Applications Of Luciferases In Studies Of Plant Promoters And Plant Stress Tolerance

The firefly luciferase isolated from North American firefly is a 61 KDa monomeric protein that does not require post-translational processing for enzymatic activity. The properties of the firefly luciferase make it a very good non-destructive reporter to quantify and image transgene promoter activity in plants. Genetic reporters are used commonly in cell biology to study gene expression and other cellular events coupled to gene expression, such as receptor activity, intracellular signal transduction, mRNA processing, protein folding and protein-protein interactions. Luciferases are nowadays successfully used as reporter genes for gene expression studies in living cells. Their application is growing in all aspects of the biological field such as animal research, environmental and plant research. In this article we studied the possible contribution of the different promoters to differences and changes in transgenic Arabidopsis luciferase activity. During high temperature stress, molecular chaperones are believed to act in preventing irreversible protein denaturation that would otherwise be detrimental to the cell. Although molecular chaperones are structurally diverse, they share the property of binding other proteins that are in non-native structural states and thereby facilitate many basic cellular processes, including protein folding, targeting, and degradation. Defining the function of molecular chaperones is clearly critical to understanding the response of plants to high temperature stress. The firefly luciferase is reported to be a good indicator of different chaperone activities both in vivo and in vitro. To test this, we developed a system to determine chaperone activities in vivo by using firefly luciferase as a reporter. The results were shown as follows:1. The construction of dual luciferase reporter vectorIn dual-luciferse reporter assay, Renilla luciferase was used as a co-reporter for normalization of experimental variations such as differences in transfection efficiencies. The ACT7-DUAL-LUC-PBI101and CaMV35S/HS-DUAL-LUC-PBI101 constructed vectors was introduced into Arabidopsis mediated by Agrobacterium tumefactions, and the transgenic Arabidopsis with dual-luciferase reporter vectors was generated. PCR analyses confirmed that the vectors have been integrated into transgenic Arabidopsis. No significant differences were detected in FLUC to RLUC ratios between different transgenic lines. Position effect was eliminated by using dual-luciferase reporter vectors. Here we have presented a simple and reliable method using stable expression of genes in Arabidopsis to test for a range of promoter activities.2. Firefly luciferase was used to analyze chaperone activityWe isolated the 1784 mutants based on their loss of thermotolerance phenotype. The constructed CaMV35S-FLUC-3301 and HS-FLUC-3301 vectors were introduced into wild type and Arabidopsis mutants mediated by Agrobacterium tumefactions, and the transgenic Arabidopsis with CaMV35S-FLUC-3301 and HS-FLUC-3301 vectors was generated. PCR analyses confirmed that the CaMV35S-FLUC-3301 and HS-FLUC-3301 vectors have been integrated into transgenic Arabidopsis. Firefly luciferase was highly expressed in transgenic Arabidopsis, which is critical for chaperone activity studies.