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

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.

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。

花粉发育对有性生殖和品种选育至关重要。在花粉发育过程中涉及到多个多糖代谢基因的参与。类纤维素合成酶基因参与了植物雄配子体的发育，但其调控机理尚不明确。课题组前期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。在此基础上对甘蓝核雄性不育两用系形态学观察发现不育株系花丝短小、花药薄而干瘪，花药表面无明显的花粉附着；通过石蜡切片的细胞学分析发现，在花蕾发育的第五阶段小孢子发育开始出现异常，药室壁没有开裂。对甘蓝核雄性两用系花蕾基于Nanopore RNA-seq全长转录组结合ITRAQ蛋白组的联合分析，发现戊糖、葡萄糖醛酸转换、淀粉和蔗糖代谢过程及植物激素信号转导与花药发育过程中细胞壁的物质、能量代谢密切相关；关键候选基因主要包括参与淀粉和糖代谢的基因；在获得基因全长序列的基础上，通过RT-PCR检测发现BoCSLD1，BoCSLD2在可育系的4级，5级花蕾和开放的花中表达，在不育系花蕾中表达下调；组织原位杂交结果进一步验证了RT-PCR的表达模式的检测结果。对类纤维素合成酶基因在芥蓝中进行了克隆、表达、亚细胞定位及系统进化等功能研究；在研究中发现类纤维素合成酶通过与E3泛素连接酶协同作用参与花药发育的调控，因此对E3泛素连接酶基因进行了功能分析；扩增了BoCSLD1和BoCSLD2的启动子序列，基因枪介导的瞬时表达发现pCSLD1::GUS和pCSLD2::GUS启动子均能激活GUS的表达，且融合蛋白表达在整个细胞中; 以甘蓝叶片为外植体，建立了稳定的甘蓝再生体系。以甘蓝花蕾为材料，成功构建了酵母cDNA文库，重组率约98% ，文库库容150万克隆基数。为进一步明晰类纤维素合成酶基因在花药发育中的转录调控关系奠定基础。该研究为花粉、花粉壁发育基因调控网络构建提供新的理论支撑，也为有效控制甘蓝育性表现、加快杂种优势育种的进程提供应用指导。