一氧化氮参与氢气诱导万寿菊不定根发生的机理研究

The formation of adventitious roots is a common vegetative propagation practice in forestry, horticulture, and fruit crops, which is a complex genetic process regulated by both environmental and endogenous factors. Adventitious root formation has many practical implications in horticulture and agronomy and there is a lot of commercial interest.It has now become well accepted that hydrogen and nitric oxide may have key roles to play in the signalling that takes place in plant cells. The signal roles of hydrogen and nitric oxide in plants become a hot topic in the academic community of the biological study. Our previous results found that both hydrogen gas and nitric oxide may induce adventitious root development in plants. However, the signal interation between them and its mechanism involved are not elucidated. On the basis of our previous studies, the signal roles of nitric oxide in hydrogen-induced adventitious rooting will be observed and analysized systematically by morphological, anatomical and pharmacological studies. In addition, with the methods of physiology, cell biology and molecular biology, the changes of cell cycle phase, antioxidant enzymes system, endogenous hydrogen and nitric oxide, and representative target genes related to adventitious rooting will also be studied, in order to reveal the mechanism of biochemistry and molecular during hydrogen-induced adventitious rooting involving the nitric oxide pathway. Expected results of the project will provide some academic reference to the study of plant growth and development regulated by hydrogen and nitric oxide, expand and enrich the knowledge of the mechanism of adventitious root formation in plant, and also provide theoretical basis for the application of hydrogen and nitric oxide in agricultural production.

不定根途径是园艺植物和林木生产中苗木快速繁育的重要方法，对农、林和园艺业的发展具有重要经济价值。氢气和一氧化氮在植物中的信号分子功能研究是当前国际生物学界的热点。我们前期研究发现氢气和一氧化氮均能诱导植物不定根发生，但两者的信号互作及其机制尚不清楚。本研究拟以万寿菊（Tagetes erecta L.）为试材，运用形态解剖学和药理学的方法系统观察和分析一氧化氮在氢气诱导万寿菊不定根发生中的信号功能；在此基础上，采用生理学、细胞生物学和分子生物学的方法研究一氧化氮参与氢气诱导不定根发生过程中细胞周期时相、抗氧化酶系统、内源氢气和一氧化氮以及生根靶基因表达等的变化，以揭示不定根发生中一氧化氮参与氢气信号过程的生理生化和分子生物学机制。为氢气和一氧化氮信号调控植物生长发育研究补充新资料，拓展对植物不定根发生调控机制的认识，以期为氢气和一氧化氮在不定根发生等农业生产实践中的应用提供新资料和新思路。

通过一系列的生理生化和分子生物学的实验，研究了氢气（H2）和一氧化氮（NO）在不定根形成中的作用及其关系。取得主要结果与结论：.1. 50%的富氢水（HRW）可以促进不定根的发生； 50 μM的NO供体（SNP）可以显著促进不定根的发生。此外NO清除剂cPTIO和NO抑制剂L-NAME和NaN3可抑制H2诱导不定根的形成，说明内源NO参与H2诱导植物不定根发生。.2. H2通过保护膜的通透性降低气孔开度增加水分含量，从而为不定根的发生过提供充足的水分供给。H2通过调节可溶性糖、淀粉和可溶性蛋白的含量变化，为不定根的形成提供充足的物质和能量需求。.3. H2通过调节POD、PPO和IAAO的活性诱导万寿菊不定根根原基的形成。并且NO在不定根形成过程中参与H2对POD、PPO和IAAO活性的调控。.4. H2诱导NOS-like和NR活性的表达，同时具有浓度和时间依赖效应。然而L-NAME抑制NOS-like活性的增加，NaN3抑制NR活性的增加。H2增加NOS-like和NR基因的表达水平，说明H2从活性和转录水平调节NOS-like和NR基因表达诱导不定根发生。.5. H2和NO处理激活细胞周期，细胞从G1期向S期转移，同时H2和NO也诱导细胞周期相关基因CycA、CycB、CDKA和CDKB基因的表达。cPTIO，L-NAME和NaN3逆转H2对细胞周期时相和相关基因的表达诱导。H2和NO增加生根靶基因CsCDNAJ-1、CsCDPK1和CsCDPK5的表达，而cPTIO，L-NAME和NaN3抑制H2的诱导作用，说明内源NO参与H2对生根靶基因表达的调节。.6. H2和NO共处理的质膜H+-ATPase和14-3-3蛋白的蛋白表达和相关基因表达均显著高于H2和NO单独处理；H2+cPTIO处理可以逆转NO对质膜H+-ATPase和14-3-3蛋白的蛋白表达和相关基因表达的效果。说明NO通过调控质膜H+-ATPase和14-3-3蛋白的蛋白表达和相关基因表达来参与H2诱导不定根发生。.7. H2和NO共处理的质膜H+-ATPase和14-3-3蛋白的蛋白互作程度均显著高于H2和NO单独处理；cPTIO可以逆转NO对质膜H+-ATPase和14-3-3蛋白的蛋白互作程度。可见，NO通过调控质膜H+-ATPase和14-3-3蛋白的蛋白互作来参与H2诱导不定根发生。