Isolation Of △～5 Fatty Acid Desaturase Gene From Two Marine Microalga

Polyunsaturated fatty acids (PUFAs) are now recognized as having a number of important nutritional and pharmaceutical values. The principal dietary source of PUFAs (especially DHA and EPA) is fish. However, fish accumulate pollutants, the extracted oil has unpleasant odor and the proportion of specific fatty acids in the lipids from this source are difficult to control. Therefore, new sources of PUFAs are needed. Microalga have been recently shown to be good alternative sources for their ability to synthesis and accumulate great amounts of of PUFAs. What's more, in order to demonstrate the mechanism of manipulation of PUFAs production, currently there is interest in identification of the genes encoding the enzymes involved in PUFAs biosynthesis (fatty acid desaturases and elongase) in many organisms, including several microalga.Nitzschia closterium f. minutissima and Phaeodastylum tricornutum, two marine diatoms, are rich in EPA (20:55,8,11,14,17) (up to 30% of total fatty acids). The 5 desaturase is responsible for the conversion of 20:4 8,11,14,17 to EPA, which is the key process in EPA biosynthesis. To obtain the genes of the 5 desaturase from both diatoms, PCR were performed on genome DNA with degenerate primer sequences designed to the conserved histidine box residues of 5 desaturases. Several PCR products were generated, and those of the expected length (500-600bp) were isolated from agarose gel and sequenced. The predicted amino acid sequences showed some similarity to other 5 desaturases, including the presence of the second and third characteristic histidine boxes. Then two pairs of primers were designed according to the known sequences to perform inverse PCR to amplify theflanking regions.The total sequence of the putative 5 desaturase gene of Nitzschia closterium f. minutissima is 1559bp. It is probable that this gene contains an intron (131bp) and the rest 1428bp encode 475 amino acids. It is deduced that the protein sequence contains three conserved histidine boxes characteristic for all membrane-bound desaturases. It also containsa cytochrome 65 domain fused at the N-terminus and an H to Q substitution in the third histidine-box, both of these features being typical of front-end desaturases. Hydrophobic plot had indicated the presence of distinct four potential transmembrane helices. The overall sequence was aligned with A5 desaturases isolated from other organisms and shares the highest identity (52%) with the P. tricornutum A5 desaturase.P. tricornutum A5 desaturase gene identified from genome DNA has 1520bp. It was found to contain an intron (HObp) by aligning with mRNA sequence published in NCBI (AY082392). Domergue et al. have analyzed its protein sequence.In this study two genome DNA sequences encoding A5 fatty acid desaturase were isolated from two marine diatoms. So far, no sequence isolated from Nitzschia dosterium f. minutissima has been published in NCBI. Therefore, this is the first reported sequence probably. Nitzschia dosterium f. minutissima was regarded as another form of P. tricornutum previously. However, their sequences of A5 fatty acid desaturase are not completely identity (52%), which implies they are not the same species.