小麦TaMBF1c基因的功能鉴定及其调控机制

Multiprotein bridging factor 1 (MBF1) is known as a transcriptional co-activator that enhances transcription of its target genes and involves in vital processes occurring in living organisms. Previously, we found an ethylene-responsive transcriptional co-activator TaMBF1c, which was significantly induced by heat stress (HS) and shown heat tolerant function in transgenic lines. The purpose of the present study is further functional characterization of this gene, with respect to its role as a transcriptional activator of HS-induced genes and its regulation by other proteins. To understand how TaMBF1c modulates wheat heat tolerance, we compared the transcriptomes of wild-type and the transgenic plants grown under heat stress conditions. By comparing the RNA-Seq results, the heat-responsive pathway will be identified. To further investigate whether TaMBF1c bound preferentially to cis-elements of downstream genes, we will profile the Chromatin immunoprecipitation (ChIP) assays under heat stress treatments. We also use the yeast two-hybrid system to identify potential TaMBF1c-interacting proteins. After screening, the interactions of TaMBF1c will corroborate by bimolecular fluorescence complementation (BiFC). Importantly, the association between the genetic variations of TaMBF1c gene with heat tolerance will evaluate using a diverse population of wheat consisting of 256 varieties from different regions. Moreover, a favorable allele of the TaMBF1c gene will identify in heat-tolerant varieties. These studies will provide a certain basis for the use of TaMBF1c gene in breeding high temperature resistant wheat.

Multiprotein bridging factor 1（MBF1）是一类特殊的转录因子，可以激活目的基因的转录从而调节生物体的各种生命活动。本实验室前期筛选到一个小麦热胁迫响应的转录共激活子基因TaMBF1c，超表达转基因株系耐热性明显提高。本项目在此基础上，进一步开展TaMBF1c基因的功能验证和参与耐热调控的机制研究。通过TaMBF1c小麦转基因株系及受体的表达谱比较获得其参与的耐热途径。通过染色质免疫共沉淀技术寻找其调控的靶基因和作用元件。另外还用酵母双杂方法筛选TaMBF1c的互作蛋白并进行验证。最后，对256份以不同来源的小麦微核心种质进行耐热表型性状分析，与TaMBF1c序列变异和表达差异进行关联，筛选出最优等位基因及其功能分子标记，为利用TaMBF1c基因进行小麦耐热性的分子育种奠定良好的基础。

Multiprotein bridging factor 1（MBF1）是一类特殊的转录因子，可以激活目的基因的转录从而调节生物体的各种生命活动。本实验室前期筛选到一个小麦热胁迫响应的转录共激活子基因TaMBF1c，超表达转基因株系耐热性明显提高。本项目在此基础上，进一步克隆了TaMBF1c三个部分同源基因，均受热胁迫诱导表达。超表达TaMBF1c-7B可以提高小麦的耐热性；而抑制TaMBF1c表达导致小麦的热敏感性增加，说明TaMBF1c参与调控小麦的热胁迫响应过程。通过酵母双杂交实验筛选到了TaMBF1c-7B的一个互作蛋白TaG3BP，同源比对预测其可能是应激颗粒（SG）的一个组成成分。利用35S:TaMBF1c-7B-MYC转基因拟南芥材料进行IP实验结合质谱分析的方法，获得了23个与TaMBF1c-7B共沉淀的SG组分。进一步通过烟草细胞内瞬时共表达实验发现，热胁迫条件下TaMBF1c-7B可以与拟南芥SG的marker蛋白AtRBP47共定位于SG中。另外，通过核糖体分离技术和Western Blot实验，证明了热胁迫条件下TaMBF1c可以与核糖体40S小亚基共沉淀。多聚核糖体图谱结合高通量测序（RNA-seq）的方法，发现热胁迫后小麦TaMBF1c基因缺失突变体中微管运输、翻译起始、胁迫响应以及蛋白质加工和代谢等途径中相关基因的翻译效率降低。为利用TaMBF1c基因进行小麦耐热性的分子育种奠定了基础。