GhMYC3调控棉花体细胞胚胎发生的分子机制

Although somatic embryogenesis (SE) is the main way to achieve transgenic cotton plant, genotype limitation,developmental asynochronization, the long time-consuming and low conversion rate of somatic embryos into plantlets in SE greatly delay the regeneration of transgenic cotton plant. Exogenous Jasmonic acid (JA) treatment and transcriptome analysis have proved that JA plays vital roles in cotton SE. We focused on functional identification of transcription factors involved JA signaling pathway, and found that the GhMYC3-overexpressed plants reduced significantly the growth of calli and inhibited the formation of embryogenic calli, knockdown of GhMYC3 increased significantly the calli growth and promoted the formation of embryogenic calli, while the underlying mechanism of GhMYC3 regulating SE remains unclear. In this project, electrophoretic mobility shift assay,luciferase Assay , ChIP-qPCR assay, yeast two-hybrid screen and CoIP assay will be employed to explore GhMYC3 targeted genes and its interacting proteins, finally to uncover the GhMYC3 molecular mechanism in SE. These results will provide new evidence regarding the mechanism of JA regulation of plant SE, and facilitate optimization of the cotton SE system for increasing the transformation efficiency.

体细胞胚胎发生是获得转基因棉花的主要途径，但基因型限制、发育不同步、遗传转化周期长和效率低等问题严重影响转基因再生苗的产生。外源处理和基因表达谱分析均已证实茉莉酸（JA）对棉花体细胞胚胎发生有重要调节作用，对JA信号途径的转录因子进行功能验证，发现过量表达GhMYC3基因抑制棉花愈伤组织生长和胚性愈伤组织形成，干涉GhMYC3基因促进棉花愈伤组织生长和胚性愈伤组织形成，但该基因调控棉花体细胞胚胎发生的分子机制仍不清楚。本项目拟通过凝胶阻滞电泳、萤光素酶检测、ChIP-qPCR、酵母双杂交和免疫共沉淀等多种技术，鉴定GhMYC3调控的靶标基因和互作蛋白，阐明GhMYC3基因在棉花体细胞胚胎发生过程中的调控机制。本研究为JA调控植物体细胞胚胎发生的分子机理提供新的实验证据，为优化棉花体细胞胚胎发生体系和提高棉花遗传转化效率提供理论支撑。

体细胞胚胎发生是获得转基因棉花的主要途径，但基因型限制、发育不同步、遗传转化周期长和效率低等问题严重影响转基因再生苗的产生。本研究解析了GhMYC3调控棉花体细胞胚胎发生的潜在机制，鉴定出GhMYC3的互作蛋白GhRCD1，GhWRKYs等，通过创制突变体材料证实GhRCD1调控愈伤细胞增值和胚性细胞的分化，初步证实GhMYC3在蛋白和转录水平上调控RCD1。此外，根据转录组和突变体材料的数据证实GhMYC3调控的体细胞胚胎发生与棉酚含量有关，GhMYC3与GhWRKYs互作后可能影响棉酚合成基因的表达，进而影响棉酚含量，调控愈伤增值和分化。本研究以MYC3为切入点，筛选了MYC3互作的蛋白和其调控的靶标基因，为解析棉花体细胞胚胎发生调控通路提供了基础，有助于筛选棉花体胚发生调控的关键基因，进而通过修饰关键基因的表达水平优化棉花遗传转化体系。