Gallinacin 3

β Gallinacin-3 (β Gal-3) is an antimicrobial peptide with strong antibacterial activity against Escherichia coli, Staphylococcus aureus and Salmonella typhimurium. In this study, the β Gal-3 gene was transferred into a plant genome by genetic engineering techniques. These transgenic plants can be used as feed additives to prevent poultry diseases and them might replace the antibiotics used in poultry industry. To ensure the β Gal-3 expresses effectively in Arabidopsis seeds, the expression was driven by promoter Ppha cloned from the β-phaseolin storage protein gene. A total of 294 transgenic lines were obtained by Agrobacterium-mediated transformation into Arabidopsis, and five transgenic lines were selected in which the expression levels of β Gal-3 were more than 0.10% of the total soluble proteins. The transgenic lines with single locus were identified by Southern blotting. The expression of β Gal-3 and the highest protein accumulation level (about 4.76 mg/g fresh weight with a maximum of 0.27% of total soluble proteins) was measured by Western blotting and ELISA, respectively. After ultrafiltration by centrifugation, the purity of recombinant β Gal-3 was up to 73%. Taken together, our data showed that expression of β Gal-3 with antimicrobial activity is possible and effective in Arabidopsis seeds.

Gallinacin-3 and gallopavin-1 (GPV-1) are newly characterized, epithelial beta-defensins of the chicken (Gallus gallus) and turkey (Meleagris gallopavo), respectively. In normal chickens, the expression of gallinacin-3 was especially prominent in the tongue, bursa of Fabricius, and trachea. It also occurred in other organs, including the skin, esophagus, air sacs, large intestine, and kidney. Tracheal expression of gallinacin-3 increased significantly after experimental infection of chickens with Haemophilus paragallinarum, whereas its expression in the tongue, esophagus, and bursa of Fabricius was unaffected. The precursor of gallinacin-3 contained a long C-terminal extension not present in the prepropeptide. By comparing the cDNA sequences of gallinacin-3 and GPV-1, we concluded that a 2-nucleotide insertion into the gallinacin-3 gene had induced a frameshift that read through the original stop codon and allowed the chicken propeptide to lengthen. The striking structural resemblance of the precursors of beta-defensins to those of crotamines (highly toxic peptides found in rattlesnake venom) supports their homology, even though defensins are specialized to kill microorganisms and crotamines are specialized to kill much larger prey.

Gallinacin-3 (Gal-3) is a newly discovered epithelial beta-defensin that acts as cationic antimicrobial peptides, and plays an important role in chicken innate immunity. However, the gallinacin-3 precursor containeda lengthy C-terminal region, which often hindered itsexpression. After codon optimization of Gal-3 and construction of an expression vector, the transgenic plants of Medicago sativa were obtained. Transgenic plants were validated and expression of proteins was detected. The antimicrobial activity of chicken β Gal-3 was analyzed and effects of chicken β Gal-3 on the body weight and intestinal microflora of mice were described. Our results demonstrated that the codon optimized chicken Gal-3 was stably expressed in transgenic Medicago sativa using the pCAMBIA3301 expression vector under the control of protein phosphatase (Ppha) promoter. Five transgenic plants with the highest expression of chicken β Gal-3 were selected, and were evaluated for the in vitro antimicrobial activity against Escherichia coli, Staphylococcus aureus and Salmonella typhi. Our findings confirmed that the Minimum Inhibitory Concentration (MIC) of the three bacterial strains were 32, 16 and 128 μg/mL, respectively. In addition, the effect of chicken Gal-3 on the body weight of mice fed with transgenic plants showed no significant deviation compared with that of the control group. Similarly, no loss of intestinal microflora was evident in the experimental group compared with the control group. Together, our findings demonstrate an alternative method for the stable expression of chicken Gal-3 withsignificant antibacterial effects and potential probiotics uses. In addition, this study may also be useful in the development of resistant M. sativa plants against pathogenic bacteria in future studies.