Transgenic tobacco plants expressing endo-β-mannanase gene from deep-sea Bacillus sp. JAMB-602 strain confer enhanced resistance against fungal pathogen (Fusarium oxysporum)

Abstract

Genes encoding pathogenesis-related proteins, such as degrading enzymes of fungal cell wall polysaccharides, have been used to confer enhanced resistance to fungal pathogens of various plants. A new type of endo-β-mannanase gene, amn5A, was isolated from alkaliphilic Bacillus strain (JAMB-602) found in deep-sea sediment. The AMN5A mannanase is active over a wide pH range (pH 7–10) and stable at high temperature. In this study, transgenic tobacco plants expressing the amn5A gene were generated using Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) analysis revealed that the amn5A gene was integrated into the genome of transgenic tobacco plants. Southern blot analysis showed that transgenic plants contained 1–6 copies of amn5A transgenes in their genome. Expression of the amn5A transgene was confirmed by reverse transcription-PCR analysis. Leaf extracts from the transgenic plants showed degradation activity of Konjak mannan. Antifungal assay of detached leaves and in vitro whole plantlets indicated that transgenic plants expressing amn5A gene acquired enhanced resistance to the soil-borne pathogenic fungus, Fusarium oxysporum, compared to untransformed control plants.