两个类纤维素合成酶基因调控甘蓝GMS花粉败育的机理研究

花粉发育对有性生殖和品种选育至关重要。本项目以甘蓝花药败育突变体为材料，前期的形态学、细胞学和转录组等分析结果发现了两个花粉特异的类纤维素合成酶基因BoCSLD1和BoCSLD2与花药发育密切相关。现已发现多个多糖代谢基因包括类纤维素合成酶基因参与花粉的发育，但其调控机理尚不明确。据此，本项目拟采用启动子的GUS基因融合表达、亚细胞定位、组织原位杂交结合qRT-PCR鉴定其时空表达模式；利用CRISPR/Cas9敲除技术、过表达、拟南芥突变体互补验证及荧光纤维技术探明其功能。透射电镜结合TUNEL检测分析花药组织在发育过程中的结构差异；通过酵母单杂交及染色体免疫共沉淀技术挖掘两个基因的转录因子，结合酵母双杂交、双分子荧光互补技术对BoCSLD1和BoCSLD2作用关系的分析，阐释其调控关系。本项目的完成将阐明类纤维素合成酶基因调控花粉发育的分子机制，为有效、精准地遗传改良提供技术支持。

Pollen development is crucial for plant reproduction and variety breeding. This study was based on anther abortion mutant in Brassica oleracea . The morphology, cytology and transcriptome analysis revealed that two cellulose synthase-like protein genes BoCSLD1 and BoCSLD2 were closely related to pollen development. It has been found that many genes including the cellulose synthase genes involved in the metabolism of polysaccharides are participated in the development of pollen. However, the molecular mechanism of these genes in regulation of pollen and pollen wall development remains to be elucidated. On this base, GUS fusion expression system, subcellular localization and in situ hybridization combined with real-time quantitative PCR will be adopted to analyze temporal and spatial expression patterns of the two genes. We plan to confirm their biological functions in pollen development by using CRISPR/Cas9 knockout technology , over expression and Arabidopsis mutant complementary technique combined with fluorescence microscopy. Furthermore, transmission electron microscope combined with TdT mediated dUTP Nick End Labeling will be used to distinguish differences in structure of anther tissue during pollen development. In order to interpret regulatory relationship between them, yeast one hybrid and chromatin immunoprecipitation will be employed to identify transcription factor, yeast two hybrid and bimolecular fluorescence complementation technology were used to investigate the interaction between BoCSLD1 and BoCSLD2. This accomplishment of this project will elucidate the molecular mechanism of cellulose synthase-like protein genes regulation of pollen development, and also to provide technical support for effective and precise genetic improvement in Brassica oleracea.