P1-Bh: A Maize Regulatory Gene Controlled by DNA Methylation

这个项目将集中在Pl-Bh的分子和遗传分析上，Pl-Bh是一个控制玉米植株中紫色花青素合成的基因。初步分析表明，该基因在核苷酸水平上与野生型Pl基因几乎相同，但与该基因不同的是，该基因高度甲基化。计划中的实验将检验这一假设，即Pl-Bh DNA的甲基化是导致该等位基因表达模式多样化或组织特异性改变的原因，或者两者兼而有之。甲基化状态将使用基因组测序方法确定，并将与植物和籽粒组织中浅色和深色细胞中的基因表达水平相关。染色质构型和/或蛋白质-DNA接触的可能变化将通过DNA酶足迹进行分析。通过基因枪法将野生型和突变型基因导入不同的组织，其表达与表型差异有关。我们将寻找突变等位基因的遗传修饰物，并将确定该修饰是否是Pl-Bh等位基因甲基化模式改变的结果。DNA甲基化在许多真核生物的基因表达调控中起着重要作用，通常通过沉默基因表达来实现。仅在少数情况下，它被证明影响表达的组织特异性。这些实验的结果应该有助于加强我们对这一机制和其他调控植物基因表达的表观遗传机制的理解。$$$

This project will focus upon the molecular and genetic analysis of Pl-Bh, a gene which controls the synthesis of purple anthocyanin pigments in the maize plant. Preliminary analysis indicates that at the nucleotide level, this gene is nearly identical to the wild- type Pl gene but, unlike that gene, is heavily methylated. The planned experiments will test the hypothesis that methylation of the Pl-Bh DNA is responsible for the variegated pattern of expression or the altered tissue-specificity of this allele or both. The methylation status will be determined using genomic sequencing methods and will be correlated with the level of gene expression in lightly pigmented and darkly pigmented cells from plant and kernel tissues. Possible alterations in chromatin configuration and/or protein-DNA contacts will be analyzed by DNase footprinting. Both the wild-type and mutant genes will be introduced into various tissues by microprojectile bombardment and their expression correlated with phenotypic differences. We will look for genetic modifiers of the mutant allele and it will be determined if the modification is a result of alterations in the methylation pattern of the Pl-Bh allele. %%% DNA methylation plays an important role in the regulation of gene expression in many eukaryotes, usually by silencing gene expression. In only a few cases has it been shown to influence the tissue-specificity of expression. The results of these experiments should serve to enhance our understanding of this and other epigenetic mechanisms which modulate plant gene expression. $$$