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作物学报 ›› 2020, Vol. 46 ›› Issue (8): 1283-1290.doi: 10.3724/SP.J.1006.2020.94192

• 研究简报 • 上一篇    下一篇

紫苏二酰基甘油酰基转移酶2基因克隆与功能研究

鲁庚,唐鑫,陆俊杏,李丹,胡秋芸,胡田,张涛()   

  1. 重庆师范大学生命科学学院, 重庆 401331
  • 收稿日期:2019-12-11 接受日期:2020-03-24 出版日期:2020-08-12 网络出版日期:2020-04-26
  • 通讯作者: 张涛
  • 作者简介:E-mail: 778448973@qq.com
  • 基金资助:
    国家自然科学基金项目(31171588);重庆市技术创新与应用发展项目(cstc2019jscx-msxm0612)

Cloning and function analysis of a type 2 diacylglycerol acyltransferase (DGAT2) from Perilla frutescens

LU Geng,TANG Xin,LU Jun-Xing,LI Dan,HU Qiu-Yun,HU Tian,ZHANG Tao()   

  1. College of Life Sciences, Chongqing Normal University, Chongqing 401331, China
  • Received:2019-12-11 Accepted:2020-03-24 Published:2020-08-12 Published online:2020-04-26
  • Contact: Tao ZHANG
  • Supported by:
    National Natural Science Foundation of China(31171588);Chongqing Technology Innovation and Application Development Project(cstc2019jscx-msxm0612)

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摘要：

二酰基甘油酰基转移酶(diacylglycerol acyltransferase, DGAT)是植物合成三酰甘油(TAG)最后一步的关键酶, 其中DGAT2在某些植物的种子油中能选择性积累更多不饱和脂肪酸。本文成功克隆了紫苏二酰基甘油酰基转移酶2基因(PfDGAT2), 并进行生物信息学分析。PfDGAT2实时荧光定量结果表明, 不同器官中PfDGAT2基因均有表达, 10 d种子的表达量最高, 在根中的表达量次之,在种子发育中后期, PfDGAT2表达量逐渐降低。与野生型拟南芥相比, 过表达PfDGAT2拟南芥种子含油率提高了21.68%~77.89%, 其中种子含油率增加最多的4个株系, 其亚麻酸(C18:3)增加4.57%, 花生一烯酸(C20:1)增加7.44%, 花生二烯酸(C20:2)增加5.4%, 二十二一烯酸(C22:1)增加10.37%, 而棕榈酸(C16:0)、硬脂酸(C18:0)和亚油酸(C18:2)含量分别降低了3.47%、6.64%和4.83%, 油酸(C18:1)和花生酸(C20:0)分别只降低了0.18%和1.91%。本研究结果表明, 紫苏PfDGAT2基因不仅能提高种子含油率, 还能促进亚麻酸、花生一烯酸等不饱和脂肪酸的积累, 这为研究植物不饱和脂肪酸的合成积累提供了参考及理论依据。

关键词: 二酰基甘油酰基转移酶(DGAT2), 种子含油率, 不饱和脂肪酸, 紫苏

Abstract:

Diacylglycerol acyltransferase (DGAT) is a key enzyme in the final step of triacylglycerol (TAG) synthesis in plant. In seed oil of certain plants, DGAT2 can selectively accumulate more unsaturated fatty acids. In this paper, we successful cloned PfDGAT2 from Perilla frutescens and performed bioinformatics analysis. Real-time fluorescence quantitative analysis showed that PfDGAT2 was expressed in different organs, with the highest in seeds at 10 d after anthesis, the medium in roots, and gradual decrement in the middle and late stages of seed. Compared with the wild-type, the oil content of seeds in PfDGAT2 transgenic Arabidopsis was increased by 21.68%-77.89%. The fatty acid components of the four strains with the largest increase in seed oil content were analyzed. Compared with the control, linolenic acid (C18:3), arachidonic acid (C20:1), arachidonic acid (C20:2), docosaenoic acid (C22:1) increased significantly by 4.57%, 7.44%, 5.40%, and 0.37%, respectively. Palmitic acid (C16:0), stearic acid (C18:0), and linoleic acid (C18:2) were obviously reduced by 3.47%, 6.64%, and 4.83%, respectively. Oleic acid (C18:1) only decreased by 0.18% and arachidic acid (C20:0) by 1.91%. In conclusion that PfDGAT2 gene can not only increase the oil content, but promote the accumulation of unsaturated fatty acids such as linolenic acid and arachidonic acid, which provides a reference and theoretical basis for studying the synthesis and accumulation of unsaturated fatty acid in plants.

Key words: diacylglycerol acyltransferase, seed oil content, unsaturated fatty acid, Perilla frutescens

表1

PCR引物"

引物名称
Primer name		 引物序列
Sequences (5'-3')
PfDGAT2 F CTCGCTTACTGCTACTTCAATG
PfDGAT2 R CGACAATTAGAGAATCCTGAGC
PfDGAT2-DL F AGTCCGAGCCCAACGGCGATGTCAG
PfDGAT2-DL R GGATGCCCAACCCTTGCTTTGTGCC
Actin F AGACCTTCAATGTGCCAGCCA
Actin R CACGACCAGCAAGATCCAACC
18S RNAF CGGCGACGCATCATTCAAA
DGAT2-BglII F ACTCTTGACCATGGTAGATCTGGAGTCCGAGCCCAACGGCG
DGAT2-BglII R GGACGTAAACTAGTCAGATCTAGAATCCTGAGCTCTAAGTCG
JD-F TTTCATTTGGAGAGAACACGGGGGA
JD-R CGCTGATCAATTCCACAGTTTTCGC

图1

PfDGAT2基因的克隆与分析 A: PfDGAT2的PCR扩增产物, M: DL2000 DNA marker。B: PfDGAT2 保守结构域的预测结果。"

图2

不同植物DGAT2氨基酸序列的系统进化树 分支上的数字表示Bootstrap验证中基于1000次重复该节点的可信度。"

图3

不同植物DGAT2蛋白序列比对分析 PfDGAT2: 紫苏; SiDGAT2: 芝麻(XP_011098009.1); StDGAT2: 马铃薯(XP_006365015.1); AtDGAT2: 拟南芥(OAP06431.1); GmDGAT2: 大豆(NP_001299586.1); NtDGAT2: 烟草(AGL46984.1); CsDGAT2: 亚麻荠 Camelina sativa(XP_010426724)。黑色方框内依次为DGAT2蛋白的YFP、EPHS、GGVQE、RXGFX(K/R)XAXXXGXX(L/V)VPXXXFG(E/Q)和VVGRPI的保守结构域。"

图4

PfDGAT2基因不同组织和种子不同时期的相对表达量 柱值标以不同字母表示在P < 0.05水平差异显著性。数据点为平均值±标准误(n = 3)。"

图5

转PfDGAT2拟南芥种子含油率 柱值标以不同字母表示在P < 0.05水平差异显著性。数据点为平均值±标准误(n = 3)。"

图6

转 PfDGAT2 拟南芥种子脂肪酸相对含量分析 图中*为PD2-1、PD2-3、PD2-5、PD2-5与野生型Col-0之间各组分显著性分析。*代表在P < 0.05时显著性的差异, **代表在P < 0.01时显著性的差异, ***代表在P < 0.001时显著性的差异。数据点为平均值±标准误(n = 3)。"

[1] Dyer J M, Stymne S, Green A G, Carlsson A S. High-value oils from plants. Plant J, 2008,54:640-655.
doi: 10.1111/j.1365-313X.2008.03430.x pmid: 18476869
[2] Xu C, Shanklin J. Triacylglycerol metabolism, function, and accumulation in plant vegetative tissues. Annu Rev Plant Biol, 2016,67:179-206.
doi: 10.1146/annurev-arplant-043015-111641 pmid: 26845499
[3] 陶国琴, 李晨. α-亚麻酸的保健功效及应用. 食品科学, 2004,21(12):140-143.
Tao G Q, Li C. Health effects and application of α-linolenic acid. Food Sci, 2004,21(12):140-143 (in Chinese).
[4] 彭小平, 熊劲松. 我国紫苏产业化研究现状与展望. 安徽农业科学, 2010,38(16):439-441.
Peng X P, Xiong J S. Research present situation in industrialization and development prospect of Perilla frutescens (Linn.) Britt in China. J Anhui Agric Sci, 2010,38(16):439-441(in Chinese with English abstract).
[5] Yoon S H, Noh S. Positional distribution of fatty acids in Perilla ( Perilla frutescens L.) oil. J Am Oil Chem Soc, 2011,88:157-158.
doi: 10.1007/s11746-010-1646-2
[6] Liu Q, Guo Q G, Akbar S, Zhi Y, Anna E T, Madeline M, Li Z Y, Pushkar S, Thomas V, Jean P R, Liang G L, Wang M B, Rosemary W, Philip L, Surinder S, James P. Genetic enhancement of oil content in potato tuber ( Solanum tuberosum L.) through an integrated metabolic engineering strategy. Plant Biotechnol J, 2017,15:56-67.
doi: 10.1111/pbi.12590 pmid: 27307093
[7] Weselake R, Taylor D C, Rahman M H, Shah S, André L, Mcvetty P B E, Harwood J L. Increasing the flow of carbon into seed oil. Biotechnol Adv, 2009,27:866-878.
doi: 10.1016/j.biotechadv.2009.07.001
[8] Zou J, Wei Y, Jako C, Kumar A, Taylor D C. The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J, 1999,19:645-653.
doi: 10.1046/j.1365-313x.1999.00555.x pmid: 10571850
[9] Saha S, Enugutti B, Rajasekharan R R. Cytosolic triacylglycerol biosynthetic pathway in oil seeds. Molecular cloning and expression of Peanut cytosolic diacylglycerol acyltransferase. Plant Physiol, 2006,141:1533-1543.
doi: 10.1104/pp.106.082198 pmid: 16798944
[10] Lehner R, Kuksis A. Biosynthesis of triacylglycerols. Prog Lipid Res, 1996,35:169-201.
doi: 10.1016/0163-7827(96)00005-7 pmid: 8944226
[11] Cases S, Smith S J, Zheng Y W, Myers H M, Lear S R, Sande E, Novak S, Colins C, Welch C B, Lusis A J, Erickson S K, Farese R V. Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proc Natl Acad Sci USA, 1998,95:13018-13023.
doi: 10.1073/pnas.95.22.13018 pmid: 9789033
[12] Liu Q, Siloto R M P, Lehner R, Stone S J, Weselake R J. acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology. Prog Lipid Res, 2012,51:350-377.
doi: 10.1016/j.plipres.2012.06.001 pmid: 22705711
[13] Shockey J M. Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum. Plant Cell, 2006,18:2294-2313.
doi: 10.1105/tpc.106.043695 pmid: 16920778
[14] Li-Beisson Y, Shorrosh B, Beisson F, Andersson M X, Ohlrogge J. Acyl-lipid metabolism. Arabidopsis Book, 2013,11:e0161.
pmid: 23505340
[15] Kalscheuer R, Alexander S. A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in acinetobacter calcoaceticus, ADP1*. J Biol Chem, 2003,278:8075-8082.
doi: 10.1074/jbc.M210533200 pmid: 12502715
[16] Li F, Wu X, Lam P, David B, Zheng H Q, Samuels L, Jetter R, Kunst L. Identification of the wax ester synthase/acyl-coenzyme A:aiacylglycerol acyltransferase WSD1 required for stem wax ester biosynthesis in Arabidopsis. Plant Physiol, 2008,148:97-107.
pmid: 18621978
[17] King A, Nam J W, Han J, Jaworski H J G. Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters. Planta, 2007,226:381-394.
pmid: 17323080
[18] Katavic V, Reed D W, Taylor D C, Giblin E M, Barton D L, Zou J T, MacKenzie S L, Covello P S, Kunst L. Alteration of seed fatty acid composition by an ethyl methanesulfonate-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity. Plant Physiol, 1995,108:399-409.
doi: 10.1104/pp.108.1.399 pmid: 7784510
[19] Jako C, Kumar A, Wei Y, Zou J T, Barton D L, Giblin E M, Covello P S. Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiol, 2001,126:861-874.
doi: 10.1104/pp.126.2.861 pmid: 11402213
[20] Chapman K D, Ohlrogge J B. Compartmentation of triacylglycerol accumulation in plants. J Biol Chem, 2012,287:2288-2294.
doi: 10.1074/jbc.R111.290072 pmid: 22090025
[21] Burgal J, Shockey J, Lu C, Dyer J, Larson T, Graham I, Browse J. Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil. Plant Biotechnol J, 2008,6:819-831.
doi: 10.1111/j.1467-7652.2008.00361.x pmid: 18643899
[22] Oelkers P. Alecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast. J Biol Chem, 2000,275:15609-15612.
doi: 10.1074/jbc.C000144200 pmid: 10747858
[23] Li R, Yu K, Hildebrand D F. DGAT1, DGAT2 and PDAT expression in seeds and other tissues of epoxy and hydroxy fatty acid accumulating plants. Lipids, 2010,45:145-157.
doi: 10.1007/s11745-010-3385-4 pmid: 20101470
[24] Jin Y H, Yuan Y J, Gao L C, Sun R H, Chen L Z, Li D D, Zheng Y S. Characterization and functional analysis of a type 2 diacylglycerol acyltransferase (DGAT2) gene from oil palm(Elaeis guineensis Jacq.) mesocarp in saccharomyces cerevisiae and transgenic Arabidopsis thaliana. Front Plant Sci, 2017,8:1791.
doi: 10.3389/fpls.2017.01791 pmid: 29089956
[25] Zheng Y, Jin Y, Yuan Y, Feng D, Chen L, Li D, Zhou P. Identification and function analysis of a type 2 diacylglycerol acyltransferase (DGAT2) from the endosperm of coconut ( Cocos nucifera L.). Gene, 2019,702:75-82.
doi: 10.1016/j.gene.2019.03.060 pmid: 30928362
[26] Ezaki O, Takahashi M, Shigematsu T, Shimamura K, Kimura J, Ezaki H, Gotoh T. Long-term effects of dietary. ALPHA-linolenic acid from Perilla oil on serum fatty acids composition and on the risk factors of coronary heart disease in Japanese elderly subjects. J Nutr Sci Vitaminol, 1999,45:759-772.
doi: 10.3177/jnsv.45.759 pmid: 10737229
[27] Renaud S, Lanzmann-Petithory D. Dietary fats and coronary heart disease pathogenesis. Curr Atheroscler Rep, 2002,4:419-424.
doi: 10.1007/s11883-002-0045-z pmid: 12361488
[28] 梁倩, 李璐, 周雅莉, 安茜, 王计平. 紫苏PfDGAT2基因生物信息学及表达特性分析. 华北农学报, 2017,32(5):90-94.
Liang Q, Li L, Zhou Y L, An Q, Wang J P. Bioinformatics and expression analysis of PfDGAT2 gene in Perilla frutescens. Acta Agric Boreali-Sin, 2017,32(5):90-94 (in Chinese with English abstract).
[29] Liao B N, Hao Y J, Lu J X, Bai H Y, Guan L, Zhang T. Transcriptomic analysis of Perilla frutescens seed to insight into the biosynthesis and metabolic of unsaturated fatty acids. BMC Genomics, 2018,19:213.
doi: 10.1186/s12864-018-4595-z pmid: 29562889
[30] Li S S, Yuan R Y, Chen L G, Wang L S, Hao X H, Wang L J, Zheng X C, Du H. Systematic qualitative and quantitative assessment of fatty acids in the seeds of 60 tree peony ( Paeonia section Moutan DC.) cultivars by GC-MS. Food Chem, 2015,173:133-140.
doi: 10.1016/j.foodchem.2014.10.017 pmid: 25466004
[31] 付松, 徐先顺, 向奋飞. 保健油脂中多不饱和脂肪酸的GC/MS分析. 中国卫生检验杂志, 2005,15:1042-1044.
Fu S, Xu X S, Xiang F F. Analysis of the multi-unsaturated fatty acids in healthy oils by GC/MS. Chin J Heaith Labor Technol, 2005,15:1042-1044 (in Chinese with English abstract).
[32] Stone S J, Levin M C, Farese R V. Membrane topology and identification of key Functional amino acid residues of murine acyl-CoA:diacylglycerol acyltransferase-2. J Biol Chem, 2006,281:40273-40282.
doi: 10.1074/jbc.M607986200 pmid: 17035227
[33] Liu Q, Siloto R M P, Snyder C L, Randall J. Weselake functional and topological analysis of yeast acyl-CoA:diacylglycerol acyltransferase 2, an endoplasmic reticulum enzyme essential for triacylglycerol biosynthesis. J Biol Chem, 2011,286:13115-13126.
doi: 10.1074/jbc.M110.204412 pmid: 21321129
[34] 郑玲, 史灵敏, 田海莹, 单雷, 边斐, 郭峰, 宣宁, 万书波, 彭振英. 花生AhDGAT2a基因启动子的克隆和功能验证. 作物学报, 2016,42:1094-1099.
doi: 10.3724/SP.J.1006.2016.01094
Zheng L, Shi L M, Tian H Y, Shan L, Bian F, Guo F, Xuan N, Wan S B, Peng Z Y. Cloning and functional analysis of peanut AhDGAT2a promoter. Acta Agron Sin, 2016,42:1094-1099 (in Chinese with English abstract).
doi: 10.3724/SP.J.1006.2016.01094
[35] He X, Turner C, Chen G Q, Lin J T, McKeon T A. Cloning and characterization of a cDNA encoding diacylglycerol acyltransferase from castor bean. Lipids, 2004,39:311-318.
doi: 10.1007/s11745-004-1234-2 pmid: 15357018
[36] Bourgis F, Kilaru A, Cao X, Frank G, Ebongue N, Drira N, Ohlrogge J B, Arondel V. Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning. Proc Natl Acad Sci USA, 2011,108:12527-12532.
doi: 10.1073/pnas.1106502108 pmid: 21709233
[37] Tranbarger T J, Dussert S, Joet T, Argout X, Summo M, Champion A, Cros D, Omore A, Nouy B, Morcillo F. Regulatory mechanisms underlying Oil Palm fruit mesocarp maturation, ripening, and functional specialization in lipid and carotenoid metabolism. Plant Physiol, 2011,156:564-584.
doi: 10.1104/pp.111.175141 pmid: 21487046
[38] Chen B, Wang J, Zhang G, Liu J, Manan S, Hu H, Zhao J. Two types of soybean diacylglycerol acyltransferases are differentially involved in triacylglycerol biosynthesis and response to environmental stresses and hormones. Sci Rep, 2016,6:28541.
doi: 10.1038/srep28541 pmid: 27345221
[39] Sambanthamurthi R. Chemistry and biochemistry of palm oil. Prog Lipid Res, 2000,39:507-558.
doi: 10.1016/s0163-7827(00)00015-1 pmid: 11106812
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