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Acta Agron Sin ›› 2016, Vol. 42 ›› Issue (07): 1094-1099.doi: 10.3724/SP.J.1006.2016.01094

• RESEARCH NOTES • Previous Articles    

Cloning and Functional Analysis of Peanut AhDGAT2a Promoter?

ZHENG Ling1,2,SHI Ling-Min1,3,TIAN Hai-Ying1,2,SHAN Lei1,2,BIAN Fei1,GUO Feng1, XUAN Ning1,WAN Shuo-Bo1,2,*,PENG Zhen-Ying1,2,3,*   

  1. 1 Biotechnology Research Center, Shandong Academy of Agricultural Sciences / Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan 250100, China; 2 College of Life Science, Shandong University, Jinan 250100, China; 3 College of Agronomy, Xinjiang Agricultural University, Urumqi 830000, China
  • Received:2016-01-11 Revised:2016-05-09 Online:2016-07-12 Published:2016-05-11
  • Contact: 万书波, E-mail: wansb@saas.ac.cn, Tel: 0531-83178335; 彭振英, E-mail: pengzhenying2005@126.com
  • Supported by:

    This study was supported by Natural Science Foundation of Shandong (ZR2013CM036) and Shandong Province Germplasm Innovation and Utilization Project (2014-2017).


Diacylglycerol acyltransferase (DGAT) is a rate-limiting enzyme in triacylglycerol (TAG) biosynthesis pathway. In this study, GenomeWalking method was used for cloning the promoter sequence of AhDGAT2a gene from Luhua 14, and finally a 1200 bp fragment flanking 5′-upstream of AhDGAT2a was obtained and named as pAhDGAT2a. The crucial regulatory elements in pAhDGAT2a were further analyzed with software PlantCARE. There were many TATA-box, CAAT-box, light regulation, stress and defense response and hormone response elements. To assess the activity of pAhDGAT2a, we constructed pAhDGAT2a:GUS cassettes and introduced it into the tobacco SR1 genome by Agrobacterium-mediated transformation. Expression pattern was monitored by histochemical staining. Results showed that GUS activity driven by the pAhDGAT2a was detected in almost all vegetative and reproductive tissues, with a higher expression level in stigma, anther and young seeds than in the other organs, indicating thatpAhDGAT2a has a constitutive promoter activity.

Key words: Arachis hypogaea L., DGAT2a gene, Promoter, Function analysis

[1] Broun P, Gettner S, Somerville C. Genetic engineering of plant lipids. Annu Rev Nutr, 1999, 19: 197–216
[2] Jung S, Swift D, Sengoku E, Patel M, Teulé F, Powell G, Moore K, Abbott A. The high oleate trait in the cultivated peanut [Arachis hypogaea L.]: I. Isolation and characterization of two genes encoding microsomal oleyl-PC desaturases. Mol Gen Genet, 2000, 263: 796–805
[3] Yu Y H, Ginsberg H N. The role of acyl-CoA: diacylglycerol acyilransferase (DGAT) in energy metabolism. Ann Med, 2004, 36: 252–261
[4] Zou J, Wei Y D, Jako C, Kumar A, Selvaraj G, Taylor D C. The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J, 1999, 19: 645–653
[5] Feussner I, Kuhn H, Wasternack C. Lipoxygenase-dependent degradation of storage lipids. Trends Plant Sci, 2001, 6: 268–273
[6] Lu C F, Hills M J. Arabidopsis mutants deficient in diacylglycerol acyltransferase display increased sensitivity to abscisic acid, sugars and osmotic stress during germination and seedling development. Plant Physiol, 2002, 129: 1352–1358
[7] He X, Chen G Q, Lin J T, Mckeon T A. Diacylglycerol acyltransferase activity and triacylglycerol synthesis in germinating castor seed cotyledons. Lipids, 2006, 41: 281–285
[8] Weiss S B, Kennedy E P, Kiyasu J Y. The enzymatic synthesis of triglycerides. J Biol Chem, 1960, 235: 40–44
[9] Saha S, Enugutti B, Rajakumari S, Rajadekharan R. Cytosolic triacylglycerol biosynthetic pathway in oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase. Plant Physiol, 2006, 141: 1533–1543
[10] Lehner R, Kuksis A. Biosynthesis of triacylglycerols. Prog Lipid Res, 1996, 35: 169–201
[11] Hofmann K. A superfamily of membrane-bound O-acyltransferases with implications for WNT signaling. Trends Biochem Sci, 2000, 25: 111–112
[12] Cases S, Smith S J, Zheng Y W. 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
[13] Bouvier P, Benveniste P, Oelkers P, Sturley S L, Schaller H. Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase. Eur J Biochem, 2000, 267: 85–96
[14] Sandager L, Gustsvsson M H, Stehl U, Dahlqvist A, Wiberg E, Banas A, Lenman M, Ronne H, Stymne S. Storage lipid synthesis is non-essential in yeast. J Biol Chem, 2002, 277: 6478–6482
[15] Stone S, Levin M, 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
[16] Shockey J, Gidda S D, Chapital, Kuan J, Dhanoa P, Bland J, Rothstein S, Mullen R, Dyer J. 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
[17] Hobbs D H, Lu C, Hills M J. Cloning of a cDNA encoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression. FEBS Lett, 1999, 452: 145–149
[18] Lu C L, Noyer S B, Hobbs D H, Kang J, Wen Y, Kratchus D, Hills M J. Expression pattern of diacyl-glycerol acyltransferase-1, an enzyme involved in triacylglycerol biosynthesis, in Arabidopsis thaliana. Plant Mol Biol, 2003, 52: 31–41
[19] Jako C, Kumar A, Wei Y D, Zou J, Barton D L, Giblin E M, Covello P S, Taylor D C. Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhance seed oil content and seed weight. Plant Physiol, 2001, 126: 861–874
[20] Zheng P, Allen W, Roesler K, Williams M, Zhang S, Li J, Glassman K, Ranch J, Nubel D, Solawetz W, Bhattramakki D, Llaca V, Deschamps S, Zhong G Y, Tarczynski, M C, Shen B. A phenylalanine in DGAT is a key determinant of oil content and composition in maize. Nat Genet, 2008, 40: 367–372
[21] Lardizabal K, Effertz R, Levering C, Jennifer M, Pedroso M C, Tom J, Eric A, Ken G, Kristen B. Expression of Umbelopsis ramanniana DGAT2A in seed increases oil in soybean. Plant Physiol, 2008, 148: 89–96
[22] 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, 46: 1502–1511
[23] 万书波主编. 花生品质学. 北京: 中国农业科学技术出版社, 2005. pp 2–10
Wan S B. Peanut Quality. Beijing: China Agricultural Science and Technology Publishers, 2005. pp 2–5 (in Chinese)
[24] 廖伯寿. 中国花生油脂产业竞争力浅析. 花生学报, 2003, 32: 11–15
Liao B S. Analysis the competitiveness of Chinese peanut oil industry. J Peanut Sci, 2003, 32: 11–15 (in Chinese)
[25] 王龙龙. 花生二酰甘油酰基转移酶(DGAT)基因的克隆与分析. 山东师范大学, 山东济南, 2010
Wang L L. Cloning and Characterization of Diacylglycerol Acyltransferase (DGAT) Gene in Peanut (Arachis hypogaea L.). MS Thesis of Shandong Normal University, Jinan, China, 2010 (in Chinese with English abstract)
[26] Peng Z Y, Li L, Yang L Q, Zhang B, Chen G, Bi Y P. Overexpression of peanut diacylglycerol acyltransferase2 in Escherichia coli. PLoS One, 2013, 8: e61363
[27] Chi X, Hu R, Zhang X, Chen M, Chen N, Pan L, Wang T, Wang M, Yang Z, Wang Q F, Yu S L. Cloning and Functional Analysis of Three Diacylglycerol Acyltransferase Genes from Peanut (Arachis hypogaea L.). PLoS One, 2014, 9: e105834
[28] Shockey J M, Gidda S K, Chapital D C, Kuan J C, Dhanoa P K, Bland J M, Rothstein S J, Mullen R T, Dyera 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
[29] Perry H J, Harwood J L. Changes in the lipid content of developing seeds of Brassica napus. Phytochemistry, 1993, 32: 1411–1415
[30] Lock Y Y, Snyder C L, Zhu W, Siloto R M, Weselake R J, Shah S. Antisense suppression of type 1 diacylglycerol acyltransferase adversely affects plant development in Brassica napus. Physiol Plant, 2009, 137: 67–74
[31] Donald R G, Cashmore A R. Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1 Apromoter. EMBO J, 1990, 9: 1717–1726
[32] Puente P, Wei N, Deng X W. Combinatorial interplay of promoter elements constitutes the minimal determinants for light and developmental control of gene expression in Arabidopsis. EMBO J, 1996, 15: 3732–3743
[33] Lam E, Chua N H. Gt1 binding site confers light-responsive expression in transgenic tobacco. Science, 1990, 248: 471–474
[34] Chen P W, Lu C A, Yu T S, Teng T H, Wang C S, Yu S M. Rice alpha-amylase transcriptional enhancers direct multiple mode regulation of promoters in transgenic rice. J Biol Chem, 2002, 277: 13641–13649
[35] 杨予涛. 一个光合组织特异表达启动子的克隆、功能分析及其转录因子的鉴定. 山东农业大学博士学位论文, 山东泰安, 2005
Yang Y T. Isolation and Characterization of A Strong Specific Promoter in Photosynthetic Tissues and Identification of a bZIP Transcription Factor. PhD dissertation of Shandong Agricultural University, Tai’an, China, 2005 (in Chinese with English abstract)
[36] Hoffmann M, Binder S. Functional importance of nucleotide identities within the peat p9 promoter sequence. Mol Biol, 2002, 320: 943–950
[37] Yadav V, Kundu S, Chattopadhyay D, Negi P, Wei N, Deng X W, Chattopadhyay S. Light regulated modulation of Z-box containing promoters by photoreceptors and downstream regulatory components, COP1 and HY5, in Arabidopsis. Plant J, 2002, 31: 741–753
[38] Tao Y B, Luo L, He L L, Ni J, Zeng F X. A promoter analysis of MOTHER OF FT AND TFL1 (JcMFT1), a seed-preferential gene from the biofuel plant Jatropha curcas. Plant Res, 2014, 127: 513–524
[39] Xu W, Yu Y, Ding J, Hua Z, Wang L. Characterization of a novel stilbene synthase promoter involved in pathogen- and stress-inducible expression from Chinese wild Vitis pseudoreticulata. Planta, 2010, 231: 475–487
[40] Wang H, Zhang J, Gai J, Chen S. Cloning and comparative analysis of the gene encoding diacylglycerol acyltransferase from wild type and cultivated soybean. Theor Appl Genet, 2006, 112: 1086–1097
[41] Xu J, Francis T, Mietkiewska E, Giblin E M, Barton D L, Zhang Y, Zhang M, Taylor D C. Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferasel (DGATl) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content. Plant Biotechnol J, 2008, 6: 799–818
[42] Li R Z, Yu K S, 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

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