[A method using long primers for cloning the upstream sequence of delta-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 y-linolenic acid. In this process, delta6-Fatty acid desaturase (D6D) plays a key role due to its enzymatic properties that catalyze the delta6 site dehydrogenation of precursor linoleic acid (18:2delta(9, 12) n-6) and a-linolenic acid (18:3delta(9, 12, 15) n-3). This reaction is the first and rate-limiting step of highly unsaturated fatty acids (HUFA) synthesis pathways. After we have isolated and cloned the gene coding delta6-fatty acid desaturase from Thamnidium elegans As3.2806 (GenBank accession number DQ099380), our interest focuses on the promotion and regulation of the gene transcription. To achieve this aim, we designed long primers and used nested inverse PCR to amplify 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 ligase at low concentration which is propitious for linear DNA to joint intromolecule. According to the sequence of delta6-fatty acid desaturase gene of Thamnidium elegans, we designed a couple of 35nt 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, and the product was used as the template of the secondary reaction, the tertiary reaction was achieved in the same way. After all the three rounds of reactions, we got a nice product about 4 kb from the EcoR I digested sample, in which a 1.3kb 5' upstream sequence (GenBank accession number DQ309425) of delta6-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. All of these implied intensely that this 1.3kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans detla6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3 kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans delta6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome.

[Cloning and expression of delta6-desaturase gene from Thamnidium elegans in Saccharomyces cerevisiae].

A 459 bp DNA fragment was amplified from Thamnidium elegans As3.2806 with degenerate oligonucleotides primers designed based on the sequences information from the conserved histidine-rich motifs II and near III of fungal delta6-fatty acid desaturases genes by RT-PCR and sequenced. Gene specific primers designed according to this partial sequence were used for the amplification of the 3'- and 5'- end of this cDNA by RACE method, and sequence information coming from these two ends were used to design two GSPs to clone the 1504bp full-length cDNA. Sequence analysis showed this cDNA sequence had an open reading frame (ORF) of 1377bp encoding 458 amino acids of 52.4kD. The deduced amino acid sequence of the ORF showed similarity to those of the above delta6-fatty acid desaturases which comprise the characteristics of membrane-bound desaturases, including three conserved histidine-rich boxes and hydropathy profile. A cytochrome b5-like domain was observed at the N-terminus. To elucidate the function of the protein, two specific primers corresponding to the nucleotide sequences of start and stop codons were used to amplify the coding sequence. The amplified cDNA TED6 was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYTED6, which was subsequently transformed into Saccharomyces cerevisiae strain INVScl for heterologous expression by lithium acetate method. Grown to logarithmic phase at 30 degrees C, the transformed cells were supplemented with 0.5 mmol/L Linoleic acid and induced by 2% galactose for a further 48 h of cultivation at 20 degrees C. Total fatty acids were extracted from the induced cell and subjected to methyl-esterification. The resultant fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC). A novel peak corresponding to gamma-linolenic acid (GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The percentage of this new fatty acid to total fatty acids was 7.5%. Gas chromatography-mass spectrometry (GC-MS) analysis of this fatty acid methyl derivative demonstrated that the novel peak was GLA methyl ester. These results showed that the coding product of this sequence exhibited the activity of converting linoleic acid (LA) to gamma-linolenic (GLA), and indicated that amino-acid sequence exhibited delta6-fatty acid desaturase activity.