Molecular Biology Reports ( IF 2.8 ) Pub Date : 2020-11-25 , DOI: 10.1007/s11033-020-06013-1 Buddhini Ranawaka , Milos Tanurdzic , Peter Waterhouse , Fatima Naim
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.
中文翻译:
一种优化本生烟草淀粉叶染色质免疫沉淀(ChIP)方法,以研究异源四倍体植物的组蛋白修饰
在整个进化过程中,所有开花植物都经过多轮多倍体进化。多倍体植物中亚基因组之间的基因组相互作用预计会诱导染色质修饰,例如组蛋白修饰,从而调节基因同源物的表达。烟草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。对这一独特集合的染色质景观进行研究可能会揭示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。通常,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。但是,由于成熟的本氏烟草叶片的淀粉性,因此以前发布的协议不适合。与核共沉淀的叶片中较高的淀粉含量阻碍了DNA的下游加工。在这里,我们提出了针对本氏烟草的优化ChIP协议可以方便地比较两种紧密相关的生态型中的染色质修饰。对ChIP的几个步骤进行了优化,包括组织收获,细胞核分离,细胞核存储,DNA剪切和DNA回收。使用了针对组蛋白3赖氨酸4三甲基化(H3K4me3)和组蛋白3赖氨酸9甲基化(H3K9me2)组蛋白修饰的常用抗体,通过PCR和下一代测序证实了ChIP的成功。总而言之,我们的优化方法是针对本氏烟草成熟淀粉叶的首个综合ChIP方法,从而能够在全基因组范围内研究染色质景观。