1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Cloning and expression of black pepper(piper nigrum L) defensin(pndef1) and assessment of its antimicrobial properties(Department of Molecular Biology and Biotechnology, College of Agriculture ,Vellayani, 2024-10-25) Arya Anilkumar.; Anuradha, TPlant defensins are cysteine-rich antimicrobial peptides that function in the innate immune response in plants. Many plant defensins with antimicrobial activity have been molecularly characterized in different plant species. The study entitled “Cloning and expression of black pepper (Piper nigrum L.) defensin (PnDef1) and assessment of its antimicrobial properties” was conducted in the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram from 2023- 2024. In a previous study here, the complete genomic DNA of the defensin gene from black pepper variety Panniyur-1 was cloned and characterized. In-silico analysis showed that the coding sequence of PnDef1 contains 231 bp open reading frame, encoding a protein of 76 amino acid residues, which exhibits antibacterial, antiviral, and antifungal properties. However, its protein purification and antimicrobial characteristics are yet to be determined. Hence the present study focuses on cloning and expression of defensin gene (PnDef1) isolated from black pepper (Piper nigrum L.); purification of its protein product and assessment of its antimicrobial properties. The full-length cDNA encoding PnDef1 gene was synthesised by RT PCR, using total RNA isolated from the leaves of black pepper var Panniyur-1 as template. The cDNA was confirmed by gene sequencing and further validated using ORF finder to ensure the integrity of the open reading frame (ORF) of PnDef1 gene. The cDNA encoding mature peptide of PnDef1 gene was inserted into prokaryotic expression vector pET-28a(+) through restriction enzyme-based cloning. The constructed recombinant pET-28a(+) with PnDef1 gene was transformed into BL21(DE3) pLysS cells using heat shock method and the recombinant colonies were confirmed by plasmid PCR. Recombinant protein expression in BL21(DE3)pLysS cells was successfully induced with 1.0 mM IPTG and subsequently incubated at 37°C for 4 hours. The expressed recombinant protein, produced as a fusion protein, was purified using a His tagged Purification Miniprep Kit, and further analysis using 12% SDS-PAGE demonstrated a molecular weight below 12 kDa and a protein concentration of 0.5 mg/mL. The study assessed the antimicrobial efficacy of both crude protein and purified PnDef1 against Phytophthora capsici and Colletotrichum gloeosporioides using the Agar well diffusion assay. Purified PnDef1 exhibited significant antimicrobial activity 65 specifically against Phytophthora capsici, with an inhibition rate of 55.5%, whereas no antimicrobial activity was observed against Colletotrichum gloeosporioides. This study marks the first successful expression and purification of the PnDef1 defensin gene from black pepper in the E. coli system. Additionally, it provides the first evidence of the antimicrobial activity of a plant defensin against Phytophthora capsici, highlighting its potential for developing strategies to enhance crop resistance against pathogens. Future studies will focus on several key objectives related to PnDef1. These include determining the optimal concentration of PnDef1 for effective inhibition of Phytophthora capsici, elucidating its antifungal mechanism and optimizing its expression and production for agricultural crop protection.