欢迎访问作物学报,今天是

作物学报 ›› 2014, Vol. 40 ›› Issue (01): 86-92.doi: 10.3724/SP.J.1006.2014.00086

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

超长链多不饱和脂肪酸在棉花中的异源合成

刘江1**,马燕斌2**,孙全喜1,3,吴霞2,李雪滢1,孙美红1,李燕娥2,李新征1*,亓宝秀1*   

  1. 1作物生物学国家重点实验室 / 山东农业大学生命科学学院, 山东泰安271018; 2山西省农业科学院棉花研究所, 山西运城044000; 3山东省花生研究所, 山东青岛266100
  • 收稿日期:2013-05-18 修回日期:2013-09-16 出版日期:2014-01-12 网络出版日期:2013-10-23
  • 通讯作者: 亓宝秀, E-mail: qbx126@sdau.edu.cn, Tel: 0538-8246205; 李新征, E-mail: lxz@sdau.edu.cn, Tel: 0538-8246205
  • 基金资助:

    本研究由国家转基因生物新品种培育科技重大专项(2009ZX08005-024B)和国家自然科学基金项目(30970222)和山东省现代农业产业技术体系建设专项资金资助。

Production of Very Long Chain Polyunsaturated Fatty Acids in Cotton

LIU Jiang1,**,MA Yan-Bin2,**,SUN Quan-Xi1,3,WU Xia2,LI Xue-Ying1,SUN Mei-Hong1,LI Yan-E2,LI Xin-Zheng1,*,QI Bao-Xiu1,*   

  1. 1 State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian 271018; 2 Coton Research Institute, Shanxi Academy of Agricultural Sciences, Yuncheng 044000, China; 3 Shandong Peanut Research Institute, Qingdao 266100, China
  • Received:2013-05-18 Revised:2013-09-16 Published:2014-01-12 Published online:2013-10-23
  • Contact: 亓宝秀, E-mail: qbx126@sdau.edu.cn, Tel: 0538-8246205; 李新征, E-mail: lxz@sdau.edu.cn, Tel: 0538-8246205

摘要:

从球等鞭金藻、眼虫、高山被孢霉和拟南芥中分别克隆到Δ9链延长酶、Δ8去饱和酶Δ5去饱和酶和Δ15去饱和酶基因, 利用我们的多基因聚合方法, 将这4个基因聚合到植物表达载体pCambia2300, 其中每个基因都含有独立的CaMV35S启动子和Tnos终止子。利用农杆菌介导法将该表达载体转入棉花, 通过卡纳霉素和PCR筛选获得转基因阳性植株, 提取转基因阳性植株叶片总脂肪酸, 用气相色谱分析法检测到花生四烯酸(AA, 20:4Δ5,8,11,14)和二十碳五烯酸(EPA, 20:5Δ5,8,11,14,17), 含量分别达1.0%5.0%。表明通过基因代谢工程在棉花中异源合成EPA是可行的, 为进一步在棉籽中生产VLCPUFAs奠定了基础。

关键词: 去饱和酶, 链延长酶, 转基因, EPA, 棉花

Abstract:

We have isolated four genes encoding a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase, and a Δ15 desaturase from Isochrysis galbana, Euglena gracilis, Mortierella alpina and Arabidopsis thaliana respectively. Using a multigene transfer technology that we developed, these genes were stacked together in the plant expression vector pCambia2300. Each gene contained its own CaMV35S promoter and Tnos terminator.This plant expression vector was then transferred into cotton by Agrobacterium-mediated transformation method. Transgenic cotton seedlings were first identified by screening them based on kanamycin-containing media and followed by PCR with gene-specific primers of the four transgenes. Finally, these transgenic plants were subjected to gas liquid chromatography analysis for their fatty acid composition and the results showed that the contents of arachidonic acid (ARA, 20:4Δ5,8,11,14) and eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) were 1.0% and 5.0% respectively in the leaves of the transgenic plants, indicating that the four genes were expressed incotton. Therefore, our data clearly demonstrated the feasibility for the heterologous production of EPA in cotton and this will lay a foundation for the production of VLCPUFAs, including EPA and DHA in cotton seed through transgenic technology in the future.

Key words: Desaturase, Elongase, Transgenes, Eicosapentaenoic acid, Cotton

[1]Abbadi A, Domergue F, Bauer J, Napier J A, Welti R, Zahringer U, Cirpus P, Heinz E. Biosynthesis of very long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation. Plant Cell, 2004, 16: 2734–2748



[2]Funk C D. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science, 2001, 294: 1871–1875



[3]Simopoulos A P. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr, 2002, 21: 495–505



[4]Uauy R, Hoffman D R, Peirano P, Birch D G, Birch E E. Essential fatty acids in visual and brain development. Lipids, 2001, 36: 885–895



[5]Qi B X, Fraser T, Mugford S, Dobson G, Sayanova O, Butler J, Napier J A, Lazarus C M. Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants. Nat Biotechnol, 2004, 22: 739–745



[6]Lu C F, Napier J A, Clemente T E, Cahoon E B. New frontiers in oilseed biotechnology: meeting the global demand for vegetable oils for food, feed, biofuel, and industrial applications. Curr Opin Plant Biol, 2011, 22: 252–259 



[7]Venegas-Calerón M, Sayanova O, Napier J A. An alternative to fish oils: metabolic engineering of oil-seed crops to produce omega-3 long chain polyunsaturated fatty acids. Prog Lipid Res, 2010, 49: 108–119



[8]Wu G, Truksa M, Datla N, Vrinten P, Bauer J, Zank T, Cirpus P, Heinz E, Qiu X. Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants. Nat biotechnol, 2005, 23: 1013–1017



[9]Hoffmann M, Wagner M, Abbadi A, Fulda M, Feussner I. Metabolic engineering of ω3-very long chain polyunsaturated fatty acid production by an exclusively acyl-CoA-dependent pathway. J Biol Chem, 2008, 283: 22352–22362



[10]杨艳, 王贤磊, 李冠. 六种新疆陆地棉棉籽脂肪酸成分分析. 生物技术, 2009, 19(4): 54–56



Yang Y, Wang X L, Li G. Analysison fatty acid composition of six species of upland cotton. Biotechnology, 2009, 19(4): 54–56 (in Chinese with English abstract)



[11]Qi B X, Beaudoin F, Fraser T, Stobart A K, Napier J A, Lazarus C M. Identification of a cDNA encoding a novel C18-Delta(9) polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana. FEBS Lett, 2002, 510: 159–165



[12]Wallis J G, Browse J. The Delta8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids. Arch Biochem Biophys, 1999, 365: 307–316



[13]Michaelson L V, Lazarus C M, Griffiths G, Napier J A, Stobart A K. Isolation of a Delta5-fatty acid desaturase gene from Mortierella alpina. J Biol Chem, 1998, 273: 19055–19059



[14]Yadav N S, Wierzbicki A, Aegerter M, Caster C S, Perez-Grau L, Kinney A J, Hitz W D, Booth J R, Schweiger B, Stecca K L. Cloning of higher plant omega-3 fatty acid desaturases. Plant Physiol, 1993, 103: 467–476



[15]Sun Q X, Liu J, Li Y X, Zhang Q, Shan S H, Li X Z, Qi B X. Creation and validation of a widely applicable multiple gene transfer vector system for stable transformation in plant. Plant Mol Biol, 2013, DOI:10.1007/s11103-013-0096-2



[16]秦永华, 乔志新, 刘进元. 转基因技术在棉花育种上的应用. 棉花学报, 2007, 19: 482–488



Qin Y H, Qiao Z X, Liu J Y. Application of genetic transformation in cotton breeding. Cotton sci, 2007, 19: 482–488 (in Chinese with English abstract)



[17]Halpin C. Gene stacking in transgenic plants--the challenge for 21st century plant biotechnology. Plant Biotechnol, 2005, 3: 141–155



[18]Sun Q, Liu J, Zhang Q, Qing X, Dobson G, Li X Z, Qi B X. Characterization of three novel desaturases involved in the delta-6 desaturation pathways for polyunsaturated fatty acid biosynthesis from Phytophthora infestans. Appl Microbiol Biotechnol, 2012, DOI: 10.1007/s00253-012-4613-z



[19]Cheng B F, Wu G H, Vrinten P, Falk K, Bauer J, Qiu X. Towards the production of high levels of eicosapentaenoic acid in transgenic in plants: the effects of different host species, genes and promoters. Transgenic Res, 2010, 19: 221–229



[20]Lu C F, Napier J A, Clemente T E, Cahoon E B. New frontiers in oilseed biotechnology: meeting the global demand for vegetable oils for food, feed, biofuel, and industrial applications. Curr Opin Plant Biol, 2011 22: 252–259 



[21]Domergue F, Abbadi A, Heinz E. Relief for fish stocks: oceanic fatty acids in transgenic oilseeds. Trends Plant Sci, 2005, 10: 112–116

[1] 周静远, 孔祥强, 张艳军, 李雪源, 张冬梅, 董合忠. 基于种子萌发出苗过程中弯钩建成和下胚轴生长的棉花出苗壮苗机制与技术[J]. 作物学报, 2022, 48(5): 1051-1058.
[2] 单露英, 李俊, 李亮, 张丽, 王颢潜, 高佳琪, 吴刚, 武玉花, 张秀杰. 转基因玉米NK603基体标准物质研制[J]. 作物学报, 2022, 48(5): 1059-1070.
[3] 孙思敏, 韩贝, 陈林, 孙伟男, 张献龙, 杨细燕. 棉花苗期根系分型及根系性状的关联分析[J]. 作物学报, 2022, 48(5): 1081-1090.
[4] 闫晓宇, 郭文君, 秦都林, 王双磊, 聂军军, 赵娜, 祁杰, 宋宪亮, 毛丽丽, 孙学振. 滨海盐碱地棉花秸秆还田和深松对棉花干物质积累、养分吸收及产量的影响[J]. 作物学报, 2022, 48(5): 1235-1247.
[5] 郑曙峰, 刘小玲, 王维, 徐道青, 阚画春, 陈敏, 李淑英. 论两熟制棉花绿色化轻简化机械化栽培[J]. 作物学报, 2022, 48(3): 541-552.
[6] 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.
[7] 张特, 王蜜蜂, 赵强. 滴施缩节胺与氮肥对棉花生长发育及产量的影响[J]. 作物学报, 2022, 48(2): 396-409.
[8] 王渭霞, 赖凤香, 胡海燕, 何佳春, 魏琪, 万品俊, 傅强. 超低温11年保存期对转基因作物基体标准样品核酸检测的影响[J]. 作物学报, 2022, 48(1): 238-248.
[9] 赵文青, 徐文正, 杨锍琰, 刘玉, 周治国, 王友华. 棉花叶片响应高温的差异与夜间淀粉降解密切相关[J]. 作物学报, 2021, 47(9): 1680-1689.
[10] 岳丹丹, 韩贝, Abid Ullah, 张献龙, 杨细燕. 干旱条件下棉花根际真菌多样性分析[J]. 作物学报, 2021, 47(9): 1806-1815.
[11] 曾紫君, 曾钰, 闫磊, 程锦, 姜存仓. 低硼及高硼胁迫对棉花幼苗生长与脯氨酸代谢的影响[J]. 作物学报, 2021, 47(8): 1616-1623.
[12] 李杰华, 端群, 史明涛, 吴潞梅, 柳寒, 林拥军, 吴高兵, 范楚川, 周永明. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定[J]. 作物学报, 2021, 47(5): 789-798.
[13] 马欢欢, 方启迪, 丁元昊, 池华斌, 张献龙, 闵玲. 棉花GhMADS7基因正调控棉花花瓣发育[J]. 作物学报, 2021, 47(5): 814-826.
[14] 许乃银, 赵素琴, 张芳, 付小琼, 杨晓妮, 乔银桃, 孙世贤. 基于GYT双标图对西北内陆棉区国审棉花品种的分类评价[J]. 作物学报, 2021, 47(4): 660-671.
[15] 周冠彤, 雷建峰, 代培红, 刘超, 李月, 刘晓东. 棉花CRISPR/Cas9基因编辑有效sgRNA高效筛选体系的研究[J]. 作物学报, 2021, 47(3): 427-437.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!