The isolation of early nuclear endosperm of Oryza sativa to facilitate gene expression analysis and screening imprinted genes

Abstract

BackgroundSince the quality and yield of rice production depends on endosperm development, previous studies have focused on the molecular mechanism that regulates this developmental process. Recently, how this process is epigenetically regulated has become an important topic. However, the gene expression analysis and screening imprinted genes during early endosperm development remain challenging since the isolation of early endosperm has not been possible. Here, we report a procedure for the isolation of endosperm at 24 or 48 HAP (hours after pollination) during the free nuclear stage of endosperm development.ResultsThis technique allows for rapid and convenient collection of pure free nuclear endosperm. Early endosperm RNA can then be extracted from the isolated endosperm cells using dynabeads. Our results showed that the quality of RNA is satisfactory for gene expression analysis and screening the parental-of-origin specific genes in early endosperm.ConclusionsThus, we offer a reliable method to overcome one of the major obstacles in the investigation of the molecular mechanisms of early endosperm development. Our approach can be used for accurate gene expression analysis and screening of imprinted genes, and facilitates the confirmation of endosperm-specific gene expression at the very early stages of endosperm development. This method could also be used in other species to collect early free nuclear endosperm.

Detailed observation on expression dynamics of Polycomb group genes during rice early endosperm development in subspecies hybridization reveals their characteristics of parent-of-origin genes

“…To explore which allele of PcG gene in endosperm was transcribed, the expression pattern of each PcG gene in endosperms at different stages (from 1 dap to 7 dap) were carefully examined. The endosperm at different stages were isolated according to our previous method (Kuang et al 2015). cDNA was prepared using mRNA extracted from endosperm at different stages.…”

“…Early researches indicated that several key endosperm developmental events including the onset of primary endosperm nucleus division, the compartmentalization of endosperm nuclei and the initiation of endosperm cellularization occur within 3 dap (Brown et al 1996; Kuang et al 2015). In addition, the effect of imprinting gene on endosperm development in A. thaliana is majorly on early endosperm development (Baroux et al 2006; Jullien et al 2006; Luo et al 2000; Yadegari et al 2000).…”

“…In addition, the effect of imprinting gene on endosperm development in A. thaliana is majorly on early endosperm development (Baroux et al 2006; Jullien et al 2006; Luo et al 2000; Yadegari et al 2000). Hence, to analyze allelic expression patterns of PcG genes comprehensively, hybrid endosperms were isolated at six key stages of endosperm development including stage 1 (1 dap), stage 2 (2 dap), stage 3 (3 dap), stage 4 (7 dap), stage 5 (10 dap) and stage 6 (15 dap), and cDNA samples from hybrid endosperms were prepared according to the previous protocol (Kuang et al 2015).…”

“…The collection of hybrid ovaries at the same stages, endosperm isolation and mRNA extraction were carried out according to the previous protocol (Kuang et al 2015). For the crosses, the flowers were previously labeled, emasculated, and hand-pollinated.…”

An Efficient Antipodal Cell Isolation Method for Screening of Cell Type-Specific Genes in Arabidopsis thaliana

“…Prior to antipodal cell isolation, enzyme buffer, razor blades, glass capillaries, a Petri dish (Φ 3.5 cm), and 2x lysis buffer should be prepared for use, as described previously [12]. Enzyme buffer contains 10.5% (w/v) mannitol with 1% (w/v) cellulose (Yakult Honsha Co. Ltd, Tokyo, Japan) and 0.8% (w/v) Macerozyme (Yakult Honsha), pH 5.8.…”