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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (8): 1283-1290.doi: 10.3724/SP.J.1006.2020.94192

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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 Online:2020-08-12 Published:2020-04-26
  • Contact: Tao ZHANG E-mail:zht2188@126.com
  • Supported by:
    National Natural Science Foundation of China(31171588);Chongqing Technology Innovation and Application Development Project(cstc2019jscx-msxm0612)

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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

Table 1

Primers used for PCR"

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

Fig. 1

Cloning and analysis of the PfDGAT2 gene A: PCR amplification product of PfDGAT2, M: DL2000 DNA marker. B: conserved domains analysis of PfDGAT2 protein."

Fig. 2

Phylogenetic tree of different plants based on amino acid of DGAT2 The number on the branches represents the reliability percent of bootstraps values based on 1000 replications."

Fig. 3

Multiple sequence alignment analysis of DGAT2 in different plants PfDGAT2: Perilla frutescens; SiDGAT2: Sesamum indicum (XP_011098009.1); StDGAT2: Solanum tuberosum (XP_006365015.1); AtDGAT2: Arabidopsis thaliana (OAP06431.1); GmDGAT2: Glycine max (NP_001299586.1); NtDGAT2: Nicotiana tabacum (AGL46984.1); CsDGAT2: Camelina sativa (XP_010426724). In the black box there are the YFP, EPHS, GGVQE, RXGFX(K/R)XAXXXGXX(L/V) VPXXXFG(E/Q), and VVGRPI conserved domains in turn."

Fig. 4

Analysis of relative expression of PfDGAT2 gene in different tissues and different growth stages of seeds Bars superscripted by different lowercase letters are significantly different at P < 0.05. Data points are means±SE (n = 3)."

Fig. 5

Seeds oil content of transgenic PfDGAT2 Arabidopsis thaliana Bars superscripted by different lowercase letters are significantly different at P < 0.05. Data are means±SE (n = 3)."

Fig. 6

Relative fatty acid content in PfDGAT2 transgenic Arabidopsis thaliana seeds In the figure, * shows significant difference of each component of PD2-1, PD2-3, PD2-5 with wild-type Col-0. * represents the significant difference at P < 0.05, ** represents significant difference at P < 0.01, *** represents significant difference at P < 0.001. Data are means±SE (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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