RESUMO
To engineer endophytic Enterobacter cloacae as a biocontrol agent against banana fusarium wilt, a promoter-probe plasmid pUCK was constructed to identify a strong promoter to express disease resistance genes. Using a kanamycin resistance gene for selection, 10 fragments with strong promoter activity were identified from the genome of the E. cloacae KKWB-10 strain. The regions of these 10 fragments that were the primary contributors to the promoter function were identified, and their promoter activities were further evaluated using green fluorescent protein (GFP) as a reporter gene. Fragment 132aâ³ drove the highest level of GFP activity when the bacteria bearing the fragments were cultured in Luria-Bertani and banana stem extract media. The GFP-expressing strain harboring fragment 132aâ³ (K-pUCK7-132aâ³-GT) was then inoculated into banana plantlets (about 1 × 10(7) CFU per plant) to verify the activity of fragment 132aâ³ in planta. Ten days after inoculation, tissue sections of these banana plantlets were observed by laser confocal scanning microscope. Green fluorescence was observed in the tissues of banana plantlets inoculated with K-pUCK7-132aâ³-GT but not in uninoculated controls. These results suggest that fragment 132aâ³ possesses strong promoter activity when its host strain colonizes the banana plants and can be used to engineer endophytic E. cloacae KKWB-10 for biocontrol.
