玉米ZmbZIP1a基因在淀粉合成调控途径中的分子机制研究

Seed starch content and composition are the direct factors affecting maize yield and quality. Presently, the function of key genes in starch synthesis have been frequently studied, but the regulation of the starch synthesis and regulation network is still unclear. In our preliminary work, we selected a starch regulatory gene, ZmbZIP1a. The gene has the ability of regulating starch synthesis accroding to the preliminary analysis of mutant and overexpression material, but the specific regulating mechanism is still incompletely understood. Our project will clear the physiological and biochemical characteristics further by analyzing starch content, starch structure and enzyme activities of starch synthesizing relating enzymes in ZmbZIP1a maize mutant and overexpression materials; we will determine ZmbZIP1a regulation of starch synthesis function by analyzing differential genes between mutant and overexpression seed endosperm using RNA-sequencing; in order to observe the specific regulation genes of ZmbZIP1a, we screen the regulating downstream genes through chromosome immune coprecipitation (ChIP-seq) and futher validate through Yeast one-hybrid and Electrophoretic Mobility Shift Assay (EMSA) assay in vitro. The project will clear starch synthesis molecular regulation mechanism of ZmbZIP1a, which lays the foundation for analyzing the biological metabolic pathways of corn starch.

种子淀粉含量和组分是影响玉米产量和品质的直接因素，当前关于淀粉合成途径中关键酶基因的功能研究已有大量报道，但其调控及调控网络仍然不清晰。项目组前期筛选得到了一个与淀粉调控相关的基因ZmbZIP1a，并对其突变体和过表达植株初步分析，发现该基因具有调节淀粉合成的能力，但其具体的分子调控机制依然不明确。本项目拟通过对ZmbZIP1a玉米过表达和突变体材料籽粒中淀粉含量、淀粉结构及淀粉合成过程中相关基因酶活等分析，以明确其调控淀粉合成的生理生化特性，利用RNA-seq对突变体和过表达植株籽粒胚乳中差异基因分析，确定ZmbZIP1a调控淀粉合成的功能，然后通过ChIP-seq技术确定ZmbZIP1a下游调控基因，结合酵母单杂和EMSA体外实验进一步验证，以明确ZmbZIP1a具体调控的基因。本项目研究将明晰ZmbZIP1a基因调控淀粉合成的分子机制，为玉米淀粉合成代谢途径的解析奠定基础。

bZIP转录因子家族在植物生长发育具有重要的作用。 项目组筛选的玉米淀粉调控因子bZIP1a 能与淀粉合成途径中相关基因相互作用，能调控淀粉合成基因的表达水平，特别能显著降低直链淀粉合成基因GBSSI (wx) 表达量。在对玉米和水稻中过表达bZIP1a 基因转基因材料分析发现，过表达该基因籽粒中直链淀粉含量降低，籽粒和淀粉颗粒就减小，淀粉化学结构发生改变，醇溶蛋白含量变化不大，而可溶性糖和还原糖含量显著提高，增加了对外界环境的抗逆性。同时，我们创建了ZmbZIP1a基因CRISPR编辑材料，该基因编辑后，淀粉合成途径中GBSSI基因表达水平提高，伴随着籽粒中直链淀粉含量提高；醇溶蛋白和非醇溶蛋白含量没有受到显著的影响，但对醇溶蛋白基因表达量分析发现α-19和γ-50表达量微微降低，γ-27显著降低，说明ZmbZIP1a基因能调控γ-27醇溶蛋白的表达。以上说明，ZmbZIP1a基因是一个负向调控因子，能直接作用于直链淀粉合成基因，影响淀粉含量，通过调控γ-27醇溶蛋白的表达量，影响籽粒中蛋白组分，改变淀粉颗粒大小和化学结构，从而影响籽粒品质。该项目研究结果不仅为玉米淀粉合成调控机理提供新证据，也为创建玉米优质新品种奠定理论基础。