紫斑牡丹种子发育期油脂合成积累的源汇基因协同表达
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摘要
为研究紫斑牡丹种子油脂合成积累与相关基因(GPD1, DGAT1和DGAT2)表达间的关系,以四个不同发育期(6月20日, 7月7日, 7月17日和7月27日)的种子为材料,氯仿甲醇法测定种子含油率,实时荧光定量PCR方法分析基因的表达模式,分析相关基因在不同发育期种子中的表达差异及其对油脂合成积累的影响。结果表明:(1)不同发育期紫斑牡丹种子含油率呈不断升高趋势,在6月20日至7月27日的38 d内,从4.21%上升到18.38%;(2)调控油脂合成的源基因GPD1和汇基因DGAT1与DGAT2在发育前期的紫斑牡丹种子中的表达呈快速上升趋势,且表达量均在7月7日达到最高值;随后源汇基因的表达均不断降低。紫斑牡丹发育前期种子中源(GPD1)汇(DGAT1和DGAT2)基因的协同高表达,既促进合成了更多的TAG前体G3P,又促进了TAG积累。源基因GPD1表达量增加了2.44倍,汇基因DGAT1和DGAT2分别增加5.16倍和3.83倍,促成种子含油量增加了2.77倍。这可为深入理解紫斑牡丹种子油脂合成积累提供理论依据,对后续开展高油紫斑牡丹的分子育种具有重要意义。
In order to explore the relationship between the oil biosynthesis and accumulation and the expression of related genes in seeds of Paeonia suffruticosa, the seeds harvested at four different developmental stages(June20, July 7, July 17 and July 27) were selected as the material. The seed oil contents were tested by the method of chloroform methanol. The different expression patterns of genes were determined using qRT-PCR, and the effects of these genes on lipid biosynthesis and accumulation were analyzed. The results showed that the seed oil contents appeared in continuous increase during seed development, and it increased from 4.21% to 18.38% in only 38 days from June 20 to July 27. The expression of source gene 'GPD1' and sink genes 'DGAT1' and 'DGAT2' increased rapidly during early seed development, which reached the highest values at July 7. After this, the expression of source and sink genes decreased gradually. The high coordinate expressions of source gene GPD1 and sink genes DGAT1 and DGAT2 not only promoted the synthesis of more TAG precursors G3 P, but also promoted the accumulation of TAG. The expression of GPD1 increased by 2.44 times, the expression of DGAT1 and DGAT2 increased by 5.16 times and 3.83 times respectively, resulting in the increase of seed oil content by 2.77 times. These results could provide a theoretical basis for further understanding of oil synthesis and accumulation in seeds of Paeonia suffruticosa, and would be of great significance for subsequent molecular breeding of Paeonia suffruticosa with high oil content.
In order to explore the relationship between the oil biosynthesis and accumulation and the expression of related genes in seeds of Paeonia suffruticosa, the seeds harvested at four different developmental stages(June20, July 7, July 17 and July 27) were selected as the material. The seed oil contents were tested by the method of chloroform methanol. The different expression patterns of genes were determined using qRT-PCR, and the effects of these genes on lipid biosynthesis and accumulation were analyzed. The results showed that the seed oil contents appeared in continuous increase during seed development, and it increased from 4.21% to 18.38% in only 38 days from June 20 to July 27. The expression of source gene 'GPD1' and sink genes 'DGAT1' and 'DGAT2' increased rapidly during early seed development, which reached the highest values at July 7. After this, the expression of source and sink genes decreased gradually. The high coordinate expressions of source gene GPD1 and sink genes DGAT1 and DGAT2 not only promoted the synthesis of more TAG precursors G3 P, but also promoted the accumulation of TAG. The expression of GPD1 increased by 2.44 times, the expression of DGAT1 and DGAT2 increased by 5.16 times and 3.83 times respectively, resulting in the increase of seed oil content by 2.77 times. These results could provide a theoretical basis for further understanding of oil synthesis and accumulation in seeds of Paeonia suffruticosa, and would be of great significance for subsequent molecular breeding of Paeonia suffruticosa with high oil content.
引文
Chen Y.C.,Cui Q.Q.,Xu Y.J.,Yang S.S.,Gao M.,and Wang YD.,2015,Effects of tung oil seed FAD2 and DGAT2 genes on unsaturated fatty acid accumulation in Rhodotorula glutinis and Arabidopsis thaliana,Mol.Genet.Genomics,290(4)1605-1613
Chhikara S.,Abdullah H.M.,Akbari P.,Schnell D.,and Dhankher O.P.,2017.,Engineering Camelina sativa(L.)Crantz for enhanced oil and seed yields by combining diacylglycerol acyltransferase1 and glycerol-3-phosphate dehydrogenase expression,Plant Biotechnol.J.,48(8):1-12
Ding J.,R uan C.J.,Guan Y.,Guan W.K.,Shan J.Y.,Wu Y.X.,and Wu T.Z.,2017,Coordinated expression of source and sink genes involved in lipid biosynthesis and accumulation during seabuckthorn pulp development,Linye Kexue Yanjiu(Forest Research),30(6):902-907(丁健,阮成江,关莹,管文柯,单金友,吴雨蹊,吴天忠,2017,沙棘果肉发育期油脂合成积累的源汇基因协同表达,林业科学研究,30(6):902-907)
Ding J.,Ruan C.J.,Shan J.Y.,and Guan Y.,2016,Expression of key genes involved in lipid biosynthesis and accumulation during seeds formation and development in Hippophae rhamnoides,Xibei Zhiwu Xuebao(Acta Botanica Boreali-occidentalia Sinica),36(8):1642-1647(丁健,阮成江,单金友,关莹,2016,沙棘种子形成发育过程中油脂合成积累关键基因的表达分析,西北植物学报,36(8):1642-1647)
Gao T.T.,2012,Analysis of peony seed oil composition and storage conditions research,Thesis for M.S.,Beijing Forestry U-niversity,Supervisor:Ren J.W.,pp.13(高婷婷,2012,牡丹籽油成分分析及储藏条件研究,硕士学位论文,北京林业大学,导师:任建武,pp.13)
Gibon Y.,Vigeolas H.,Tiessen A.,Geigenberger P.,and Stitt M.,2002,Sensitive and high throughput metabolite assays for inorganic pyrophosphate,ADPGlc,nucleotide phosphates,and glycolytic intermediates based on a novel enzymic cycling system,Plant J.,30(2):221-235
Hobbs D.H.,Lu C.F.,and Hills M.J.,1999,Cloning of a cDNAencoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression,FEBS Letters,452(3):145-149
Jako C.,Kuma A.,Wei Y.D.,Zou J.T.,Barton D.L.,Giblin E.M.,Covello P.S.,and Taylor D.C.,2001,Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight,Plant Physiol.,126(2):861-874
Liu C.J.,Wang S.L.,Xue J.Q.,Zhu F.Y.,Ren X.X.,Li M.Y.,and Zhang X.X.,2015,Molecular cloning of ubiquitin protein gene and study on this gene as reference gene in tree peony Yuanyi Xuebao(Acta Horticulturae Sinica),42(10):1983-1992(刘传娇,王顺利,薛璟祺,朱富勇,任秀霞,李名扬张秀新,2015,牡丹泛素延伸蛋白基因ubiquitin的克隆及其作为内参基因的研究,园艺学报,42(10):1983-1992)
Liu M.,2014,Expression and function of DGAT1 in Camellia oleifera,Thesis for M.S.,Central South University of Forestry&Technology,Supervisor:Zhang L.,pp.5-8(刘敏,2014,油茶DGAT1基因的表达和功能研究,硕士学位论文,中南林业科技大学,导师:张琳,pp.5-8)
Lu L.C.F.,Noyer S.B.D.,Hobbs D.H.,Kang J.L.,Wen Y.C.Krachtus D.,and Hills M.J.,2003,Expression pattern of diacylglycerol acyltransferase-1,an enzyme involved in triacylglycerol biosynthesis,in Arabidopsis thaliana,Plant Mol.Biol.,52(1):31-41
Remize F.,Barnavon L.,and Dequin S.,2001,Glycerol expor and glycerol-3-phosphate dehydrogenase,but not glycero phosphatase,are rate limiting for glycerol production in Saccharomyces cerevisiae,Metab.Eng.,3(4):301-312
Ruan C.J.,and Li Q.,2008,Status of oil content control in seed by genetic regulation and the strategy for improving the gene of Kosteletzkya for biodiesel production,Kezaisheng Nengyuan(Renewable Energy Resources),26(4):35-40(阮成江李群,2008,基因调控种子含油量研究进展及生物柴油用海滨锦葵遗传改良策略,可再生能源,26(4):35-40)
Shi G.A.,Guo X.F.,Jin B.L.,Huang H.X.,Wang W.,Zhang S.X.and Wang F.L.,2013,Optimization of supercritical CO2extraction and analysis of antioxidation activity of peony seed oil,Zhongguo Liangyou Xuebao(Journal of the Chinese Cereals and Oils Association),28(4):47-50(史国安,郭香凤金宝磊,黄海霞,王玮,张淑霞,王凤楼,2013,牡丹籽油超临界CO2萃取工艺优化及抗氧化活性的研究,中国粮油学报,28(4):47-50)
Shi G.A.,Jiao F.X.,Jiao Y.P.,Yang H.A.,Han M.W.,Wu Y.Q.and Shi B.R.,2014,Development prospects and strategies of oil tree peony industry in China,Zhongguo Liangyou Xuebao(Journal of the Chinese Cereals and Oils Association),29(9):124-128(史国安,焦封喜,焦元鹏,杨会安,韩孟伟,吴育谦,史秉锐,2014,中国油用牡丹的发展前景及对策,中国粮油学报,29(9):124-128)
Slocombe S.P.,Cornah J.,Pinfield-Wells H.,Soady K.,Zhang QY.,Gilday A.,Dyer J.M.,and Graham I.A.,2009,Oil accumulation in leaves directed by modification of fatty acid breakdown and lipid synthesis pathways,Plant Biotechnol.J.7(7):694-703
Snyder C.L.,Yurchenko O.P.,Siloto R.M.P.,Chen X.,Liu Q.,Mietkiewska E.,and Weselake R.J.,2009,Acyltransferase action in the modification of seed oil biosynthesis,New Biotechnol.,26(1):11-16
Vigeolas H.,and Geigenberger P.,2004,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,219(5):827-835
Vigeolas H.,Waldeck P.,Zank T.,and Geigenberger P.,2007,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 Biotechnol.J.,5(3):431
Wu B.,Ruan C.J.,Ding J.,Du W.,Li J.B.,Yan R.,and Liu L.Y.,2017,Coordinated expression of source and sink genes involved in lipid biosynthesis and accumulation during camellia oleifera seed development,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(12):2483-2488(吴波,阮成江,丁健,杜维,李景滨,闫蕊,刘祾悦,2017,油茶种子发育期油脂合成积累的源汇基因协同表达,西北植物学报,37(12):2483-2488)
Xu J.Y.,Francis T.,Mietkiewska E.,Giblin E.M.,Barton D.L.,Zhang Y.,Zhang M.,and Taylor D.C.,2008,Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1(DGAT1)gene from Tropaeolum majus,and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content,Plant Biotechnol.J.,6(8):799
Yang Z.S.,Zhang Y.J.,Gao W.Y.,Lv Y.W.,and Wang Y.J.,2017,Construction of gpd1 gene binary vector and agrobacterium-mediated transformation of Chlamydomonas reinhardtii,Xibei Daxue Xuebao(Journal of Northwest University(Natural Science Edition)),47(1):75-81(杨泽熵,张雨靖,高文英,吕亚维,王亚娟,2017,gpd1基因莱茵衣藻表达载体构建及农杆菌介导转化莱茵衣藻,西北大学学报(自然科学版),47(1):75-81)
Zou B.J.,Lv L.T.,and Zhao D.G.,2016,Effect of overpression of AtBAS1 gene regulated vascular development in Nicotianatabacum L.,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(6):1487-1492(邹冰杰,吕立堂,赵德刚,2016,超量表达拟南芥油菜素内酯基因BAS1对烟草维管组织发育的影响,基因组学与应用生物学,35(6):1487-1492)
Zou J.T.,Wei.Y.D.,Jako C.,Kumar A.,Selvaraj G.,and Taylor D.C.,1999,The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene,Plant J.,19(6):645-653
Chhikara S.,Abdullah H.M.,Akbari P.,Schnell D.,and Dhankher O.P.,2017.,Engineering Camelina sativa(L.)Crantz for enhanced oil and seed yields by combining diacylglycerol acyltransferase1 and glycerol-3-phosphate dehydrogenase expression,Plant Biotechnol.J.,48(8):1-12
Ding J.,R uan C.J.,Guan Y.,Guan W.K.,Shan J.Y.,Wu Y.X.,and Wu T.Z.,2017,Coordinated expression of source and sink genes involved in lipid biosynthesis and accumulation during seabuckthorn pulp development,Linye Kexue Yanjiu(Forest Research),30(6):902-907(丁健,阮成江,关莹,管文柯,单金友,吴雨蹊,吴天忠,2017,沙棘果肉发育期油脂合成积累的源汇基因协同表达,林业科学研究,30(6):902-907)
Ding J.,Ruan C.J.,Shan J.Y.,and Guan Y.,2016,Expression of key genes involved in lipid biosynthesis and accumulation during seeds formation and development in Hippophae rhamnoides,Xibei Zhiwu Xuebao(Acta Botanica Boreali-occidentalia Sinica),36(8):1642-1647(丁健,阮成江,单金友,关莹,2016,沙棘种子形成发育过程中油脂合成积累关键基因的表达分析,西北植物学报,36(8):1642-1647)
Gao T.T.,2012,Analysis of peony seed oil composition and storage conditions research,Thesis for M.S.,Beijing Forestry U-niversity,Supervisor:Ren J.W.,pp.13(高婷婷,2012,牡丹籽油成分分析及储藏条件研究,硕士学位论文,北京林业大学,导师:任建武,pp.13)
Gibon Y.,Vigeolas H.,Tiessen A.,Geigenberger P.,and Stitt M.,2002,Sensitive and high throughput metabolite assays for inorganic pyrophosphate,ADPGlc,nucleotide phosphates,and glycolytic intermediates based on a novel enzymic cycling system,Plant J.,30(2):221-235
Hobbs D.H.,Lu C.F.,and Hills M.J.,1999,Cloning of a cDNAencoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression,FEBS Letters,452(3):145-149
Jako C.,Kuma A.,Wei Y.D.,Zou J.T.,Barton D.L.,Giblin E.M.,Covello P.S.,and Taylor D.C.,2001,Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight,Plant Physiol.,126(2):861-874
Liu C.J.,Wang S.L.,Xue J.Q.,Zhu F.Y.,Ren X.X.,Li M.Y.,and Zhang X.X.,2015,Molecular cloning of ubiquitin protein gene and study on this gene as reference gene in tree peony Yuanyi Xuebao(Acta Horticulturae Sinica),42(10):1983-1992(刘传娇,王顺利,薛璟祺,朱富勇,任秀霞,李名扬张秀新,2015,牡丹泛素延伸蛋白基因ubiquitin的克隆及其作为内参基因的研究,园艺学报,42(10):1983-1992)
Liu M.,2014,Expression and function of DGAT1 in Camellia oleifera,Thesis for M.S.,Central South University of Forestry&Technology,Supervisor:Zhang L.,pp.5-8(刘敏,2014,油茶DGAT1基因的表达和功能研究,硕士学位论文,中南林业科技大学,导师:张琳,pp.5-8)
Lu L.C.F.,Noyer S.B.D.,Hobbs D.H.,Kang J.L.,Wen Y.C.Krachtus D.,and Hills M.J.,2003,Expression pattern of diacylglycerol acyltransferase-1,an enzyme involved in triacylglycerol biosynthesis,in Arabidopsis thaliana,Plant Mol.Biol.,52(1):31-41
Remize F.,Barnavon L.,and Dequin S.,2001,Glycerol expor and glycerol-3-phosphate dehydrogenase,but not glycero phosphatase,are rate limiting for glycerol production in Saccharomyces cerevisiae,Metab.Eng.,3(4):301-312
Ruan C.J.,and Li Q.,2008,Status of oil content control in seed by genetic regulation and the strategy for improving the gene of Kosteletzkya for biodiesel production,Kezaisheng Nengyuan(Renewable Energy Resources),26(4):35-40(阮成江李群,2008,基因调控种子含油量研究进展及生物柴油用海滨锦葵遗传改良策略,可再生能源,26(4):35-40)
Shi G.A.,Guo X.F.,Jin B.L.,Huang H.X.,Wang W.,Zhang S.X.and Wang F.L.,2013,Optimization of supercritical CO2extraction and analysis of antioxidation activity of peony seed oil,Zhongguo Liangyou Xuebao(Journal of the Chinese Cereals and Oils Association),28(4):47-50(史国安,郭香凤金宝磊,黄海霞,王玮,张淑霞,王凤楼,2013,牡丹籽油超临界CO2萃取工艺优化及抗氧化活性的研究,中国粮油学报,28(4):47-50)
Shi G.A.,Jiao F.X.,Jiao Y.P.,Yang H.A.,Han M.W.,Wu Y.Q.and Shi B.R.,2014,Development prospects and strategies of oil tree peony industry in China,Zhongguo Liangyou Xuebao(Journal of the Chinese Cereals and Oils Association),29(9):124-128(史国安,焦封喜,焦元鹏,杨会安,韩孟伟,吴育谦,史秉锐,2014,中国油用牡丹的发展前景及对策,中国粮油学报,29(9):124-128)
Slocombe S.P.,Cornah J.,Pinfield-Wells H.,Soady K.,Zhang QY.,Gilday A.,Dyer J.M.,and Graham I.A.,2009,Oil accumulation in leaves directed by modification of fatty acid breakdown and lipid synthesis pathways,Plant Biotechnol.J.7(7):694-703
Snyder C.L.,Yurchenko O.P.,Siloto R.M.P.,Chen X.,Liu Q.,Mietkiewska E.,and Weselake R.J.,2009,Acyltransferase action in the modification of seed oil biosynthesis,New Biotechnol.,26(1):11-16
Vigeolas H.,and Geigenberger P.,2004,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,219(5):827-835
Vigeolas H.,Waldeck P.,Zank T.,and Geigenberger P.,2007,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 Biotechnol.J.,5(3):431
Wu B.,Ruan C.J.,Ding J.,Du W.,Li J.B.,Yan R.,and Liu L.Y.,2017,Coordinated expression of source and sink genes involved in lipid biosynthesis and accumulation during camellia oleifera seed development,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(12):2483-2488(吴波,阮成江,丁健,杜维,李景滨,闫蕊,刘祾悦,2017,油茶种子发育期油脂合成积累的源汇基因协同表达,西北植物学报,37(12):2483-2488)
Xu J.Y.,Francis T.,Mietkiewska E.,Giblin E.M.,Barton D.L.,Zhang Y.,Zhang M.,and Taylor D.C.,2008,Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1(DGAT1)gene from Tropaeolum majus,and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content,Plant Biotechnol.J.,6(8):799
Yang Z.S.,Zhang Y.J.,Gao W.Y.,Lv Y.W.,and Wang Y.J.,2017,Construction of gpd1 gene binary vector and agrobacterium-mediated transformation of Chlamydomonas reinhardtii,Xibei Daxue Xuebao(Journal of Northwest University(Natural Science Edition)),47(1):75-81(杨泽熵,张雨靖,高文英,吕亚维,王亚娟,2017,gpd1基因莱茵衣藻表达载体构建及农杆菌介导转化莱茵衣藻,西北大学学报(自然科学版),47(1):75-81)
Zou B.J.,Lv L.T.,and Zhao D.G.,2016,Effect of overpression of AtBAS1 gene regulated vascular development in Nicotianatabacum L.,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(6):1487-1492(邹冰杰,吕立堂,赵德刚,2016,超量表达拟南芥油菜素内酯基因BAS1对烟草维管组织发育的影响,基因组学与应用生物学,35(6):1487-1492)
Zou J.T.,Wei.Y.D.,Jako C.,Kumar A.,Selvaraj G.,and Taylor D.C.,1999,The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene,Plant J.,19(6):645-653