Departamento de Fisiología, Biología Molecular y Celular, IFIByNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Plant Methods. 2010 Apr 9;6:11. doi: 10.1186/1746-4811-6-11.
Searching thoroughly for plant cis-elements corresponding to transcription factors is worthwhile to reveal novel gene activation cascades. At the same time, a great deal of research is currently focused on epigenetic events in plants. A widely used method serving both purposes is chromatin immunoprecipitation, which was developed for Arabidopsis and other plants but is not yet operational for tomato (Solanum lycopersicum), a model plant species for a group of economically important crops.
We developed a chromatin immunoprecipitation protocol suitable for tomato by adjusting the parameters to optimise in vivo crosslinking, purification of nuclei, chromatin extraction, DNA shearing and precipitate analysis using real-time PCR. Results were obtained with two different antibodies, five control loci and two normalisation criteria.
Here we provide a chromatin immunoprecipitation procedure for tomato leaves that could be combined with high-throughput sequencing to generate a detailed map of epigenetic modifications or genome-wide nucleosome positioning data.
彻底搜索与转录因子对应的植物顺式元件对于揭示新的基因激活级联是值得的。同时,大量的研究目前集中在植物的表观遗传事件上。一种广泛用于这两个目的的方法是染色质免疫沉淀,它是为拟南芥和其他植物开发的,但对于番茄(Solanum lycopersicum)这种经济作物的模式植物物种来说,还不可行。
我们通过调整参数,优化体内交联、核纯化、染色质提取、DNA 片段化和使用实时 PCR 分析沉淀,为番茄开发了一种适合的染色质免疫沉淀方案。该方案使用了两种不同的抗体、五个对照基因座和两个标准化标准。
本文为番茄叶片提供了一种染色质免疫沉淀程序,该程序可与高通量测序相结合,生成详细的表观遗传修饰图谱或全基因组核小体定位数据。