假俭草EoMYBs转录因子调控根系发育关键基因EoSINAT5的分子机制研究

Centipede grass is the best warm-season turfgrass originated from China, with high ornamental value, low maintenance and resistance to leanness. Stolon is the main carrier of turfgrass vegetative propagation because of its easy rooting characteristics. However, the difficulties in rooting of the centipede stolon led to the relatively difficult establishment of lawn, and the lack of related rooting molecular mechanisms, making a bottleneck for centipede grass exploitation and utilization. Previous studies showed that overexpression of EoSINAT5 gene can significantly inhibit the root development of Arabidopsis and shorten the root. Sequence analysis of the EoSINAT5 gene promoter revealed the presence of multiple MYB transcription factor associated binding elements. However, the regulatory mechanism of EoSINAT5 gene by EoMYBs remains unknown. This project mainly uses transgenic technology and molecular biology methods to study three major issues: one is to clarify the binding characteristics of EoMYBs and EoSINAT5 promoter; the other is to clarify the regulatory properties of EoMYBs and EoSINAT5 promoter; the third is to elucidate the molecular mechanism of EoMYBs regulating EoSINAT5 expression. The results of this project can not only further explain the mechanism of adventitious root development, but also reveal the molecular mechanism of the rooting difficulties of centipede grass. It will also provide important theoretical guidance for the improvement of centipede grass species and have practical value.

假俭草是起源于我国最好的暖季型草坪草，坪用价值高。匍匐茎是草坪草营养体繁殖的主要器官，但是假俭草匍匐茎生根困难导致了该草坪建植相对困难，且相关分子机理研究缺乏，成为其持续发掘利用的瓶颈。前期研究发现超表达EoSINAT5基因能够显著抑制拟南芥的根系发育，使根系变短。对EoSINAT5基因启动子序列分析显示存在多个MYB转录因子相关的结合元件。然而，EoMYBs对EoSINAT5基因的调控机制仍然未知。本项目主要利用转基因技术和分子生物学等手段，研究三个主要问题：一是明确EoMYBs与EoSINAT5启动子的结合特性；二是明确EoMYBs对EoSINAT5启动子的调控特性；三是阐明EoMYBs调控EoSINAT5基因表达的分子机制。该项目不仅能进一步解析不定根发育机制，揭示假俭草生根困难的分子机制，也将为假俭草品种改良提供重要理论指导。

假俭草是起源于我国最好的暖季型草坪草，坪用价值高。匍匐茎是草坪草营养体繁殖的主要器官，但是假俭草匍匐茎生根困难导致了该草坪建植相对困难，且相关分子机理研究缺乏，成为其持续发掘利用的瓶颈。申请人前期研究发现EoSINAT5基因能够调控拟南芥和假俭草的根系发育，但是其分子调控机制仍然未知。因此本项目通过对EoSINAT5基因启动子序列进行分析，利用酵母单杂交筛库技术筛选出了启动子上游结合的转录因子EoBBR基因，经过酵母验证和EMSA验证明确EoBBR基因能够与EoSINAT5启动子上的多个GAGA元件结合，包括E1（GAGAGAGAGA，-1995bp~-1986bp）、E2（GAGA，-1984bp~-1980bp）和E3（GAGA，-1764bp~-1760bp）；亚细胞定位分析显示，EoBBR定位在细胞核上；时空表达模式分析显示，EoBBR基因在根系水培0 d的表达量最高，之后表达水平降低；组织表达模式分析显示，EoBBR基因在叶中具有最高的表达量，其次是节和幼穗；假俭草同源转基因结果显示，在EoBBR超表达株系中，EoSINAT5基因表达量均出现了明显的下降；通过酵母双杂交筛库技术，筛选出了EoBBR的互作蛋白EoPROG1，经过酵母一对一与BiFC验证，确认了EoBBR与EoPROG1存在互作关系。基于以上研究结果，本项目初步构建了调控假俭草根系发育的EoPROG1/EoBBR-EoSINAT5分子模块。该项目的顺利实施，不仅揭示了假俭草根系发育的分子调控机制，丰富了不定根发育机制研究，也为通过分子手段对假俭草进行品种改良提供重要的理论指导。