Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants.
Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants.
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DOI：
10.1186/1746-4811-1-13
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发表时间：
2005-12-18
期刊：
影响因子：
5.1
通讯作者：
Laing WA
中科院分区：
文献类型：
--
作者：
Hellens RP;Allan AC;Friel EN;Bolitho K;Grafton K;Templeton MD;Karunairetnam S;Gleave AP;Laing WA
我们描述了新的质粒载体瞬时基因表达，利用农杆菌，渗入烟草本塞姆安叶片。我们已经产生了一系列的pGreenII克隆载体，非常适合于瞬时基因表达，通过去除稳定转化所需的常规二元载体的元素，如转化选择基因。我们给出了一个表达血红素硫醇盐P450的例子来证明这个系统的有效性。我们还设计了利用双荧光素酶测定系统来分析启动子序列或基因表达的转录后调控的载体。我们已经证明了它们的效用，通过共表达的假定的转录因子和潜在的靶基因的启动子序列，并显示如何orthopathic启动子序列响应这些基因。最后，我们构建了一个载体，使我们能够研究发夹构建体与其启动RNA沉默能力相关的设计特征，并使用这些工具研究含内含子基因构建体的顺式调节效应。在开发一系列非常适合瞬时表达分析的载体的过程中，我们提供了进一步推进该技术应用的资源。这些最小载体非常适合常规克隆方法，我们已经使用它们来证明它们在瞬时测定中研究酶活性、转录调节和转录后调节过程的灵活性。
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.
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影响因子：
7.4
作者：
Blount, JW;Korth, KL;Dixon, RA
通讯作者：
Dixon, RA
DOI：
10.1007/bf00331014
发表时间：
1986-01-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
KONCZ, C;SCHELL, J
通讯作者：
SCHELL, J
DOI：
10.1073/pnas.96.24.14147
发表时间：
1999-11-23
影响因子：
11.1
作者：
Voinnet, O;Pinto, YM;Baulcombe, DC
通讯作者：
Baulcombe, DC
影响因子：
4.5
作者：
Rose, AB
通讯作者：
Rose, AB
影响因子：
7.2
作者：
Wesley, SV;Helliwell, CA;Waterhouse, PM
通讯作者：
Waterhouse, PM