细胞壁蛋白NtProRP1对烟草早期胚胎发生的作用与调控机制研究

Early embryogenesis is the starting point of higher plants’ development. Therefore, the regulatory mechanism underlying the process has been a matter of great concern in this field. Recently, signals that located in the extracellular microenvironment of the embryo were proofed to be obviously effective on early embryogenesis. However, the nature of extracellular signals and their regulatory mechanisms involved in early embryogenesis were largely unknown. In our previous work, a protein called NtProRP1 was verified elementarily to be secreted to the extracellular space of the pollen tube, and also it highly expressed in the zygote to regulate the pattern formation of the early embryo. Thus it provided a unique opportunity to seek the role of extracellular signals in early embryogenesis. In this work, we attempt to: (1) further investigate the expression pattern and the secretory of NtProRP1 in the zygote and early embryo, and the secretory pathway of NtProRP1; (2) clarify the mechanism that NtProRP1 regulates early embryogenesis by functional analysis of its mutant with diverse techniques; (3) discuss the significance of NtProRP1’s secretory in early embryogenesis by its mutant complementation with ectopic expression of NtProRP1 or NtProRP1 without a signal peptide; (4) confirm the predicted upstream regulator of NtProRP1. These results will verify a new type of extracellular signal involved in early embryogenesis, and then be benefit to extensive understanding of the role of extracellular microenvironment in early embryogenesis.

早期胚胎发生是高等植物发育的起点，其调控机制历来备受关注。最近的研究证实胞外微环境中的信号分子对早期胚胎发生具有明显的调控作用。但相关胞外信号具体包含哪些类型？涉及的调控机理有哪些？迄今尚无明确答案。前期工作初步显示在花粉管中具有分泌性的烟草细胞壁蛋白NtProRP1可同时在合子中高量表达，并影响早期胚胎的模式建成，为研究胞外信号调控早期胚胎发生提供了绝佳的切入点。本项目拟：(1)进一步确认NtProRP1在胚胎发生中的表达模式、分泌性和分泌途径；(2)通过突变体功能分析，详细探究NtProRP1在早期胚胎发生中的具体作用；(3)突变体背景下，异位表达、去信号肽后表达NtProRP1以探讨其分泌性对早期胚胎发生的影响；(4)验证推测的NtProRP1上游调控因子。本课题的完成将证实一种调控早期胚胎发生的新型胞外信号分子，并对全面理解胞外微环境对早期胚胎发生的作用提供有价值的实验证据。

近年来的研究已发现诸多胞内蛋白参与高等植物早期胚胎发生，而胞外微环境中的信号分子通过何种途径调控早期胚胎发生，到目前为止所知甚少。细胞壁作为胞外基质的重要组成部分，参与塑造了胞外微环境。因此，对来自细胞壁的胞外信号进行研究对于完整了解胞外微环境中的胞外信号调控植物早期胚胎发生的分子机制而言是一个很好的切入点。. 在前期的实验中我们发现了一种烟草细胞壁蛋白NtProRP1。我们依托业已获得的NtProRP1基因主要做了以下几个方面的工作：(1)验证NtProRP1基因在早期胚胎发生时期的表达模式，利用NtProRP1的GFP融合蛋白确认其在早期胚胎中的亚细胞定位；(2)创制NtProRP1基因的突变体植株并分析其表型，提取突变体早期胚胎的转录组进行高通量分析，分析NtProRP1基因调控早期胚胎发生的分子机制；(3)对在转录组中出现的一些差异显著基因进行功能分析；(4)验证NtProRP1基因的上游调控因子。. 实验结果表明：(1)在早期胚胎发生中NtProRP1是一种胚胎特异的基因，且其编码的蛋白定位在胚胎细胞壁上发挥作用；(2)NtProRP1的突变体植株的2胞原胚表现出顶基轴向的异常偏折；(3)分离突变体植株2胞原胚和野生型植株2胞原胚进行转录组高通量分析，发现突变体的转录组中细胞壁多糖相关的合成和修饰基因几乎一致性上调或下调，暗示了突变体2胞原胚的表型极可能是细胞壁成分和结构的变化所导致；(4)发现核糖体蛋白基因和热激蛋白基因是转录组中差异表达最多的两类基因。对拟南芥中的同源基因进行分析，发现核糖体蛋白基因AtRPL14B和热激蛋白基因AtHSP90.6的突变体中都存在胚胎异常发育；(5)酵母单杂实验表明与渗透压应答相关的转录因子Tsi1并非设想的NtProRP1基因的上游调控因子。对NtProRP1基因的上游调控机制还需进一步探索。. 由此本研究证实烟草细胞壁蛋白NtProRP1作为一种新型胞外信号参与调控了早期胚胎发生，并阐明了其作为胞外信号调控早期胚胎发生的分子机制。研究结果为全面、完整的了解胞外微环境中的胞外信号分子调控植物早期胚胎发生的途径与机理提供了线索。