FG1调控花粉管珠柄导向的分子机理

In angiosperms, the immobile sperm cells are delivered to the embryo sac by the pollen tubes which respond to the attractants secreted from the embryo sac to complete double fertilization. The delivery process that is guided by different female tissues is called pollen tube guidance. Multistep controls of pollen tube guidance can be separated into two steps: preovular guidance and ovular guidance. The ovular guidance refers to the pollen tube emergence from the transmitting tract, migration on the septum, and growth on the surface of the funiculus and then to the micropyle to contact the embryo sac. The process from the transmitting tract to the funiculus is called funicular guidance and that from the funiculus to the micropyle is micropylar guidance. In the past years, the embryo sac-derived peptides attracting pollen tubes and their regulation have been reported to regulate micropylar guidance. However, although theoretically funicular guidance is proposed for more than two decades based on the phenotypes of several ovule mutants, no direct molecular evidence has been unveiled. In previous work, we found that a new transcription factor-encoding gene FG1 exhibits dramatic down-regulation in pollen tube guidance mutant central cell guidance (ccg). Surprisingly, FG1 is predominantly expressed in on the funiculus. Although fg1 loss-of-function mutants exhibit morphologically normal embryo sac and funiculus structure, they display high proportion of ovule abortion due to failed funicular guidance. We aim to figure out the molecular mechanism of FG1 in regulating funiculus development and funicular guidance. Meanwhile, we will explore how spatially and temporally the embryo sac and funiculus crosstalk to guide the pollen tube through dissecting the interrelation between CCG and FG1. This study will fill in the gap of our understanding of funicular guidance and provide evidences to a new layer of regulation of male-female interaction.

被子植物的精细胞不能移动，需依赖花粉管导向过程将其传递到胚囊。花粉管穿出引导组织，爬上珠柄的过程为珠柄导向；之后，从珠柄进入珠孔的过程则为珠孔导向。虽然，胚囊分泌的珠孔吸引信号及其调控已有报道，但珠柄在导向过程中的作用机制，以及珠柄导向的吸引信号的性质一直处于猜测阶段，缺少直接的分子证据。我们的前期工作发现，在中央细胞中表达的CCG突变后，可以同时破坏珠柄和珠孔导向。通过转录组分析发现，一个转录因子编码基因FG1的表达量在ccg中明显下调，但该基因在珠柄中优势表达，且呈动态定位。fg1敲除突变体胚囊可以发育至成熟期，但表现为珠孔导向失败。本项目将以上述研究基础为切入点，旨在研究FG1调控珠柄导向的作用机理，并通过分析CCG与FG1间的调控关系，探究胚囊和珠柄共同参与珠柄导向的空间调控机制。该结果将填补花粉管珠柄导向的研究空白，为受精过程中雌雄互作存在的又一环节的调控提供证据。

被子植物的精细胞不能自主运动，需依赖花粉管将其传递到胚囊。花粉管正确进入胚囊需受到来自不同组织的引导，即花粉管导向过程。花粉管穿出引导组织，爬上珠柄的过程为珠柄导向；花粉管离开珠柄表面进入珠孔的过程则为珠孔导向。目前，珠孔导向的调控因子已有诸多报道。而珠柄导向的概念虽已提出二十余年，其调控机制仍不清楚。我们发现中央细胞特异表达的CCG突变后，花粉管的珠柄导向和珠孔导向同时被破坏。通过比较转录组发现，编码转录因子的基因FG1的表达量在ccg中显著下调, FG1-GFP融合荧光蛋白特异地在珠柄中表达。因此研究FG1在株柄导向过程中的功能和机制，有望为株柄导向的机制提供分子见解。主要研究结果如下：.1. 首先构建FG1的CRISPR/Cas9系统，在野生型中将FG1进行敲除。经多代筛选得到3个稳定遗传的编辑系。统计结实率发现，fg1的育性正常。观察并统计不同授粉时间的花粉管导向发现，与野生型相比，fg1的花粉管导向效率无显著差异。以上结果表明FG1单个基因的敲除，不会引起任何生殖异常，需要构建高阶突变体。我们通过构建系统发育树发现，FG1与FG2同源性较高，且是双子叶植物所特有的。这表明FG1/FG2是在物种演化过程中新产生的，它们可能参与双子叶植物珠柄特有的形态发育和功能调控。.2. 利用FG1pro:FG1-GFP植株观察FG1-GFP的定位发现，FG1特异地在珠柄高表达，其表达模式是一个动态变化过程。从花发育第10期到第15期，FG1的信号会出现从无到有，再由强转弱的变化过程。我们还发现，在FG1的表达早期，珠被上也有较弱的信号。.3. 在观察FG1动态变化过程时，我们发现FG1早期主要在细胞质表达，随后会迁移入核，同时在细胞质和细胞核表达。随后，我们观察了FG1在去雄和30℃逆境情况下的表达，发现其能正常入核。用中央细胞缺陷突变体ccg和胚囊发育缺陷突变体spl观察，发现FG1的表达模式亦未出现异常。另外，剪去柱头实验证明FG1是否入核与柱头毛发育状态无关。.4. 酵母文库筛选和酵母双杂实验发现其与AtVDAC家族能发生体外互作。

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