Investigating and modulating recombination control in Arabidopsis thaliana using synthetic biology

交叉(Cos)是减数分裂前期I期非姐妹同源染色体之间的相互链交换。CoS的频率和分布受到高度调控，其中一个CO是保证中期I分离的保真性所必需的。在许多作物中，CoS的频率和分布往往受到限制。这对于培育具有优势等位基因的新型作物是有问题的。相比之下，芽殖酵母的一氧化碳含量相对较高，对一氧化碳的分布没有同样的限制。此外，CO的形成在真核生物之间是高度保守的。在这个项目中，我们将使用合成生物学来研究和调控拟南芥的减数分裂重组控制。这种方法将被用于研究已知的减数分裂基因和新的候选基因。我们将产生嵌合的酵母-拟南芥蛋白结构物-与酵母交换拟南芥蛋白基序或结构域-以及从从头开始的序列组装的新的蛋白质结构物。使用CRISPR/Cas9，这些构建物将被转化到拟南芥和酵母中。我们将使用最先进的荧光显微镜(包括共聚焦和超分辨率)以及涉及下一代测序和生物信息学分析的分子标记技术来分析产品。通过这个合成生物学系统，我们的目标是研究减数分裂调节蛋白的功能，并调节Cos的施加。这项工作将测试CO控制是否可以通过人工工程进行，并跨越王国的障碍。通过这一点，我们将进一步了解重组控制，这将有助于开发新的植物育种工具。

Crossovers (COs) are the reciprocal strand exchange between non-sister homologous chromosomes during prophase I of meiosis. The frequency and distribution of COs is highly regulated with one CO being essential for fidelity in segregation at metaphase I. In many crop plants, the frequency and distribution of COs is often restricted. This is problematic for generating novel crop plants with advantageous alleles. In contrast Budding yeast has proportionally high levels of COs and does not show the same restrictions to CO distribution. Furthermore, CO formation is highly conserved between eukaryotes. In this project we will use synthetic biology to investigate and modulate meiotic recombination control in Arabidopsis thaliana. This approach will be used to investigate known meiotic genes and novel candidates. We will generate both chimeric Yeast-Arabidopsis protein constructs - exchanging Arabidopsis protein motifs or domains with yeast - and novel protein constructs from de-novo sequence assemblies. These constructs will be transformed into Arabidopsis and Yeast - using CRISPR/Cas9. We will analyse the products using state-of-the-art fluorescence microscopy (including confocal and super-resolution) as well as molecular marker techniques involving next generation sequencing and bioinformatics analysis. Through this synthetic biology system, we aim to investigate the function of meiotic regulatory proteins and modulate the imposition of COs. This work will test if CO control can be both artificially engineered and transferred across kingdom barriers. Through this we will further understanding of recombination control which will facilitate the development of novel tools for plant breeding.