小麦DOF转录因子调控谷蛋白合成与面筋品质形成的分子机制

项目名称： 小麦DOF转录因子调控谷蛋白合成与面筋品质形成的分子机制

项目编号： No.31471485

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 食品科学、农学基础与作物学

项目作者： 晏月明

作者单位： 首都师范大学

项目金额： 90万元

中文摘要： 利用本课题组创制和引进的小麦贮藏蛋白亚基缺失突变系、近等基因系、染色体代换系和谷蛋白超表达系等一系列特殊遗传材料，以高分子量谷蛋白亚基和醇溶蛋白基因沉默的二穗短柄草作对照，克隆鉴定小麦种子特异表达的DNA结合锌指蛋白(DOF)基因，分析其分子结构特征、功能结构域及其系统进化关系；结合面筋和面包品质测定、磷酸化蛋白质组和qRT-PCR分析，研究籽粒不同发育时期DOF蛋白的磷酸化修饰特征、不同DOF 基因的动态表达模式及其与蛋白体发育和面筋品质形成的关系；利用酵母单杂交（Y1H）和凝胶阻滞电泳（EMSA）技术确定DOF蛋白与谷蛋白基因启动子的特异结合位点及其对谷蛋白合成的调控；通过酵母双杂交（Y2H）和Pull-down方法研究DOF蛋白与SPA等其他转录因子的相互作用及其对谷蛋白合成与面筋品质形成的影响，为小麦品质改良提供理论依据。

中文关键词： 普通小麦；DOF转录因子；基因克隆；功能分析；面筋品质

英文摘要： In this study,various wheat genetic resources are used as materials, including a set of complete Triple HMW-GS deletion lines, glutenin near-isogenic lines, chromosome substitution lines and glutenin over-expression lines. Brachypodium distachyon accessions are used as control, in which HMW-GS and gliadin genes are silent. DNA binding with one finger (DOF) protein genes specifically expressed in seeds are cloned and characterized by allelic-specific PCR and rapid amplification of cDNA ends (RACE), and their molecular structures, functional domains and phylogenetic evolutionary relationships are investigated; In combination with the results of gluten and bread-making quality testing, phosphoproteome approaches and real-time quantitative reverse transcriptional PCR (qRT-PCR), the phosphorylation modification features of DOF proteins, dynamic transcriptional expression patterns during grain development and their relations with protein body development and gluten quality comformation are studied; The specific binding sites of DOF proteins to the promoters of glutenin genes as well as their regulations to glutenin protein synthesis are determined by yeast one hybrid (Y1H）and electrophoretic mobility shift assay (EMSA). The interacts of DOF proteins with storage protein activator (SPA) and other transcript factors, and their effects on glutenin synthesis and gluten quality conformation are explored by yeast two hybrid (Y2H) and Pull-down. The results obtained in this project will provide important theoretical basis for wheat quality improvement.

英文关键词： Common wheat;DOF;Gene cloning;Functional analysis;Gluten quality