蛋白质N-连糖基化修饰调控小麦籽粒发育与品质形成的分子机制

In this project, we will use bread wheat cultivars distinct in gluten quality properties and various glutenin near-isogenetic lines, deletion lines and specific chromosome substitution lines as materials. On the basis of previous modification proteomics research in our group, different methods for enriching glycosylated proteins such as lectin affinity chromatography (LAC), zwitterionic-hydrophilic interaction liquid chromatography (ZIC-HILIC) and hydrazide chemistry are optimized and then glycosylated peptides are enriched on a large scale by the optimized method. The glycosylated sites and glucoside compositions are identified by LC-MS/MS. The N-linked glycosyproteome characterization is studied by combining iTRAQ and bioinformatic methods. The conserved glycosylated motifs and their functions for regulating storage protein and starch biosynthesis are analyzed, and then the interaction nets of key glycosylated proteins involved in various metabolic pathways are constructed. Meanwhile, the dynamic expression characteristics of important glycoslated proteins during grain development from transcriptional and translational levels is investigated by qRT-PCR and 2D-DIGE proteome approach. The identified glycosylated proteins and their interaction proteins are further varified by Pro-Q Emerald 488 dyes, Western-blot, Co-IP and Y2H. On the basis of these studies from field and greenhouse experiments of consecutive three years, the molecular mechanisms of glycosylated proteins regulating wheat seed development and quality formation are deciphered. This study will provide new insights into the molecular mechanisms of wheat quality formation and quality improvement.

本课题拟选用不同品质特性的小麦品种和不同谷蛋白组成的近等基因系、缺失系和特异染色体代换系为材料，通过凝集素亲和层析法、两性离子亲水互作色谱法、酰肼化学法等方法的优化，高通量富集糖基化肽段和LC-MS/MS鉴定糖基化修饰蛋白，并结合iTRAQ定量研究小麦种子发育过程中N-连糖基化蛋白质组特征及其与品质的关系，分析糖基化修饰蛋白的分子结构特征及其调控贮藏蛋白和淀粉生物合成的互作网络与代谢通路，同时采用qRT-PCR和蛋白质组学方法从转录和翻译水平分析重要糖基化蛋白在种子发育过程中的动态表达特征，进一步通过Pro-Q Emerald 488荧光染色、Western-blot、Co-IP、Y2H等方法，对鉴定的重要糖基化蛋白及其互作蛋白进行进一步验证。结合连续三年大田和温室试验结果，解析蛋白质N-连糖基化修饰调控小麦籽粒发育与品质形成的分子机制，为深入了解小麦品质形成机理和品质改良奠定理论基础。

本项目着重解析小麦N-连糖基化修饰蛋白质组特征及其在生长发育与淀粉生物合成中的作用。通过HILIC 糖基化富集、化学去糖基化、HPLC分离与串联质谱鉴定，首次解析了三叶期幼苗叶片以及干旱胁迫下全蛋白和质膜蛋白N-连糖基化蛋白质组特征。全蛋白中鉴定到316个糖基化位点和246个糖蛋白，其中干旱胁迫下11个糖蛋白显著上调表达，特别是细胞壁转化酶（CWIN）上调表达2倍，另外有9个糖蛋白显著下调表达，鉴定到的糖蛋白主要参与信号传导和细胞壁代谢。亚细胞定位分析发现25.6%和16.8%的糖蛋白分别定位于细胞膜和细胞壁上。叶片质膜蛋白糖基化组分析鉴定到312个糖蛋白，其中在干旱胁迫下显著上调表达173个，主要与蛋白激酶活性相关，参与胞外信号感知与传导以及细胞壁重构。显著上调的N-连糖基化蛋白多富集[NxT] 基序，其中79.5%位于蛋白表面和刚性结构的无规卷曲上，这有利于蛋白糖基化修饰，并通过降低蛋白质刚性提高蛋白稳定性。PNGase F 酶消化与糖基化位点定点突变分析进一步证实N-连糖基化修饰可增加蛋白质稳定性。因此，N-连糖基化修饰主要通过提高细胞壁重构相关质膜蛋白的结构稳定性来参与植物干旱胁迫响应。. 初步解析了细胞壁转化酶（TaCWIN2）糖基化修饰的功能，TaCWIN2属于CWIN基因家族、位于细胞壁上的酸性蔗糖转化酶，参与植物蔗糖代谢，是蔗糖卸载的关键酶，对蔗糖合成转运与籽粒淀粉生物合成具有重要作用。在N-292和N-338的Asn鉴定到两个糖基化位点，定点突变发现糖基化修饰影响TaCWIN2蛋白的稳定性，并显著提高转基因拟南芥植株的抗旱性，并通过与分子伴侣蛋白HSP81-3和HSP90相互作用促进蔗糖代谢，从而增强植物抗旱性。另外，初步解析了类受体蛋白激酶TaFER19糖基化修饰特征与功能，质膜N-连糖基化蛋白组分析鉴定到一个糖基化修饰水平显著上调的类受体蛋白激酶蛋白TaFER19，在N-126和N-170位点发生糖基化修饰，两位点的糖基化修饰不影响TaFER19在质膜上的定位及其蛋白质稳定性，但通过FERONIA功能缺失拟南芥fer4突变体互补实验表明，N-126和N-170的糖基化修饰是TaFER19发挥生物学功能所必须的。