Transgenic expression and evaluation of tobacco (Nicotiana tabacum) transformed with a synthetic analog of magainin

Magainins 1 and 2 belong to a new class of peptides that have antimicrobial properties. These peptides are derived from the skin of the African frog Xenopus laevis. Magainin 2 is a 23 amino acid peptide that inhibits the growth of various species of bacteria and fungi. The activity of magainin can be attributed to its affinity to bilayer membranes. An analog of magainin was engineered into tobacco plants and resistance was assessed against selective pathogenic microbes.; Isolated chloroplasts from pea plants were subjected to either 0.1 mug/ul or 0.2 mug/ul of magainin to study the effect of magainin on chloroplasts. Magainin did not have an effect on the isolated chloroplasts as compared to the untreated controls. The magainin gene was amplified by polymerase chain reaction (PCR). Following synthesis it was cloned into the pBIN vector and mobilized into Agrobacterium tumefaciens. Presence of the gene fragment was verified by PCR on DNA from transformed Agrobacterium cells. Tobacco leaf discs were transformed by co-cultivation with Agrobacterium. Shoots were selected on kanamycin plates and were transferred to rooting media. Mature plants were transferred to soil. Putative transformants were screened for the transgene by PCR. The gene integration event into the plant genome was analyzed by Southern blots. RT-PCR and Northern blots demonstrated the mRNA for the magainin gene.; Four transgenic plants, arising from separate transformation events, were selected and their extracts were tested for antimicrobial activity. Plants transformed with the pBIN vector without the magainin fragment served as controls. Rhizoctonia solani and Verticillium dahliae were used for bioassays of efficacy. Fungal homogenates (50 mul) were incubated with plant extracts and cell viability was measured by counting the colony forming units (cfus) obtained after plating the treated cells on PDA plates. Extracts from transformed plants decreased cell viability by 31--55% of the control. In a similar experiment, Xanthomonas campestris suspensions (50 mul; 106 cfu/ml) were treated with 75 or 100 mul of transformed plant extract. Cell viability was reduced by 18--26% and 46--52%, respectively. The successful integration and expression of the magainin gene in tobacco offers numerous possibilities for its use in cotton as well as other crop species.