Isolation and In Silico Characterization of Defensin Genes from Some Indigenous Plant Species of Pakistan

Aims: Damage of crop plants due to pathogenic attacks, postharvest crops spoilage and lethal effects of chemical pesticides has enforced scientists to find some potential natural alternative. Plants produce different types of antimicrobial peptides including defensins in response to biotic and abiotic stresses. Defensins are small cysteine rich, cationic peptide with 40-45 amino acid residues with a variety of biological activities. Hence defensins have pharmaceutical and agricultural significance especially wide range of antifungal activities. Methodology: Nine plant species viz Brassica napus, Brassica nigra, Conyza bonariensis, Alhagi marorum, Sonchus arvensis, Brassica compestris, Eruca sativa, Cirsium arvensis and Brassica juncea were selected for the study. Four set of primers were applied on extracted genomic DNAs and four amplified genes were isolated from different plants species by PCR. The amplified genes were cloned via pTZ57R/T in E.coli. The cloned fragments were sequenced and characterized by different bioinformatics tool such as coding length and peptide sequence, site of cell aggregation, peptide physico-chemical properties, antimicrobial properties and gene expression pattern. Original Research Article Aslam et al.; JPRI, 34(41B): 12-21, 2022; Article no.JPRI.87867 13 Result: The amplified products from B. napus, B. nigra, B. compestris, E. sativa and B. juncea were about 330 bp in length and showed upto 85% nucleotide homology to the reported defensins. Deduced amino acid sequence from Bn-Def, Es-Def Bj-Def and BcDef showed the conserved defensin domain. These sequences were also characterized for different characteristic like charged amino acids, pI value, shelflife and stability. Sonchus arvensis didn’t give any specific product by prescribed set of primers. However the products from Alhagi marorum was about 770 bp and B. napus was of 270 bp but both give homology below 30%. Conclusion: This study showed that in silico characterization of deduced antimicrobial peptides from different plant species has depicted these as an effective alternative to communal therapeutics. However, further work is needed to verify defensins specific activity at protein level.


INTRODUCTION
Antimicrobial resistance is a great threat to the human race as stated by UN assembly in 2015. Defensin peptides can be used to counter the antimicrobial resistance along with other antimicrobial agents against pathogens. Use of defensins as natural analog to design and synthesize new molecules can be very optimistic approach to develop new field of applications including antimicrobial therapy and use of possible vaccine adjuvants [1]. In addition antimicrobial resistant defensins also assist in abiotic stress. By using their properties new opportunities of biomedical strategies and crop improvement can be developed with a great impact on humankind [2]. Novel antifungals are in high demand due to the challenges associated with resistant, persistent and systemic fungal infections.
Defensins are ubiquitous in plants [3], insects [4], animals [5,6,3], human [7] and mediate innate nonspecific response [3]. They are classified into different categories according to their amino acid composition, size, and conformational structures [8]. Defensins are divided at least into five classes. First three classes α, β, and θdefensins belong to birds and mammals whereas defensins from insects and plants constitute fourth and fifth classes [9].
These small cationic peptides with highly conserved signature tertiary structure [5] consists of one α-helix and a triple-stranded antiparallel βsheet stabilized by the disulfide bridges into a compact shape named as "cysteine-stabilized αhelix β-sheet motif (CSα/β)" [4,10]. Expression of defensins increases in response to abiotic stress, injury and pathogen attack [3,11]. These properties have made this peptide an attractive effective tool [12]. Many defensin and defensin like peptides have been isolated from medicinal plants [13,14].
Herbs and plants species are endowed with a variety of clinically important compounds and have been extensively used locally against different diseases but need further exploration especially. Here we report defensin genes from Brassicacea plant species with potential antimicrobials.

Amplification of Defensin Genes
Amplification of candidate genes was carried out by polymerase chain reaction Defensin genes specific primers were designed using the nucleotide sequences available in database of National Commission on Biotechnology Information (NCBI) in Genbank. Four sets of primers were designed (Table 1) with the help of different primer designing bioinformatics tools. PCR reaction for defensin gene was carried by using 2X Green dream Taq master mix (Thermo scientific) in the thermocycler (BioRad T100). PCR conditions (Table 2) and annealing temperature (Table 3) were optimized for optimal amplification of the genes. After confirmation by agarose the amplified products were purified from primer dimers and other impurities by ISOLATE ll PCR purification kit (Bioline) following user defined protocol.

Cloning of Amplified Product
The purified PCR product was ligated into TAcloning vector pTZ57R/T. Transformation of E. coli competent cells was performed by heat shock method [16].
The colonies were screened by Blue /White screening and colony PCR. Plasmid DNA was extracted by Plasmid DNA extraction kit (ThermoScientific#k0512) following manufacturer's instructions. To confirm the recombinant pTZ57R/T, restriction digestion analysis was done by using PvuII and product sizes were analyzed according to the vector maps. Taq DNA polymerase 1U/L 0.5 µL 8

Sequence Analysis
The nucleotide sequence of all cloned products was analyzed using different bioinformatics tools. and ExPASY tool ((http://www.expasy.org/translate/). The predicted protein sequence was again BLAST to determine similarity using two database NCBI GenBank database and UniProt database. This analysis also predicted the function of protein to be expressed. Characterization of deduced amino acid sequence (amino acid composition, ratio of positively charged amino acids to negatively charged amino acids, pI value, shelflife and stability) was made by ProtParam tools of ExPASY Proteomics server (http://www.expasy.org/cgibin/protparam/protparam/).
The conserved regions of plant defensins were located among deduced amino acid sequence by the conserved domain finder tool of NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/). Signal sequence was determined by predisi (http://www.predisi.de/predisi) and SignalP 4.0 [16]. The cleavage site of the deduced amino acid sequences were also predicted in signal peptide.

Multiple Sequence Alignment
To findout the comparison between species, these nucleotide sequences of different defensins were aligned individually with defensins sequences from other species through Clustal Omega tool (http://www.ebi.ac.uk/Tools/msa/clustalo/).

Isolation of Defensin Genes
Genomic DNA was successfully isolated by following the modified CTAB method from medicinal plant speiecs. Four sets of primers (Table 1) were designed from the sequences that showed highest homology. Selectivity and specificity of these primers was confirmed by aligning these primers sequences to different reported defensin sequences. Recommended parameters for primer designing like GC%, melting temperature, GC clamp, primer length, absence of hairpin and self/cross dimerization and annealing temperature were calculated using different online available bioinformatics tools e.g; primer 3.0 and hosted tools of justbio.com. Seven out of these nine plants species yielded positive amplification of the defensin genes.
Ampliconse from Brassica napus, Brassica compestris, Eruca sativa, Brassica nigra and Brassica juncea were of the size 330 bp (Fig. 1). Amplification of single band for all plants indicated there was no isoform of these defensins genes. Defensin gene sequence from Brassica juncea with 300 bp is analogous to that reported by Oguro et al. [18].

Fig. 1. PCR amplifications of defensin gene (330 bp) from. Brassica nigra, Brassica compestris, Eruca sativa, Brassica juncea, Brassica napus
As no defensin has been reported until now from Eruca sativa besides its diversely reported activities against microbes, this is the first reported sequence of the defensin gene from this species. Similarly, no defensin sequence has been reported from genomic DNA/ complete coding sequence of mRNA of Brassica rapa with subspecies compestris and Brassica nigra.

TBLASTN analysis of amplified fragment from
Alhagi marorum showed only 10% homology to antifungal genes from fabaceae family. However, its deduced amino acid sequence analysis TBLASTP did not show considerable identity to reported defensin on NCBI databank. So this sequence was not used for further proceedings. Amplified products exhibited DNA band of about 300 bp approximately for Cirsium arvensis and no specific product was obtained for S. arvensis though tried with varied PCR conditions. It might be due to the absence of that specific defensin in that particular plant species.
Up till now no defensin has been reported form Cirsium arvensis however other plant species of astereraceae have been used for defensin gene isolation. Defensin genes from fabaceae and asteraceae family showed efficient amplification at 1 mM of MgCl 2 with 10 ng/µL of genomic DNA. When PCR reaction mixtures for defensin amplification from fabaceae and brassicaceae were analyzed on 1% agarose gel, showed clear single bands without any smear. The amplified products were pure but Cirsium arvensis showed primer dimer very close to its product size in reaction mixture. Hence this PCR product was purified using Bioline PCR purification kit following the instructions of manufacturer.
Confirmation of the purified product was made on 1.2% agarose gel. Rest of the two plants species did not result in amplification although tried with different primer sets at different annealing conditions and reagent concentration. Absence of expression of some defensin gene fragments in some species could be explained by the differential presence of defensin genes in the defensin multigene family in different species or tissue specific expression [19].

Cloning of Amplified Defensin Genes
The defensin genes form all three families corresponding to six plant species were amplified in thermocycler using heat stable Taq DNA polymerase. The PCR amplified gene fragments of Brassicaceae and Fabaceae were ligated into pTZ57R/T vector whereas PCR purified product of Cirsium arvensis was used.
The plasmids were screened for recombinant plasmid by restriction analysis. Although blue white screening is efficient method for screening of recombinant plasmid but presence of false positives in blue white screening has minimized its efficiency [20].

Sequence Analysis of Cloned Defensin Genes
Positive clones after confirmation by restriction digestion and colony PCR were sequenced from CAMB, Lahore, Pakistan (http://www.camb.edu.pk/dnacf.asp). Bioedit and SnapGene viewer softwares [21] were used to remove unusual sequences/plasmid portion and developed corresponding contigs. The sequences thus obtained were used to make nucleotide and amino acid based studies. Nucleotide sequence of B. nupus amplicone yielded homology upto 30% to defensin sequences. Presence of signal peptide of 22 amino acids was also predicted but its deduced amino acid sequence showed absence of conserved domain for defensin. So the amplicone was not complete coding sequence of defensin gene.

PCR based screening of genomic DNA of
BLAST results revealed that nucleotide sequence of Brassica juncea, Brassica compestris, Brassica nigra and Eruca sativa had more than 65% homology to the reported defensin sequences. Mostly reported defensin peptide sequences are mRNA based and the number for DNA based sequences is extremely low. Among all the plant species used in this study only B. juncea had two reported deoxyribonucleotide sequences for defensin and revealed highest homology(>85%) to our said plant deoxyribonucleotide sequences. However similarity index of nucleotide sequence for defensins of Brassica campestris, Brassica nigra, Eruca sativa, to other brassicaceae species was quite distinct. BLAST tool of both databases NCBI and UniProt to screen the biological sequences for local similarity to their database sequences revealed statistical significance [22]. Use of two databases to find out the nucleotide similarity was made to ensure our work output as recommended by Silverstein and colleagues [23].
The deduced amino acid sequences of these gene products were analyzed by TBLASTp of NCBI and BLAST of UniProt. Mature peptide coding sequence revealed highest similarity to defensin and antifungal peptides with different species of brassicaceae. Bn-Def and Bj-Def amino acid sequences had significant similarity to defensin and antifungal peptides from model plant Arabidopsis thaliana that itself hosts 306 defensin like peptides [23]. Bn-Def also indicated homology to defensin from Arabidpsis hellari involved in its zinc tolerance ability. Schutte et al. [24] discovered five conserved β-defensin gene clusters using computational tools whom products were confirmed by following this strategy.
Weerden and Anderson in [25] depicted that the plant defensins had very limited conservation in amino acid sequences except 6-8 cysteine, one glutamic acid, one histidine residue and two glycine residues at specific positions. This specific position of cysteine residues signature a conserved γ-core motif. This γ-core motif has significant effect in antimicrobial activities of cysteine rich peptides [26,27] and consists of almost 14 residues (roughly) in length [4]. All sequences containing BLAST hits for defensins were studied further with NCBI conserved domain finder. For the functionality of defensin peptides, conserved domain γ-core motif (GXCX 3-9 C) is crucial. Studies on plant defensins reveal that γ-core domain is primarily located at C-terminal of active peptide. The only genes Bc-Def, Bn-Def, Es-Def and Bj-Def indicated the presence of Gamma-thionin conserved domain. Plant defensins were originally called γ-thionin because of their size and cysteine content (6-8) similar to the thionin [28]. Later identification of two antifungal peptides Rs-AFP1 and Rs-AFP2 by Terras and his colleagues (1995) which were structurally and functionally related to defensins from insects and mammals, hence this family was renamed as plant defensin.
Deduced amino acid alignment results showed that γ-core motif (GXCX 3-9 C) (14 amino acid) had 100% homology in their amino acid sequence for Bn-Def, Es-Def and Bj-Def but Bc-Def had 4 different amino acid in this region. Among these 4, two have cationic replacement to cationic ones as Bj-Def have lysine and Bc-Def had arginine in replacement at position 2 and in second position the Histidine in Bc-Def is replaced by arginine, and in remaining two, one hydrophobic amino acids alanine in Bj-Def is replaced to hydrophilic serine residue and one hydrophobic asparagine in Bj-Def is replaced to hydrophilic glutamine in Bc-Def. Difference in amino acid sequence contributes to difference in antifungal activity and their morphogenecity. [29]. Even a single amino acid variation in γ-core motif can change the target and mode of action of defensins [13]. This 24 amino conserved region "LCERPSGTWSGVCGNNNACKNQCI" of Bj-Def, Bn-Def, and Es-Def had 100% homology to antimicrobial protein (27 amino acids only) with fungicidal characteristic from brassica 'rapa (UniProtKB/Swiss-Prot: P30227.2), antifungal protein (25 amino acids) from Sinapis alba (AAB25088.1) antifungal protein (27 amino acid) from Arabidopsis thaliana (AAB25092.1) and antifungal protein from Raphanus sativa (AAB22710.1) predicted for its fungicidal characteristic.
The deduced amino acid sequences peptide gives 100% homology to peptide sequence which were isolated in functional form. Antifungal activity of antifungal proteins mainly depends on the net charge of peptide of this amino acid in this region [26]. Mostly plant defensins are positively charged peptides as increase in positive charge increase their binding towards negatively charged pathogens. However, anionic defensins have been reported with increased pore formation ability as compared to cationic peptides [30,31].
Another semiconserved region of plant defensins reported by Picart et al. in 2012 is α-core GXC(X 3-5 ) C. This region is not hallmark structure of plant antimicrobial peptides like γ-core and similarly have no contribution to their characteristic function/activity. This region had shown 100% homology for Bc-Def and Bj-Def but among four only a few residues are common. Moreover, Wu and colleagues [30] has explained the multitasking characteristics of defensins lies in its different regions.
As γ-core is responsible for antimicrobial activity similarly their ability to effect K + , Na + and Cl channels is due to CysX 3 Cys/CysX 1 Cys motif [31]. This motif was founded in Bj-Def only and can propose its biological activity in addition to antimicrobial activity. Amplicone sequences from Brassica napus and Cirsium arvensis missed the conserved domain. Genes with less homology can show activity but absence of conserved domain lose their function and results in pseudogenes [32]. Besides γ-thionin domain, Bj-Def showed an additional domain for Knot1 and NCBI conserved domain finder has classified this peptide to Knot1 domain containing peptide under pfam00304. Knot1 domain proteins are restricted to eukaryotes and are called Knottins or cysteine-knot1 peptides. Such proteins have protease and insecticidal activity like scorpion toxin along with antibacterial/antifungal activity [33]. pfam database has collection of γ -thionin conserved domain sequences from about 70 plant species and our amplicon showed significant homology to their database sequences.
The subcellular location of these protein sequences was predicted by PrediSI and SignalP. According to PrediSI, almost all transcripts including Brassica napus were predicted to be secretory except Cirsium Arvensis. However, the SignalP has predicted Bc-Def in contrast to PrediSI and amplicon from Cirsium arvensis as non-secretory peptides. All transcripts with reliable predictions were gone along with localization of proteins to secretory pathway. Secretory pathway means the proteins will be located in apoplast of the cells. Custers; [34] used this strategy to verify peroxidase products.

CONCLUSION
Defensins are natural antibiotics and have a great potential for therapeutic use in agriculture and healthcare. These are good alternative to their chemical counterparts to protect plants, animals and human from disease. Antimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi. Besides antimicrobial resistance (AMR) to some existing antibiotics an increasingly serious threat to global public health requires action across all government sectors and society. So to protect human, animals and plants there is a need to develop inexpensive antimicrobials. In this study, with the use of EST databases, primers were design for conserved defensin gene sequences to isolate and amplify defensin genes from different plant species. The gene sequence were further characterized by various bioinformatics tools. High grade identity of Brassica juncea to Brassica rapa depicts its prophylactic origin as stated by Kaur et al. [35] and shows that defensin sequence remained preserve. Presence of gamma-core region in Brassica juncea, Brassica compestris, Brassica nigra and Eruca sativa indicates the presence of functional defensin genes. Low grade identity of defensin gene sequence in B. napus and Alhagi marorum might be due to pseudo defensin genes. Conclusively the study showed that the defensin genes from some plant species share functional characteristics with other reported defensins, Hence have a great antimicrobial potential but further work is needed to verify defensins specific activity at protein level.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.