Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane
© Chou and Moyle; licensee BioMed Central Ltd. 2014
Received: 16 January 2014
Accepted: 31 March 2014
Published: 8 April 2014
An Expression of Concern has been published on this article.
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.
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.
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.
Molecular analysis of transgene expression can be hampered by events that lead to erratic, diminished or absolute loss of expression [1, 2]. Early attempts to express foreign transgenes frequently resulted in low levels of protein accumulation in transgenic plants. Vaeck et. al. introduced the bt2 gene from Bacillus thuringiensis into tobacco and detected the expressed protein at only 0.0002-0.02% of total soluble protein . Furthermore the majority of the bt2 transcripts were shorter than expected, presumed to be due to premature polyadenylation  and/or posttranscriptional processing and transcript instability . Similarly, the GFP reporter gene from Aequorea victoria is not expressed well in certain plant species. Little or no GFP fluorescence could be detected in Arabidopsis or tobacco transgenic plants despite the use of strong promoters such as CaMV 35S [6, 7].
Plant coding sequences generally have a codon bias relatively high in G + C content compared to bacteria, insects and other sources of foreign transgenes. A lower percentage G + C content and associated codon bias in foreign transgenes appears to have a major influence on transgene expression. Low transgene expression correlating with differences in codon bias has been hypothesised to be due to lower availability of specific tRNAs encoded by rare codons. The presence of these rare codons may cause stalling during translation, thereby destabilizing the transcripts by leaving the mRNA exposed to components of the RNA degradation machinery. Furthermore, A + T rich transgene sequences may provide motifs that will function as splice sites, polyadenylation sequences and RNA destabilizing elements in plants [4, 8, 9]. Thus using the redundancy of the genetic code to design synthetic copies of foreign transgenes with higher G + C contents can potentially not only increase translational efficiency, but can also remove deleterious A + T rich sequence motifs responsible for mRNA instability . Both GFP and bt2 transgene expression were greatly enhanced in transgenic plants when the codon usage was optimized, increasing the overall G + C content [5, 7]. A modified firefly luciferase gene with improved codon usage for mammalian cells (1.8% higher G + C content) had 14-23× increased activity in maize suspension cells and 53-59× increased activity in wheat scutellum .
Sugarcane species are tall perennial grasses of the genus Sacharrum. The jointed fibrous sugarcane stalks are rich in sugar and can measure up to six meters tall. Modern cultivars are highly polyploidy hybrids of S. officinarum and S. spontaneum with 2n = 100–130 chromosomes . Sugarcane is an important economic crop, responsible for the majority of the world’s sugar production and is also recognised as the most sustainable of the current generation of biofuel crops. As such there is a large degree of interest in researching and engineering sugarcane varieties using molecular and biotechnology approaches . Substantial progress has been made to develop the enabling technologies and tools necessary for molecular analysis and applied biotechnology in sugarcane. An extensive EST collection has been amassed , small RNA developmental profiles analyzed [14–17], (Sternes and Moyle: Deep sequencing reveals divergent expression patterns within the small RNA transcriptomes of cultured and vegetative tissues of sugarcane, under review) transformation systems developed [18, 19] and promoter sequences isolated [[20–24]]. However, molecular analysis in transgenic sugarcane has been hampered by events that have led to erratic, diminished or absolute loss of foreign transgene expression. Past studies aimed at the functional analysis of promoter sequences targeted for use in sugarcane biotechnology have reported aggressive silencing of reporter transgenes. Expression of the GUS reporter gene under the control of the ubi4 or ubi9 promoter was aggressively repressed in transgenic sugarcane plants . Nuclear run-off assays prove the repression of the GUS transgene expression was due to post-transcriptional silencing (PGTS) . Mudge et al. (2009)  reported expression of both the GUS-Plus and firefly luciferase reporter genes were similarly repressed in mature sugarcane plants, even under the control of endogenous MYB gene promoters. Indeed the firefly luciferase gene was found to be strongly down-regulated in transgenic sugarcane lines under the control of a range of endogenous, foreign and recombinant promoters [26, 27]. Recently a synthetic GUS transgene, codon optimised for use in sugarcane, was shown to generate significantly higher levels of GUS activity than the native GUS reporter gene . Similar design rules were applied to synthesize a silencing resistant version of a sucrose isomerase transgene, used in metabolic engineering of sugarcane to produce alternative sucrose isomers [19, 28].
Firefly luciferase has been used as a reporter gene of promoter function in many plant species [29–35]. The luciferase reaction emits light that can be detected under long exposure camera images or quantified using a luminometer. Together with the Renilla luciferase gene, dual luciferase assays are particularly useful in promoter analysis studies, where the Renilla luciferase gene is typically used to normalise expression across different co-bombardments, transfections or treatments [35, 36]. However, the propensity for firefly luciferase transgene expression to be aggressively repressed in transgenic sugarcane is a major obstacle to the usefulness of luciferase as a reporter of promoter function in sugarcane .
We have investigated designing synthetic versions of both the firefly luciferase and Renilla luciferase reporter transgenes as a strategy to combat silencing mechanisms in sugarcane. By synthesising a higher G + C content in line with sugarcane codon usage and avoiding known RNA instability motifs, we successfully designed firefly and Renilla luciferase transgenes that yield significantly higher activity levels in both transient assays and in stable transgenic lines of sugarcane. Additionally, we used a novel transient co-bombardment and quantitative real time PCR assay to determine that the increase in expression from the optimised luciferase transgenes is at least in part due to increased mRNA stability.
Sequence analysis of synthetic firefly and Renillaluciferase coding sequences
The average G + C content of 71 Sacchurum officinarum gene coding sequences in the Kazusa database is 55.7% . In contrast, firefly luciferase (luc) and Renilla luciferase (Renluc) coding sequences have G + C contents of just 45% and 36.5% respectively. We took advantage of the redundancy of the genetic code to design new sequences encoding for synthetic versions of luc and Renluc, and to eliminate rare codons, strings of A or T bases, palindromes, polyadenylation signals, and to avoid the potential addition of an intron (refer to Methods section for details). The synthetic versions, denoted as luc* and Renluc*, have a G + C content of 55% and 52% respectively. Sequence alignment reveals the synthetic luc* and Renluc* sequences share 75% identity with the native luc and Renluc sequences (Additional file 1: Figure S1 and Additional file 2: Figure S2). The sequence of luc* and Renluc* can be found in GenBank accessions KC147725 and KJ140114, respectively.
luc* and Renluc*generate higher transient expression levels than the native firefly and Renillaluciferase genes
Using the pUbi-luc* construct to normalise Renilla luciferase expression across each co-bombardment, dual luciferase activity measurements showed the pUbi-Renluc* construct produced on average 15-fold higher luciferase activity than pUbi-Renluc (Figure 2B).
Synthetic luc* and Renluc* generate higher steady state transcript levels than the native luc and Renluctransgenes
Equal quantities of pUbi-luc and pUbi-luc* (or pUbi-Renluc and pUbi-Renluc*) plasmid were subsequently co-bombarded at sugarcane suspension cell cultures. RNA was extracted 24 h post-bombardment and cDNA synthesised. qRT-PCR reactions using the cDNA as a template revealed a shift in the amplification curve between reactions using either the native luc or synthetic luc* primer pairs (Figure 3B and E). As each primer pair amplifies at an equal rate, the shift in the amplification curve is due to a differential abundance of luc transcript relative to the luc* transcript in the co-bombarded suspension cell cDNA. The co-bombardment and qRT-PCR assay reveals ~9-fold higher luc* transcript levels, on average, relative to luc (Figure 3C). Using the same co-bombardment and qRT-PCR approach, we detected ~6.5-fold higher Renluc* transcript levels, on average, relative to Renluc (Figure 3F). This co-bombardment and qRT-PCR approach assumes there is no gene specific bias during cDNA synthesis.
The synthetic luc* transgene generates substantially higher luciferase activity than the native firefly luctransgene in stably transformed lines
Fold expression changes in multiple tissues assayed from pUbi- luc and pUbi- luc* transgenic lines of sugarcane
pUbi-lucMean +/- SD
pUbi- luc* Mean +/- SD
Mean fold difference
20040 +/- 8311
2215000 +/- 979000
264000 +/- 205500
4829000 +/- 1770000
52730 +/- 23730
7030000 +/- 2913000
139.4 +/- 105.8
1031000 +/- 447200
1455000 +/- 869400
33430000 +/- 10180000
302800 +/- 82140
42080000 +/- 12360000
6.619 +/- 3.353
5312 +/- 3249
56.70 +/- 19.09
6430 +/- 2925
144.3 +/- 64.86
6033 +/- 2029
165.2 +/- 68.15
5680 +/- 1937
231.6 +/- 85.04
4203 +/- 1077
323.6 +/- 109.7
3470 +/- 813.3
440.3 +/- 137.4
3045 +/- 663.4
397.8 +/- 123.9
3014 +/- 698.4
441.3 +/- 116.5
3296 +/- 737.6
421.5 +/- 107.8
4047 +/- 1093
414.4 +/- 90.91
3757 +/- 1057
3rd basal Internode
601.5 +/- 155.5
5472 +/- 1287
Firefly luciferase is a reporter gene commonly used in promoter functional analysis studies. Firefly luciferase converts the substrate luciferin to oxyluciferin and releases light during the reaction. The light emitted can be detected and quantified using a luminometer or visualised using long exposure cameras. Renilla luciferase converts coelenterazine to coelenteramide in a reaction that also emits light. Together, firefly luciferase and Renilla luciferase form the basis of a dual luciferase assay that provides researchers with a sensitive and normalised method to quantify promoter activity. However, the application of luciferase genes as reporters of promoter function in sugarcane has been thwarted by aggressive down-regulation and silencing .
Synthesising foreign transgene sequences to contain optimized codon usage and G + C contents closer to that of the host species has been an effective way to increase transgene expression in many plant species . Modifying a foreign transgene sequence to mimic coding sequences endogenous to the host plant can be thought of as a ploy to prevent triggering the host plant’s defence mechanisms against foreign gene sequences.
In this study we modified the firefly luciferase and Renilla luciferase reporter gene sequences and assessed any enhanced effects on transgene expression in sugarcane. One cautionary note when using firefly luciferase as a reporter in sugarcane comes from the discovery that certain sugarcane tissue extracts inhibit luciferase activity and that some tissue extracts confer stronger inhibition than others . Fortunately, the inhibitory effect can be diluted out and we recommend utilising a 50-100× dilution of tissue extracts (at the concentrations specified in the material and methods) prior to measurement of luciferase activity using a luminometer.
Transient assays in sugarcane suspension cell cultures revealed the synthetic luc* gene produced on average 60-fold higher luciferase activity relative to the native firefly luciferase gene. The synthesized Renluc* gene yielded on average 15-fold higher luciferase activity than the native Renilla luciferase gene. Thus, modifying the codon usage of luciferase transgenes to mimic endogenous sugarcane genes and avoiding known destabilizing motifs proved to have a profound positive effect on transient luciferase transgene expression in sugarcane. Such transient assays may provide a fast and efficient preliminary screen for testing the effectiveness of synthetic transgene variants prior to undergoing analysis in stable transgenic lines.
Modifying codon usage has been postulated to increase translational efficiency by avoiding stalling at the ribosome due to uncommon codons encoding rare species of tRNA. However, our design rules also include avoiding polyadenylation signals and known RNA destabilizing motifs. Indeed increasing the G + C content may have also eliminated, by chance, unknown A + T rich RNA destabilizing sequences. Therefore it is of interest to determine if the increase in luciferase activity of our synthesized genes is entirely due to increased translational efficiency or whether our sequence modifications have also increased the stability of the transcript. To compare the steady state transcript levels between the native luciferase genes and our synthesized versions, we developed a novel co-bombardment and qRT-PCR protocol. This transient assay allowed us to detect and quantify ~6.5-fold and ~9-fold increases in the transcript levels derived from both the synthesized Renluc* and luc* transgenes respectively, indicating our sequence modifications have had a positive effect on mRNA steady state levels.
Luciferase assays on populations of stable transgenic sugarcane lines further confirms improved luciferase activity from luc* over the native firefly luciferase gene. luc* activity was significantly higher across each tissue type and stage of development tested. However, it is interesting to note that the fold enhancement of activity by luc* varied considerably from tissue to tissue. An apparent trend that emerged from the data was that fold difference in expression between luc* and luc was progressively stronger in the younger tissues than in the older tissue types tested. It is possible that this sequence-specific tissue-dependent down-regulation of the native firefly luciferase transgene may be due to the action of temporally expressed endogenous regulatory small RNA species such as microRNA’s, for example. Alternatively, silencing mechanisms involving RNA destabilizing motifs, polyadenylation signals or translational inhibition mechanisms may be more pronounced in the younger and more metabolically active tissue types. Other possible explanations include that younger plant tissues have higher metabolic rates which might be reflected in higher transgene expression levels, or that different tissues in different developmental stages may have different populations of tRNA’s or different levels of specific tRNA’s which could potentially alter the codon usage, as has been documented in mammalian systems .
The development of synthetic luciferase genes that resist transgene silencing in sugarcane demonstrates the importance of performing careful codon/coding sequence optimization for plant transgene expression, using design rules that have evolved over a number of years from research in various plant species. These optimized luciferase transgenes will likely have great utility across research and applied studies in sugarcane and other monocots. For example, they will benefit future research aimed at characterizing expression patterns conferred by promoters as molecular tools used in basic and applied research. The synthetic luciferase reporter genes could also be used as markers of expression in emerging research fields. For example, we have tagged regulatory small RNA target sequences to the synthetic luc* coding sequence to investigate developmental patterns conferred by small RNAs in sugarcane (Moyle RL, Sternes PR and Birch RG: Incorporating target sequences of developmentally-regulated small RNAs into transgenes to enhance tissue specificity of expression in plants, submitted). These luciferases may also be useful in many dicot plant systems, although this aspect needs to be tested on a case-by-case basis.
The strategy of optimizing transgenes for expression in sugarcane and the molecular techniques developed to quantify enhanced expression characteristics could be applied to many other transgenes of interest in basic and applied sugarcane research. For example, there is much interest in metabolic engineering sugarcane to produce higher sugar yields, healthier sugars and other value-added products [12, 28, 39].
In conclusion, we demonstrate that designing transgenes to mimic codon usage in sugarcane and avoid known destabilizing motifs is an effective strategy to optimise transgene expression and prevent transgene silencing in sugarcane. We designed synthetic Renilla luciferase and firefly luciferase transgenes that were resistant to silencing in transient assays and in stable transgenic lines. The development of these silencing resistant luciferase reporter genes enables the functional analysis of promoter sequences for use in basic and applied sugarcane research. The application of our strategy to optimise transgene expression and avoid transgene silencing could be applied to other transgenes and advances the prospect of applying genetic engineering strategies to the improvement of sugarcane varieties.
Design and synthesis of luc* and Renluc* coding sequences
Our process for the design of sugarcane optimised synthetic luciferase transgenes used software tools to help eliminate sequence motifs with the potential to cause instability or generate silencing triggers. The design process involved generating sugarcane codon usage tables from the Kazusa database . We also generated a list of specified motifs to be excluded based on potential unintended intron splice signals, known triggers of RNA instability  and bioinformatic analysis of rice polyadenylation signals  (Additional file 6: Table S1). Candidate sequences were generated using the program Gene Designer . This program excludes codons below our specified threshold value of 20% and then uses a Monte Carlo algorithm based on the probabilities obtained from the codon usage table. Initial candidates were then filtered in an iterative process that attempts to meet additional design criteria including exclusion of the aforementioned motifs and certain DNA repeats, including those generating any mRNA structures with double-stranded RNA stems of 12 bp or more. The program typically was unable to converge on a solution that incorporated all of our specifications as the degeneracy of the excluded motifs in Additional file 6: Table S1 is very demanding. Nonetheless, it provided useful output for further manual refinement to eliminate features of concern such as residual AT-rich strings and repeats. The designed sequences (Additional file 1: Figure S1 and Additional file 2: Figure S2) were synthesised using Genscript Inc (http://www.gencript.com) as the service provider, including two tandem stop codons and flanking NotI and PacI restriction sites to facilitate cloning. The synthetic luc* and Renluc* sequences are available in the GenBank database (accession numbers KC147725 and KJ140114, respectively).
Construct design and preparation
Plasmid was prepared using the plasmid maxiprep kit (QIAGEN) according to the manufacturer’s instructions. The firefly luciferase, Renilla luciferase and synthesised luc* and Renluc* coding sequences were cloned into the pUbi T3 background plasmid , using unique NotI and PacI restriction sites (Figure 1). The pUKR plasmid, containing an nptII kanamycin resistance marker gene under the control of the pUbi promoter , was used for selection and for generating negative control lines.
Dual luciferase assays
Sugarcane suspension cell cultures were prepared for particle bombardment as previously described . Plasmid DNA (5 μg) was coated onto tungsten particles and co-bombarded onto plates of suspension cells as previously described . The bombarded suspension cell plates were incubated in the dark for 24 h. The suspension cells were harvested and ground in the lysis buffer provided in the dual luciferase reporter assay system kit (Promega). Dual luciferase assay reactions were prepared using the dual luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Luciferase activity was quantified using a BMG POLARstar OPTIMA luminometer.
Quantitative real time PCR
Equimolar quantities of synthesised and native luciferase constructs were co-bombarded onto suspension cell cultures as described above. Co-bombarded suspension cell cultures (100-200 mg) were ground in 1 mL of TRIzol® reagent (Invitrogen) and RNA extracted following the manufacturer’s instructions. Each RNA preparation was treated with 2 units of DNase I (New England Biolabs) at 37°C for 30 min followed by inactivation at 70°C for 10 min. cDNA was synthesized using Oligo(dT)20, as previously described [45, 46].
qRT-PCR primers were designed to the 3’ region of the target coding sequence (listed in supplementary Figure S3), taking advantage of transgene specific sequence polymorphisms, according to previously described methods . qRT-PCR was performed using FastStart Universal SYBR Green Master (ROX) (Roche Diagnostics) in a Rotorgene 3000 thermocycler (Corbett Research). After denaturation at 95°C for 10 min, the qRT-PCR cycles consisted of denaturation at 95°C for 15 s followed by annealing/extension at 62°C for 60 s for a total of 45 cycles. Differences in expression were calculated using the comparative CT method.
Generation of stable transgenic lines
Callus initiation from sugarcane cultivar Q117 and callus proliferation was performed as previously described . The aforementioned pUbi-luc or pUbi-luc* plasmids were co-bombarded with the selection plasmid pUKR, containing the nptII selection marker under the control of the pUbi promoter, as previously described [18, 27]. Lines of transgenic calli were selected, regenerated and grown in the glasshouse as previously described . Negative control plant lines were regenerated from calli bombarded with pUKR only .
Molecular analysis of independent transgenic lines
Multiple plants regenerated from each selected transgenic line of calli were analysed for relative copy number according to the methods described by Basnayake et al. 2011 , with the exception of using a different GAPDH reference gene 3’ primer (GAPDHrevb ACGGGATCTCCTCAGGGTTC). In cases where two or more clearly distinct copy numbers were identified among the population of plants regenerated from a single selected transgenic line of calli, those plants were separated and relabelled as independent transgenic lines.
Luminometer assay of firefly luciferase activity
Transgenic sugarcane tissues were harvested and snap frozen in liquid nitrogen prior to grinding into powder form using a ball mill apparatus (Retsch). Luminometer assays were performed on ground tissue extracts in CCLR* reagent using a BMG POLARstar Optima luminometer . We typically mixed ~15-20 mg of leaf tissue, ~15 mg of meristem-internode 2 tissue, ~50-100 mg of young internode tissue, ~100-200 mg of mature internode tissue and ~100 mg of root tissue with 100 μl of CCLR* to obtain each tissue extract. After thorough mixing, the extracts are centrifuged to pellet insoluble material. As luciferase activity can be inhibited by undiluted sugarcane tissue extracts , we typically dilute an aliquot of the soluble fraction of each tissue extract by 100x in CCLR* reagent prior to performing luciferase assay measurements in a luminometer. The output measurement readings from the luminometer are referred to as the relative light units (RLU). Bradford assays using protein assay dye reagent (Bio-Rad) were utilised to quantify the soluble protein concentration within the soluble fraction of each undiluted tissue extract .
In vivofirefly luciferase activity camera assay
Luciferase activity in plated and bombarded suspension cells or transverse internode sections was imaged by first immersing samples in 0.4 mM luciferin as previously described [44, 48]. The light emission was measured using a PIXIS 102A camera over a 500 second exposure time. WinView32 software (Princeton Instruments) was used to measure the average pixel intensities (background subtracted) across each internode section.
Luciferase activity data were log transformed to more closely approximate the mathematical assumption of normality and equality variance in the experimental data, and analysed using GraphPad Prism 5 software.
The authors acknowledge the assistance of Michael Graham in the design of luc* and Renluc* sequences. The authors acknowledge Robert Birch for critical assessment during preparation of the manuscript. This research was supported through a collaboration between CSR Sugar Limited (Sucrogen) and The University of Queensland under the Australian Research Council’s Linkage scheme.
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