Cloning and characterization of an asparagus wound-induced gene.
thesisposted on 19.11.2015, 09:10 by Simon A. J. Warner
Following previous studies, Asparagus officinalis single cell suspensions were hypothesized to be a rich source of wound-inducible mRNAs. A previously isolated clone, DDl-34, was shown to hybridize to wound-inducible transcript. This sequence was used to isolate the AoPR1 (Asparagus officinalis Pathogenesis Related cDNA clone 1). Data from the isolation and analysis of genomic clones hybridizing to DDl-34 probe suggested that these clones were unlikely to contain the upstream regulatory sequences of the AoPR1 gene and that the genomic arrangement of these sequences is complex. Inverse polymerase chain reactions (IPCR) were used to amplify AoPR1 genic sequences directly from the asparagus genome. Two products were cloned and sequenced, demonstrating that the correct sequences, upstream and downstream of the primers, had been amplified. The downstream IPCR product's sequence overlaps with AoPR1 coding sequence and contains an intron sequence. The upstream IPCR product partially overlaps with the start of AoPR1 coding sequence and was successfully used in transcript mapping experiments. Translational fusions were constructed between this fragment and the -glucuronidase (gus) reporter gene. GUS analysis demonstrated that this fragment, containing the AoPR1 promoter, was sufficient to drive wound-inducible transcription in transgenic tobacco. A smaller upstream fragment was insufficient to drive wound-inducible transcription. GUS expression was also detectable in tissues such as the xylem parenchyma, mature pollen and coloured regions of the petal. AoPR1-gus transgene expression correlates with the spatial expression patterns of phenylpropanoid biosynthesis pathway genes. The nature of the fusion suggested that the AoPR1 protein is intracellular. This is the first example of the cloning and analysis of a monocotyledon gene belonging to the 'intracellular pathogenesis related protein' class. The analysis and application of AoPR1 sequences are discussed.