Iraninan Biology Society Journal of Cellular and Molecular Research (Iranian Journal of Biology) 2383-2738 38 3 2025 11 22 Cloning and transient and constitutive expression of human antimicrobial peptide Cathelicidin LL-37 in tobacco for its influence on bacterial canker agent in In vitro condition Cloning and transient and constitutive expression of human antimicrobial peptide Cathelicidin LL-37 in tobacco for its influence on bacterial canker agent in In vitro condition 258 273 2342 FA Aboalfazl Eftekhari Cis Biotechnology Department, Faculty of Agriculture and Natural Sciences, Imam Khomeini International University (IKIU), Qazvin, Iran 0009-0003-7354-4703 Fatemeh Dehghan Nayeri Biotechnology Department, Faculty of Agriculture and Natural Sciences, Imam Khomeini International University (IKIU), Qazvin, Iran 0000-0002-1281-1690 Journal Article 2023 09 02 Plant bacterial diseases, including bacterial canker, are the cause of reduced yields in agriculture. Antimicrobial peptides are among the defense lines of living organisms against various pathogens. In this study, first the human antimicrobial peptide LL-37 was synthesized as an optimized codon for the plant expression system and cloned into pCAMBIA1304 plasmid. Then the gene construct was expressed transiently and constitutively in tobacco, variety Xanthi, using Agrobacterium LBA4404 respectively by leaf microinjection and agroinfiltration methods. The expression level of LL-37 gene in transgenic tobacco leaves was measured by RT-PCR and qRT-PCR methods. The results showed the successful expression of LL-37 gene in tobacco plant in both transient and constitutive gene expression systems. Polyacrylamide gel electrophoresis of recombinant protein revealed a protein fragment related to peptide LL-37 with a size of 5 kilodaltons. The antibacterial effect of recombinant protein on Xanthomonas citri was confirmed by the formation of growth inhibition zone in vitro condition. The antimicrobial effect shows the value of further study on this peptide as a new antibiotic and for the production of transgenic plants resistant to plant pathogens. Plant bacterial diseases, including bacterial canker, are the cause of reduced yields in agriculture. Antimicrobial peptides are among the defense lines of living organisms against various pathogens. In this study, first the human antimicrobial peptide LL-37 was synthesized as an optimized codon for the plant expression system and cloned into pCAMBIA1304 plasmid. Then the gene construct was expressed transiently and constitutively in tobacco, variety Xanthi, using Agrobacterium LBA4404 respectively by leaf microinjection and agroinfiltration methods. The expression level of LL-37 gene in transgenic tobacco leaves was measured by RT-PCR and qRT-PCR methods. The results showed the successful expression of LL-37 gene in tobacco plant in both transient and constitutive gene expression systems. Polyacrylamide gel electrophoresis of recombinant protein revealed a protein fragment related to peptide LL-37 with a size of 5 kilodaltons. The antibacterial effect of recombinant protein on Xanthomonas citri was confirmed by the formation of growth inhibition zone in vitro condition. The antimicrobial effect shows the value of further study on this peptide as a new antibiotic and for the production of transgenic plants resistant to plant pathogens. https://cell.ijbio.ir/article_2342_2321994d85d661d792223f647000c65f.pdf