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

阐明拟南芥中 83 kb 大细胞色素 P450 基因簇的生物学功能

• 批准号: 463197382
• 负责人: Professor Dr. Erich Glawischnig
• 金额: --
• 依托单位: Professur für Mikrobielle Biotechnologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/463197382?language=en
• 关键词: Elucidation; biological; function; 83; kb

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.

植物的特征是存在重复基因簇。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，植物基因组编辑技术已经建立，这也允许多个基因发生突变。然而，靶向删除较大的基因簇仍然具有挑战性。作为概念验证研究，我们一直关注拟南芥中最大的重复细胞色素 P450 基因簇（属于 CYP71B 亚家族），该基因簇跨度为 83 kb。通过基于 CRISPR/Cas9 的方法，我们生成了删除该簇的纯合突变体。这些植物具有完全的生命力，但与防御相关的代谢物的组成发生了改变。为了具体鉴定簇基因的体内功能，还将通过不同组织中的比较代谢组学以及对病原体和非生物胁迫的反应来生成和分析部分缺失和互补系。这将通过酵母和本塞姆氏烟草中表达的各自 P450 酶的表征得到补充。然后，我们将在其他 CYP71B 基因中生成删除和插入突变的组合，以增强我们对这个大型多样化亚家族的功能理解。