作物学报 ›› 2019, Vol. 45 ›› Issue (6): 839-847.doi: 10.3724/SP.J.1006.2019.84157
侯智红,吴艳,程群,董利东,芦思佳,南海洋,甘卓然,刘宝辉()
Zhi-Hong HOU,Yan WU,Qun CHENG,Li-Dong DONG,Si-Jia LU,Hai-Yang NAN,Zhuo-Ran GAN,Bao-Hui LIU()
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。
[1] |
Thelen J J, Ohlrogge J B . Metabolic engineering of fatty acid biosynthesis in plants. Metaba Eng, 2002,4:12-21.
doi: 10.1006/mben.2001.0204 pmid: 11800570 |
[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.
doi: 10.1111/j.1445-5994.1992.tb00480.x pmid: 1449442 |
[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.
doi: 10.1136/bmj.306.6893.1648 pmid: 8518602 |
[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.
doi: 10.1073/pnas.76.3.1194 pmid: 286305 |
[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 J Bot, 2001,88:92-102.
doi: 10.2307/2657130 pmid: 11159130 |
[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.
doi: 10.1105/tpc.6.1.147 pmid: 7907506 |
[12] |
Zhang D, Irma L P, Stacy J P, Mongkol N, Purnima N, Sylvia W W, 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.
doi: 10.1016/j.plaphy.2008.12.024 pmid: 19217793 |
[13] |
Hongtrakul V, Slabaugh M, Knapp S J . A seed specific Δ 12 oleate desaturase is duplicated, rearranged, and weakly expressed in high oleic acid sunflower lines . Crop Sci, 1998,38:1245-1249.
doi: 10.2135/cropsci1998.0011183X003800050022x |
[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.
doi: 10.1016/j.jplph.2006.08.007 pmid: 17141918 |
[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.
doi: 10.1104/pp.110.1.311 pmid: 8587990 |
[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.
doi: 10.1080/10425170701207208 pmid: 18300159 |
[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.
doi: 10.1186/1471-2229-10-195 pmid: 20828382 |
[18] | Pham A T, Lee J D, Shannon J G, Bilyeu K D . A novel FAD2-1A allele 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.
doi: 10.1007/s00299-008-0535-8 pmid: 18347801 |
[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.
doi: 10.1155/2014/921950 pmid: 4147191 |
[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GmFAD2-1B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.
doi: 10.1111/pbi.12201 pmid: 24851712 |
[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.
doi: 10.1038/cr.2013.114 pmid: 23958582 |
[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.
doi: 10.1016/j.jgg.2013.12.001 pmid: 24576457 |
[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.
doi: 10.1038/nbt.2969 pmid: 25038773 |
[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.
doi: 10.1038/nbt.2650 pmid: 23929338 |
[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/Cas9 technology. 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.
doi: 10.1186/s12896-015-0131-2 pmid: 4365529 |
[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.
doi: 10.1111/pbi.12758 pmid: 28509421 |
[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 . A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant, 2015,8:1274-1284.
doi: 10.1016/j.molp.2015.04.007 pmid: 25917172 |
[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.
doi: 10.1385/1-59745-130-4:385 pmid: 16988361 |
[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 medium- chain-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.
doi: 10.1007/s11745-998-0231-9 pmid: 9625595 |
[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.
doi: 10.1016/S0735-1097(98)00681-0 pmid: 10091835 |
[36] |
Billek G . Health aspects of thermoxidized oils and fats. Eur J Lipid Sci Technol, 2000,102:587-593.
doi: 10.1002/1438-9312(200009)102:8/9<587::aid-ejlt587>3.0.co;2-# |
[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.
doi: 10.1016/0304-4211(80)90084-X |
[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.
doi: 10.1007/s00425-002-0922-2 pmid: 12624759 |
[1] | 陈玲玲, 李战, 刘亭萱, 谷勇哲, 宋健, 王俊, 邱丽娟. 基于783份大豆种质资源的叶柄夹角全基因组关联分析[J]. 作物学报, 2022, 48(6): 1333-1345. |
[2] | 杨欢, 周颖, 陈平, 杜青, 郑本川, 蒲甜, 温晶, 杨文钰, 雍太文. 玉米-豆科作物带状间套作对养分吸收利用及产量优势的影响[J]. 作物学报, 2022, 48(6): 1476-1487. |
[3] | 王炫栋, 杨孙玉悦, 高润杰, 余俊杰, 郑丹沛, 倪峰, 蒋冬花. 拮抗大豆斑疹病菌放线菌菌株的筛选和促生作用及防效研究[J]. 作物学报, 2022, 48(6): 1546-1557. |
[4] | 于春淼, 张勇, 王好让, 杨兴勇, 董全中, 薛红, 张明明, 李微微, 王磊, 胡凯凤, 谷勇哲, 邱丽娟. 栽培大豆×半野生大豆高密度遗传图谱构建及株高QTL定位[J]. 作物学报, 2022, 48(5): 1091-1102. |
[5] | 李阿立, 冯雅楠, 李萍, 张东升, 宗毓铮, 林文, 郝兴宇. 大豆叶片响应CO2浓度升高、干旱及其交互作用的转录组分析[J]. 作物学报, 2022, 48(5): 1103-1118. |
[6] | 彭西红, 陈平, 杜青, 杨雪丽, 任俊波, 郑本川, 罗凯, 谢琛, 雷鹿, 雍太文, 杨文钰. 减量施氮对带状套作大豆土壤通气环境及结瘤固氮的影响[J]. 作物学报, 2022, 48(5): 1199-1209. |
[7] | 王好让, 张勇, 于春淼, 董全中, 李微微, 胡凯凤, 张明明, 薛红, 杨梦平, 宋继玲, 王磊, 杨兴勇, 邱丽娟. 大豆突变体ygl2黄绿叶基因的精细定位[J]. 作物学报, 2022, 48(4): 791-800. |
[8] | 李瑞东, 尹阳阳, 宋雯雯, 武婷婷, 孙石, 韩天富, 徐彩龙, 吴存祥, 胡水秀. 增密对不同分枝类型大豆品种同化物积累和产量的影响[J]. 作物学报, 2022, 48(4): 942-951. |
[9] | 杜浩, 程玉汉, 李泰, 侯智红, 黎永力, 南海洋, 董利东, 刘宝辉, 程群. 利用Ln位点进行分子设计提高大豆单荚粒数[J]. 作物学报, 2022, 48(3): 565-571. |
[10] | 周悦, 赵志华, 张宏宁, 孔佑宾. 大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析[J]. 作物学报, 2022, 48(3): 590-596. |
[11] | 王娟, 张彦威, 焦铸锦, 刘盼盼, 常玮. 利用PyBSASeq算法挖掘大豆百粒重相关位点与候选基因[J]. 作物学报, 2022, 48(3): 635-643. |
[12] | 董衍坤, 黄定全, 高震, 陈栩. 大豆PIN-Like (PILS)基因家族的鉴定、表达分析及在根瘤共生固氮过程中的功能[J]. 作物学报, 2022, 48(2): 353-366. |
[13] | 张艳波, 王袁, 冯甘雨, 段慧蓉, 刘海英. 棉籽油分和3种主要脂肪酸含量QTL分析[J]. 作物学报, 2022, 48(2): 380-395. |
[14] | 张国伟, 李凯, 李思嘉, 王晓婧, 杨长琴, 刘瑞显. 减库对大豆叶片碳代谢的影响[J]. 作物学报, 2022, 48(2): 529-537. |
[15] | 石磊, 苗利娟, 黄冰艳, 高伟, 张忠信, 齐飞艳, 刘娟, 董文召, 张新友. 花生AhFAD2-1基因启动子及5'-UTR内含子功能验证及其低温胁迫应答[J]. 作物学报, 2021, 47(9): 1703-1711. |
|