花粉表面蛋白的功能,一直是植物生殖生物学研究的重要问题。我们发现,玉米半胱氨酸蛋白酶（cysteine protease,CP）大量存在于花粉表面和内部,与花丝（柱头）表面蛋白存在互作;CP抗体抑制花粉管顶端分泌囊泡的形成、延缓花粉管生长。然而,玉米花粉CP的功能尚未明确。本项目以玉米B73和Mo17为材料,研究CP的酶学特性、降解花丝表面蛋白的特异性;利用免疫共沉淀和酵母双杂交,研究CP与花丝表面蛋白的互作,鉴定其分子作用靶点;利用单克隆抗体和抑制剂,研究CP功能干扰下的花粉萌发和花粉管生长;通过转基因技术创制功能缺失的Cp-花粉,研究Cp功能缺失对花粉萌发和授粉竞争力的影响。本项目旨在阐明CP参与玉米花粉萌发和花粉管生长的分子生理机制,揭示玉米花粉迅速萌发的分子基础,为分子调控玉米授粉过程、培育优异授粉性状(花粉萌发快)的新品种,进而改善玉米生产上授粉效率低的问题,奠定理论和应用基础。

Maize (Zea mays L.) is one of the most important grain crops worldwide. Being a wind-pollinated species, maize produces a large quantity of pollen grains and has elongated silks (stigma/style). Thus, maize is an important model of monocot species for studying plant reproduction. The pollen surface, consisting of an outermost coat and an underlying wall, plays a vital role in pollen-stigma interaction, pollen germination, and successful fertilization. Surface proteins of pollen, which confer species specificity, are required for pollen adhesion, recognition, hydration, and germination on the stigma. The mechanism by which the pollen surface proteins are involved in pollen-stigma interaction and pollen germination is one of the central issues in plant reproductive biology.. Recently, we found that cysteine protease (CP, EC3.4.22) exists in substantial abundance on the surface and inside of pollens in maize and can interact with the surface proteins released from the pistil and silk. The presence of the monoclonal antibody against CP reduces the number of apical vesicles in pollen tubes and slows down the in vitro growth of pollen tubes in maize. However, the role of CP in maize pollen germination and tube growth, especially in pollen-stigma interaction, remains unclear. We propose that the CP present on the surface of the pollen may play an important role during hydrolysis or through specific binding proteins existing on the surface of the stigmatic cells. Thus, it is required to further clarify how the CP present inside the pollen is involved in the regulation of pollen tube growth.. In the present project, we will use maize B73 and Mo17 to investigate: a) the enzymatic characteristics, ability of purified CP to degrade stigma/silk surface proteins, and its effect on the surface structure of the stigma/silk; b) the interaction between CP and stigma/silk surface proteins using immunoprecipitation, pull-down technique and yeast two-hybrid; c) the behavior of germination and the ability of pollen grains to pollinate under the conditions where the function of CP is interfered by its monoclonal antibody and inhibitors; and d) the effect of loss-of-function of CP on pollen germination and pollination through anti-sense nucleic acid approaches.. Through the molecular, cellular and proteomic studies in vitro and in vivo, we will elucidate the molecular and physiological mechanism of CP involved in regulation of maize pollen germination and tube growth, reveal the molecular basis for the rapid germination of maize pollen, which is imporatnt for providing molecular means to control and solve the problem of the low pollination in maize production, and finally to breed new maize lines with better pollination traits.