巨龙竹茎秆发育的呼吸代谢机制研究

The mechanism of the rapid development of bamboo culms has not been clearly revealed until now. Bamboo culms during development do not form leaves, and was covered by a layer of sheath, which is etiolated and lack of the autotrophic photosynthesis. Its nutrients are mainly originated from other photosynthetic tissues. Thus, respiratory metabolism plays a decisive role in the culm development. So, in-depth study of regulation process of glycolysis, citric acid cycle and oxidative phosphorylation involved in respiratory metabolism during culm development will be a breakthrough to reveal the problem of culm development. Dendrocalamus sinicus is the largest bamboo species in the world. In this project, bamboo culm material collected from different developmental stages was investigated based on comparative proteomics methods. Firstly, the proteins of chloroplast stoma and mitochondria were separated by 2D-PAGE and BN-PAGE, respectively. Then, MALDI-TOF/TOF-MS and HPLC-ESI-MS/MS were used to identification of proteins. The differential expressed proteins that located at chloroplast stoma and involved in glycolysis, and that located at mitochondria and related to citric acid cycle and oxidative phosphorylation were analyzed. Also, we have got some related data of transcriptome and differential expression profile before this study. Finally, the all data that come from proteome and transcriptome were integrated to get functional proteins/or genes that regulate the respiratory metabolism. Thus, the mechanism of respiratory metabolism during culm development of Dendrocalamus sinicus was disclosed, which will provide theoretical basis for other studies of bamboo development.

目前，竹子茎秆快速发育机理仍未被阐明。竹秆在发育过程中还未形成叶片，被竹鞘包被呈黄化状态，不具备光合自养能力，其营养物质主要源自其他光合组织，由此呼吸代谢在整个竹秆发育过程必然起到决定性作用。所以，深入研究竹秆发育过程中与呼吸代谢相关的糖酵解、三羧酸循环和氧化磷酸化的调控过程是解读竹秆发育的一个突破口。巨龙竹（Dendrocalamus sinicus）是世界上最高大的竹种。本研究拟以不同发育阶段的巨龙竹茎秆为试材，基于比较蛋白组学手段，采用双向凝胶电泳和蓝绿温和胶凝胶电泳分别对叶绿体基质蛋白和线粒体蛋白分离，经质谱鉴定，对定位于叶绿体基质参与糖酵解、定位于线粒体参与三羧酸循环和氧化磷酸化的差异表达蛋白进行分析，最后把蛋白组和已有的转录组、表达谱数据耦合，以期获得参与和调控巨龙竹茎秆呼吸代谢的功能基因/蛋白，揭示巨龙竹茎秆发育过程中呼吸代谢机制，为其他竹子发育研究提供理论依据。

目前，竹类植物茎秆发育的机制有待于阐明。竹秆在发育过程中还不具备独立的光合自养能力，其营养物质来源主要是从其他光合组织获得，呼吸代谢在整个竹秆发育过程必然起到决定作用。因此，研究竹秆发育过程中与呼吸代谢相关的糖酵解、三羧酸循环和氧化磷酸化的调控过程是解读竹秆发育的一个突破口。巨龙竹（Dendrocalamus sinicus）是世界上最高大的竹种。本研究以不同发育阶段的巨龙竹茎秆为试材，基于比较蛋白组学手段，采用双向凝胶电泳和蓝绿温和胶凝胶电泳分别对叶绿体基质蛋白和线粒体蛋白分离，经质谱鉴定，对定位于叶绿体基质参与糖酵解、定位于线粒体参与三羧酸循环和氧化磷酸化的差异表达蛋白进行分析，随后把蛋白组和已有的转录数据耦合，另外结合酶活测定和基因表达分析。酶活测定结果表明，参与淀粉合成的腺苷二磷酸葡萄糖焦磷酸化酶、淀粉合成酶、淀粉分支酶在不同茎秆节间存在显著的丰度差异，表明茎秆发育过程中涉及到大量的淀粉代谢。在蛋白水平，双向电泳结合飞行时间质谱技术鉴定到36个差异表达蛋白，通过功能分类，最大的类为能量代谢（27.78%）、其次为中间代谢（22.22%），表明碳水化合物代谢在茎秆发育中发挥了重要作用。尤其是四个糖酵解途径中的差异蛋白显著表达。另外，一个原叶绿素酸酯还原酶被鉴定到，它与茎秆叶绿素合成有关，这表明茎秆属于光合组织，木质组织光合作用参与了茎秆发育。在成熟茎秆中苹果酸脱氢酶显著上调，它广泛的调控了茎秆能量代谢。一些基因出现转录和蛋白水平不同步变化，表明茎秆发育中涉及到大量的转录后修饰事件。同时我们构建了差异蛋白的互作网络，发现Os10g0483400蛋白处在中心位置，这暗示其在茎秆发育过程中的独特作用。本研究获得参与和调控巨龙竹茎秆呼吸代谢的功能蛋白，揭示了巨龙竹茎秆发育过程中呼吸代谢机制，并为其他竹类植物发育研究提供理论依据。