21ENGBIO - Engineering Nucleosome Positioning in Plants

植物生物技术在全世界29个国家种植的生物技术作物方面取得了巨大成功，并与改善小农福利和减少农药使用有关。此外，植物栽培的低成本以及它们积累的天然产物的丰富性和多样性导致它们发展为用于生产在健康和工业中使用的有价值分子的光合平台。为了改良作物并提高有用分子的产量，我们需要详细了解调节功能如何编码到DNA序列中以及控制基因表达的方法。为了将染色体装入细胞核，真核生物的DNA被包装到染色质中。染色质的基本结构单位是核小体，由一段DNA缠绕在8个组蛋白周围组成。核小体的位置和稳定性可以决定DNA对蛋白质和其他因子的可接近性，因此在DNA复制和修复以及基因表达中具有重要作用。有证据表明，DNA序列的编码可以影响核小体的位置和稳定性。在酵母中的演示表明，重新编码DNA以影响核小体位置或稳定性可用于调节基因表达，但在植物中还没有进行类似的工作。在初步研究中，我们发现，植物调控序列的设计，以指定脆弱的核小体和增加的DNA的可及性增加表达。在这个提议中，我们将进一步研究这一点，目的是获得直接的证据，核小体定位可以用来调整植物合成启动子的水平。以前，我们和其他人也发现，合成基因的表达水平往往受到邻近基因表达的显著影响。在这里，我们还将研究使用核小体不利序列来减轻这些不希望的背景效应。

Plant biotechnology has been enormously successful with biotech crops grown in 29 countries worldwide and associated with improved welfare of smallholder farmers and reduced use of pesticides. In addition, the low cost of plant cultivation together with the abundance and diversity of natural products that they accumulate has led to their development as photosynthetic platforms for producing valuable molecules used in health and industry. For both crop improvement and to improve the yields of useful molecules, we require a detailed understanding of how regulatory function is encoded into DNA sequences together with methods to control gene expression.To fit their chromosomes into the nucleus, the DNA of eukaryotes is packaged into chromatin. The basic structural unit of chromatin is the nucleosome, consisting of a segment of DNA wound around eight histone proteins. The position and stability of nucleosomes can dictate the accessibility of DNA to proteins and other factors and, therefore, have important roles in DNA replication and repair as well as in gene expression. There is evidence that DNA sequences can be coded to influence the position and stability of nucleosomes. Demonstrations in yeast have shown that recoding DNA to influence nucleosome position or stability can be used to modulate gene expression but similar work has not been done in plants. In preliminary studies, we found that plant regulatory sequences designed to specify fragile nucleosomes and increase the accessibility of the DNA gave increased expression. In this proposal, we will investigate this further with the aim of obtaining direct evidence that nucleosome positioning can be used to tune the level of plant synthetic promoters. Previously, we and others have also found that expression levels from synthetic genes are often significantly affected by the expression of closely located genes. Here, we will also investigate the use of nucleosome disfavouring sequences to mitigate these undesired context effects.