Abstract
Genetic manipulation of the oil-yielding crop plants for better oil quality through biotechnological methods is an important aspect of crop improvement. Due to the inherent absence of the Δ6-desaturase (d6D) function, Brassica juncea, an oil-yielding crop plant, is unable to synthesize γ-linolenic acid (GLA), a nutritionally important fatty acid although the crop plant synthesizes the precursor fatty acids required for GLA production. Cyanobacterial d6D introduces carbon–carbon double bond onto linoleic acid (C18:2) and α-linolenic acid (C18:3) by desaturation processes for production of GLA and octadecatetraenoic acid (OTA) respectively. In the present investigation, d6D coding sequence from Synechocystis sp. PCC6803 was cloned by polymerase chain reaction and introduced into B. juncea through Agrobacterium-mediated transformation technique. Both cytosolic as well as seed-specific expression of d6D were attempted. The transformed plants show production of GLA and OTA in contrast to their absence in the untransformed control plants adducing evidence for introgression and functional expression of the cyanobacterial d6D gene in B. juncea.
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Abbreviations
- ALA:
-
α-linolenic acid
- BAP:
-
6-benzylaminopurine
- CaMV:
-
Cauliflower mosaic virus
- d6D:
-
Δ6-desaturase
- 2,4,-D:
-
2,4,-dichlorophenoxyacetic acid
- FAMEs:
-
fatty acid methyl esters
- GLA:
-
γ-linolenic acid
- GLC:
-
gas liquid chromatography
- GUS:
-
β-glucuronidase
- hptII:
-
hygromycin phosphotransferase gene
- MS:
-
Murashige and Skoog
- nos :
-
nopaline synthase gene
- OTA:
-
octadecatetraenoic acid
- PCR:
-
polymerase chain reaction
- SD:
-
standard deviation
- TAG:
-
triacyl glycerol
- TLC:
-
thin layer chromatography
References
Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer U, Cirpus P, Heinz E (2004) Biosynthesis of very-long-chain-ployunsaturated fatty acids in transgenic oilseed: constraints on their accumulation. Plant Cell 16:2734–2748
Agnihotri A, Kaushik N (1998) Transgressive segregation and selection of zero erucic acid strains from intergeneric crosses of Brassica. Indian J Plant Genet Resour 11:251–255
Agnihotri A, Kaushik N (2000) Incorporation of superior nutritional quality traits in Indian B. juncea. Indian J Plant Genet Resour 12:352–358
Agriculture and Agri-food Canada Bi-weekly bulletin (2004) Overview: world oilseed sector and Canadian marketing opportunities. 17(6):1–4
Barfield DG, Pua EC (1991) Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation. Plant Cell Rep 10:308–314
Barre DE (2001) Potential of evening primrose, borage, black currant, and fungal oils in human health. Ann Nutr Metab 45:47–57
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917
Burton WA, Salisbury P, Potts D (2003) The potential of canola quality Brassica juncea as an oilseed crop for Australia. In: Proceedings of the 13th biennial Australian research assembly on Brassicas. Tamworth, NSW, Australia, pp 62–64
Cardoza V, Stewart CN Jr (2004) Brassica biotechnology: progress in cellular and molecular biology. In Vitro Cell Dev Biol-Plant 40:542–551
Clemente R, Walker DJ, Bernal MP (2005) Uptake of heavy metals and As by Brassica juncea grown on contaminated soil in Aznalcollar (Spain): the effects of soil amendments. Environ Pollut 138:46–58
Cook D, Grierson D, Jones C, Wallace A, West G, Tucker G (2002) Modification of fatty acid composition in tomato (Lycopersicon esculentum) by expression of a borage delta6-desaturase. Mol Biotechnol 21:123–128
Dellaporta SL, Wood J, Hiks JB (1983) A Plant DNA minipreparatio; version II. Plant Mol Biol Rep 1:19–21
Denis M, Delourme R, Gourret JP, Mariani C, Renard M (1993) Expression of engineered nuclear male sterility in Brassica napus (genetics, morphology, cytology and sensitivity to temperature). Plant Physiol 101: 1295–1304
Goffman FD, Galletti S (2001) Gamma-linolenic acid and tocopherol contents in the seed oil of 47 accessions from several Ribes species. J Agric Food Chem 49:349–354
Grison R, Grezes-Besset B, Schneider M, Lucante N, Olsen L, Leguay JJ, Toppan A (1996) Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene. Nat Biotechnol 14:643–646
Gupta V, Mukhopadhyay A, Arumugal N, Sodhi YS, Pental D, Pradhan AK (2004) Molecular tagging of erucic acid trait in oilseed mustard (Brassica juncea) by QTL mapping and single nucleotide polymorphisms in FAE1 gene. Theor Appl Genet 108:743–749
Gyves de EM, Sparks CA, Sayanova O, Lazzeri P, Napier JA, Jones HD (2004) Genetic manipulation of γ-linolenic acid (GLA) synthesis in a commercial variety of evening primrose (Oenothera sp.). Plant Biotechnol 2:351
Hegde DM (2003) Well oiled solutions: extensive application of emerging technologies will help India make a qualitative leap in the oilseeds sector. Times Agric J 2:43–47
Henz BM, Jablonska S, van-de-kerkhof PC, Stingl G, Blaszczyk M, Vandervalk PG, Veenhuizen R, Muggli R, Raederstorff D (1999) Double-blind, multicentre analysis of the efficacy of borage oil in patients with atopic eczema. Br J Dermatol 140:685–688
Hong H, Dalta N, Reed DW, Covello PS, MacKenzie SL, Qiu X (2002) High-level production of γ-linolenic acid in Brassica juncea using a Δ6 desaturase from Pythium irregulare. Plant Physiol 129:354–362
Jefferson RA (1987) Assaying chimeric genes in plant: GUS gene fusion system. Plant Mol Biol Rep 5:387–405
Kates M (1986) Techniques of lipidology: isolation, analysis and identification. Elsevier, Amsterdam
Kaushik N, Agnihotri A (2003) Fatty acid variability in Indian Mustard (Brassica juncea) lines generated through inter- and/or intra-specific hybridization. In: Proceedings of the 11th international rapeseed congress, Copenhagen, Denmark, 2003, vol 1, pp 274–276
Kinney JA (1994) Genetic modification of the storage lipids of plants. Curr Opin Biotechnol 5:144–151
Knutzon DS, Thomson GA, Radke SE, Johnson WB, Knauf VC, Kridl JC (1992) Modification of Brassica seed oil by antisense expression of a stearoyl-ACP desaturase gene. Proc Natl Acad Sci USA 89:2624–2628
Knutzon D, Chan GM, Mukerji P, Thurmond JM, Chaudhary S, Huang Y-S (1999) Genetic engineering of seed oil fatty acid composition. In: Altman A, Ziv M, Izhar S (eds) Plant biotechnology and in vitro biology in the twenty-first century. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 575–578
Laoteng K, Mannontarat R, Tantichareon M, Cheevadhanarak S (2000) Δ6-desaturase of Mucor rouxii with high similarity to plant Δ6-desaturase and its heterologous expression in Saccharomyces cerevisiae. Biochem Biophys Res Commun 279:17–22
Liu J-W, Huang Y-S, DeMicheles S, Bergana M, Mobike E, Hastilow C, Chuang L-T, Mukherjee P, Knutzon D (2001) Evaluation of seed oil from a canola plant genetically transformed to produce high level of γ-linolenic acid. In: Huang Y-S, Ziboh A (eds) γ-Linolenic acid: recent advances in biotechnology and clinical applications. AOCS Press, Champaign IL, pp 61–71
Mackey M (2002) The application of biotechnology to nutrition: an overview. J Am Coll Nutr 21(3):157S–160S
Majur BJ, Rice D, Haselkorn R (1980) Identification of blue green algal nitrogen fixation genes by using heterologous DNA hybridization probes. Proc Natl Acad Sci USA 77:186–190
Mitchell AG, Martin CE (1995) A novel cytochrome b5-like domain is linked to the carboxy terminus of the Saccharomyces cerevisiae Δ9-fatty acid desaturase. J Biol Chem 270:29766–29772
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15:173–193
Napier JA, Michaelson LV, Stobart AK (1999a) Plant desaturases: harvesting the fat of the land. Curr Opin Plant Biol 2:123–127
Napier JA, Sayanova O, Sperling P, Heinz E (1999b) A growing family of Cytochrome b 5 -domain fusion proteins. Trends Plant Sci 4:2–4
Palombo JD, DeMichele SJ, Liu JW, Huang YS (2000) Comparison of growth and fatty acid metabolism in rat fed diets containing equal levels of γ-linolenic acid from high γ-linolenic acid canola or borage oil. Lipids 35:975–981
Purdy RH (1986) High oleic sunflower: physical and chemical characteristics. J Am Oil Chem Soc 63:773–775
Qi B, Fraser T, Mugford S, Dobson G, Sayanova O, Butler J, Napier JA, Stobart AK, Lazarus CM (2004) Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants. Nat Biotechnol 22:739–745
Qiu X, Hong HP, Datla N, MacKenzie SL, Tayler CD, Thomas LT (2002) Expression of borage Δ6-desaturase in Saccharomyces cerevisiae and oilseed crops. Can J Bot 80:42–49
Reddy AS, Thomas TL (1996) Expression of a cyanobacterial Δ6-desaturase gene results in γ-linolenic acid production in transgenic plants. Nat Biotechnol 14:639–642
Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Genetic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–6
Rogers SO, Bendich AJ (1994) Plant molecular biology manual D1. In: Gelvin SB, Scilperoot RA (eds) Extraction of total cellular DNA from plants, algae and fungi. Kluwer Academic Publishers, New York, pp 1–8
Sayanova O, Davies GM, Smith MA, Griffiths G, Stobart AK, Shewry PR, Napier JA (1999a) Accumulation of Δ6-unsaturated fatty acids in transgenic tobacco plants expressing a Δ6-desaturase from Borago officinalis. J Exp Bot 50:1647–1652
Sayanova O, Shewry PR, Napier JA (1999b) Histidine-41 of the cytochrome b5 domain of the borage Δ6 fatty acid desaturase is essential for enzyme activity. Plant Physiol 121:641–646
Sayanova O, Napier JA (2004) Eicosapentaenoic acid: biosynthetic routes and the potential for synthesis in transgenic plants. Phytochemistry 65:147–158
Shiba H, Kimura N, Takayama S, Hinata K, Suzuki A, Isogai A (2000) Alteration of the self-incompatibility phenotype in Brassica by transformation of the antisense SLG gene. Biosci Biotechnol Biochem 64:1016–1024
Singh S, Green A, Stoutjesdijk P, Liu Q (2000) Invert-repeat DNA: a new gene silencing tool for seed lipid composition. Biochem Soc Trans 28:925–927
Singh SP, Zhou XR, Liu Q, Stymne S, Green AG (2005) Metabolic engineering of new fatty acids in plants. Curr Opin Plant Biol 8:197–203
Stahl RJ, Arnoldo M, Glavin TL, Goring DR, Rothstein SJ (1998) The self-incompatibility phenotype in Brassica is altered by the transformation of a mutant S locus receptor kinase. Plant Cell 10:209–218
Stoutjesdijk PA, Hurlestone C, Singh SP, Green AG (2000) High-oleic acid Australian Brassica napus and Brassica juncea varieties produced by co-suppression of endogenous delta12-desaturase. Biochem Soc Trans 28:938–940
Tsang EW, Yang J, Chang Q, Nowak G, Kolenovsky A, McGregor DI, Keller WA (2003) Chorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene. Plant Mol Biol 51:191–201
Tzen JTC, Cao Y, Laurent P, Ratnayake C, Huang AHC (1993) Lipids, proteins, and structure of seed oil bodies from diverse species. Plant Physiol 101:267–276
Verwoert II, van der Linden KH, Walsh MC, Nijkamp HJ, Stuitje AR (1995) Modification of Brassica napus seed oil by expression of the Escheresia coli fabH gene, encoding 3-ketoacyl-acyl carrier protein synthase III. Plant Mol Biol 27:875–886
Vitha S, Benes K, Philips JP, Gartland KMA (1995) Histochemical GUS analysis. In: Gartland KMA, Davey MR (eds) Agrobacterium protocols. Humana Press, Totowa, NJ, pp 185–193
Acknowledgements
BD, LG and SR are supported by the Council of Scientific and Industrial Research, New Delhi, India through Research Associateship and Senior Research Fellowships, respectively. BD is thankful to Dr. Gourab Gangopadhyay, Department of Botany, Bose Institute for helpful suggestions. The work is partly supported by research grants to ALM from the Department of Biotechnology, Government of India.
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Das, B., Goswami, L., Ray, S. et al. Agrobacterium-mediated Transformation of Brassica juncea with a Cyanobacterial (Synechocystis PCC6803) Delta-6 Desaturase Gene Leads to Production of Gamma-linolenic Acid. Plant Cell Tiss Organ Cult 86, 219–231 (2006). https://doi.org/10.1007/s11240-006-9111-5
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DOI: https://doi.org/10.1007/s11240-006-9111-5