蓖麻三磷酸甘油脱氢酶（RcGPDH）基因克隆与功能鉴定
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摘要
蓖麻(Ricinus communis L.)为大戟科蓖麻属一年生或多年生草本植物,其种子含油量可高达60%。蓖麻油的主要成分是羟基化的蓖麻油酸(顺式-12-羟基十八碳-9-烯酸),其特殊的羟基化结构决定了蓖麻油具有特殊的性能及广泛的工业用途,蓖麻因此而成为重要的工业油源作物,也是世界十大油料作物之一。目前,针对植物种子油累积的分子机制的报道主要研究了参与三酰甘油(TAG)合成的Kennedy途径中相关酰基转移酶的作用,而为Kennedy途径提供甘油骨架的三磷酸甘油脱氢酶(glycerol-3-phosphate dehydrogenase)在TAG合成中的作用一直不太受关注。本研究主要探讨蓖麻三磷酸甘油脱氢酶基因(RcGPDH)在TAG合成上的功能,进而阐明在蓖麻油累积过程中的作用。
根据已报道的蓖麻种子表达序列标签(Expressed Sequence Tags, EST)设计引物,通过RACE (rapid-amplification of cDNA ends)方法克隆了蓖麻RcGPDH基因并对其序列及所编码的蛋白质结构进行了分析；荧光定量PCR分析结果表明,该基因在蓖麻种子发育后期有较强的表达。为了研究该基因的功能,构建了该基因的酵母表达载体PYES2.1/V5-His -RcGPDH,转化酵母野生型菌株BY4742,运用分光光度法测定转基因酵母的生长曲线,通过香草醛法测定稳定生长期的转基因酵母的油脂含量；构建了在种子特异表达启动子NAPIN启动下的植物表达载体,并通过遗传转化使其在野生型拟南芥中过量表达。结果表明转基因酵母菌株比转空载体对照菌株生长慢,两者均在培养18小时后进入平台期,但油脂含量及脂肪酸组成没有显著的变化。RcGPDH基因在拟南芥中过量表达使转基因拟南芥的种子油脂含量减少而种子增大,同时,转基因拟南芥饱和脂肪酸减少,而多不饱和脂肪酸增加,由此推测该基因可能与蓖麻适应低温胁迫有关,而对油脂累积没有积极的作用。为了能更全面的研究该基因的功能,同时开展了建立蓖麻遗传转化体系的实验。
The castor plant (Ricinus communis), a species of Euphorbiaceae family, is a perennial shrub. Its seeds accumulate 60% oil in the form of triacylglycerol (TAG). Castor oil is unique in that 90% of its fatty acid content is ricinoleate (12-hydroxy-9-cis-octadecenoic acid), which make castor oil a vital industrial raw material for numerous products. Now, the castor plant is an important industrial oil crop of the world. Rencnetly, many researches focused on the acyltransferase in the Kennedy pathway of triacylgycerol biosynthesis were reported. A few studies were performed to analysis the function of the glycerol-3-phosphate dehydrogenase which catalyses dihydroxyacetone phosphate to produce the glycerol-3-phosphate, a glycerol backbone in TAG biosynthesis. The function of glycerol-3-phosphate dehydrogenase in plant oil biosynthesis is unclear. In present study, a glycerol-3-phosphate dehydrogenase gene (RcGPDH) was cloned from castor bean and characterized.
According to the Express Sequence Tags (EST) of castor bean, primers were designed and the full cDNA of RcGPDH gene was cloned by RACE (rapid-amplification of cDNA ends). High levels of expression were detected by real-time PCR in the castor seeds during later stages of developments. To elucidate the function of RcGPDH gene, a yeast expression vector PYES2.1/V5-His-TOPO-RcGPDH was constructed and transformed to the wild yeast strain BY4742. The growth curve of transgenic yeasts was figured using spectrophotometry, and the oil contents and fatty acid compositions were measured using vanillin method and GC (gas chromatography) respectively. Meanwhile, a plant expression vector, which contained a seed specific promoter, was constructed and transformed to Arabidopsis thaliana. Results showed that the transgenic yeasts grew stably after cultured 18 hours, but the strain with RcGPDH gene grew slower than the control strain. The oil content and the fatty acids composition weren't various between the two yeast strains, which suggested that RcGPDH gene didn't play a positive role on the yeast oil accumulation. Total lipid content decreased and seeds weight increased in RcGPDH-overexpressing plants, and apparent changes in total fatty acid composition were also detected in the transgenic seeds, saturated fatty acids reduced and polyunsaturated fatty acids increased. So we speculated that RcGPDH gene has no positive contribution to oil accumulation, however maybe has positive role to adapt to low temperature stress. To further verify the function of RcGPDH gene in the oil accumulation, in vitro propagation also was performed to establish a stable genetic transformation system of castor.
根据已报道的蓖麻种子表达序列标签(Expressed Sequence Tags, EST)设计引物,通过RACE (rapid-amplification of cDNA ends)方法克隆了蓖麻RcGPDH基因并对其序列及所编码的蛋白质结构进行了分析；荧光定量PCR分析结果表明,该基因在蓖麻种子发育后期有较强的表达。为了研究该基因的功能,构建了该基因的酵母表达载体PYES2.1/V5-His -RcGPDH,转化酵母野生型菌株BY4742,运用分光光度法测定转基因酵母的生长曲线,通过香草醛法测定稳定生长期的转基因酵母的油脂含量；构建了在种子特异表达启动子NAPIN启动下的植物表达载体,并通过遗传转化使其在野生型拟南芥中过量表达。结果表明转基因酵母菌株比转空载体对照菌株生长慢,两者均在培养18小时后进入平台期,但油脂含量及脂肪酸组成没有显著的变化。RcGPDH基因在拟南芥中过量表达使转基因拟南芥的种子油脂含量减少而种子增大,同时,转基因拟南芥饱和脂肪酸减少,而多不饱和脂肪酸增加,由此推测该基因可能与蓖麻适应低温胁迫有关,而对油脂累积没有积极的作用。为了能更全面的研究该基因的功能,同时开展了建立蓖麻遗传转化体系的实验。
The castor plant (Ricinus communis), a species of Euphorbiaceae family, is a perennial shrub. Its seeds accumulate 60% oil in the form of triacylglycerol (TAG). Castor oil is unique in that 90% of its fatty acid content is ricinoleate (12-hydroxy-9-cis-octadecenoic acid), which make castor oil a vital industrial raw material for numerous products. Now, the castor plant is an important industrial oil crop of the world. Rencnetly, many researches focused on the acyltransferase in the Kennedy pathway of triacylgycerol biosynthesis were reported. A few studies were performed to analysis the function of the glycerol-3-phosphate dehydrogenase which catalyses dihydroxyacetone phosphate to produce the glycerol-3-phosphate, a glycerol backbone in TAG biosynthesis. The function of glycerol-3-phosphate dehydrogenase in plant oil biosynthesis is unclear. In present study, a glycerol-3-phosphate dehydrogenase gene (RcGPDH) was cloned from castor bean and characterized.
According to the Express Sequence Tags (EST) of castor bean, primers were designed and the full cDNA of RcGPDH gene was cloned by RACE (rapid-amplification of cDNA ends). High levels of expression were detected by real-time PCR in the castor seeds during later stages of developments. To elucidate the function of RcGPDH gene, a yeast expression vector PYES2.1/V5-His-TOPO-RcGPDH was constructed and transformed to the wild yeast strain BY4742. The growth curve of transgenic yeasts was figured using spectrophotometry, and the oil contents and fatty acid compositions were measured using vanillin method and GC (gas chromatography) respectively. Meanwhile, a plant expression vector, which contained a seed specific promoter, was constructed and transformed to Arabidopsis thaliana. Results showed that the transgenic yeasts grew stably after cultured 18 hours, but the strain with RcGPDH gene grew slower than the control strain. The oil content and the fatty acids composition weren't various between the two yeast strains, which suggested that RcGPDH gene didn't play a positive role on the yeast oil accumulation. Total lipid content decreased and seeds weight increased in RcGPDH-overexpressing plants, and apparent changes in total fatty acid composition were also detected in the transgenic seeds, saturated fatty acids reduced and polyunsaturated fatty acids increased. So we speculated that RcGPDH gene has no positive contribution to oil accumulation, however maybe has positive role to adapt to low temperature stress. To further verify the function of RcGPDH gene in the oil accumulation, in vitro propagation also was performed to establish a stable genetic transformation system of castor.
引文
[1]Baud S, Lepiniec L. Physiological and developmental regulation of seed oil production. Progress in Lipid Research,2010,49(3):235-249
[2]Broun P, Somerville C. Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean. Plant Physiology,1997,113(3):933-938
[3]Burgal J, Shockey J, Lu C, et al. Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil. Plant Biotechnology Journal, 2008,6(8):819-831
[4]Chaofu L, James W. An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library.2007,34(3):23-25
[5]Dahlqvist A, St hl U, Lenman M, et al. Phospholipid:diacylglycerol acyltransferase:an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proceedings of the National Academy of Sciences of the United States of America,2000,97(12):64-87
[6]Gibon Y, Vigeolas H, Tiessen A, et al. Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system. The Plant Journal,2002,30(2):221-235
[7]He X, Turner C, Chen GQ, et al. Cloning and characterization of a cDNA encoding diacylglycerol acyltransferase from castor bean. Lipids,2004,39(4):311-318
[8]He Y, Meng X, Fan Q, et al. Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis. Plant Molecular Biology,2009, 71(1):193-205
[9]Hills MJ. Control of storage-product synthesis in seeds. Current opinion in plant biology,2004,7(3): 302-308
[10]Jain R, Coffey M, Lai K, et al. Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes. Biochemical Society Transactions,2000,28(6):959-960
[11]Jako C, Kumar A, Wei Y, et al. Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiology,2001, 126(2):861-864
[12]Kennedy EP. Biosynthesis of complex lipids. Journal,1961,20(3):934-936
[13]Kroon J, Wei W, Simon WJ, et al. Identification and functional expression of a type 2 acyl-CoA: diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals. Phytochemistry,2006,67(23): 541-549
[14]Lu C, Fulda M, Wallis JG. A high throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. The Plant Journal,2006,45(5):847-856
[15]Lung SC, Weselake RJ. Diacylglycerol acyltransferase:a key mediator of plant triacylglycerol synthesis. Lipids,2006,41(12):1073-1088
[16]Malathi B, Ramesh S, Rao KV, et al. Agrobacterium-mediated genetic transformation and production of semilooper resistant transgenic castor (Ricinus communis L.). Euphytica,2006,147(3):441-449
[17]Mhaske V, Beldjilali K, Ohlrogge J, et al. Isolation and characterization of an Arabidopsis thaliana knockout line for phospholipid:diacylglycerol transacylase gene (At5g13640). Plant Physiology and Biochemistry,2005,43(4):413-417
[18]Petschnigg J, Wolinski H, Kolb D, et al. Good fat, essential cellular requirements for triacylglycerol synthesis to maintain membrane homeostasis in yeast. Journal of Biological Chemistry,2009. 284(45):30-38
[19]Roesler K, Shintani D, Savage L, et al. Targeting of the Arabidopsis homomeric acetyl-coenzyme A carboxylase to plastids of rapeseeds. Plant Physiology,1997,113(1):75-78
[20]Shanklin J, Somerville C. Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs. Proceedings of the National Academy of Sciences of the United States of America,1991,88(6):25-30
[21]Shen W, Li JQ, Dauk M, et al. Metabolic and Transcriptional Responses of Glycerolipid Pathways to a Perturbation of Glycerol 3-Phosphate Metabolism in Arabidopsis. Journal of Biological Chemistry, 2010,285(30):229-257
[22]Smith MA, Moon H, Chowrira G, et al. Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta,2003,217(3):507-516
[23]Stahl U, Carlsson AS, Lenman M, et al. Cloning and functional characterization of a phospholipid: diacylglycerol acyltransferase from Arabidopsis. Plant Physiology,2004,135(3):13-24
[24]Sujatha M, Sailaja M. Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds. Plant Cell Reports,2005,23(12):803-810
[25]Thelen JJ, Ohlrogge JB. Metabolic engineering of fatty acid biosynthesis in plants. Metabolic Engineering,2002,4(1):12-21
[26]Van de Loo FJ, Turner S, Somerville C. Expressed sequence tags from developing castor seeds. Plant Physiology,1995,108(3):11-14
[27]Vigeolas H, Geigenberger P. Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of Brassica napus L. in planta. Planta,2004,219(5):827-835
[28]Vigeolas H, Waldeck P, Zank T, et al. Increasing seed oil content in oil seed rape (Brassica napus L.) by overexpression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed specific promoter. Plant Biotechnology Journal,2007a,5(3):431-441
[29]Vigeolas H, Waldeck P, Zank T, et al. Increasing seed oil content in oil seed rape (Brassica napus L.) by over expression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed specific promoter. Plant Biotechnology Journal,2007b,5(3):431-441
[30]Voelker T, Kinney AJ. Variations in the biosynthesis of seed-storage lipids. Annual Review of Plant Biology,2001,52(1):335-361
[31]Wang J, Li R, Lu D, et al. A quick isolation method for mutants with high lipid yield in oleaginous yeast. World Journal of Microbiology and Biotechnology,2009,25(5):921-925
[32]Zheng P, Allen WB, Roesler K, et al. A phenylalanine in DGAT is a key determinant of oil content and composition in maize. Nature Genetics,2008,40(3):367-372
[33]Zou J, Katavic V, Giblin EM, et al. Modification of seed oil content and acyl composition in the Brassicaceae by expression of a yeast sn-2 acyltransferase gene. The Plant Cell Online,1997,9(6): 909-912
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[35]王莉衡.关于开发能源植物问题的思考.陕西农业科学,2010,2(4)：123-126
[36]白建明,刘树清.中国蓖麻杂种优势育种研究的现状及建议.云南农业科技,2007,2(1)：58-61
[37]朱保葛,李文彬.蓖麻生物工程研究进展.中国生物工程杂志,2007,27(10)：98-102
[38]衣海会,李凤山,贾娟霞,等蓖麻矮化育种研究现状及前景展望.安徽农学通报,2008,14(007)：66-67
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[2]Broun P, Somerville C. Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean. Plant Physiology,1997,113(3):933-938
[3]Burgal J, Shockey J, Lu C, et al. Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil. Plant Biotechnology Journal, 2008,6(8):819-831
[4]Chaofu L, James W. An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library.2007,34(3):23-25
[5]Dahlqvist A, St hl U, Lenman M, et al. Phospholipid:diacylglycerol acyltransferase:an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proceedings of the National Academy of Sciences of the United States of America,2000,97(12):64-87
[6]Gibon Y, Vigeolas H, Tiessen A, et al. Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system. The Plant Journal,2002,30(2):221-235
[7]He X, Turner C, Chen GQ, et al. Cloning and characterization of a cDNA encoding diacylglycerol acyltransferase from castor bean. Lipids,2004,39(4):311-318
[8]He Y, Meng X, Fan Q, et al. Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis. Plant Molecular Biology,2009, 71(1):193-205
[9]Hills MJ. Control of storage-product synthesis in seeds. Current opinion in plant biology,2004,7(3): 302-308
[10]Jain R, Coffey M, Lai K, et al. Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes. Biochemical Society Transactions,2000,28(6):959-960
[11]Jako C, Kumar A, Wei Y, et al. Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiology,2001, 126(2):861-864
[12]Kennedy EP. Biosynthesis of complex lipids. Journal,1961,20(3):934-936
[13]Kroon J, Wei W, Simon WJ, et al. Identification and functional expression of a type 2 acyl-CoA: diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals. Phytochemistry,2006,67(23): 541-549
[14]Lu C, Fulda M, Wallis JG. A high throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. The Plant Journal,2006,45(5):847-856
[15]Lung SC, Weselake RJ. Diacylglycerol acyltransferase:a key mediator of plant triacylglycerol synthesis. Lipids,2006,41(12):1073-1088
[16]Malathi B, Ramesh S, Rao KV, et al. Agrobacterium-mediated genetic transformation and production of semilooper resistant transgenic castor (Ricinus communis L.). Euphytica,2006,147(3):441-449
[17]Mhaske V, Beldjilali K, Ohlrogge J, et al. Isolation and characterization of an Arabidopsis thaliana knockout line for phospholipid:diacylglycerol transacylase gene (At5g13640). Plant Physiology and Biochemistry,2005,43(4):413-417
[18]Petschnigg J, Wolinski H, Kolb D, et al. Good fat, essential cellular requirements for triacylglycerol synthesis to maintain membrane homeostasis in yeast. Journal of Biological Chemistry,2009. 284(45):30-38
[19]Roesler K, Shintani D, Savage L, et al. Targeting of the Arabidopsis homomeric acetyl-coenzyme A carboxylase to plastids of rapeseeds. Plant Physiology,1997,113(1):75-78
[20]Shanklin J, Somerville C. Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs. Proceedings of the National Academy of Sciences of the United States of America,1991,88(6):25-30
[21]Shen W, Li JQ, Dauk M, et al. Metabolic and Transcriptional Responses of Glycerolipid Pathways to a Perturbation of Glycerol 3-Phosphate Metabolism in Arabidopsis. Journal of Biological Chemistry, 2010,285(30):229-257
[22]Smith MA, Moon H, Chowrira G, et al. Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta,2003,217(3):507-516
[23]Stahl U, Carlsson AS, Lenman M, et al. Cloning and functional characterization of a phospholipid: diacylglycerol acyltransferase from Arabidopsis. Plant Physiology,2004,135(3):13-24
[24]Sujatha M, Sailaja M. Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds. Plant Cell Reports,2005,23(12):803-810
[25]Thelen JJ, Ohlrogge JB. Metabolic engineering of fatty acid biosynthesis in plants. Metabolic Engineering,2002,4(1):12-21
[26]Van de Loo FJ, Turner S, Somerville C. Expressed sequence tags from developing castor seeds. Plant Physiology,1995,108(3):11-14
[27]Vigeolas H, Geigenberger P. Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of Brassica napus L. in planta. Planta,2004,219(5):827-835
[28]Vigeolas H, Waldeck P, Zank T, et al. Increasing seed oil content in oil seed rape (Brassica napus L.) by overexpression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed specific promoter. Plant Biotechnology Journal,2007a,5(3):431-441
[29]Vigeolas H, Waldeck P, Zank T, et al. Increasing seed oil content in oil seed rape (Brassica napus L.) by over expression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed specific promoter. Plant Biotechnology Journal,2007b,5(3):431-441
[30]Voelker T, Kinney AJ. Variations in the biosynthesis of seed-storage lipids. Annual Review of Plant Biology,2001,52(1):335-361
[31]Wang J, Li R, Lu D, et al. A quick isolation method for mutants with high lipid yield in oleaginous yeast. World Journal of Microbiology and Biotechnology,2009,25(5):921-925
[32]Zheng P, Allen WB, Roesler K, et al. A phenylalanine in DGAT is a key determinant of oil content and composition in maize. Nature Genetics,2008,40(3):367-372
[33]Zou J, Katavic V, Giblin EM, et al. Modification of seed oil content and acyl composition in the Brassicaceae by expression of a yeast sn-2 acyltransferase gene. The Plant Cell Online,1997,9(6): 909-912
[34]丁兰,王涛,景宏伟,等.蓖麻的组织培养.西北师范大学学报,2010,12(1)：79-83
[35]王莉衡.关于开发能源植物问题的思考.陕西农业科学,2010,2(4)：123-126
[36]白建明,刘树清.中国蓖麻杂种优势育种研究的现状及建议.云南农业科技,2007,2(1)：58-61
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