长链非编码RNA（lncRNA）在生物体的特定组织和发育阶段表达，或者对环境信号应答后特异表达，调控各种生物过程。lncRNA在植物生长发育和环境胁迫应答中起调节因子作用。低温驯化特性是温带和寒带植物适应低温冷冻胁迫的内在生理机制，分子调控网络复杂。CBF途径是至今刻画比较清楚的低温驯化响应分子机制, 但是调节CBF转录因子表达的上游组分还不清楚。本项目拟利用申请人构建的豆科模式植物蒺藜苜蓿低温驯化响应lncRNA数据库和前期结果，筛选低温驯化响应关键lncRNA及其靶基因，研究它们与靶基因之间的关系。通过构建调节CBF转录因子表达的lncRNA超表达/沉默株系，结合其它分子生物学和生理学手段，研究lncRNA调节CBF表达和植物抗冻的作用机理，为发现新的低温驯化响应功能分子元件及由其引发的新的低温驯化响应分子机制研究提供关键信息。研究结果有助于揭示lncRNA在植物逆境胁迫中的作用模式。

Emerging evidence shows that long non-coding RNA (lncRNA) with length greater than 200 bp regulate numerous biological processes by specifically expressing in the particular organs and developmental stages, as well as in response to abiotic stresses. Plants grown in temperate and cold climates evolved an effective mechanism to enhance their freezing tolerance known as cold acclimation by exposure to low, non-freezing temperatures. Numerous molecular and physiological processes are involved in the cold acclimation-induced increase in plant freezing tolerance. The CBF-dependent network is an important responsible mechanism for the enhancement of freezing tolerance by cold acclimation. However, the upstream components regulating the expression of CBF transcription remain unclear. Recent studies demonstrated that a large number of non-coding RNAs including lncRNAs that are responsive to abiotic stress in general and to cold stress in particular have been identified. However, there have been no detailed studies to evaluate the roles of lncRNAs in the regulation of CBF expression during cold acclimation-dependent freezing tolerance in plants. A comprehensive set of lncRNAs that are responsive to cold acclimation in the legume model plant Medicago truncatula was identified by genome-wide high-throughput sequencing. We further obtained a putative network associated with MtCBFs and their related lncRNAs by analyzing lncRNAs and their potential target genes. Based on these results, the proposed project aims to identify and functionally characterize the key lncRNAs and their target genes invovled in the regulation of cold acclimation-dependent freezing tolerance. Furthermore, the function of lncRNAs in the regulation of CBFs during cold acclimation will be studied by generating mutants and transgenic M. truncatula lines overexpression of lncRNAs and CBF genes. The outcome of the project will contribute to discovery of novel molecular components in the cold acclimation-dependent network for freezing tolerance in plants, and advance our knowledge on the function of lncRNAs in plant abiotic stress.