HECT类E3连接酶GhUPL7调控GhASN-like对棉花产量的影响

Plants utilize the ubiquitin-proteasome system (UPS) to modulate every aspect of growth and development. Ubiquitin is covalently attached to a substrate through the action of three enzymes: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase enzyme (E3). Single subunit ubiquitin ligase E3s can be subdivided into three basic groups dependent on the HECT, RING or U-box domain. Genome analysis has identified seven HECT E3 ligases in Arabidopsis that can be grouped four subfamilies (UPL1/2, UPL3/4, UPL5, and UPL6/7)..UPL3 plays a specific role in trichome development, and UPL5 negatively regulates leaf senescence through degradation of the transcription factor WRKY53 in Arabidopsis. Except UPL5, the substrates for other members of HECT E3 ligase are still unknown. The other UPLs function are also still unknown. The previous data indicated that GhASN-like contributes to improve cotton high yield. Using GhASN-like in a yeast two-hybrid, we isolated the HECT domain E3 ligase GhUPL7 from cotton ovule library previously. Furthermore, the BiFC result strongly suggested that GhASN-like interacts with GhUPL7. In order to investigate whether the GhASN-like protein can be marked for degradation by 26S proteasome through polyubiquitination by interaction with GhUPL7, and unravel the mechanism to affect cotton yield through ubiquitination of GhASN-like mediated by GhUPL7. Here we carry on the GhASN-like ubiquitination by GhUPL7 in vitro and in vivo; identification the target substrate of GhUPL7; investigation yield trait of up- and down-regulated GhUPL7 transgenic cotton plants. These results will contribute to the understanding regulation pathway of GhASN-like ubiquitination and cotton yield by E3 ligase. It has important theoretical and practical significance in knowledge of protein ubiquitination in cotton yield trait and high yield breeding.

泛素连接酶E3参与植物生长发育等调控，但植物中HECT类E3连接酶家族成员除UPL5外，其余靶标底物仍不清楚。项目组从棉花胚珠文库中筛选出HECT类E3连接酶基因GhUPL7与影响棉花产量基因GhASN-like 存在相互作用，并经BiFC验证。为进一步明确GhUPL7与GhASN-like间的关系，及其对棉花产量形成的影响，本项目拟采用免疫共沉淀验证两个蛋白间相互作用，进行体内和体外泛素化分析，研究GhUPL7对棉花产量性状的影响。鉴定GhASN-like是否GhUPL7的重要靶标，弄清该连接酶对GhASN-like的调控作用，解析HECT类E3连接酶通过泛素化GhASN-like影响作物产量形成的调控机制。该项目研究不仅在了解调控蛋白泛素化影响作物产量形成的机制方面具有科学意义，也将为棉花高产育种提供重要的理论依据。

泛素连接酶E3参与植物生长发育等调控，但植物中HECT类E3连接酶家族成员除UPL5外，其余靶标底物仍不清楚。项目组从棉花胚珠文库中筛选出HECT类E3连接酶基因GhUPL7与影响棉花产量基因GhASN-like 存在相互作用，并经BiFC验证。为进一步明确GhUPL7与GhASN-like间的关系，及其对棉花产量形成的影响，本项目首先分析和鉴定出陆地棉中的HECT基因，并描绘它们的结构特征。试验获得GhUPL7的表达特征以及其上游序列在烟草和拟南芥中的表达模式，检测和观察反义抑制、RNA干扰转基因棉花植株的胚珠、纤维和叶片表皮毛的变化，从而了解GhUPL7对棉花部分器官生长发育的影响，分析了转基因棉花T2、T3代的产量性状变化。采用免疫共沉淀验证两个蛋白间的相互作用，在转基因拟南芥和转基因棉花中进行体内和体外泛素化分析，分析了异源表达GhUPL7 对拟南芥生长发育的影响，研究GhUPL7对棉花产量性状的影响。通过Western blot等检测了转基因拟南芥和转基因棉花中GhUPL7和GhASN-like（亦称GhAILP1）蛋白的表达量，明确了两种蛋白间存在相互作用的关系，研究结果鉴定GhASN-like是GhUPL7的重要靶标，弄清了该连接酶对GhASN-like的调控作用，解析了HECT类E3连接酶通过泛素化GhASN-like影响作物产量形成的调控机制。该项目的研究不仅在了解调控蛋白泛素化影响作物产量形成的机制方面具有科学意义，也将为棉花高产育种提供重要的理论依据。