Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/12/2003
Publication Date: 9/20/2003
Citation: Cazzonelli, C.I., Velten, J.P. 2003. Construction and testing of an intron-containing luciferase reporter gene from renilla reniformis. Plant Molecular Biology Reporter. p. 100-106. Interpretive Summary: The gene encoding a bioluminescence enzyme (luciferase) from the sea pansy has been used as a reporter gene in plants, indicating, through the production of light, when the gene is active. However, the original luciferase gene was also active within the bacteria (Agrobacteria) normally used to move genes into plants, creating an undesirable background of light in tissues that were infected with Agrobacteria during the process of genetic engineering. We have modified the sea pansy luciferase gene by the introduction of DNA sequence containing an intron. The intron sequence is removed during gene expression in plants but not in bacteria. Therefore, the new intron-containing luciferase gene can only be expressed within plant cells, eliminating the problems associated with background light production from bacteria. The new luciferase gene is a significant improvement for assays using luciferase expression to measure gene activity within plant cells.
Technical Abstract: A new Renilla reniformis luciferase reporter gene, RiLUC, designed to allow detection of luciferase activity in Agrobacterium-based transient expression studies is described. The RLUC gene was altered to contain a modified intron from the castor bean catalase gene, while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome. Both the native RLUC and intron-modified RiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activities within Agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, Agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these reporter gene fusions were co-transformed with a compatible normalization plasmid containing a Cauliflower Mosaic Virus-35S promoter (CaMV) joined to the Firefly Luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. Comparison of the kinetics of luciferase production from the RLUC or RiLUC reporters were consistent with expression of the RiLUC gene being limited to transiently transformed plant cells. RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either the SUPER-RLUC or SUPER-RiLUC reporter gene fusions showed that the presence of the intron did not alter the levels of luciferase activity or tissue specific expression patterns driven by the SUPER promoter. These results demonstrate that the RiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating Agrobacteria.