Cloning and expression studies of osmotin like protein gene from Solanum nigrum in Escherichia coli

doi:10.21203/rs.3.rs-4989893/v1

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Cloning and expression studies of osmotin like protein gene from Solanum nigrum in Escherichia coli

https://doi.org/10.21203/rs.3.rs-4989893/v1

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Plants usually face different types of stresses both biotic and abiotic. To combat against these stresses, they have defensive proteins. That only induce when there is a need of them. Plant pathogenesis related proteins (PR) is a group of proteins that help plants to fight against the stresses. Osmotin like protein is one of them and belongs to superfamily PR 5. In this study, OLP gene was isolated from the DNA of a medicinal plant Solanum nigrum. The plant was cultured in tissue culture laboratory under specific conditions. Plant genomic DNA was isolated by following a modified protocol. To isolate the gene, primers set was designed by Primer3 software with retrieved gene sequence from NCBI data base. The OLP gene was amplified by gradient PCR at specific set conditions. The annealing temperature range was set at 50°C-60°C. The primers to be optimized showed the optimum annealing temperature at 58.3°C to 60°C. The gel eluted amplified PCR product was cloned in cloning vector pTZ57R/T by using a cloning kit. The transformed plasmid DNA was sequenced to confirm the insertion of gene. The homology of sequenced gene was 98% with the reported sequences. To study the expression of the gene, the OLP gene was cloned in an expression vector pET15b. The construct was transformed into BL21 DE3 (E. coli strain). The expression of protein was analyzed through12% SDS- PAGE after inducing the cells for 3 h at 37°C with 1mM final concentration of IPTG as inducer. The clear difference was observed between induced and un-induced cells through protein profile. The induced OLP was at the right size of 26 kDa. Protein inclusions were made and protein blotting was done by Protein Dot Blot method. By using anti histidine antibodies and color reaction, the clear result of induced osmotin like protein was observed.

Biological sciences/Molecular biology

Biological sciences/Physiology

Biological sciences/Plant sciences

Plants usually face various stresses both biotic and abiotic in their life cycle that cause solid acidification, toxicity, and numerous diseases¹. In response to these environmental stresses, different complex and fine defense mechanisms have been developed by plants. The defenses are both constitutive and inducible. The primary or constitutive defense contains diverse preformed barriers like cell wall strengthening, bark, and epidermal waxy cuticles. These components help plants by giving rigidity and strength against any attack. Moreover, the secondary defense mechanism employed by plants is the activation of immune response via receptors that recognize and inhibit pathogen-associated molecular patterns ². This phenomenon triggers a cascade of responses including the hypersensitive reaction by salicylic acid (SA) and jasmonic acid (JA) signaling that unregulated pathogene related (PR) gene. These genes encode defense proteins including the expression of 17 different mechanisms, PR protein against attacks of fungi or other pathogens. This protein is expressed in all parts of a plant, ranging from 5-43kDa³.

This family forms an assembly of exclusive proteins that show antifungal and antifreeze activity. osmotin and osmotin-like proteins (OLP)from PR-5 proteins are those defensive proteins that are rich in cysteine residues. They assist in the maintenance of protein molecules as well as permit their correct folding. In this way, the proteins become resistant to extreme conditions and promote stress tolerance in plants. This activity was studied in different plant organs such as Solanum nigrum, Arabidopsis thaliana, and many others including crops. First, the PR-5 proteins were known and expressed in young leaves of tobacco. Under the influence of such environmental stresses, the PR-5 proteins accumulated fast at higher concentrations ⁴.

From the different explants of Solanum nigrum, various works of micro propagation have been done. Got regeneration of plant from stem, shoot tip, root segments, and leaf of Solanum nigrum via callus culture. The shoots were cultured from germinated seeds of the black nightshade plant (Solanum nigrum) ⁵.

After isolating the gene, it is cloned into a cloning vector. Gene cloning is a very significant technique in which we insert or ligate the gene of interest into a self-replicating system, such as a plasmid of bacteria. The objectives of molecular gene cloning involve the insertion of a gene of interest into a cloning vector or a receiving plasmid vector, then transformation of the plasmid in E. coli, recovery of the plasmid DNA, and checking of the accurate insertion of the particular gene of interest. Research has been successfully done related to OLP transformation into e. coli, cloned PnOLP1 gene from Panax notogineng using RACE technology⁶.

There are different cloning vectors used for this purpose. We use the pTZ57R/T cloning vector here. The length of this cloning vector is 2886 bp. It has two origins of replication, multiple cloning sites, and a T7 promotor, and its selective marker is Ampicillin. It has a high copy number. The gene sequence can be analyzed by using different bioinformatics tools like ExPASy to translate the DNA sequence into a protein sequence. Through the Clustal W program, multiple alignments of gene and amino acid sequences can be performed. The 3D structure of the protein (OLP) can be developed by SWISS Model software⁷.

Different expression studies about OLP have been reported already that discuss abiotic stress tolerance. According to Weber et al, overexpression of this protein might enhance the drought tolerance in plants as salt tolerance is enhanced with the expression of osmotin in tobacco plant ⁸. The major abiotic stress faced by plants is mainly salinity, which results in toxicity of ions (Na+), adverse effects on DNA methylation, and oxidative damage, and that would reduce via salt-induced 26kDa protein, a first time characterized as osmotin protein in tobacco. OLPs aid in regulating cellular osmolarity by solute compartmentalizing via metabolic alternations⁹. Here pET15b expression vector is used having size of 5.7 kbp with the selection marker of Ampicillin. So one of the key objectives of the conducted study is the study of the expression of OLP from S. nigrum after transforming into the host cell- E. coli¹⁰.

Explant Collection and Culture Conditions in Solanum nigrum Tissue Culture:

The disease free, healthy and fresh explants of Solanum nigrum were collected from the fields of university campus. After washing, explants were put is a sterilized jar containing commercial bleach and shaken for 20 min and washed to remove any bleaching agent. The shoots (explants) were cultured on the autoclaved MS (Murashige & Skoog) basal culture medium in sterilized conditions.

Explants inoculated by cutting shoots into 1–2 cm lengths in a laminar hood and placing them in medium with sterilized forceps. For culture conditions, Jars and tubes placed in a 25°C room with fluorescent lights for 16 hours and 8 hours dark. Explants re cultured in new media every 1 or 2 weeks Fig. 1. (Cloning of full length Solanum nigrum OLP gene 1). Tissue cultured sample isolation involved growing plants and cutting young leaves for DNA isolation.

Plant Genomic DNA Isolation

Genomic DNA was isolated leaves by CTAB method¹¹ and quality of DNA was checked by 0.8% agarose gel electrophoresis.

Digestion of RNA from Isolated DNA using RNase enzyme

After DNA isolation, 150 µL of TE buffer and 3 µL of RNase enzyme were added and incubated at 37°C for 15 min. Then, 20 µL potassium acetate and 200 µL of chloroform isoamyl alcohol (24:1) were added, followed by centrifugation at 8000 rpm for 10 min. After adding 200 µL of pre-chilled 2-propanol and incubating at -20°C for 10 min, the mixture was centrifuged at 14000 rpm for 10 min. Washing with 70% ethanol was done by centrifuging at 14000 rpm for 10 min, and the pellet was re-suspended in 50 µL of sterile water.

Amplification of Osmotin-like Protein Gene and cloning

The Osmotin-like Protein (OLP) gene from Solanum nigrum was amplified using gene-specific primers in Table 1. The polymerase chain reaction (PCR) was optimized via gradient PCR for the OLP gene from S. nigrum. The reaction mixture, prepared for 20 µL total in 14 reaction tubes ,12 gradient PCR tubes and other 2 for control (+ ve, -ve). PCR was done using 50 ng of S. nigrum genomic DNA, 1 mM dNTPs mix, and 5 U of DNA polymerase in a final volume of 20 µl. The conditions were 95°C for 3 min, followed by 30 cycles of 45 sec at 94°C, 1 min at 52°C, and 1 min at 72°C, with a final extension of 1 min at 72°C. The amplified product was eluted from gel by using Gel extraction kit (Axy Prep Kit) as per instructions of manufacturer. The product was cloned by using Thermo Scientific Ins. TA clone PCR Cloning Kit was used, employing the cloning vector pTZ57R/T (2887 bp) in E. coli (competent Top 10F´ Cell) and recombinants were screened through PCR. Both strands of cloned DNA were sequenced by ABI sequencer (Applied Biosystems 3100 DNA Analyzer).

Table 1

List of forward and reverse primer used for cloning
Primer Name	Sequence
SnOLP F2 (Forward)	5´CATATGACCTCTTTCGAGGTCCGA3´
SnOLP R2 (Reverse)	5´GAATTCTTTTACTTAGCCACTTCATCAG3´

Cloning of OLP gene in expression vector

Cloning in expression vector involved digesting the pET-15b vector (5.7 kbp) with NdeI, rendering it linearized for gene product ligation. The ligation product was transformed in Competent BL21 (DE3) by using standard CaCl₂ transformation protocol.

Expression and purification of Recombinant OLP gene

For expression studies, transformed BL21 (DE3) cells were cultured overnight in LB medium with antibiotics at 37°C and 250 rpm. The culture was then diluted 1:10 and incubated for 3 hours under the same conditions. Induction was initiated by adding 1 mM IPTG, followed by incubation for 3 hours at 37°C with shaking. The cells were re-suspended in ice-cold lysis buffer (50 mM potassium phosphate buffer pH 7.8, 100 mM KCl, 400 mM NaCl, 10% glycerol, 0.5% Triton X-100, 10 mM imidazole) and sonicated for the release of proteins under native conditions. The protein was purified by using Columns. The purity of fractions was checked by 12% SDS-PAGE and Dot blot was further performed for Protein purification and quantification Fig. 4.

DNA Translation into amino acid sequence:

The DNA sequence was input in FASTA format into the ExPASy Translate tool for conversion into an amino acid sequence.

https://web.expasy.org/translate/

In-silico physiochemical analysis of SnOLP-Z

For in silico analysis, the physicochemical properties were calculated using the free web tool available at https://web.expasy.org/protparam. The following parameters were assessed for the amino acid sequence of the SnOLP-Z gene: amino acid percentage, molecular weight, isoelectric point, total negatively and positively charged amino acids, instability index, aliphatic index, and GRAVY (grand average of hydropathicity).

Gene Sequence Retrieval and Alignment

Gene sequences for AGH14263.1, SNOLP-Z, AAL87640.1, and AAL79832.2 were obtained from relevant genomic databases. These sequences were aligned using Clustal software (CLUSTAL O(1.2.4)) to perform a multiple sequence comparison. This alignment process facilitated the identification of conserved regions and variations, offering insights into the evolutionary relationships among the sequences.

Cloning and Sequence analysis of OLP gene:

We cloned complete open reading frame of OLP gene of Solanum nigrum, 744 bp in length, coding a polypeptide of 247 amino acids. Cloning of OLP was confirmed by PCR and double digestion of vector by Eco RI/NdeI (Fig. 2a,b).

Sequence analysis of our gene showed more exons and less introns which is the feature of PR-5 proteins, which code 210–250 amino acids and have molecular mass of range 24–28 KDa ¹². At nucleotide level it showed 98% homology with OLP gene (AF 473702.2) from solanum nigrum.

General protein mass analysis for windows (GPMAW) was used for amino acid analysis which predicted mature protein with molecular mass of 26.5 kDa. The hydrophobicity index of protein is -0.3 and theoretical PI = 7.84. Mature protein has cleavage signal at N-terminal of 21 amino acids and probability of cleavage site is between 21st and 22nd amino acid. Kyte-Doolittle algorithm of Expasy was use to generate hydropathy plot which predicts that OLP has six transmembrane helices (Fig. 2c).

Expression of OLP gene in E. coli and its Purification

The gene was cloned in pET15b expression vector through ligation and transformation in chemically E.coli (competent cells of Top10F´ cells) as shown in Fig. 3. Colonies appeared on selection plates of Ampicillin (100µg/mL).

The pET15b-OLP construct was transformed in the competent cells of strain BL21 DE3. The transformed bacteria were grown on 1mM IPTG for induction and total protein was extracted by native buffers and avoiding denaturation. The total protein extracted from BL21 (DE3) harboring pET15b-OLP showed clear band in SDS –PAGE at 26kDa molecular mass. The semi- quantitative analysis of protein is done by performing Dot Blot method (Fig. 4).

Translation of DNA sequence into amino acid and amino acid composition of SnOLP-Z protein

The graph illustrates the amino acid composition of the SnOLP-Z protein. Each bar in the chart represents a different amino acid, with the height of each bar indicating the percentage of that amino acid within the protein. For example, alanine (Ala) constitutes approximately 5% of the protein, while glycine (Gly) comprises around 10%. Additionally, the chart displays the total count of each amino acid found in SnOLP-Z. Overall, this visual representation highlights the relative abundance of the various amino acids that form the SnOLP-Z protein (Fig. 5a, b).

GC Content Distribution of the SnOLP-Z Gene

The line graph illustrates the GC content of the SnOLP-Z gene, with a total length of 743 base pairs (bp). The base composition is as follows: Adenine (A) accounts for 25.3% (188 bases), Cytosine (C) and Guanine (G) each constitute 23.42% (174 bases each), and Thymine (T) makes up 27.86% (207 bases). The graph reveals fluctuations in GC content throughout the gene, with peaks and troughs indicating regions of higher and lower GC concentrations. The average GC content for the SnOLP-Z gene is approximately 47%. These fluctuations are likely due to the presence of various DNA sequences, such as coding regions or regulatory elements, which can influence the distribution of GC content along the gene (Fig. 6).

Computing a multiple Sequence Alignment of AGH14263.1, SNOLP-Z, AAL87640.1, and AAL79832.2

The image displays an amino acid sequence alignment of four proteins: AGH14263.1, SNOLP-Z, AAL87640.1, and AAL79832.2. The sequences are aligned vertically, with identical amino acids highlighted by the same color to emphasize regions of similarity and dissimilarity. For instance, the sequence.

"MGYSRSSFVFFLLTFVTYTYATSFEVRNNCPYTVWAASTPIGGGRRLDRGQTWVINAPRG" is conserved across all four proteins, as evidenced by the uniform coloring of the amino acids. This conserved region suggests that the proteins share a common evolutionary origin and may have similar functional roles (Fig. 7).

Phylogenetic Analysis: Insights into Evolutionary Relationships

The phylogenetic tree presented delineates the evolutionary relationships among the four sequences: AGH14263.1, AAL79832.2, SnOLP-Z, and AAL87648.1. Rooted to reveal a common ancestor for all sequences, the tree provides several key insights. The lengths of the branches in the tree correspond to evolutionary distances, with longer branches indicating greater genetic divergence. Notably, the tree’s topology shows that SnOLP-Z and AAL87648.1 are more closely related to each other, sharing a more recent common ancestor compared to their relationships with AGH14263.1 and AAL79832.2. AGH14263.1, positioned as the outgroup, is the most distantly related sequence in this analysis, highlighting its more remote evolutionary lineage relative to the other sequences Fig. 8.

Physicochemical Properties of the SnOLP-Z Protein Analyzed Using Protparam

The Table 2 provides a detailed analysis of the physicochemical properties of the SnOLP-Z protein, computed using the Protparam tool. The protein consists of 247 amino acids and has a molecular weight of 26,743.10 Da. Its theoretical isoelectric point (pI) is 5.79, indicating the pH at which the protein has no net charge. The protein contains 17 negatively charged residues (aspartic acid and glutamic acid) and 16 positively charged residues (arginine and lysine), which contribute to its overall charge distribution.

Table 2

Physicochemical Properties of the SnOLP-Z Protein Computed Using the Protparam Tool
Parameters	value
No. of amino acids	247
Molecular weight	26743.10
Theoretical pI	5.79
No. Of -Ve Charged residues (Asp + Glu)	17
No. Of + Ve charged residues (Arg + Lys)	16
Ext. coefficient	40910
Instability index (II)	41.11
Aliphatic index (AI)	48.18
Grand average of hydropathicity (GRAVY)	-0.315

The extinction coefficient, which measures the protein's absorbance at 280 nm, is 40,910. The instability index (II) is 41.11, suggesting that the protein may be moderately unstable. Its aliphatic index (AI) of 48.18 reflects the relative volume occupied by aliphatic side chains, influencing the protein’s stability in different temperatures. Finally, the Grand Average of Hydropathicity (GRAVY) is -0.315, indicating that the protein is relatively hydrophilic, which affects its solubility in aqueous environments.

Plants usually face various stresses both biotic and abiotic in their life cycle that cause different diseases¹³. In response to these environmental stresses, different complex and fine defense mechanisms have been developed by plants¹⁴. To combat the physical, biotic, and abiotic stresses, plants produce defensive proteins. The defensive proteins belong to group of protein families called PR proteins. One family which is considered super family is PR 5 protein family and concerned osmotin like protein belongs to this PR 5 family.

OLP gene was isolated from a medicinal plant Solanum nigrum that was cultured through tissue culturing. The gene was amplified through PCR with specific primer set for mature peptide. Optimization of PCR indicated the optimum temperature for annealing of primers that was 58.3°C to 60°C. In another study, for obOLP (from Ocimum basilicum) gene amplification the optimum temperature for annealing was 52°C ¹⁵. This difference shows that primers for a same gene but different gene source anneals at different temperature. This argument is further supported by the study conducted ¹⁶. They reported the annealing temperature of 52°C for OLP gene obtained from S. nigrum L (var indica).

The complete ORF of OLP gene from S. nigrum having length of 744 bp, encoding 247 amino acid polypeptide (NCBI) (GenBank: Accession ID: AF450276.1) was cloned in cloning vector pTZ57R/T with restriction sites of EcoRI and NdeI. The restriction sites are added according to the vector used for cloning or expression. It has to be ensured that particular restriction enzymes could cut the cloning vector or not. As in case of pTZ57R/T, Eco32I, NheI restriction enzymes cannot cut the vector. In another research, pBSKSII cloning vector with restriction sites BamHI and HindIII attached with OLP was used for cloning ¹⁷. As these sites are compatible with the pBSKSII cloning vector.

The cloning of OLP gene was confirmed by PCR and double digestion by NdeI and EcoRI restriction enzymes. The OLP gene sequential analysis indicated that the gene consisted of coding sequence only with no intron region. Having no introns in the gene sequence, is the distinctive feature of proteins from PR 5 family and typically have a polypeptide of up to 250 amino acids with molecular weight 24 to 28 kDa ¹⁸. The amplified OLP gene sequence exhibited a high degree of similarity, showing 98% homology with both the OLP precursor gene from Solanum nigrum (Accession: AF450276.1) (NCBI) and the complete coding sequence of the OLP gene from the same species (Accession: AF473702.2) (NCBI). This close match underscores the accuracy of the amplified sequence and its alignment with known genetic data for Solanum nigrum.

While the sequenced OLP gene in research conducted by ¹⁷showed 95% homology with the same OLP precursor gene from Solanum nigrum (Accession: AF450276.1) (NCBI). The difference of homology of our sequenced gene and the gene of cited research indicated that our sequenced gene has more accuracy than this. The expression of SnOLP was studied by using expression vector pET15b. The construct (gene insert in vector) was developed by cutting the vector through restriction enzyme EcoRI (single restriction digestion). After linearizing the plasmid, gene inserted was ligated and cells were transformed. From the colonies of 53 transformed cells, plasmid DNA was isolated and gene insertion was confirmed by PCR.

For the target protein expression, the construct pET15b-SnOLP was transformed in BL21 (DE3) competent cells. The transformed bacterial cells were incubated in the presence of inducer IPTG with the final concentration of 1mM for 3 h at 37°C. The total protein was analyzed through 12% SDS- PAGE after lysis of the cells and total protein was loaded. The induced band appeared significantly as compared to un-induced. Camos expressed the SniOLP from S. nigrum var americanum in E. coli ¹².

The construct was pQE30-SnOLP and they induced the cells for the expression of protein with 0.4 mM of IPTG final concentration for 2 h. The bands of induced total protein appeared in this study were light as compared to our expressed protein. This might be due to the reason of low concentration of IPTG and less time for induction. With the increase in time the expression of protein can be enhanced but too much induction might degrade the protein and hence its expression would be minimized. After analyzing the total protein profile on SDS- PAGE, the inclusions were made to blot the proteins. Protein blotting was done by Protein dot blot method. The anti his antibody was used along with the secondary antibody. But the protein expression can also be confirmed by the western blot. In transgenic soybean plants, the band of SnOLP at 27 kDa size was observed in western blotting by using polyclonal antibody ¹⁹. The alignment of gene sequences for AGH14263.1, SNOLP-Z, AAL87640.1, and AAL79832.2 was performed using Clustal software, revealing both conserved regions and variations among the sequences. This analysis provides valuable insights into their evolutionary relationships, as conserved regions often indicate functional or structural importance. Variations, on the other hand, can highlight areas of divergence and adaptation. Similar methodologies have been successfully utilized in previous studies, such as those by Di Tommaso²⁰, where T-Coffee was employed for the multiple sequence alignment of protein and RNA sequences, incorporating structural information and homology extension. Their work underscores the importance of accurate sequence alignment in understanding evolutionary relationships and functional genomics.

The physicochemical analysis of the SnOLP-Z gene, based on its translated amino acid sequence, provides significant insights into the characteristics of the cloned gene. The ProtParam tool computed various parameters that are crucial for understanding the protein's properties²¹. For example, the molecular weight and number of cysteine residues can be used to predict the potential disulfide bridges within the protein sequence, which are important for protein stability and function. Additionally, parameters such as the instability index, aliphatic index, and Grand Average of Hydropathicity (GRAVY) offer insights into the protein’s stability, solubility, and overall behavior in biological systems. These computed values are essential for guiding further wet lab analyses, including experiments designed to assess protein stability, functionality, and interaction with other molecules ^22,23.

In conclusion, this study effectively isolated and characterized the OLP gene from the medicinal plant Solanum nigrum, revealing its potential role in plant defense mechanisms. Utilizing a range of molecular techniques such as PCR amplification, gene cloning, and protein expression analysis the researchers confirmed the presence and stress-responsive expression of the OLP gene. The physicochemical analysis of the SnOLP-Z gene, derived from its translated amino acid sequence, offers valuable insights into the gene's characteristics and properties. These findings enhance our understanding of plant-pathogen interactions and offer valuable insights for future research aimed at leveraging PR proteins for agricultural and medicinal applications.

Pathogenesis related proteins (PR)

Salicylic acid (SA)

Jasmonic acid (JA)

Osmotin

like proteins (OLP)

Funding: Researchers supporting the project (RSP2024R393) at King Saud University, Riyadh, Saudi Arabia.

Conflict of interest: All authors declare no conflict of interest

Authors Contributions: [MZS], [FA]; idea formulation and design the experiment. [NA], [AS]; Performed experimental work. [GZJ], [AZ]; Data curation and writing. [MZA]; methodology. [AAS], [MI], contributed various inputs during the preparation and designing the manuscript. Validation, funding contributed by [SS], [MKG]. [MKG], [AAS] approved the final version to be published. All the authors read and approved the finalized manuscript for publication.

Ethics approval and consent to participate

Not applicable

Consent for publication

All authors give permission to the publisher to publish this research work.

Availability of data and materials

All data is presented in this manuscript. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. More, the accession number (Accession ID: AF450276.1), (Accession: AF473702.2) data is stored in the given link.

https://www.ncbi.nlm.nih.gov/nuccore/AF473702.2/, https://www.ncbi.nlm.nih.gov/nuccore/AF450276.1

Competing interests

The authors declare no known competing financial interest.

Acknowledgments: The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R393) of King Saud University, Riyadh, Saudi Arabia.

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Cloning and expression studies of osmotin like protein gene from Solanum nigrum in Escherichia coli

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Abstract

Figures

Introduction

Materials and Methods

Plant Genomic DNA Isolation

Digestion of RNA from Isolated DNA using RNase enzyme

Amplification of Osmotin-like Protein Gene and cloning

Cloning of OLP gene in expression vector

Expression and purification of Recombinant OLP gene

DNA Translation into amino acid sequence:

https://web.expasy.org/translate/

In-silico physiochemical analysis of SnOLP-Z

Gene Sequence Retrieval and Alignment

Results

Cloning and Sequence analysis of OLP gene:

Translation of DNA sequence into amino acid and amino acid composition of SnOLP-Z protein

GC Content Distribution of the SnOLP-Z Gene

Computing a multiple Sequence Alignment of AGH14263.1, SNOLP-Z, AAL87640.1, and AAL79832.2

Phylogenetic Analysis: Insights into Evolutionary Relationships

Physicochemical Properties of the SnOLP-Z Protein Analyzed Using Protparam

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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