基于靶向突变体的甘蔗TB1转录因子分蘖调控互作蛋白筛选及其功能验证

In sugarcane, tillering affects cane yield as a crucial factor to productive stalk number, therefore takes an important position in architecture optimization and yield potential improvement. Tillering is a trait strictly regulated by molecular interaction network. For sugarcane, however, the genetic regulation network of tillerring is far from well-studied. Transcription factor TB1 has significant and conservative function as a negative regulator for tillering. It has been identified to be an integrator of multiple tillering regulatory pathways . As a regulatory center , TB1 provides a promising breakthrough to illuminate the genetic network of sugarcane tillering . However, the TB1-interacting proteins are not clear yet, systemic screening is thus badly desired. As a preliminary basis, targeted knockout of TB1 gene in Black Cheribon (Saccharum officinarum) has been conducted by CRISPR/Cas9 system. In this study, interacting proteins of TB1 involved in tillering regulation are to be screened through proteomic dissection of the targeted-mutant. Comparetive proteomic analysis with the wild-type will be conducted to profiling the expression pattern of the mutant in tiller outgrowth. Module analysis of the co-expression network of the differently expressed protein, interaction prediction, and Yeast Two-Hybrid assays, will be performed successively to detect TB1-interacting proteins responsible for tillering regulation. In the final, gene knockout and over expression assays will be carried out to verify the function of the interactor identified. The results will provide not only data and clues for further illumination of the tillering regulating network, but also novel gene resources benifiting architecture optimization and yield improvement for sugarcane.

甘蔗分蘖通过有效茎数量影响蔗茎产量，是株型优化和产量提升的关键。分蘖性状受到分子互作网络的严格调控，但甘蔗分蘖调控网络尚缺少研究。TB1转录因子具有显著和保守的分蘖负调控功能，在分蘖遗传调控网络中居于核心地位，因此成为解析甘蔗分蘖调控网络的有利突破口。然而，目前TB1甘蔗分蘖调控互作蛋白尚不清楚，亟需开展全面系统的筛选鉴定工作。项目组前期利用CRISPR/Cas9系统成功敲除了甘蔗TB1基因，现拟采取靶向突变体定量蛋白质组学分析的策略，系统发掘TB1在甘蔗分蘖调控中的互作蛋白。通过与野生型对照的对比分析，明确该突变体分蘖生长的蛋白质组学特征；通过差异蛋白共表达网络分析和蛋白互作关系预测，结合酵母双杂交实验，发现甘蔗TB1分蘖调控互作蛋白；最终，通过基因敲除和过表达分析，验证该蛋白的甘蔗分蘖调控功能。研究结果可为甘蔗分蘖调控网络解析提供重要线索，并为甘蔗株型和产量遗传改良提供基因资源。

分蘖是甘蔗产量最活跃的影响因子。TB1为植物分蘖调控网络的核心转录因子，明确其互作蛋白，可为甘蔗分蘖调控机制研究和遗传改良提供重要基因资源。项目以甘蔗热带种黑车里本为材料，取得了以下研究结果：1）构建了涵盖其地下非延长节分蘖芽发育全过程的酵母双杂交文库，利用Y2H-seq高通量检测技术对TB1转录因子互作蛋白进行了蛋白组水平的全局筛选，共鉴定TB1互作蛋白1288个，注释、富集和蛋白互作网络分析结果显示，TB1互作蛋白主要参与了分蘖芽萌发伸长过程中细胞前体代谢物和能量的产生；2）选择MADS57为TB1转录因子分蘖调控互作候选蛋白，克隆获得了其编码基因；分析其表达特征，发现其与已知具有分蘖负调控功能的D14基因的表达整体呈负相关关系，初步验证了其分蘖正调控功能；构建其过表达和基因编辑载体，并进行遗传转化，获得了相应的转基因苗；3）借助优化的技术体系，补充创制了TB1基因的靶向突变蔗株，突变株初始发株数增加、分蘖数提高、分蘖起始时间缩短，整体分蘖性能提升显著；4）发现短期诱导产生的愈伤组织即可高效分化再生；利用这一特征，结合GFP可视化绿色荧光标记，建立了甘蔗快速遗传转化技术体系，压缩流程耗时30%；5）通过浸染方法、分化培养基组分、筛选方法等三方面的系统优化，建立了农杆菌介导甘蔗高频遗传转化技术体系，令转化效率从原来的1.12%跃升至7.17%，提升幅度达6.4倍；6）借助高频转化技术体系和热激处理等方式，优化了甘蔗CRISPR/Cas9基因编辑技术，令突变效率和基因编辑效率均提升了2倍以上。本项目研究成果为甘蔗分蘖的精细调控提供了重要的研究技术和基因资源储备，为甘蔗分蘖性状靶向遗传改良奠定了基础。通过本项目的开展，课题组发表SCI论文2篇；授权国家发明专利1项、新增国家发明专利申请1项；1名成员破格晋升副高级职称；优化建立的遗传转化和基因编辑技术体系已应用于甘蔗基因工程育种实践。

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