Xiao Wenyan
Department of Biology, Saint Louis University, St. Louis, MO, USA.
Methods Mol Biol. 2012;925:231-47. doi: 10.1007/978-1-62703-011-3_16.
Plants are excellent systems for discovering and studying epigenetic phenomena, such as transposon silencing, RNAi, imprinting, and DNA methylation. Imprinting, referring to preferential expression of maternal or paternal alleles, plays an important role in reproduction development of both mammals and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA (MEA), a SET domain Polycomb group gene, was the first plant gene shown to be imprinted in endosperm, and its maternal expression is controlled by DNA methylation and demethylation. Recently there has been significant progress in identifying imprinted genes as well as understanding molecular mechanisms of imprinting in plants. Up to date, approximately 350 genes were found to have differential parent-of-origin expression in plant endosperm (Arabidopsis, corn, and rice). In Arabidopsis, many imprinted genes are regulated by the DNA METHYLTRANSFERASE1 (MET1) and the DNA-demethylating glycosylase DEMETER (DME), and/or their chromatin states regulated by Polycomb group proteins (PRC2). There are also maternally expressed genes regulated by unknown mechanisms in endosperm. In this protocol, we describe in detail how to perform a genetic cross, isolate the endosperm tissue from seed, determine the imprinting status of a gene, and analyze DNA methylation of imprinted genes by bisulfite sequencing in Arabidopsis.
植物是发现和研究表观遗传现象的优秀系统，例如转座子沉默、RNA干扰、印记和DNA甲基化。印记是指母本或父本等位基因的优先表达，在哺乳动物和植物的生殖发育中都起着重要作用。DNA甲基化对于确定印记基因的母本或父本等位基因是表达还是沉默至关重要。在开花植物中，生殖过程存在双受精事件：一个精子使卵细胞受精形成胚胎，另一个精子与中央细胞融合形成胚乳。胚乳是植物中发生印记的组织。MEDEA（MEA）是一个含有SET结构域的多梳家族基因，是第一个被证明在胚乳中印记的植物基因，其母本表达受DNA甲基化和去甲基化控制。最近，在鉴定印记基因以及了解植物印记的分子机制方面取得了重大进展。截至目前，在植物胚乳（拟南芥、玉米和水稻）中发现约350个基因具有不同的亲本来源表达。在拟南芥中，许多印记基因受DNA甲基转移酶1（MET1）和DNA去甲基化糖基化酶DEMETER（DME）调控，和/或其染色质状态受多梳家族蛋白（PRC2）调控。胚乳中也存在受未知机制调控的母本表达基因。在本实验方案中，我们详细描述了如何在拟南芥中进行遗传杂交、从种子中分离胚乳组织、确定基因的印记状态以及通过亚硫酸氢盐测序分析印记基因的DNA甲基化。
