Cloning and Expression of Antimicrobial Peptides from Vigna subterranea (Bambara Groundnut)

Abstract

Antimicrobial Peptides (AMPs) are short peptides of about 45 - 54 amino acids that exhibit antibacterial and antifungal activities. Plant defensin is a type of AMP in plants which belong to a family of cationic peptides with a characteristic 3D folding pattern held in place by four disulfide bridges. AMPs especially defensins have been identified to have a huge biotechnological potential and are being patented for many applications. The aim of this work was to clone an antimicrobial peptide from Vigna subterranea and characterise it with bioinformatics analysis. 4 sets of primers were synthesized according to the sequences of conserved regions in AMPs i.e. defensins from legumes like Vigna unguiculata, Vigna radiata, Cicer arietinum and Cajanus cajan, amongst others, which have defensins with only a few sequence differences. The primers were designated VsDef P1 to P4. Using Vigna subterranea total genomic DNA as a template, fragments of expected sizes were successfully amplified and cloned into the pDRIVE vector and used to transform Escherichia coli JM109 cells in each case. Representative clones were sequenced and analysed using BLAST from National Center for Biotechnology Information. However, only the VIG clone was shown to be a bona fide defensin (over 90% identity, E-value of 1ex102, 99% query coverage of the nucleotide sequence, compared to Vigna unguiculata defensin). Based on this high sequence identity, a new pair of primers VsDef P5 was designed based on the Vigna unguiculata defensin sequence to specifically amplify the complete Vigna subterranea defensin gene, hereafter called VsDef1. Attempts to clone VsDef1 were however unsuccessful, and evidence of clone deletion and insert re-arrangement of insert DNA was observed. Direct sequencing of the PCR product demonstrated that it was indeed the complete VsDef1 pre-protein, composed of 433 nucleotides. In silico translation and analysis showed that VsDef1 has an intron at position 105 − 259 of the nucleotide sequences and encodes for a 78 amino acid peptide. Phylogenetic analysis revealed to be similar to the sequence of the defensins for Vigna unguiculata (96%), Vigna radiata (95%), Vigna angularis (95%) and Phaseolus vulgaris (93%) on the NCBI database. The three - dimensional structure of the peptide was modelled with SWISS-MODEL expasy and the structure was found to include one α- and three β domains, similar to those of other defensins. The failure to identify VsDef1 clone in a V. subterranea library and the failure to recover its cDNA clone are consistent with the hypothesised toxicity of VsDef1 to Escherichia coli. It is suggested that a different host, such as yeast, should be used in the future. The VsDef1 mRNA levels in germinating V. subterranea seeds was however successfully investigated using real-time reverse transcription quantitative PCR. VsDef1 mRNA is present in both the testa and embryo of dry seed and will persist through the early stages of seedling growth. This demonstrates the importance of VsDef1 in fighting off infection during germination in order to ensure successful germination. It is therefore essential to characterise more antimicrobial peptides from V. subterranea. The diversity of AMPs and their patterns of expressed genes will enable understanding of complex regulatory networks, which will likely enable identifying of genes involved in diseases and new biological processes.