Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants
Roger P. Hellens,Andrew C. Allan,Ellen N. Friel,Karen Bolitho,Karryn Grafton,Matthew D. Templeton,Sakuntala Karunairetnam,Andrew P. Gleave,William A. Laing +8 more
TLDR
In this article, a series of plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves, are described and compared to conventional binary vectors for stable transformation such as transformation selection genes.Abstract:
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.read more
Citations
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Journal ArticleDOI
Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10
Richard V. Espley,Roger P. Hellens,Joanna Putterill,David E. Stevenson,Sumathi Kutty-Amma,Andrew C. Allan +5 more
TL;DR: In this article, the authors reported the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple compared with a white-fleshhed cultivar.
Journal Article
Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10
Richard V. Espley,Roger P. Hellens,Joanna Putterill,David E. Stevenson,Sumathi Kutty-Amma,Andrew C. Allan +5 more
TL;DR: The strong correlation between the expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this transcription factor is responsible for controlling anthocianin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdmyB10 expression concurrent with colour formation during development.
Journal ArticleDOI
pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants
TL;DR: V vectors have been developed which allow the direct cloning of genes into the binary plasmid by both restriction enzyme-based cloning and GATEWAY recombination and N- or C-terminal histidine tags may be fused to the target sequence as required.
Journal ArticleDOI
Photoexcited CRY2 Interacts with CIB1 to Regulate Transcription and Floral Initiation in Arabidopsis
TL;DR: The identification and characterization of the Arabidopsis CIB1 (cryptochrome-interacting basic-helix-loop-Helix) protein is reported, and it is proposed that the blue light–dependent interaction of cryptochrome(s) with C IB1 and CIB 1-related proteins represents an early photoreceptor signaling mechanism in plants.
Journal ArticleDOI
Brassinosteroid, gibberellin and phytochrome impinge on a common transcription module in Arabidopsis.
Ming-Yi Bai,Jian Xiu Shang,Jian Xiu Shang,Eunkyoo Oh,Min Fan,Yang Bai,Rodolfo Zentella,Tai-ping Sun,Zhi-Yong Wang +8 more
TL;DR: It is shown that BR and GA act interdependently through a direct interaction between the BR-activated BZR1 and GA-inactivated DELLA transcription regulators, which defines a core transcription module that mediates coordinated growth regulation by GA, BR and light signals.
References
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An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.
TL;DR: A system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus, that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression is described.
Journal ArticleDOI
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