An exploration of the rapid transformation method for Dunaliella salina system
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As a new expression system, Dunaliella salina (D. salina) has bright prospects and applications in various fields. However, its application is currently restricted because of the low expression and instability of foreign gene in D. salina cells. During genetic operation, transformation is a crucial step for genes expression in D. salina system. Although several transformation methods are existing currently, many inherent deficiencies and limitations still can be found in actual practice. Thus, we attempted to set up a rapid transformation method using the change of salt concentrations for D. salina. Based on osmotic pressure difference, exogenous genes can be spontaneously transferred into D. salina cells. After that, transformed D. salina cells were subjected to histochemical and molecular analysis. The results showed that the reporter gene, beta-glucuronidase genes were successfully expressed in the positive transformants, and detected in all of transformed cells by PCR analysis. Moreover, different transformation parameters, containing the salt gradient, time, dye dosage and Triton X-100 concentration, were optimized to obtain an optimal transformation result. Taken together, we preliminarily established a rapid transformation method with the features of fast, simple, economic, and high-efficient. This method will provide a strong genetic manipulation tool for the future transformation of D. salina system.
KeywordsDunaliella salina Transformation Salt gradient High efficiency Rapid method
- D. salina
Currently, microalga as a versatile expression system that has been widely used in the fields of vaccine (Dehghani et al. 2018), bio-based chemicals (Ng et al. 2017), metabolic engineering (Anila et al. 2016), pharmaceutical engineering (Zhang et al. 2018), and so on. Among them, since Dunaliella salina (D. salina) offers numerous special advantages, it has been exploited as a novel expression system for production of recombinant proteins (Poungpair et al. 2014; Feng et al. 2014a, b, c). Although several exogenous genes have been transformed into D. salina, such as the human canstatin (Feng et al. 2014a), soybean kunitz trypsin inhibitor (Chai et al. 2012), and white spot syndrome virus VP28 (Feng et al. 2014b), and interferon-thymosin fusion proteins (Zhang et al. 2018), most of them exhibited the low and instable expression in nuclear system of D. salina. Thus, improving the expression level of foreign gene in D. salina cells is an urgent issue for current study.
In the genetic operation of D. salina, transformation is a key step for expression of exogenous genes. So far, several transformation methods have been established for D. salina system, like electroporation (Lü et al. 2009), bombardment particle (Tan et al. 2005), and glass beads (Feng et al. 2009). Each of them has some inherent disadvantages with respect to simplicity, economy, operability and reproducibility, which made them have great limitations in the practical transformation of D. salina. Given this, more efficient and rapid approach should be established for future transformation of D. salina.
Due to it can grow in 0.1–5 M salt concentration, D. salina has the remarkable halotolerant ability in practical culture (Feng et al. 2014b). Based on this feature, we attempted to establish a rapid transformation method using the difference of salt gradients. When salt gradient decreased from high to low, cells permeability was increased instantaneously and then exogenous plasmids introduced simultaneously into D. salina cells. Moreover, to obtain an optimal transformation rate, we optimized the different transformation parameters which including salt gradient, time, dye dosage and Triton X-100 concentration. Under the optical transformation conditions, exogenous genes could be high-efficiently transferred into D. salina cells. Our study showed that a simple and rapid transformation method successfully established for transformation of D. salina cells.
Materials and methods
Algal strain and culture condition
The D. salina strain UTEX-1644 was purchased from the Culture Collection of Algae (University of Texas, USA). Under light intensity of 50 µM photon m−2s−1 with a 12 h-light/day, D. salina cells were cultured in 2 L beaker with the modified PKS medium at 26 °C (Feng et al. 2009). The modified liquid medium consist of 1.5 M NaCl, 1 µM CuCl2·2H2O, 10 mM KNO3, 50 mM NaHCO3, 0.4 mM KH2PO4, 185 µM H3Bo3, 2 µM FeCl3·6H2O, 5 mM MgSO4·7H2O, 5 µM EDTA, 1 µM (NH4) Mo7O24·4H2O, 7 µM MnCl2·4H2O, 1 µM ZnCl2, 1 µM CoCl2·6H2O, and 0.2 mM CaCl2. When growth reached logarithmic phase (about 105–106 cells/mL), D. salina cells were harvested by centrifugation for future use. After washed three times with the fresh medium, D. salina cells were re-suspended and the cells concentration was adjusted to about 106 cells/mL for further use.
Manipulation protocols with the salt gradients
Ethidium bromide (EB) was purchased from Solarbio Science & Technology Co. Ltd (Beijing, China); and was formulated into different concentration gradients. Triton X-100 was purchased from Baoxin biotechnological Co. Ltd (Luoyang, China), and was made as a 0.1% stock solution. After 8 h cultured in 1 M medium, D. salina cells were harvested and then re-suspended with the 0.1 M fresh medium. Meanwhile, different concentrations of Triton X-100 (5, 10, 15, 20, 25 and 30 µL) and EB (1.25%, 2.5%, 3.75%, 5%, 6.25% and 7.5%) were immediately added to 1 mL D. salina separately. And then, this mixture was blended briefly using inverting tube. D. salina cells were observed quickly at the point in time of 60 s, 90 s, 120 s, 150 s and 180 s under a fluorescence microscope. In this experiment, four factors were determined orderly which including salt gradient, time, TritonX-100 concentration and EB amount. When one variable of four variables was optimized, the other three parameters were unchanged as described above. The independent experiments were repeated three times at least.
Transformation of D. salina cells with the plasmids
In this study, all the numbers came from independently repeated experiments at least three times. The transformants were counted by the number of blue cells in total transformed cells. Using SPSS version 17.0, all data were carried out with one-way analysis of variance. In which, the value P < 0.05 was considered statistically significant in all statistical analyses.
Morphology of stained D. salina cells with EB
Optimization of different parameters
Detection of GUS gene expression
To improve the gene expression in D. salina cells, and then facilitate the maturation of D. salina system, a robust transformation tool is essential for production of recombinant proteins. Since the shortcomings of current methods, we attempted to explore a simple and rapid transformation method using salt gradient for D. salina. Using EB as the staining dye, different transformation parameters were fast determined in the study. Among them, effect of salt concentration is the most obvious factor on the transformation rate. The transformation rate increased significantly along with the decrease of salt concentration. When salt concentration was less than 0.1 M, the transformation rate greatly reduced due to the water absorption of cells that ended up with the cells rupture. Although followed the increasing of time extension, the transformation rate declined distinctly owing to cells lysis when time was more than 120 s. Because Triton X-100 can dissolve the lipid bilayer of cells membrane, the tremendous destruction of Triton X-100 leads to a remarkable decrease of the number of transformants when its concentration was over than 15 µL. Collectively, in present study, the optimal transformation conditions was determined as follows: adding 15 µL 0.1% Triton X-100 and 10% EB to 1 mL culture (0.1 M salt concentration) with the treating time of 90–120 s.
In this study, relied on the features of quick, high sensitive, efficient staining, EB was used to rapidly identify the transformation results. Although the transformation of EB is different from that of plasmids, its transformation processes and parameters can be referred to the next transformation work of D. salina system. Here, we preliminarily stated the feasibility of this method for transformation of D. salina cells. Compared with the other methods, it has a significant transformation efficiency and the minimal damage to cells (Lü et al. 2009; Tan et al. 2005; Feng et al. 2009; Chai et al. 2012). However, the other transformation parameters were not optimized for transformation of plasmids into D. salina cells, which would be deeply studied in next works. The positive transformants were only identified at the level of cells and nucleic acid. The bio-activities of recombinant proteins were still not analyzed, which was the work in progress. Taken together, all the results demonstrated that an alternative rapid method has been successfully established for D. salina transformation. And it will provide a strong genetic manipulation tool for the future transformation of D. salina system.
The authors gratefully acknowledge Prof. Russell Carlson of Department of Biochemistry & Molecular Biology of University of Georgia for modifying and polishing this manuscript.
GN, WW, LN carried out the experiments of EB staining and drafted the manuscript. YL, AF, JX optimized the transformation parameters of this study. JW, MY, SY made contribution to the transformation of foreign genes into D. salina cells. SY supervised the project and revised the manuscript. All authors read and approved the final manuscript.
This work was funded by the National Natural Science Foundation of China (Nos. 31571289, U1804112), the Young Backbone Teacher Project of Henan Province Universities, China (No. 2012GGJS-080).
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The authors declare that they have no competing interests.
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