Breed High-yielding Polyunsaturated Fatty Acid Rhodospridium Toruloides And Clone Delta6Fatty Acid Desaturase Gene

The result showed that Rhodosporidium toruloides AS2.1389and Mortierellaisabellina AS3.3410were oleaginous microorganism by detecting fatty acid of fivedifferent strains, using Soxhlet extraction and gas chromatography. It was found out thatRhodosporidium toruloides contains α-linolenic acid, using Gas chromatography–massspectrometry technology.The original strain Rhodospridium toruloides AS2.1389was treated by NTG.Thequantity of the lipids of screening strains was analyzed by phosphoric acid-vanillinreaction. A high-yielding quantity of the lipids mutant NTG-7was obtained. The resultsshowed that the fatty acid was improved47%, up to7.63g/L.Selection of α-linolenic acid high-producing Rhodospridium toruloides strain bytreating Rhodospridium toruloides with UV and LiCl. The primary screening was carriedout by using96plates with TTC as a spectrophotometric reagent，and the secondaryscreening was carried out by GC detection，In the end, a high-yielding α-linolenic acid(ALA) mutant strain D2was obtained. The result showed that α-linolenic acid content wasincrease by2.59times compare to the original strain, up to0.508g/L.Total RNA in Mortierella isabellina AS3.3410was obtained by using Trizol, andcDNA was reached by revering PCR.A partial gene sequence of delta6fatty aciddesaturase gene (D6D gene) was amplified by using degenerate primers which werederived from conserved region in the D6D gene. The up and down sequence of D6D genewere amplified by High-efficiency Thermal asymmetric interlaced PCR technology, usingspecific primers in the conserved region which was obtainded. At last the complete D6Dgene was successfully cloned from Mortierella isabellina by using the specific primers inthe front and end sequences.