Construction and testing of an intron-containing luciferase reporter gene from<Emphasis Type="Italic">Renilla reniformis</Emphasis> |
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Authors: | Christopher?Ian?Cazzonelli Email author" target="_blank">Jeff?VeltenEmail author |
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Institution: | (1) United States Department of Agriculture-Agricultural Research Services, USDA-ARS, USA;(2) Present address: 3810 4th Street, 79415 Lubbock, TX, USA |
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Abstract: | We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC 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.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity 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 gene fusions were cotransformed 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. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC 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 reporter gene fusion showed that the intron altered neither the levels
of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that
theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference
from contaminating agrobacteria. |
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Keywords: | dual luciferase assay firefly intron luciferase Photinus pyralis Renilla reniformis reporter gene sea pansy |
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