基于高通量测序的棉花DNase I 超敏感位点的鉴定与分析

DNase I hypersensitive sites (DHSs) are specific chromatin regions, which bind with regulatory factors and cause the hypersensitivity of DNase I cleavage.Therefore, DHSs have been identified as the maker of regulatory element in chromatin. Based on the accuracy and reliability in identification of regulatory element, DHSs mapping has been long considered the gold-standard for identification of various classes of regulatory elements. In early studies,DHSs were identified using traditional Southern blot assay, which has a low through-put nature and limits analysis to small regions of the genome. Recently, a new strategy based on high-throughput sequencing (DNase-seq) was developed and used to simultaneously identify thousands of DHSs from the whole-genome-level. Results showed that it had a high efficiency and reliability in the identification of DHSs and regulatory elements. In this study, we will conduct the identification and analysis of cotton DHSs based on DNase-seq combining with the characterizing of histone modification, transcription and DNase I footprints. By comparison and analysis, we will elucidate the association between DHSs and their corresponding regulatory elements. To check its reliability, we will check the function of candidate regulatory elements by transgenic assay. We believe that this study will provide useful information and a powerful tool for identification of regulatory elements and further genetic improvement in cotton.

DNase Ⅰ超敏感位点（DH位点）是染色质上对DNase I 高度敏感的特殊区域,它通常是由于调控因子的结合导致，因此与各种调控元件紧密关联。通过它可以准确地预测不同基因调控元件，也因此被称为调控元件鉴定的"金标准"。早期的DH位点研究主要基于southern杂交技术，由于检测效率低，不适合大量或基因组水平的DH位点分析。最近，基于高通量测序技术，研究者开发出一种兼具高效性和高灵敏度的DH位点分析方法-DNase-seq。这一方法实现了人们从全基因组水平高效、准确的进行DH位点鉴定及相应调控元件分析的目标。本研究将以DNase-seq为基础，结合组蛋白修饰、转录组及DNase I印记等数据，开展棉花DH位点的鉴定与分析工作。希望通过比较分析，明确不同调控元件与DH位点的内在联系；并通过预测及验证不同调控元件来检验该方法的可靠性，为棉花基因调控元件的发掘以及遗传改良应用奠定基础。

DNase I超敏感位点（DH位点）与基因表达调控元件有着高度的关联性。基于高通量测序的DNase-seq技术的开发为从基因组水平快速、准确的进行调控元件鉴定开辟了一条新途径。为了能够从全基因组水平发掘分析棉花基因表达调控元件，发掘棉花重要农艺性状基因调控因子，我们开展棉花了DH 位点的鉴定与分析工作。分别构建了陆地棉及其理论祖先种中棉与雷蒙德氏棉的DH图谱；结合转录组、组蛋白修饰分析，明确了不同棉种DH位点的特征；结合H3K27Ac与H3K27Me3两种组蛋白修饰特征，分离获得了具有增强基因表达活性的增强子DH；结合转录因子RNA polymerase II subunit B1全基因组分布分析，筛选出具有启动子活性的DH位点，并通过功能验证证实预测的可靠性；与之同时，系统开展了DH技术体系的改良探索，建立了简单、可靠的新的DH技术方法；并在不同单双子叶物种开展了尝试，构建了适合不同物种的成熟的DH试验技术及生物信息分析方法；结合未来深入研究需要，进一步探索开展了染色质空间分布、着丝粒DNA组成及结构以及染色体特异细胞学标记开发的分析工作，为未来DH位点所代表的调控元件的功能研究奠定了基础；研究相关结果截至目前，已发表文章10篇，其中SCI 杂志文章8篇；英文专著两个章节；申请专利4项，已授权1项。本研究为棉花基因组中基因表达调控元件的发掘提供了新的策略和数据，在棉花遗传育种改良上有着重要的应用潜力。