RUB类泛素激活酶E1调控玉米种子发育的机理研究

种子发育是高等植物完成世代繁衍和粮食作物获取高产和高品质的关键过程。种子发育受到生长素信号的精确调控，而该信号阻遏蛋白的泛素化降解受到RUB类泛素化修饰的调控，但RUB修饰对种子发育的调控机理并不清楚。RUB修饰激活酶E1由ECR1和AXR1亚基组成，本项目前期克隆了一个小粒玉米种子突变体，编码ZmECR1亚基，并发现ZmAXR1亚基基因突变也导致小粒种子表型。胚乳细胞数量和大小、生长素响应基因表达等在zmecr1突变体中显著下降，推测RUB修饰在玉米种子发育中发挥重要作用。本项目将通过基因表达、蛋白互作、转基因等技术验证ZmECR1和ZmAXR1的功能，然后构建这两个基因的单/双基因突变体和单/双基因超表达系，创制RUB修饰活性受损和增强程度不同的材料，通过组织学观察、细胞分裂速率测定、以及底物蛋白的含量、泛素化程度和稳定性测定等阐明RUB修饰在调控玉米种子发育过程中的作用机理。

Seed development is a pivotal process in plant life cycle, as well as in cereal grain yield and quality improvement. Seed development was precisely regulated by auxin signal, which was controlled by ubiquitination-mediated degradation of the signal inhibitor protein, and the degradation activity was further regulated by RUB modification (RUBylation). In Arabidopsis, mutations in RUBylation related genes resulted in impaired degradation of auxin signal inhibitor protein, leading to auxin signal deficient and auxin resistant phenotypes, but showing no obvious change in seed development. In the preliminary work of this project, we cloned the ZmECR1 gene from a small seed maize mutant, which encodes a subunit protein of RUBylation activating enzyme E1, and found that the mutant of the other subunit ZmAXR1 showed similar small seed phenotype. In addition, cell size and number in endosperm of Zmecr1 mutant was decreased, accompanied with reduced expression of several auxin regulated genes. These evidences suggested an important role of RUBylation in maize seed development. In the following study, we aim to confirm the functions of ZmRCE1 and ZmAXR1, and explore the mechanism of RUBylation in regulating seed development of maize by diverse methods such as transgenic techniques, histological observation, and protein content and degradation rate measurements.