Articles
EFFECT OF MULTIPLE TRANSGENES ON RESISTANCE TO FIRE BLIGHT OF ‘GALAXY’ APPLE
Genes encoding the lytic proteins, attacin E (att E) from Hyalophora cecropia, and T4 lysozyme (T4L) from T4 bacteriophage, with different promoters and expression regulatory sequences, were cloned into 5 different plasmid binary vectors for use in Agrobacterium-mediated transformation of apple (Ko et al., 1997), with the goal of enhancing resistance to fire blight.
All 5 constructs containing att E, T4L, or both, and pinattE (from pLDB15 (Destéfano Beltrán, 1991; Norelli et al., 1994)) were successfully transferred to ‘Galaxy’ using Agrobacterium. Transformation of ‘Galaxy’ was confirmed by PCR, NPTII ELISA, and western analysis.
The transformation rate varied among the different plasmid constructs.
Variation in attacin expression levels were observed among transgenic lines containing different constructs, and also containing the same construct.
Generally, transgenic lines containing att E under the control of the potato protease inhibitor II (pin) promoter had higher attacin expression than those under the control of the enhanced CaMV 35S promoter.
The AMV translation enhancer sequence increased attacin expression levels, while a signal peptide sequence resulted in lower attacin levels in transgenic lines.
Attacin was degraded in intercellular fluid extract, indicating that reduction of attacin level could be explained by intercellular degradation.
Disease evaluation in controlled environment chambers showed that some transgenic lines had significantly higher disease resistance than the nontransgenic parent.
When transgenic lines contained a signal peptide sequence, attacin levels were negatively correlated with % lesion length, supporting the hypothesis that the attacin protein increased resistance to E. amylovora. Transgenic lines containing both T4L and att E are being evaluated for synergism between the two proteins against Erwinia amylovora.