Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum
Introduction
Diatoms are thought to be the most successful and diverse group of unicellular photosynthetic eukaryotes, with perhaps as many as 100,000 extant species (Round et al., 1990) found throughout the oceans and other aquatic systems. Their contribution to global CO2 fixation represents around 40% of marine organic carbon production (Nelson et al., 1995). In addition to their crucial role in carbon cycling, an unusual feature of diatoms is their ability to precipitate soluble silicic acid into highly elaborate nanostructures. This unique silicon metabolism is of major importance for the biogeochemical cycling of this element in the oceans (Tréguer et al., 1995). A range of other novel metabolic pathways are also found in diatoms, presumably acquired during their evolution (Armbrust et al., 2004), and so they are of major interest for the discovery of novel biological processes not present in more intensively studied model organisms.
Despite their abundance in the oceans, the molecular mechanisms underlying the ecological success of diatoms are largely unexplored. Nevertheless, tremendous progress has been made very recently with the whole genome sequencing of the centric diatom Thalassiosira pseudonana (Armbrust et al., 2004), the completion of the genome of the pennate diatom Phaeodactylum tricornutum (http://genome.jgi-psf.org/Phatr2/Phatr2.home.html), and the generation of an Expressed Sequence Tag (EST) database for P. tricornutum (Maheswari et al., 2005, Montsant et al., 2005) (http://avesthagen.sznbowler.com). In addition, the molecular tools required for functional genomics, such as genetic transformation, are available for P. tricornutum (Apt et al., 1996, Falciatore et al., 1999), as well as for other non-sequenced diatom species (Dunahay et al., 1995, Fischer et al., 1999), and more recently also for T. pseudonana (Poulsen et al., 2006).
This recent availability of diatom whole genome sequences provides new opportunities for large scale gene expression analyses. Such studies have previously been performed in diatoms by northern blotting, RNA dot blot (Oeltjen et al., 2004) and semi-quantitative RT-PCR (Leblanc et al., 1999). However, in the last ten years, qRT-PCR has become the method of choice because it combines high-throughput procedures with the accurate expression profiling of selected genes. The qRT-PCR method in fact provides possibilities for the simultaneous analysis of transcript levels for many genes in many different samples, and is especially suitable when biological material is only available in limited amounts (Fink et al., 1998, Heid et al., 1996, Higuchi et al., 1993). Furthermore, the technique has the advantage of speed, throughput and a high degree of potential automation compared to more classical quantification methods, and is often used to validate expression data obtained from EST or microarray analyses.
Two different strategies to analyze qRT-PCR data exist: absolute quantification or relative quantification. Absolute quantification is well adapted to study a small set of genes and determines the initial copy number of the transcript molecules of interest. Relative quantification is the more commonly used method and is based on the normalized expression of a target gene versus an internal stable reference gene. Although relative expression analysis removes the laborious preparation steps of absolute quantification, the selection of a proper internal reference gene expressed at a relatively stable level throughout the different samples being examined is an absolute necessity for reliable results. For many years, housekeeping genes have been used as references in traditional mRNA quantification methods, and were subsequently used to normalize qRT-PCR data without further statistical analyses (Andersen et al., 2004, Brunner et al., 2004, Bustin, 2002, Dheda et al., 2004, Goidin et al., 2001, Kim et al., 2003, Radonic et al., 2004). However, several articles have shown how some of the most commonly used housekeeping genes are not reliable controls in all tested conditions (Dheda et al., 2005, Lee et al., 2002, Suzuki et al., 2000, Thellin et al., 1999), emphasizing the importance of preliminary evaluation studies to identify the most stable housekeeping genes in the selected experimental conditions. To investigate the expression stability of commonly used reference genes, it is necessary to have gene expression data for many expressed genes in various experimental conditions. In this normalization strategy, internal controls are subjected to the same conditions as the mRNA of interest and their expression is measured by qRT-PCR. To determine which reference genes are best suited for transcript normalization in a given subset of biological samples, various statistical algorithms, such as geNorm or BestKeeper, have been developed (Pfaffl, 2001, Pfaffl et al., 2004, Vandesompele et al., 2002).
Typical of other photosynthetic organisms, in diatoms such as P. tricornutum, metabolism is highly dependent on the light source (Leblanc et al., 1999). To examine the influence of light on transcriptional regulation in this diatom, it is therefore of interest to develop reliable qRT-PCR-based methods. For this, we selected 12 potential housekeeping genes for qRT-PCR data normalization and examined their expression levels during two complete light–dark cycles in standard P. tricornutum culture conditions. Several genes are reported that will serve as useful references for physiological studies of gene expression in P. tricornutum grown in these conditions.
Another important methodology for reverse genetics comprises the expression of a cloned gene of interest in homologous and heterologous systems in order to identify the function of the protein it encodes. In a homologous context, the protein can be (i) tagged to an epitope that permits the characterization of the protein by immunological techniques, or (ii) fused to a fluorescent protein to allow its subcellular localization to be visualized in living cells. In addition, the overexpression of an endogenous protein in the native or mutated form can induce the appearance of a novel phenotype allowing studies of its function. In a heterologous context, the expression of the gene in bacteria or yeast can provide a simple methodology for the purification of the corresponding protein, or for complementation of known mutants. The engineering of multiple expression constructs to accomplish these goals for each target gene is time-consuming and laborious using traditional cloning methods. We have therefore eliminated this bottleneck by designing multiple chimeric diatom expression vectors based on the Gateway cloning technology (Walhout et al., 2000) developed by Invitrogen (Carlsbad, USA). Briefly, an open reading frame is first inserted into a commercially available Entry vector (pENTR) using a topoisomerase and then recombined into a series of diatom Destination vectors (pDEST).
In the current work, we describe seven diatom Destination vectors that have been designed for a variety of specific purposes including gene overexpression, protein localization (by fusion to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP)), and production of an epitope (haemagglutinin (HA))-tagged protein for immunodetection and affinity purification. Representative protein localization and immunoblotting data are presented in order to validate these Gateway-compatible diatom expression vectors.
Section snippets
Cell culture
Axenic cultures of P. tricornutum Bohlin clone Pt1 8.6 (CCMP632) and clone Pt8 (CCMP2560) were obtained from the culture collection of the Provasoli-Guillard National Center for Culture of Marine Phytoplankton, Bigelow Laboratory for Ocean Sciences, USA. Cultures were grown in f/2 medium (Guillard, 1975) made with 0.2-μm-filtered and autoclaved local seawater supplemented with f/2 vitamins and inorganic nutrients (Guillard, 1975) (filter sterilized and added after autoclaving). Cultures were
Selection of housekeeping genes
The successful use of qRT-PCR is highly dependent on the use of constitutive reference genes. To determine a set of suitable genes for data normalization during a 12 h photoperiod, we followed the expression of a range of commonly used housekeeping genes. As shown in Table 1, we evaluated a total of 12 diatom genes involved in (presumably constitutive) basic cellular processes (histone H4, TATA box binding protein (TBP), elongation factor 1α (EF1α), 30S ribosomal protein subunit (RPS), 18S
Conclusion
The availability of whole genome sequences from diatoms has represented a major advance that will serve diatom research well through the beginning of the 21st century. Notwithstanding, these sequences represent only static resources without the development of complementary tools to permit investigation of diatom biology at the cellular level. In this report, we have examined the utility of a range of reference genes to study gene expression by qRT-PCR and have developed a series of generic
Acknowledgements
We would like to thank Benjamin Mathieu for technical assistance with confocal microscopy in Paris, Marco Borra and the SBM (Servizio di Biologia Molecolare) in Naples for technical assistance with the real time PCR machine, Nazanine Ezdiari for her pictures of the GS clones, and Mariella Ragni for her help and enriching discussions. M.S. was supported by a post doctoral fellowship from the EU-FP6 Diatomics project (LSHG-CT-2004-512035). M.H. was supported by a doctoral fellowship from the
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- 1
Current address: CEA, DSV, IBEB, SBVME, UMR 6191 CNRS-CEA, Aix-Marseille II, Laboratoire de Bioénergétique et Biotechnologie des Bactéries et Microalgues, Cadarache, Saint-Paul les Durance, F-13108 France.
- 2
These authors contributed equally to this work.
- 3
These authors contributed equally to the coordination of this work.