CoMKK6基因参与高温诱导油茶2n花粉形成的作用机制

高温已成功应用于诱导林木花粉染色体加倍的育种实践。申请人前期研究发现油茶可产生天然2n花粉，且得率在高温条件下显著提高。同时，转录组分析表明油茶CoMKK6基因可能参与高温诱导2n花粉形成的调控，但其作用机制尚不明确。因此，本项目以普通油茶‘华硕’为试材，在筛选高温处理产生2n花粉的适宜时期、温度和持续处理时间范围的基础上，应用醋酸洋红、苯胺蓝、免疫荧光染色等方法，探究高温条件下CoMKK6基因的差异表达与2n花粉形成、小孢子母细胞染色体分离、微管骨架动态变化、胞质分裂、辐射状微管定位和细胞板形成等相关异常细胞学行为的关系。另外，通过CoMKK6基因功能验证并结合酵母杂交、凝胶阻滞实验、双荧光素酶报告基因检测系统、双分子荧光互补实验，明确油茶CoMKK6基因功能与调控模式，进而揭示CoMKK6基因参与高温诱导油茶2n花粉形成的作用机制，为进一步利用2n花粉开展油茶遗传改良工作奠定基础。

High temperature has been successfully applied to the breeding practice of inducing forest tree pollen chromosomes doubling. The preliminary research indicated that Camellia oleifera can produce natural 2n pollen, and the yield is significantly improved under high-temperature conditions. At the same time, transcriptome analysis showed that Camellia oleifera CoMKK6 gene may be involved in the regulation of 2n pollen formation induced by high temperature, but the action mechanism of CoMKK6 gene is unclear. Therefore, this project uses Camellia oleifera ‘hua shuo’ as the test material, and on the basis of screening the suitable period, temperature and continuous processing time for producing 2n pollen by high-temperature treatment, using aceto-carmine staining, aniline blue staining, immunofluorescence staining, etc. to explore the relationship between the differential expression of CoMKK6 gene and 2n pollen formation, abnormal cytological behavior during meiosis of microspore mother cells including chromosome segregation, dynamic changes of microtubule skeleton, cytokinesis, and cell plate formation, etc. during high-temperature treament. In addition, the CoMKK6 gene function verification combines with Yeast hybridization, Electrophoretic mobility shift assay (EMSA), Dual-Luciferase reporter assay and Bimolecular fluorescence complementation (BiFC) to clarify the function and regulation model of CoMKK6 gene. Furthermore, the results in this study will reveal the action mechanism of the CoMKK6 gene involved in high temperature-induced 2n pollen formation of Camellia oleifera, and lay the foundation for further use of 2n pollen to carry out genetic improvement of Camellia oleifera.