半胱氨酸蛋白酶参与玉米花粉萌发和花粉管生长的分子生理机制

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.

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。

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。半胱氨酸蛋白酶ZmCP03在花粉蛋白质组高水平存在，但该蛋白在花粉发育及萌发过程中的作用尚不清楚。本项目系统研究了ZmCP03的分子进化、亚细胞定位、表达规律以及基因过表达及缺失对花粉发育及花粉萌发及育性的影响。.首先，通过生物信息学工具分析了ZmCP03及38个同源蛋白的序列特征、基因结构、分子进化；并与模式植物中该基因家族进行了比较分析。ZmCP03基因在进化过程中相对保守和稳定的，在花药和花粉中高水平表达，为阐明ZmPCP在玉米花粉的发育和萌发中的功能提供了线索。.通过荧光显微镜和免疫金电镜技术研究了ZmCP03在花粉粒中的亚细胞定位。利用单克隆抗体进行免疫荧光定位，发现ZmCP03大量存在于花粉表面，也存在于花粉细胞质中；免疫金电镜定位进一步证实了该结论。.创制了ZmCP03基因过表达及敲除突变植株，研究了ZmCP03基因表达水平对花粉发育、花粉粒结构、花粉萌发及育性的影响。ZmCP03的敲除或过表达对玉米田间表型没有显著影响，可能的原因是该基因主要在花粉表面以及内部表达。透射电镜观察发现，缺失突变植株花粉中淀粉粒含量显著增加、且花粉外壁表面附着物明显增多，暗示ZmCP03可能参与玉米花粉中淀粉合成的调控，且影响花粉表面结构。.ZmCP03缺失植株的花粉活性显著高于过表达植株的花粉活性；离体萌发0.5~1.5 h的花粉管长度显著长于对照，表明该基因影响花粉萌发及前期生长。ZmCP03敲除植株的花粉中淀粉粒显著增加，结实率显著提高，暗示ZmCP03在玉米花粉发育及有性生殖过程中可能起负调节作用。.上述研究结果为分子调控玉米花粉育性、改善玉米生产上授粉效率低的问题提供了线索。发表相关成果 SCI 论文6篇(其中影响因子>5.0的2篇、>3.0 的3篇),培养毕业博士生3名、硕士生3名。