沙棘果实不同器官脂肪酸差异的多基因协同调控
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摘要
为探讨沙棘果肉高积累碳16脂肪酸和种子高积累碳十八不饱和脂肪酸的基因调控差异,以沙棘品系"TF-23"4个不同发育期(7月10日, 7月26日, 8月11日, 8月26日)的果肉和种子为研究材料,采用气相色谱飞行时间质谱法测定干果肉和种子油脂的脂肪酸组份及不同组份的相对百分含量,运用qRT-PCR分析脂肪酸形成相关基因KAR、FATB、Δ9D、KCS1、KASⅡ、SAD、FAD2、FAD3、FAD7和FAD8的表达模式,验证多基因表达对果实不同器官脂肪酸形成积累差异的影响。结果表明:(1)沙棘干果肉油和种子油中共检测出8种主要脂肪酸,果肉油脂中棕榈酸(35.15%)、棕榈油酸(26.21%)和异油酸(24.22%)为主要组份,种子油脂中异油酸(15.98%)、亚油酸(43.43%)和亚麻酸(26.70%)为主要组份。(2)沙棘果肉高积累棕榈酸、棕榈油酸和异油酸源于KAR、FATB、Δ9D和KCS1基因的高表达协同KASⅡ基因的低表达。(3)沙棘种子油脂高富集亚油酸、亚麻酸和异油酸源于种子发育过程中KCS1、KASⅡ、SAD、FAD2和FAD3基因的协同高表达。首次揭示出调控沙棘果肉和种子富集不同脂肪酸的关键基因是KASⅡ基因,种子中KASⅡ基因高表达为亚油酸和亚麻酸的富集提供了充足底物C18:0,果肉中KASⅡ基因低表达协同FATB和Δ9D基因高表达促进了棕榈酸和棕榈油酸的富集。这可为进一步遗传改良沙棘果肉和种子油脂肪酸组成提供科学参考,对沙棘开发利用具有重要意义。
In order to st udy the difference of genes regulation for high accumulation of C16 fatty acids in sea buckthorn( Hippophae L.) pulp and C18 unsaturated fatty acids in seeds, pulp and seeds of the line TF-23 harvested at four different development stages(July 10 th, July 26 th, August 11 th, August 26 th) were selected as the material. The fatty acids and relative percentage of different components in dried fruit pulp and seed oil were determined by gas chromatography-time of flight mass spectrometry. The expression patterns of fatty acid formation related genes KAR, FATB, Δ9 D, KCS1, KASⅡ, SAD, FAD2, FAD3, FAD7 and FAD8 were analyzed by qRT-PCR. The effects of polygene expression on fatty acid formation and accumulation in different organs of fruits were verified. The result showed that eight fatty acids were detected in dried pulp and seed oils of line TF-23. Palmitic acid(35.15%), palmitoleic acid(26.21%) and isooleic acid(24.22%) were the major fatty acids in pulp oils, and linoleic acid(43.43%), linolenic acid(26.70) and isooleic acid(24.22%) were the major fatty acids in seed oils. High accumulations of palmitic acid, palmitoleic acid and isooleic acid in pulp were from high expressions of KAR, FATB, Δ9 D and KCS1 genes, which coordinated with the low expression of KAS Ⅱ gene.High accumulations of linoleic acid, linolenic acid and isooleic acid in seeds were from high co-expressions of KCS1, KASⅡ, SAD, FAD2 and FAD3 genes. It was revealed for the first time that KASⅡ gene was the key gene regulating the accumulation of different fatty acids in seabuckthorn pulp and seeds. The high expression of KASⅡgene in seeds provided sufficient substrate C18:0 for the enrichment of linoleic acid and linolenic acid. The low expression of KASⅡ gene in pulp promoted the enrichment of palmitic acid and palmitoleic acid with the high expression of FATB and Δ9 D genes. This could provide scientific reference for genetic improvement of fatty acids in pulp and seeds of sea buckthorn fruits, which was significant for exploration and utilization of this species.
In order to st udy the difference of genes regulation for high accumulation of C16 fatty acids in sea buckthorn( Hippophae L.) pulp and C18 unsaturated fatty acids in seeds, pulp and seeds of the line TF-23 harvested at four different development stages(July 10 th, July 26 th, August 11 th, August 26 th) were selected as the material. The fatty acids and relative percentage of different components in dried fruit pulp and seed oil were determined by gas chromatography-time of flight mass spectrometry. The expression patterns of fatty acid formation related genes KAR, FATB, Δ9 D, KCS1, KASⅡ, SAD, FAD2, FAD3, FAD7 and FAD8 were analyzed by qRT-PCR. The effects of polygene expression on fatty acid formation and accumulation in different organs of fruits were verified. The result showed that eight fatty acids were detected in dried pulp and seed oils of line TF-23. Palmitic acid(35.15%), palmitoleic acid(26.21%) and isooleic acid(24.22%) were the major fatty acids in pulp oils, and linoleic acid(43.43%), linolenic acid(26.70) and isooleic acid(24.22%) were the major fatty acids in seed oils. High accumulations of palmitic acid, palmitoleic acid and isooleic acid in pulp were from high expressions of KAR, FATB, Δ9 D and KCS1 genes, which coordinated with the low expression of KAS Ⅱ gene.High accumulations of linoleic acid, linolenic acid and isooleic acid in seeds were from high co-expressions of KCS1, KASⅡ, SAD, FAD2 and FAD3 genes. It was revealed for the first time that KASⅡ gene was the key gene regulating the accumulation of different fatty acids in seabuckthorn pulp and seeds. The high expression of KASⅡgene in seeds provided sufficient substrate C18:0 for the enrichment of linoleic acid and linolenic acid. The low expression of KASⅡ gene in pulp promoted the enrichment of palmitic acid and palmitoleic acid with the high expression of FATB and Δ9 D genes. This could provide scientific reference for genetic improvement of fatty acids in pulp and seeds of sea buckthorn fruits, which was significant for exploration and utilization of this species.
引文
Bona venture G.,Salas J.J.,Pollard M.R.,and Ohlrogge J.B.,2003,Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growth,Plant Cell,15(4):1020-1033
Cenkowski S.,Yakimishen R.,Przybylski R.,and Muir W.E.,2006,Quality of extracted sea buckthorn seed and pulp oil,Canadian Biosystem Engineering,48(3):9-16
Chen Y.C.,Cui Q.Q.,Xu Y.J.,Yang S.,Gao M.,and Wang Y.,2015,Effects of tung oilseed FAD2 and DGAT2 genes on unsaturated fatty acid accumulation in Rhodotorula glutinis and Arabidopsis thaliana,Mol.Genet.Genomics,290(4):1605-1613
Dellapenna D.,2001,Plant metabolic engineering,Plant Physiol.,125(1):160-163
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
Jadhav A.,Katavic V.,Marillia E.F.,Michael G.E.,Barton D.L.,Kumar A.,Sonntaq C.,Babic V.,Keller W.A.,and Taylor D.C.,2005,Increased levels of erucic acid in Brassica carinata 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 con tribution of leaf isoforms to oleic acid synthesis,Plant Mol.Biol.,63(2):257-271
Knutzon D.S.,Thompson 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
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.2-5(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:廖伯寿,pp.2-5)
Li S.S.,Wang L.S.,Shu Q.Y.,Wu J.,Chen L.G.,Shao S.,and Yin D.D.,2015,Fatty acid composition of developing tree peony(Paeonia section Moutan DC.)seeds and transcriptome analysis during seed development,BMC Genomics,16(1):208
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
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
Sanchez J.,Cucillo M.T.,and Harwood J.L.,1992,Glycolipid biosynthesis by microsomal fractions from olive fruits,Phytochemistry,31(1):129-134
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
Simopoulos A.P.,2006,Evolutionary aspects of diet,the omega-6/omega-3 ratio and genetic variation:nutritional implications for chronic deseases,Biomed.Pharmacother.,60(9):502-507
Yang B.R.,and Kallio H.P.,2001,Fatty acid composition of lipids in sea buckthorn(Hippophae rhamnoides L.)berries of different origins,J.Agric.Food Chem.,49(4):1939-1947
Cenkowski S.,Yakimishen R.,Przybylski R.,and Muir W.E.,2006,Quality of extracted sea buckthorn seed and pulp oil,Canadian Biosystem Engineering,48(3):9-16
Chen Y.C.,Cui Q.Q.,Xu Y.J.,Yang S.,Gao M.,and Wang Y.,2015,Effects of tung oilseed FAD2 and DGAT2 genes on unsaturated fatty acid accumulation in Rhodotorula glutinis and Arabidopsis thaliana,Mol.Genet.Genomics,290(4):1605-1613
Dellapenna D.,2001,Plant metabolic engineering,Plant Physiol.,125(1):160-163
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
Jadhav A.,Katavic V.,Marillia E.F.,Michael G.E.,Barton D.L.,Kumar A.,Sonntaq C.,Babic V.,Keller W.A.,and Taylor D.C.,2005,Increased levels of erucic acid in Brassica carinata 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 con tribution of leaf isoforms to oleic acid synthesis,Plant Mol.Biol.,63(2):257-271
Knutzon D.S.,Thompson 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
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.2-5(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:廖伯寿,pp.2-5)
Li S.S.,Wang L.S.,Shu Q.Y.,Wu J.,Chen L.G.,Shao S.,and Yin D.D.,2015,Fatty acid composition of developing tree peony(Paeonia section Moutan DC.)seeds and transcriptome analysis during seed development,BMC Genomics,16(1):208
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
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
Sanchez J.,Cucillo M.T.,and Harwood J.L.,1992,Glycolipid biosynthesis by microsomal fractions from olive fruits,Phytochemistry,31(1):129-134
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
Simopoulos A.P.,2006,Evolutionary aspects of diet,the omega-6/omega-3 ratio and genetic variation:nutritional implications for chronic deseases,Biomed.Pharmacother.,60(9):502-507
Yang B.R.,and Kallio H.P.,2001,Fatty acid composition of lipids in sea buckthorn(Hippophae rhamnoides L.)berries of different origins,J.Agric.Food Chem.,49(4):1939-1947