甘蓝型油菜3D基因组学的初步研究与应用

发布时间：2018-05-01 16:01

本文选题：甘蓝型油菜 + 3D基因组　；参考：《华中农业大学》2016年博士论文

【摘要】：三维(three dimensional,3D)基因组学是在传统的一维线性序列的基础上研究染色质三维空间结构的一门学科。染色质构象捕获技术(chromatin conformation capture,3C)及其衍生技术用于测定特定的点到点之间的染色质交互作用,是3D基因组领域近年来发展起来的一类技术。随着染色质构象捕获技术的迅速发展,3D基因组学不仅被运用于基因表达、转录调控的机制研究,还被用于基因组组装(传统基因组组装、单倍型组装、宏基因组组装等)的研究当中。目前,3D基因组学的研究主要集中在小鼠、果蝇、酵母等模式生物和人类中,并取得了一系列丰硕的成果,但其在植物中鲜有报道,仅在模式物种拟南芥中有部分研究。而油菜是我国第一大油料作物,在食用油生产中占有重要地位。近期,国际上完成了甘蓝型油菜基因组的测序和草图的绘制工作,这是首次对传统的多倍体作物复杂的基因组进行的完整测序和组装,并且为多倍体植物三维基因组学的研究提供了数据资源。为了研究三维基因组在多倍体物种中的应用,本文从如下几个方面开展了工作:首先,本研究选取甘蓝型油菜品种中双11号(ZS11)作为实验对象,采用高通量染色质构象捕获技术(high-throughput chromosome conformation capture,Hi-C),通过甲醛交联、酶切、末端补齐及生物素标记、连接、DNA纯化、超声打断及生物素富集等步骤,完成了甘蓝型油菜染色质交互文库的构建。实验共获得了约110 Gb油菜基因组染色质交互的原始测序数据,通过质量控制、过滤及重复性检验等生物信息处理步骤,结果显示数据质量各项指标(比对率,无效连接及可重复性等)均符合预期结果,表明了Hi-C技术在传统多倍体植物中的可操作性。基于上述染色质交互数据,本研究构建了甘蓝型油菜中高质量高分辨率的全基因组交互矩阵,用于后续油菜基因组组装完善和三维基因组学转录调控功能研究。然后,为了检验基于染色质交互在多倍体植物组装中的可行性,首先利用上述染色质交互数据将未挂载到甘蓝型油菜染色体上的scaffolds进行锚位并进行方向的确定,从而提高甘蓝型因组参考序列的完整性。组装后有91.05 Mb scaffolds能被分配到相应的染色体上,并且111.70 Mb scaffolds能成功确定其在染色体上的位置和方向(现有草图中染色体号未知的scaffolds和没有确定具体位置方向的scaffolds分别为135.94 Mb,202.91 Mb),染色体号未知的scaffolds和没有确定具体位置方向的scaffolds分别从全基因组长度的16.03%和23.92%下降到5.29%和10.75%。通过预测已组装的scaffolds,显示该方法的预测甘蓝型油菜scaffolds的染色体号、位置和方向的正确率分别为98.85%、90.15%和90.39%。甘蓝型油菜基因组的完善对后续优良品种的选育和品质的改良具有重要的理论和实践意义。另外,本研究的开展不仅检验了基于染色质三维结构的组装方法在多倍体植物基因组辅助组装的可行性和有效性,而且为复杂植物基因组组装提供了新的思路。最后,本研究利用甘蓝型油菜的交互数据研究了其蛋白-蛋白相互作用(protein-protein interactions,PPIs)基因在染色质3D结构上的特征。通过基于直系同源的方法,本研究鉴定了甘蓝型油菜中163,475个PPIs,将PPIs基因对与随机基因对进行比较。结果显示蛋白-蛋白互作基因对的相互作用的可能性和交互频率都显著大于随机选取的基因对。上述结果表明形成PPIs的基因在空间上更容易形成共定位,在空间上靠的更近,同时还说明了在空间上距离较近的基因之间更利于形成蛋白-蛋白相互作用。结果暗示了植物基因组中染色质3D的组织和PPIs的形成可能存在一定联系,为解析甘蓝型油菜基因的转录调控机制提供了新的线索。
[Abstract]:Three dimensional (3D) genomics is a subject of study on the three-dimensional spatial structure of chromatin based on the traditional one-dimensional linear sequence. The chromatin conformation capture (chromatin conformation capture, 3C) and its derivatization technique are used to determine the interaction of chromatin between specific points and points, which is the near field of the 3D genome. With the rapid development of chromatin conformation capture technology, 3D genomics has been used not only in gene expression, in the study of transcriptional regulation, but also in the study of genome assembly (traditional genome assembly, haplotype assembly, macrogenome assembly, etc.). At present, research on 3D genomics is the main collection. A series of fruitful results have been achieved in mice, Drosophila, yeast and other models, but there are few reports in plants, only part of the study of Arabidopsis in the pattern species. And rape is the first major oil crop in China, and occupies an important position in the production of edible oil. Sequencing and drawing, this is the first complete sequencing and assembly of the complex genome of a traditional polyploid crop, and provides a data resource for the study of the three dimensional genomics of polyploid plants. In order to study the use of the three dimensional genome in polyploid species, this paper works from the following aspects: First, we selected ZS11 (ZS11) in Brassica napus (Brassica napus) as the experimental object, using high throughput chromatin conformation capture (high-throughput chromosome conformation capture, Hi-C), through the crosslinking of formaldehyde, enzyme cutting, end filling and biotin labeling, connection, DNA purification, ultrasonic interruption and biotin enrichment, etc. The construction of the chromatin interactive Library of rapeseed was constructed. The original sequencing data of the chromatin interaction of some 110 Gb rapeseed genome were obtained. The results showed that the data quality indexes (ratio, non effective connection and repeatability, etc.) were all conformed to the expected results. The maneuverability of Hi-C technology in the traditional polyploid plants. Based on the above-mentioned chromatin interaction data, a high quality and high resolution whole genome interaction matrix in Brassica napus was constructed, which was used for the completion of the subsequent rape genome assembly and the study of the three dimensional genomics transcriptional control power. The feasibility of interacting with the polyploid plants is first used to anchorage and determine the direction of scaffolds, which is not mounted on the chromosome of Brassica napus, in order to improve the integrity of the reference sequence of the Brassica napus group. After assembly, 91.05 Mb scaffolds can be assigned to the corresponding chromosomes, and 1 11.70 Mb scaffolds can successfully determine its location and direction on the chromosome (the unknown scaffolds of the chromosome number in the present sketch, and the scaffolds that does not determine the specific location of the scaffolds, respectively, 202.91 Mb), the unknown scaffolds of the chromosome number, and the scaffolds that do not determine the specific location of the scaffolds from the total genome length of 16, respectively. 3% and 23.92% decreased to 5.29% and 10.75%. by predicting the assembled scaffolds. The method showed that the correct rate of chromosome number, location and direction of Brassica napus scaffolds was 98.85%, 90.15% and 90.39%. of Brassica napus, respectively, had important theory and improvement on the selection and quality improvement of subsequent fine varieties. In addition, the development of this study not only examined the feasibility and effectiveness of the assembly method based on the three-dimensional structure of chromatin in the genome assisted assembly of polyploid plants, but also provided new ideas for the assembly of complex plants. Finally, the protein protein phase was studied by the interaction data of Brassica napus. The characteristics of protein-protein interactions (PPIs) gene in the structure of chromatin 3D. Through the direct homologous method, this study identified 163475 PPIs in Brassica napus and compared the PPIs gene to the random gene pair. The results showed the possibility and frequency of interaction between protein protein interaction gene pairs. Both were significantly greater than the random selection of gene pairs. The results showed that the gene forming PPIs was more likely to form co loci in space, closer in space, and also indicated that protein protein interaction was more conducive to the formation of protein protein interaction in space, which suggested the tissue and PPIs of the chromatin 3D in the plant genome. It may provide some new clues for analyzing the transcriptional regulation mechanism of Brassica napus.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S565.4

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