油茶种子油酸合成积累相关基因的表达模式分析
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摘要
油茶是中国南方的重要木本油料树种,但其种子油富含油酸的多基因协同调控机制仍不清楚。以高油酸品系‘MY003’和低油酸品系‘MY008’的不同发育期(6月2日, 7月4日, 8月5日, 9月3日)种子为材料,利用三氟化硼—甲醇法对样品进行甲酯化,采用GC-TOF/MS方法测定所提取油茶种子油的脂肪酸组份及不同组份的相对百分比;采用qRT-PCR方法分析油酸合成积累相关基因KAR、HAD、EAR、KASI、KASII、FATB、SAD、FAD2等的表达模式,并分析油酸合成积累上下游基因表达对油茶种子富积油酸的影响。结果显示:品系‘MY003’和‘MY008’种子油中共检测出6种主要脂肪酸,其中油酸为主要组份,分别占总脂肪酸的82.51%和76.64%;种子发育期间,油酸含量一直呈上升趋势,棕榈酸、亚油酸和亚麻酸含量呈下降趋势,而硬脂酸和花生烯酸含量变化不明显。HAD、EAR和KASI基因协同高表达为油酸合成前体C16:0-ACP的合成提供了基础;FATB基因下调表达减弱C16:0-ACP向棕榈酸的转化,而KASII基因持续高表达为油酸合成提供了充足原料。SAD基因持续高表达及FAD2、FAD3、FAD7、FAD8和FAE1基因协同低表达,既加快油酸的合成积累,同时也降低了油酸的去饱和作用,这种上下游多基因的协同调控促成和保证了油茶种子油酸的高积累。本研究可为后续开展油茶种子油酸含量遗传改良研究提供科学依据。
Camellia oleifera is an important woody oil tree in southern China, its seed oil contains rich oleic acid,but the coordinated regulation mechanism of multigenes is still unclear. In this research, the line 'MY003' with high oil content in the seeds and the line 'MY008' with low oil content in the seeds were selected as sample trees.Their seeds were harvested on June 2 nd, July 4 th, August 5 th, and September 3 rd. These samples were methylated by method of boron trifluoride-methyl alcohol. Changes of the fatty acid components and relative contents in oils extracted from different developmental stages of these two lines were determined by method of GC-TOF/MS. The expression patterns of KAR, HAD, EAR, KASI, KASII, FATB, SAD, FAD2 genes involved in fatty acid biosynthesis were tested by using q RT-PCR analysis, and the effects of expression changes of multigenes on the synthesis and accumulation of oil acids were analyze d. The results showed that six fatty acids were detected in seed oils of'MY003' and 'MY008', among which oleic acid was the main component, accounting for 82.51% and 76.64% of the total fatty acids, respectively. Stearic acid and arachidonic acid had no significant changes. The co-expression of HAD, EAR and KASI genes directly increased the relative contents of C16:0-ACP, which provided enough precursor resources for the synthesis of oil acid in the seeds of C. oleifera; the down-regulation expression of FATB weakened the conversion of C16:0-ACP to palmitic acid, the high expression of KASII contributed to promote the stearic acid synthesis, which improved enough resources for the synthesis of oil acids. The continuous high expression of SAD gene contributed to accelerate the synthesis and accumulation of oleic acid, and the coordinated low expressions of FAD2, FAD3, FAD7, FAD8 and FAE1 also reduced the desaturation of oleic acid. The coordinated regulation of these multigenes contributed to ensure the high accumulation of oleic acid in seeds of C.oleifera. This research could provide scientific bases for the study of genetic improvement of oleic acid content in seeds of C. oleifera.
Camellia oleifera is an important woody oil tree in southern China, its seed oil contains rich oleic acid,but the coordinated regulation mechanism of multigenes is still unclear. In this research, the line 'MY003' with high oil content in the seeds and the line 'MY008' with low oil content in the seeds were selected as sample trees.Their seeds were harvested on June 2 nd, July 4 th, August 5 th, and September 3 rd. These samples were methylated by method of boron trifluoride-methyl alcohol. Changes of the fatty acid components and relative contents in oils extracted from different developmental stages of these two lines were determined by method of GC-TOF/MS. The expression patterns of KAR, HAD, EAR, KASI, KASII, FATB, SAD, FAD2 genes involved in fatty acid biosynthesis were tested by using q RT-PCR analysis, and the effects of expression changes of multigenes on the synthesis and accumulation of oil acids were analyze d. The results showed that six fatty acids were detected in seed oils of'MY003' and 'MY008', among which oleic acid was the main component, accounting for 82.51% and 76.64% of the total fatty acids, respectively. Stearic acid and arachidonic acid had no significant changes. The co-expression of HAD, EAR and KASI genes directly increased the relative contents of C16:0-ACP, which provided enough precursor resources for the synthesis of oil acid in the seeds of C. oleifera; the down-regulation expression of FATB weakened the conversion of C16:0-ACP to palmitic acid, the high expression of KASII contributed to promote the stearic acid synthesis, which improved enough resources for the synthesis of oil acids. The continuous high expression of SAD gene contributed to accelerate the synthesis and accumulation of oleic acid, and the coordinated low expressions of FAD2, FAD3, FAD7, FAD8 and FAE1 also reduced the desaturation of oleic acid. The coordinated regulation of these multigenes contributed to ensure the high accumulation of oleic acid in seeds of C.oleifera. This research could provide scientific bases for the study of genetic improvement of oleic acid content in seeds of C. oleifera.
引文
Bonaventu re G.,Salas J.J.,Pollard M.R.,and Ohlrogge J.B.,2003,Disruption of the FATB gene in Arabidopsis demon strates an essential role of saturated fatty acids in plant growth,Plant Cell,15(4):1020-1033
DellaPenna D.,2001,Plant metabolic engineering,Plant Physiol.,125(1):160-163
Ding J.,Ruan C.J.,and Guan 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,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)
D?rmann P.,Voelker T.A.,and Ohlrogge J.B.,2000,Accumulation of palmitate in Arabidopsis mediated by the acyl-acyl carrier protein thioesterase FATB1,Plant Physiol.,123(2):637-644
Hitz W.D.,Yadav N.S.,Reiter R.J.,Mauvis C.J.,and Kinney A.J.,1995,Reducing polyunsaturation in oils of transgenic canola and soybean,In:Kader J.C.,and Mazliak P.(eds.),Plant Lipid Metabolism,Kluwer,Dordrecht,The Netherland,pp.506-508
Jadhav A.,Katavic V.,Marillia E.F.,Michael Giblin E.,Barton D.L.,Kumar A.,Sonntag C.,Babic V.,Keller W.A.,and Taylor D.C.,2005,Increased levels of erucic acid in Brassica car inata by co-suppression and antisense repression of the endogenous FAD2 gene,Metab.Eng.,7(3):215-220
Kachroo A.,Shanklin J.,Whittle E.,Lapchyk L.,Hildebrand D.,and Kachroo P.,2007,The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis,Plant Mol.Biol.,63(2):257-271
Knutzon D.S.,Tho mpson G.A.,Radke S.E.,Johnson W.B.,Knauf V.C.,and Kridl J.C.,1992,Modification of brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene,Proc.Natl.Acad.Sci.USA,89(7):2624-2628
Lei Y.,2010,Molecular mechanism and genetic enhancement technology for high oleic acid in peanut(Arachis hypogaea L.),Dissertation for Ph.D.,Chinese Academy of Agricultural Sciences,Supervisor:Liao B.S.,pp.1-20(雷永,2010,花生高油酸的分子遗传机制及其高效遗传改良体系构建,博士学位论文,中国农业科学院,导师:廖博寿,pp.1-20)
Liao S.J.,Ji D.L.,and Tong H.R.,2005,Study on fatty acid composition and nutrition health protection function of the oiltea Camellia seed oil,Liangshi Yu Youzhi(Journal of Cereals&Oils),(6):7-9(廖书娟,吉当玲,童华荣,2005,茶油脂肪酸组成及其营养保健功能,粮食与油脂,(6):7-9)
Liu Q.,Singh S.P.,and Green A.G.,2002,High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing,Plant Physiol.,129(4):1732-1743
Martz F.,Kiviniemi S.,Palva T.E.,and Sutinen M.L.,2006,Contribution of omega-3 fatty acid desaturase and 3-ketoacyl-ACP synthaseⅡ(KASⅡ)genes in the modulation of glycerolipid fatty acid composition during cold acclimation in birch leaves,J.Exp.Bot.,57(4):897-909
Millar A.A.,and Kunst L.,1997,Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme,Plant J.,12(1):121-131
Niu B.,Guo L.,Zhao M.,Luo T.,Zhang R.,Zhang F.,Hou P.,Zhang Y.,Xu Y.,Wang S.,and Chen F.,2008,Molecular cloning,characterization,and expression of an omega-3 fatty acid desaturase gene from Sapium sebiferum,J.Biosci.Bioeng.,106(4):375-380
Ohlrogge J.,and Browse J.,1995,Lipid biosynthesis,Plant Cell,7(7):957-970
Sánchez-Salcedo E.M.,Sendra E.,Carbonell-Barrachina譧.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
Sasaki Y.,and Nagano Y.,2004,Plant acetyl-CoA carboxylase:structure,biosynthesis,regulation,and gene manipulation for plant breeding,Biosci.Biotechnol.Biochem.,68(6):1175-1184
Schmittgen T.D.,and Livak K.J.,2008,Analyzing real-time PCRdata by the comparative C(T)method,Nat.Protoc.,3(6):1101-1108
Stoutjesdijk P.A.,Hurlestone C.,and Singh S.P.,2001,High oleic acid Australian Brassica napus and B.juncea varieties produced by co-suppression of endogenous△I 2-desaturases,Biochemical Society Transactions,28(6):938-940
Sun P.G.,Xi R.C.,Niu.S.H.,Pian R.Q.,and Chen X.Y.,2010,Establishment and optimization of SRAP-PCR reaction system in Camellia oleifera,Jiy inzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(6):1192-1199(孙佩光,奚如春,钮世辉,骈瑞琪,陈晓阳,2010,油茶SRAP-PCR反应体系的建立和优化,基因组学与应用生物学,29(6):1192-1199)
Wu L.,Jia Y.L.,Wu G.,and Lu C.M.,2015,Molecular evidence for blocking erucic acid synthesis in rapeseed(Brassica napus L.)by a two-base-pair deletion in FAE1(fatty acid elongase 1),Journal of Integrative Agriculture,14(7):1251-1260
Ye J.,Qu J.,Bui H.T.,and Chua N.H.,2009,Rapid analysis of Jatropha curcas gene functions by virus-induced gene silencing,Plant Biotechnol.J.,7(9):964-976
Yu Y.C.,Pan X.C.,Xu M.J.,Zhang H.C.,Wang L.D.,Wei Y.,and Wu X.X.,2016,Cloning and expression analysis of regeneration related gene GmRUB1 in soybean,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3465-3473(于以成,潘校成,徐梦洁,张赫纯,王领东,魏怡,武小霞,2016,大豆再生相关基因GmRUB1的克隆及表达分析,基因组学与应用生物学,35(12):3465-3473)
Yuan Z.H.,2014,Cloning and expression analysis of fatty acid desaturase genes from Perilla frutescens,Thesis for M.S.,Chongqing Normal University,Supervisor:Zhang T.,pp.28-38(袁中厚,2014,紫苏ω-3脂肪酸脱氢酶基因的克隆与表达分析,硕士学位论文,重庆师范大学,导师:张涛,pp.28-38)
Zaborowska Z.,Starzycki M.,Femiak I.,Swiderski M.,and Legocki A.B.,2002,Yellow lupine gene encoding stearoylACP desaturase-organization,expression and potential application,Acta Biochim.Pol.,49(1):29-42
Zhang W.L.,Pu J.Y.,Zhu M.Y.,Niu J.,Feng Q.M.,Hu H.W.,Hou Q.,Guo J.,Yuan Y.M.,Zhang Z.X.,and Lin S.Z.,2013,Cloning and analysis of PcSAD gene from Pistacia chinensis Bunge,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),2(4):503-509(章文乐,蒲婧懿,朱梦媛,钮俊,冯清茗,胡惠雯,侯琦,郭静,袁一鸣,张志翔,林善枝,2013,黄连木PcSAD基因的克隆及分析,基因组学与应用生物学,2(4):503-509)
Zhao C.G.,Liu D.,Li F.L.,and Guo H.H.,2010,Advances in research on seed oil biosynthesis and basal metabolism,Zhongzi(Seed),29(4):56-62(赵翠格,刘頔,李凤兰,郭惠红,2010,植物种子油脂的生物合成及代谢基础研究进展,种子,29(4):56-62)
Zhou C.F.,2013,Changes of ingredients and genes expression of lipid metabolism associated with seeds development of Cameilla olelfera Abel,Dissertation for Ph.D.,Chinese A-cademy of Forestry,Supervisor:Yao X.H.,pp.1-5(周长富,2013,油茶种子发育过程组分及脂类代谢相关基因表达变化研究,博士学位论文,中国林业科学研究院,导师:姚小华,pp.1-5)
DellaPenna D.,2001,Plant metabolic engineering,Plant Physiol.,125(1):160-163
Ding J.,Ruan C.J.,and Guan 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,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)
D?rmann P.,Voelker T.A.,and Ohlrogge J.B.,2000,Accumulation of palmitate in Arabidopsis mediated by the acyl-acyl carrier protein thioesterase FATB1,Plant Physiol.,123(2):637-644
Hitz W.D.,Yadav N.S.,Reiter R.J.,Mauvis C.J.,and Kinney A.J.,1995,Reducing polyunsaturation in oils of transgenic canola and soybean,In:Kader J.C.,and Mazliak P.(eds.),Plant Lipid Metabolism,Kluwer,Dordrecht,The Netherland,pp.506-508
Jadhav A.,Katavic V.,Marillia E.F.,Michael Giblin E.,Barton D.L.,Kumar A.,Sonntag C.,Babic V.,Keller W.A.,and Taylor D.C.,2005,Increased levels of erucic acid in Brassica car inata by co-suppression and antisense repression of the endogenous FAD2 gene,Metab.Eng.,7(3):215-220
Kachroo A.,Shanklin J.,Whittle E.,Lapchyk L.,Hildebrand D.,and Kachroo P.,2007,The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis,Plant Mol.Biol.,63(2):257-271
Knutzon D.S.,Tho mpson G.A.,Radke S.E.,Johnson W.B.,Knauf V.C.,and Kridl J.C.,1992,Modification of brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene,Proc.Natl.Acad.Sci.USA,89(7):2624-2628
Lei Y.,2010,Molecular mechanism and genetic enhancement technology for high oleic acid in peanut(Arachis hypogaea L.),Dissertation for Ph.D.,Chinese Academy of Agricultural Sciences,Supervisor:Liao B.S.,pp.1-20(雷永,2010,花生高油酸的分子遗传机制及其高效遗传改良体系构建,博士学位论文,中国农业科学院,导师:廖博寿,pp.1-20)
Liao S.J.,Ji D.L.,and Tong H.R.,2005,Study on fatty acid composition and nutrition health protection function of the oiltea Camellia seed oil,Liangshi Yu Youzhi(Journal of Cereals&Oils),(6):7-9(廖书娟,吉当玲,童华荣,2005,茶油脂肪酸组成及其营养保健功能,粮食与油脂,(6):7-9)
Liu Q.,Singh S.P.,and Green A.G.,2002,High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing,Plant Physiol.,129(4):1732-1743
Martz F.,Kiviniemi S.,Palva T.E.,and Sutinen M.L.,2006,Contribution of omega-3 fatty acid desaturase and 3-ketoacyl-ACP synthaseⅡ(KASⅡ)genes in the modulation of glycerolipid fatty acid composition during cold acclimation in birch leaves,J.Exp.Bot.,57(4):897-909
Millar A.A.,and Kunst L.,1997,Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme,Plant J.,12(1):121-131
Niu B.,Guo L.,Zhao M.,Luo T.,Zhang R.,Zhang F.,Hou P.,Zhang Y.,Xu Y.,Wang S.,and Chen F.,2008,Molecular cloning,characterization,and expression of an omega-3 fatty acid desaturase gene from Sapium sebiferum,J.Biosci.Bioeng.,106(4):375-380
Ohlrogge J.,and Browse J.,1995,Lipid biosynthesis,Plant Cell,7(7):957-970
Sánchez-Salcedo E.M.,Sendra E.,Carbonell-Barrachina譧.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
Sasaki Y.,and Nagano Y.,2004,Plant acetyl-CoA carboxylase:structure,biosynthesis,regulation,and gene manipulation for plant breeding,Biosci.Biotechnol.Biochem.,68(6):1175-1184
Schmittgen T.D.,and Livak K.J.,2008,Analyzing real-time PCRdata by the comparative C(T)method,Nat.Protoc.,3(6):1101-1108
Stoutjesdijk P.A.,Hurlestone C.,and Singh S.P.,2001,High oleic acid Australian Brassica napus and B.juncea varieties produced by co-suppression of endogenous△I 2-desaturases,Biochemical Society Transactions,28(6):938-940
Sun P.G.,Xi R.C.,Niu.S.H.,Pian R.Q.,and Chen X.Y.,2010,Establishment and optimization of SRAP-PCR reaction system in Camellia oleifera,Jiy inzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(6):1192-1199(孙佩光,奚如春,钮世辉,骈瑞琪,陈晓阳,2010,油茶SRAP-PCR反应体系的建立和优化,基因组学与应用生物学,29(6):1192-1199)
Wu L.,Jia Y.L.,Wu G.,and Lu C.M.,2015,Molecular evidence for blocking erucic acid synthesis in rapeseed(Brassica napus L.)by a two-base-pair deletion in FAE1(fatty acid elongase 1),Journal of Integrative Agriculture,14(7):1251-1260
Ye J.,Qu J.,Bui H.T.,and Chua N.H.,2009,Rapid analysis of Jatropha curcas gene functions by virus-induced gene silencing,Plant Biotechnol.J.,7(9):964-976
Yu Y.C.,Pan X.C.,Xu M.J.,Zhang H.C.,Wang L.D.,Wei Y.,and Wu X.X.,2016,Cloning and expression analysis of regeneration related gene GmRUB1 in soybean,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3465-3473(于以成,潘校成,徐梦洁,张赫纯,王领东,魏怡,武小霞,2016,大豆再生相关基因GmRUB1的克隆及表达分析,基因组学与应用生物学,35(12):3465-3473)
Yuan Z.H.,2014,Cloning and expression analysis of fatty acid desaturase genes from Perilla frutescens,Thesis for M.S.,Chongqing Normal University,Supervisor:Zhang T.,pp.28-38(袁中厚,2014,紫苏ω-3脂肪酸脱氢酶基因的克隆与表达分析,硕士学位论文,重庆师范大学,导师:张涛,pp.28-38)
Zaborowska Z.,Starzycki M.,Femiak I.,Swiderski M.,and Legocki A.B.,2002,Yellow lupine gene encoding stearoylACP desaturase-organization,expression and potential application,Acta Biochim.Pol.,49(1):29-42
Zhang W.L.,Pu J.Y.,Zhu M.Y.,Niu J.,Feng Q.M.,Hu H.W.,Hou Q.,Guo J.,Yuan Y.M.,Zhang Z.X.,and Lin S.Z.,2013,Cloning and analysis of PcSAD gene from Pistacia chinensis Bunge,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),2(4):503-509(章文乐,蒲婧懿,朱梦媛,钮俊,冯清茗,胡惠雯,侯琦,郭静,袁一鸣,张志翔,林善枝,2013,黄连木PcSAD基因的克隆及分析,基因组学与应用生物学,2(4):503-509)
Zhao C.G.,Liu D.,Li F.L.,and Guo H.H.,2010,Advances in research on seed oil biosynthesis and basal metabolism,Zhongzi(Seed),29(4):56-62(赵翠格,刘頔,李凤兰,郭惠红,2010,植物种子油脂的生物合成及代谢基础研究进展,种子,29(4):56-62)
Zhou C.F.,2013,Changes of ingredients and genes expression of lipid metabolism associated with seeds development of Cameilla olelfera Abel,Dissertation for Ph.D.,Chinese A-cademy of Forestry,Supervisor:Yao X.H.,pp.1-5(周长富,2013,油茶种子发育过程组分及脂类代谢相关基因表达变化研究,博士学位论文,中国林业科学研究院,导师:姚小华,pp.1-5)