灌浆期籽粒淀粉合成关键酶活性决定了作物的灌浆速率。同时提高多个淀粉合成关键酶基因的表达是提高作物灌浆速率的合理途径。本项目选取灌浆快、慢的两个玉米骨干自交系Mo17和黄早四为材料，通过淀粉积累动态测定、RNA-seq、比较基因组学以及顺式作用元件和转录因子预测等，筛选能同时调控多个胚乳淀粉合成关键酶基因表达的转录因子；分析可能调控淀粉合成限速酶基因sh2、bt2表达的转录因子的功能。通过酵母单杂交和凝胶阻滞实验验证候选转录因子与关键酶基因启动子的结合特性；通过胚乳瞬时表达分析转录因子对关键酶基因启动子活性的影响；再通过转基因过表达和敲除分析转录因子对胚乳淀粉合成和籽粒灌浆的作用。以此筛选出能同时调控多个玉米淀粉合成关键酶基因表达、控制玉米灌浆速率的重要转录因子，为开展高效灌浆速率玉米分子育种设计，增加玉米单产提供理论支持。

The key enzymes’ activities involved in starch synthesis in kernel during grain filling period determine the grain filling rate of crops. Simultaneously increasing the expression of several key enzymes’ genes is a feasible way to improve grain filling rate of crops. In this project, a fast and a slow grain filling elite maize inbred line Huangzaosi and Mo17 were selected as materials, transcription factors of coregulators of several key starch synthetic genes will be identified by starch accumulation dynamic measurement, RNA-seq, comparative genomics analysis and cis element and transcription factors prediction. potential regulators of the sh2 and bt2 genes, the rate-limiting enzyme coding genes of starch biosynthesis, will be selected as the key research object. Then, the binding properties of transcription factors and key enzyme genes’ promoters were identified by yeast one hybrid and electrophoretic mobility shift assay (EMSA), the effect of transcription factors on key enzyme genes' promoters' activities were analyzed by maize endosperm transient expression assay, the effect of transcription factors on starch synthesis in endosperm and grain filling were analyzed by over-expressing and knocking out of transcription factor genes in maize. Accordingly, the most important transcription factors which could regulate several maize starch synthesis genes’ expression and control maize grain filling rate would be selected. The results of this project would provide theoretical support for the molecular breeding design of efficient filling rate of maize and improving the yield of corn.

灌浆期籽粒淀粉合成关键酶活性决定了作物的灌浆速率，同时提高多个淀粉合成关键酶基因的表达是提高作物灌浆速率的合理途径。本项目选取灌浆快、慢的玉米骨干自交系Mo17，黄早四，R08等为材料，通过淀粉积累动态测定、RNA-seq、比较基因组学以及顺式作用元件和转录因子预测等，筛选鉴定了能同时调控多个胚乳淀粉合成关键酶基因表达的转录因子并对其调控机制进行了深入研究： 通过鉴定影响Sh2启动子活性的关键顺式作用元件，发现胚乳异源表达转录因子VP1能直接与RY repeat元件相结合调控Sh2基因的表达。由于前期报道VP1能与RY repeat结合调控基因表达，为了鉴定能同时调控多个胚乳淀粉相关基因表达的异源转录因子，我们对玉米淀粉合成关键基因Sh2的启动子进行了分析并进一步鉴定了调控玉米胚乳淀粉合成关键基因的转录因子VP1，同时构建其植物表达载体，以R08为受体材料，获得了VP1转基因自交系，并通过连续回交，获得了性状稳定的转基因材料，籽粒淀粉含量显著提高。同时研究了蔗糖和ABA协同调控玉米胚乳淀粉合成的分子机制，筛选了ZmWRKY82.2转录因子能介导蔗糖和ABA协同调控淀粉合成关键基因表达，并对ZmWRKY82.2的作用机制进行了深入分析。通过对胚乳灌浆期间淀粉合成相关基因表达谱进行测定，同时测定糖和激素对胚乳淀粉合成相关基因表达的影响，分析胚乳淀粉合成相关基因表达规律。结合灌浆期间淀粉合成相关基因表达规律，采用共相关分析筛选调控淀粉合成关键候选转录因子。重点对候选转录因子ZmbZIP91进行鉴定，分析了ZmbZIP91表达特性，利用瞬时表达体系鉴定ZmbZIP91对淀粉合成相关基因的调控功能。并通过各中分子生物学实验技术对ZmbZIP91调控玉米淀粉合成相关基因表达分子机理进行了解析。 本项目研究成果克隆鉴定了VP1，ZmbZIP91，ZmWRKY82.2 三个调控玉米淀粉多个功能基因表达的转录因子，深入解析了玉米籽粒淀粉的生物合成分子机制，并获得了VP1转基因玉米高淀粉材料，研究成果为进一步通过分子手段培育高淀粉玉米新品种提供了候选材料和基因。