芝麻硬脂酸脱饱和酶基因SiSAD的克隆及功能验证
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摘要
【目的】对芝麻△9硬脂酰-ACP脱饱和酶基因SiSAD(△9 stearoyl acyl-carrier-protein desaturase)进行克隆与表达分析,并转入拟南芥,探究其在油酸合成过程中的作用,为芝麻油酸含量的遗传改良提供分子基础。【方法】提取中芝13叶片的总RNA,反转录为cDNA。根据芝麻基因组数据库中的SiSAD序列信息(序列号为SIN_1008977)设计引物,以cDNA为模板,通过RT-PCR克隆获得SiSAD编码区序列,并与参考基因组序列进行比较。利用InterPro进行保守结构域分析,获得SiSAD蛋白的保守结构域。利用BLAST对SiSAD蛋白进行同源对比,获得SiSAD的同源蛋白质。采用邻接法构建系统进化树,获得芝麻SAD蛋白与橄榄、牵牛花、蓖麻、莴苣、葡萄、柑橘、拟南芥等植物SAD蛋白的亲缘关系。通过荧光定量PCR检测SiSAD在2个芝麻品种中芝33和中丰芝一号的根、茎、叶、蕾和种子中的相对表达量,分析SiSAD的表达特异性。将SiSAD连接过表达载体,通过农杆菌介导法转化野生型拟南芥(Col-0),筛选阳性后代,对T3代转基因和野生型的拟南芥种子中硬脂酸和油酸相对含量进行测定,分析SiSAD的功能。【结果】成功获得SiSAD的编码区序列,与参考基因组序列一致,全长为1 152 bp,编码383个氨基酸,SiSAD蛋白的分子量为43 kD,等电点为6.18。发现SiSAD蛋白含有一个保守结构域,属于脂肪酸去饱和酶家族成员,与其他植物的SAD蛋白质序列的同源性较高,暗示SiSAD在不同物种中的功能可能比较保守。系统进化分析显示,芝麻SAD蛋白与牵牛花和橄榄的SAD蛋白处于同一分支,进化关系较近,与蓖麻、拟南芥、柑橘的SAD蛋白亲缘关系较远。荧光定量PCR结果表明,SiSAD在芝麻种子中的表达量远远高于其他组织,有显著的组织特异性。成功构建了SiSAD的过表达载体,通过农杆菌介导法转化拟南芥,结果表明SiSAD成功导入拟南芥中,而且转录水平很高。对T3代转基因拟南芥种子中硬脂酸和油酸的相对含量分析表明,与野生型拟南芥比较,3个转SiSAD拟南芥株系中硬脂酸(C18:0)含量分别降低了3.0%、4.8%和6.1%,而油酸(C18:1)含量分别升高了2.8%、4.3%和7.8%,平均升高4.97%。【结论】克隆获得芝麻SiSAD的全长cDNA序列,鉴定了SiSAD的功能,发现SiSAD在油酸合成代谢过程中正向增加油酸含量,可应用于高油酸芝麻新品种培育。
【Objective】 Sesame SiSAD(△9 stearoyl acyl-carrier-protein desaturase) gene was cloned and the expression of it was detected. It was transformed into Arabidopsis to investigate its role in the oleic acid synthesis. This study aims to provide molecular basis for the genetic improvement of sesame oleic acid content. 【Method】Total RNA was extracted from leaf of the variety Zhongzhi13 and then was reverse transcripted into cDNA. Using the primers that designed according to the reference genome,the coding region sequence of SiSAD was obtained by RT-PCR. The sequence was further compared with the reference genome. The conserved motifs of SiSAD protein were identified by InterPro and the homologous proteins of SiSAD were recognized by BLAST.A phylogenetic tree of Si SAD from sesame, Olea europaea var. sylvestris, Ipomoea nil, Ricinus communis, Lactuca sativa, Vitis vinifera, Citrus sinensis and Arabidopsis thaliana was constructed by neighbor-joining method to reveal the relationship of SiSAD protein in these species. Expression profiles of SiSAD in roots, stems, leaves, buds and seeds at two varieties Zhongzhi33 and Zhongfengzhi No.1 were investigated. The SiSAD gene was linked to a 35 S vector and transformed into Arabidopsis by the Agrobacterium tumefaciens-mediated floral dip method. Based on the qRT-PCR detection, successful transformed Arabidopsis individuals were selected from the progenies. The stearic acid and oleic acid content in the seeds of transgenic T3 Arabidopsis seeds and Col-0 were detected and function of SiSAD was concluded. 【Result】 Total coding region sequence of SiSAD was cloned and the sequence was the same as the reference genome. It consisted of 1 152 nucleotides encoding a protein of 383 amino acids with a calculated molecular mass of 43 kD and a predicted pI of 6.18. We found that SiSAD gene contained one conserved function domain, which had been identified as a signature motif within the fatty acid desaturase family members. The similarity of SiSAD proteins from different species was quite high, indicating that SiSAD in different plant might had conserved function. The phylogenetic tree composed of SAD proteins showed that SiSAD, InSAD and OeSAD had been grouped together, suggested a close relationship of SiSAD protein among sesame, O. europaea var. sylvestris and Ipomoea nil. In contrast, SiSAD had a far relationship to AtSAD, CsSAD and RcSAD. qRT-PCR results showed that SiSAD is organ-specific expressed and had a highest expression level in seeds. We successfully constructed the overexpression vector of SiSAD and introduced the vector into Arabidopsis by Agrobacterium-mediated transformation. qRT-PCR was used to test the transcription of SiSAD in transgenic Arabidopsis plants.Compared with the Arabidopsis wild type Col-0, stearic acid content of 3 transgenic lines with overexpressed SiSAD gene was decreased by 3.0%, 4.8% and 6.1%, respectively. Which oleic acid content in these lines was increased by 2.8%, 4.3% and 7.8%(4.97% in average). 【Conclusion】 In this study, the total coding region sequence of SiSAD was cloned and function of SiSAD was characterized. SiSAD might plays important roles in improving oleic acid content, which could be used in the genetic improvement of oleic acid content in sesame seeds.
【Objective】 Sesame SiSAD(△9 stearoyl acyl-carrier-protein desaturase) gene was cloned and the expression of it was detected. It was transformed into Arabidopsis to investigate its role in the oleic acid synthesis. This study aims to provide molecular basis for the genetic improvement of sesame oleic acid content. 【Method】Total RNA was extracted from leaf of the variety Zhongzhi13 and then was reverse transcripted into cDNA. Using the primers that designed according to the reference genome,the coding region sequence of SiSAD was obtained by RT-PCR. The sequence was further compared with the reference genome. The conserved motifs of SiSAD protein were identified by InterPro and the homologous proteins of SiSAD were recognized by BLAST.A phylogenetic tree of Si SAD from sesame, Olea europaea var. sylvestris, Ipomoea nil, Ricinus communis, Lactuca sativa, Vitis vinifera, Citrus sinensis and Arabidopsis thaliana was constructed by neighbor-joining method to reveal the relationship of SiSAD protein in these species. Expression profiles of SiSAD in roots, stems, leaves, buds and seeds at two varieties Zhongzhi33 and Zhongfengzhi No.1 were investigated. The SiSAD gene was linked to a 35 S vector and transformed into Arabidopsis by the Agrobacterium tumefaciens-mediated floral dip method. Based on the qRT-PCR detection, successful transformed Arabidopsis individuals were selected from the progenies. The stearic acid and oleic acid content in the seeds of transgenic T3 Arabidopsis seeds and Col-0 were detected and function of SiSAD was concluded. 【Result】 Total coding region sequence of SiSAD was cloned and the sequence was the same as the reference genome. It consisted of 1 152 nucleotides encoding a protein of 383 amino acids with a calculated molecular mass of 43 kD and a predicted pI of 6.18. We found that SiSAD gene contained one conserved function domain, which had been identified as a signature motif within the fatty acid desaturase family members. The similarity of SiSAD proteins from different species was quite high, indicating that SiSAD in different plant might had conserved function. The phylogenetic tree composed of SAD proteins showed that SiSAD, InSAD and OeSAD had been grouped together, suggested a close relationship of SiSAD protein among sesame, O. europaea var. sylvestris and Ipomoea nil. In contrast, SiSAD had a far relationship to AtSAD, CsSAD and RcSAD. qRT-PCR results showed that SiSAD is organ-specific expressed and had a highest expression level in seeds. We successfully constructed the overexpression vector of SiSAD and introduced the vector into Arabidopsis by Agrobacterium-mediated transformation. qRT-PCR was used to test the transcription of SiSAD in transgenic Arabidopsis plants.Compared with the Arabidopsis wild type Col-0, stearic acid content of 3 transgenic lines with overexpressed SiSAD gene was decreased by 3.0%, 4.8% and 6.1%, respectively. Which oleic acid content in these lines was increased by 2.8%, 4.3% and 7.8%(4.97% in average). 【Conclusion】 In this study, the total coding region sequence of SiSAD was cloned and function of SiSAD was characterized. SiSAD might plays important roles in improving oleic acid content, which could be used in the genetic improvement of oleic acid content in sesame seeds.
引文
[1]林平,姜玉梅,陈瑛.几种油料作物中脂肪酸组成的研究及探讨.江西科学,2000,18(2):116-119.LIN P,JIANG Y M,CHEN Y.A study of the fatty acid content in several oil_bearing crops.JiangXi Science,2000,18(2):116-119.(in Chinese)
[2]BOLTON G E,SANDERS T H.Effect of roasting oil composition on the stability of roasted high-oleic peanuts.Journal of the American Oil Chemists'Society,2002,79:129-132.
[3]CHANG S,PETERSON R J,Ho C T.Chemical reactions involved in the deep-fat frying of foods.Journal of the American Oil Chemists'Society,1978,55(10):718-727.
[4]杜海,郎春秀,王伏林,陈锦清,吴关庭.油菜种子油酸含量的遗传改良.核农学报,2011,25(6):1179-1183.DU H,LANG C X,WANG F L,CHEN J Q,WU G T.Genetic improvement of oleic acid content in rapeseed.Journal of Nuclear Agricultural Science,2011,25(6):1179-1183.(in Chinese)
[5]SLEIGHT P.Cholesterol and coronary heart disease mortality.Australian&New Zealand Journal of Medicine,1992,22(5):576-579.
[6]SMITH D G,Song F,SHELDON T A.Cholesterol lowering and mortality:The importance of considering initial level of risk.British Medical Journal,1993,306(6889):1367-1373.
[7]王景梓,徐贵发.单不饱和脂肪酸与冠心病的关系.食品与药品,2005,7(10A):21-23.WANG J Z,XU G F.Monounsaturated fatty acid in relation to coronary heart disease.Food and Drug,2005,7(10A):21-23.(in Chinese)
[8]蒋秀琴,刘立成,赵福忠,刘光前,王旭.常见植物油脂肪酸含量的分析.饲料博览,2010(3):27-30.JIANG X Q,LIU L C,ZHAO G Z,LIU G Q,WANG X.Analysis of fatty acid in vegetable oils.Feed Review,2010(3):27-30.(in Chinese)
[9]袁利文.植物油中主要脂肪酸含量的分析.中国检验检测,2018(1):18-21.YUAN L W.Analysis of main fatty acid in vegetable oil.China Inspection Body and Laboratory,2018(1):18-21.(in Chinese)
[10]杨帆,薛长勇.常用食用油的营养特点和作用研究进展.中国食物与营养.2013,19(3):63-66.YANG F,XUE C Y.Research advancement of nutritional characteristics and functions of common edible oils.Food and Nutrition in China,2013,19(3):63-66.(in Chinese)
[11]BROADWATER J A,AI J,LOEHR T M.Peroxodiferric intermediate of stearoyl-acyl carrier protein A9-desaturase:Oxidase reactivity during single turn over and implications for the mechanism of desaturation.Biochemistry,1998,37:14664-14671.
[12]ROUGHAN P G,SLACK C R.Cellular organization of glycerolipid metabolism.Annual Reviews Plant Physiology,1982,33:97-132.
[13]DAMUDE H G,ZHANG H,FARRALL L,RIPP K G,TOMB J F,HOLLERBACH D,YADAV N S.Identification of bifunctional delta12/omega3 fatty acid desaturases for improving the ratio of omega3 to omega6 fatty acids in microbes and plants.Proceedings of the National Academy of Sciences of the United States of America,2006,103(25):9446-9451.
[14]PAUL R,REBECCA W,ANDREA C,REOBERT G U,LILIAN M.Effect ofΔ9-stearoyl-ACP-desaturase-C mutants in a high oleic background on soybean seed oil composition.Theoretical and Applied Genetics,2014,127(2):349-358.
[15]ZHANG Y F,MAXIMOVA S N,GUILTINAN M J.Characterization of a stearoyl-acyl carrier protein desaturase gene from potential biofuel plant.Frontiers in Plant Science,2015,6:239.
[16]BYFIELD G E,XUE H,UPCHURCH R G.Two genes from soybean encoding solubleΔ9 stearoyl-ACP desaturases.Crop Science,2006,46(2):840-846.
[17]FOFANA B,DUGUID S,CLOUTIER S.Cloning of fatty acid biosynthetic genesβ-ketoacyl CoA synthase,fatty acid elongase,stearoyl-ACP desaturase,and fatty acid desaturase and analysis of expression in the early developmental stages of flax(Linum usitatissimum L.)seeds.Plant Science,2004,166(6):1487-1496
[18]LIU Z,YANG X,FU Y.SAD,a stearoyl-acyl carrier protein desaturase highly expressed in high-oil maize inbred lines.Russian Journal of Plant Physiology,2009,56(5):709-715.
[19]SHANG X,CHENG C,DING J,GUO W.Identification of candidate genes from the SAD gene family in cotton for determination of cottonseed oil composition.Molecular Genetics and Genomics,2017,292:173-186.
[20]KNUTZON D S,SCHERER D E,SCHRECHENGOST W E.Nucleotide sequence of a complementary DNA clone encoding stearoyl-ACP desaturase from castor bean,Ricinus communis.Plant Physiology,1991,96:344-345
[21]KLINKENBERG J,FAIST H,SAUPE S,LAMBERTZ S,KRISCHKE M,STINGL N,FEKETE A,MUELLER M J,FEUSSNER I,HEDRICHR,DEEKEN R.Two fatty acid desaturases,stearoyl-acyl carrier proteinΔ9-desaturase 6 and fatty acid desaturase 3 are involved in drought and hypoxia stress signaling in Arabidopsis crown galls.Plant Physiology,2014,164:570-583.
[22]WENDY C,PAOLO L,NUNZIA S,MONICA D P,PAOLA S,VIRGINIA C,NOREEN M C,ALAN M M,PETER M,TONY A K,PHILIP J D,STEFANIA G,TEODORO C.Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance.Transgenic Research,2008,17:769-782.
[23]KNUTZON D S,THOMPSON G A,RADKE S E.Modification of brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene.Plant Biology,1992,89:2624-2628.
[24]WANG L,YU S,TONG C,ZHAO Y,LIU Y,SONG C,ZHANG Y,ZHANG X,WANG Y,HUA W,LI D,LI D,LI F,YU J,XU C,HANX,HUANG S,TAI S,WANG J,XU X,LI Y,LIU S,VARSHNEY R,WANG J,ZHANG X.Genome sequencing of the high oil crop sesame provides insight into oil biosynthesis.Genome Biology,2014,15(2):1-13.
[25]WANG L,YU J,LI D,ZHANG X.Sinbase:an integrated database to study genomics,genetics and comparative genomics in Sesamum indicum.Plant&Cell Physiology,2015,56(1):e2.
[26]罗通,邓骛远,张富丽.植物硬脂酰-酰基载体蛋白脱饱和酶.生命的化学,2006,26(2):133-136.LUO T,DENG W Y,ZHANG F L.The stearoyl-acyl carrier protein desaturase in plants.Chemistry of Life,2006,26(2):133-136.(in Chinese)
[27]FOFANA B,CLOUTIER S,DUGUID S,CHING J,RAMPITSCH C.Gene expression of stearoyl-ACP desaturase and delta12 fatty acid desaturase 2 is modulated during seed development of flax(Linum usitatissimum).Lipids,2006,41(7):705-712.
[28]DU H,MIN H,HU J,LI J.Modification of the fatty acid composition in Arabidopsis and maize seeds using a stearoyl-acyl carrier protein desaturase-1(ZmSAD1)gene.BMC Plant Biology,2016,16(1):137.
[29]AARDRA K,JOHN S,EDWARD W,LUDMILA L,DAVID H,PARDEEP K.The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis.Plant Molecular Biology,2007,63,2:257-271.
[30]LIU Q,SINGH S P,GREEN A G.High-stearic and high-oleic cottonseed oils produced by hpRNA-mediated post-transcriptional gene silencing.Plant Physiology,2002,129:1732-1743.
[2]BOLTON G E,SANDERS T H.Effect of roasting oil composition on the stability of roasted high-oleic peanuts.Journal of the American Oil Chemists'Society,2002,79:129-132.
[3]CHANG S,PETERSON R J,Ho C T.Chemical reactions involved in the deep-fat frying of foods.Journal of the American Oil Chemists'Society,1978,55(10):718-727.
[4]杜海,郎春秀,王伏林,陈锦清,吴关庭.油菜种子油酸含量的遗传改良.核农学报,2011,25(6):1179-1183.DU H,LANG C X,WANG F L,CHEN J Q,WU G T.Genetic improvement of oleic acid content in rapeseed.Journal of Nuclear Agricultural Science,2011,25(6):1179-1183.(in Chinese)
[5]SLEIGHT P.Cholesterol and coronary heart disease mortality.Australian&New Zealand Journal of Medicine,1992,22(5):576-579.
[6]SMITH D G,Song F,SHELDON T A.Cholesterol lowering and mortality:The importance of considering initial level of risk.British Medical Journal,1993,306(6889):1367-1373.
[7]王景梓,徐贵发.单不饱和脂肪酸与冠心病的关系.食品与药品,2005,7(10A):21-23.WANG J Z,XU G F.Monounsaturated fatty acid in relation to coronary heart disease.Food and Drug,2005,7(10A):21-23.(in Chinese)
[8]蒋秀琴,刘立成,赵福忠,刘光前,王旭.常见植物油脂肪酸含量的分析.饲料博览,2010(3):27-30.JIANG X Q,LIU L C,ZHAO G Z,LIU G Q,WANG X.Analysis of fatty acid in vegetable oils.Feed Review,2010(3):27-30.(in Chinese)
[9]袁利文.植物油中主要脂肪酸含量的分析.中国检验检测,2018(1):18-21.YUAN L W.Analysis of main fatty acid in vegetable oil.China Inspection Body and Laboratory,2018(1):18-21.(in Chinese)
[10]杨帆,薛长勇.常用食用油的营养特点和作用研究进展.中国食物与营养.2013,19(3):63-66.YANG F,XUE C Y.Research advancement of nutritional characteristics and functions of common edible oils.Food and Nutrition in China,2013,19(3):63-66.(in Chinese)
[11]BROADWATER J A,AI J,LOEHR T M.Peroxodiferric intermediate of stearoyl-acyl carrier protein A9-desaturase:Oxidase reactivity during single turn over and implications for the mechanism of desaturation.Biochemistry,1998,37:14664-14671.
[12]ROUGHAN P G,SLACK C R.Cellular organization of glycerolipid metabolism.Annual Reviews Plant Physiology,1982,33:97-132.
[13]DAMUDE H G,ZHANG H,FARRALL L,RIPP K G,TOMB J F,HOLLERBACH D,YADAV N S.Identification of bifunctional delta12/omega3 fatty acid desaturases for improving the ratio of omega3 to omega6 fatty acids in microbes and plants.Proceedings of the National Academy of Sciences of the United States of America,2006,103(25):9446-9451.
[14]PAUL R,REBECCA W,ANDREA C,REOBERT G U,LILIAN M.Effect ofΔ9-stearoyl-ACP-desaturase-C mutants in a high oleic background on soybean seed oil composition.Theoretical and Applied Genetics,2014,127(2):349-358.
[15]ZHANG Y F,MAXIMOVA S N,GUILTINAN M J.Characterization of a stearoyl-acyl carrier protein desaturase gene from potential biofuel plant.Frontiers in Plant Science,2015,6:239.
[16]BYFIELD G E,XUE H,UPCHURCH R G.Two genes from soybean encoding solubleΔ9 stearoyl-ACP desaturases.Crop Science,2006,46(2):840-846.
[17]FOFANA B,DUGUID S,CLOUTIER S.Cloning of fatty acid biosynthetic genesβ-ketoacyl CoA synthase,fatty acid elongase,stearoyl-ACP desaturase,and fatty acid desaturase and analysis of expression in the early developmental stages of flax(Linum usitatissimum L.)seeds.Plant Science,2004,166(6):1487-1496
[18]LIU Z,YANG X,FU Y.SAD,a stearoyl-acyl carrier protein desaturase highly expressed in high-oil maize inbred lines.Russian Journal of Plant Physiology,2009,56(5):709-715.
[19]SHANG X,CHENG C,DING J,GUO W.Identification of candidate genes from the SAD gene family in cotton for determination of cottonseed oil composition.Molecular Genetics and Genomics,2017,292:173-186.
[20]KNUTZON D S,SCHERER D E,SCHRECHENGOST W E.Nucleotide sequence of a complementary DNA clone encoding stearoyl-ACP desaturase from castor bean,Ricinus communis.Plant Physiology,1991,96:344-345
[21]KLINKENBERG J,FAIST H,SAUPE S,LAMBERTZ S,KRISCHKE M,STINGL N,FEKETE A,MUELLER M J,FEUSSNER I,HEDRICHR,DEEKEN R.Two fatty acid desaturases,stearoyl-acyl carrier proteinΔ9-desaturase 6 and fatty acid desaturase 3 are involved in drought and hypoxia stress signaling in Arabidopsis crown galls.Plant Physiology,2014,164:570-583.
[22]WENDY C,PAOLO L,NUNZIA S,MONICA D P,PAOLA S,VIRGINIA C,NOREEN M C,ALAN M M,PETER M,TONY A K,PHILIP J D,STEFANIA G,TEODORO C.Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance.Transgenic Research,2008,17:769-782.
[23]KNUTZON D S,THOMPSON G A,RADKE S E.Modification of brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene.Plant Biology,1992,89:2624-2628.
[24]WANG L,YU S,TONG C,ZHAO Y,LIU Y,SONG C,ZHANG Y,ZHANG X,WANG Y,HUA W,LI D,LI D,LI F,YU J,XU C,HANX,HUANG S,TAI S,WANG J,XU X,LI Y,LIU S,VARSHNEY R,WANG J,ZHANG X.Genome sequencing of the high oil crop sesame provides insight into oil biosynthesis.Genome Biology,2014,15(2):1-13.
[25]WANG L,YU J,LI D,ZHANG X.Sinbase:an integrated database to study genomics,genetics and comparative genomics in Sesamum indicum.Plant&Cell Physiology,2015,56(1):e2.
[26]罗通,邓骛远,张富丽.植物硬脂酰-酰基载体蛋白脱饱和酶.生命的化学,2006,26(2):133-136.LUO T,DENG W Y,ZHANG F L.The stearoyl-acyl carrier protein desaturase in plants.Chemistry of Life,2006,26(2):133-136.(in Chinese)
[27]FOFANA B,CLOUTIER S,DUGUID S,CHING J,RAMPITSCH C.Gene expression of stearoyl-ACP desaturase and delta12 fatty acid desaturase 2 is modulated during seed development of flax(Linum usitatissimum).Lipids,2006,41(7):705-712.
[28]DU H,MIN H,HU J,LI J.Modification of the fatty acid composition in Arabidopsis and maize seeds using a stearoyl-acyl carrier protein desaturase-1(ZmSAD1)gene.BMC Plant Biology,2016,16(1):137.
[29]AARDRA K,JOHN S,EDWARD W,LUDMILA L,DAVID H,PARDEEP K.The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis.Plant Molecular Biology,2007,63,2:257-271.
[30]LIU Q,SINGH S P,GREEN A G.High-stearic and high-oleic cottonseed oils produced by hpRNA-mediated post-transcriptional gene silencing.Plant Physiology,2002,129:1732-1743.