被子植物的精细胞不能自由运动，且与雌配子体的物理距离较远，需通过花粉管的生长将精细胞运送到雌配子体中完成双受精。因此精细胞在花粉管中成功迁移是被子植物完成双受精作用的前提，对其分子调控机理的深入研究具有重要意义。花粉中两个精细胞和营养核紧密连接在一起组成雄性生殖单位，当其连接被破坏时，精细胞在花粉管中的迁移受到干扰导致双受精不能顺利完成，说明雄性生殖单位的维持和迁移具有重要意义。目前LINC复合体是唯一被报道的定位于营养核核膜并在雄性生殖单位的维持和迁移中起重要作用的调控因子，但具体分子调控机理有待深入研究。AtRAN1和AtRAN2是拟南芥小G蛋白RAN家族的成员，在花粉中高表达。本实验室前期研究发现，AtRAN1与LINC复合体成员存在互作，暗示了AtRAN1可能参与调控雄性生殖单位的维持和迁移。

本研究通过CRISRP Cas9技术构建了双突变体ran1ran2，观察到ran1ran2中植株育性显著下降，通过正反交实验进一步验证了育性受损表型与雄配子体和雌配子体有关。之后深入分析了ran1ran2花粉中雄性生殖单位的迁移动态，在花粉长时间培养5小时后，观察到在花粉管存在暂停生长一段时间然后恢复生长的现象，在此期间雄性生殖单位会表现出剧烈向前、向后的振荡运动，而ran1ran2的雄性生殖单位与野生型相比表现出更紊乱的运动状态，营养核与花粉管顶端最近和最远位置间的距离更长，振荡运动的次数更多、距离更长、速度更快，说明在ran1ran2中雄性生殖单位的稳定迁移受到了影响。之后进一步探究了AtRAN1活性对雄性生殖单位维持和迁移的影响，利用实验室保存的AtRAN1的组成型激活 (CA) 和显性抑制 (DN) 株系，观察到AtRAN1CA特异定位于营养核核膜上，而AtRAN1DN定位于胞质和营养核，其中AtRAN1CA会导致雄性生殖单位与花粉管顶端的距离显著增加，说明AtRAN1的活性状态影响雄性生殖单位在花粉管中稳定位置的维持。与此同时，本论文还探究了RAN的活性调节蛋白AtRanGAP1对雄性生殖单位迁移的影响，发现在rangap1和r1-4rangap1中，营养核与精细胞发生倒置的比例与野生型相比显著增加，但不影响植株育性，说明了AtRanGAP1可能参与调节雄性生殖单位的稳定迁移过程。此外还探究了AtRAN1与LINC复合体的互作关系，观察到在wit1wit2双突中，花粉粒中AtRAN1CA活性蛋白含量显著增加，其定位从核膜特异定位变成了胞质和细胞核的弥散定位，表明 WIT蛋白参与调控AtRAN1-GTP的含量和定位。

上述结果表明，AtRanGAP1以及受调控的AtRAN1、AtRAN2可能参与调控雄性生殖单位的维持和稳定迁移过程，为深入研究雄性生殖单位维持和迁移的分子机理提供了新的实验证据，对于深入了解植物的有性生殖以及细胞器的运动模式具有重要科学意义。

The sperm cells of angiosperms cannot move freely and are far away from the female gametophyte physically, so the sperm cells need to be transported to the female gametophyte through the growth of pollen tubes to complete double fertilization process. Therefore, the successful migration of sperm cells in pollen tube is the premise for angiosperms to complete double fertilization, and it is of great significance to further study its molecular regulation mechanism. The two sperm cells in the pollen are closely connected with the vegetative nucleus to form the male germ unit. When their connection is destroyed, the migration of sperm cells in the pollen tube is interfered, resulting in the failure of double fertilization, which indicates that the maintenance and migration of the male germ unit is of great significance. At present, the LINC complex is the only regulatory factor reported to be located in the nuclear membrane of the vegetative nucleus and plays an important role in the maintenance and migration of male germ unit, but the specific molecular regulatory mechanism needs to be further studied. AtRAN1 and AtRAN2 are members of Arabidopsis small G protein RAN family, which are highly expressed in pollen. Our laboratory's previous research found that AtRAN1 interacts with members of the LINC complex, suggesting that AtRAN1 may be involved in regulating the maintenance and migration of male germ unit.

In this study, we constructed a double mutant ran1ran2 through CRISRP Cas9 method, and observed that the plant fertility in ran1ran2 was significantly reduced, and further verified that the fertility impaired phenotype was related to male gametophyte and female gametophyte through reciprocal cross experiments. Afterwards, an in-depth analysis was conducted on the migration dynamics of male germ unit in ran1ran2 pollen. After long-term cultivation of pollen for 5 hours, it was observed that the growth of pollen tubes was suspended for a period of time and then resumed. During this period, male germ unit exhibited intense forward and backward oscillatory movements, while the male germ unit in ran1ran2 exhibited a more chaotic movement state compared to the wild type. The distance between the vegetative nucleus’s closest and farthest position to the pollen tube tip was longer. The oscillatory movements were more frequent, the distance was longer and the speed was faster, which indicates that the stable migration of male germ unit in ran1ran2 is affected. Subsequently, the impact of AtRAN1 activity on the maintenance and migration of male germ unit was studied. Using the constitutive activated (CA) and dominant negative (DN) point mutation plants of AtRAN1 preserved in the laboratory, it was observed that AtRAN1CA was specifically located on the nuclear membrane of the vegetative nucleus, while AtRAN1DN was located in the cytoplasm and vegetative nucleus. Compared with the wild type, AtRAN1CA significantly increased the distance between male germ unit and the tip of the pollen tube. The active state of AtRAN1 affects the maintenance of stable positions of male germ unit in pollen tubes. At the same time, this study also explored the effect of AtRanGAP1, the active regulator of RAN, on the migration of male germ unit. It was found that in rangap1 and r1-4rangap1, the proportion of inversion of vegetative nucleus and sperm cells significantly increased compared with the wild type, indicating that AtRanGAP1 may be involved in regulating the stable migration of male germ unit. In addition, the interaction between AtRAN1 and LINC complex was explored, and it was observed that in the wit1wit2 double mutant, the content of AtRAN1CA protein in pollen grains significantly increased. Its localization shifted from nuclear membrane specific localization to diffuse localization of cytoplasm and nucleus, indicating that WIT protein is involved in regulating the content and localization of AtRAN1-GTP.

The above results indicate that AtRanGAP1 and the regulated AtRAN1 and AtRAN2 may be involved in the process of regulating the maintenance and stable migration of male germ unit, providing new experimental evidence for in-depth research on the molecular mechanism of male germ unit maintenance and migration, and having important scientific significance for in-depth understanding of plant sexual reproduction and the movement mode of organelle.