不同的浮萍ADP葡萄糖焦磷酸化酶与淀粉快速积累的关系研究

ADP-glucose pyrophosphorylase (AGPase) is the first and the most important key enzyme involved in starch biosynthesis in plants. It is a heterotetramer consisting of two identical large subunits and two identical small subunits. Usually, there are various combinations of different large and small subunits, which will form different AGPases. Different AGPases have different spatial and temporal expression patterns. Duckweed is a new energy crop, developing of this crop can integrates the wastewater treatment with bioenergy development. Duckweed can accumulate high starch content to more than 45% (dry weight) in 7 d. However, the relationship between the AGPases and starch accumulation has not yet been clarified. This project will focus on investigating the interaction and enzymatic activity of different large and small subunit combinations of duckweed (Landoltia punctata) by using the yeast two hybrid, Pull-down and bacterial co-expression systems. To elucidate the function of different AGPases in the process of starch accumulation stimulated by nutrient starvation, the temporal expression patterns of different AGPases will be analyzed by using qRT-PCR for nutrient starvation treated duckweed samples. This project will provide guidance for the further studies focus on revealing starch accumulation mechanism in the future.

ADP-葡萄糖焦磷酸化酶（ADP-glucose pyrophosphorylase，AGPase）是植物体内淀粉合成途径的第一个也是最重要的限速酶，由2个相同的小亚基和2个相同的大亚基组成异源四聚体。植物体内可有多种大/小亚基组合形成不同的AGPase，这些AGPase有不同的时空表达规律。浮萍作为一种新的集污水处理与生物能源开发于一体的能源作物，其淀粉含量可在7天内快速积累到45%以上（干重），但浮萍体内多个AGPase与淀粉积累的关系尚未解析。本项目将以酵母双杂交、Pull-down、细菌共表达等方法重点研究少根紫萍AGPase不同大/小亚基间的相互作用及组合体的酶学活性，确定不同大/小亚基的组合关系。通过荧光定量PCR技术分析寡营养胁迫条件下少根紫萍淀粉快速积累过程中不同AGPase的表达模式，以揭示不同AGPase的表达与淀粉积累的关系，为解析淀粉积累的分子机理奠定理论基础。

浮萍是世界上最小、最简单的开花植物，在适宜的生长条件下其生长速度几乎比其他任何高等植物都快。少根紫萍经寡营养或烯效唑处理后可快速积累淀粉和类黄酮，但目前有关其分子机制的研究未见报道。通过高通量RNA测序和蛋白组分析方法，发现寡营养处理后淀粉和类黄酮代谢流发生明显变化，导致淀粉和类黄酮同时大量积累，寡营养处理可作为同时快速积累淀粉和类黄酮的培养方法。本研究以植物体内淀粉合成途径的第一个也是最重要的限速酶ADP-葡萄糖焦磷酸化酶（AGPase）为研究对象，克隆少根紫萍的5个AGPase编码基因（LpAGPS1、LpAGPS2、LpAGPL1、LpAGPL2、LpAGPL3），其中S1与L3存在互作且具备AGP酶活性，S2与L1、L2、L3均存在互作并有AGP酶活性；S1、S2亚基蛋白定位于叶绿体内，L1、L2、L3定位于叶绿体内和细胞膜上。LpAGPL2、LpAGPL3在寡营养和烯效唑处理后表达量明显上调，在淀粉快速积累过程中发挥重要作用。该研究初步解析了少根紫萍AGPase酶的组合及起在淀粉积累过程中的作用。