Establishment of a micro-particle bombardment transformation system for Dunaliella salina
Abstract
In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. salina with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 microg/ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.
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- Various other studies have reported that D. salina cells are extremely sensitive to Basta®. Jiang et al. (2005) reported that the concentrations inhibiting the growth of D. salina were 0.5 μg/ml in liquid medium and 1 μg/ml in solid medium, and Tan et al. (2005) reported that a concentration of 20 μg/ml was sufficient to completely inhibit the growth of D. salina. Our findings suggest that the appropriate concentrations of chloramphenicol and Basta® for the selection of transgenic D. salina are 1500 and 5 μg/ml, respectively, and that D. salina cells are more sensitive to Basta® than to chloramphenicol.
[Show abstract] [Hide abstract] ABSTRACT: Background The objective of this study was to develop an efficient selectable marker for transgenic Dunaliella salina. ResultsTests of the sensitivity of D. salina to the antibiotic chloramphenicol and the herbicide Basta® showed that cells (1.0 × 106 cells/ml) treated with 1000 or 1500 μg/ml chloramphenicol died in 8 or 6 days, respectively, whereas D. salina cells (1.0 × 106 cells/ml) treated with 5, 10, 20, or 40 μg/ml Basta® died in 2 days. Therefore, D. salina is more sensitive to Basta® than to chloramphenicol. To examine the possibility of using the phosphinothricin N-acetyltransferase (pat) gene as a selectable marker gene, we introduced the pat genes into D. salina with particle bombardment system under the condition of helium pressure of 900 psi from a distance of 3 cm. PCR analysis confirmed that the gene was stably inserted into the cells and that the cells survived in 5 μg/ml Basta®, the medium used to select the transformed cells. Conclusions The findings of this study suggest that the pat gene can be used as an efficient selectable marker when producing transgenic D. salina.- Several species of Chlorella have been transformed as well, most notably Chlorella vulgaris [87]. Green algae in the Dunaliella genus, which have significant commercial potential and have been transformed in both the nuclear and chloroplast genomes, include Dunaliella salina [88, 89] and Dunaliella tertiolecta [90]. Two species of dinoflagellates have been transformed using silicon carbide whiskers [91], as well as two species of red algae, including chloroplast transformation in Porphyridium spp.
[Show abstract] [Hide abstract] ABSTRACT: Algae encompass an enormously diverse set of photosynthetic organisms, which have relatively recently been investigated for industrial&;#x02010;scale production of bioproducts. Their fast growth and minimal nutritional requirements make them suitable candidates for large&;#x02010;scale applications such as biofuel production, yet they are also capable of producing complex, high&;#x02010;value products such as therapeutic proteins, vaccines, and nutritional supplements. As advances are made in the toolkit for algal genetic manipulation and metabolic engineering, algae are likely to emerge as a viable production platform for a wide range of products.- Although research has yielded several expression systems for exploitation of the algae for its products, hitherto available systems possess several disadvantages in the production of recombinant products, nutraceutical by-products, biodiesel or other value added products. Regardless of the existence of several techniques for gene transformation in D. salina, such as electroporation [1], glass bead [2] , biolistic gun [3], silicon carbide whiskers [4], protoplast transformation [5], microinjection [6] and PEG-mediated transformation [7], Agrobacterium-mediated transformation remains one of the most promising method for transient expression and stable integration of foreign gene into the algal host. Extensive work has been carried out in the development of efficient Agrobacterium-mediated transformation protocols for microalgae with various types of inducers[8, 9].
[Show abstract] [Hide abstract] ABSTRACT: An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.- Also the endogenous arylsulfatase (ars) gene is a suitable reporter gene in microalgae [200, 294]. The Escherichia coli β-glucuronidase (GUS) uidA reporter gene has been used successfully in several algal species such as Dunaliella salina [232], Chlorella kessleri [237], Chlorella vulgaris [241], Porphyra yezoensis [206], Porphyra miniata [244], Thalassiosira weissflogii [247], Cylindrotheca fusiformis [247] and Phaeodactylum tricornutum [249, 292]. The Escherichia coli β-galactosidase gene (lacZ) was shown to be a valuable reporter in the red alga Gracilaria changii [208] as well as in the brown algae Laminaria japonica [210] and Undaria pinnatifida [211].
[Show abstract] [Hide abstract] ABSTRACT: BACKGROUND: Recent years have witnessed fast growing developments in algae biotechnology. There is a broad range of diversity in algae biotechnology research and industry. METHODS: A general literature review on all aspects of algae biotechnology was conducted. The main findings are summarized and the relevance for further research and biotechnological applications is discussed. RESULTS: Algae are used as bioreactors for producing bioproducts such as pharmaceuticals, nutraceuticals, cosmetics, pigments and other useful chemicals, algal-based biomaterials, feed and more. Light-sensitive proteins from algae represent a cornerstone in the emerging field of optogenetics. In addition, many efforts are currently being undertaken to make algae competitive for production of bioenergy and biofuels, aiming to evolve into integrated biorefineries. Applied research approaches require mass-culture strategies for algae including bioprocess engineering, fermentation, harvesting, and downstream processing. Some algal-based strategies also meet the requirements for use in bioremediation, biodegradation or other environmental applications. A powerful driving force in algae biotechnology is the enticing option to use genetically improved organisms. Selectable marker genes, reporter genes, promoters, transformation techniques and other genetic tools and methods are already available for several few algae species and this molecular toolbox is becoming increasingly powerful. Quite a few algae genome sequencing projects are completed and others are in progress or planned facilitating genetic engineering. Transgenic algae promise a much broader field of application than unmodified organisms or breeds, e.g., through additionally acquired physiological capabilities and new biochemical reactions, and open the door to improved algal bioproducts and molecular farming. CONCLUSION: Algae are an extremely diverse group of organisms and therefore provide a substantial reservoir of biomolecules, cellular functions and physiological characteristics. Insight into cellular and molecular mechanisms and the opportunity to use algae as green cell-factories have resulted in a constantly growing economic importance of algae technologies and products.- Many attempts to optimize the genetic transformation of new microalgae species have been based on heterologous promoters typically used in higher plants, such as the 35S promoter from the cauliflower mosaic virus (CaMV35Sp) or the nopaline synthase promoter (NOSp) from Agrobacterium tumefaciens. In many cases, typical plant plasmids, such as pBI121 (Talebi et al. 2013), pBI221 (Tan et al. 2005), or those from pCAMBIA series (Kathirsesan et al. 2009; Guo et al. 2013; Úbeda-Mínguez et al. 2015) have been used, directly or with minor modifications, for transformation of microalgae. These heterologous promoters have the enormous advantage of being universal.
[Show abstract] [Hide abstract] ABSTRACT: The choice of strong efficient promoters is a critical step in the development of efficient transformation systems for microalgae; however, the physiological and genetic diversity among microalgae groups makes very difficult to develop standard universal plasmids for a wide number of microalgal species as has been achieved for higher plants. Here, we propose a new approach to express transgenes in microalgae: cotransformation with two naked promoterless genes, a selectable antibiotic-resistant gene and a gene of our interest. These genes are randomly inserted into the nuclear genome, where their transcription relies on their adequate insertion in a region adjacent to an endogenous genomic promoter or in frame with a native gene. In a high percentage of the transformants obtained, both genes are, not only adequately incorporated in the nuclear genome, but also efficiently transcribed and translated. This transformation method is validated in the model microalga Chlamydomonas reinhardtii with the bleomycin-resistant gene from Streptoalloteichus hindustanus (ShBLE) as gene of interest, and it is employed to express a flocculin gene from Saccharomyces bayanus (SbFLO5), which is responsible for the flocculation process in yeasts. Chlamydomonas reinhardtii transformants exhibited self-flocculation abilities between 2- and 3.5-fold higher than the control untransformed strain. The successful cotransformation of C. reinhardtii with two promoterless genes opens doors for the establishment of a universal transformation system based on endogenous promoters, applicable to any microalgal species.- Further the alga being halophilic and autotrophic is also suitable for outdoor cultivation. Also genetic transformation procedures in the alga have been established through various methods (Tan et al. 2005; Geng et al. 2003; Walker et al. 2005a, b; Degui et al. 2002; Sun et al. 2005; Jin et al. 2001; Fig. 1 Schematic representation of the various intermediates that are formed upon the action of b-carotene ketolase and b-carotene hydroxylase in different transgenic systems Photosynth Res Anila et al. 2011). Essentially, the metabolic engineering discussed in the current study is an attempt to extend the existing carotenoid pathway in D. salina through a single gene (bkt) for production of ketocarotenoids including astaxanthin from b-carotene.
[Show abstract] [Hide abstract] ABSTRACT: Dunaliella is a commercially important marine alga producing high amount of β-carotene. The use of Dunaliella as a potential transgenic system for the production of recombinant proteins has been recently recognized. The present study reports for the first time the metabolic engineering of carotenoid biosynthesis in Dunaliella salina for ketocarotenoid production. The pathway modification included the introduction of a bkt gene from H. pluvialis encoding β-carotene ketolase (4,4′β-oxygenase) along with chloroplast targeting for the production of ketocarotenoids. The bkt under the control of Dunaliella Rubisco smaller subunit promoter along with its transit peptide sequence was introduced into the alga through standardized Agrobacterium-mediated transformation procedure. The selected transformants were confirmed using GFP and GUS expression, PCR and southern blot analysis. A notable upregulation of the endogenous hydroxylase level of transformants was observed where the BKT expression was higher in nutrient-limiting conditions. Carotenoid analysis of the transformants through HPLC and MS analysis showed the presence of astaxanthin and canthaxanthin with maximum content of 3.5 and 1.9 µg/g DW, respectively. The present study reports the feasibility of using D. salina for the production of ketocarotenoids including astaxanthin.
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