Key Laboratory of Eutrophication and Control of HAB of Guangdong Higher Education Institute, Jinan University, Guangzhou, China
*Y.F.N. and Z.K.Y. contributed equally to this work
BioTechniques, Vol. , No. , June 2012, pp. 1–3
The nitrate reductase promoter/terminator of the model diatom Phaeodactylum tricornutum was cloned and used to control the expression of the chloramphenicol acetyltransferase (CAT) reporter gene. The construct was electroporated into P. tricornutum grown in f/2 medium minus silicon. Expression of CAT in diatom conferred resistance to chloramphenicol and the expression of CAT transcript and protein was demonstrated by reverse transcription PCR and Western blotting, respectively. CAT expression was induced by nitrate and repressed when ammonium was the sole nitrogen source.
Diatoms are important primary producers in the marine ecosystem. Currently it is difficult to genetically transform diatoms due to the technical limitations of existing methods. The promoter/terminator of the nitrate reductase gene of the model diatom Phaeodactylum tricornutum was cloned and used to drive chloramphenicol acetyltransferase (CAT) reporter gene expression. The construct was transferred by electroporation into P. tricornutum grown in medium lacking silicon. CAT expression was induced in transformed diatoms in the presence of nitrate, enabling growth in selective medium, and was repressed when ammonium was the only nitrogen source. Expression of CAT transcript and protein were demonstrated by RT-PCR and Western blot analysis, respectively. Our study is the first to report a successful genetic transformation of diatom by electroporation in an economical and efficient manner and provides a tightly regulated inducible gene expression system for diatom.
Although diatoms are important primary producers in the marine ecosystem, genetic engineering of diatom has been slow compared with higher plants due to the technical limitations of existing methods. An efficient genetic transformation system for diatom is urgently needed. The marine diatom Phaeodactylum tricornutum, which is enriched with proteins and lipids, is utilized in aquaculture as live feed for fishery. Due to its fast growth rate and sequenced genome, it has become a model species for diatom research. Genetically engineered P. tricornutum has been obtained by microprojectile bombardment (1), where a variety of selectable markers and reporter genes were tested. To date, diatom transformation has solely relied on microprojectile bombardment. However, microprojectile bombardment is not routinely available, and costly consumables also limit its application in many laboratories. In contrast, electroporation is commonly used without the need for special consumables. Electroporation introduces foreign DNA into cells by temporarily opening pores in the cell membrane (2). This method has been widely used for the genetic transformation of microbial and animal cells and has also attracted attention as a means of introducing DNA into algal cells. Success was first reported for a green alga, Chlorella ellipsoidea, where a foreign gene was transferred by electroporation to manipulate algal biosynthetic pathways (3). Subsequently, additional green microalgae species such as Chlamydomonas reinhardtii (4) and Chlorella vulgaris (5) were transformed by electroporation. Recently, an efficient transformation method based on electroporation has been developed for the oil-producing alga Nannochloropsis sp. (6). In this study, we report for the first time the transformation of diatoms by electroporation.
An appropriate promoter is critical for a transformation vector. Good inducible gene expression systems are desirable because transgenic lines with inducible phenotypes are as useful as conditional mutants isolated by traditional genetics. Expression of nitrate reductase (NR) has been shown to be induced by nitrate in higher plant. The NR promoter from the microalga Cylindrotheca fusiformis is inducible and can be used for the controllable expression of foreign genes (7, 8). Since nitrogen is a component of culture medium, a nitrate-inducible promoter could be ideal for foreign gene expression. The NR promoter from P. tricornutum has been tested in the green microalga C. vulgaris (5) and is tested in this work for diatom-inducible expression.
P. tricornutum Bohlin was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, China (no. FACHB-863). Seawater from the Gulf of Dayawan was filter-sterilized and supplemented with f/2 nutrients as medium. Diatom cells were grown as batch cultures in flasks containing f/2-Si medium (prepared as for f/2 medium, but omitting Na2SiO3 ·9H2O), thus minimizing the capability of creating silicon-based extracellular skeleton that could be a barrier to electroporation. Cultures were grown at 23° ± 1°C in an artificial climate incubator. Cool-white fluorescent tubes provided an irradiance of 200 µmol photons m-2 s-1 under long-day light conditions (15 h/9 h light/dark regime). For nitrate induction, cells were inoculated in media containing different nitrogen sources: either 1.5 mM NH4Cl or 2.5 mM NaNO3.
To generate transgenic P. tricornutum, plasmid pHY11 (Figure 1A) containing the CAT reporter gene controlled by the NR promoter/terminator of P. tricornutum was used for electroporation with a GenePulser Xcell apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Diatom cells in exponential phase (0.5 × 107 cells) were collected by centrifugation at 1350× g for 10 min, resuspended with 150 µL 1.0 M NaCl, and then mixed with 150 µL 0.1 M mannitol and kept on ice for 30 min. The salt concentration used here is higher than that used in other microalgae, such as the 10 mM CaCl2 used for C. reinhardtii (9), which contributes to the permeability of the diatom cells during the pulse phase. A suspension aliquot of 0.4 mL was mixed with 0.4 µg plasmid and then transferred into an electroporation cuvette (Gene Pulser/MicroPulser Cuvette, 0.4 cm gap; Bio-Rad Laboratories). By observing electroporated cells under microscope to examine their mortality ratio, electroporation parameters were set as 1.5 kV, 25 µF, and 400 Ohm, to keep the ratio of normal cells to damaged cells at about 3:1. To achieve high transformation efficiency, the pulsed diatoms were kept in nonselective medium for 24 h to allow recovery before spreading on selection plates. After electroporation, cells were transferred into 10 mL f/2-Si medium, kept in the dark for 2 h, and then incubated in nonselective medium at 23°C for 24 h (12L:12D) under a photon flux density of 200 mol m-2 s-1. Cells were then collected by centrifugation at 1500× g for 5 min and resuspended in 1 mL medium. Transformed cells were selected on selection plates with nitrate as the nitrogen source. The sensitivity of P. tricornutum to chloramphenicol was tested beforehand, and it could not survive ≥200 mg/L chloramphenicol, which is consistent with a previous report (10). Hence, chloramphenicol at 200 mg/L was used for selection of transformants. Surviving cells on selection plates were streaked and later cultured in liquid medium supplemented with chloramphenicol, while untransformed cells inoculated in parallel could not grow even after 2 weeks, indicating that CAT was successfully expressed and resulted in chloramphenicol resistance. The overall transformation efficiency was estimated to be approximately one transgenic colony per 105 cells, similar to those reported for other P. tricornutum transformation systems (7, 10). Transformed cell lines cultured in liquid medium were subjected to PCR screening using primers CATf and CATr, which can amplify full-length coding sequence of CAT gene, and an expected 0.7-kb band was present in the transgenic cell line while absent in wild-type (Figure 1B).
CAT transcription was shown by RT-PCR. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and finally treated with RNase-free DNase I (New England BioLabs, Ipswich, MA, USA) for 30 min at 37°C. First-strand synthesis was carried out using the Superscript First-Strand Synthesis system (Invitrogen). The CAT transcript was amplified using primers CATf and CATr. Actin16 was used as an internal control, and primers were designed according to Siaut et al. (11) to amplify a 156-bp fragment. The 0.7-kb CAT transcript was detected in transgenic cells (Figure 1C, left, lane 1) but not in wild-type cells (Figure 1C, left, lane 2). A 156-bp actin16 band was present in both wild-type and transgenic cells (Figure 1C, middle).
For Western blot analysis, total protein was extracted, and the protein concentration was determined using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories). Protein (20 µg/well) was separated by SDS-PAGE and electrophoretically transferred to PVDF membrane (Pall, Pensacola, FL, USA). The blot was blocked in Tris-buffered saline with Tween (TBST) containing 5% BSA for 2 h and incubated for an additional 2 h with rabbit anti-chloramphenicol antibodies (Bluegene Biotech Ltd, Shanghai, China) at 1:300 dilution. The blot was washed three times with TTBS and then incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5000) (Beijing ComWin Biotech Ltd., Beijing, China) for 1 h. The BeyoECL Plus (Beyotime, Jiangsu, China) kit was used to detect cross-reacting bands. Although the antibodies showed several nonspecific bands, the cross-reacting 29-kDa CAT band (arrowhead) was detected in transgenic cells (Figure 1D, lane 2) while absent in wild-type (Figure 1D, lane 1).
For nitrogen induction, cells were tested for growth up to 3 weeks in chloramphenicol-containing media with either nitrate or ammonium as the nitrogen source. Transgenic cells streaked on the plate containing NaNO3 as the sole nitrogen source could grow well, while no growth occurred on the plate containing NH4Cl as the sole nitrogen source even after a 3-week incubation (Figure 1E). Wild-type cells could not grow in both cases (Figure 1E). These results indicated that CAT expression under NR promoter control was repressed in the presence of ammonium (NH4Cl) and induced by nitrate (NaNO3).
In addition to pHY11, we have successfully transformed constructs incorporating an additional cassette containing either enhanced GFP (eGFP, 6.3-kb construct; Supplementary Figure S1) or phospholipid:diacylglycerol acyltransferase gene (6.9-kb construct, unpublished) driven by the fcpC promoter/fcpA terminator of P. tricornutum. In conclusion, we have demonstrated that electroporation is a convenient method for the transformation of the diatom P. tricornutum and have also presented a tight gene regulatory system useful for conditional gene expression in P. tricornutum.
This work was supported by the National Science and Technology Program (2011BAD14B03), Science and Technology Project of Guangdong (2009B020301002, 2009B050600005, 2010B030600005), and the Fundamental Research Funds for the Central Universities.
The authors declare no competing interests.
Address correspondence to Hong-Ye Li, Key Laboratory of Eutrophication and Control of HAB of Guangdong Higher Education Institute, Jinan University, Guangzhou 510632, China. e-mail: [email protected]
1.) Zaslavskaia, L., and J. Lippmeier. 2000. Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. J. Phycol. 36:379-386.
2.) Chang, D., and T. Reese. 1990. Changes in membrane-structure induced by electroporation as revealed by rapid-freezing electron-microscopy. Biophys. J. 58:1-12.
3.) Dünahay, T.G., E.E. Jarvis, K.G. Zeiler, P.G. Roessler, and L.M. Brown. 1992. Genetic engineering of microalgae for fuel production. Appl. Biochem. Biotechnol. 34-35:331-339.
4.) Brown, L., S. Sprecher, and L. Keller. 1991. Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation. Mol. Cell. Biol. 11:2328-2332.
5.) Niu, Y.F., M.H. Zhang, W.H. Xie, J.N. Li, Y.F. Gao, W.D. Yang, J.S. Liu, and H.Y. Li. 2011. A new inducible expression system in a transformed green alga, Chlorella vulgaris. Genet. Mol. Res. 10:3427-3434.
6.) Kilian, O., C.S. Benemann, K.K. Niyogi, and B. Vick. 2011. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Proc. Natl. Acad. Sci. USA 108:21265-21269.
7.) Miyagawa, A., T. Okami, N. Kira, H. Yamaguchi, K. Ohnishi, and M. Adachi. 2009. High efficiency transformation of the diatom Phaeodactylum tricornutum with a promoter from the diatom Cylindrotheca fusiformis. Phycol. Res. 57:142-146.
8.) Poulsen, N., and N. Kröger. 2005. A new molecular tool for transgenic diatoms: control of mRNA and protein biosynthesis by an inducible promoter-terminator cassette. FEBS J. 272:3413-3423.
9.) Ladygin, V.G. 2004. Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation. Process Biochem. 39:1685-1691.
10.) Apt, K.E., P.G. Kroth-Pancic, and A.R. Grossman. 1996. Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol. Gen. Genet. 252:572-579.
11.) Siaut, M., M. Heijde, M. Mangogna, A. Montsant, S. Coesel, A. Allen, A. Manfredonia, A. Falciatore, and C. Bowler. 2007. Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene 406:23-35.