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Showing 2 results for Antimicrobial Peptide
Mitra Khademi, Farhad Nazarian-Firouzabadi,
Volume 6, Issue 1 (9-2019)
Abstract
Recently, new molecular breeding and genetic engineering approaches have emerged to overcome the limitations of conventional breeding methods in generating disease-resistance transgenic plants. The use of antimicrobial peptides (AMPs) to produce transgenic plants resistant to a wide range of plant pathogens has achieved great success. Among huge number of AMPs, Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acids peptide, exhibits significant antimicrobial activities towards a wide range of pathogens. In order to increase the antimicrobial efficacy of DrsB1, the DrsB1 encoding DNA sequence was either fused to the N- or C-terminus of the sequence encoding chitin-binding domain (CBD) of Avr4 gene from Cladosporium fulvum and constructs (CBD-DrsB1 and DrsB1-CBD) were used for tobacco leaf disk Agrobacterium-mediated transformation. Polymerase chain reaction (PCR), semi-quantitative RT-PCR and SDS-PAGE analysis indicated the integration of transgenes in tobacco genome and expression of the recombinant genes in transgenic plants, respectively. The antimicrobial activity of extracted recombinant peptides were assessed against a number of plant and human pathogens. Both recombinant peptides had statistically significant (P<0.01) inhibitory effects on the growth and development of fungi pathogens. Also, CFU test result showed that extracted recombinant peptides from transgenic plants, had a relatively high inhibitory effect on plant pathogens. The CBD-DrsB1 recombinant peptide demonstrated a higher antibacterial activity, whereas the DrsB1-CBD recombinant peptide performed a greater antifungal activity. In addition, the expression of DrsB1-CBD recombinant peptide significantly inhibited R.solani fungal infection in comparison with Pythium sp. interestingly, fungi with a higher amount of cell wall chitin were more vulnerable to recombinant peptides, suggesting recombinant peptides present a higher affinity for cell wall chitin. Owing to the high antimicrobial activity and novelty of recombinant peptides, this strategy for the first time, could be used to generate transgenic crop plants resistant to devastating plant pathogens.
Zahra Zarindast , Farhad Nazarian-Firouzabadi, Mitra Khademi,
Volume 10, Issue 1 (9-2023)
Abstract
Expression of antimicrobial peptides (AMPs) in plants to resist plant pathogens as well as to produce novel AMPs for pharmaceutical applications has recently received much consideration. alfAFP, a defensin cationic peptide synthesizing in alfalfa seeds, exhibits a strong antimicrobial activity. In order to facilitate alfAFP access to the pathogen’s membrane and increase the activity of the alfAFP peptide, the alfAFP encoding sequence was fused to the C-terminal of a chitin-binding domain (CBD) from a rice chitinase encoding gene. First, the antimicrobial properties of the recombinant peptide were assessed using bioinformatics tools. Next, the pGSA1285 expression vector harboring the CBD-alfAFP heterologous DNA was transformed into Agrobacterium rhizogenes for hairy root (HR) production in tobacco. The presence of transgene, transcription, and the expression of recombinant peptide in the HRs were confirmed by PCR and semi-quantitative RT-PCR analysis, respectively. Bioinformatic analysis was used to predict the antimicrobial activity of the alfAFP recombinant peptide. The results of the 3D structure analysis revealed a β-sheet and an α-helix structure that corresponded well with the structure of plant defensins. A Knottin functional domain was also recognized, suggesting that the recombinant peptide retains its antimicrobial activity. The results of the in vitro antimicrobial activity of the alfAFP recombinant peptide using CFU test showed that the recombinant peptide had significant inhibitory effects on Pseudomonas syringae pathogen. Therefore, the chitin-binding domain provided a better access of the recombinant peptide to the pathogenic bacterial cell wall through binding to peptidoglycan, and probably the recombinant peptide was able to target the plasma membrane with better efficiency. The results of this study suggested that the expression of the CBD-alfAFP recombinant peptide in crop plants and HRs can be a promising approach to producing pathogen-resistant plants as well as to produce new recombinant pharmaceutical AMPs.
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