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Abstract：
All flowering plants have evolved through multiple rounds of polyploidy throughout the evolutionary process. Intergenomic interactions between subgenomes in polyploid plants are predicted to induce chromatin modifications such as histone modifications to regulate expression of gene homoeologs. Nicotiana benthamiana is an ancient allotetraploid plant with ecotypes collected from climatically diverse regions of Australia. Studying the chromatin landscape of this unique collection will likely shed light on the importance of chromatin modifications in gene regulation in polyploids as well its implications in adaptation of plants in environmentally diverse conditions. Generally, chromatin immunoprecipitation and high throughput DNA sequencing (ChIP-seq) is used to study chromatin modifications. However, due to the starchy nature of mature N. benthamiana leaves, previously published protocols were unsuitable. The higher amounts of starch in leaves that co-precipitated with nuclei hindered downstream processing of DNA. Here we present an optimised ChIP protocol for N. benthamiana leaves to facilitate comparison of chromatin modifications in two closely related ecotypes. Several steps of ChIP were optimised including tissue harvesting, nuclei isolation, nuclei storage, DNA shearing and DNA recovery. Commonly available antibodies targeting histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 dimethylation (H3K9me2) histone modifications were used and success of ChIP was confirmed by PCR and next generation sequencing. Collectively, our optimised method is the first comprehensive ChIP method for mature starchy leaves of N. benthamiana to enable studies of chromatin landscape at the genome-wide scale.
摘要：
所有开花植物在整个进化过程中都经过多轮多倍体进化。预测多倍体植物中亚基因组之间的基因组间相互作用会诱导染色质修饰，例如组蛋白修饰，以调节基因同源物的表达。Nicotianabethamiana是一种古老的异源四倍体植物，具有从澳大利亚气候不同地区收集的生态型。研究这个独特集合的染色质景观可能会阐明染色质修饰在多倍体基因调控中的重要性，以及其在环境多样化条件下对植物适应的影响。一般来说,染色质免疫沉淀和高通量DNA测序（ChIP-seq）用于研究染色质修饰。然而,由于成熟N.benthamiana叶子的淀粉性质，以前发表的协议是不合适的。叶片中与核共沉淀的较高量的淀粉阻碍了DNA的下游加工。在这里，我们提出了一种优化的ChIP方案，以促进两种密切相关的生态型中染色质修饰的比较。优化了ChIP的几个步骤，包括组织收获，细胞核分离,原子核储存，DNA剪切和DNA回收。使用靶向组蛋白3赖氨酸4三甲基化(H3K4me3)和组蛋白3赖氨酸9二甲基化(H3K9me2)组蛋白修饰的常用抗体,并通过PCR和下一代测序确认ChIP的成功。总的来说，我们的优化方法是第一个全面的ChIP方法，用于N.benthamiana的成熟淀粉叶，使染色质景观的研究在全基因组规模。
