Sequencing of alliinase gene from Allium stipitatum REGEL subgenus Melanocrommyum

The genus Allium contains about 800 species divided into different subgenera [1]. All species have an enzyme called alliinase, which catalyzes the cleavage of cysteine sulfoxides forming bioactive aroma compounds [2]. Common species like Allium sativum or Allium cepa are well characterized by secondary plant compounds as well as their alliinases [3,4]. Allium stipitatum is well established as a condiment in Central Asian cuisine. However no research has been done on its alliinase yet. Besides, A. stipitatum contains a unique pyridinyl cysteine N-oxide, which is subjected to alliinase [5].

A. stipitatum and A. sativum display a different protein pattern on SDS-gels (Fig.). A protein extract of A. sativum has a high amount of alliinase (monomers at 50 kDa), whereas A. stipitatum's protein extracts only show a faint protein spot up there. Highly conserved sequences of alliinase genes were used to design primers for amplification of the corresponding alliinase gene in A. stipitatum via PCR. Thus it was possible to obtain a part of an alliinase gene. Using the method of restriction enzyme site-directed amplification [6], the gene was extended to the flanking regions leading to the whole alliinase gene of A. stipitatum, which will be the first complete gene of subgenus Melanocrommyum in data bases. Investigation of mRNA proved the gene expression in A. stipitatum bulbs. The gene consists of five exons and four introns. Despite the wide differences in protein expression scheme of A. stipitatum and A. sativum, their alliinases amino acid sequences are 83% alike and only differ four amino acids in length.

Fig. 1

References:

[1] Fritsch RM et al. Phyton (Horn, Austria) (2010) 49:145 – 220

[2] Stoll A, Seebeck E, Helvet Chim Acta. (1949) 32: 197 – 205

[3] Nock LP, Mazelis M, Plant Physiol. (1987) 85:1079 – 1083

[4] Kuettner EB, Hilgenfeld R, Weiss MS, J Biol Chem. (2002) 277:46402 – 7

[5] Kubec R et al. J Agric and Food Chem. (2011) 59: 5763 – 5770

[6] Gonzáles-Ballester D et al. Anal Biochem. (2005) 340:330 – 5