Cloning and functional expression of a cDNA encoding a metabolic acyl-CoA Delta 9-desaturase of the cabbage looper moth, Trichoplusia ni

Acyl-CoA Delta 9-desaturases play essential roles in fatty acid metabolism and the regulation of cell membrane fluidity. In this research, a cDNA sequ ence was obtained from Trichoplusia ni adult fat body mRNA by using RT-PCR with degenerate primers based on other characterized Delta 9-desaturase seq uences. The remainder of the sequence was amplified using 3'- and 5'-RACE. A 1439 bp cDNA reconstructed from three overlapping PCR products contains a n ORF encoding a 353-amino acids (aa) protein that shows clear homology (gr eater than 50% aa identity and greater than 65% aa similarity to characteri zed insect and vertebrate desaturases). The ORF of this cDNA was subcloned into an expression vector, which relieved the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient yeast strain following genetic transfo rmation. The newly characterized desaturase from T. ni produced fatty acids Delta 9-16 and Delta 9-18 in a 1:6 ratio, compared to a 5:1 ratio, respect ively, with the yeast Delta 9 desaturase. A Northern blot hybridization and a RT-PCR experiment showed that temporal and tissue-specific patterns of e xpression of the corresponding mRNA are distinct from those of the Delta 11 -desaturase mRMA present in the pheromone glands of adult females. Based on its homology to other desaturases, the widespread distribution of its corr esponding mRNA in various tissues, and its functional assay, we conclude th at this cDNA encodes the apoprotein corresponding to the desaturase compone nt of the metabolic Delta 9-desaturase complex of T. ni. (C) 1999 Elsevier Science Ltd. All rights reserved.