Molecular cloning and in silico analysis of novel oleate desaturase gene homologues from Brassica juncea through sub-genomic library approach

Suresha, G. S.; Santha, I. M.

Plant Omics 6(1): 55-64

2013


ISSN/ISBN: 1836-0661
Accession: 066271423

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Abstract
The oleate desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18: 1) to linoleic acid (18: 2). Using the sequence information of Bjfad2 (EF639848) gene that was isolated and characterized earlier from Brassica juncea, three additional novel fad2 gene homologues designated as Bjfad2a (FJ696650), Bjfad2b (FJ696651) and Bjfad2c (FJ696652) were isolated from sub-genomic library of B. juncea. Sequence analysis of these genes revealed the sequence size of 2526 bp (Bjfad2a), 2326 bp (Bjfad2b) & 2325 bp (Bjfad2c), an open reading frame of 1155 bp that encodes a single polypeptide of 384 amino acids. Sequence comparison of isolated Bjfad2 gene sequences shared 99% (Bjfad2a), 96% (Bjfad2b) and 95% (Bjfad2c) sequence similarity to reported B. juncea fad2 gene (EF639848). The deduced amino acids of the these gene homologues displayed the typical three histidine boxes, a characteristic of all membrane bound desaturases along with five membrane spanning domains that are different from known plant desaturases. The signature amino acid sequence 'YNNKL' was found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the genomic structures alignment grouped them within the cluster of fad2 gene(s) of Brassicacea and are distinct from fad2 gene sequences from sesame (42%), soybean (38%) and sunflower (19%). Comparison of the Bjfad2 genomic structures with other plant fad2 genes revealed the presence of single large intron in their 5'UTR region which is evolutionarily conserved. However, the length of the intron varies across the plant species. Variation in the restriction sites of all three Bjfad2 genes and its further confirmation through restriction analysis showed that the isolated genes are different homologues but code for the same enzyme, oleate desaturase. These observations confirm that atleast four copies of fad2 gene exist which is consistent with the tetraploid nature of B. juncea genome.