本文選題：水稻 + 轉(zhuǎn)錄組　； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：H2A.Z是一種重要且十分保守的組蛋白變體,在許多生命活動(dòng)中具有重要的功能,例如轉(zhuǎn)錄調(diào)控、減數(shù)分裂、DNA修復(fù)、染色質(zhì)動(dòng)態(tài)變化等。相對(duì)于酵母和動(dòng)物而言,植物中關(guān)于H2A.Z的研究較為薄弱,僅在模式植物擬南芥中有一些報(bào)道,而在重要的糧食作物和模式植物水稻中鮮有H2A.Z的相關(guān)報(bào)道。因此,本研究利用表觀基因組學(xué)與生物信息學(xué)整合分析的方法,對(duì)水稻中組蛋白變體H2A.Z的分布特征及其與基因表達(dá)的關(guān)系進(jìn)行了初步探索。本研究首先通過(guò)蛋白質(zhì)免疫印跡實(shí)驗(yàn)證明了 H2A.Z抗體的有效性,然后利用染色質(zhì)免疫共沉淀結(jié)合高通量測(cè)序的方法首次在水稻中獲得了組蛋白變體H2A.Z的全基因組圖譜,其中包括了不同組織(愈傷組織和幼苗地上部分)、不同的時(shí)間點(diǎn)(10:00 am和10:00 pm)以及野生型和轉(zhuǎn)基因株系中的數(shù)據(jù)。通過(guò)對(duì)H2A.Z數(shù)據(jù)的分析,發(fā)現(xiàn)其富集區(qū)域顯著分布于5'UTR和外顯子區(qū)域,與轉(zhuǎn)錄組學(xué)數(shù)據(jù)進(jìn)行整合分析還發(fā)現(xiàn)H2A.Z在轉(zhuǎn)錄起始位點(diǎn)下游的富集程度與基因表達(dá)水平呈現(xiàn)類似拋物線的關(guān)系。同時(shí),我還注意到在表達(dá)較高的基因附近H2A.Z主要富集于5'端,而在表達(dá)水平較低的基因中H2A.Z傾向于覆蓋整個(gè)基因區(qū)域。通過(guò)與H3K4me3、H3K27me3和DHSs整合分析發(fā)現(xiàn),H2A.Z在基因組中既與H3K4me3和DHSs共同出現(xiàn),又與H3K27me3具有一定的相關(guān)性。然而,H2A.Z與DNA甲基化卻呈現(xiàn)明顯的負(fù)相關(guān)。通過(guò)對(duì)不同組織(愈傷組織和幼苗地上部分)以及不同時(shí)間點(diǎn)(10:00am和10:00 pm)的H2A.Z數(shù)據(jù)和RNA-seq數(shù)據(jù)進(jìn)行差異分析,發(fā)現(xiàn)H2A.Z與轉(zhuǎn)錄激活和抑制都有一定的關(guān)系,但是更加傾向于與基因表達(dá)的變化方向呈負(fù)相關(guān),并且通過(guò)GO富集分析發(fā)現(xiàn)H2A.Z富集程度與表達(dá)變化方向相反的基因在水稻生長(zhǎng)發(fā)育和晝夜交替變化的過(guò)程中發(fā)揮著重要的功能。除了對(duì)生長(zhǎng)發(fā)育和環(huán)境變化過(guò)程中H2A.Z的作用進(jìn)行研究之外,本研究還對(duì)H2A.Z在信號(hào)傳導(dǎo)過(guò)程中發(fā)揮的作用進(jìn)行了初步探索。在擬南芥中研究發(fā)現(xiàn)H2A.Z與磷的感受基因AtSPX1具有密切的關(guān)系。在水稻中AtSPX1的同源基因OsSPX1被認(rèn)為與磷饑餓響應(yīng)、花粉發(fā)育和氧化脅迫具有密切的聯(lián)系,并且具有晝夜節(jié)律性表達(dá)模式。因此,我對(duì)水稻中OsSRX1基因下調(diào)后H2A.Z與基因表達(dá)的關(guān)系進(jìn)行了初步探索。通過(guò)對(duì)野生型Nipponbare和OsSPX1-antisense反義轉(zhuǎn)基因株系的H2A.Z測(cè)序數(shù)據(jù)進(jìn)行分析,發(fā)現(xiàn)H2A.Z富集程度與基因表達(dá)既有正相關(guān)的關(guān)系,又有負(fù)相關(guān)的關(guān)系。利用GO富集分析,發(fā)現(xiàn)OsSPX1-antisense株系中表達(dá)上調(diào)并且H2A.Z富集具有差異的基因在磷信號(hào)傳導(dǎo)和磷代謝等生物學(xué)過(guò)程中發(fā)揮著重要的作用,而OsSPX1-antisense株系中表達(dá)下調(diào)且H2A.Z差異富集的基因與能量代謝、氧化還原反應(yīng)和物質(zhì)運(yùn)輸?shù)壬飳W(xué)過(guò)程具有密切聯(lián)系,說(shuō)明H2A.Z在水稻中可能對(duì)基因的轉(zhuǎn)錄具有雙重功能。為了從多個(gè)層面對(duì)H2A.Z進(jìn)行研究,本研究通過(guò)收集水稻多種表觀基因組數(shù)據(jù)(DNaseI超敏感位點(diǎn)、組蛋白修飾、DNA甲基化等)對(duì)染色質(zhì)狀態(tài)進(jìn)行識(shí)別和分析,從染色質(zhì)水平上對(duì)H2A.Z的分布特征進(jìn)行了進(jìn)一步的解析。利用基于隱馬爾可夫模型(HMM)的生物信息學(xué)工具,將染色質(zhì)初步定義為13種狀態(tài),其中包含了 452,202個(gè)染色質(zhì)片段。為了更好的對(duì)這13種狀態(tài)的染色質(zhì)狀態(tài)的片段進(jìn)行挖掘和分析,利用基于自組織映射神經(jīng)網(wǎng)絡(luò)算法(SOM)的工具對(duì)452,202個(gè)染色質(zhì)片段進(jìn)行聚類分析和可視化。在不同表觀基因組的SOM圖譜比較中,可以發(fā)現(xiàn)H2A.Z與H3K4me3和H3K27me3有共同出現(xiàn)的分布特征。然后,我搭建了基于染色質(zhì)狀態(tài)的水稻表觀基因組數(shù)據(jù)平臺(tái)RiceEpi,通過(guò)該平臺(tái)可以查詢已有的染色質(zhì)狀態(tài)和SOM圖譜信息,并且可以對(duì)新的表觀基因組數(shù)據(jù)進(jìn)行分析,進(jìn)而對(duì)水稻基因組進(jìn)行多方面的注釋和功能元件的挖掘。最后,通過(guò)RiceEpi平臺(tái)中的分析功能對(duì)不同時(shí)間點(diǎn)(10:00 am和10:00 pm)的H2A.Z在SOM圖譜中的分布區(qū)域進(jìn)行差異分析發(fā)現(xiàn)H2A.Z可能在晝夜變化的過(guò)程中發(fā)揮著重要的作用,也進(jìn)一步證明了 RiceEpi平臺(tái)的有效性。本論文利用表觀基因組學(xué)、轉(zhuǎn)錄組學(xué)與生物信息學(xué)整合分析方法,并結(jié)合分子生物學(xué)手段探索水稻中組蛋白變體H2A.Z在基因組中的分布特征及其與基因表達(dá)的相關(guān)性,從表觀遺傳學(xué)的角度為解析基因表達(dá)的調(diào)控方式提供一定的理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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本文編號(hào)：1845852