A cyclic nucleotide sensitive promoter reporter system suitable for bacteria and plant cells

BMC Biotechnology

Janet I Wheeler¹  Lubna Freihat¹  Helen R Irving¹ 

[1] Monash Institute of Pharmaceutical Sciences, Monash University (Parkville campus), 381 Royal Parade, Parkville, VIC 3052, Australia

关键词: OPTX promoter;    Luciferase reporter;    Cyclic AMP;    Cyclic GMP;   

Others  :  835117
DOI  :  10.1186/1472-6750-13-97

 received in 2013-08-19, accepted in 2013-10-30,  发布年份 2013

【 摘 要 】

Background

Cyclic AMP (cAMP) and cyclic GMP (cGMP) have roles in relaying external signals and modifying gene expression within cells in all phyla. Currently there are no reporter systems suitable for bacteria and plant cells that measure alterations in downstream gene expression following changes in intracellular levels of cyclic nucleotides. As the plant protein OLIGOPEPTIDE TRANSPORTER X (OPTX) is upregulated by cGMP, we fused the OPTX promoter to a luciferase reporter gene (OPTX:LUC) to develop a plant cell reporter of cGMP-induced gene expression. We prepared a second construct augmented with three mammalian cGMP response elements (OPTXcGMPRE:LUC) and a third construct containing five gibberellic acid response elements (OPTXGARE:LUC). All three constructs were tested in bacteria and isolated plant protoplasts.

Results

Membrane permeable cGMP enhanced luciferase activity of OPTX:LUC and OPTXGARE:LUC in protoplasts. Treatment with the plant hormone gibberellic acid which acts via cGMP also generated downstream luciferase activity. However, membrane permeable cAMP induced similar responses to cGMP in protoplasts. Significantly increased luciferase activity occurred in bacteria transformed with either OPTXcGMPRE:LUC or OPTXGARE:LUC in response to membrane permeable cAMP and cGMP. Bacteria co-transformed with OPTXcGMPRE:LUC or OPTXGARE:LUC and the soluble cytoplasmic domain of phytosulfokine receptor1 (PSKR1; a novel guanylate cyclase) had enhanced luciferase activity following induction of PSKR1 expression.

Conclusions

We have developed promoter reporter systems based on the plant OPTX promoter that can be employed in bacteria and isolated plant cells. We have shown that it can be used in bacteria to screen recombinant proteins for guanylate cyclase activity as increases in intracellular cGMP levels result in altered gene transcription and luciferase activity.

【 授权许可】

   
2013 Wheeler et al.; licensee BioMed Central Ltd.

【 图 表 】

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