## 3 Results
### Phylogenetic Analysis  
A phylogenetic analysis was conducted to characterize VLP in _N. tabacum_, revealing its evolutionary origin, function, and relationships among different plants. The analysis elucidated how VLP has evolved, its connections to other proteins, and its distribution across plant groups, facilitating functional inference based on similarities or dissimilarities with known proteins. The ancestral lineage of VLP in _N. tabacum_ was elucidated using the ML method with the Le Gascuel 2008 model, selected based on its minimal BIC value of 59,785.02594. The phylogenetic tree for VLP in _N. tabacum_ was constructed using protein sequences, achieving the highest log likelihood of \(-\)31,553.29. This tree incorporated 417 amino acid sequences, spanning 657 positions in the final dataset. The analysis revealed that VLP in _N. tabacum_ demonstrated close phylogenetic relationships with VLP homologs from related genera within the Solanaceae family, including _Capsicum_, _Solanum_, _Datura_, _Lycium_, and _Anisodus_. These genera share a common ancestor and form distinct clades, reflecting evolutionary diversification driven by ecological adaptations and selective pressures. The close clustering of the _N. tabacum_ VLP with these homologs, as shown in Figure 2, underscores a conserved evolutionary trajectory across Solanaceae. While only a single _N. tabacum_ VLP sequence was available for this analysis, the phylogenetic positioning suggests functional conservation and potential divergence from other Solanaceae members, supporting its unique role in plant defense and adaptation. \begin{table}
\begin{tabular}{l l} \hline
**Physiochemical Properties** & **VLP** \\ \hline Number of amino acids & 417 \\ Molecular weight (kDa) & 45.145 \\ Average Residue Weight (EMBOSS-PESTATS) & 108.264 \\ Theoretical pl (ExPASy-ProtParam) & 9.81 \\ Theoretical pl (EMBOSS-PESTATS) & 10.43 \\ Negatively charged residues (Asp + Glu) & 33 \\ Positively charged residues (Arg + Lys) & 54 \\ Formula & C\({}_{1916}\)H\({}_{3048}\)N\({}_{584}\)O\({}_{642}\)S\({}_{19}\) \\ Total number of atoms & 6209 \\ Ext. coefficient (ExPASy-ProtParam) & 29,450, Abs 0.1\% (=1 g/L) 0.652, assuming all \\  & Cys residues are reduced, and 29,950, Abs 0.1\% (=1 g/L) 0.663, assuming all pairs of Cys \\  & residues form cystines \\  & 0.652 (reduced), 0.663 (cystine bridges) \\  & 30 h \\ Estimated Half-life (yeast, in vivo) (hours) & \textgreater{}20 h \\ Estimated Half-life (_E. coli_, in vivo) (hours) & \textgreater{}10 h \\ Aliphatic index & 49.81 \\ Grand average of hydropathicity (GRAVY) & \(-\)0.658 \\ Solubility & 0.345 \\ Improbability of expression in inclusion bodies & 0.979 \\ \hline \end{tabular}
\end{table} 
Table 1: Physicochemical properties of plant defense VLP of _N. tabacum_. VLP comprises 417 amino acids with a molecular weight of \(\sim\)45.1 kDa and a basic nature, as indicated by high pI values (9.81–10.43) and a greater number of positively charged residues. The protein shows moderate thermostability (aliphatic index: 49.81), good stability across expression systems, and hydrophilicity (GRAVY: \(-\)0.658). Despite limited solubility (0.345), the high improbability of inclusion body formation (0.979) suggests good expression potential for further functional applications. Figure 2: The construction of a phylogenetic tree elucidating the evolutionary relationships among VLPs in _N. tabacum_ and its homologs was performed using a rigorous computational approach. Homologous VLP sequences were retrieved from the NCBI Clustered Non-Redundant (nr) database via BLASTp, and the corresponding accession numbers are provided in Supplementary Table S2 for transparency and reproducibility. The tree was constructed using the p-distance model for substitution with amino acids as the substitution type, uniform rates and patterns, and 1000 bootstrap replications to assess branch reliability. Bootstrap values are labeled on the corresponding branches in the figure. Our analysis demonstrated that the _N.