蓖麻胚乳表观调控与基因组印迹研究

发布时间：2018-07-17 20:03

【摘要】：植物胚乳发育的研究一直是植物生殖发育,表观遗传学和作物遗传育种关注的热点领域和重要科学前沿。DNA甲基化是一种非常重要的表观修饰因子,参与异染色质的形成,转座子的沉默,基因表达的调控以及基因组印迹的发生。越来越多的证据显示DNA甲基化和基因组印迹对胚乳发育和种子大小形成具有重要的调控作用。目前已经在模式植物如拟南芥、水稻和玉米的种子中调查了DNA甲基化的水平和分布,揭示出胚乳基因组的低甲基化状态。但是,关于胚乳基因组的低甲基化状态是如何建立和维持的,是否具有广泛性以及它在胚乳发育过程中的作用,目前还不是很清楚。特别是对于大多数真双子叶植物包括拟南芥,胚乳组织是瞬时的,随着种子的发育逐渐消失,因此在真双子叶植物种子中研究胚乳DNA甲基化的调控方式以及DNA甲基化对胚乳发育和基因组印迹的调控受到了很大的限制。蓖麻种子是典型的双子叶胚乳型种子,胚乳在整个种子发育过程中持续存在,具有大的体积且易于分离,是种子生物学研究的模式材料。蓖麻为深入研究表观调控因子对胚乳发育的影响提供了理想的研究系统。本研究以蓖麻品系ZB107的种子为研究材料,结合全基因组DNA甲基化测序、small RNA测序、mRNA测序以及实验验证,全面分析了DNA甲基化的分布规律、调控方式和对基因表达的影响。主要结果如下：1.蓖麻胚乳基因组的低甲基化通过全基因组DNA甲基化测序,我们发现蓖麻胚乳基因组中CG和CHG甲基化水平分别为30.3%和18.3%,明显低于胚基因组中CG(40.7%)和CHG(24%)的甲基化水平,与拟南芥、玉米和水稻中的报道一致。但是蓖麻胚乳CHH甲基化水平(11.2%)相对于胚(12.7%)并没有发生显著下降。在检测的所有甲基化位点中,CHH甲基化类型占了大约68%。在蓖麻种子中绝大部分CG和CHG的甲基化水平维持在90%以上,而CHH的甲基化水平平均分布在30%-100%。而且胚乳基因组广泛的去甲基化作用导致了胚乳和胚间差异甲基化的形成。2.DNA甲基化的分布规律以及对基因表达的影响通过调查DNA甲基化在基因区域和转座子区域的分布,我们发现CG甲基化在整个基因组都有分布,而CHG和CHH甲基化在基因区很少分布,绝大部分分布在转座子区域。在基因区和转座子区,胚乳的DNA甲基化水平均明显低于胚。通过分析DNA甲基化与基因表达的关系,我们发现不同表达程度基因的DNA甲基化水平并没有发生明显的改变,而不表达的基因(RPKM≤1)的DNA甲基化水平最高,表明DNA甲基化的主要作用是抑制基因的表达。而对于胚乳特异表达的基因,DNA甲基化水平,特别是CG和CHG甲基化,在胚乳中发生了明显的下降,表明CG和CHG的低甲基化对胚乳特异表达基因的调控作用。3.24-siRNAs对DNA甲基化的调控为了进一步解析蓖麻种子中高比例的CHH甲基化,我们调查了蓖麻胚乳和胚中small RNA以及与DNA甲基化的关系。small RNA测序结果显示,蓖麻种子中包含丰富的24-nt small RNA,且这些24-nt siRNAs与基因组CHG和CHH甲基化的分布和水平显著相关。在胚基因组中,基因区和TE区24-nt siRNAs的丰度明显高于胚乳,且在胚和胚乳中,24-nt siRNAs很少在基因上分布,与CHG和CHH甲基化在基因区的分布一致。另外,我们发现24-nt siRNAs显著地富集在CHH的高甲基化区域；而在CHH低甲基化区域24-nt siRNAs的丰度发生了明显的下降,这些结果表明蓖麻种子中24-nt siRNAs参与了基因组CHH甲基化的维持。4.蓖麻种子DNA甲基化调控方式为了揭示蓖麻种子中DNA甲基化分布的潜在机制,我们调查了DNA甲基化相关基因在不同组织中的表达。结果表明,相对于胚组织,DNA甲基转移酶基因RcMET1和RcCMT在胚乳中的表达明显下降,而且DNA去甲基化酶基因RcDME在胚乳中也表达,共同导致了胚乳CG和CHG甲基化水平的下降;而DNA甲基化酶基因RcDRM3在胚乳中的表达并没有受到抑制。同时,在蓖麻胚和胚乳中丰富的24-siRNAs以及RdDM途径的激活维持了蓖麻种子基因组高比例的CHH甲基化。基因组印迹是一种典型的表观遗传学现象,主要发生在开花植物的胚乳中,与胚乳和种子的发育密切相关。在本研究中,我们以蓖麻品系ZB107和ZB306为亲本进行互交,对获得的杂交胚乳组织进行了深度mRNA测序和等位基因分‘离分析,调查和特征化了蓖麻胚乳中的印迹基因。主要结果如下：1.在蓖麻胚乳中鉴别到大量印迹基因通过全基因组重测序,我们在亲本ZB107和ZB306品系间鉴别了1007066个SNPs和100615个indels。结合这些SNPs和严格的筛选,我们在蓖麻胚乳中鉴别到184个母源印迹基因(maternally imprinted genes,MEGs)和9个父源印迹基因(paternally imprinted genes,PEGs)。在挑选的67个印迹基因中,57个得到了实验验证,有5个基因表现出品系依赖性印迹。在胚乳的不同发育阶段,我们发现这些基因表现出动态的印迹过程。另外,我们也鉴别到14个印迹的long non-coding RNAs,并得到了实验验证。在这些验证的印迹基因中,仅39%的基因在胚乳中特异表达,大部分基因在蓖麻各个组织中均有表达。2.印迹基因的特征化通过印迹基因的聚类分析,发现仅有少量印迹位点在基因组中发生了明显的聚类。GO功能富集分析显示出这些印迹基因显著地参与了胚乳的发育过程。另外,我们发现TE显著地富集在这些印迹基因的周围(上下游4 kb范围内),特别是LTR/Gypsy TE类型。通过比较拟南芥、水稻、玉米和蓖麻中的印迹基因,发现印迹基因在物种间的保守性比较低,说明印迹基因在物种间发生了独立分化,有较快的进化速率。3.DNA甲基化对印迹基因表达的调控通过对杂交胚和胚乳基因组DNA甲基化测序,我们发现胚乳的低甲基化区域显著地富集在印迹基因周围(上下游2 kb范围内),且这些低甲基化区域主要发生在LTR/Gypsy TE上,表明了TE的去甲基化过程对印迹基因表达的影响。另外,我们检测到6个MEGs的母源等位基因甲基化水平明显低于其父源等位基因,表明等位基因甲基化水平的差异对印迹基因的表达调控作用。
[Abstract]:The research of plant endosperm development has always been plant reproductive development. Epigenetics and crop genetics and breeding focus on the hot areas and important scientific frontiers.DNA methylation is a very important apparent modifier. It participates in the formation of heterochromatin, transposon silence, gene expression regulation and the occurrence of genomic imprinting. Many evidence shows that DNA methylation and genomic imprinting have important regulatory effects on the development of endosperm and the formation of seed size. The level and distribution of DNA methylation in the seeds of model plants, such as Arabidopsis, rice and corn, have been investigated, and the low methylation status of the endosperm genome is revealed. However, the low endosperm genome is low. How the methylation state is established and maintained, whether it is extensive and its role in the development of the endosperm, is not yet clear. Especially for most of the true dicotyledonous plants, including Arabidopsis, endosperm tissue is instantaneous, with the gradual loss of seed development, so the endosperm DN is studied in the seeds of true dicotyledonous plants. The regulation of A methylation and the regulation of DNA methylation on the development of endosperm and genomic imprinting are greatly restricted. The castor seed is a typical dicotyledonous endosperm seed. The endosperm persists in the whole process of seed development. It has large volume and is easy to separate. It is a model material for seed biological research. The effects of apparent regulatory factors on the endosperm development provided an ideal research system. This study used the seeds of ZB107 of Ricinus ricinus as the research material, combined with the whole genome DNA methylation sequencing, small RNA sequencing, mRNA sequencing and experimental verification, to comprehensively analyze the distribution of DNA methylation, the mode of regulation and the effect on the gene expression. The main results are as follows: 1. the methylation of the genomes of the castor plant endosperm through full genome DNA methylation sequencing, we found that the level of CG and CHG methylation in the endosperm genome of castor bean was 30.3% and 18.3%, respectively, significantly lower than the level of methylation of CG (40.7%) and CHG (24%) in the embryo genome, but it was consistent with the reports in Arabidopsis, corn and rice. The level of CHH methylation (11.2%) of the castor endosperm (12.7%) did not decrease significantly. In all the methylation sites, the CHH methylation type accounted for about 68%. over 90% of the methylation level of CG and CHG in Castor seeds, while the methylation level of CHH was distributed evenly in 30%-100%. and in the endosperm genome. Extensive demethylation leads to the distribution of.2.DNA methylation of differentially methylation in the endosperm and embryo, and the effect on gene expression by investigating the distribution of DNA methylation in the region of the gene and the transposon region. We found that CG methylation is distributed throughout the genome, while CHG and CHH methylation is rarely divided in the gene region. Most of the DNA methylation levels of the endosperm in the gene and transposon areas were significantly lower than those of the embryo. By analyzing the relationship between DNA methylation and gene expression, we found that the level of DNA methylation of different expression levels did not occur obviously, but the DNA methylation of the non expressed gene (RPKM < 1) The main function of DNA methylation is to inhibit the expression of gene, and for the gene of endosperm specific expression, the level of DNA methylation, especially CG and CHG methylation, is obviously decreased in the endosperm, indicating the regulation of CG and CHG low methylation on the specific expression of endosperm, the regulation of.3.24-siRNAs on DNA methylation In order to further analyze the high proportion of CHH methylation in Castor seeds, we investigated the relationship between the endosperm and small RNA in the embryo and the relationship with DNA methylation by.Small RNA sequencing results showed that the rich 24-nt small RNA contained in the Castor seeds, and these 24-nt siRNAs were significantly related to the distribution and level of genomic CHG and methylation. In the genome, the abundance of 24-nt siRNAs in the gene region and the TE region is significantly higher than that in the endosperm, and in the embryo and endosperm, the 24-nt siRNAs is rarely distributed in the gene, which is consistent with the distribution of CHG and CHH methylation in the gene region. In addition, we found that 24-nt siRNAs is significantly enriched in the high methylation region of CHH, while the CHH methylation region is abundant in the abundance of 24-nt. The results showed that the degree of 24-nt siRNAs in castor seed was involved in the DNA methylation of.4. Castor Seeds by genomic CHH methylation in order to reveal the potential mechanism of DNA methylation distribution in Castor seeds. We investigated the expression of DNA methylation related genes in different tissues. The results showed that the expression of DNA methylation related genes in different tissues was relative. The expression of DNA methyltransferase gene RcMET1 and RcCMT decreased in the endosperm, and the DNA demethylation gene RcDME was also expressed in the endosperm, which resulted in the decrease in the level of CG and CHG methylation in the endosperm, while the expression of the DNA methylation gene RcDRM3 in the endosperm was not suppressed. The activation of the rich 24-siRNAs and RdDM pathway maintains the high proportion of CHH methylation in the castor seed genome. Genomic imprinting is a typical epigenetic phenomenon, mainly occurring in the endosperm of flowering plants, closely related to the development of the endosperm and seeds. In this study, we use the castor strain ZB107 and ZB306 as parents. Cross intercross, deep mRNA sequencing and allele segregation analysis were carried out to investigate and characterize the imprinting genes in the endosperm of castor bean. The main results were as follows: 1. in the castor endosperm, a large number of imprinted genes were identified by whole genome re sequencing, and 1007066 of our parents were identified between the parent ZB107 and ZB306 lines. Combining these SNPs and 100615 indels. with these SNPs and strict screening, we identified 184 parent imprinting genes (maternally imprinted genes, MEGs) and 9 parent source imprinted genes (paternally imprinted genes, PEGs) in the castor endosperm. 57 of the 67 imprinted genes selected were tested with 5 gene expression lines. In the different stages of endosperm, we found that these genes showed a dynamic imprinting process. In addition, we also identified 14 imprinted long non-coding RNAs, which were verified by experiments. In these imprinted genes, only 39% of the genes were specifically expressed in the endosperm, and most of the genes were in the various tissues of castor. The.2. imprinting gene was characterized by the clustering analysis of the imprinted gene. It was found that only a small number of imprinted loci had a distinct clustering.GO enrichment analysis in the genome, which showed that these imprinted genes were significantly involved in the development of the endosperm. In addition, we found that TE was significantly enriched around these imprinted genes. (4 kb in the upper and lower reaches), especially the LTR/Gypsy TE type. By comparing the imprinted genes in Arabidopsis, rice, corn and castor, it is found that the conservatism of imprinted genes is relatively low among species, indicating that the imprinted gene has been independently differentiated in the species, and the rapid evolution rate of.3.DNA methylation regulates the expression of imprinted genes through the regulation DNA methylation sequencing of the hybrid embryo and endosperm genome, we found that the hypomethylation region of the endosperm is significantly enriched around the imprinted gene (2 kb in the upper and lower reaches), and these low methylation regions mainly occur on LTR/Gypsy TE, indicating the effect of TE demethylation on the expression of imprinted genes. In addition, we detected 6 MEGs The methylation level of maternal allele was significantly lower than that of its parent allele, indicating that the difference of allele methylation level played an important role in regulating the expression of imprinted genes.
【学位授予单位】：云南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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