Molecular cloning and functional analysis of two FAD2 genes from American grape (Vitis labrusca L.) |
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Authors: | Kyeong-Ryeol Lee Sun Hee Kim Young-Sam Go Sung Min Jung Kyung Hee Roh Jong-Bum Kim Mi-Chung Suh Sukchan Lee Hyun Uk Kim |
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Affiliation: | 1. Department of Agricultural Biotechnology, National Academy of Agricultural Science, RDA, Suwon 441‐707, Republic of Korea;2. Department of Plant Biotechnology, Chonnam National University, Gwangju 500‐757, Republic of Korea;3. Department of Horticultural Crop Research, National Institute of Horticultural and Herbal Science, RDA, Suwon 440‐706, Republic of Korea;4. Department of Bioenergy Science and Technology, Chonnam National University, Gwangju 500‐757, Republic of Korea;5. Department of Genetic Engineering, Sung Kyun Kwan University, Suwon 440‐746, Republic of Korea |
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Abstract: | The synthesis of polyunsaturated fatty acids (PUFAs), the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase 2 (FAD2; EC 1.3.1.35), also called microsomal oleate Δ12-desaturase. Since the FAD2 gene was first identified in Arabidopsis thaliana, FAD2 research has gained wide interest as the essential enzyme for synthesizing PUFA. Grapes are one of the most frequently cultivated fruits in the world, with most commercial growers cultivating Vitis vinifera and V. labrusca. Grapeseed oil contains a high proportion, 60–70% of linoleic acid (18:2). We cloned two putative FAD2 genes from V. labrusca cv. Campbell Early based on V. vinifera genome sequences. Deduced amino acid sequences of two putative genes showed that VlFAD2s show high similarity to Arabidopsis FAD2 and commonly contain six transmembrane domain, three histidine boxes and endoplasmic reticulum (ER) retrieval motif representing the characteristics of fatty acid desaturase. Phylogenetic analyses of various plant FAD2s showed that VlFAD2-1 and VlFAD2-2 are separately grouped with constitutive and seed-type FAD2s, respectively. Southern blot showed that one or two bands are found in each lane. Because Campbell Early is a hybrid cultivar, FAD2-1 and FAD2-2 genes may exist as one copy in V. labrusca. Expression analysis in different tissues indicated that VlFAD2-1 is a constitutive gene but VlFAD2-2 is a seed-type gene. Complementation experiments of fad2-1 mutant Arabidopsis with VlFAD2-1 or VlFAD2-2 demonstrated that VlFAD2-1 and VlFAD2-2 can restore low PUFA proportion of fad2 to normal PUFA proportion. |
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Keywords: | PUFA, polyunsaturated fatty acid PC, phosphatidylcholine BSA, bovine serum albumin EDTA, ethylenediaminetetraacetic acid SDS, sodium dodecylsulfate SSC, sodium chloride&ndash sodium citrate ORF, open reading frame cDNA, DNA complementary to RNA PPT, dl-phosphinothricin UTR, untranslated region bp, base pair kb, kilobase |
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