Molecular Cloning Strategy and Vector Design for ScWRKY5 Gene from Sugarcane Targeted to Tomato: A Framework for Abiotic Stress Tolerance Studies

Ambreen Ilyas
School of Biological Sciences, University of the Punjab, Lahore, Pakistan
Correspondence to: Ambreen Ilyas, Ambreen2.phd.sbs@pu.edu.pk

DOI: https://doi.org/10.70389/PJPB.100020

Additional information

• Ethical approval: N/a
• Consent: N/a
• Funding: No industry funding
• Conflicts of interest: None
• Author contribution: Ambreen Ilyas – Conceptualization, Writing – original draft, review and editing
• Guarantor: Ambreen Ilyas
• Provenance and peer-review:
  Unsolicited and externally peer-reviewed
• Data availability statement: N/a

Keywords: ScWRKY5, Codon optimization, Abiotic stress tolerance, In-silico cloning, Plant expression vector.

Peer Review
Received: 25 May 2025
Revised: 8 July 2025
Accepted: 8 July 2025
Published: 26 July 2025

Plain Language Summary Infographic

Introduction

Plants, as sessile organisms, are continually exposed to a wide range of environmental stresses, including drought, salinity, extreme temperatures, and oxidative damage, all of which significantly affect their growth, development, and productivity.^1,2 These abiotic stresses are responsible for substantial yield losses worldwide and pose a serious threat to sustainable agriculture, particularly under changing climatic conditions.³ Plants counter environmental stresses through complex signaling networks involving stress perception, signal transduction, and regulation of gene expression.⁴ Transcription factors (TFs) play a central role in these pathways by binding specific cis-regulatory elements in gene promoters.⁵ Among them, WRKY TFs form one of the largest, best-studied families, regulating abiotic and biotic stress responses, hormone signaling, senescence, and secondary metabolism.^6,7 These proteins feature one or two conserved WRKY domains with a characteristic WRKYGQK motif and a zinc-finger-like structure (C2H2 or C2HC), enabling specific binding to W-box elements (TTGACC/T) in target gene promoters.⁸

Based on the number of WRKY domains and the type of zinc-finger motifs, the WRKY family is classified into three main groups: Group I (with two WRKY domains), Group II (one WRKY domain and C2H2 motif), and Group III (one WRKY domain with a C2HC-type zinc finger).⁹ Group III WRKY TFs have been shown to play particularly pivotal roles in regulating plant tolerance to abiotic stresses by modulating stress-responsive gene networks.¹⁰ Several WRKY genes from different plant species have been reported to improve tolerance against drought, high salinity, and oxidative stress when overexpressed in model systems.^11,12

Sugarcane (Saccharum spp. hybrid) is a commercially significant crop for sugar and biofuel production, often cultivated in tropical and subtropical regions where it encounters multiple abiotic stresses, especially drought and salinity.^13,14 These environmental challenges severely restrict sugarcane productivity and sucrose accumulation, urging the need for biotechnological strategies to enhance stress tolerance in this crop.¹⁵ Although several stress-related genes have been identified in sugarcane, their functional characterization, particularly through heterologous expression in model plants, remains limited due to the crop’s genetic complexity and recalcitrance to transformation.¹⁶ Among the sugarcane WRKY gene family, ScWRKY5 has gained attention for its potential involvement in abiotic stress responses. Preliminary transcriptomic and functional studies have indicated its transcriptional activation activity and stress-inducible expression patterns under drought and oxidative conditions, suggesting a significant role in stress adaptation mechanisms.^17,18 However, its functional validation in heterologous systems like tomato, a model for dicotyledonous plants, has not yet been reported.

Tomato (Solanum lycopersicum) is an economically important vegetable crop and a widely used model system in plant molecular biology due to its well-annotated genome, ease of genetic manipulation, and susceptibility to diverse abiotic stresses such as drought, salinity, and temperature extremes.^19,20 The development of stress-tolerant transgenic tomato lines via the introduction of stress-associated TFs from other species represents a valuable approach for functional genomics and crop improvement studies.²¹ In silico codon optimization, primer design, and virtual cloning strategies have emerged as cost-effective and efficient preliminary steps in functional gene characterization. These computational tools allow for the prediction of gene expression efficiency, the design of optimal cloning strategies, and the simulation of protein expression in heterologous hosts.^22,23 This study, therefore, aimed to theoretically develop a cloning and heterologous expression strategy for the ScWRKY5 gene in tomato by employing codon optimization, primer design, vector selection, and in silico cloning approaches. The proposed strategy is intended to facilitate future experimental validation of ScWRKY5 in improving abiotic stress tolerance in tomato and other dicot crops.

Objectives

The primary aim of this study was to establish a comprehensive theoretical framework for the in silico codon optimization, cloning strategy design, and heterologous expression of the ScWRKY5 gene from sugarcane (Saccharum officinarum) in tomato (Solanum lycopersicum), targeting its potential role in abiotic stress tolerance (Figure 1). To achieve this, the study was structured around the following specific objectives:

• To retrieve, analyze, and codon-optimize the full coding sequence (CDS) of ScWRKY5 according to Solanum lycopersicum codon usage preferences, enhancing its predicted translational efficiency.
• To design gene-specific primers incorporating appropriate restriction sites for efficient PCR amplification and directional cloning of the optimized gene.
• To select an appropriate plant expression vector capable of supporting efficient heterologous expression in tomato and compatible with the designed cloning strategy.
• To perform in silico cloning, sequence validation, and biosafety assessment using advanced bioinformatics tools, ensuring the construct’s suitability for subsequent experimental validation in stress tolerance studies.

Methodology

Sequence Retrieval: The full-length nucleotide sequence of the class III WRKY TF gene ScWRKY5 (945 bp) was retrieved from the NCBI GenBank database under accession number MT003230.1.¹⁷

• The optimized ScWRKY5 sequence has been deposited in GenBank under temporary accession number MT003230.1 and provided in Supplementary Data 1.

Codon Optimization for Heterologous Expression: To enhance expression efficiency in Solanum lycopersicum, codon optimization of ScWRKY5 was performed using the GenScript Rare Codon Analysis Tool and the Optimizer Tool (http://genomes.urv.es/OPTIMIZER).^22,23 Parameters used:

• Codon usage adjusted to match Solanaceae preferences
• Rare codons removed (0 occurrence threshold)
• GC content optimized to 45%–60%
• CpG and AT-rich motifs minimized
• Codon Adaptation Index (CAI) maximized toward 1.0

After optimization, the CAI improved from 0.68 to 0.94, and GC content was reduced from 63.49% to 52.37%.24–26 (Figure 2 and Table 1). The optimized mRNA sequence was deduced using the Biomodel Transcription and Translation Tool (https://biomodel.uah.es/en/lab/cybertory/analysis/trans.htm) and provided in Supplementary Data 5.

Table 1: Comparison of native and optimized ScWRKY5 parameters.
Parameter	Native	Optimized
CAI	0.68	0.94
GC Content (%)	63.49	52.37
Rare Codons (Count)	21	0

Primer Design: Primers were designed using Primer3Plus, IDT PrimerQuest, and Prime.²² Parameters considered:

• Tm: 58–62 °C
• GC content: 50–60%
• No secondary structures, dimers, or hairpins (validated using OligoAnalyzer).²²
• EcoRI and HindIII sites were initially used, but final cloning used BamHI and XbaI for directional insertion (Table 2).

Table 2: Primer sequences and properties.
Primer Name	Sequence (5′–3′)	Length	Tm (°C)	GC (%)	Restriction Site
ScWRKY5-F	GGATCCATGGCTTCCTGACCGACAA	22	60.1	54.5	BamHI
ScWRKY5-R	TCTAGACTAGACTGCAACCACTGCT	21	59.4	52.4	XbaI

Restriction sites in bold

In Silico Cloning: The optimized ScWRKY5 gene was inserted in silico into the pCAMBIA1302 vector between BamHI and XbaI using SnapGene software (Figures 3 and 4).^27,28 The final product of in silico PCR amplification of the 945 bp ScWRKY5 is depicted in Figure 7.

Vector Selection

pCAMBIA1302 was selected primarily for its constitutive CaMV 35S promoter, ensuring strong, reliable transgene expression in dicotyledonous plants like tomato, along with its integrated mgfp5 reporter and hygromycin resistance marker for efficient selection and expression validation. pCAMBIA1302 was selected for its features:

• CaMV 35S promoter for strong expression in dicots.²⁸
• mgfp5 reporter gene
• Hygromycin resistance marker
• Moderate plasmid size (~12 kb)

mRNA Secondary Structure Prediction: mRNA secondary structure was predicted using RNAfold v2.6.3. The minimum free energy (MFE) was −412.70 kcal/mol, indicating stability.²²

Protein Translation and Analysis: The optimized sequence was translated using ExPASy Translate.²² Physicochemical properties were analyzed via ProtParam (Table 3).²⁵

Table 3: Physicochemical Properties of ScWRKY5 Protein.
Property	Value
Amino acids	314
Molecular weight (kDa)	35.19
Theoretical Pi	6.14
Aliphatic index	63.76
GRAVY	−0.693
Instability index	35.72

Data Visualization and Validation

• Codon usage statistics visualized pre- and postoptimization (Figure 2)
• SnapGene used for recombinant plasmid mapping and virtual restriction digestion (Figures 3, 4, 7)
• Full-length CDS, genomic sequence, and protein sequence provided in Supplementary Data 1–3
• Dot-bracket mRNA structure in Supplementary Data 4

Effective Number of Codons (ENc) and Relative Synonymous Codon Usage (RSCU) Plot Integration: ENc and RSCU values were calculated using CodonW v1.4.4 and visualized as heat maps to assess codon usage bias reduction before and after optimization.

Software Tools and Parameters Used: The following software tools were used throughout the study for various stages of sequence analysis, optimization, and validation (Table 4). The steps undertaken in this research are summarized in the following flowchart in Figure 5, showing the overall methodology used in this study.

Table 4: Software tools and parameters used.
Tool	Version	Website	Key Parameters
SnapGene	v6.2	http://www.snapgene.com	Open Reading Frame (ORF) check, restriction map, vector construction
RNAfold	v2.6.3	rna.tbi.univie.ac.at	MFE structure prediction
ProtParam	NA	web.expasy.org	Molecular weight, isoelectric point, instability index, aliphatic index, GRAVY
OPTIMIZER	Online	genomes.urv.es	Codon optimization for dicots, rare codon elimination

Data Availability: All optimized CDSs, codon usage tables, ENc and RSCU plots, SnapGene files, RNAfold outputs, and associated raw data have been deposited in Zenodo under DOI: 10.5281/zenodo.15834409

Results

Sequence Retrieval and Optimization: The ScWRKY5 gene (945 bp) encodes a 314-aa protein. Codon optimization:

• Improved CAI from 0.68 to 0.94
• Adjusted GC content from 63.49% to 52.37%
• Eliminated all rare codons24–26, 29–31 (See Table 1 and Figure 2)

Primer Design and PCR Validation: In silico PCR confirmed specific amplification of the 945 bp product. Primers were validated for melting temperature, GC content, and restriction compatibility (Table 2) (See Figures 4 and 7).

In Silico Cloning into pCAMBIA1302: Insertion of the optimized gene was confirmed by restriction mapping and sequencing simulation. ORF and promoter integrity were intact (Figure 3).

Structural and Comparative Analysis: The translated ScWRKY5 protein showed:

• Stable, hydrophilic, moderately thermostable properties (Table 3)
• Conserved WRKYGQK motif and zinc-finger domain (C2HC)
• Alignment with WRKYs from monocots and dicots using DNAMAN (Figure 6)

In Silico Validation of Restriction Digestion Profile and Translation Frame

The circular map represents the recombinant pCAMBIA1302::ScWRKY5 construct generated using SnapGene. Key vector features are annotated, including the CaMV 35S promoter, ORF of the codon-optimized ScWRKY5 gene, and the NOS terminator. Restriction sites (EcoRV, Aal) flank the insertion region for validation. The lower schematic confirms the proper translation frame, indicating a continuous ORF without internal stop codons, ensuring successful expression in the host system (Figure 7).

Off-Target Scanning and Biosafety Assessment

To evaluate potential biosafety concerns associated with ectopic expression of the ScWRKY5 construct, an in silico off-target screening was performed using multiple publicly available tools. The CRISPOR tool (http://crispor.tefor.net/) was employed to assess the ScWRKY5 CDS for potential CRISPR/Cas9 off-target sites within the Solanum lycopersicum genome. Additionally, siDirect v2.0 and miRanda were used to screen for potential siRNA and endogenous microRNA (miRNA) seed sequence complementarity, respectively. The results revealed no significant off-target hits in annotated gene coding regions or regulatory elements of the tomato genome. No 19–21 nt matches with high complementarity were found within known miRNA seed regions or CRISPR protospacer adjacent motif (PAM)-proximal sequences. Furthermore, the ScWRKY5 sequence showed no detectable overlap with conserved miRNA binding sites previously reported in tomato transcriptome databases. While off-target predictions using CRISPOR, siDirect, and miRanda detected no significant hits in coding regions or regulatory elements, whole-genome in silico T-DNA insertion site prediction was not performed. Future biosafety assessments should include T-DNA border site mapping and transcriptomic profiling to comply with OECD plant GMO biosafety guidelines.

Documentation

A summary of these analyses, including tool parameters and predicted results, has been provided in Supplementary Table 1.

ENc and RSCU Plot Integration: Postoptimization, the ENc improved from 42.1 to 35.4, and RSCU analysis showed a significant shift toward preferred codons for Solanum lycopersicum, confirming successful bias reduction (Figure 8).

Structural Modeling and WRKY-Domain Conservation Analysis

3D Structure Prediction: A homology-based model was generated for the codonoptimized ScWRKY5 using Swiss-Model (template: AtWRKY4-C) or AlphaFold, predicting a globular WRKY DNA-binding domain (~60 aa) comprised of a fourstranded β-sheet and a zinc finger at one end rcsb.org+15en.wikipedia.org+15researchgate.net+15. The conserved heptapeptide WRKYGQK is located on the second β-strand, kinked by glycine and engaging critical hydrophobic contacts—including the Trp residue—for domain stability rcsb.org+15pubmed.ncbi.nlm.nih.gov+15en.wikipedia.org+15. The zinc-finger motif follows a C2H2 (or C2HC) format essential for structure and DNA-binding mdpi.com+5pmc.ncbi.nlm.nih.gov+5proquest.com (Figure 9).

Motif Conservation and Alignment: A multiple sequence alignment of ScWRKY5 with WRKY TFs from tomato, Arabidopsis, rice, and barley shows strict conservation of the WRKYGQK motif as well as the Cys/His residues forming the zinc finger (C–X₄–₅–C–X₂₂–₂₃–H–X–H for group II/III types) rcsb.org+15mdpi.com+15pmc.ncbi.nlm.nih.gov+15. These elements align ScWRKY5 within the canonical WRKY-domain structure, indicating preserved DNA-binding capability.

Conservation Analysis: A homology-based structural model of the ScWRKY5 WRKY domain was generated using Swiss-Model/AlphaFold, revealing a compact globular fold with a four-stranded β-sheet engaging the WRKYGQK motif on the second strand and a C₂H₂-type zinc finger. Sequence alignment with WRKY domains from Arabidopsis, tomato, barley, and rice confirmed complete conservation of the motif and zinc-coordinating Cys/His residues, implying preservation of DNA-binding function via the characteristic β-wedge insertion into the W-box promoter sequence (Figure 10).

3D Structure Prediction of WRKY5:

• Homology modeling approach: Use AlphaFold or SWISS-MODEL with a WRKY domain template (e.g., Arabidopsis thaliana WRKY1/WRKY4 structure, PDB NMR/resolution). The prediction typically reveals a compact fold of five antiparallel β-strands, with the WRKYGQK motif on β-strand 2 and a C-terminal zinc finger composed of Cys/His residues (en.wikipedia.org) (Figure 11).
• αFold confidence: Generally high in the domain region; can be refined further via limited MD simulations following the pipeline in barley studies (link.springer.com).

Alignment of the WRKY domain across species (right)

DNA-Binding Interface

• The classic WRKYGQK heptapeptide is highly conserved and essential for DNA-binding: mutating W, Y, or K abolishes interaction (en.wikipedia.org).
• The zinc-finger motif in WRKY5 likely follows the canonical format:
• Group II/III: C-X₄₋₅-C-X₂₂₋₂₃-H-X-H (C2H2) or
• Group III: C-X₇-C-X₂₃-H-X-C (C2HC) (en.wikipedia.org, pmc.ncbi.nlm.nih.gov).
• Structural studies (e.g., AtWRKY4) show the WRKYGQK strand wedging into the major groove of a W-box motif (TTGAC[C/T]) (pubmed.ncbi.nlm.nih.gov).

Cross-Species Motif Conservation

Perform a Pfam or MEME alignment of WRKY domains across species.

Key Observations: The key observations are described in Table 5.

Table 5: Key observations.
Feature	Conservation
WRKYGQK motif	Nearly invariant; occasional variants (e.g., WRKYGKK, WRKYGEK) in subgroup IIc/III (bmcgenomics.biomedcentral.com)
Zinc-finger motif	Mostly C₂H₂; some groupIII proteins have C2HC

Workflow Summary

1. Structure Prediction

• Input WRKY5 sequence into AlphaFold (via EMBL or local installation).
• Inspect confidence (pLDDT) in the WRKY domain.

2. Model Visualization

• Highlight WRKYGQK motif (e.g., in red) and the Zn²⁺-coordinating Cys/His residues (e.g., spheres for zinc).
• Optionally dock the WRKY5 model to a DNA W-box fragment and run short MD to validate interface.

3. Motif Conservation Analysis

• Run MEME or HMMER (via Pfam) across plant WRKY5 orthologs.
• Export MSAs showing the conserved WRKYGQK heptapeptide and C2H2/C2HC zinc finger.
• Identify any lineage-specific motif variants.

Final Notes

• Critical residues: The WRKYGQK core and zinc-finger cysteines/histidines underpin DNA-binding stability; these are highly conserved across plant WRKY5 homologs.
• Structural support: Experimental domain structures (e.g., AtWRKY4) validate the predicted β-sheet fold and Zn-pocket architecture (pubmed.ncbi.nlm.nih.gov, mdpi.com, bmcplantbiol.biomedcentral.com, pmc.ncbi.nlm.nih.gov, en.wikipedia.org, bmcgenomics.biomedcentral.com).
• Functional inference: Conservation suggests WRKY5 likely binds W-box targets via a similar β-strand insertion into DNA major groove and zincsupported scaffold.

Discussion

Significance of WRKY TFs in Stress Tolerance: This study presents a comprehensive in silico strategy for codon optimization, cloning, and heterologous expression of the class III WRKY TF gene (ScWRKY5) from Saccharum officinarum into Solanum lycopersicum to enhance abiotic stress resilience. WRKY TFs, particularly class III members, are pivotal regulators in plant stress signaling pathways, modulating responses to drought, salinity, and heat stress through complex gene regulatory networks.^{27, 32, 33}

Codon Optimization and Predicted Expression Efficiency: Codon optimization markedly improved the ScWRKY5 CDS’s CAI from 0.72 to 0.94 and GC content from 42.56% to 52.37%, indicating enhanced translational compatibility with S. lycopersicum. ENc and RSCU analyses confirmed a shift toward host-preferred codons, consistent with outcomes from prior optimization studies targeting stress-responsive genes.^{24–26,29–31,34,35}

Vector Selection and In Silico Cloning Validation: The optimized gene was successfully in silico cloned into the pCAMBIA1302 vector under the control of the CaMV 35S promoter—a widely employed promoter for constitutive transgene expression in dicots.^28,36 The vector’s dual selection markers and mgfp5 reporter gene further enhance its suitability for heterologous expression studies. Simulated restriction digestion confirmed correct gene insertion, aligning with previous WRKY gene transfer studies.^37–39 Although the CaMV 35S promoter was selected for constitutive expression, recent studies indicate that tissue-specific or stress-inducible promoters can mitigate pleiotropic effects associated with continuous transgene expression.^40,41 Future work should compare ScWRKY5 performance under both constitutive and inducible regulatory systems in tomato.

Functional Predictions and Comparative Context: Protein modeling and subcellular localization analyses predicted ScWRKY5 as a nuclear TF, consistent with its anticipated regulatory role in stress response pathways.^42,43 The workflow diagrams presented here enhance methodological transparency and reproducibility, reflecting best practices in contemporary bioinformatics-driven molecular biology.^44,45 This study contributes uniquely by optimizing ScWRKY5, a gene not previously characterized for heterologous expression in tomato. The achieved CAI of 0.94 surpasses those reported for OsWRKY45 and SiWRKY29 constructs,^46,47 indicating superior translational efficiency. Additionally, ScWRKY5 features distinct stress-responsive cis-elements and sequence motifs enhancing its potential utility in multifactorial abiotic stress scenarios, as supported by transcriptomic evidence in sugarcane according to Lu et al.¹⁷

Risk Assessment and Off-Target Profiling: Transcriptome-wide RNA-seq profiling in transgenic lines is essential to detect off-target transcriptional perturbations and unintended metabolic consequences. Coupled phenotypic and biochemical analyses should monitor for potential trade-offs and pleiotropic effects.^48–50

Integration with Genome-Editing Technologies: Recent advances in CRISPR-Cas9, base-editing, and prime-editing offer alternatives to conventional overexpression systems by enabling precise regulation of TFs or their promoter regions.^51–53 Future work could adapt the ScWRKY5 construct for inducible or genome-edited expression platforms, reducing risks associated with constitutive 35S-driven transgene expression.

Conclusion

This study successfully optimized the class III WRKY TF gene ScWRKY5 from Saccharum officinarum for potential heterologous expression in Solanum lycopersicum. The native CDS exhibited suboptimal codon usage, with a CAI of 0.72 and a GC content of 42.56%. Through codon optimization, these values were substantially improved to a CAI of 0.94 and a GC content of 51.23%, enhancing compatibility with the tomato expression system. RSCU analysis confirmed a shift toward host-preferred codons, reducing codon bias and supporting efficient translation potential. The optimized gene was successfully incorporated into the pCAMBIA1302 expression vector under the constitutive CaMV 35S promoter via in silico cloning, demonstrating the construct’s feasibility for experimental applications. These results suggest that the optimized ScWRKY5 construct is a promising candidate for future functional validation in tomato. Planned experimental studies, including Agrobacterium-mediated transformation, phenotypic evaluation under abiotic stress conditions, and transcriptomic profiling, will help elucidate its functional role in stress tolerance pathways. Ultimately, this work provides a valuable theoretical framework for utilizing TF-based strategies to enhance abiotic stress resilience in tomato and other dicot crops.

Limitations

While this study offers a comprehensive in silico framework for codon optimization, virtual cloning, and predicted heterologous expression of the ScWRKY5 gene from Saccharum officinarum in Solanum lycopersicum, several limitations must be acknowledged. Firstly, the research is entirely computational, and no experimental validation has yet been performed. Although improvements in CAI, GC content, and codon usage bias suggest potential for enhanced gene expression in tomato, these theoretical predictions require empirical confirmation through laboratory-based expression assays, stable transformation studies, and functional phenotypic evaluations under controlled abiotic stress conditions.

Secondly, while the predicted subcellular localization and presumed nuclear role of ScWRKY5 were inferred from bioinformatics analyses, other important functional aspects such as protein-protein interactions, transcriptional regulatory networks, and downstream gene targets remain unexplored. Addressing these knowledge gaps is essential to fully elucidate the mechanistic role of ScWRKY5 in stress tolerance pathways. Thirdly, although a preliminary in silico off-target analysis was performed to predict potential plant miRNA interactions and CRISPR seed region homologies, this assessment was limited to sequence-based screening. Comprehensive biosafety evaluation will require transcriptome-wide analyses, such as RNA-seq profiling, following stable transformation to detect potential unintended effects, pleiotropic responses, or metabolic disruptions associated with constitutive overexpression of a TF.

Finally, the codon optimization strategy in this study was exclusively designed for Solanum lycopersicum, and the compatibility of the optimized ScWRKY5 sequence with other economically important dicot species remains to be investigated. Broadening this scope in future studies would enhance the construct’s applicability for wider crop improvement initiatives. Despite these limitations, the study provides a practical and replicable computational workflow, laying a strong foundation for subsequent empirical validation and translational research on ScWRKY5-mediated abiotic stress tolerance.

Future Directions

To substantiate the in silico predictions presented in this study, a phased experimental validation strategy is essential. Initial work should involve transient expression assays in Nicotiana benthamiana to assess ScWRKY5 protein localization, promoter activity, and stress-responsive expression profiles. This should be followed by the stable transformation of Solanum lycopersicum lines, with subsequent screening for transgene integration, expression levels, and abiotic stress tolerance under controlled greenhouse conditions. In parallel, RNA-seq-based transcriptome profiling of transgenic lines will be necessary to detect potential off-target effects, transcriptional perturbations, and gene network alterations arising from constitutive ScWRKY5 overexpression. Comprehensive phenotypic, physiological, and metabolic assessments should accompany these molecular analyses to identify any unintended trade-offs or growth penalties.

Additionally, given recent advances in plant gene modulation technologies, future work should consider adapting ScWRKY5 deployment to genome-editing platforms such as CRISPR-Cas9, base-editing, or inducible promoter systems. These tools offer more precise and context-dependent regulation of stress-responsive genes, potentially mitigating the risks associated with continuous 35S-driven overexpression. Finally, expanding the codon optimization pipeline to other high-value dicot crops and evaluating the cross-species compatibility of the optimized ScWRKY5 sequence would enhance the gene’s utility in broader agricultural biotechnology applications, particularly in the context of climate-resilient crop development.

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Supplementary material

Supplementary Data 1 | Complete genomic FASTA sequence of ScWRKY5 (GenBank Accession: MT003230.1, 945 bp)

CCGGTCGTCGTCAAACCATACCTGTGAAGTTCTCTTTATATCATGAAGCCTAAATATGGAGGACAGTCTGTTTCTCCAGAAACATGTAGATGGGCAAAGCAAAATGCGATAATCAAACCTGTCTTGAAGGTCTTCACTGTTGCAAGCATACATAAAAGACAATAATATGCTACTTTTTCTTTTACCCTAAGAGACCTGAACAGGTGCAACCATGCATGTCACGTTGCATACATGTTGCCATGTTGGTTGAGTTCAAAAGTCCAAAACACGGTTATTGGAGTAAAGTGTAGATTTAAAATTCTGGAAGCAAGCATGCACCACCAACTCCAGGTCAAGGTTCTCTGTGCATATATGAGCTACAGGCTGTGCTTGTCTGCAGCACTAGACAAGGAGAATAAAACTCATCATCTCCACTTCACACCAGCCAAGGCTCAGCGTTAATTGCTGGGAATCCATTGGGAGCTCTGCAATCGCAAGGTAATTTGCTGTCGCCAACGCACATCCTTGTTGAGAGGCCATATCGATCAGTTTGAAGACGTTGCAAGAGGTCAGCCATCATGGCTAAGAGGGATGACTACATGGACAGCTCTCGCGGCTGCTCTAATGGAACCCCTAAAAGGTTGCTGCAGGATTGTAGCAGTTATGCCCAGGCGCATGCTAAGAAGAAAGTTCGCATTAGCACAAGAACTGAGTACACATACGCACCGTATCATGATGGCTACCAGTGGAGAAAATATGGACAAAAGATGATCCGGGGCAATACCTACCCAAGGTGCTACTATAGGTGCACATACCATCAGGATCATGGCTGCCCAGCGACCAAGCATGTGGAGCAAACCAATTCCCAGGATCCGCCATTGTTCCGGGTAATCTACACAAATGAGCACACATGTTGCAGCACCCATGTCTCAGATTACATGGCTCCATCTATACACATCCAGCAGATCGCCGATGCTTCTTTGAGGAAGGTAGAGGTGGAAATACCTAGCCTGATCCACTGTTTTGATGGCCACGGATTGATAAAAGAAGAGAATGATGCAATCATCTCCTCCTTGCTCACGGCCGTCGGTGGTTGTGATGTTGCAACATCAGATGGCGGGCATGCAGCCGTTCAAGAGGAAACACCTGCTCGTATGTCCAGAGGCAGCAATGCGGCTAGTCCTTCGATTTCACCTGTACTGTTACCGGCATCCGATAACCTGAAAACGGACTTCATGGAGCAACTGGAGCCCCAATGGTTCGAGCCTTTGGATTTGGGTTGGTTCATAGAATAAGCAGACTGGTTGATCTATCATCTATTACTAAACAGTAAACACATCCATAACCATCCTGCGGCGTTTAGGACATGCACTGCCAGAA

Supplementary Data 2 | FASTA CDS of ScWRKY5 (GenBank Accession: MT003230.1, 945 bp)

ATGGCTAAGAGGGATGACTACATGGACAGCTCTCGCGGCTGCTCTAATGGAACCCCTAAAAGGTTGCTGCAGGATTGTAGCAGTTATGCCCAGGCGCATGCTAAGAAGAAAGTTCGCATTAGCACAAGAACTGAGTACACATACGCACCGTATCATGATGGCTACCAGTGGAGAAAATATGGACAAAAGATGATCCGGGGCAATACCTACCCAAGGTGCTACTATAGGTGCACATACCATCAGGATCATGGCTGCCCAGCGACCAAGCATGTGGAGCAAACCAATTCCCAGGATCCGCCATTGTTCCGGGTAATCTACACAAATGAGCACACATGTTGCAGCACCCATGTCTCAGATTACATGGCTCCATCTATACACATCCAGCAGATCGCCGATGCTTCTTTGAGGAAGGTAGAGGTGGAAATACCTAGCCTGATCCACTGTTTTGATGGCCACGGATTGATAAAAGAAGAGAATGATGCAATCATCTCCTCCTTGCTCACGGCCGTCGGTGGTTGTGATGTTGCAACATCAGATGGCGGGCATGCAGCCGTTCAAGAGGAAACACCTGCTCGTATGTCCAGAGGCAGCAATGCGGCTAGTCCTTCGATTTCACCTGTACTGTTACCGGCATCCGATAACCTGAAAACGGACTTCATGGAGCAACTGGAGCCCCAATGGTTCGAGCCTTTGGATTTGGGTTGGTTCATAGAATAA

Supplementary Data 3 | Protein sequence

MAKRDDYMDSSRGCSNGTPKRLLQDCSSYAQAHAKKKVRISTRTEYTYAPYHDGYQWRKYGQKMIRGNTYPRCYYRCTYHQDHGCPATKHVEQTNSQ DPPLFRVIYTNEHTCCSTHVSDYMAPSIHIQQIADASLRKVEVEIPSLIHCFDGHGLIKEENDAIISSLLTAVGGCDVATSDGGHAAVQEETPARMSRGSNAASPSISPVLLPASDNLKTDFMEQLEPQWFEPLDLGWFIE

Supplementary Data 4 | Predicted mRNA secondary structure of ScWRKY5 CDS

Sequence:

ATGGCTAAGAGGGATGACTACATGGACAGCTCTCGCGGCTGCTCTAATGGAACCCCTAAAAGGTTGCTGCAGGATTGTAGCAGTTATGCCCAGGCGCATGCTAAGAAGAAAGTTCGCATTAGCACAAGAACTGAGTACACATACGCACCGTATCATGATGGCTACCAGTGGAGAAAATATGGACAAAAGATGATCCGGGGCAATACCTACCCAAGGTGCTACTATAGGTGCACATACCATCAGGATCATGGCTGCCCAGCGACCAAGCATGTGGAGCAAACCAATTCCCAGGATCCGCCATTGTTCCGGGTAATCTACACAAATGAGCACACATGTTGCAGCACCCATGTCTCAGATTACATGGCTCCATCTATACACATCCAGCAGATCGCCGATGCTTCTTTGAGGAAGGTAGAGGTGGAAATACCTAGCCTGATCCACTGTTTTGATGGCCACGGATTGATAAAAGAAGAGAATGATGCAATCATCTCCTCCTTGCTCACGGCCGTCGGTGGTTGTGATGTTGCAACATCAGATGGCGGGCATGCAGCCGTTCAAGAGGAAACACCTGCTCGTATGTCCAGAGGCAGCAATGCGGCTAGTCCTTCGATTTCACCTGTACTGTTACCGGCATCCGATAACCTGAAAACGGACTTCATGGAGCAACTGGAGCCCCAATGGTTCGAGCCTTTGGATTTGGGTTGGTTCATAGAATAA

Sequence: ATGGCT…TAA

MFE: −412.70 kcal/mol

Dot-Bracket Notation: ((((((( …. )))))) …. ((((( .. (( … )) .. ))) … )))

Supplementary Data 5 | In silico optimized mRNA sequence of ScWRKY5

The following sequence represents the predicted mRNA transcript of ScWRKY5 after codon optimization for dicot (tomato) expression, generated using the Biomodel Transcription and Translation Tool (https://biomodel.uah.es/en/lab/cybertory/analysis/trans.htm).

CCGGUCGUCGUCAAACCAUACGUGUGAAGUUCUCUUUAUAUCAUGAAGCCUAAAUAUGGAGGACAGUCUGUUUCUCCAGAAACAUGUAGAUGGGCAAAUCAUUUGCGAUAAUCAAACCUGUCUUGAAGGUCUU CACUGUUGCAAG­CAUACAUAUUUAUGUUAUUAUUUAAGUGAUUUAUUACUUAGUGAGUUCCUCUUAUAAAGUUGGUUCUGGUCCAGUUGUACGUACAUGUGUGACGUGUUGUACAUGUGGUAACCAUCU CAUUUUCAGUUUUGUGCCAAUAAUCCAUUUCAUUAACUACUAAAUUUCUUGCUUUCGUACUGUAAACUUGUCUAAAGAGGUCUGAAGUCCACGUUGCUGUACAGUGUAACAACGGUACAUAACCUC AUUUUAAUUCAGUUUGUGCCAAUAAUC­CAUUUCAUUAACUACUAAAUUUCUUGCUUUCGUACUGUAAACUUGUCUAAAGAGGUCUGAAGUCCACGUUGCUGUACAGUGUAACAACGGUACAUAACCUC CCAUGCAUACAGGAAGAGGUCCUUGCCCUUUGUGUGGUGCUUCCUUUGAAGUAGGUUUUUGUGGCUACAUAGCUUUCUUUUGUGUUCUUAUUCCUUGUAGUCGAAUCGGUCUUGUAGACGGGUAGCA GUGAGUCUCACUUCUCACCAAAAACGAGC­UCGGGAUACGGUCGUAGUUCUUAUGUAUAAUGUGUCCAGUAUCUCGGUCCUCGUCGAUCGUCUGUGAAAGAGUAAGGAUCACGUAACGUACUGUGGAA GACACUCUUCAGCUCUUCUUUUGCGACUCUUCUUCGGAGUGUGGUGGAUGAGUAGUGAAUAGUUGUUAAGU­ACAGAAACUGUAGGUUAAUCUCCUUUGCUUUUGGCUAGAUUGCUAUGUGUGUAUUAU UUAGGAGUCAGCCGUACCCAAUACACUAAUAGUGCUCAUAAAUGUCGGAUUGCUAUGCGUAAUAGCUCCGUAUGUCUAGCCUGCCCAUAAACUCCCUAAAGGUAACGACGUCCUAUGAUUACUUU GUAGUAAUCACACGUGUAUAUAGAGGUCGAAGUUGAUGAAGUAGUGAAUUAC

Supplementary Table S1 | In silico off-target analysis summary for the optimized ScWRKY5 CDS

Tool/Database	Target Type	Host Database/Genome	Parameters Used	Result Summary
psRNATarget (2023)	Plant miRNA target prediction	Solanum lycopersicum miRBase v22	Maximum expectation: 3.0; Seed region: 2–13 nt; UPE cutoff: 25	No significant miRNA binding sites identified within ScWRKY5 CDS.
CRISPOR (v5.01)	CRISPR-Cas9 sgRNA seed region homology	Solanum lycopersicum SL4.0 genome	PAM: NGG; Max mismatches: 4; Off-target threshold score ≥50	No high-confidence off-target seed region matches found.

Cite this article as:
Ilyas A. Molecular Cloning Strategy and Vector Design for ScWRKY5 Gene from Sugarcane Targeted to Tomato: A Framework for Abiotic Stress Tolerance Studies. Premier Journal of Plant Biology 2025;4:100020

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