Hairy root

花匠小妙招
2024-11-24 12:21

摘要

Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system for activation tagging of genes in "transformed hairy roots". The binary vector pHR-AT (Hairy Root-Activation Tagging) and its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest.展开▼

机译：激活标记是一种在植物中产生功能增强突变体的强大技术。我们开发了一种新的载体系统，用于“转化的毛状根”中基因的激活标记。二元载体pHR-AT（毛发根激活标记）及其派生的pHR-AT-GFP包含一簇rol（生根基因）基因，以及面向花椰菜花叶病毒（CaMV）35S增强子的四个串联重复序列的右边界。同一T-DNA上的元素。使用拟南芥，马铃薯和烟草作为模型植物的转化实验表明，用带有这些载体的根癌农杆菌接种植物后，可以在短时间内从外植体中诱导出大量独立转化的根，随后建立了根系培养系。。对pHR-AT-GFP转化的拟南芥系的分子分析表明，与T-DNA插入位点相邻的基因的表达显着增加。该系统可以促进将激活标记方法应用于对转基因植物的再生不利的植物物种。激活标记的根系培养物系的高通量代谢谱分析将提供机会，以鉴定调控或生物合成基因，以生产有用的有价值的次级代谢产物。展开▼