系统分析水稻H2A.Z的分布特征并探索其与基因表达的相关性

发布时间：2018-05-05 02:44

本文选题：水稻 + 转录组　；参考：《中国农业大学》2017年博士论文

【摘要】：H2A.Z是一种重要且十分保守的组蛋白变体,在许多生命活动中具有重要的功能,例如转录调控、减数分裂、DNA修复、染色质动态变化等。相对于酵母和动物而言,植物中关于H2A.Z的研究较为薄弱,仅在模式植物拟南芥中有一些报道,而在重要的粮食作物和模式植物水稻中鲜有H2A.Z的相关报道。因此,本研究利用表观基因组学与生物信息学整合分析的方法,对水稻中组蛋白变体H2A.Z的分布特征及其与基因表达的关系进行了初步探索。本研究首先通过蛋白质免疫印迹实验证明了 H2A.Z抗体的有效性,然后利用染色质免疫共沉淀结合高通量测序的方法首次在水稻中获得了组蛋白变体H2A.Z的全基因组图谱,其中包括了不同组织(愈伤组织和幼苗地上部分)、不同的时间点(10:00 am和10:00 pm)以及野生型和转基因株系中的数据。通过对H2A.Z数据的分析,发现其富集区域显著分布于5'UTR和外显子区域,与转录组学数据进行整合分析还发现H2A.Z在转录起始位点下游的富集程度与基因表达水平呈现类似抛物线的关系。同时,我还注意到在表达较高的基因附近H2A.Z主要富集于5'端,而在表达水平较低的基因中H2A.Z倾向于覆盖整个基因区域。通过与H3K4me3、H3K27me3和DHSs整合分析发现,H2A.Z在基因组中既与H3K4me3和DHSs共同出现,又与H3K27me3具有一定的相关性。然而,H2A.Z与DNA甲基化却呈现明显的负相关。通过对不同组织(愈伤组织和幼苗地上部分)以及不同时间点(10:00am和10:00 pm)的H2A.Z数据和RNA-seq数据进行差异分析,发现H2A.Z与转录激活和抑制都有一定的关系,但是更加倾向于与基因表达的变化方向呈负相关,并且通过GO富集分析发现H2A.Z富集程度与表达变化方向相反的基因在水稻生长发育和昼夜交替变化的过程中发挥着重要的功能。除了对生长发育和环境变化过程中H2A.Z的作用进行研究之外,本研究还对H2A.Z在信号传导过程中发挥的作用进行了初步探索。在拟南芥中研究发现H2A.Z与磷的感受基因AtSPX1具有密切的关系。在水稻中AtSPX1的同源基因OsSPX1被认为与磷饥饿响应、花粉发育和氧化胁迫具有密切的联系,并且具有昼夜节律性表达模式。因此,我对水稻中OsSRX1基因下调后H2A.Z与基因表达的关系进行了初步探索。通过对野生型Nipponbare和OsSPX1-antisense反义转基因株系的H2A.Z测序数据进行分析,发现H2A.Z富集程度与基因表达既有正相关的关系,又有负相关的关系。利用GO富集分析,发现OsSPX1-antisense株系中表达上调并且H2A.Z富集具有差异的基因在磷信号传导和磷代谢等生物学过程中发挥着重要的作用,而OsSPX1-antisense株系中表达下调且H2A.Z差异富集的基因与能量代谢、氧化还原反应和物质运输等生物学过程具有密切联系,说明H2A.Z在水稻中可能对基因的转录具有双重功能。为了从多个层面对H2A.Z进行研究,本研究通过收集水稻多种表观基因组数据(DNaseI超敏感位点、组蛋白修饰、DNA甲基化等)对染色质状态进行识别和分析,从染色质水平上对H2A.Z的分布特征进行了进一步的解析。利用基于隐马尔可夫模型(HMM)的生物信息学工具,将染色质初步定义为13种状态,其中包含了 452,202个染色质片段。为了更好的对这13种状态的染色质状态的片段进行挖掘和分析,利用基于自组织映射神经网络算法(SOM)的工具对452,202个染色质片段进行聚类分析和可视化。在不同表观基因组的SOM图谱比较中,可以发现H2A.Z与H3K4me3和H3K27me3有共同出现的分布特征。然后,我搭建了基于染色质状态的水稻表观基因组数据平台RiceEpi,通过该平台可以查询已有的染色质状态和SOM图谱信息,并且可以对新的表观基因组数据进行分析,进而对水稻基因组进行多方面的注释和功能元件的挖掘。最后,通过RiceEpi平台中的分析功能对不同时间点(10:00 am和10:00 pm)的H2A.Z在SOM图谱中的分布区域进行差异分析发现H2A.Z可能在昼夜变化的过程中发挥着重要的作用,也进一步证明了 RiceEpi平台的有效性。本论文利用表观基因组学、转录组学与生物信息学整合分析方法,并结合分子生物学手段探索水稻中组蛋白变体H2A.Z在基因组中的分布特征及其与基因表达的相关性,从表观遗传学的角度为解析基因表达的调控方式提供一定的理论基础。
[Abstract]:H2A.Z is an important and very conservative histone variant, which has important functions in many life activities, such as transcriptional regulation, meiosis, DNA repair, and dynamic changes in chromatin. Compared with yeast and animals, the research on H2A.Z is relatively weak in plants. There are few related reports of H2A.Z in grain crops and pattern plant rice. Therefore, this study uses epigenetic and bioinformatics integration methods to explore the distribution characteristics of H2A.Z in rice medium group and its relationship with gene expression. The effectiveness of H2A.Z antibody was obtained by using chromatin immunoprecipitation combined with high throughput sequencing for the first time to obtain the entire genome map of the histone variant H2A.Z in rice, including the different tissues (callus and the aboveground part of the seedlings), different time points (10:00 am and 10:00 PM), and wild and transgenic lines Through the analysis of H2A.Z data, it was found that the enrichment region was significantly distributed in the 5'UTR and exon regions. The integration analysis with the transcriptional data also found that the concentration of H2A.Z at the downstream of the transcriptional starting site was similar to the gene expression level. At the same time, I also noticed the high expression of gene attachment. The near H2A.Z is mainly enriched at the 5'end, while H2A.Z tends to cover the whole gene region in the lower expression genes. Through the integration with H3K4me3, H3K27me3 and DHSs, it is found that H2A.Z appears in the genome not only with H3K4me3 and DHSs, but also with H3K27me3. However, H2A.Z and DNA methylation present a significant negative phase. Through the difference analysis of H2A.Z data and RNA-seq data of different tissues (callus and seedling on the ground part) and at different time points (10:00am and 10:00 PM), it is found that H2A.Z has a certain relationship with transcription activation and inhibition, but it is more inclined to be negatively correlated with the direction of gene expression, and is enriched by GO. It is found that the genes of H2A.Z enrichment and the opposite direction of expression play an important role in the process of rice growth and day and night alternation. Besides the study of the role of H2A.Z in the process of growth and environmental changes, this study also preliminarily explored the role of H2A.Z in the process of signal transduction. The study in Arabidopsis found that H2A.Z has a close relationship with the phosphorous receptor gene AtSPX1. In rice, AtSPX1 homologous gene OsSPX1 is considered to be closely related to phosphorus starvation, pollen development and oxidative stress, and has a circadian rhythmic expression pattern. Therefore, I downregulated the OsSRX1 gene in rice after H2A.Z and gene table. Through the analysis of H2A.Z sequencing data of wild type Nipponbare and OsSPX1-antisense antisense transgenic lines, it was found that the degree of H2A.Z enrichment and gene expression were both positive and negatively related. GO enrichment analysis was used to increase the expression of H2A.Z and H2A.Z. The genes with different concentrations play an important role in biological processes such as phosphorous signal transduction and phosphorus metabolism, and the genes in the OsSPX1-antisense strain and the differentially enriched H2A.Z are closely related to the biological processes such as energy metabolism, redox reaction and material transport, indicating that H2A.Z may be gene in rice. Transcription has dual functions. In order to study H2A.Z from multiple layers, this study identifies and analyzes chromatin status by collecting a variety of epigenetic data from rice (DNaseI hypersensitive loci, histone modification, DNA methylation, etc.). The distribution of H2A.Z is further analyzed from chromatin level. In the bioinformatics tool of the hidden Markov model (HMM), the chromatin was initially defined as 13 states, including 452202 chromatin fragments. In order to better explore and analyze the chromatin states of these 13 states, 452202 chromatin based on the self-organized mapping God network algorithm (SOM) was used. Cluster analysis and visualization. In the SOM map comparison of different epigenetic genomes, we can find the common distribution characteristics of H2A.Z and H3K4me3 and H3K27me3. Then, I build the rice epigenetic data platform based on the chromatin state RiceEpi, which can be used to query the existing chromatin state and SOM atlas. Information, and the analysis of the new epigenetic data, and further annotation of the rice genome and the mining of functional components. Finally, the analysis of the RiceEpi platform is used to analyze the differences in the distribution regions of H2A.Z in the SOM atlas at different time points (10:00 am and 10:00 PM). It is found that H2A.Z may be in the day. In the process of night change, it plays an important role and further proves the effectiveness of the RiceEpi platform. This paper uses epigenetic, transcriptional and bioinformatics integration and analysis, and combines molecular biology to explore the distribution characteristics of H2A.Z in the rice group and the phase of its gene expression. Guan Xing provides a theoretical basis for analyzing the regulation of gene expression from the perspective of epigenetics.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2

【参考文献】