AtNMD1和AtNMD2共同调控拟南芥雄配子体发育的分子机理研究

In higher plants, the male gametophyte development is one of the crucial steps for sexual reproduction, which is not only important for reproductive biology but also for plant breeding. Although various important genes involved in the male gametophyte development have been reported, the molecular mechanism of gene transcriptional regulation controlling this process is still unclear. Using CRIPSR/Cas9 genome editing approach, we obtained mutants for both AtNMD1 and AtNMD2, all showing male gametophyte development defects. Furthermore, the transgenic plants of RNAi for both genes also displayed similar fertility defects with their genome edited mutants, individually. The above results strongly suggested that both AtNMD1 and AtNMD2 are essential for the male gametophyte development. BiFC experimental data showed that AtNMD1 and AtNMD2 can interact in tobacco cells, indicating that they might form a complex to regulate the male gametophyte development. We also found that the AtNMD1 gene was highly expressed in pollen; and AtNMD1 protein was also located in pollen. Moreover, we detected that the expression level of various genes involved in pollen wall formation is significantly reduced in Atnmd1, implying that AtNMD1 might regulate them as downstream genes. Here，we planned to employ transcriptome, proteome, Co-IP and ChIP-seq approaches, combined with molecular genetics and cell biology analyses to comprehensively investigate the molecular mechanism of gene regulation participated by AtNMD1 and AtNMD2 during the male gametophyte development.

高等植物雄性配子体发育是有性生殖过程中的关键环节之一，对生殖发育和遗传育种极其重要。虽已发现多个影响雄配子体发育的基因，但调控其发育的基因转录机制还不清楚。我们发现利用CRIPSR/Cas9编辑系统获取的AtNMD1和AtNMD2突变体均表现出雄配子体发育缺陷表型,而且它们的RNAi转基因植株也具有相似的配子体缺陷表型，表明AtNMD1和AtNMD2为雄配子体发育所必需。双分子荧光互补实验证明它们在植物细胞体内能相互作用，提示这两个蛋白可能形成复合体共同调控配子体发育过程。AtNMD1主要在配子体中表达，其蛋白定位在花粉中。多个与花粉壁发育相关的基因在Atnmd1中表达下降，表明花粉壁相关基因位于其下游。本项目拟通过转录组、蛋白组、Co-IP、ChIP-seq等方法，并结合分子遗传和细胞生物学的多种研究手段，全面深入的揭示AtNMD1和AtNMD2共同参与雄配子体发育的基因转录调控机制。

在开花植物中，雄配子的育性对有性生殖过程的顺利完成至关重要，成熟的花粉细胞壁主要包括外层外壁和内层内壁，完整的花粉细胞壁是雄配子保持育性必不可少的部分。由于外壁坐落在最外层便于观察与研究，花粉外壁发育的相关机制目前研究得较为清楚。而完整的内壁对花粉的育性及生存起到关键作用，同时也是承担着保障成熟花粉的活性及花粉管的萌发的任务，但花粉内壁发育的分子机制还存在大量的空白。在本研究中，我们鉴定到一个新的拟南芥雄配子体缺陷基因AtNMD1（ARABIDOPSIS NOVEL MICROGAMETOPHYTE DEFECTIVE MUTANT 1），当AtNMD1发生突变或者表达量下降均会引起雄配子发育缺陷。AtNMD1编码一个被注释为转录共激活因子的蛋白，其在花药第10至12期的花粉中的表达水平较高。AtNMD1蛋白主要定位在单核小孢子细胞核中，随后特异性地定位在花粉的营养细胞核中，这暗示AtNMD1在转录调控活动中具有重要的作用。通过对AtNMD1不同等位突变类型Atnmd1-3和Atnmd1-5的遗传分析，我们发现AtNMD1是一个雄配子致死基因。在AtNMD1+/-突变体和AtNMD1 RNAi材料中，钙荧光白染色方法和透射电子显微镜分析显示花粉外壁发育正常，花粉的凋亡是由花粉内壁的发育异常导致的。转录组数据分析显示，与野生型相比，在突变体Atnmd1-5/+花药组织中有705个存在显著性的差异表达基因，其中328个基因表达下调，377个基因表达上调。基因功能富集分析表明大部分表达显著下调基因主要富集在细胞间信号途径和细胞壁修饰进程两大类上。qRT-PCR和转录组分析显示与花粉内壁发育相关的阿拉伯半乳糖蛋白及果胶甲酯酶类基因的表达显著下调。我们进一步利用免疫沉淀质谱分析方法从含AtNMD1-FLAG融合蛋白的转基因材料花序中鉴定到247个蛋白，这些蛋白显著地富集到细胞内相关的生物学进程。并从中筛选出另一个被注释为转录共激活因子AtNMD2，发现其能与AtNMD1在花粉细胞核中互作。结合转录组测序及免疫沉淀质谱分析法初步揭示了AtNMD1与AtNMD2形成异源二聚体调控花粉内壁发育相关的分子机理，拓宽了对花粉内壁发育的分子机制的理解，为将来对花粉内壁发育的进一步探究奠定了基础。