小麦A、B型淀粉粒合成相关蛋白鉴定及其分子调控机制

Starch accounts for about 70% of wheat dry grains and has important effects on wheat yield and quality， which is also the most important energy source in the human diet. In this project, several Aegilops species (Ae. peregrine,Ae. kotschyi and Ae. crassa) only containing A-granule and common wheat cultivars with significantly different starch granule compositions as well as amylose and amylopectin contents will be used and A- and B-granules are separated and purified through optimized extraction methods and scanning electronic misroscope. The global protemone mapping of A- and B-granules is constructed by using high performance capillary electrophoresis (HPCE),ultra performance liquid chromatography (UPLC) and non-linear 2-DE and the differentially expression proteins between A- and B-granules are identified by 2D-DIGE and various tandem mass spectrometry methods. The phosphorylated and glycosylated proteins related to A- and B-granule formation are enriched and identified by Pro-Q Diamond and Pro-Q Emerald 488 dyes,respectively.Their modified sites,types and levels as well as their effects on different starch granule formation are further determined by different tandem mass spectrometry methods. Meanwhile, the dynamic expression patterns in both transcriptional and translational levels and their interactions of A- and B-granules related proteins and main enzymes during grain development are investigated by qRT-PCR, Western-blotting, yest two hybridation and immune co-precipitation. According to these studies, the molecular regulating mechanisms for wheat A- and B-granule synthesis and formation are exploited.

小麦籽粒中淀粉约占干重的70%，对小麦产量和品质都有重要影响，也是人类食物消耗中最重要的能量来源。本项目拟选用只含有A型淀粉粒而B型淀粉粒缺失的山羊草属Ae. peregrine、Ae. kotschyi和Ae. crassa 以及B型淀粉粒含量和直链淀粉含量有显著差异的小麦品种中优9507、CA9632、PH82-2和不同Waxy亚基缺失的糯性小麦等作材料，通过提取方法优化和扫描电镜检测，分离纯化A、B淀粉粒并提取总蛋白，采用高分辨HPCE、UPLC和非线性2-DE等方法构建A、B淀粉粒全蛋白表达谱，在此基础上用2D-DIGE和多种串联质谱方法鉴定A、B淀粉粒合成密切相关蛋白及其磷酸化和糖基化修饰特征，分析它们对淀粉粒形成的作用；同时从转录和翻译水平研究重要淀粉粒合成酶及其相关蛋白的动态表达模式、相互作用及其与淀粉粒颗粒形成的关系，初步探明A、B淀粉粒形成的生化基础及其分子调控机制。

通过不同提取方法的优化和梯度离心参数筛选，建立了A、B淀粉粒高通量分离纯化方法，可分离纯化足够量的、可用于比较蛋白质组鉴定的高纯度A、B淀粉粒，在此基础上通过蛋白质提取纯化，可用于双向电泳的分离和质谱鉴定。以含有A、B淀粉粒的普通小麦品种CB037和只含有A淀粉粒的Aegilops crassa PI330483为材料，对分离纯化的A、B淀粉颗粒结合蛋白进行了比较蛋白质组分析，共获得119 个差异表达蛋白，多数都是与淀粉合成相关的各种酶类，其中 granule-bound starch synthase 和 starch synthaseⅡa-3具有不同磷酸化修饰，且在A、B淀粉粒中存在显著的差异表达，有可能是导致A、B淀粉粒发育差异的主要原因。首次研究了糯小麦和面包小麦淀粉颗粒结合蛋白的蛋白质组和磷酸化蛋白质组差异，鉴定了糯与非糯小麦间的40个差异表达蛋白点，主要包括淀粉合成酶SS I和SS IIa、颗粒结合SS I。淀粉颗粒结合蛋白的磷酸化蛋白质组分析表明，面包小麦的磷酸化蛋白数量和磷酸化水平均显著高于糯小麦，但调控支链淀粉合成的蛋白具有相似的磷酸化水平，说明淀粉颗粒结合蛋白的磷酸化修饰直接参与直链淀粉和支链淀粉的生物合成。对小麦（含A、B淀粉粒）、Aegilops peregrina（只含A淀粉粒）和模式种二穗短柄草Bd21（只有B淀粉粒）淀粉粒动态发育与淀粉生物合成进行了比较研究，Bd21中淀粉生物合成关键基因早期表达主要在果皮中调节淀粉合成，而在小麦中国春和Aegilops peregrina中这些基因在早期高表达，促进新的B淀粉粒生产和增加淀粉粒体积。与直链淀粉合成相关的淀粉粒结合淀粉合成酶（GBSSI）主要存在于淀粉粒中，在Bd21的B淀粉粒中表达水平低于中国春。进一步的磷酸化修饰分析显示，GBSSI在中国春和Ae. peregrina淀粉合成酶催化结构域的183位苏氨酸和185位酪氨酸发生了磷酸化修饰，但在Bd21中没有发现磷酸化修饰，暗示GBSSI的磷酸化修饰可促进淀粉生物合成。另外，还研究了面包小麦籽粒发育定量蛋白质组变化与磷酸化蛋白质组特征、小麦/二穗短柄草幼苗叶片生长、籽粒发育与干旱胁迫下磷酸化蛋白质组特征，鉴定了多个参与调控淀粉和贮藏蛋白合成的磷酸化蛋白，为进一步探讨小麦淀粉生物合成及其产量与品质形成的分子机制奠定了基础。