Galaxy Lines Transgenic for Attacin E AND T4 lysozyme Genes Have Increased Resistance to Fire Blight

Abstract

Genes encoding lytic proteins, attacin E (att E) and T4 lysozyme (T4 L), were cloned into 6 different plasmid binary vectors for use in Agrobacterium-mediated transformation of apple. All 6 constructs containing att E, T4 L, or both were successfully transferred to Galaxy by Agrobacterium-mediated transformation. Apple transgenics have been recovered with all six constructs. NPTII ELISA and transgene PCR have confirmed transgene transformation with att E and T4 L. Transformation rate varied among different plasmid constructs. The transformation rate of pCa2AMVAtt plasmid vector was lower than with other plasmids. All att E transgenic lines expressed att E. However, the protein expression level was different among transgenic lines. pCa2AMVSPAtt transgenic lines had low expression of art E compared to other att E transgenic lines. In pCa2AMVSPAtt transgenic lines, att E was detected in total apple protein but not in intercellular fluid. Inoculation of transgenic lines indicated that some lines have increased resistance to fin blight. Fusion att E was produced using the pRSET E. coli expression vector and purified. The purified fusion att E and T4 L were used successfully to obtain polyclonal antibodies for protein analysis.