A Method Using Long Primers for Cloning the Upstream Sequence of Δ-6 Fatty Acid Desaturases Gene of Thamnidium elegans by Nested Inverse PCR

Thamnidium elegans is a kind of phycomycete that produces essential unsaturated fatty acids, particularly γ-linolenic acid. In this process, Δ6-Fatty acid desaturase (D6D) plays a key role because of its enzymatic properties that catalyze the Δ6 site dehydrogenation of precursor linolenic acid (18:2Δ9,12 n-6) and γ-linolenic acid (18:3Δ9,12,15 n-3). This reaction is the first and rate-limiting step of highly unsaturated fatty acid (HUFA) synthesis pathways. After we isolated and cloned the gene coding Δ6-fatty acid desaturase from Thamnidium elegans AS3.2806 (GenBank accession number DQ099380), our interest was focused on the promotion and regulation of the gene transcription. To achieve this aim, we designed long primers and used nested inverse PCR to amplify the DNA flanking sequences. First, genome of Thamnidium elegans was extracted and digested with restriction enzymes EcoR I and Kpn I, respectively. Then we ligated the digested DNA with T4 DNA ligase at a low concentration, which is propitious for linear DNA to joint intermolecularly. According to the sequence of Δ6-fatty acid desaturase gene of Thamnidium elegans, we designed a couple of 35 nt long inverse primers and two couples of shorter inverse primers for inverse PCR. Three rounds of PCR reactions were performed. In the primary reaction, the ligated DNA was used as a template, the product was used as the template in the secondary reaction, and the tertiary reaction was achieved in the same way. After all the three rounds of reactions, we obtain a nice product, about 4 kb, from the EcoR I digested sample, in which a 1.3 kb 5′ upstream sequence (GenBank accession number DQ309425) of Δ6-fatty acid desaturase gene containing several putative regulatory elements, including TATA box, FSE-2, AP-1 sites, CCAAT cis-element site, and STRE-binding site, was derived after sequencing. These implied that this 1.3 kb fragment was a condition-regulated promoter. It is the first report about Thamnidium elegans Δ6-fatty acid desaturase gene promoter. The procedure described in this article is a rapid and simple method and particularly useful to isolate flanking sequences from the fungal genome.