Phycologia Volume 55 (5), 564–567 Published 23 June 2016

Transient expression of the enhanced green fluorescence protein (egfp) gene in subcordiformis (, ) with three exogenous promoters

1 1,2 1 YULIN CUI †,JIALIN ZHAO † AND SONG QIN * 1Key Laboratory of Coastal Biology and Biological Resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, Shandong, China 2Yantai Ocean Environmental Monitoring Central Station of State Oceanic Administration, Yantai 264006, Shandong, China 3University of Chinese Academy of Sciences, Beijing 101408, China

ABSTRACT: A stable transformation system for Tetraselmis subcordiformis had been established, but a low transformation rate limits its application to this alga. As a promoter is a key factor for transgenic research, a suitable promoter could increase the transformation rate. To improve a transformation system for T. subcordiformis, three commonly used promoters for algal transformation were selected. The CMV, CaMV35S and SV40 promoters were transferred to T. subcordiformis by glass-bead agitation and particle bombardment, with the enhanced green fluorescence protein (egfp) gene as the reporter gene. The transformation rate was calculated according to the GFP fluorescence in transformed algae. Results showed that the CaMV 35S promoter transformation efficiency was higher than that of CMV and SV40 promoters. This report will contribute to efforts to establish a high-efficiency transformation system and expands the fundamental research and applications of this alga.

KEY WORDS: Enhanced green fluorescence protein gene, Promoter, Tetraselmis subcordiformis, Transformation rate

INTRODUCTION Fundamental research into T. subcordiformis is rare, especially regarding genome information. On this basis, it Tetraselmis subcordiformis (H.J.Carter) Stein is a unicel- is possible in research to use an exogenous promoter lular marine green alga with broad application prospects rather than an endogenous promoter. In algal transfor- in aquaculture and bioenergy. Because of its rich content mation research, the commonly used efficient promoters of amino acids and fatty acid, it is used as feed for bivalve contain the CaMV 35S promoter (El-Sheekh 1999; mollusks, rotifers, fishes, and penaeid shrimp larvae. In Kathiresan et al. 2009), the CMV promoter (Cui et al. recent years, there have been many reports regarding 2010) and the SV40 promoter (Teng et al. 2002; Cui et al. hydrogen gas production by T. subcordiformis (Guan et 2012). In the present study, the focus was on screening for al. 2004; Guo et al. 2008), which demonstrates that this asuitablepromoterwithanimprovedrateinT. alga could possibly be used for sustainable energy subcordiformis to provide evidence for establishing a production (Wei et al. 2015). high-efficiency transformation system, advance funda- Genetic engineering is a basic and necessary tool for mental research, and expand potential applications of this fundamental research. In earlier research, this lab alga. established a stable, nuclear transformation system of T. subcordiformis using glass-bead agitation and particle bombardment (Cui et al. 2012). In that study, we reported MATERIAL AND METHODS that the transformation rate of particle bombardment was 1.63 3 106; whereas, that of glass beads was 6.54 3 Strains of T. subcordiformis were grown in a modified f/2 106. Thus, the transformation rates from these two medium (vitamin free; Guillard & Ryther 1962) at 238C– 2 1 methods were lower than needed, and their application 258C at 80–90 lmol photons m s and a 12:12 was hindered as a result. Thus, a more efficient light:dark photoperiod. Cells were harvested at mid–log transformation method was needed to improve the phase, and culture density was measured using a expression rate. A suitable promoter is an essential tool hemocytometer. for genetic engineering. According to reports of higher Three vectors with different promoters were adopted to transformations, the promoter is the key factor for regulate egfp gene expression (Table 1 and Fig. 1). All research on transgenes, regulation mechanisms and other vectors were amplified in the Escherichia coli Top10 areas (Li et al. 2007). strain. Plasmid DNA was isolated from E. coli with a plasmid small extraction kit (Tiangen Biotech Co., Beijing, China). * Corresponding author ([email protected]). DOI: 10.2216/15-136.1 Cells of T. subcordiformis at log phase were collected Ó 2016 International Phycological Society by centrifugation at 6000 rpm (approximately 3500 3

564 Cui et al.: Transformation of Tetraselmis 565

Table 1. Vectors used in this study.

Vector Promoter Label Resistance pEGFP-N11 CMV EGFP kanamycin pBI221-EGFP2 CaMV 35S EGFP ampicillin pCMS-EGFP1 SV40 EGFP ampicillin 1 Clontech Laboratories, Inc. (Palo Alto, California USA). 2 YouBio (Changsha, China). g) for 5 min. Then 50–100 ll of harvested algae (with about 1 3 108 cells) were spread onto the central area of f/2 agar plates to a diameter of 2 cm. Gold carrier particles were mixed with plasmid DNA, according to Jiang et al. (2003). Plates were then bombarded with Fig. 1. Structure of the plasmid pEGFP-N1, PBI221-EGFP and PCMS-EGFP used in transformation of Tetraselmis subcordiformis. 12 ll of DNA (~ 1 lg) per plate at 450, 650, 900 and Bar ¼ 100 base pairs. 1100 psi from two distances: 6 and 9 cm. After transformation, plates were regenerated in darkness for 8 h and then transferred. A plate bombarded with gold powder without plasmid DNA was used as a negative RESULTS control. Cell walls of T. subcordiformis must be degraded Detection of EGFP expression with a lytic enzyme mixture produced from abalone In cells of T. subcordiformis, the combination of green acetone powder and cellulose solutions before glass- fluorescence from EGFP and red fluorescence from chloro- bead agitation (Cui et al. 2010). The resulting phyll produced a bright greenish-yellow fluorescence. When protoplasts were harvested by centrifugation at 1000 observed with a fluorescence microscope, nontransformed rpm (approximately 150 3 g) for 10 s and washed twice cells emitted the characteristic red fluorescence of chlorophyll; in 0.5 M mannitol in seawater, and the cells whereas, transformed cells produced a bright greenish-yellow resuspended in 300 llof0.5Mmannitolinseawater. fluorescence, indicating the presence of EGFP (Figs 2–13). Next, ~ 300 mg of glass beads with a diameter of 0.425–0.600 mm (Sigma-Aldrich, St. Louis, Missouri Comparison of transformation rate USA), 1 lg of plasmid DNA, 15 lgPEGand~ 5 3 7 Using glass beads, the transformation rate with pBI221-EGFP 10 cells (final volume about 700 ll) were mixed and 3 agitated at top speed on a vortex mixer (XM-80A, was the highest, at ~ 10 . The transformation rate with the SV40 promoter was ~ 6 3 104; whereas, the rate with the Jingke Co., Shanghai, China) for 25 s. Protoplasts 4 agitated without plasmid DNA were used as blank CMV promoter was ~ 5 3 10 (Table 2). Using particle bombardment, the rate with CaMV 35S promoter was the controls, and cells without the preceding step were highest, at ~ 3.0 3 105, followed by CMV and SV40 considered negative controls. Finally, the cells were promoters, with rates of 1.0 3 105 and 2.0 3 105, collected, cultured in 20 ml of mannitol in seawater in respectively. the dark for about 8 h, collected again and resus- pended in f/2 medium. All experiments were performed in triplicate. DISCUSSION EGFP expression of transformed cells was detected 72 h after transformation (Garc´ıa-Mata et al. 1999) with a We introduced the CaMV 35S, SV40 and CMV Nikon Eclipse 50i microscope (Nikon, Tokyo, Japan) promoters into cells of T. subcordiformis by glass-bead using blue-light excitation (450–520 nm). The volume of agitation and particle bombardment and detected GFP each sample was ~ 200 ll. Images were obtained with 5-s fluorescence by fluorescence microscopy, with the fluo- exposure time and analyzed with Adobe Photoshop rescence indicating successful transformation. According software. The transformed algae were monitored per the to a comparison of transformation rates, the transfor- above manipulation for ~ 15 d. mation rate of the CaMV 35S promoter was the highest, After counting the cell density of each culture with a and the transformation rates of the SV40 and CMV hemocytometer, a portion of algal culture (~ 10% by promoters were almost the same. volume) from each sample was removed for detection The CaMV 35S promoter is widely used for algal according to the following formula (Kindle 1990): transgenic processes and is a constitutive promoter for highly efficient gene expression. The SV40 promoter, Transformation rate from both monkeys and humans, exhibits an astonish- Positive cells ¼ : ing performance when applied to transgenic brown Detected cells 3 Proportion of detected cells algae (Teng et al. 2002; Ladygin 2003). It is quite 566 Phycologia, Vol. 55 (5)

common for the CMV promoter to be used in animal genetic engineering; whereas, there are few reports of its application to algae. In early research here, it performed efficiently in T. subcordiformis. The results of this study showed that the CaMV 35S promoter was quite suitable for the transformation of T. subcordiformis.Yooet al. (2005) reported that the 35S promoter effectively places its downstream gene outside any regulatory control by the host genome and, as a result, expresses the gene at approximately two to three orders of magnitude higher than normal; furthermore, the CaMV 35S promoter sequence has been found in Cauliflower mosaic virus (Franck et al. 1980). While the SV40 and CMV promoters come from animal viruses, as a plant promoter, the CaMV 35S promoter appears to be more easily identified in this alga. In this study, the transformation rate of CaMV 35S promoter was higher than that of CMV and SV40. Furthermore, this study provided evidence that CMV promoters could be applied to genetic transformations of . But the endogenous promoters always showed a higher rate than the exogenous ones in higher and some algae (Apt et al. 1996). Some reports regarding tandem promoters driving foreign gene expressions have indicated that their expression is significantly greater than with a single promoter driving gene expression in plants (Wu et al. 2008). Thus, further research efforts will aim to improve the genetic transformation rate in the two effects for T. subcordiformis.

Fig. 2. Transformed Tetraselmis subcordiformis with the CaMV 35S promoter by particle bombardment under white light. Bar ¼ 10 lm. Fig. 3. Transformed T. subcordiformis with the CaMV 35S promoter by particle bombardment under blue light excitation. Bar ¼ 10 lm. Fig. 4. Transformed T. subcordiformis with the CaMV 35S promoter by glass bead under white light. Bar ¼ 10 lm. Fig. 5. Transformed T. subcordiformis with the CaMV 35S promoter by glass bead under blue light excitation. Bar ¼ 10 lm. Fig. 6. Transformed T. subcordiformis with the CMV promoter by particle bombardment under white light. Bar ¼ 10 lm. Fig. 7. Transformed T. subcordiformis with the CMV promoter by particle bombardment under blue light excitation. Bar ¼ 10 lm. Fig. 8. Transformed T. subcordiformis with the CMV promoter by glass bead under white light. Bar ¼ 10 lm. Fig. 9. Transformed T. subcordiformis with the CMV promoter by glass bead under blue light excitation. Bar ¼ 10 lm. Fig. 10. Transformed T. subcordiformis with the SV40 promoter by particle bombardment under white light. Bar ¼ 10 lm. Fig. 11. Transformed T. subcordiformis with the SV40 promoter by particle bombardment under blue light excitation. Bar ¼ 10 lm. Fig. 12. Transformed T. subcordiformis with the SV 40 promoter by glass bead under white light. Bar ¼ 10 lm. Fig. 13. Transformed T. subcordiformis with the SV 40 promoter by glass bead under blue light excitation. Bar ¼ 10 lm. Cui et al.: Transformation of Tetraselmis 567

Table 2. Transformation rate in Tetraselmis subcordiformis with three vectors with glass beads agitation and particle bombardment.

Transformation rate with Transformation rate with glass beads agitation particle bombardment Vector Promoter (three repeats with nine samples) (three repeats with nine samples) pBI221-EGFP CaMV35S (1.23 6 0.32) 3 103 (3.32 6 0.51) 3 105 pCMS-EGFP SV40 (6.17 6 0.41) 3 104 (1.36 6 0.37) 3 105 pEGFP-N1 CMV (5.01 6 0.28) 3 104 (2.47 6 0.34) 3 105

ACKNOWLEDGEMENTS GUO Z., CHEN Z., ZHANG W., YU X. & JIN M. 2008. Improved hydrogen photoproduction regulated by carbonylcyanide m- chlorophenylhrazone from marine green alga Platymonas sub- This work was supported by National Natural Science cordiformis grown in CO2-supplemented air bubble column Foundation of China (41406192, 41176144, 41376139), bioreactor. Biotechnology Letters 30: 877–883. National Key Technology R&D Program of China JIANG P., QIN S., & TSENG C.K. 2003. Expression of the lacZ (2013BAB01B01), National High-tech R&D Program of reporter gene in sporophytes of the seaweed Laminaria japonica China (2014AA093505) and Ocean Public Welfare Scientific (Phaeophyceae) by gametophyte-targeted transformation. Plant Research Project, State Oceanic Administration of China Cell Reports 21: 1211–1216. KATHIRESAN S., CHANDRASHEKAR A., RAVISHANKAR G.A. & SARADA (201205027). R. 2009. Agrobacterium-mediated transformation in the green alga Haematococcus pluvlalis (, Volvocales). Jour- nal of Phycology 45: 642–649. REFERENCES KINDLE K.L. 1990. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences 87: 1228–1232. APT K.E., KROTH-PANIC P.E. & GROSSMAN A.R. 1996. Stable LADYGIN V.G. 2003. The transformation of the unicellular alga nuclear transformation of the diatom Phaeodactylum tricorntum. Chlamydomonas reinhardtii by electroporation. Microbiology 72: Molecular and General Genetics 252: 572–579. 658–665. CUI Y.L., WANG J.F., JIANG P., BIAN S.G. & QIN S. 2010. LI J., QU D.J., LIU L.L, FENG S.Y. & XUE L.X. 2007. Comparison Transformation of Platymonas (Tetraselmis) subcordiformis of stable expression of foreign genes driven by different (, Chlorophyta) by agitation with glass beads. promoters in transgenic Dunaliella salina. China Biotechnology World Journal of Microbiology and Biotechnology 26: 1653–1657. 27: 47–53. CUI Y.L., JIANG P., WANG J.F., LI F.C., CHEN Y.J., ZHENG G.T. & TENG C.Y., QIN S., LIU J.G., YU D.Z., LIANG C.W. & TSENG C.K. IN Platymonas Tetraselmis Q S. 2012. Genetic transformation of ( ) 2002.Transient expression of lacZ in bombarded unicellular green subcordiformis (Prasinophyceae, Chlorophyta) using particle alga Haematococcus pluvialis. Journal of Applied Phycology 14: bombardment and glass-bead agitation. Chinese Journal of 495–500. Oceanology and Limnology 30: 471–475. WEI L.K., HUANG X.X. & HUANG Z.Z. 2015. Temperature effects on EL-SHEEKH M.M. 1999. Stable transformation of the intact cells of Chlorella kessleri with high velocity microprojectiles. Biologia lipid properties of microalgae Tetraselmis subcordiformis and Plantarum 42: 209–216. Nannochloropsis oculata as biofuel resources. Chinese Journal of FRANCK A., GUILLEY H., JONARD G., RICHARDS K. & HIRTH L. 1980. Oceanology and Limnology 33: 99–106. Nucleotide sequence of Cauliflower mosaic virus DNA. Cell 21: WU J.X., HU Z.L., WANG C.G., LI S.F. & LEI A.P. 2008. Efficient 285–294. expression of green fluorescent protein (GFP) mediated by a GARCI´A-MATA R., BEBO¨ K Z, SORSCHER E.J. & SZTUL E.S. 1999. chimeric promoter in Chlamydomonas reinhardtii. Chinese Journal Characterization and dynamics of aggresome formation by a of Oceanology and Limnology 26: 242–247. cytosolic GFP-chimera. Journal of Cell Biology 146: 1239–1254. YOO S.Y., BOMBLIES K., YOO S.K., YANG J.W. & CHOI M.S. 2005. GUAN Y.F., ZHANG W., DENG M.C., JIN M.F. & YU X.J. 2004. The 35S promoter used in a selectable marker gene of a plant Significant enhancement of photobiological H2 evolution by transformation vector affects the expression of the transgene. carbonylcyanide m-chlorophenylhydrazone in the marine green Planta 221: 523–530. alga Platymonas subcordiformis. Biotechnology Letters 26: 1031– 1035. GUILLARD R.R.L. AND RYTHER J.H. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula Received 30 November 2015; accepted 13 April 2016 confervacea Cleve. Canadian Journal of. Microbiology 8: 229–239. Associate Editor: Elena Tarakhovskaya