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Showing 2 results for Hairy Root
Mitra Khademi, Marzih Varasteh-Shams, Farhad Nazarian-Firouzabadi,
Volume 9, Issue 1 (9-2022)
Abstract
Hairy and adventitious roots are efficient systems for expressing recombinant proteins. In the present study, the amount of DrsB1-CBDAvr4 recombinant protein in hairy and adventitious root systems was compared. To this end, the effect of different factors on the optimization of culture conditions to obtain adventitious and hairy roots was evaluated in three separate experiments by assessment of biomass production in T1 transgenic plants expressing DrsB1-CBDAvr4 recombinant protein. The efficacy of Agrobacterium rhizogenesis in producing hairy roots was ensured using rolC gene specific primers. The insertion of DrsB1-CBDAvr4 recombinant peptide transgene in the genome of hairy and adventitious roots was confirmed by PCR analysis. Also, the level of DrsB1-CBDAvr4 protein was measured in hairy and adventitious roots by ELISA analysis. Analysis of the variance of data showed that the highest number of roots and the longest roots were obtained in MS media supplemented with 1 mg/L NAA and 0.5 mg/L IBA. The results of adventitious root biomass showed that liquid MS medium containing 1 mg/L of NAA hormone had a significant effect (P <0.01) on biomass production. More biomass was obtained in MS medium supplemented with 1mg/L NAA, whereas a lower fresh and dry weight was obtained in a 1/4 MS medium with no NAA. The results also showed that ATCC15834 strain with MS media supplemented with 3% sucrose, 10 minutes inoculation time was high efficiency to induce hairy roots in tobacco plants. The results of ELISA analysis showed that clones obtained from both roots showed a significant difference in terms of total protein content. The amount of recombinant protein from hairy roots was much higher than that of adventitious roots.
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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