Expression of recombinant human lactoferrin in transgenic alfalfa plants

Abstract

In order to produce human lactoferrin (Lf) in alfalfa (Medicago sativa L.), a construct containing human Lf cDNA under the control of cauliflower mosaic virus 35S promoter was engineered. As a selectable marker bar gene whose expression in plant cells confers tolerance to L-phosphinothricin (ppt) was used. Plants from a highly embryogenic alfalfa clone from the Bulgarian cultivar Obnova 10 were transformed using Agrobacterium tumefaciens mediated leaf disc method. Transgenic alfalfa plants were established from ppt-resistant calli via indirect somatic embryogenesis. The presence of human Lf cDNA in the genome of the selected regenerants was confirmed by polymerase chain reaction (PCR). Reverse transcriptase (RT)-PCR and Western blot showed expression of human Lf in leaf tissue. Studies on antibacterial effect of the recombinant glycoprotein were conducted and resistance of the transgenic alfalfa plants to two phytopathogens, Pseudomonas syringae pv. syringae and Clavibacter michiganensis, was demonstrated. The obtained results suggest that the expression of human Lf in alfalfa could be beneficial not only for producing recombinant protein for clinical application but also for crop quality improvement.

Molecular improvement of alfalfa for enhanced productivity and adaptability in a changing environment

“…The heterologous expression of genes encoding various other antimicrobial/antifungal proteins, such as β‐1,3‐glucanase and lactoferrin, in alfalfa has also demonstrated potential in terms of reducing the severity of infection by a range of plant pathogens, such as Phytophthora or Pseudomonas syringae pv. syringae and C. michiganensis , respectively (Masoud, Zhu, Lamb, & Dixon, ; Stefanova, Slavov, Gecheff, Vlahova, & Atanassov, ).…”

Plant‐based biopharming of recombinant human lactoferrin

“…(Min et al, 2006), eleuthero (Acanthopanax senticosus (Rupr. (Nguyen et al, 2011), barley (Hordeum vulgare L.) (Tanasienko et al, 2011), wheat (Triticum aestivum L.) (Han et al, 2012) and alfalfa (Medicago sativa L.) (Stefanova et al, 2013), insects fall armyworm (Spodoptera frugiperda Smith.) (Nguyen et al, 2011), barley (Hordeum vulgare L.) (Tanasienko et al, 2011), wheat (Triticum aestivum L.) (Han et al, 2012) and alfalfa (Medicago sativa L.) (Stefanova et al, 2013), insects fall armyworm (Spodoptera frugiperda Smith.)…”

“…Microalgae (Franklin and Mayfield, 2005), barley (Kamenarova et al, 2007;Tanasienko et al, 2009Tanasienko et al, , 2011Yemets et al, 2012) and alfalfa (Stefanova et al, 2013) could also be used as alternative platforms for the accumulation of high levels of the recombinant proteins. Plant-based expression systems have a set of advantages such as sufficient polypeptides expression, eukaryotic protein folding and relatively simple process of target protein purification in comparison with yeast and fungal systems, allowing the development of highly efficient technologies for the largescale production of Lf.…”

“…& Maxim) Harms) (Jo et al, 2006), Thale cress (Arabidopsis thaliana (L.) Heynh.) (Nguyen et al, 2011), barley (Hordeum vulgare L.) (Tanasienko et al, 2011), wheat (Triticum aestivum L.) (Han et al, 2012) and alfalfa (Medicago sativa L.) (Stefanova et al, 2013), insects fall armyworm (Spodoptera frugiperda Smith.) (Zhang et al, 1998a) and silkworm (Bombyx mori L.) (Liu et al, 2005); mammals: cow (Bos sp.…”

“…Among all engineered plant-systems for Lf synthesis the most promising ones are based on rice. Microalgae (Franklin and Mayfield, 2005), barley (Kamenarova et al, 2007;Tanasienko et al, 2009Tanasienko et al, , 2011Yemets et al, 2012) and alfalfa (Stefanova et al, 2013) could also be used as alternative platforms for the accumulation of high levels of the recombinant proteins.…”