Isolation and characterisation of genomic clones of alliinase from Allium cepa L.

Abstract

The culinary and medicinal attributes of Alliums are derived from the high levels of nonprotein sulfur compounds which they contain. When onion cells are disrupted, the vacuolar enzyme alliinase hydrolyses the cytoplasmic S-alk(en)yl cysteine sulfoxide flavour precursors to produce pyruvate, ammonia and the many volatile sulphur compounds associated with flavour and odour. cDNA clones of alliinase have been previously isolated by immunoscreening an onion cDNA expression library. In this thesis, a genomic library of A. cepa was constructed and screened using the alliinase cDNA clone as a probe. Positively hybridising clones were screened initially using alliinase specific primers. Sequencing of PCR products, intact lambda and plasmid clones was performed to determine the sequence of the clones. One of the clones isolated encoded the previously isolated cDNA clones, while another contained a number of nucleotide differences. Both clones contained four small introns within the coding region. The predicted proteins of each clone are very similar, but nucleotide differences within the upstream, downstream, intron and mRNA regions may influence gene expression. An alliinase promoter from one of the genomic clones was cloned adjacent to a β-glucuronidase gene to determine the promoter functionality. These constructs were used for Agrobacterium mediated transformation of tobacco, and microprojectile bombardment of onion mini-bulbs. In tobacco, the alliinase promoter was expressed to a limited extent in some vascular tissue. Expression in onion minibulbs, although only evaluated transiently, indicated that this is a functional monocotyledonous promoter, which may have many applications in the genetic manipulation of onions. The expression level of the alliinase promoter was approximately half that of CaMV 35S promoter suggesting that features of the mRNA and protein are mainly responsible for the high expression level of alliinase