needed to prevent self-crossing. The identity of sperm-specific membrane molecules will be useful in future genetic studies. Undergraduate students will gain valuable insight into active research. By solving problems and assisting in attainment of goals, they will gain experience in terms of understanding the approach to research and in development of technical skills which will facilitate their development as scientists.