Elucidation of the biological function of an 83 kb large cytochrome P450 gene cluster in Arabidopsis

植物的特征是存在大量重复的基因。通常这些基因在功能上是冗余的，这使得很难或不可能从单插入突变体的表型分析中得出生物学功能的结论。最近，植物基因组编辑已经建立，它也允许多个基因突变。然而，靶向删除较大的基因簇仍然具有挑战性。作为一项概念验证研究，我们一直专注于拟南芥中最大的复制细胞色素P450基因集群（属于CYP71B亚家族），其跨度为83 kb。通过基于CRISPR/ cas9的方法，我们产生了纯合突变体，其中该簇被删除。这些植物完全是至关重要的，但显示出与防御相关的代谢产物的修改组成。为了明确集群基因在体内的功能，还将在不同组织中产生部分缺失和互补系，并通过比较代谢组学分析它们对病原体和非生物胁迫的反应。这将与酵母和烟叶中各自表达的P450酶的特性相补充。然后，我们将在其他CYP71B基因中产生缺失和插入突变的组合，以增强我们对这个大的多样化亚家族的功能理解。

Characteristic for plants is the presence of clusters of duplicated genes. Often these genes are functionally redundant, which makes it difficult or impossible to draw conclusions on biological functions from the phenotypic analysis of single insertion mutants. Recently, genome editing has been established for plants, which also allows mutation of more than one gene. However, targeted deletion of larger gene clusters is still challenging. As a proof of concept study, we have been focusing on the largest cluster of duplicated cytochrome P450 genes (belonging to the CYP71B subfamily) in Arabidopsis thaliana, which spans 83 kb. By a CRISPR/Cas9-based approach we have generated homozygous mutants in which this cluster is deleted. These plants are fully vital, but show a modified composition of defence-related metabolites. To specifically identify in vivo functions of the cluster genes, also partial deletion and complementation lines will be generated and analysed by comparative metabolomics in different tissues and in response to pathogens and abiotic stresses. This will be complemented with the characterisation of the respective P450 enzymes expressed in yeast and Nicotiana benthamiana. We will then generate combinations with deletion and insertion mutations in other CYP71B genes to enhance our functional understanding of this large diversified subfamily.