Identification of a novel delta9-fatty acid desaturase gene and its promoter from oil-producing fungus Rhizopus arrhizus.

Reverse Transcriptional Polymerase Chain Reaction (RT-PCR), Rapid Amplification of the cDNA ends (RACE) and Thermal asymmetric interlaced (TAIL)-PCR were used successfully to clone the open reading frame (1,377 bp) of delta9-fatty acid desaturase gene (named as RAD9) and its promoter region from oil-producing fungi Rhizopus arrhizus. Functional identification of the protein was done by sub-cloning RAD9 into the expression vector pYES2.0 to generate a recombinant plasmid pYRAD9, which was then subsequently transformed into Saccharomyces cerevisiae delta9-fatty acid desaturase mutation strain L8-14C to be expressed under the control of GAL1 promoter. The transformant containing RAD9 named as L8-14C-pYRAD9 could grow on the synthetic minimal medium plate with out oleic acid supplement. Fatty acid analysis also showed that the transformant contained 16:1 and 18:1. This indicated that pYRAD9 could successfully complement the mutation of L8-14C. Computational analysis of the nucleotide sequence of RAD9 promoter showed several basic transcriptional elements including a CAAT box, a GC box, a CACCC box, two TATA boxes and also several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Preliminary functional analysis of this promoter in S. cerevisiae was done using lacZ report gene.