CmLEC1调控菊花胚胎发育的机理研究

There are thousands of chrysanthemum cultivars in China now, but most of them are poor in biotic and abiotic resistance. However, many chrysanthemum wild resources usually have strong biotic and abiotic resistance. Distant hybridization between chrysanthemum cultivars and the wild resources is an effective way to improve chrysanthemum resistance. Previous studies have shown that embryo abortion occurs frequently in chrysanthemum distant hybridization, and seriously affects its breeding effect. We invesitigated the mechanism of embryo abortion in chrysanthemum distant hybridization from 2012 to 2015 under the support of a project from National Natural Science Foundation of China, and some stage results have been published in Scientific Reports and some other journals. In particular, we found that one gene, CmLEC1, is closely related to chrysanthemum embryo development, while its function and mechanism is unclear. Based on our good foundation,we will clone CmLEC1, screen its interaction proteins, and study its mechanism and function by RACE, RT-PCR, yeast two hybrid, bimolecular fluorescence complementation, and in vivo transient expression system. In addition, we will screen downstream key functional genes regulated by CmLEC1 and carry out internal and external validation expreiments. Results will shed light on the mechanism of CmLEC1 regulating chrysanthemum embryo development, and provide theoretical foundation and excellent gene pool for molecular design breeding during chrysanthemum distant hybridization embryo development. Therefore, the study in this project is of great scientific significance and prospective application.

目前国内菊花品种高达数千个，但多数品种抗性差，而许多菊花野生资源抗性强，运用栽培品种与野生资源进行杂交是改良菊花抗性的有效途径。研究表明菊花远缘杂交中胚胎大量败育经常发生，严重影响育种效果。在刚结题国家基金资助下，我们针对菊花远缘杂交胚胎败育现象进行了研究，阶段性结果已在Scientific Reports等期刊上发表，并发现CmLEC1与菊花胚胎发育密切相关，但其作用机制尚不清楚。为此，本项目在良好研究基础上，采用RACE、RT-PCR技术克隆该基因，利用酵母双杂技术筛选互作蛋白，运用蛋白体外表达、BiFC及体内瞬时表达系统研究其机制和功能；通过超表达、RNAi转基因植株筛选出受CmLEC1调控的下游关键功能基因，并进行体内外验证。结果将揭示CmLEC1调控菊花胚胎发育的作用机制，为菊花远缘杂交胚胎发育的分子设计育种提供理论基础和优异基因储备。因此，本研究具有重要的科学意义和应用前景。

目前国内菊花品种高达数千个，但多数品种抗性差，而许多菊花野生资源抗性强，运用栽培品种与野生资源进行杂交是改良菊花抗性的有效途径。我们前期研究发现，菊花远缘杂交中胚胎败育经常发生，严重影响育种效果；同时CmLEC1与菊花胚胎发育密切相关，但其作用机制尚不清楚。为此，在本项目中，我们采用RACE、RT-PCR、酵母单杂与双杂、双分子荧光互补、转基因、凝胶迁移等技术克隆了CmLEC1基因，研究了该基因的表达特性、亚细胞定位、功能及作用机制等。结果发现，CmLEC1具有促进菊花胚胎发育、提高菊花远缘杂交结实率的功能。CmLEC1与CmLEA、CmC3H、CmERF12形成基因网络共同调控菊花胚胎发育，其中CmLEC1通过与CmLEA上游启动子结合增强CmLEA表达来促进菊花胚胎发育，并在与CmC3H互作下，进一步增强了CmLEC1功能，而CmERF12负调控胚胎发育。此外，通过本项目研究，建立了地被菊‘雨花落英’品种的转基因技术体系，获得了一批转基因菊花新种质和菊花杂交育种后代。项目负责人以通讯作者发表了6篇研究论文，培养了5名研究生，申请了3项国家发明专利，1项发明专利获得授权，以第五完成人获得了1项国家技术发明奖二等奖。综上，本项目揭示了CmLEC1调控菊花胚胎发育的机制，不仅丰富了植物胚胎发育研究的理论知识，而且将为利用分子设计育种技术提高菊花、乃至其它花卉育种效率提供科学指导和优异基因储备。