紫斑牡丹种子高积累碳十八不饱和脂肪酸的多基因调控
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摘要
为研究紫斑牡丹种子C18不饱和脂肪酸的高积累与相关基因SAD、FAD2、FAD3、FAD7和FAD8表达间的关系,以四个不同发育时期(6月20日, 7月7日, 7月17日和7月27日)的种子为材料,利用气相色谱飞行时间质谱测定种子油脂的脂肪酸组成,采用实时荧光定量PCR方法分析相关基因的表达模式,分析相关基因在不同发育期种子中的表达差异对种子油脂中碳十八不饱和脂肪酸积累的影响。结果表明:(1)紫斑牡丹种子油脂的不饱和脂肪酸占总脂肪酸的94.09%,其中碳十八不饱和脂肪酸占93.84%;不同种子发育期的油酸和亚麻酸呈一直上升趋势,而亚油酸则呈一直下降趋势;(2)种子发育过程中,KAR基因的持续上调表达合成了更多的C16:0-ACP;FATB和Δ9D基因下调表达,分别减弱了C16:0-ACP向棕榈酸和棕榈油酸的转化,KASⅡ基因的高表达促进了C16:0-ACP向C18:0-ACP的转化,为C18脂肪酸合成提供了充足原料;(3)种子发育过程中,SAD基因持续上调表达加快了种子中油酸的合成积累;FAD2基因表达量一直维持在相对较高水平,保证了种子油脂亚油酸占比一直在20%以上;发育初期FAD3基因表达量和发育末期FAD8基因表达量的迅速上升,加快了亚麻酸的合成。紫斑牡丹种子C18不饱和脂肪酸的高积累主要来源于SAD、FAD2、FAD3和FAD8基因的协同高表达。这为理解种子油富集碳18不饱脂肪酸的基因调控机制提供了理论依据,对木本油料油脂脂肪酸组份的遗传改良具有重要意义。
In order to study the relationship between the high accumulation of carbon 18 unsaturated fatty acids and the related genes of SAD, FAD2, FAD3, FAD7 and FAD8 in seeds of Paeonia suffruticosa, the seeds of four different development periods(June 20 th, July 7 th, July 17 thand July 27 th) were used as materials. The fatty acid compositions and their relative contents of seed oils were determined by GC-TOF/MS. The expression patterns of related genes were analyzed by real-time fluorescence quantitative PCR, and the effect of the expression difference of related genes at different seed developments on accumulation of carbon 18 unsaturated fatty acids were analyzed.The results showed that the unsaturated fatty acid in the seed oils accounted for 94.09% of the total fatty acid.Among them, C18 unsaturated fatty acids accounted for 93.84%. The oleic acid and linolenic acid showed an upward trend at different seed developments, while linoleic acid showed a downward trend. During seed development, the continuous up-regulated expression of KAR gene produced more C16:0-ACP. The down-regulation of FATB and Δ9 D gene reduced the conversion of C16:0-ACP to palmitic acid and palmitoleic acid,respectively. The high expression of KASⅡ gene promoted the conversion of C16:0-ACP to C18:0-ACP, which provided sufficient raw materials for the synthesis of C18 fatty acids. During seed development, the continuous up-regulated expression of SAD gene accelerated the synthesis and accumulation of oleic acid in seeds. FAD2 gene expression had been maintained at a relatively high level, guaranteeing that the proportion of linoleic acid in seed oil had been more than 20%. Fast increases of FAD3 gene expression at the early developmental stages and FAD8 gene expression at the late developmental stages promoted the biosynthesis and accumulation of linolenic acid.The high accumulation of C18 unsaturated fatty acids was mainly from coordinated high expression of SAD,FAD2, FAD3 and FAD8 genes in the seed of P. suffruticosa. This could provide a theoretical basis for understanding the regulative mechanism of multigenes for high accumulation of C18 unsaturated fatty acids in seed oils,which could be significant for genetic improvement of fatty acid compositions of woody-oil trees.
In order to study the relationship between the high accumulation of carbon 18 unsaturated fatty acids and the related genes of SAD, FAD2, FAD3, FAD7 and FAD8 in seeds of Paeonia suffruticosa, the seeds of four different development periods(June 20 th, July 7 th, July 17 thand July 27 th) were used as materials. The fatty acid compositions and their relative contents of seed oils were determined by GC-TOF/MS. The expression patterns of related genes were analyzed by real-time fluorescence quantitative PCR, and the effect of the expression difference of related genes at different seed developments on accumulation of carbon 18 unsaturated fatty acids were analyzed.The results showed that the unsaturated fatty acid in the seed oils accounted for 94.09% of the total fatty acid.Among them, C18 unsaturated fatty acids accounted for 93.84%. The oleic acid and linolenic acid showed an upward trend at different seed developments, while linoleic acid showed a downward trend. During seed development, the continuous up-regulated expression of KAR gene produced more C16:0-ACP. The down-regulation of FATB and Δ9 D gene reduced the conversion of C16:0-ACP to palmitic acid and palmitoleic acid,respectively. The high expression of KASⅡ gene promoted the conversion of C16:0-ACP to C18:0-ACP, which provided sufficient raw materials for the synthesis of C18 fatty acids. During seed development, the continuous up-regulated expression of SAD gene accelerated the synthesis and accumulation of oleic acid in seeds. FAD2 gene expression had been maintained at a relatively high level, guaranteeing that the proportion of linoleic acid in seed oil had been more than 20%. Fast increases of FAD3 gene expression at the early developmental stages and FAD8 gene expression at the late developmental stages promoted the biosynthesis and accumulation of linolenic acid.The high accumulation of C18 unsaturated fatty acids was mainly from coordinated high expression of SAD,FAD2, FAD3 and FAD8 genes in the seed of P. suffruticosa. This could provide a theoretical basis for understanding the regulative mechanism of multigenes for high accumulation of C18 unsaturated fatty acids in seed oils,which could be significant for genetic improvement of fatty acid compositions of woody-oil trees.
引文
Bhunia R.K.,Chakraborty A.,Kaur R.,Maiti M.K.,and Sen S.K.,2016,Enhancement ofα-linolenic acid content in transgenic tobacco seeds by targeting a plastidialω-3 fatty acid desaturase(fad7)gene of Sesamum indicum to ER,Plant Cell Rep.,35(1):213-226
Bryant F.M.,Munoz-Azcarate O.,Kelly A.A.,Beaudoin F.,Kurup S.,and Eastmond P.,2016,Acyl-acyl carrier protein desaturase2 and 3 are responsible for making omega-7 fatty acids in the Arabidopsis aleurone,Plant Physiol.,172(1):154-162
Chung C.H.,Kim J.L.,Lee Y.C.,and Choi Y.L.,1999,Cloning and characterization of a seed-specific omega-3 fatty acid desaturase cDNA from Perilla frutescens,Plant Cell Physiol.,40(1):114-118
Connor W.E.,1999,Alpha-linolenic acid in health and disease,Am.J.Clin.Nutr.,69(5):827-828
Dellapenna D.,2001,Plant metabolic engineering,Plant Physiol.,125(1):160-163
Ding J.,Ruan C.J.,Guan Y.,Wu L.R.,and Shan J.Y.,2017,Coordinated regulation mechanism of multigenes involved in high accumulation of C18 unsaturated fatty acids in sea buckthorn seed,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(6):1080-1089(丁健,阮成江,关莹,吴立仁,单金友,2017,沙棘种子高积累碳十八不饱和脂肪酸的多基因协同调控机制,西北植物学报,37(6):1080-1089)
Fu Z.H.,2017,Study on cloning and functional expression of KASⅡgene in Vernicia fordii,Thesis for M.S.,Central South University of Forestry&Technology,Supervisor:Tan X.F.,pp.19-21(付子豪,2017,油桐KASII基因的克隆与功能研究,硕士学位论文,中南林业科技大学,导师:谭晓风,pp.19-21)
Harbige L.S.,2003,Fatty acids,the immune response,and autoimmunity:a question of n-6 essentiality and the balance between n-6 and n-3,Lipids,38(4):323-341
He C.,Peng Y.,Xiao W.,Liu H.,and Xiao P.G.,2013,Determination of chemical variability of phenolic and monoterpene glycosides in the seeds of Paeonia species using HPLC and profiling analysis,Food Chem.,138(4):2108-2114
Huang J.Q.,Zhang T.,Zhang Q.X.,Chen M.,Wang Z.J.,Zheng B.S.,Xia G.H.,Yang X.Y.,Huang C.Y.,and Huang Y.J.,2016,The mechanism of high contents of oil and oleic acid revealed by transcriptomic and lipidomic analysis during embryogenesis in Carya cathayensis Sarg.,BMC Genomics,17(1):113
Jia Y.L.,Wu L.,and Lu C.M.,2014,Stearoyl-acyl carrier protein desaturase(SAD)gene cloning and expression in Brassica napus,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),36(2):135-141(贾艳丽,吴磊,卢长明,2014,甘蓝型油菜Δ9硬脂酰ACP脱氢酶(SAD)基因的克隆与表达分析,中国油料作物学报,36(2):135-141)
Li S.S.,Chen L.G.,Xu Y.J.,Wang L.J.,and Wang L.S.,2012,I-dentification of floral fragrances in tree peony cultivars by gas chromatography-mass spectrometry,Sci.Hortic.,142(4):158-165
Li S.S.,Wang L.S.,Shu Q.Y.,Chen L.G.,Shao S.,and Yin D.D.,2015a,Fatty acid composition of developing tree peony(Paeonia section Moutan DC.)seeds and transcriptome analysis during seed development,BMC Genomics,16(1):208
Li S.S.,Yuan R.Y.,Chen L.G.,Wang L.S.,Hao X.H.,Wang L.J.,Zheng X.C.,and Du H.,2015b,Systematic qualitative and quantitative assessment of fatty acids in the seeds of 60 tree peony(Paeonia section Moutan DC.)cultivars by GC-MS,Food Chem.,173:133-140
Li Z.X.,Qin G.W.,He J.H.,and Cao X.Y.,2010,Comparative analysis of fatty acid composition in seed kernel and coat of Paeonia rockii seeds,Zhongzi(Seed),29(1):34-36(李子璇,秦公伟,何建华,曹小勇,2010,紫斑牡丹种仁种皮中脂肪酸组成比较分析,种子,29(1):34-36)
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.L.,2015,Study on cloning and functional expression of KAR gene in Vernicia fordii,Thesis for M.S.,Central South University of Forestry&Technology,Supervisor:Tan X.F.,pp.18-19(刘美兰,2015,油桐KAR基因的克隆与功能研究,硕士学位论文,中南林业科技大学,导师:谭晓风,pp.18-19)
Nehdi I.A.,2013,Cupressus sempervirens var.horizentalis seed oil:chemical composition,physicochemical characteristics,and utilizations,Ind.Crops Prod.,41(1):381-385
Nehdi I.A.,Sbihi H.,Tan C.P.,and Al-Resayes S.I.,2012,Garden cress(Lepidium sativum Linn.)seed oil as a potential feedstock for biodiesel production,Bioresour.Technol.,126(4):193-197
Sánchez-Salcedo E.M.,Sendra E.,Carbonell-Barrachina魣.A.,Martínez J.J.,and Hernández F.,2016,Fatty acids composition of Spanish black(Morus nigra L.)and white(Morus alba L.)mulberries,Food Chem.,190:566-571
Simopoulos A.P.,2003,Importance of the ratio of omega-6/omega-3 essential fatty acids:evolutionary aspects,World Rev.Nutr.Diet.,92(8):1-22
Simopoulos A.P.,2006,Evolutionary aspects of diet:the omega-6/omega-3 ratio and genetic variation:nutritional implications for chronic diseases,Biomed.Pharmacother.,60(9):502-507
Yang B.,and Kallio H.P.,2001,Fatty acid composition of lipids in sea buckthorn(Hippopha觕rhamnoides L.)berries of different origins,J.Agric.Food Chem.,49(4):1939-1947
Yu L.,and Zuo L.J.,2017,Development and utilization research progress of oil tree peony,Beijing Nongye Zhiye Xueyuan Xuebao(Journal of Beijing Vocational College of Agriculture),31(1):23-31(于玲,左利娟,2017,油用牡丹开发利用研究进展,北京农业职业学院学报,31(1):23-31)
Yurchenko O.P.,Park S.,Ilut D.C.,Inmon J.J.,Millhollon J.C.,Liechy Z.,Page J.T.,Jenks M.A.,Chapman K.D.,Udall J.A.,Gore M.A.,and Dyer J.M.,2014,Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium,BMC Plant Biol.,14(1):312
Zaborowska Z.,Starzycki M.,Femiak I.,Swiderski M.,and Legocki A.B.,2002,Yellow lupine gene encoding stearoyl-A-CP desaturase-organization,expression and potential application,Acta Biochim.Pol.,49(1):29-42
Zhang Q.Y.,Yu R.,Xie L.H.,Rahman M.M.,Kilaru A.,Niu L.X.,and Zhang Y.L.,2018,Fatty acid and associated gene expression analyses of three tree peony species reveal key genes forα-linolenic acid synthesis in seeds,Front.Plant Sci.,9:106
Zhou S.L.,Zou X.H.,Zhou Z.Q.,Liu J.,Xu C.,Yu J.,Wang Q.,Zhang D.M.,Wang X.Q.,Ge S.,Sang T.,Pan K.Y.,and Hong D.Y.,2014,Multiple species of wild tree peonies gave rise to the'king of flowers',Paeonia suffruticosa Andrews,Proc.Biol.Sci.,Doi:10.1098/rspb.2014.1687
Bryant F.M.,Munoz-Azcarate O.,Kelly A.A.,Beaudoin F.,Kurup S.,and Eastmond P.,2016,Acyl-acyl carrier protein desaturase2 and 3 are responsible for making omega-7 fatty acids in the Arabidopsis aleurone,Plant Physiol.,172(1):154-162
Chung C.H.,Kim J.L.,Lee Y.C.,and Choi Y.L.,1999,Cloning and characterization of a seed-specific omega-3 fatty acid desaturase cDNA from Perilla frutescens,Plant Cell Physiol.,40(1):114-118
Connor W.E.,1999,Alpha-linolenic acid in health and disease,Am.J.Clin.Nutr.,69(5):827-828
Dellapenna D.,2001,Plant metabolic engineering,Plant Physiol.,125(1):160-163
Ding J.,Ruan C.J.,Guan Y.,Wu L.R.,and Shan J.Y.,2017,Coordinated regulation mechanism of multigenes involved in high accumulation of C18 unsaturated fatty acids in sea buckthorn seed,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(6):1080-1089(丁健,阮成江,关莹,吴立仁,单金友,2017,沙棘种子高积累碳十八不饱和脂肪酸的多基因协同调控机制,西北植物学报,37(6):1080-1089)
Fu Z.H.,2017,Study on cloning and functional expression of KASⅡgene in Vernicia fordii,Thesis for M.S.,Central South University of Forestry&Technology,Supervisor:Tan X.F.,pp.19-21(付子豪,2017,油桐KASII基因的克隆与功能研究,硕士学位论文,中南林业科技大学,导师:谭晓风,pp.19-21)
Harbige L.S.,2003,Fatty acids,the immune response,and autoimmunity:a question of n-6 essentiality and the balance between n-6 and n-3,Lipids,38(4):323-341
He C.,Peng Y.,Xiao W.,Liu H.,and Xiao P.G.,2013,Determination of chemical variability of phenolic and monoterpene glycosides in the seeds of Paeonia species using HPLC and profiling analysis,Food Chem.,138(4):2108-2114
Huang J.Q.,Zhang T.,Zhang Q.X.,Chen M.,Wang Z.J.,Zheng B.S.,Xia G.H.,Yang X.Y.,Huang C.Y.,and Huang Y.J.,2016,The mechanism of high contents of oil and oleic acid revealed by transcriptomic and lipidomic analysis during embryogenesis in Carya cathayensis Sarg.,BMC Genomics,17(1):113
Jia Y.L.,Wu L.,and Lu C.M.,2014,Stearoyl-acyl carrier protein desaturase(SAD)gene cloning and expression in Brassica napus,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),36(2):135-141(贾艳丽,吴磊,卢长明,2014,甘蓝型油菜Δ9硬脂酰ACP脱氢酶(SAD)基因的克隆与表达分析,中国油料作物学报,36(2):135-141)
Li S.S.,Chen L.G.,Xu Y.J.,Wang L.J.,and Wang L.S.,2012,I-dentification of floral fragrances in tree peony cultivars by gas chromatography-mass spectrometry,Sci.Hortic.,142(4):158-165
Li S.S.,Wang L.S.,Shu Q.Y.,Chen L.G.,Shao S.,and Yin D.D.,2015a,Fatty acid composition of developing tree peony(Paeonia section Moutan DC.)seeds and transcriptome analysis during seed development,BMC Genomics,16(1):208
Li S.S.,Yuan R.Y.,Chen L.G.,Wang L.S.,Hao X.H.,Wang L.J.,Zheng X.C.,and Du H.,2015b,Systematic qualitative and quantitative assessment of fatty acids in the seeds of 60 tree peony(Paeonia section Moutan DC.)cultivars by GC-MS,Food Chem.,173:133-140
Li Z.X.,Qin G.W.,He J.H.,and Cao X.Y.,2010,Comparative analysis of fatty acid composition in seed kernel and coat of Paeonia rockii seeds,Zhongzi(Seed),29(1):34-36(李子璇,秦公伟,何建华,曹小勇,2010,紫斑牡丹种仁种皮中脂肪酸组成比较分析,种子,29(1):34-36)
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.L.,2015,Study on cloning and functional expression of KAR gene in Vernicia fordii,Thesis for M.S.,Central South University of Forestry&Technology,Supervisor:Tan X.F.,pp.18-19(刘美兰,2015,油桐KAR基因的克隆与功能研究,硕士学位论文,中南林业科技大学,导师:谭晓风,pp.18-19)
Nehdi I.A.,2013,Cupressus sempervirens var.horizentalis seed oil:chemical composition,physicochemical characteristics,and utilizations,Ind.Crops Prod.,41(1):381-385
Nehdi I.A.,Sbihi H.,Tan C.P.,and Al-Resayes S.I.,2012,Garden cress(Lepidium sativum Linn.)seed oil as a potential feedstock for biodiesel production,Bioresour.Technol.,126(4):193-197
Sánchez-Salcedo E.M.,Sendra E.,Carbonell-Barrachina魣.A.,Martínez J.J.,and Hernández F.,2016,Fatty acids composition of Spanish black(Morus nigra L.)and white(Morus alba L.)mulberries,Food Chem.,190:566-571
Simopoulos A.P.,2003,Importance of the ratio of omega-6/omega-3 essential fatty acids:evolutionary aspects,World Rev.Nutr.Diet.,92(8):1-22
Simopoulos A.P.,2006,Evolutionary aspects of diet:the omega-6/omega-3 ratio and genetic variation:nutritional implications for chronic diseases,Biomed.Pharmacother.,60(9):502-507
Yang B.,and Kallio H.P.,2001,Fatty acid composition of lipids in sea buckthorn(Hippopha觕rhamnoides L.)berries of different origins,J.Agric.Food Chem.,49(4):1939-1947
Yu L.,and Zuo L.J.,2017,Development and utilization research progress of oil tree peony,Beijing Nongye Zhiye Xueyuan Xuebao(Journal of Beijing Vocational College of Agriculture),31(1):23-31(于玲,左利娟,2017,油用牡丹开发利用研究进展,北京农业职业学院学报,31(1):23-31)
Yurchenko O.P.,Park S.,Ilut D.C.,Inmon J.J.,Millhollon J.C.,Liechy Z.,Page J.T.,Jenks M.A.,Chapman K.D.,Udall J.A.,Gore M.A.,and Dyer J.M.,2014,Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium,BMC Plant Biol.,14(1):312
Zaborowska Z.,Starzycki M.,Femiak I.,Swiderski M.,and Legocki A.B.,2002,Yellow lupine gene encoding stearoyl-A-CP desaturase-organization,expression and potential application,Acta Biochim.Pol.,49(1):29-42
Zhang Q.Y.,Yu R.,Xie L.H.,Rahman M.M.,Kilaru A.,Niu L.X.,and Zhang Y.L.,2018,Fatty acid and associated gene expression analyses of three tree peony species reveal key genes forα-linolenic acid synthesis in seeds,Front.Plant Sci.,9:106
Zhou S.L.,Zou X.H.,Zhou Z.Q.,Liu J.,Xu C.,Yu J.,Wang Q.,Zhang D.M.,Wang X.Q.,Ge S.,Sang T.,Pan K.Y.,and Hong D.Y.,2014,Multiple species of wild tree peonies gave rise to the'king of flowers',Paeonia suffruticosa Andrews,Proc.Biol.Sci.,Doi:10.1098/rspb.2014.1687