兜兰PaCSE在木质素合成代谢及种子萌发中的功能研究

Paphiopedilum is an important genus of flowering plant family Orchidaceae with high horticultural value. The wild populations are under the threat of extinction because of over-collection and habitat destruction. Paphiopedilum seeds are tiny and contain no endosperm. In the natural habitat, Paphiopedilum seeds are generally unable to germinate on their own. They need to engage in a mycorrhizal relationship with a fungus that helps to feed the emerging seedling. Even feeding enough nutrient supply in asymbiotic germination, mature seeds of most Paphiopedilum species are still difficult to germinate, which severely restricts the germplasm resources protection and commercial production. Our preliminary studies suggested that C lignin and H lignin were synthesized during embryo development, and their deposition was strongly related to the germination inhibition process. However, the mechanism of how this specialized lignin inhibits germination and its biosynthetic machinery remains unclear. We found a potential key gene involved in the lignin synthesis through transcriptome analysis. In this proposal, we are going to investigate the biochemical function of PaCSE and its effect on seed mechanics、permeability and other germination related parameters. The results from this study will provide theoretical guidance for conservation and commercial production of this endangered species.

兜兰观赏价值高，繁殖难度大，人工过度采挖和生境破坏导致许多野生种濒临灭绝。在野外条件下需与真菌共生才能萌发，且萌发率低。即使在提供充足营养的无菌播种条件下，大部分兜兰成熟种子依然萌发困难，这成为制约兜兰种质资源保护和规模化生产的瓶颈。我们的前期研究结果表明杏黄兜兰种子中存在结构特殊的H型和C型木质素，其积累量与萌发率存在巨大的相关性。本项目通过转录组测序筛选到可能参与调控木质素合成的关键基因-PaCSE。在前期工作的基础上，将通过在拟南芥、苜蓿和兜兰中进行转基因功能验证和PaCSE酶活分析进一步研究PaCSE在兜兰种子木质素合成代谢中的作用，明确其对种子的机械强度、通透性等与萌发相关特性的作用，从而通过调控PaCSE的表达来部分或完全解除萌发的抑制。本项目的实施不仅在兜兰种子萌发和木质素调控机制的基础研究方面会有突破，也对兜兰种苗的繁殖和生产有重要的指导意义。

兜兰种子萌发困难是制约其种质资源保护和商品化生产的瓶颈。兜兰种子成熟后木质素开始大量合成，其积累量与萌发率存在显著的相关性。经过3年研究，本项目获得以下结果：（1）兜兰种子萌发是一个复杂的生物学过程，受到内源激素和木质素共同调控。成熟兜兰种子难萌发的主要原因是非甲基化的木质素的积累引起的种子物理休眠。（2）PaCSE作为参与兜兰木质素单体合成的重要酶之一，在种皮木质化过程中发挥重要作用。通过基因功能互补实验分析，明确PaCSE通过调控种皮非甲基化木质素的合成来影响种子萌发速度和通透性。（3）对PaCSE开展了酶学性质研究，结果表明PaCSE具有较强的咖啡酰莽草酸酯酶的催化能力。对于底物咖啡酰莽草酸， PaCSE的Km和Vmax值分别为67.5 uM和17.8 nM min-1， Kcat/Km=0.0072 s-1uM-1 。结合兜兰体内酶活检测实验的结果，表明PaCSE在体内和体外均具有较强的催化功能。此外，在本项目的开展过程中，还优化了兜兰遗传转化体系，发表SCI研究论文3篇，申请并获得授权国家专利1项，培养研究生3名。本项目的实施不仅丰富了兜兰种子萌发机制和木质素合成调控机制的理论研究，也对兜兰种苗繁殖和生产具有重要的指导意义。