Dual-luciferase reporter gene assay

Service Introduction

Luciferase refers to a group of enzymes in organisms that catalyze the oxidation of luciferin or fatty aldehydes to produce light, found in naturally luminescent organisms. Currently, the luciferase gene derived from the North American firefly (Photinus pyralis) is the most widely used. By utilizing the property of luciferase to undergo a chemiluminescent reaction with its substrate, the regulatory elements of the gene of interest can be cloned upstream or downstream of the firefly luciferase gene to construct a luciferase reporter plasmid. The plasmid is then transfected into cells, and after appropriate stimulation or treatment, the cells are lysed to measure luciferase activity. The effect of different stimuli or conditions on the regulatory elements of interest can be determined by comparing luciferase activity before and after stimulation.

Promoter structure analysis:

The promoter region sequence (usually around 2 kb) is segmented and truncated, or specific sites are mutated, and then each version is constructed into a luciferase reporter vector to replace the promoter sequence to assess its promoter activity. Analysis of specific transcription factor and promoter interactions. The sequence (usually the promoter region) is inserted into a reporter gene vector, and the transcription factor is co-overexpressed in protoplasts, to analyze whether overexpression of the transcription factor increases or decreases luciferase activity.

Analysis of interactions between transcription factors and regulatory sequences:

By inserting the sequence (usually the promoter region) into a reporter gene vector, and co-transfecting its transcription factor into experimental cells, it is possible to analyze whether the transcription factor increases luciferase activity.

miRNA Target Gene Interaction Analysis:

Since miRNAs mainly function by acting on the 3’UTR of target genes, the 3’UTR of a gene of interest can be constructed downstream of a reporter gene, luc. By comparing the changes in reporter gene expression after overexpressing or inhibiting the miRNA, the inhibitory effect of the miRNA on the target gene can be quantitatively assessed; methods such as point mutations at the functional site can further identify the specific binding site of the miRNA on the target gene's 3’UTR.

Service Advantages

▶High sensitivity, over 1000 times more sensitive than Western blot;

▶Allows for preliminary testing of promoter activity;

▶Includes positive controls to ensure the reliability of the experimental process; 

▶In vivo experiments, providing a more accurate reflection of interactions within the plant.

Service Process 

Article Case

(1) Analysis of interactions between transcription factors and regulatory sequences

Petal size is controlled by the MYB73/TPL/HDA19-miR159-CKX6 module regulating cytokinin catabolism in Rosa hybrida

academic journal：nature communications

Repression of transactivation of the MIR159 promoter by RhMYB73. Left, diagrams of reporter and effectorconstructs. Middle images, brightfield and LUC activity. Right, quantitative analysis.Data are shown as means ± SD (n = 3). The experiments were performed independently twice with similar results, and one representative result is shown. Differentlowercase letters (in (d) and (e)) indicate significant differences according to oneway ANOVA with Tukey’s multiple comparisons test (P < 0.05). Asterisks (in (f))indicate statistically significant differences (two-sided Student’s t-test; **P < 0.01).

(2) Analysis of miRNA target gene interactions

Transcriptome and miRNAome analyses uncover the regulatory role of miR6155 in trichome development of tobacco

academic journal：Chemical and Biological Technologies in Agriculture

Validation of miRNAs and their targets in vivo. A Heatmap of miRNA fold change between trichome and leaf-trichome samples with respect to indicated target genes. Log2fold changes are shown here. B–F The validation of interactions between five known and novel miRNAs and their targets. Above, the target site between miRNA and corresponding targets. Below, photographs of the co-infiltrated N. benthamiana leaves on the third day after infiltration under UV light. Top left, pGreenII 62-SK+pGreenII 0800-target; top right, pGreenII 62-SK-miRNA+pGreenII0800-target; negative Control, pGreenII 62-SK+pGreenII 0800-LUC; lower right, pGreenII 62-SK-miRNA+pGreenII 0800-LUC. B NAC021 with its target miR164, C AP2 with its target miR172, D MYB36 with its target miR6145, E WRKY6 with its target miR6155, and F TIFY10B with its target novelmiRNA-9.

Applicable Fields

▶Promoter structure analysis: Segment and truncate the promoter region sequence (usually around 2 kb), or mutate specific sites, then construct them into luciferase reporter vectors separately to detect their promoter activity.▶Promoter SNP analysis: Some genes have single nucleotide polymorphisms in the promoter region, which can be analyzed for relative activity using a luciferase reporter system.

▶Verification of specific transcription factor interactions with regulatory sequences: Insert the sequence (usually the promoter region) into a reporter gene vector, and co-transfect the transcription factor in experimental cells to analyze whether overexpression of the transcription factor increases luciferase activity.

▶Analysis of signaling pathway activation: Insert the downstream response element sequence of the signaling pathway into the reporter gene vector. Under different upstream signaling conditions, luciferase activity represents the downstream response of the pathway. For example, in GPCR research, insert the cAMP response element (CRE) into a reporter gene vector and establish a stable expressing cell line to analyze GPCR activation and screen for inhibitors. Similarly, insert the hypoxia-responsive element (HRE), which is the response element of HIF1α, into a luciferase reporter vector to construct a stable cell line for studying hypoxia-related pathways.

▶Verification of microRNA target sequences: Insert the 3’UTR sequence to be tested into a reporter gene vector, then co-transfect with the microRNA. A decrease in luciferase activity indicates that it is a target sequence.