Biosci. Biotechnol. Biochem., 77 (4), 874–876, 2013
Note
Highly Efficient Transformation of the Diatom Phaeodactylum tricornutum by Multi-Pulse Electroporation Mado M IYAHARA,1 Masaki A OI,1 Natsuko I NOUE-K ASHINO,2 Yasuhiro K ASHINO,2 and Kentaro IFUKU1; y 1 2
Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan Graduate School and Faculty of Science, University of Hyogo, 3-2-1 Kohto, Ako-gun, Hyogo 678-1297, Japan
Received December 6, 2012; Accepted January 10, 2013; Online Publication, April 7, 2013 [doi:10.1271/bbb.120936]
A highly efficient nuclear transformation method was established for the pennate diatom Phaeodactylum tricornutum using an electroporation system that drives multi-sequence pulses to introduce foreign DNAs into the cells. By optimizing pulse conditions, the diatom cells can be transformed without removing rigid silicabased cell walls, and high transformation efficiency (about 4,500 per 108 cells) is achieved. Key words:
diatom; electroporation; Phaeodactylum tricornutum
multi-pulse;
Diatoms are important primary producers in the ecosystem that have crucial roles in the global carbon cycle.1) The complex evolutional history of diatoms as secondary endosymbionts is providing new insight into host-endosymbiont relationships.2) In addition, their unique abilities to form silica-based cell walls and to produce valuable lipids have attracted attention to them as economically important resources.3,4) Both for basic biological research and for commercial exploitation of diatoms, an efficient genetic transformation system is very important. Transformation of ditoms has been reported for several diatom species, including pennate diatoms Phaeodactylum tricornutum,5–8) Cylindrotheca fusiformis,9,10) and Navicula saprophila, and centric diatoms Cyclothella cryptica,11) Thalassiosira pseudonana,12) and Chaetoceros sp.13) In general, delivery of foreign DNAs into diatom cells has been achieved by microprojectile bombardment, but this is not suited to high-throughput transformation of diatom cells due to the time required for sample preparation, costly consumables, and limited transformation efficiency. There is only one report that describes the genetic transformation of P. tricornutum by electroporation,14) and this is still at a rudimental stage of development. In this study, we applied a multi-pulse electroporation system to achieve nuclear transformation of P. tricornutum. We observed that increases in the number of pulses significantly improved transformation efficiency. The transformation efficiency reached 4,500 per 108 cells, which is higher than any previously reported diatom transformation system. P. tricornutum Bohlin (UTEX 642) was derived from the University of Texas Culture Collection. It was
cultured in Daigo’s IMK culture medium (Nihon Pharmaceutical, Osaka, Japan) supplemented with sea salts (Sigma, St. Louis, MO) and 0.2 mM Na2 SiO3 . Cultures were grown at 20 C in an artificial climate incubator. Cool-white fluorescent tubes provided an irradiance of 30 mmol photons m2 s1 under continuous light conditions. To generate transgenic P. tricornutum, plasmid pPhaT1, which has a bleomycin-resistant gene cassette with the fcpB promoter,7) was used. The sGFP gene (sgfp) and the GUS (uidA) gene were amplified by PCR using primer pairs, 50 -GCCGTTTCGAGAATTCATGGTGAGCAAGGGCGAGG-30 and 50 -CACGCTTCTGAAGCTTACTTGTACAGCTCGTCCATGC-30 for sgfp, and 50 -AGAATTCTCCCCGGGTGGTCAG-30 and 50 -AAAAGCTTTGGTGCGCCAGGAGA-30 for uidA. Plasmids CaMV35S-sGFP(S65T)-nos15) and pBI221 were used as templates for PCR. The amplified sgfp and uidA cDNA fragments were inserted between the EcoRI and the HindIII site located in the multi-clonig site of pPha-T1, which is located downstream of the fcpA promoter. Electroporation was done by NEPA21 apparatus (NEPAGENE, Chiba, Japan). Diatom cells at exponential growth phase (OD700 ¼ 0.2–0.4, 5:9 106 cells) were collected by centrifugation at 700 g for 4 min, washed, and resuspended with 0.77 M mannitol. A suspension aliquot of 0.15 mL was mixed with 1–10 mg of plasmid that had been linearized by NdeI, and then transferred to an electroporation cuvette with 0.2 cm gap. After electroporation, the cells were transferred into 4 mL of IMK medium and then incubated to allow recovery in nonselective medium at 20 C for 16–20 h under a photon flux density of 30 mmol photons m2 s1 . Considering the slower growth rate of P. tricornutum cells,16) most of the cells did not be divide during this incubation period. Cells were collected by centrifugation at 700 g over 4 min and resuspended in 0.2 mL of IMK medium. The transformed cells were selected on IMK agar plates containing 1% agar and 100 mg/mL of zeocin (Life Technologies, Carlsbad, CA). To examine the integration of the reporter genes in the diatom genome, genomic DNA was isolated from transformed cells that were subcultured 2–3 times on selective medium. The protocol described by Kim et al.17) was used to isolate genomic DNA. Primer pairs
y To whom correspondence should be addressed. Fax: +81-75-753-6398; E-mail:
[email protected] Abbreviations: GFP, green fluorescent protein; GUS, -glucuronidase
Voltage (V)
P.P.
T.P.
A
Time (ms)
Number of transformants / 10 8 cells
Highly Efficient Transformation of a Diatom by Electroporation
875
5,000 4,000 3,000 2,000 1,000 0 0
1
2
3
4
5
6
7
8
9 10
B
+ 0
− Fig. 1. Illustration of Pulse Regime of Multi-Pulse Electroporation. Multiple high-voltage poring pulses (P.P.) facilitate the formation of temporary pores, and subsequent multiple low-voltage transferring pulses (T.P.) facilitate the introduction of DNA into the cells.
0
0
0
5 -CCGCTTTACTTGTACAGCTCG-3 and 5 -CCATCTTCTTCAAGGACG-30 for sgfp, 50 -GCCACTGGCGGAAGC-30 and 50 -CGCCAACGCGCAATA-30 for uidA, and 50 -TGACAGAGCGTGGTTACTCG-30 and 50 -AGGTCCACATGGACTGGAAG-30 for actin were used in genomic PCR. The GFP fluorescence of the transformed diatom cells was analyzed under a BZ-9000 fluorescent microscope (KEYENCE, Osaka, Japan). The field of cells was excited at 490 nm, and fluorescence emission was detected at 510 nm. GUS staining was performed as described previously.18) Figure 1 illustrates a muti-pulse sequence generated by the electroporation system (NEPA21). Multiple highvoltage pulses of short duration and with 10% voltage decay (poring pulses) facilitate the formation of temporary pores. Subsequent multiple low-voltage pulses (transferring pulses) of long duration and with 40% voltage decay facilitate the delivery of DNA into the cells. During the transfer pulse regime, the electrode polarities are reversed to make the DNA move in the opposite direction, as this increases transformation efficiency. This electroporation system was applied for the efficient transformation of various types of animal cells (http://www.nepagene.jp/E/Eindex.htm). This 2-step multi-pulse electroporation was applied to diatom transformation. It was found that the critical parameter was the number of poring pulses. For direct comparison of transformation efficiency, we used plasmid vector pPha-T1, widely used in previous studies. The GFP (S65T) and the GUS gene (sgfp and uidA respectively) were included in pPha-T1 as reporter genes. The voltage of the poring pulses should be higher than 250 V to achieve zeocin-resistant colonies. The pulse duration can be either of 2.5 ms or 5 ms, whereas a duration of 5 ms was preferred for higher transformation efficiency (data not shown). Figure 2A shows the dependence of the transformation efficiency on the number of poring pulses applied. Transformation efficiency reached a maximum when seven pulses were applied, and further increases in the number of pulses did not improve efficiency.
Number of transformants / 10 8 cells
Number of poring pulses
4,000 3,000 2,000 1,000 0 0
2
4
6
DNA amount [µg / cuvette]
Fig. 2. Optimization of Pulse Conditions for Electroporation. (A) Dependence of transformation efficiency on pulse numbers. Square electric poring pulses were applied at 300 V (pulse duration, 5 ms; 1–9 pulses; interval 50 ms; 10% decay rate), followed by transferring pulses at 8 V (pulse duration, 50 ms; 5 pulses; interval 50 ms; 40% decay rate). (B) Dependence of transformation efficiency on DNA amounts. Square electric poring pulses were applied at 300 V (pulse duration, 5 ms; 8 pulses; interval 50 ms; 10% decay rate), followed by transferring pulses at 8 V (pulse duration, 50 ms; 5 pulses; interval 50 ms; 40% decay rate).
We also examined the buffer for electroporation, and found that complete replacement of IMK medium with 0.77 M mannitol gave good results. Furthermore, increases in the amount of DNA up to 3–5 mg per cuvette improved transformation efficiency (Fig. 2B). It is very important to use a linearized vector to obtain high transformation efficiency. Without linearization, an approximately 10-fold reduction in transformation efficiency was observed (data not shown). The optimized transformation efficiency reached about 4,500 transgenic colonies per 108 cells, 10–100 times higher than reported for other P. tricornutum transformation systems, using microprojectile bombardment and the same pPha-T1 vector.7,8) An additional advantage of the method described here is the shorter period required to obtain zeocin-resistant colonies. Usually it takes 3–4 weeks to observe zeocin-resistant colonies by microprojectile bombardment. In our system, zeocin-resistant colonies started to appear 10–15 d after electroporation. This might have been due to less damage to diatom cells, with intact cell walls. We examined the introduction and expression of the sgfp and uidA genes in transgenic diatom cells. It has been reported that some GFP derivatives do not accumulate efficiently in diatom cells.7) In general, the eGFP gene is used for diatom study because it has a number of silent mutations that ensure optimum codon usage in human cells, and they are coincidentally preferred by P. tricornuntum. In this study, we used the sGFP (S65T) gene, widely used in plant research.
876
M. MIYAHARA et al.
A
wt
G1 G2 G3 G4
wt
U1 U2 U3 U4
sgfp Actin
uidA Actin
C
B
D
staining in the cells containing uidA (Fig. 3D), confirming that expression of the reporter genes was maintained stably during the repeated segregation process. Expression level of sgfp in each transgenic line was estimated from the intensity of GFP fluorescence (Fig. 3E). GFP fluorescence levels were different among the transgenic cell lines and this was probably caused by differences in copy number or the position of the reporter gene in the genome. In conclusion, the application of the multi-pulse electroporation markedly improved the transformation efficiency of the diatom P. tricornuntum. We found that this system was also effective for other algal cells, such as the green algae Chlamydomonas reinhardtii with cell walls (Aoi and Ifuku, unpublished data). The method described here is fast and economical, and should contribute to academic and commercial exploitation of the organism.
Acknowledgments This work was supported by the Japan Science and Technology Agency, Advanced Low Carbon Technology Research and Development Program (to K. I. and Y. K.).
E
References
Fluorescence (Rel. units)
5.0
1)
4.0
2)
3.0 2.0
3)
1.0
4) 5)
0.0 1 2 3 4 5 6 7 8 9 10 11 Transformant
Fig. 3. Integration and Expression of the Reporter Genes in Transgenic Phaeodactylum tricornutum Cells. (A) PCR amplification of the sgfp, uidA, and actin genes in transgenic P. tricornutum genomic DNA. Isolated genomic DNAs from the wild type (wt) and four zeocin resistant clones (G1–G4 for sgfp-transformed cells and U1–U4 for uidA-transformed cells) were used for PCR analysis. Fluorescent (B) and Light (C) photomicrographs of P. tricornutum cells expressing sGFP (S65T). The presence of the sGFP (S65T) protein was indicated by bright-green fluorescence. (D) Light photomicrograph of P. tricornutum cells expressing GUS. GUS activity was detected as blue staining. Scale bar is 10 mm. (E) Fluorescence levels of transformed P. tricornutum cells expressing sGFP. The relative fluorescence levels of the cells in the transgenic lines were estimated by the Image J program.19) Values are means SD for 5–6 cells.
6) 7) 8) 9) 10) 11) 12) 13) 14) 15)
Genomic DNA was isolated from transgenic diatom cells that had been subcultured 2–3 times on fresh selective media after transformation. The presence of the introduced sgfp or uidA gene in the genome was detected by PCR in all the transgenic diatom cells examined (Fig. 3A). Observation under a fluorescent microscope showed bright GFP fluorescence in the cells containing the sgfp gene (Fig. 3B, C) and strong GUS
16) 17) 18) 19)
Falkowski PG, Barber RT, and Smetacek VV, Science, 281, 200–207 (1998). Prihoda J, Tanaka A, de Paula WB, Allen JF, Tirichine L, and Bowler C, J. Exp. Bot., 63, 1543–1557 (2012). Ramachandra TV, Mahapatra DM, Karthick B, and Gordon R, Ind. Eng. Chem. Res., 48, 8769–8788 (2009). Gordon R, Losic D, Tiffany MA, Nagy SS, and Sterrenburg FA, Trends Biotechnol., 27, 116–127 (2009). Apt KE, Kroth-Pancic PG, and Grossman AR, Mol. Gen. Genet., 252, 572–579 (1996). Falciatore A, Casotti R, Leblanc C, Abrescia C, and Bowler C, Mar. Biotechnol. (NY), 1, 239–251 (1999). Zaslavskaia LA, Lippmeier JC, Kroth PG, Grossman AR, and Apt KE, J. Phycol., 36, 379–386 (2000). Miyagawa A, Okami T, Kira N, Yamaguchi H, Ohnishi K, and Adachi M, Phycol. Res., 57, 142–146 (2009). Fischer H, Robl I, Sumper M, and Kro¨ger N, J. Phycol., 35, 113–120 (1999). Poulsen N and Kro¨ger N, FEBS J., 272, 3413–3423 (2005). Du¨nahay TG, Jarvis EE, Zeiler KG, Roessler PG, and Brown LM, Appl. Biochem. Biotechnol., 34–35, 331–339 (1992). Poulsen N, Chesley PM, and Kro¨ger N, J. Phycol., 42, 1059– 1065 (2006). Miyagawa-Yamaguchi A, Okami T, Kira N, Yamaguchi H, Ohnishi K, and Adachi M, Phycol. Res., 59, 113–119 (2012). Niu YF, Yang ZK, Zhang MH, Zhu CC, Yang WD, Liu JS, and Li HY, Biotechniques, 0, 1–3 (2012). Chiu WL, Niwa Y, Zeng W, Hirano T, Kobayashi H, and Sheen J, Curr. Biol., 6, 325–330 (1996). Johnston AM and Raven JA, Mar. Ecol. Prog. Ser., 87, 295–300 (1992). Kim BH, Ramanan R, Cho DH, Choi GG, La HJ, Ahn CY, Oh HM, and Kim HS, PLoS ONE, 7, e37770 (2012). Jefferson RA, Kavanagh TA, and Bevan MW, EMBO J., 6, 3901–3907 (1987). Schneider CA, Rasband WS, and Eliceiri KW, Nat. Methods, 9, 671–675 (2012).
