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Construction and testing of an intron-containing luciferase reporter gene fromRenilla reniformis

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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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Abbreviations

CaMV 35S:

cauliflower mosaic virus 35S RNA promoter

DTT:

dithiothreitol

GUS :

β-glucuronidase

Km:

kanamycin

LB:

Luria-Bertani

FLUC :

Photinus pyralis (firefly) luciferase

FiLUC :

Photinus pyralis (firefly) intron-modified luciferase

LARII:

luciferase reagent II (Promega)

MgCl2 :

magnesium chloride

MS:

Murashige and

Skoog:

ng, nanograms

Nost :

nopaline synthase terminator

NPTII :

neomycin phosphotransferase II

pBS:

pBluescript II (SK+)

S&G:

stop and glo (Promega)

R0 :

primary transformant

R1 :

progeny of self-fertilized R0 plant

RLUC :

Renilla reniformis (sea pansy) luciferase

RiLUC :

Renilla reniformis (sea pansy) intron-modified luciferase

RLU:

relative light units

TBE:

tris-borate EDTA

T-DNA:

transferred-DNA

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  1. Jeff Velten

    Present address: , 3810 4th Street, 79415, Lubbock, TX, USA

Authors and Affiliations

  1. United States Department of Agriculture-Agricultural Research Services, USDA-ARS, USA

    Christopher Ian Cazzonelli & Jeff Velten

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  1. Christopher Ian Cazzonelli
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  2. Jeff Velten
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Correspondence to Jeff Velten.

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Cazzonelli, C.I., Velten, J. Construction and testing of an intron-containing luciferase reporter gene fromRenilla reniformis . Plant Mol Biol Rep 21, 271–280 (2003). https://doi.org/10.1007/BF02772802

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