植物对低温的胁迫响应依赖众多的基因调控通路。目前已发现花粉响应低温胁迫的机理可能不同于营养生长时期，但对其认识极为有限。lncRNA被证实参与幼苗低温响应相关基因的表达调控，但迄今为止尚未有lncRNA参与调控花粉响应低温胁迫的相关报道。我们前期在一个受低温诱导后于白菜花蕾中上调表达的转录因子BrCBF4的启动子区域筛选得到一个与之负相关表达的反义lncRNA（lncCBF4）。本项目拟进一步通过构建表达抑制和/或过表达转基因白菜植株分析lncCBF4和BrCBF4的生物学功能；通过转录组测序、荧光定量PCR和印记杂交实验明确两者之间的调控关系；通过RNA纯化的染色质分离、RNA沉降、Northern杂交和核糖核酸酶保护实验明确lncCBF4的作用机制；通过染色质免疫共沉淀测序结合基因功能研究鉴定BrCBF4靶基因，以此疏通花粉响应低温胁迫的lncRNA-BrCBF4-COR基因调控通路。

Cold response in plants relies on reprogramming gene expression and triggering a variety of regulatory pathways. Knowledge on the cold response pathway and mechanism in pollen development process, which was proposed to be other than that in vegetative growth, is still elusive. Effects of long non-coding RNAs (lncRNAs) have so far been associated with the expression regulation of target cold-regulated (COR) genes in seedling. However, it is currently unknown if response to cold temperatures in pollen is mediated by lncRNAs. Previously, we identified a natural antisense lncRNA (lncCBF4) in the promoter region of a transcription factor-encoding gene BrCBF4 in Brassica rapa. In addition, we found that BrCBF4 is induced and up-regulated by cold stress in the floral buds, whereas the expression of lncCBF4 negatively correlates with BrCBF4. Here, we intend to focus on studying the cold response pathway and molecular mechanism during pollen development process of B. rapa in this proposal. Firstly, the function of lncCBF4 and BrCBF4 will be explored via gene silencing and/or overexpression technologies in B. rapa. Subsequently, high-throughput transcriptome sequencing (RNA-seq), qRT-PCR, Northern blot, and Western blot will be employed to investigate the regulatory relationship and regulatory level between lncCBF4 and its sense gene BrCBF4. Chromatin isolation by RNA purification (ChIRP), RNA pull-down, Northern blot, and ribonuclease protection assays (RPA) will be applied to identify lncRNA-associated DNAs and proteins and to investigate the regulatory mechanism of lncCBF4. Meanwhile, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) will be utilized to check the binding of BrCBF4 to the promoter of downstream target COR genes. Moreover, biological function of one of the downstream target COR genes will be further revealed. These results will provide a new insight into the regulatory pathway of lncRNA-BrCBF4-COR genes in cold response during pollen development process in B. rapa.