植物整合子调控花粉发育机制的研究。

The male gametophyte (pollen) development of flowering plant is a critical step for completing life cycle, expression of genes and regulation network in the developing pollen has been well studied. Although it would appear that a large number of genes are involve in pollen development from studies in the literature, unfortunately, our knowledge about pollen development including the regulation mechanism is not profound..The integrator was generally believed to be involved with the procession and maturation of snRNA. Beside its function in snRNA, the emerging reports about impact of integrators on various regulation processes has aroused great attention over the last decade, and has become one research hotspot. None of integrator genes was identified as being involved in male gametophyte development, and our results demonstrate a novel critical role of two plant integrators in pollen development. It will be of interest to identify the functions of those pollen-related integrator genes, and this project intends to solve the following problems: defining the defective stage or subcellular organ during pollen development of mutants; identifying the synthesis stage and localization of integrators protein at different periods of pollen development; verifying whether the pollen defective phenotype was caused by accumulation of pre-snRNA/snRNA, comparing pre-snRNA/snRNA of pollen in mutant and wild type plant; performing chromatin immunoprecipitation (ChIP)-sequencing to analysis the target genes of integrator and further investigating their relationship with known pollen regulation network; investigating the interacted protein with integrator using Co-IP-MS and yeast two hybridization; identification of transcriptome changes in mutants pollen by deep sequencing. .With the regulation network of integrator during pollen development, this project likely to further our understanding of pollen development in the foreseeable future and uncovers a previously unknown function of integrator.

花粉作为雄配子是植物完成有性繁殖必要的一环，一直以来花粉发育的研究就备受关注。以往研究表明花粉发育受到多方面因素影响，而至今没有关于整合子与花粉发育有关的报道。整合子起初被认为只参与snRNA的生物合成，但近几年不断发现其在生命发育过程中的新功能，并逐渐引起关注。在植物整合子研究过程中，本项目申请者首次发现整合子影响花粉发育，这将是关于花粉发育调控途径的新发现。本项目拟研究以下科学问题：研究整合子突变导致花粉发育异常的具体阶段或具体器官；分析整合子在花粉不同发育阶段中的时空表达特性；解析突变体花粉中snRNA/pre-snRNA含量变化对花粉发育的影响；寻找植物整合子在花粉中的互作因子以及受其调控的靶基因；描绘植物整合子对于花粉转录组的整体影响。最终揭示植物整合子对花粉发育调控的作用机理和网络，延伸对整合子功能的认识。

花粉作为雄配子是植物完成有性繁殖必要的一环，花粉发育的影响因素被不断的研究报道，但是至今没有关于整合子与花粉发育有关的研究。整合子主要功能是参与细胞核小（snRNA）的生物合成，但近几年不断发现整合子的新功能。我们在之前的研究中发现植物snRNA合成相关的DSP1整合子复合体包括至少5个成员，他们对植物生长发育至关重要。但是他们之间的协作关系并不清楚。在本项目研究中发现整合子DSP4与DSP1相互作用，并且DSP4基因突变会影响诸多植物功能异常，包括snRNA转录和成熟异常，植物雄孢子发育异常及花粉育性降低。同时，dsp1 dsp4的双突变体导致更严重的植物表型，包括更低的snRNA转录和成熟活性，以及完全的花粉败育现象，同时我们发现snRNA的剪切位点在双突变体中发生错误的位移。我们发现DSP4在植物的雄性孢子中存在特异表达，但是在雌性孢子中没有表达，这与DSP4影响花粉发育而不影响雌配子发育的现象相吻合。我们的研究结果表明DSP4是DSP整合子复合体中的重要成分，并且影响了snRNA的成熟效率。同时发现DSP1和DSP4在功能上存在协同效应，他们共同促进snRNA的转录合成以及加工成熟，同时它们在花粉发育中起到重要功能。