二穗短柄草CBFⅢd基因应答低温胁迫的分子调控机理

The mechanisms of plants in response to cold stress has made considerable progress in Arabidopsis, while that in monocots is relatively slow. Our team screened a CBF transcription factor from Brachypodium distachyon, a new monocot model plant in the early of this project. This gene was named as CBFⅢd by us and was induced significantly by cold stress. The overexpression transgenic lines of CBFⅢd was obtained using our well-built genetic transformation system of Brachypodium. Based on our present experiment, we will focus on the downstream transcriptional regulatory mechanism, transcriptional regulation, and the upstream regulatory factors of CBFⅢd in response to stresses. It mainly includes the following contents: Identify the downstream regulary elements of CBFⅢd and their related downstream genes with ChIP-Seq technology using the transgenic Brachypodium, determine the transcriptional regulatory networks of CBFⅢd with the expression profiles of the transgenic and wild-type Brachypodium, obtain the upstream regulatory factors of CBFⅢd using its upstream promoter element and yeast one-hybrid assay, verify the interactions through tobacco transient expression system for the transcription factors and cis-acting elements identified from ChIP-Seq and yeast one-hybrid, reveal the physiological mechanism of associated phenotype resulted from CBFⅢd by other physiological and biochemical means. Finally, the molecular regulation mechanism and regulatory network underlying CBFⅢd could be systematically illuminated in Brachypodium through the above studies.

植物应答低温胁迫机制的研究在拟南芥中已经取得长足进展，而在单子叶植物中的研究进展却相对缓慢。项目组前期从新型单子叶模式植物二穗短柄草中筛选到的一个响应冷较显著的CBFⅢd基因，利用已建好的短柄草遗传转化体系，获得了该基因的超表达转基因株系。我们将在现有的实验基础上，对CBFⅢd转基因材料进行ChIP-Seq分析，确定其所调控的下游元件及关联的下游靶基因。用高通量测序技术对转基因短柄草与野生型进行表达谱分析，确定CBFⅢd可能介导的表达调控网络。分析CBFⅢd上游启动子元件，利用酵母单杂研究其上游调控因子。对于ChIP-Seq和酵母单杂鉴定出的转录因子及顺式作用元件，利用烟草瞬时表达系统在烟草中进行相互作用验证。并借助于其它生理生化手段研究与低温及其它表型相关的生理变化机理。通过以上研究最终阐明CBFⅢd基因介导短柄草抗逆的分子调控机理及其调控网络。

CBF转录因子在应答植物低温、干旱和高盐等多种逆境胁迫过程中起着重要作用， 其在双子叶植物中的研究已取得了较好的进展，而在单子叶植物中的研究却相对缓慢。项目前期我们从新型单子叶模式植物二穗短柄草中筛选到的一个响应冷较显著的CBFⅢd基因，把该基因构建到酵母诱饵载体上，利用酵母单杂筛选其上游调控因子；把该的基因的上游启动子区不同长度的片段分别构建到GUS基因的上游，研究其上游启动子区响应逆境胁迫的主要调控元件；把该基因构建到植物表达载体上，获得超表达转基因烟草，然后对转基因株系进行多种逆境胁迫，通过其表型、生理生化及转录组分析来阐明CBFⅢd基因介导植物抗逆的分子调控机理。