应用纳米孔测序技术对拟南芥转录终止和表观基因组的研究

纳米孔测序技术可以在单分子水平获得 DNA 和 RNA 的序列及其修饰信息。近年来，该技术快速发展，在测序数据准确性、读长和测序通量等方面都有了显著提高，推动了多领域的研究进展。通过不断开发新的基于纳米孔测序技术的方法和生物信息学分析流程，不仅可以突破二代测序技术读长的限制，还可以从多维度，多层次对序列进行分析，从而获得新的视角和发现。本论文基于纳米孔测序技术，利用单分子新生 RNA 测序技术 FLEP-seq（Full-length elongating and polyadenylated RNA sequencing）来分析拟南芥转录终止，和开发了单分子靶向染色质开放状态和甲基化测序方法 STAM-seq（Single-molecule targeted accessibility and methylation sequencing）用于研究高度重复区域的表观遗传特征。主要结果如下：

1. 利用 FLEP-seq 技术，对拟南芥基因进行了转录终止研究。通过单分子测序的优势，本研究区分了转录终止过程中产生的 RNA 中间产物，包括顺读（Readthrough） 转录本、5'切割产物、3'切割产物和 polyA 转录本，并利用这些序列信息对转录过程进行了分析，绘制了转录终止图谱。研究数据表明，拟南芥RNA聚合酶II在经过polyA位点后会继续一段距离的转录才从DNA模板解离。该距离（即转录终止窗口）分布范围广泛，从 50 nt 到 1,000 nt 不等。蛋白编码基因下游紧邻（200 bp内）的同向tRNA 可以高效促进上游基因的转录终止。通过突变体的研究，本研究发现在5'→3'核酸外切酶xrn3突变体中，累积了大量的3'切割产物，个别基因的3'切割产物甚至延伸至下游基因，并可进行剪接和多聚腺苷酸化。在3'末端加工因子fpa突变体中，少数基因的转录终止延迟，转录本延伸至下游基因并形成嵌合转录本。然而，在DNA甲基转移酶met1突变体中并未发现明显的转录终止缺陷，说明CG甲基化不影响转录终止。

2. 综合利用了纳米孔测序的长读长、可直接检测DNA分子碱基修饰以及可靶向富集基因组目标区间的优势，同时结合外源标记染色质开放区域的策略，开发了STAM-seq方法，从单分子水平研究了拟南芥基因组高度重复区域（着丝粒、端粒和 45S rDNAs） 的表观遗传特征。结果表明，拟南芥的着丝粒区域表现出链特异的染色质开放状态；在rRNA 基因中，DNA 甲基化水平与染色质的开放程度呈负相关关系；在端粒下游 1 kb 区域具有链特异的 DNA 甲基化分布。

综上所述，本研究利用基于纳米孔测序平台的两种单分子测序技术 FLEP-seq 和 STAM-seq，分别对拟南芥转录终止和表观基因组进行了深入研究，为探究基因表达和基因组表观遗传的调控机制提供了新的视角。此外，本研究创新性的技术和生物信息学分析流程也可以用于其他物种，为解决复杂生物学问题提供更加完整和准确的信息。

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