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Cloning of a Novel Omega-6 Desaturase from Flax (Linum usitatissimum L.) and Its Functional Analysis in Saccharomyces cerevisiae

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Abstract

The Δ12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Δ12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids—putative membrane-bound Δ12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Δ12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence ‘YNNKL’ was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.

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Abbreviations

ALA:

Alpha linolenic acid; 18:3

ER:

Endoplasmic reticulum

FAD:

Fatty acid desaturase

FAD2:

Omega-6 desaturase

LA:

Linoleic acid; 18:2

PUFA:

Polyunsaturated fatty acid 16:0; Palmitic acid, 18:0; Stearic acid, 18:1; Oleic acid

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Acknowledgments

The work in this manuscript is an outcome of the R&D project on flax supported financially by Department of Biotechnology, Government of India. The inputs provided by BVU also were crucial for completing the project. We thank Dr. P. B. Ghorpade, Flax Breeder, Nagpur for supplying seeds of the flax variety NL 97. Technical support by Ms. Ankita Khare and Mrs. Ashwini Rajwade is acknowledged.

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  1. Interactive Research School for Health Affairs, Medical College Campus, Bharati Vidyapeeth University, Pune-Satara Road, Pune, 411043, India

    Rupali M. Khadake, Prabhakar K. Ranjekar & Abhay M. Harsulkar

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  1. Rupali M. Khadake
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  2. Prabhakar K. Ranjekar
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Correspondence to Abhay M. Harsulkar.

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Khadake, R.M., Ranjekar, P.K. & Harsulkar, A.M. Cloning of a Novel Omega-6 Desaturase from Flax (Linum usitatissimum L.) and Its Functional Analysis in Saccharomyces cerevisiae . Mol Biotechnol 42, 168–174 (2009). https://doi.org/10.1007/s12033-009-9150-3

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