Research article
Isolation and characterization of an oil palm constitutive promoter derived from a translationally control tumor protein (TCTP) gene

https://doi.org/10.1016/j.plaphy.2011.04.003Get rights and content

Abstract

We have characterized an oil palm (Elaeis guineensis Jacq.) constitutive promoter that is derived from a translationally control tumor protein (TCTP) gene. The TCTP promoter was fused transcriptionally with the gusA reporter gene and transferred to monocot and dicot systems in order to study its regulatory role in a transient expression study. It was found that the 5’ region of TCTP was capable of driving the gusA expression in all the oil palm tissues tested, including immature embryo, embryogenic callus, embryoid, young leaflet from mature palm, green leaf, mesocarp and stem. It could also be used in dicot systems as it was also capable of driving gusA expression in tobacco leaves. The results indicate that the TCTP promoter could be used for the production of recombinant proteins that require constitutive expression in the plant system.

Highlights

► We identified constitutively expressing genes in oil palm. ► We then isolate the promoter from one of the constitutively expressing gene. ► We test the constitutive nature of promoter via transient expression.

Introduction

Constitutive promoters are essential to facilitate the genetic improvement of oil palm and other genetically engineered plants. The promoters are required to produce compounds that require ubiquitous activity in the plant and are required at all stages of plant development. A good example is the use of a constitutive promoter to drive a selectable marker gene to establish a reliable transformation system for a particular plant species. Most studies indicate that constitutive promoters are derived from genes that are constitutively expressed, and are involved in a wide range of physiological processes. The constitutive nature of these genes accounts for the ability of their promoter to drive high and strong constitutive expression of the transgenes in transgenic plants. Examples of gene promoters that are derived from constitutively expressing genes are ubi1 from maize [1], [2], Act1 [3], [4], OsTubA1 [5], OsCc1 [6], RUBQ1 and 2 [7], rubi3 [8] and OsAct2 [9] from rice, and pEmu [10]. These promoters are highly active in monocot crops but are distinct in various ways [11]. The cauliflower mosaic virus CaMV35S is the most common constitutive promoter used in dicots and monocots. Despite its advantages in enhancing the expression of transgenes, the use of this viral promoter is limited by the perception of risk to human health [12], [13], [14].

Translationally control tumor protein (TCTP) is a growth-related protein that is constitutively expressed and distributed in eukaryotes [15]. Although the function of this protein remains elusive, TCTP has been implicated in diverse cellular processes, such as apoptosis, microtubule organization, or ion homeostatis, and is also associated with several interacting proteins such as polo kinase, tubulin and Na+/K+-ATPase. Recent studies have also shown that TCTP is an important component of the TOR (target of rapamycin) signaling pathway, which is known as a major regulator of cell growth [16]. Although this gene was initially found in cancerous mammalian tissues, there is evidence to indicate that TCTP is not a tumor-specific protein. In fact, its expression has been found in healthy animal and plant tissues [17]. For example, high expression of the TCTP transcript was detected in all parts of the plants examined such as in Arabidopsis [16], Hevea brasiliensis [18], Pharbitis [19] and Pisum sativum [20]. This gene was also found to be very stable against heat, pH, ionic strength and even against protease, suggesting that it has a very compact globular structure [17].

A promoter of TCTP was isolated from Arabidopsis, and it was found to be capable of driving high expression of transgenes in transgenic plants [17]. Most studies indicate that plants such as pea, tobacco, Arabidopsis and Japanese morning glory have either a single or at the most two copies of the TCTP gene [17]. However, in oil palm, detailed analyses using databases developed by the Genomics Group of MPOB reveal that there are three isoforms of the TCTP gene. Their amino acid sequences are highly identical at about 97–98% similarity, but markedly different in the 5′ and 3′ untranslated regions. Oil palm TCTP (GenBank Accession No. EU284975) was shown to be constitutively expressed in all the tissues tested. We decided to further characterize the promoter region of the gene to examine its regulatory role in gene expression, and to develop a useful system to be used in genetic engineering of oil palm or other plants, including monocots and dicots.

Section snippets

Reverse northern analyses

Reverse northern analyses were performed according to the manufacturer’s instructions (Bio-Dot® Microfiltration Apparatus, BIO-RAD). Wells of dot blots were rinsed with 300 μl 2X SSC (3 M NaCl, 300 mM tri-sodium citrate, pH 7.0). About 200 μg of the amplified cDNA products were added to 0.4 N NaOH, denatured by boiling for 10 min and then immediately chilled on ice. About 100 μl of the prepared amplicons were dot-blotted onto a nylon membrane, and aspirated through the membrane under a vacuum. The

Identification of TCTP as constitutively expressing gene from oil palm

The TCTP gene was identified through screening the expression pattern of 73 EST clones using reverse northern analysis (Fig. 2). In this study, a cDNA clone, namely pOP-RD00041, was shown to produce a strong signal in all the tissues tested. The analysis was carried out by normalizing the intensities of cDNA expression to the intensity of ribosomal DNA. The lowest value of cDNA expression obtained from the normalization in any specific tissue was set as 1 fold. The cDNA was expressed in all the

Conclusions

In summary, the findings of TCTP increase the wider range of alternative promoter for introducing the multiple transgene into plant. This promoter was found to be active in most oil palm tissues although with slight variations in activity. Modification of this promoter by the addition of an intron region might increase its strength and effectiveness, and finally would serve as a crucial biotechnological tool for genetic engineering of oil palm or other plant with agronomically beneficial trait.

Acknowledgement

The authors wish to thank the Director-General of MPOB for permission to publish this paper. We wish to thank the members of the Genomic Group, MPOB, for their kind consent in providing EST clones of oil palm. Special thanks to the Breeding Group for providing oil palm tissues, and also to Dr Noor Azmi Shaharudin of the Gene Expression Group, MPOB. We thank Dr. Omar Rasid and Dr. Abrizah Othman of MPOB for critically reviewing the manuscript. Finally, the authors would like to acknowledge all

References (35)

  • A.H. Christiensen et al.

    Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer of protoplast by electroporation

    Plant Mol. Biol.

    (1992)
  • M.J. Cornejo et al.

    Activity of maize ubiquitin promoter in transgenic rice

    Plant Mol. Biol.

    (1993)
  • D. McElroy et al.

    Isolation of an efficient actin promoter for used in rice transformation

    Plant Cell

    (1990)
  • D. McElroy et al.

    Construction of expression vectors based on the rice actin (Act1) 5′ region for use in monocot transformation

    Mol. Gen. Genet.

    (1991)
  • J.S. Jeon et al.

    Tissue preferential expression of a rice α-tubulin gene, OsTubA1, mediated by the first intron

    Plant Physiol.

    (2000)
  • I.C. Jang et al.

    High level and ubiquitous expression of the rice cyctochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for transgenesis of monocots

    Plant Physiol.

    (2002)
  • J. Wang et al.

    Rice ubiquitin promoters: deletion analysis and potential usefulness in plant transformation systems

    Plant Cell Rep.

    (2003)
  • J. Lu et al.

    Activity of the 5′ regulatory regions of the rice polyubiquitin rubi3 gene in transgenic rice plants as analysed by both GUS and GFP reporter genes

    Plant Cell Rep.

    (2008)
  • C. He et al.

    Identification of rice Actin2 gene regulatory region for high-level expression of transgene in monocots

    Plant Biotechnol. J.

    (2009)
  • D.L. Last et al.

    pEmu: an improved promoter for gene expression in cereal

    Theor. Appl. Genet.

    (1991)
  • S.H. Park et al.

    Analysis of five novel putative constitutive gene promoters in transgenic rice plants

    J. Exp. Bot.

    (2010)
  • M.W. Ho et al.

    Cauliflower mosaic virus promoter – a recipe for disaster?

    Microb. Ecol. Health Dis.

    (1999)
  • J. Hodgson

    Scientists avert new GMO crisis

    Nat. Biotechnol.

    (2000)
  • C. Potenza et al.

    Targeting transgene expression in research, agricultural and environmental applications: promoters used in plant transformation

    In Vitro Cell Dev. Biol. Plant

    (2004)
  • U.A. Bommer et al.

    The translationally controlled tumor protein (TCTP)

    Int. J. Biochem. Cell Biol.

    (1993)
  • O. Berkowitz et al.

    Characterization of TCTP, the transcriptionally control tumor protein, from Arabidopsis thaliana

    Plant Cell

    (2008)
  • J.G. Kang, J. Yun, K.S. Chung, P.S. Song, C.M. Park, Promoter system of plant translationally control tumor protein...
  • Cited by (20)

    • Biotechnology of oil palm: Current status of oil palm genetic transformation

      2018, Biocatalysis and Agricultural Biotechnology
      Citation Excerpt :

      The standard protocol is being used for obtaining stable transgenic oil palm (Parveez et al., 2015a, 2015b; Manaf et al., 2017; Masura et al., 2017). Furthermore, the protocol has been frequently used in transient gene expression system for characterization of oil palm promoters such as a mesocarp-specific promoter (MSP1) (Siti Nor Akmar and Zubaidah, 2008), leaf-specific promoter (LSP1) (Chan et al., 2008), constitutive promoters i.e. ubiquitin extension protein (UEP1) (Masura et al., 2010) and translationally control tumor protein (TCTP) (Masura et al., 2011), and evaluation of effectiveness of marker genes for oil palm genetic transformation such as green fluorescence protein (GFP) gene (Majid and Parveez, 2007, 2016; Parveez and Majid, 2008), phosphomannose isomerase (PMI) gene (Bahariah et al., 2013) and hygromycin phosphotransferase (HPT) gene (Bahariah et al., 2017). Moreover, the standard protocol has been used as a guideline for improvements of oil palm genetic transformation (Kanchanapoom et al., 2008) and also for development of transformation system for other palm species such as date palm (Mousavi et al., 2009, 2014).

    • Green fluorescent protein as a visual selection marker for oil palm transformation

      2018, Industrial Crops and Products
      Citation Excerpt :

      A new transformation vector carrying the mgfp gene driven by double CaMV35S (2XCaMV35S) promoter was recently constructed and showed transient gfp expression in oil palm (Bahariah et al., 2017). Other constitutive promoters from oil palm (Masura et al., 2010; Masura and Parveez, 2011; Zubaidah et al., 2018) could also be considered for expressing gfp gene in oil palm. As the biolistic method used in this study was known to introduce high copy number that could induce gene silencing, Agrobacterium-mediated transformation (Masli et al., 2009) could be considered.

    • Tissue Culture and Genetic Engineering of Oil Palm

      2012, Palm Oil: Production, Processing, Characterization, and Uses
    View all citing articles on Scopus
    View full text