Cloning And Heterologous Expression Of The △6-Fatty Acid Desaturase Gene From Oenothera Biennis

γ-linolenic acid(GLA) is one of the essential fatty acids in mammals.It is involved in membrane structure in various cells and in the biosynthesis of prostaglandins. GLA is commonly obtained from plants such as evening primrose. However, it is evident that GLA production from current sources is inadequate for supplying the expanding market.Therefore, seeking for alternative sources are demanding.△6-fatty acid desaturase is the rate-limiting enzyme for the biosynthesis of polyunsaturated fatty acids, which catalyses the conversion of (?)inoleic acid andα-linolenic acid toγ-linolenic acid and octadecatetraenoic acid respectively.So people want to produce GLA using some microorganisms and oil crops transformed△6-fatty acid desaturase gene. The seed of Oenothera biennis is rich in GLA, but△6-fatty acid desaturase gene of Oenothera biennis has not been cloned.In this research, a 565bp DNA fragment was amplified from Oenothera biennis with degenerate oligonucleotides primers designed based on the sequences information from the conserved histidine-rich motifs II and III of plant△6-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 the 1941bp full-length cDNA sequence was firstly obtained from Oenothera biennis. Sequence analysis showed this cDNA sequence had an open reading frame (ORF) of 1344bp encoding 447 amino acids of 51.1 kDa. The deduced amino acid sequence of the ORF showed similarity to those of the△8-Sphingolipid desaturases and A 6-fatty acid desaturases from plants 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 0bD6D was subcloned into the plasmid pYES2 to generate a recombinant expression vector pYES2-D6D, which was subsequently transformed into Saccharomyces cerevisiae strain W303a for heterologous expression by lithium acetate method and the protein exhibited A6-fatty acid desaturase activity. Subsequently, the full-length cDNA sequence was submitted to GenBank (accession number EU416278). PCR products of Oenothera biennis genomic DNA template were sequenced. The result shows 0bD6D genomic gene is intronless and in-frame tandem stop codons of 9 UGA are as termination signals at the end of the ORF. Phylogenetic analysis reveals that ObD6D is the transition state between the plant△8-Sphingolipid desaturases and△6-fatty acid desaturases during the evolution period by CLUSTAL W. In addition, RT-PCR result indicates the transcript level of 0bD6D in developing seeds is higher than in leaves or developed seeds.The promoter region of a seed-specific gene napin was isolated by PCR from genomic DNA of Brassica napus strain taikong3-19 and shares homology of over 98.5% with the corresponding sequences. Subsequently, the fragment was submitted to GenBank (accession number EU416279). Two recombinant expression vectors pPZP221 (35S-D6D-NOS) and pPZP221 (NAPIN-D6D-NOS) were generated and subsequently transformed into Brassica napus for heterologous expression by pollen-tube pathway method.2000 of T0generation seeds were obtained respectively. Then five positive T0generation plants with pPZP221 (35S-D6D-NOS) and six plants with pPZP221 (NAPIN-D6D-NOS) were screened by gentamycin and PCR method.