Isolation, cloning and bioinformatics analyses of the Delta 15 desaturase gene from the yeast Pichia pastoris GS115 in order toincreasing of alpha-linolenic acid content of oilseeds

Alpha-linolenic acid is an omega-3 fatty that is essential for maintaining a healthy body but can't produce in the human body. The oils of some oilseeds, as well as some plants are the main sources of alpha-linolenic acid but the content of alpha-linolenic acid, in important oilseed plants like soybean and rapeseed are relatively low. The aim of this study was the cloning of Delta 15 Desaturase gene to increase alpha-linolenic acid production in the oilseed plants. For this purpose, after extracting total RNA and cDNA synthesisfrom Pichia pastoris GS115, the genewas amplified using gene-specific primers containing Kozak sequence and then cloned in PTZ57R / T vector and transformed into E.coli strain DH5α. After sequencing, the recombinant fragment was isolated from TA vector and was cloned into pBluescript KS(+) vector containing Napin promoter. The designed gene construct was cloned in pBI121 binary vector and then was transformed into Agrobacterium tumefaciens strain LBA4404. Bioinformatics characterization of the target gene was investigated by servers TopPred, TMHMM, ProtParam and SOPMA. The results of the colony PCR and amplification of the 1246 bp fragment, confirmed the accuracy of the gene cloning. The amplification of a 1210 bp fragment using an internal primer of napin promoter and Delta 15 desaturase, as well as the production of the 2145 bp fragment in enzymatic digestion confirmed the correct incorporation of the gene along with Napin promoter. Nucleotide sequencing results showed that the cloned CDS include 1246 nucleotides and translated to a protein with 415 amino acids. The amino acid sequence analysis confirmed the presence of two domains and five Transmembrane helices. Also, prediction of the protein's properties, signal peptides, and the second structure, proved that this enzyme is stable transmembrane enzymes.