Skip to main content
SpringerLink
Log in

Isolation of the endosperm-specific LPAAT gene promoter from coconut (Cocos nucifera L.) and its functional analysis in transgenic rice plants

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

As one of the key tropical crops, coconut (Cocos nucifera L.) is a member of the monocotyledonous family Aracaceae (Palmaceae). In this study, we amplified the upstream region of an endosperm-specific expression gene, Lysophosphatidyl acyltransferase (LPAAT), from the coconut genomic DNA by chromosome walking. In this sequence, we found several types of promoter-related elements including TATA-box, CAAT-box and Skn1-motif. In order to further examine its function, three different 5′-deletion fragments were inserted into pBI101.3, a plant expression vector harboring the LPAAT upstream sequence, leading to pBI101.3-L1, pBI101.3-L2 and pBI101.3-L3, respectively. We obtained transgenic plants of rice by Agrobacterium-mediated callus transformation and plant regeneration and detected the expression of gus gene by histochemical staining and fluorometric determination. We found that gus gene driven by the three deletion fragments was specifically expressed in the endosperm of rice seeds, but not in the empty vector of pBI101.3 and other tissues. The highest expression level of GUS was at 15 DAF in pBI101.3-L3 and pBI101.3-L2 transgenic lines, while the same level was detected at 10 DAF in pBI101.3-L1. The expression driven by the whole fragment was up to 1.76- and 2.8-fold higher than those driven by the −817 bp and −453 bp upstream fragments, and 10.7-fold higher than that driven by the vector without the promoter. Taken together, our results strongly suggest that these promoter fragments from coconut have a significant potential in genetically improving endosperm in main crops.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

CTAB:

Cetyltriethylammnonium Bromide

GUS:

β-glucuronidase

LPA:

Lysophosphatidic acid

LPAAT:

(1-acyl-sn-glycerol-3-acyltransferase)

MU:

4-Methylumbelliferone

MUG:

4-Methylumbelliferyl-β-d-glucuronide

PA:

Phosphatidic acid

TGA:

Triacylglycerol

X-Gluc:

5-bromo-4-chloro-3-indolyl-β-d-glucuronide

References

  • Ahlandsberg S, Sun C, Jansson C (2005) An intronic element directs endosperm-specific expression of the sbellb gene during barley seed development. Plant Cell Rep 20:864–868

    Google Scholar 

  • Bajaj S, Mohanty A (2005) Recent advances in rice biotechnology—towards genetically superior transgenic rice. Plant Biotechnol J 3:275–307

    Article  CAS  PubMed  Google Scholar 

  • Bhullar S, Chakravarthy S, Advani S, Datta S, Pental D, Burma PK (2003) Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping. Plant Physiol 132:988–998

    Article  CAS  PubMed  Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Charrier B, Scollan C, Ross S, Zubko E, Meyer P (2000) Co-silencing of homologous transgenes in tobacco. Mol Breed 6:407–419

    Article  CAS  Google Scholar 

  • Choi HW, Lemaux PG, Cho MJ (2003) Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley. Plant Cell Rep 21:1108–1120

    Article  CAS  PubMed  Google Scholar 

  • Coleman RA, Lee DP (2004) Enzymes of triacylglycerol synthesis and their regulation. Prog Lipid Res 43:134–176

    Article  CAS  PubMed  Google Scholar 

  • Daniel ZV, Miguel FB, Nelson TH, Patricia CG (2005) Morphological variation of fruit in Mexican populations of Cocos nucifera L. (Arecaceae) under in situ and ex situ conditions. Genet Resour Crop Evol 52:421–434

    Article  Google Scholar 

  • Frisch DA, van der Geest AHM, Dias K, Hall TC (1995) Chromosomal integration is required for spatial regulation of expression from the β-phaseolin promoter. Plant J 7:503–512

    Article  CAS  Google Scholar 

  • Furtado A, Henry RJ, Takaiwa F (2008) Comparison of promoters in transgenic rice. Plant Biotechnol J 6:679–693

    Article  CAS  PubMed  Google Scholar 

  • Giovinazzo G, Manzocchi LA, Bianchi MW, Coraggio I, Viotti A (1992) Functional analysis of the regulatory region of a zein gene in transiently transformed protoplasts. Plant Mol Biol 19:257–263

    Article  CAS  PubMed  Google Scholar 

  • Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of boundaries of the T-DNA. Plant J 6:271–282

    Article  CAS  PubMed  Google Scholar 

  • Hiroi T, Takaiwa F (2006) Peptide immunotherapy for allergic diseases using a rice-based edible vaccine. Curr Opin Allergy Clin Immunol 6:455–460

    Article  CAS  PubMed  Google Scholar 

  • Huang JQ, Wei ZM, An HL, Zhu YX (2001) Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaf folder and striped stem borer. Cell Res 11:149–155

    Article  CAS  PubMed  Google Scholar 

  • Hwang YS, Ciceri P, Parsons RL, Moose SP, Schmidt RJ, Huang N (2004) The maize O2 and PBF proteins act additively to promote transcription from storage protein gene promoters in rice endosperm cells. Plant Cell Physiol 45:1509–1518

    Article  CAS  PubMed  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed  Google Scholar 

  • Joersbo M, Marcussen J, Brunstedt J (2001) In vivo modification of the cell wall polysaccharide galactomannan of guar transformed with a α-galactosidase gene cloned from senna. Mol Breed 7:211–219

    Article  CAS  Google Scholar 

  • Kawakatsu T, Yamamoto MP, Hirose S, Yano M, Takaiwa F (2008) Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm. J Exp Bot 59:4233–4245

    Article  CAS  PubMed  Google Scholar 

  • Kim HU, Huang AH (2004) Plastid lysophosphatidyl acyltransferase is essential for embryo development in Arabidopsis. Plant Physiol 134:1206–1216

    Article  CAS  PubMed  Google Scholar 

  • Kluth A, Sprunck S, Becker D, Lorz H, Lutticke S (2002) 5′ deletion of a gbss1 promoter region from wheat leads to changes in tissue and developmental specificities. Plant Mol Biol 49:669–682

    Article  CAS  PubMed  Google Scholar 

  • Knutzon DS, Lardizabal KD, Nelsen JS, Bleibaum JL, Davies HM, Metz JC (1995) Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-alycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates. Plant Physiol 109:999–1006

    Article  CAS  PubMed  Google Scholar 

  • Lamacchia C, Shewry PR, Fonzo ND, Forsyth JL, Harris N, Lazzeri PA, Napier JA, Halford NG, Barcelo P (2001) Endosperm-specific activity of a storage protein gene promoter in transgenic wheat seed. J Exp Bot 52:243–250

    Article  CAS  PubMed  Google Scholar 

  • Li DD, Fan YM (2008) Construction and characterization of a cDNA library from the pulp of coconut (Cocos nucifera L.). Agric Sci China 7:101–105

    Article  Google Scholar 

  • Li M, Singh R, Bazanova N, Milligan AS, Shirley N, Langridge P, Lopato S (2008) Spatial and temporal expression of endosperm transfer cell-specific promoters in transgenic rice and barley. Plant Biotechnol J 6:465–476

    Article  CAS  PubMed  Google Scholar 

  • Lin YJ, Zhang Q (2005) Optimizing the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep 23:540–547

    Article  CAS  PubMed  Google Scholar 

  • Liu XL, Tang H, Li DD, Wang Q, Lin YQ, Zhou R (2008) Genetic diversity of coconut cultivars from Hainan province on SSR markers. Acta Hortic Sin 35:1199–1204

    CAS  Google Scholar 

  • Muller M, Knudsen S (1993) The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box. Plant J 4:343–355

    Article  CAS  PubMed  Google Scholar 

  • Nath UK, Wilmer JA, Wallington EJ, Becker HC, Möllers C (2009) Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT + Bn-fae1 transgenes in rapeseed (Brassica napus L.). Theor Appl Genet 118:765–773

    Article  CAS  PubMed  Google Scholar 

  • Okazaki K, Sato N, Tsuji N, Tsuzuki M, Nishida I (2006) The significance of C16 fatty acids in the sn-2 positions of glycerolipids in the photosynthetic growth of Synechocystis sp. PCC6803. Plant Physiol 141:546–556

    Article  CAS  PubMed  Google Scholar 

  • Qu LQ, Takaiwa F (2004) Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice. Plant Biotechnol J 2:113–125

    Article  CAS  Google Scholar 

  • Qu LQ, Xing YP, Liu WX, Xu XP, Song YR (2008) Expression pattern and activity of six glutelin gene promoters in transgenic rice. J Exp Bot 59:2417–2424

    Article  CAS  Google Scholar 

  • Rasmussen TB, Donaldson IA (2006) Investigation of the endosperm-specific sucrose synthase promoter from rice using transient expression of reporter genes in guar seed tissue. Plant Cell Rep 25:1035–1042

    Article  CAS  PubMed  Google Scholar 

  • Rivera R, Edwards KJ, Barker JHA, Arnold GM, Ayad G, Hodgkin T (1999) Isolation and characterization of polymorphic microsatellites in Cocos nucifera L. Genome 42:668–675

    Article  CAS  PubMed  Google Scholar 

  • Russell DA, Fromm ME (1997) Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice. Transgenic Res 6:157–168

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Takaiwa F, Takagi H, Hirose S, Wakasa Y (2007) Endosperm tissue is a good production platform for artificial recombinant proteins in transgenic rice. Plant Biotechnol J 5:84–92

    Article  CAS  PubMed  Google Scholar 

  • Thomas MS, Flavell RB (1990) Identification of an enhancer element for the endosperm-specific expression of high molecular weight glutenin. Plant Cell 2:1171–1180

    Article  CAS  PubMed  Google Scholar 

  • Washida H, Wu CY, Suzuki A, Yamanouchi U, Akihama T, Harada K, Takaiwa F (1999) Identification of cis-regulatory elements required for endosperm expression of the rice storage protein glutelin gene Glub-1. Plant Mol Biol 40:1–12

    Article  CAS  PubMed  Google Scholar 

  • Wiley PR, Tosi P, Evrard A, Lovegrove A, Jones HD, Shewry PR (2007) Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain. Plant Mol Biol 64:125–136

    Article  CAS  PubMed  Google Scholar 

  • Wu J, Yu L, Li L, Hu J, Zhou J, Zhou X (2007) Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens. Plant Biotechnol J 5:570–578

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This research was supported by the National Natural Science Foundation of China (NSFC) (No: 30560092 and 30860149), the National High Technology Research and Development Program of China (863 Program) and National Nonprofit Institute Research Grant of CATAS-ITBB.

Author information

Authors and Affiliations

  1. Key Laboratory of Tropical Crops Germplasm Resources Utilization of Ministry of Agriculture, Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, 571737, Danzhou, Hainan, China

    Li Xu

  2. Key Laboratory of Tropical Crop Biotechnology, Institute of Tropical Bioscience and Biotechnology, Ministry of Agriculture, Chinese Academy of Tropical Agricultural Sciences, 571737, Danzhou, Hainan, China

    Peng Zhou

  3. Key Laboratory of Tropic Biological Resources of Ministry of Education, Hainan University, 570228, Haikou, Hainan, China

    Yusheng Zheng, Zhekui Wang & Dongdong Li

  4. National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, 430070, Wuhan, China

    Rongjian Ye & Yongjun Lin

Authors
  1. Li Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rongjian Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yusheng Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhekui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peng Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yongjun Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dongdong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yongjun Lin or Dongdong Li.

Additional information

Communicated by M. Jordan.

L. Xu and R. Ye contributed equally to the work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xu, L., Ye, R., Zheng, Y. et al. Isolation of the endosperm-specific LPAAT gene promoter from coconut (Cocos nucifera L.) and its functional analysis in transgenic rice plants. Plant Cell Rep 29, 1061–1068 (2010). https://doi.org/10.1007/s00299-010-0892-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-010-0892-y

Keywords

Search

Navigation