Cloning and characterization of full length CDS of delta-6 desaturase (d6d) from Iranian borage (Echium amoenum)

Document Type : Research Paper

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Abstract

Delta-6 desaturase (D6D), an enzyme of a lipid methabolic pathway, has been found as active form in some microorganisms and plants like different species of the genus Echium from family Boraginaceae. The enzyme converts essential fatty acids linoleic acid and α-linolenic acid into long-chain polyunsaturated ones, γ-linolenic acid and stearidonic acid, respectively. In this research, to isolate the d6d gene from Iranian borage (Echium amoenum), we followed total RNA isolation from immature seeds, total cDNA synthesis and PCR amplification using the gene specific primers designed according to the sequence of d6d in the other Echium species. Approximately a 1350 bp amplified fragment was inserted into the vector pBluscript SK(+) and cloned in Escherichia coli strain DH5α. Nucleic acid sequence analysis of the cloned fragment verified the successful isolation and cloning of the full length CDS of d6d gene from E. amoenum. The cloned CDS include 1347 nucleotides coding for 448 amino acids. The translated protein possessed high similarity of 94–96% to its counterparts previously reported in other Echium species. All the expected domains, i.e., cytochrome b5, fatty acid desaturase, HPGG motif and histidine rich conserved motifs were found in the sequence of translated protein. Furthermore, secondary structure, transmembrane domains and the hydropathy profile of the protein were highly similar to its counterparts in the other Echium species. The d6d gene is a promising gene to engineer the fatty acid metabolism in crop plants. Gene expression studies are necessary for further characterization of the cloned gene and its encoded enzyme. The sequence of the coding region of d6d gene from E. amoenum was submitted to GenBank under the accession number GU237486.

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