Abstract
Immunolocalization is a necessary technique in the study of chromosome structure and function. It can provide important information for better understanding the relation between epigenetic modifications and chromosome condensation. In this protocol, we describe a way to localize and detect the distribution of histone H3 methylated at lysine 9 (inactive heterochromatin) in embryos at a period of 14 day-after-pollination (DAP). The protocol allows the observation of somatic cells in mitotic chromosome spreads.
Keywords: Immunolocalization, Mitotic, Maize, Plant, Embryo, Chromosome spread
Background
Immunolocalization, a technique widely applied in cytogenetics, offers a great contribution to explore the structure of chromosomes. The development of cytogenetic techniques enables us to gain knowledge about spatial and temporal proceedings of a given protein in the whole cell (Fransz et al., 1998; De Paula and Techio, 2014). Epigenetic modifications regulate gene expression by affecting chromatin structure and function (Bannister and Kouzarides, 2011). Moreover, a number of studies indicated that pathways and patterns of epigenetic modification exhibit distinct properties in different species, including Arabidopsis, rice, and maize (Kim et al., 2009; Chen and Zhou, 2013; Capuano et al., 2014; Springer and Schmitz, 2017). Immunolocalization techniques benefit greatly to decipher the morphology and structure of chromosomes during each step of plant meiosis (Pawlowski et al., 2013). However, many proteins associated with chromatin are not expressed in tassels or ears during meiosis. Thus, somatic cells as materials were used to analyze the localization of proteins that are expressed in other plant tissues during mitosis. Analyzing the changes in those proteins and in epigenetic modifications between wild type and a mutant allows us to gain a deeper understanding in their functions. In summary, this method allows us to detect the spatial or temporal distribution of a given protein during mitosis, and identify the distribution of various epigenetic modifications attributing to the molecular function via mitotic chromosome spreads.
Materials and Reagents
Equipment
Software
Procedure
Notes
Recipes
Acknowledgments
This work was supported by grants from National Natural Science Foundation of China (31671277). This protocol is adapted from Chromatin immunoprecipitation for studying chromosomal localization of meiotic proteins in maize (He Y, Sidhu G, Pawlowski WP. Methods Mol Biol. 2013; 990:191-201. doi: 10.1007/978-1-62703-333-6_19).
Competing interests
The authors declare that they have no competing interests.
References
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