拟南芥花粉萌发及花粉管伸长过程中关键质外体蛋白的鉴定及功能研究

Plant apoplast is the region outside the plasma membrane including cell wall matrix as well as gas and liquid phases of intercellular space. Due to limited techniques and knowledge, apoplast is still not well studied. However, increasing evident reveal apoplast plays an irreplaceable role in plant development and stress response. On the other side, since pollen is the germ cell of plant, pollen germination and pollen tube growth are always hotspot research areas. This proposal aims to find the key apoplastic proteins or peptides involved in pollen germination and pollen tube growth through efficiently extracting the apoplastic proteins of pollen or pollen tube in different developmental stages in Arabidopsis. Then 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) will be applied to identify differentially expressed proteins or peptides being selected for further subcellular localization and function analysis. These results could give us a complete picture of apoplastic proteins changes during pollen germination and pollen tube growth and discover novel candidate proteins participated in pollen germination, interaction with pistls and pollen tube growth, thereby providing new sights on these processes. Meanwhile, this work could lead to a better understanding on the function of apoplastic proteins in plant and also has high value in basic theories research.

质外体是植物细胞质膜以外的部分，包括细胞壁和植物细胞间的气相及液相组份。由于对其认识的不足及研究方法上存在较大难度，关于植物质外体功能研究报道较少。近年来，研究表明，植物质外体可能作为植物细胞的重要组分广泛参与了植物生长发育及逆境反应等多种生理过程。而花粉作为植物生殖细胞，其萌发和花粉管伸长过程一直是植物细胞生物学的研究热点之一。本实验通过提取拟南芥花粉萌发及花粉管伸长不同阶段的质外体蛋白，进行基于荧光差异胶电泳(DIGE)的差异蛋白质组学分析, 进而对所获取的重要差异蛋白进行亚细胞定位和功能分析。该研究可以全貌地反映在花粉萌发及花粉管伸长过程中质外体蛋白的变化，为进一步研究花粉萌发及其与柱头互作、花粉管生长提供重要的候选蛋白，进而为花粉萌发和花粉管伸长机理的研究提供新的思路。同时该研究可扩展和加深人们对植物质外体功能的认识，具有重要的理论研究价值。

质外体是植物细胞的一个重要组成部分，越来越多的实验证据证明其在植物的生长发育过程中发挥着重要的生理功能。为了研究花粉质外体在花粉萌发及花粉管生长中的作用，我们利用荧光差异双向凝胶电泳饱和标记技术，比较了拟南芥花粉萌发过程中可溶性质外体蛋白的差异表达，并找到103个差异蛋白点，通过质谱技术成功鉴定出98个点，代表了71种蛋白质。进而我们还利用同样的技术手段比较分析了不同时间进程下花粉萌发过程中主动分泌到液体培养基介质中的分泌蛋白，并找共得到174的差异蛋白点，通过质谱技术成功鉴定出148个，代表94中蛋白质。本项目利用蛋白质组学技术首次对拟南芥花粉质外体蛋白进行了全貌性分析，并比较了萌发过程中差异表达的可溶性质外体蛋白和分泌蛋白，同时还对差异表达蛋白的功能类群特征进行了分析，最后根据上述分析结果，我们对可溶性质外体蛋白参与花粉萌发和花粉管生长的机制做出了一个推测，这为进一步揭示花粉萌发和花粉管生长的分子机制提供了重要的信息，同时为我们更好的理解花粉的受精过程也打下了良好的基础。