Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean

Abstract

In plants, the endoplasmic reticulum (ER)-associated oleate desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues. In soybean three FAD2-like genes have been reported including two seed-specific genes, FAD2-1A and FAD2-1B, and a house-keeping gene FAD2-2. In this study, we isolated a novel gene encoding FAD2 isoform, designated as FAD2-3. The deduced amino acid sequences of the FAD2-3 displayed the typical three histidine boxes characteristic of all membrane-bound desaturases, and possessed a C-terminal signal for ER retention. Phylogenetic analysis showed that FAD2-3 is grouped within plant house-keeping FAD2 sequences. Yeast cells transformed with a plasmid construct containing the FAD2-3 coding region accumulated a considerable amount of linoleic acid (18:2), normally not present in wild-type yeast cells, suggesting that the isolated gene encodes a functional FAD2 enzyme. Semi-quantitative RT-PCR and in silico analysis showed that FAD2-3 gene is constitutively expressed in both vegetative tissues and developing seeds. In soybean leaves, the level of linolenic acid (18:3) increases with the decrease of linoleic aicd (18:2) under cold treatment. However, no significant change of transcript levels of FAD2-2 and FAD2-3 genes was detected. These results indicated that the altered polyunsaturated fatty acid levels in leaves treated with cold stress have no direct correlation with the expression of these two microsomal oleate desaturase genes.