球等鞭金藻中Δ5 去饱和酶基因的克隆与功能鉴定
ISOLATION AND FUNCTIONAL ANALYSIS OF A Δ5 DESATURASE GENE FROM ISOCHRYSIS GALBANA
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摘要: 球等鞭金藻(Isochrysis galbana)是一类单细胞海洋微藻, 富含二十二碳六烯酸(DHA, 22: 6Δ4, 7, 10, 13, 16, 19)。我们利用RACE 的方法从球等鞭金藻cDNA 文库中同源克隆到一个大小为1329 bp 的cDNA 片段, 编码442个氨基酸的多肽, 分子量约49.9 kD。生物信息学分析表明, 其编码产物N 端具有细胞色素b5 结构域, 以及与电子传递有关的三个富含组氨酸的结构域, 与Pavlova salina Δ5 去饱和酶同源性最高, 达56%, 故将该基因命名为IgD5。酿酒酵母功能鉴定实验表明, 其编码的蛋白质具有Δ5 去饱和酶活性, 能够将二高-γ-亚麻酸(DGLA, 20:3Δ8, 11, 14)转化成花生四烯酸(AA, 20:4Δ5, 8, 11, 14), 转化效率平均为34.6%, 最高可达40.3%。
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关键词:
- 球等鞭金藻 /
- Δ5 去饱和酶 /
- 超长链多不饱和脂肪酸
Abstract: The very long chain (≥C20) polyunsaturated fatty acids (VLCPUFA), such as arachidonic acid (AA; 20:4ω6), eicosapentaenoic acid (EPA; 20:5ω3) and docosapentaenoic acid (DHA; 22:6ω3) are essential for human health and nutrition. Dietary supplementation with these fatty acids is not only helpful for fetal neuronal, but also can reduce the risk of cardiovascular disease, hypertension, inflammatory and other diseases. Human beings can synthesize these fatty acids from the two essential fatty acid, linoleic acid (LA; 18:2ω6) and α-linolenic acid (ALA; 18:3ω3), which must be obtained from the diet. However, the synthetic efficiency is somewhat limited, and can not meet our daily requirement. AA can be obtained easily from meat, egg and milk, often in excessive amounts, EPA and DHA, however, can only be obtained from marine fish that are so often omitted from the modern diet, in much lower amounts than required. This shortage is further worsened by the fact that the natural marine fish resources have been depleting fast in recent years. In addition, the recent findings of toxic chemicals in fish oil has created fears for the consumption of fish products hence reduced the intake of EPA and DHA even further. Therefore, alternative source of these VLCPUFAs are therefore desirable. To obtain them from oil plants in commercial and sustainable quantities is particularly attractive. However, no oil-seed species produce such products naturally. The VLCPUFA biosynthesis pathways in organisms such as filamentous fungus and marine microalgae have been elaborated. Accordingly, genes encoding for elongases and desaturases involved in their metabolic pathway have been cloned from a variety of organisms including algae, mosses, fungi, nematodes and humans in the last 10 years. The reconstruction of the VLCPUFAs metabolic pathway into higher plants has been achieved in Arabidopsis, linseed, mustard and soya bean by introducing a set of 3—9 fatty acid desaturase and elongase genes. However, the production of VLCPUFAs, especially DHA, in these transgenic plants is somewhat low, much lower than that found in the EPA and DHA producing microorganisms from which these genes were originally isolated. This may be attributed to the usual ‘pick-and-mix’ strategies to choose the gene set to produce these transgenics. We aim to mine the whole set of enzymes from the same organism and use them as a gene set for the production of EPA and DHA in oilseed plants. Isochrysis galbana, a marine microalga, rich in docosahexaenoic acid (DHA, 22:6n-3, Δ4, 7, 10, 13, 16, 19), was chosen in this study. The Δ8 desaturation pathway may be operating in the production of EPA/DHA in this organism, and this pathway was thought to be more efficient over the conventional Δ6 desaturation pathway. Previously, two of the five genes involved in the conversion of C18 fatty acid substrates to the final product DHA have been isolated. The first one was the Δ9 elongase gene that catalyses the first step in the DHA biosynthesis pathway, the elongation of LA to EDA and A LA to ETrA. The second one was a Δ4 desaturase, which was involved in the final step of the biosynthesis of DHA. Both enzymes showed high catalytic activities and also had restricted substrate specificity. Here, we reported the isolation of the third gene, a Δ5 desaturase gene that was capable to convert DGLA to AA. That was achieved by the RACE strategy using different degenerate primers based on the conserved motifs of known desaturase sequences to isolate a partial cDNA from an I. galbana cDNA library. The full length cDNA was subsequently assembled and it consisted of 1329 nucleotides, encoding a protein of 442 amino acids with predicted molecular mass of 49.9 kD. Bioinformatics analysis showed that it shared homology with other functionally known front-end fatty acid desaturases and the highest homology of 56% was found with a Δ5 desaturase from Pavlova salina. As characterized by this family of desaturases, it contained an N-terminal cytochrome b5 domain, and three histidine rich motifs (his-boxes) related to electron transfer. Functional analysis by expression in Saccharomyces cerevisiae revealed that it could convert DGLA (20:3Δ8, 11, 14) to AA (20:4Δ5, 8, 11, 14) by introducing a double bond in the acyl chain at the Δ5 position, indicating that this newly isolated cDNA sequence encodes a protein that specifically catalyzes for the conversion of C20-Δ5-polyunsaturated fatty acid, AA, hence it was designated as IgD5. -
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[1] Crawford M. Placental delivery of arachidonic and docosahexaenoic acids: implications for the lipid nutrition of preterm infants [J]. American Journal of Clinical Nutrition, 2000, 71, 275—284
[2] Thies F, Garry J M, Yaqoob P, et al. Association of n-3 polyunsaturated fatty acids with stability of atherosclerotic plaques: a randomised controlled trial [J]. Lancet, 2003, 361(9356): 477—485
[3] Kinsella J E, Lokesh B, Broughton S, et al. Dietary polyunsaturated fatty acids and eicosanoids: potential effects on the modulation of inflammatory and immune cells: an overview[J]. Nutrition, 1990, 6(1): 24—44
[4] Pauly D, Alder J, Bennett E, et al. The future for fisheries [J]. Science, 2003, 302(5649): 1359—1361
[5] Yokoo E M, Valente J G, Grattan L, et al. Low level methylmercury exposure affects neuropsychological function in adults [J]. Environmental Health, 2003, 2(1): 8
[6] Drexler H, Spiekermann P, Meyer A, et al. Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results [J]. Journal of Plant Physiology, 2003, 160(7): 779—802
[7] Li X B, Xu X D, Kong R Q. Studies on the production of oil and polyunsaturated fatty acids in five species of Nannochloropsis[J]. Acta Hydrobiologica Sinica, 2010, 34(5): 893—897 [李秀波, 徐旭东, 孔任秋. 五种微绿球藻产油和产多不饱和脂肪酸的研究. 水生生物学报, 2010, 34(5): 893—897]
[8] Yadav N S, Wierzbicki A, Aegerter M, et al. Cloning of higher plant omega-3 fatty acid desaturases [J]. Plant Physiology, 1993, 103(2): 467—476
[9] Pereira S L, Huang Y S, Bobik E G, et al. A novel omega3- fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid [J]. Biochemical Journal, 2004, 378(2): 665—671
[10] Yuan D J, Zhou K Y, Kang J X. The cloning and preliminary characterization of a C18:0 Δ9 desaturase gene from marine microalgae Pavlova viridis [J]. Acta Hydrobiologica Sinica, 2009, 33(4): 732—739 [元冬娟, 周克元, 康景轩. 绿色巴夫藻脂肪酸去饱和酶的克隆和初步研究. 水生生物学报, 2009, 33(4): 732—739]
[11] Wallis J G, Browse J. The Delta8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids [J]. Archives of Biochemistry and Biophysics, 1999, 365(2): 307—316.
[12] Qi B, Beaudoin F, FraseR T, et al. Identification of a cDNA encoding a novel C18-Delta(9) polyunsaturated fatty acidspecific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana [J]. FEBS Letters, 2002, 510(3): 159—165
[13] Michaelson L V, Lazarus C M, Griffiths G, et al. Isolation of a Delta5-fatty acid desaturase gene from Mortierella alpina[J]. Journal of Biological Chemistry, 1998, 273(30): 19055— 19059
[14] Sayanova O, Haslam R, Qi B, et al. The alternative pathway C20 Delta8-desaturase from the non-photosynthetic organism Acanthamoeba castellanii is an atypical cytochrome b5-fusion desaturase [J]. FEBS Letters, 2006, 580(8): 1946—1952
[15] Kajikawa M, Yamato K T, Sakai Y, et al. Isolation and functional characterization of fatty acid delta5-elongase gene from the liverwort Marchantia polymorpha L. [J]. FEBS Letters, 2006, 580(1): 149—154
[16] Cho H P, Nakamura M T, Clarke S D. Cloning, expression, and nutritional regulation of the mammalian Delta-6 desaturase[J]. Journal of Biological Chemistry, 1999, 274(1): 471— 477
[17] Sperling P, Lee M, Girke T, et al. A bifunctional delta-fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus. A new member of the cytochrome b5 superfamily [J]. European Journal of Biochemistry, 2000, 267(12): 3801— 3811
[18] Leonard A E, Kelder B, Bobik E G, et al. cDNA cloning and characterization of human Delta5-desaturase involved in the biosynthesis of arachidonic acid [J]. Biochemical Journal, 2000, 347(3): 719—724
[19] Watts J L, Browse J. Isolation and characterization of a Delta 5-fatty acid desaturase from Caenorhabditis elegans [J]. Archives of Biochemistry and Biophysics, 1999, 362(1): 175— 182
[20] Abbadi A, Domergue F, Bauer J, et al. Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation [J]. Plant Cell, 2004, 16(10): 2734—2748
[21] Qi B, Fraser T, Mugford S, et al. Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants [J]. Nature Biotechnology, 2004, 22(6): 739—745
[22] Wu G, Truksa M, Datla N, et al. Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants [J]. Nature Biotechnology, 2005, 23(8): 1013— 1017
[23] Pereira S L, Leonard A E, Huang Y S, et al. Identification of two novel microalgal enzymes involved in the conversion of the omega3-fatty acid, eicosapentaenoic acid, into docosahexaenoic acid [J]. Biochemical Journal, 2004, 384(2): 357— 366
[24] Zhou X R, Robert S S, Petrie J R, et al. Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis [J]. Phytochemistry, 2007, 68(6): 785—796
[25] Venegas-caleron M, Beaudoin F, Sayanova O, et al. Cotranscribed genes for long chain polyunsaturated fatty acid biosynthesis in the protozoon Perkinsus marinus include a plant-like FAE1 3-ketoacyl coenzyme A synthase [J]. Journal of Biological Chemistry, 2007, 282(5): 2996—3003
[26] Qiu X, Hong H, Mackenzie S L. Identification of a Delta 4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea[J]. Journal of Biological Chemistry, 2001, 276(34): 31561— 31566
[27] Elble R. A simple and efficient procedure for transformation of yeasts [J]. Biotechniques, 1992, 13(1): 18—20
[28] Sperling P, Heinz E. Desaturases fused to their electron donor[J]. European Journal of Lipid Science and Technology, 2001, 103: 158—180
[29] Tonon T, Sayanova O, Michaelson L V, et al. Fatty acid desaturases from the microalga Thalassiosira pseudonana [J]. FEBS Journal, 2005, 272(13): 3401—3412
[30] Kaewsuwan S, Cahoon E B, Perroud P F, et al. Identification and functional characterization of the moss Physcomitrella patens delta5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis [J]. Journal of Biological Chemistry, 2006, 281(31): 21988—21997
[31] Domergue F, Lerchl J, Zahringer U, et al. Cloning and functional characterization of Phaeodactylum tricornutum frontend desaturases involved in eicosapentaenoic acid biosynthesis[J]. European Journal of Biochemistry, 2002, 269(16): 4105—4113
[32] Iskandarov U, Khozin-goldberg I, Cohen Z. Identification and characterization of Delta12, Delta6, and Delta5 Desaturases from the green microalga Parietochloris incisa [J]. Lipids, 2010, 45(6): 519—530
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期刊类型引用(1)
1. 刘江，孙全喜，李新征，亓宝秀. 球等鞭金藻Δ5去饱和酶基因IgD5在拟南芥中的功能鉴定. 作物学报. 2013(05): 928-934 . 
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