本实验室前期研究结果表明，MtCBF4正向调节截型苜蓿耐冷和结瘤固氮两个生物学过程，并通过EMSA和ChIP确定了MtCAS15是MtCBF4的一个直接正调控靶基因。在此基础上，本课题拟挖掘MtCBF4的上、下游基因，深入研究其提高植物耐冷性和促进结瘤的分子机制。主要研究内容包括：通过分别筛选截型苜蓿冷胁迫和根瘤两个酵母单杂交cDNA文库，寻找MtCBF4上游转录因子；通过ChIP-Seq技术高通量筛选MtCBF4下游靶基因。进一步通过EMSA、ChIP、截型苜蓿瞬时转化系统、截型苜蓿稳定转化系统、荧光定量PCR等体外、体内实验确证MtCBF4上、下游关键基因的功能。综合以上研究结果，解析MtCBF4提高植物耐冷性和促进结瘤的分子调控路径，并结合MtCBF4及其两条通路的上游关键基因对乙烯、细胞分裂素等激素的响应，探讨这两条路径的交叉点。本课题的研究结果对豆科植物抗逆优质育种具有重要价值。

Our previous studies showed that Medicago truncatula MtCBF4 positively controlled the cold tolerance and nodulation. The MtCAS15 was the target gene of MtCBF4 by EMSA and ChIP. On these basis, this project intends to study the molecular mechanism on MtCBF4 improves cold tolerance and promotes nodulation. The main research contains three aspects. Two yeast single hybrid cDNA libraries will be screened to look for the MtCBF4 upstream transcription factor. High throughput ChIP - Seq will be used to search MtCBF4 downstream target genes. Furthermore, EMSA, ChIP, plant transcient and stable transformation system, quantitative PCR will be used to confirm the gene function in vitro and in vivo. Integrated above research results, we expect to uncover the MtCBF4's molecular regulation pathway on cold tolerance and nodulation respectively. The crosstalk between the two pathways will be discussed combining with MtCBF4 and upstream key genes in response to plant hormones.These results will provide important value for leguminous plant quality breeding.