利用CRISPR/Cas9技术创制大豆高油酸突变系
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摘要
大豆是重要的油料作物,其种子脂肪酸中油酸含量是评价大豆油脂品质的重要指标之一。本研究设计了分别由AtU3d、AtU3b和AtU6-1启动子驱动、长20 bp的guide RNA (gRNA)靶点以靶向编辑GmFAD2-1A基因的外显子区,首先将这3个靶点一起组装到pYLCRISPR/Cas9-DB载体上,然后利用农杆菌介导的方法转化大豆材料华夏3号。通过PCR技术及测序分析对T1代转基因大豆植株靶点编辑情况进行检测,获得纯合GmFAD2-1A大豆突变体。GmFAD2-1A突变大豆植株在株高、主茎节数、单枝分枝数、叶形、花色、种皮色、种脐色、生育期等方面与对照大豆植株没有显著差异;而GmFAD2-1A突变体大豆种子油酸含量显著高于对照大豆品种华夏3号,说明GmFAD2-1A是油酸代谢过程中的关键基因。本研究利用CRISPR/Cas9技术成功对控制大豆油酸基因GmFAD2-1A进行编辑,获得稳定的纯合GmFAD2-1A大豆突变体材料,为高油酸育种提供了新的种质资源并创建了方法。
Oleic acid content is one of the essential indicators to evaluate quality of oil in soybean. Three sites of 20 nt guide RNA(gRNA) targeted to the exon of Gm FAD2-1 A were designed and transcribed from the AtU3 d, AtU3 b, and AtU6-1 promoters, respectively. The three target sites of gRNA were ligated to the vector pYLCRISPR/Cas9-DB, and then the recombinant plasmid was transformed into a soybean cultivar Huaxia 3 by Agrobacterium-mediated transformation. The sequences near the editing site were analyzed by the PCR method and sequencing from T1 transgenic soybean plants, homozygous Gm FAD2-1 A mutants were obtained using CRISPR/Cas9 technology. The agronomic traits such as plant height, main stem number, branching number per plant, leaf shape, flower color, seed coat color, hilum color and growth period were no significant difference between the transgenic soybeans and non-transformed controls. However, the content of oleic acid in the transgenic soybean seed was significantly higher than that of the control cultivar Huaxia 3, indicating that Gm FAD2-1 A was a key gene during synthesis of oleic acid. We succeeded in editing the Gm FAD2-1 A by CRISPR/Cas9 technology in soybean and obtained homozygous mutant materials, which provides new germplasm resources and method for the breeding of high oleic acid.
Oleic acid content is one of the essential indicators to evaluate quality of oil in soybean. Three sites of 20 nt guide RNA(gRNA) targeted to the exon of Gm FAD2-1 A were designed and transcribed from the AtU3 d, AtU3 b, and AtU6-1 promoters, respectively. The three target sites of gRNA were ligated to the vector pYLCRISPR/Cas9-DB, and then the recombinant plasmid was transformed into a soybean cultivar Huaxia 3 by Agrobacterium-mediated transformation. The sequences near the editing site were analyzed by the PCR method and sequencing from T1 transgenic soybean plants, homozygous Gm FAD2-1 A mutants were obtained using CRISPR/Cas9 technology. The agronomic traits such as plant height, main stem number, branching number per plant, leaf shape, flower color, seed coat color, hilum color and growth period were no significant difference between the transgenic soybeans and non-transformed controls. However, the content of oleic acid in the transgenic soybean seed was significantly higher than that of the control cultivar Huaxia 3, indicating that Gm FAD2-1 A was a key gene during synthesis of oleic acid. We succeeded in editing the Gm FAD2-1 A by CRISPR/Cas9 technology in soybean and obtained homozygous mutant materials, which provides new germplasm resources and method for the breeding of high oleic acid.
引文
[1]Thelen J J,Ohlrogge J B.Metabolic engineering of fatty acid biosynthesis in plants.Metaba Eng,2002,4:12-21.
[2]任波,李毅.大豆种子脂肪酸合成代谢的研究进展.分子植物育种,2005,3:301-306.Ren B,Li Y.Research advances on fatty acid biogynthesis metabolism in soybean seed.Mol Plant Breed,2005,3:301-306(in Chinese with English abstract).
[3]Clemente T E,Cahoon E.Soybean oil:genetic approaches for modification of functionality and total content.Plant Physiol,2009,151:1030-1040.
[4]邹筱,韩粉霞,陈明阳,孙君明,南金平,闫淑荣,杨华.大豆脂肪酸主要组分含量QTL定位.作物学报,2014,40:1595-1603.Zou X,Han F X,Chen M Y,Sun J M,Nan J P,Yan S R,Yang H.Quantitative trait loci associated with major fatty acid components in soybean.Acta Agron Sin,2014,40:1595-1603(in Chinese with English abstract).
[5]宋晓昆,张颖君,闫龙,杨春燕,郑艳艳,蒋春志,荆慧贤,张孟臣,黄占景.大豆脂肪酸组份相关、变异特点分析.华北农学报,2010,25(增刊):68-73.Song X K,Zhang Y J,Yan L,Yang C Y,Zheng Y Y,Jiang C Z,Jing H X,Zhang M C,Huang Z J.A Study on correlation and variability of fatty acid composition contents of soybean cultivars.Acta Agric Boreali-Sin,2010,25(suppl):68-73(in Chinese with English abstract).
[6]Sleper D A,Shannon J G.Role of public and private soybean breeding programs in the development of soybean varieties using biotechnology.AgBioForum,2003,6:27-32.
[7]Sleight P.Cholesterol and coronary heart disease mortality.Aust N Z J Med,1992,22:576-579.
[8]Smith G D,Song F,Sheldon T A.Cholesterol lowering and mortality:the importance of considering initial level of risk.BMJ,1993,306:1367-1373.
[9]Ohlrogge J B,Kuhn D N,Stumpf P K.Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea.Proc Natl Acad Sci USA,1979,76:1194-1198.
[10]Liu Q,Brubaker C L,Green A G,Marshall D R,Sharp P J,Singh S P.Evolution of the FAD2-1 fatty acid desaturase 5'UTR intron and the molecular systematics of Gossypium(Malvaceae).Am JBot,2001,88:92-102.
[11]Okuley J,Lightner J,Feldmann K,Yadav N,Lark E,Browse J.Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis.Plant Cell,1994,6:147-158.
[12]Zhang D,Irma L P,Stacy J P,Mongkol N,Purnima N,Sylvia WW,Robert M P,Kent D C.Identification and expression of a new delta-12 fatty acid desaturase(FAD2-4)gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants.Plant Physiol Biochem,2009,47:462-471.
[13]Hongtrakul V,Slabaugh M,Knapp S J.A seed specificΔ12 oleate desaturase is duplicated,rearranged,and weakly expressed in high oleic acid sunflower lines.Crop Sci,1998,38:1245-1249.
[14]Li L Y,Wang X L,Gai J Y,Yu D.Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean.J Plant Physiol,2007,64:1516-1526.
[15]Heppard E P,Kinney A J,Stecca K L,Miao G H.Developmental and growth temperature regulation of two different microsomalω-6saturase genes in soybeans.Plant Physiol,1996,110:311-319.
[16]Li L Y,Wang X L,Gai J Y,Yu D Y.Isolation and characterization of a seed-specific isoform of microsomal omega-6 fatty acid desaturase gene(FAD2-1B)from soybean.DNA Seq,2008,19:28-36.
[17]Pham A T,Lee J D,Shannon J G,Bilyeu K D.Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait.BMC Plant Biol,2010,10:195,doi:10.1186/1471-2229-10-195.
[18]Pham A T,Lee J D,Shannon J G,Bilyeu K D.A novel FAD2-1Aallele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85%oleic acid content.Theor Appl Genet,2011,123:793-802.
[19]Wang G L,Xu Y N.Hypocotyl-based Agrobacterium-mediated transformation of soybean(Glycine max)and application for RNA interference.Plant Cell Rep,2008,27:1177-1184.
[20]Zhang L,Yang X D,Zhang Y Y,Yang J,Qi G X,Guo D Q,Xing G J,Yao Y,Xu W J,Li H Y,Li Q Y,Dong Y S.Changes in oleic acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing.Int J Genomics,2014,2014:921-950.
[21]杨静,邢国杰,牛陆,贺红利,杜茜,郭东全,袁英,杨向东.反义RNA介导Gm FAD2-1B基因沉默增强大豆种子中油酸的高效积累.作物学报,2017,43:1588-1595.Yang J,Xing G J,Niu L,He H L,Du Q,Guo D Q,Yuan Y,Yang X D.Antisense RNA-mediated GmFAD2-1B gene silencing enhances accumulation of oleic acid in transgenic soybean seeds.Acta Agron Sin,2017,43:1588-1595(in Chinese with English abstract).
[22]Haun W,Coffman A,Clasen B M,Demorest Z L,Lowy A,Ray E,Retterath A,Stoddard T,Juillerat A,Cedrone F,Mathis L,Voytas D F,Zhang F.Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family.Plant Biotechnol J,2014,12:934-940.
[23]Jinek M,Chylinski K,Fonfara I,Hauer M,Doudna J A,Charpentier E,A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science,2012,337:816-821.
[24]Feng Z Y,Zhang B T,Ding W N,Liu X D,Yang D L,Wei P L,Cao F Q,Zhu S H,Zhang F,Mao Y F,Zhu J K.Efficient genome editing in plants using a CRISPR/Cas system.Cell Res,2013,23:1229-1232.
[25]Liang Z,Zhang K,Chen K L,Gao C X.Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.J Genet Genomics,2014,41:63-68.
[26]Wang Y P,Cheng X,Shan Q W,Zhang Y,Liu J X,Gao C X,Qiu J L.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.Nat Biotechnol,2014,32:947-951.
[27]Shan Q W,Wang Y P,Li J,Zhang Y,Chen K L,Liang Z,Zhang K,Liu J X,Liu J X,Jeff Xi J Z,Qiu J L,Gao C X.Targeted genome modification of crop plants using a CRISPR/Cas system.Nat Biotechnol,2013,31:686-688.
[28]王加峰,郑才敏,刘维,罗文龙,王慧,陈志强,郭涛.基于CRISPR/Cas9技术的水稻千粒重基因tgw6突变体的创建.作物学报,2016,42:1160-1167.Wang J F,Zheng C M,Liu W,Luo W L,Wang H,Chen Z Q,Guo T.Construction of tgw6 mutants in rice based on CRISPR/Cas9technology.Acta Agron Sin,2016,42:1160-1167(in Chinese with English abstract).
[29]Jacobs T B,LaFayette P R,Schmitz R J,Parrott W A.Targeted genome modifications in soybean with CRISPR/Cas9.BMC Biotechnol,2015,15:16,doi:10.1186/s12896-015-0131-2.
[30]Cai Y P,Chen L,Liu X J,Chen G,Sun S,Wu C X,Jiang B J,Han T F,Hou W S.CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean.Plant Biotechnol J,2018,16:176-185.
[31]Ma X L,Zhang Q Y,Zhu Q L,Liu W,Chen Y,Qiu R,Wang B,Yang Z F,Li H Y,Lin Y R,Xie Y Y,Shen R X,Chen S F,Wang Z,Chen Y L,Guo J X,Chen L T,Zhao X C,Dong Z C,Liu Y G.Arobust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants.Mol Plant,2015,8:1274-1284.
[32]Olhoft P M,Donovan C M,Somers D A.Soybean(Glycine max)transformation using mature cotyledonary node explants.Methods Mol Biol,2006,343:385-396.
[33]Poirier Y,Ventre G,Caldelari D.Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing mediumchain-length fatty acids.Plant Physiol,1999,121:1359-1366.
[34]Chang N W,Huang P C.Effects of the ratio of polyunsaturated and monounsaturated fatty acid to saturated fatty acid on rat plasma and liver lipid concentrations.Lipids,1998,33:481-487.
[35]Williams M J,Sutherland W H,Mccormick M P,De Jong S A,Walker R J,Wilkins G T.Impaired endothelial function following a meal rich in used cooking fat.J Am Coll Cardiol,1999,33:1050-1055.
[36]Billek G.Health aspects of thermoxidized oils and fats.Eur J Lipid Sci Technol,2000,102:587-593.
[37]Paz M M,Martinez J C,Kalvig A B,Fonger T M,Wang K.Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation.Plant Cell Rep,2006,25:206-213.
[38]Cheng T Y,Saka T,Voqui-Dinh T H.Plant regeneration from soybean cotyledonary node segments in culture.Plant Sci Lett,1980,19:91-99.
[39]Olhoft P M,Flagel L E,Donovan C M,Somers D A.Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method.Planta,2003,216:723-735.
[2]任波,李毅.大豆种子脂肪酸合成代谢的研究进展.分子植物育种,2005,3:301-306.Ren B,Li Y.Research advances on fatty acid biogynthesis metabolism in soybean seed.Mol Plant Breed,2005,3:301-306(in Chinese with English abstract).
[3]Clemente T E,Cahoon E.Soybean oil:genetic approaches for modification of functionality and total content.Plant Physiol,2009,151:1030-1040.
[4]邹筱,韩粉霞,陈明阳,孙君明,南金平,闫淑荣,杨华.大豆脂肪酸主要组分含量QTL定位.作物学报,2014,40:1595-1603.Zou X,Han F X,Chen M Y,Sun J M,Nan J P,Yan S R,Yang H.Quantitative trait loci associated with major fatty acid components in soybean.Acta Agron Sin,2014,40:1595-1603(in Chinese with English abstract).
[5]宋晓昆,张颖君,闫龙,杨春燕,郑艳艳,蒋春志,荆慧贤,张孟臣,黄占景.大豆脂肪酸组份相关、变异特点分析.华北农学报,2010,25(增刊):68-73.Song X K,Zhang Y J,Yan L,Yang C Y,Zheng Y Y,Jiang C Z,Jing H X,Zhang M C,Huang Z J.A Study on correlation and variability of fatty acid composition contents of soybean cultivars.Acta Agric Boreali-Sin,2010,25(suppl):68-73(in Chinese with English abstract).
[6]Sleper D A,Shannon J G.Role of public and private soybean breeding programs in the development of soybean varieties using biotechnology.AgBioForum,2003,6:27-32.
[7]Sleight P.Cholesterol and coronary heart disease mortality.Aust N Z J Med,1992,22:576-579.
[8]Smith G D,Song F,Sheldon T A.Cholesterol lowering and mortality:the importance of considering initial level of risk.BMJ,1993,306:1367-1373.
[9]Ohlrogge J B,Kuhn D N,Stumpf P K.Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea.Proc Natl Acad Sci USA,1979,76:1194-1198.
[10]Liu Q,Brubaker C L,Green A G,Marshall D R,Sharp P J,Singh S P.Evolution of the FAD2-1 fatty acid desaturase 5'UTR intron and the molecular systematics of Gossypium(Malvaceae).Am JBot,2001,88:92-102.
[11]Okuley J,Lightner J,Feldmann K,Yadav N,Lark E,Browse J.Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis.Plant Cell,1994,6:147-158.
[12]Zhang D,Irma L P,Stacy J P,Mongkol N,Purnima N,Sylvia WW,Robert M P,Kent D C.Identification and expression of a new delta-12 fatty acid desaturase(FAD2-4)gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants.Plant Physiol Biochem,2009,47:462-471.
[13]Hongtrakul V,Slabaugh M,Knapp S J.A seed specificΔ12 oleate desaturase is duplicated,rearranged,and weakly expressed in high oleic acid sunflower lines.Crop Sci,1998,38:1245-1249.
[14]Li L Y,Wang X L,Gai J Y,Yu D.Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean.J Plant Physiol,2007,64:1516-1526.
[15]Heppard E P,Kinney A J,Stecca K L,Miao G H.Developmental and growth temperature regulation of two different microsomalω-6saturase genes in soybeans.Plant Physiol,1996,110:311-319.
[16]Li L Y,Wang X L,Gai J Y,Yu D Y.Isolation and characterization of a seed-specific isoform of microsomal omega-6 fatty acid desaturase gene(FAD2-1B)from soybean.DNA Seq,2008,19:28-36.
[17]Pham A T,Lee J D,Shannon J G,Bilyeu K D.Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait.BMC Plant Biol,2010,10:195,doi:10.1186/1471-2229-10-195.
[18]Pham A T,Lee J D,Shannon J G,Bilyeu K D.A novel FAD2-1Aallele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85%oleic acid content.Theor Appl Genet,2011,123:793-802.
[19]Wang G L,Xu Y N.Hypocotyl-based Agrobacterium-mediated transformation of soybean(Glycine max)and application for RNA interference.Plant Cell Rep,2008,27:1177-1184.
[20]Zhang L,Yang X D,Zhang Y Y,Yang J,Qi G X,Guo D Q,Xing G J,Yao Y,Xu W J,Li H Y,Li Q Y,Dong Y S.Changes in oleic acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing.Int J Genomics,2014,2014:921-950.
[21]杨静,邢国杰,牛陆,贺红利,杜茜,郭东全,袁英,杨向东.反义RNA介导Gm FAD2-1B基因沉默增强大豆种子中油酸的高效积累.作物学报,2017,43:1588-1595.Yang J,Xing G J,Niu L,He H L,Du Q,Guo D Q,Yuan Y,Yang X D.Antisense RNA-mediated GmFAD2-1B gene silencing enhances accumulation of oleic acid in transgenic soybean seeds.Acta Agron Sin,2017,43:1588-1595(in Chinese with English abstract).
[22]Haun W,Coffman A,Clasen B M,Demorest Z L,Lowy A,Ray E,Retterath A,Stoddard T,Juillerat A,Cedrone F,Mathis L,Voytas D F,Zhang F.Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family.Plant Biotechnol J,2014,12:934-940.
[23]Jinek M,Chylinski K,Fonfara I,Hauer M,Doudna J A,Charpentier E,A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science,2012,337:816-821.
[24]Feng Z Y,Zhang B T,Ding W N,Liu X D,Yang D L,Wei P L,Cao F Q,Zhu S H,Zhang F,Mao Y F,Zhu J K.Efficient genome editing in plants using a CRISPR/Cas system.Cell Res,2013,23:1229-1232.
[25]Liang Z,Zhang K,Chen K L,Gao C X.Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.J Genet Genomics,2014,41:63-68.
[26]Wang Y P,Cheng X,Shan Q W,Zhang Y,Liu J X,Gao C X,Qiu J L.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.Nat Biotechnol,2014,32:947-951.
[27]Shan Q W,Wang Y P,Li J,Zhang Y,Chen K L,Liang Z,Zhang K,Liu J X,Liu J X,Jeff Xi J Z,Qiu J L,Gao C X.Targeted genome modification of crop plants using a CRISPR/Cas system.Nat Biotechnol,2013,31:686-688.
[28]王加峰,郑才敏,刘维,罗文龙,王慧,陈志强,郭涛.基于CRISPR/Cas9技术的水稻千粒重基因tgw6突变体的创建.作物学报,2016,42:1160-1167.Wang J F,Zheng C M,Liu W,Luo W L,Wang H,Chen Z Q,Guo T.Construction of tgw6 mutants in rice based on CRISPR/Cas9technology.Acta Agron Sin,2016,42:1160-1167(in Chinese with English abstract).
[29]Jacobs T B,LaFayette P R,Schmitz R J,Parrott W A.Targeted genome modifications in soybean with CRISPR/Cas9.BMC Biotechnol,2015,15:16,doi:10.1186/s12896-015-0131-2.
[30]Cai Y P,Chen L,Liu X J,Chen G,Sun S,Wu C X,Jiang B J,Han T F,Hou W S.CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean.Plant Biotechnol J,2018,16:176-185.
[31]Ma X L,Zhang Q Y,Zhu Q L,Liu W,Chen Y,Qiu R,Wang B,Yang Z F,Li H Y,Lin Y R,Xie Y Y,Shen R X,Chen S F,Wang Z,Chen Y L,Guo J X,Chen L T,Zhao X C,Dong Z C,Liu Y G.Arobust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants.Mol Plant,2015,8:1274-1284.
[32]Olhoft P M,Donovan C M,Somers D A.Soybean(Glycine max)transformation using mature cotyledonary node explants.Methods Mol Biol,2006,343:385-396.
[33]Poirier Y,Ventre G,Caldelari D.Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing mediumchain-length fatty acids.Plant Physiol,1999,121:1359-1366.
[34]Chang N W,Huang P C.Effects of the ratio of polyunsaturated and monounsaturated fatty acid to saturated fatty acid on rat plasma and liver lipid concentrations.Lipids,1998,33:481-487.
[35]Williams M J,Sutherland W H,Mccormick M P,De Jong S A,Walker R J,Wilkins G T.Impaired endothelial function following a meal rich in used cooking fat.J Am Coll Cardiol,1999,33:1050-1055.
[36]Billek G.Health aspects of thermoxidized oils and fats.Eur J Lipid Sci Technol,2000,102:587-593.
[37]Paz M M,Martinez J C,Kalvig A B,Fonger T M,Wang K.Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation.Plant Cell Rep,2006,25:206-213.
[38]Cheng T Y,Saka T,Voqui-Dinh T H.Plant regeneration from soybean cotyledonary node segments in culture.Plant Sci Lett,1980,19:91-99.
[39]Olhoft P M,Flagel L E,Donovan C M,Somers D A.Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method.Planta,2003,216:723-735.