木薯叶绿体与淀粉体中调控淀粉含量的蛋白酶关联性研究

As the signature crop in tropical agriculture economics, obtaining new varieties with high yield and high starch content is the mainly goal of cassava breeding. Starch mainly exists in amyloplast in form of starch granules, the source is from for photosynthesis production in chloroplast, so the starch accumulation relationship between chloroplast and amyloplasts plays an important role in mining potential production of cassava. In this study, wild relative W14 (M. esculenta ssp. flabellifolia) and SC205（M. esculenta cv. SC No.205）were used as materials, chloroplasts and amyloplasts were separated at different stages of root development, characteristics of starch grains in the chloroplasts and amyloplasts were observed, and its starch content were measured. Using Western Blot and RT-PCR were used to analyze expression level of protease involved in starch accumulation, through statistical methods to find a correlation between the organelles, while using 2-DE establish protein expression profiles of chloroplast and amyloplast, analyzing and screening proteins which participated in starch accumulation indirectly. Based on the these datas, establish the proteins regulation network to illustrate the interaction mechanism of proteins in chloroplast and amyloplast. These datas can be used as a reference for selecting cassava germplasm with high yield and high starch.

木薯为热带经济作物的典型研究材料，获得高产、高淀粉木薯品种是育种追求的主要目标。淀粉主要以淀粉颗粒形式存在于块根淀粉体，其来源为叶绿体中光合作用产物，因此研究叶绿体与淀粉体中淀粉积累的关联性对挖掘木薯的生产潜力具有重要作用。本项目以野生近缘种W14和栽培种华南205为材料，分别在块根发育的不同时期分离叶绿体与淀粉体，观察叶片叶绿体及块根淀粉体中淀粉颗粒形态结构、数量、空间排布等特征并测定其淀粉含量。采用Western Blot及RT-PCR技术对参与淀粉积累的蛋白酶在翻译及转录水平的表达进行分析，通过统计学方法分析这些蛋白酶系在不同亚细胞器间的关联性；同时利用2-DE技术构建叶绿体及淀粉体蛋白质表达谱，分析并筛选间接参与淀粉积累的蛋白质；在此基础上建立蛋白质相互作用调控网络来阐述叶绿体与淀粉体中调控淀粉含量的蛋白质间作用机制，为选育高产高淀粉木薯种质提供理论依据。

为了研究叶绿体与淀粉体中淀粉积累的关联性，挖掘木薯的生产潜力，本项目以木薯栽培品种SC205及其野生祖先种Manihot esculenta ssp flabellifolia（W14）为材料，分别在块根发育的不同时期分离叶绿体与淀粉体，观察叶片叶绿体及块根淀粉体中淀粉颗粒形态结构、数量、空间排布等特征并测定其淀粉含量。发现SC205 叶片拥有“独特”的绿色维管束鞘细胞结构，但没有C4 植物典型的“花环结构”；W14 叶片海绵组织叶肉细胞围绕维管束鞘细胞排列，但发达程度略低，整体形状略似“花环结构”，但没有典型C4 植物的“花环结构”明显；采用Western Blot及RT-PCR技术对参与淀粉积累的蛋白酶在翻译及转录水平的表达进行分析，发现与W14相比，SC205块根中MeSPS2、Meα-amylase和Meβ-amylase基因的表达显著上调，表明W14块根淀粉的分解能力较SC205较强，而MeAGPase、MeSSS1、MeSP1和MeSP2基因的表达显著上调，表明W14块根淀粉的合成能力较SC205弱；在此基础上构建SC205高光合效率的蛋白质相互作用调控网络，网络显示41个差异蛋白之间的相互作用对光合作用途径的调节作用，而PGK 1（磷酸甘油酸激酶1）是枢纽蛋白。项目研究结果可为选育高产高淀粉木薯种质提供理论依据。