悬铃叶苎麻无融合生殖的发生及其分子机制

Apomixis is a naturally occurring mode of asexual reproduction in flowering plants. This process allows clonal reproduction through seeds, without meiosis and fertilization, resulting in offspring that are genetically identical to the maternal plant. The use of apomixis technology in major crop plants could potentially revolutionize agriculture. Over the last two decades, different experimental techniques have been used to investigate the genetic factors regulating apomixis and have led to the identification of many candidate genes. These genes participated in many processes potentially central to apomixis, including protein degradation, transcription, stress response, and cell-to-cell signalling. However, an understanding of its genetic control and molecular regulation remains very incomplete. In this study, next-generation sequencing will be used to suquence the flower-specific transcriptomes of five genotypes of Boehmeria tricuspis, representing three ploidy level (diploidy, triploidy and tetraploidy) and three reprodutive mode (sexual reprodution, obligate apomixis, and facultative apomixis). We will analyze the differentially expressed genes at diplosporous apomeiosis and multiple gene ontology terms and KEGG pathway enriched in apomictic genotypes. And we will analyze the asexual genome evolution through the orthologous genes between apomictic and sexual genotypes and make it clear that if hybridization and polyploidization may induce apomixis. By analyzing the results, we will get some key genes and pathways related to apomixis. Then we will employ the technologies such as qRT-PCR , RNA in situ hybridization, subcellular localization, and yeast two-hybridiztion to identify the key gene ontology and pathway. Our work will promote understanding of apomictic genetic control and molecular regulation and will provide theoretical basis for creating apomictic materials in the future.

无融合生殖是自然界中存在的一种不发生核融合的生殖方式，能产生遗传上与母本完全一致的后代，在农业上具有潜在的巨大价值。虽然研究者证明了不少无融合生殖相关基因，但对于基因如何控制无减数分裂、孤雌生殖、胚胎发育共同完成整个无融合生殖过程的分子机制依然知之甚少。本研究拟利用本团队发现的悬铃叶苎麻五种不同倍性（二、三、四倍体）、不同生殖型（有性生殖、兼性二倍体孢子生殖、专性二倍体孢子生殖）材料进行进化转录组分析，通过分析转录区序列变异（SNP、Indel）研究杂合、基因突变积累等在无融合发生机制中的作用，鉴定出无融合生殖型中快速进化和显著性聚集的基因；与五种生殖型悬铃叶苎麻雌花功能大孢子形成期的转录组测序结果进行比较后，利用qRT-PCR、原位杂交、亚细胞定位和酵母双杂交等技术发掘关键基因功能和通路，在此基础上解析悬铃叶苎麻无融合生殖的发生及其分子机制，为今后创制无融合生殖材料提供理论依据和思路。

无融合生殖是自然界中存在的一种不发生核融合的生殖方式，能产生遗传上与母本完全一致的后代，在农业生产上具有极其重要的价值。虽然研究者证明了不少无融合生殖相关基因，但对于基因如何控制无减数分裂、孤雌生殖、胚胎发育共同完成整个无融合生殖过程的分子机制依然知之甚少。在本研究中，通过进化转录组、差异转录组和蛋白组分析挖掘了多个重要的无融合生殖候选基因。我们发现刺激反应特别是较低水平的氧化还原压（ROS）诱导了无减数分裂的发生。而且，研究表明在悬铃叶苎麻中，倍性的提高并不足以诱导无融合的产生，它的作用可能与提高植株本身对DNA损伤和致死突变的忍耐有关。我们研究发现无融合生殖材料中通过蛋白磷酸化修饰发生了明显有别于有性生殖材料的基因转录后的剪辑、基因沉默、蛋白的加工。结果表明，蛋白磷酸化修饰在悬铃叶苎麻无融合生殖的发生中起重要作用。我们还发现一个转录因子蛋白TCP20与无减数分裂密切相关，酵母双杂试验发现其与多个具重要功能的蛋白互作。我们推测由于在无融合生殖型和有性生殖型间雌花中存在氧化还原压的差异，引起了TCP20特定位点不同程度的磷酸化或者氧化，改变了它和目标DNA序列的结合，从而引起了调控基因的差异表达，再加上TCP20与互作蛋白共同协作，最终导致了生殖型的差异。综上所述，本研究扩展了我们对悬铃叶苎麻无融合生殖的发生及其分子机制的了解，为今后创制无融合生殖材料提供理论依据和思路。