Identification of seed-borne lipoxygenase genes and the determination of changes in sequence in faba bean lines

Lipoxygenase enzymes, which contribute significantly to storage protein in legume seeds have been reported to cause the emission of volatile compounds associated with the generation of off-flavours. This is an are important factor limiting the acceptance of faba bean (Vicia faba) I foods. This study aimed at using bioinformatic tools to identify seed-borne lipoxygenase (LOX) genes and to design a biological tool using molecular techniques to find changes in sequence in faba bean lines. LOX gene mining by Exonerate sequence comparison on the whole genome sequence of faba bean was used to identify six LOX genes containing Polycystin-1, Lipoxygenase, Alpha-Toxin (PLAT) and/or LH2 LOX domains. Their sequence properties, evolutionary relationships, important conserved LOX motifs and subcellular location were analysed. The LOX gene proteins identified contained 272 – 853 amino acids (aa). The molecular weight ranged from 23.67 kDa in Gene 6 to 96.45 kDA in Gene 1. All the proteins had isoelectric points in the acidic range except Genes 6 and 7 which were alkaline. Only one gene had both LOX conserved domains with aa sequence length similar with that found in soybean and pea LOX genes and isoelectric properties with soybean LOX3. Phylogenetic analysis indicated that the genes were clustered into 9S LOX and 13S LOX types alongside other seed LOX genes in some legumes. Five motifs were found, and sequence analysis showed that three genes (Gene 1, 2 and 3) contained the 38-aa residue motif that includes five histidine residues [His-(X)4-His-(X)4-His-(X)17-His-(X)8-His]. The subcellular localization of the lipoxygenase proteins was predicted to be primarily the cytoplasm and chloroplast. Primers covering ~1.2 kb were designed, based on the conserved region of Genes 1, 2 and 3 nucleotide sequences. Gel electrophoresis showed the PCR amplification of the seed LOX gene at the expected region for twelve faba bean lines. Phylogenetic analysis showed evolutionary divergence among faba bean lines for sequenced and amplified region of their respective seed LOX alleles