Hellens Roger P, Allan Andrew C, Friel Ellen N, Bolitho Karen, Grafton Karryn, Templeton Matthew D, Karunairetnam Sakuntala, Gleave Andrew P, Laing William A
HortResearch, Mt Albert Research Centre, Private Bag 92169, Auckland, New Zealand.
Plant Methods. 2005 Dec 18;1:13. doi: 10.1186/1746-4811-1-13.
We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.
We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs.
In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.
我们描述了用于瞬时基因表达的新型质粒载体，该载体通过农杆菌渗入本氏烟草叶片中。我们通过去除稳定转化所需的传统二元载体元件（如转化选择基因），构建了一系列非常适合瞬时基因表达的pGreenII克隆载体。
我们给出了血红素硫醇盐P450表达的例子，以证明该系统的有效性。我们还设计了利用双荧光素酶检测系统来分析启动子序列或基因表达转录后调控的载体。我们通过共表达假定的转录因子和潜在靶基因的启动子序列证明了它们的实用性，并展示了直系同源启动子序列对这些基因的反应。最后，我们构建了一个载体，使我们能够研究与发夹结构启动RNA沉默能力相关的设计特征，并利用这些工具研究含内含子基因构建体的顺式调控效应。
在开发一系列非常适合瞬时表达分析的载体过程中，我们提供了一种进一步推动该技术应用的资源。这些最小化载体非常适合传统克隆方法，我们已用它们证明了其在瞬时分析中研究酶活性、转录调控和转录后调控过程的灵活性。
