在前期工作基础上，用Northern blot和RT-PCR研究Knob串联重复序列在冷处理的玉米不同组织中的表达。用染色质修饰酶抑制剂和冷处理玉米，ChIP-PCR和重亚硫酸盐测序技术分析Knob染色质修饰状况的变化。用3D-Immuno-FISH方法在间期核定位重复序列确定它们的核空间分布、与染色中心和环的位置关系、染色质浓缩和脱浓缩状况及与不同染色质修饰和相关蛋白的联系。利用RT-PCR分析Knob序列和冷反应转录因子基因如ZmDREB1等随时间的表达和转录因子免疫沉淀-PCR研究转录因子和Knob序列之间的关系。RNA原位杂交分析Knob序列的RNA在间期核中的分布, RT-PCR和Southern blot分析冷压下重复序列拷贝数或染色体外DNA的形成。本研究试图阐明这些序列在染色质水平上的表达机制，将加深人们对玉米基因组重复序列的认识，为玉米基于重复序列的抗性改良奠定理论基础。

Based on the previous studies, we will analyze the expression of Knob tandem repeats in maize roots, shoots and leaves by using Northern blot and RT-PCR. Following treatment with inhibitors of epigenetic enzymes and cold stress, the epigenetic modification status will be comparatively analyzed by ChIP-PCR and Bis-Seq. 3-Inmmuno-FISH will be used to map these DNAs in nuclei, determine their relationship with chromatin center and loops, detect the status of chromatin condensation and decondensation, and understand possible association with chromatin modifications and HP1 after cold treatment. Following treatment with inhibitors of epigenetic enzymes, ABA and cold stress, the expression time course of knob repeats and a series of cold responsive transcription factors will be studied by RT-PCR and the possible action between knob repeats and these factors will be further analyzed by ChIP with antibodies against theses factors. RNA in situ hybridization will be applied to localize the RNAs expressed by knob repeats in nuclei and RT-PCR and Southern blot will be used to detect copies of these tandem repeats and formation of extrachromosomal DNAs after cold treatment. These studies will elucidate the possible association of nucleus location, chromatin structure and function of these repeats and illustrate regulation mechanism of Knob tandem repeats at chromatin levels in maize during cold acclimation,which will lay a foundation for maize breeding based on the repetitive sequences.