棉子糖代谢调控玉米种子活力的生化与分子机制

Seed vigor is critical for crop yield. Raffinose plays an important role in seed desiccation-tolerance and high raffinose has been positively correlated with seed vigor. Raffinose synthase (RS) is responsible for raffinose biosynthesis and alkaline α galactosidase (AGA) is responsidble for raffinose hydrolysis. We have found that the ZmRS muant seeds are more sensitive to artificial aging treatment than the wild type control. We have also characterized that, the embryos from ZmAGA4 mutant kernels contain less glucose in the embryo than WT. The reduction of glucose in ZmAGA4 mutant seed embryo is associated with significantly higher seed vigor in 3 independent ZmAGA4 mutant lines. These findings provide a clue to investigate the effects of raffinose on seed vigor. The first aim of this proposal is to determine whether less glucose or more raffinose in seed embryo, or both, would inhibit respiration and generate less reactive oxygen species, thus, caused the greater seed vigor, by characterizing the knock out / over-expression lines of ZmRS or ZmAGA4 genes. The 2nd aim of this proposal is to determine the correlation of the ZmRS variants and the corn seed vigor. The correlation of seed vigor and the raffinose content in seed embryo, or the ZmRS gene expression level in late stage development seeds, or the polymorphisms of the coding region and 5’ regulatory region of ZmRS gene of 200 maize inbred lines will be investigated. This research will not only elucidate the mechanism of raffinose metabolism on seed vigor，but will also benefit the maize molecular breeding by utilization of ZmRS or ZmAGA4 genes.

玉米种子活力直接影响出苗率，是玉米能否单粒播种的关键。棉子糖与玉米种子活力密切相关。我们前期工作发现玉米棉子糖合成酶（ZmRS）和碱性半乳糖苷酶（ZmAGA4）是控制棉子糖代谢及调控玉米种子活力的关键酶，但其机理有待阐明。本项目将以ZmRS及ZmAGA4基因突变体和超表达株系为材料，通过测定种子整个生理周期胚中棉子糖代谢变化以及人工老化前后的活性氧水平、呼吸速率、种子活力，解析棉子糖代谢调控种子活力的生化与分子机制。同时对200个玉米自交系进行种子活力、糖组分、ZmRS基因表达水平、ZmRS基因编码区和调控区核苷酸多态性进行鉴定和关联分析，明确ZmRS等位基因变异与种子活力的关系。该研究对利用棉子糖代谢关键基因提高玉米种子活力的分子育种具有重要意义。

种子活力是制约玉米生产的关键因素。棉子糖与种子活力密切相关。棉子糖合成酶(ZmRAFS)是玉米棉子糖合成的关键酶，而半乳糖苷水解酶（ZmAGA）负责棉子糖的水解。我们利用玉米ZmRAFS基因突变体和过表达材料研究了棉子糖调控玉米种子活力的分子机制，发现了棉子糖和蔗糖的比例是调控玉米种子活力的关键因素，通过调控棉子糖代谢可以提高种子活力；半乳糖苷水解酶ZmAGA1能够水解棉子糖，我们发现ZmAGA1在种子吸涨后表达量提高，ZmAGA1过表达降低了种子中棉子糖含量，提高了种子萌发的速率但降低了种子的耐老化能力。在种子引发过程中抑制ZmAGA1的表达，能够提高种子的萌发速率，抑制种子耐老化能力的降低。我们发现了转录因子ZmDREB2A通过调控生长素降解酶基因ZmGH3.2的表达，抑制玉米幼苗的生长，同时ZmDREB2A正调控ZmRAFS基因的表达提高棉子糖的合成，提高玉米种子的耐老化能力。此外，我们发现过表达ZmRAFS基因虽然在种子中合成棉子糖，但在叶片中，由于其底物蔗糖不足，ZmRAFS水解肌醇半乳糖苷产生了肌醇，并提高了植株的抗旱能力。上述研究结果为通过调控棉子糖代谢通路提高玉米种子活力和抗逆性的生物技术育种奠定了理论基础。