Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

Chou, Ting-Chun and Moyle, Richard L. (2014) Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. BMC Plant Biology, 14 1: 92.1-92.8. doi:10.1186/1471-2229-14-92


Author Chou, Ting-Chun
Moyle, Richard L.
Title Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane
Formatted title
Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane
Journal name BMC Plant Biology   Check publisher's open access policy
ISSN 1471-2229
Publication date 2014-04-08
Year available 2014
Sub-type Article (original research)
DOI 10.1186/1471-2229-14-92
Open Access Status DOI
Volume 14
Issue 1
Start page 92.1
End page 92.8
Total pages 8
Place of publication London, United Kingdom
Publisher BioMed Central
Language eng
Formatted abstract
Background: Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene.

Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane.

Results: The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences.

Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures.

Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes.

In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues.

Conclusions: We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: School of Agriculture and Food Sciences
Official 2015 Collection
 
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