长链非编码RNA（lncRNA）调控蒺藜苜蓿低温胁迫响应的研究

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.

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。

长链非编码RNA（lncRNA）在植物生长发育和环境胁迫应答中起调节作用。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。.本项目创建豆科模式植物蒺藜苜蓿低温胁迫响应lncRNA高通量测序数据库，并进行生物信息学分析，发现大量低温胁迫响应lncRNAs。通过构建lncRNAs与其靶基因表达关系图谱，结合MtCBFs 在染色体上的分布，我们找到了4个可能调控MtCBFs转录因子表达的lncRNAs。利用遗传、分子和生理学手段研究了其中两个lncRNAs（lncRNA86和lncRNA208）对CBF途径和蒺藜苜蓿抗冻性的调节。发现lncRNA86上调下游MtCBFs和MtCASs基因表达，增加植物的抗冻性。lncRNA208作用于糖代谢和蛋白质磷酸化相关基因，增强蒺藜苜蓿抗冻性。.表达分析发现，渗透和盐胁迫都能显著抑制lncRNA86和lncRNA208表达。由此我们推测，lncRNA86和lncRNA208可能对蒺藜苜蓿渗透和盐胁迫抗性具有负调控作用，这为以后研究lncRNA86和lncRNA208对蒺藜苜蓿渗透和盐胁迫抗性调节作用和机理提供研究思路。 ABA处理强烈抑制lncRNA86和lncRNA208表达，说明ABA可能通过影响lncRNA86和lncRNA208表达，对植物逆境胁迫抗性、特别是低温冷冻胁迫抗性进行调节。.本研究所取得的成果为发现新的低温胁迫响应功能分子元件及由其引发的新的低温胁迫响应分子机制研究提供重要信息。