Molecular cloning of cDNA encoding alliinase from onion (Allium cepa L.)
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Alliinase catalyses the release of a range of volatile sulphur compounds which are responsible for the distinctive flavour and odour of Allium species. Clones encoding this vacuolar enzyme were isolated by immunoscreening a cDNA expression library constructed in the vector λZAPII with mRNA extracted from sprouting A. cepa bulb shoots. Six clones were characterized by DNA sequencing. Four of the cDNA sequences (Alli6, 7, 8B and 9) were found to be identical apart from variation in the length of their 5' ends. These sequences contained three putative polyadenylation signals. A fifth clone, Alli4B, was also very similar but displayed a truncated 3' end with six divergent bases just prior to the polyadenylate tail and lacked the most 3' polyadenylation signal present within the sequences of the other clones. It was evident from these differences that the clones were unique. However, their high sequence homology suggested they were encoded by a single gene, or two that are very closely related. The sixth clone sequenced, Alli4A, showed no homology to the other five clones, nor to any sequences within the GenBank database. The five highly homologous cDNA sequences ranged in length from 1604 bp to 1757 bp, and all appeared to contain a complete longest open reading frame encoding a polypeptide with a predicted size of 54 884 Da. Four peptide sequences derived from purified A. cepa alliinase were aligned, and showed 93% homology with the corresponding amino acid sequences deduced from the cDNA clones. Alignment of the native alliinase N-terminal peptide to the cDNA-inferred protein sequence predicted a hydrophobic 34-residue prepeptide sequence terminating in a peptidase cleavage site. Given the vacuolar location of this enzyme, it is probable that this region functions in targeting of the alliinase precursor to the endoplasmic reticulum. The inferred mature alliinase subunit polypeptide contained sequence motifs compatible with both Asn-linked glycosylation and pyridoxal phosphate cofactor binding. Conversion of clones to the phagemid form allowed the protein expressed in Escherichia coli from Alli6 to be western blotted. This analysis revealed protein moieties with molecular masses of 47 and 41.4 kDa. Southern hybridization analysis of A. cepa genomic DNA using an alliinase cDNA probe demonstrated the presence of a small multigene family with at least four members. Further genomic DNA hybridization analysis using a probe encompassing the 3' untranslated region of the alliinase clones demonstrated that the highest identity was to a single fragment, suggesting some alliinase gene family members may be transcriptionally inactive in sprouting bulb shoot tissue. A 1.7 kb transcript was detected by northern analysis of RNA extracted from developing A. cepa seedlings, indicating that the cDNA clones were near-full-length. Alliinase mRNA could be easily detected in seedlings up to 14 days after germination. Visual assessment and densitometer analysis of hybridization intensities suggested that alliinase transcripts were most abundant two to six days after germination and declined rapidly over the ensuing eight days. This indicated that alliinase expression is regulated at the transcriptional level during this period.