在开花植物中，精细胞与营养核连接以雄性生殖单位的方式随花粉管的生长进行迁移，最终到达胚珠完成双受精，这对于有性生殖至关重要。然而在这个过程中，精细胞迁移的动力学机制，目前仍不清楚。实验室前期研究发现：小G蛋白AtRAN1定位于花粉营养核核膜，AtRAN1的突变导致雄性生殖单位迁移异常，与文献报道的植物LINC复合体突变的表型较为相似。由此推测AtRAN1可能与LINC复合体协同调控花粉雄性生殖单位的迁移，因此本文利用多种实验方法对AtRAN1与LINC复合体之间的互作关系进行了深入分析。

为了验证AtRAN1与LINC复合体成员AtWIP1以及AtWIT1之间是否存在直接相互作用，本论文进行了Pull Down以及烟草萤火素酶互补实验，发现AtRAN1与AtWIP1以及AtWIT1之间存在着直接的相互作用。为了进一步确定AtWIP1与AtRAN1互作的关键结构域，对AtWIP1蛋白进行截短，并分别与AtRAN1进行互作验证，发现只有coiled-coil结构域与AtRAN1存在相互作用。之后，进一步探究了AtRAN1的活性形式是否影响其与AtWIP1以及AtWIT1结合，结果发现AtRAN1的组成型激活形式（AtRAN1Q72L）与显性抑制形式（AtRAN1T27N）都可以与AtWIP1和AtWIT1结合，说明这种结合不受AtRAN1活性形式的影响。本研究使用一系列体外生化实验，证明AtRAN1可以直接结合AtWIP1以及AtWIT1，且不受AtRAN1活性形式的影响。暗示着在花粉管中，AtRAN1可能与LINC复合体协同作用，共同调控雄性生殖单位的迁移。

花粉萌发与花粉管极性生长对于开花植物有性生殖至关重要。实验室前期研究发现：拟南芥Formin家族蛋白AtFH5在花粉萌发过程中发挥关键作用。细胞学和遗传学数据发现：AtFH5定位于分泌囊泡并促进微丝组装，微丝组装驱动AtFH5定位的分泌囊泡在花粉粒中定向运输，进而决定花粉萌发位置。然而目前关于AtFH5介导的微丝组装及其推动分泌囊泡定向运输的动力学机制，仍不清楚。鉴定参与该动态的调控蛋白、解析其分子机理，对于理解花粉细胞中微丝驱动分泌囊泡的动态过程具有重要的理论价值。

借助于邻近标记技术可以探究活细胞生理条件下膜蛋白的互作蛋白以及邻近蛋白、检测瞬时蛋白互作的特点，我们首先构建了带有TurboID标签的AtFH5-TbID载体，获得拟南芥稳定转化株系，通过生物素处理以及Western Blot检测，发现在AtFH5-TbID样品中存在特异性生物素标记条带，说明AtFH5-TbID可以响应外源生物素处理，进行互作蛋白标记。虽然目前未能获取大量样品进行质谱检测，但是该实验的前期方法建立和优化为之后寻找参与调控微丝组装以及推动分泌囊泡定向运输的蛋白奠定基础。

In flowering plants, sperm cells connected to the vegetative nucleus migrate with the growth of pollen tube in the form of male germ unit, and finally reach the ovule to complete double fertilization, which is critical for sexual reproduction. However, the kinetic mechanism of sperm cells migration in this process is still unclear. Previous laboratory studies found that the small G protein AtRAN1 is located on the nuclear membrane of pollen vegetative nucleus. The mutation of AtRAN1 lead to abnormal migration of male germ unit, which is similar to the mutation of plant LINC complex reported in the literature. Therefore, it is speculated that AtRAN1 and LINC complex may regulate the migration of male germ unit together. So, we analysis the interaction between AtRAN1 and LINC complex deeply by various experimental methods.

In order to verify whether there is a direct interaction between AtRAN1 and LINC complex members AtWIP1 and AtWIT1, Pull Down and tobacco luciferase complementarity experiment were carried out in this paper. It was found that there is a direct interaction between AtRAN1 and AtWIP1 and AtWIT1. In order to further determine the key domain of the interaction between AtWIP1 and AtRAN1, the AtWIP1 protein was truncated and verified with AtRAN1 respectively. It was found that only the coiled-coil domain interacts with AtRAN1. After that, we further explored whether the active form of AtRAN1 affects its binding to AtWIP1 and AtWIT1. The results showed that both the constitutive activation form (AtRAN1Q72L) and the dominant negative form (AtRAN1T27N) of AtRAN1 could bind to AtWIP1 and AtWIT1, indicating that this binding is not affected by the active form of AtRAN1. This study used a series of biochemical experiments in vitro to prove that AtRAN1 can directly bind to AtWIP1 and AtWIT1, and is not affected by the active form of AtRAN1. It is suggested that AtRAN1 may cooperate with LINC complex to regulate the migration of male germ unit in pollen tube.

Pollen germination and pollen tube polar growth are essential for flowering plants. Previous laboratory studies found that Arabidopsis Formin family protein AtFH5 plays a key role in pollen germination. Cytological and genetic data show that AtFH5 is located at secretory vesicles and promotes the polymerization of actin filaments. In turn, the polymerization of actin filaments drives the directional transportation of AtFH5-localized secretory vesicles in pollen grains, and then determines the site of pollen germination. However, the kinetic mechanism of AtFH5 mediated actin assembly and the directional transport of secretory vesicles is still unclear. Identifying the regulatory proteins involved in this dynamic process and analyzing its molecular mechanism have important theoretical value.

With the help of proximity labeling technology, we can explore the interacting proteins and adjacent proteins of membrane proteins under the physiological conditions of living cells, and detect the transient protein interactions. Firstly, we constructed the AtFH5-TbID vector and obtained the stable transformation strains of Arabidopsis thaliana. Through biotin treatment and Western Blot, we found some specific biotin labeled bands in AtFH5-TbID samples, indicating that AtFH5-TbID can respond to exogenous biotin and label interacting proteins. Although a large number of samples have not been obtained for mass spectrometry, the establishment and optimization of the preliminary method of this experiment lay the foundation for looking for proteins involved in regulating actin assembly and promoting the directional transport of secretory vesicles.