Abstract
Genetic manipulation of Haematococcus pluvialis is difficult because of the lack of a stable and convenient transformation system. The pH124-EGFP-Ble vector containing ble as a selective gene and EGFP as a reporter gene was constructed and employed for effective transformation. H. pluvialis protoplasts were obtained by treating with cellulase and macerozeme. Then polyethylene glycol-mediated transformation was established by incubating the protoplast with the vector. To improve the transformation efficiency of H. pluvialis protoplasts, the transformation system was optimized in consideration of different influencing factors, including zeomycin concentration, growth stage, amount of transformed vector, linearization of the vector, and duration of low-intensity illumination. The integration and expression of ble and EGFP was confirmed in the transformants. Moreover, the optimal combination for protoplast transformation of H. pluvialis was determined to be 5 µg of the linearized vector used to transform cells in the log growth phase, and then the transformed protoplasts allowed to recover under low-intensity illumination for 6 h. This study represents and describes the successful development of an H. pluvialis transformation protocol using protoplasts, which will enable convenient genetic manipulation of this important algal species.
Highlights
IntroductionHuman health can be improved and various diseases potentially may be prevented by astaxanthin, such as cancer (Emiko et al 2008), diabetes, diabetic nephropathy, eye fatigue and chronic inflammatory, cardiovascular, gastrointestinal, liver, neurodegenerative, and ophthalmic diseases (Chew et al 1999; Wang et al 2000; Gross and Lockwood 2005; Izumi-Nagai et al 2008; Chang et al 2010; Curek et al 2010)
The cell wall is thickened which greatly hinders the extraction of astaxanthin and the transformation of H. pluvialis (Damiani et al 2006)
Following the transformation of pH124-EGFP-Ble into H. pluvialis protoplasts, which were treated with cellulase and macerozeme, transformed cells were poured onto the ESP culture containing zeomycin (5 μg mL−1)
Summary
Human health can be improved and various diseases potentially may be prevented by astaxanthin, such as cancer (Emiko et al 2008), diabetes, diabetic nephropathy, eye fatigue and chronic inflammatory, cardiovascular, gastrointestinal, liver, neurodegenerative, and ophthalmic diseases (Chew et al 1999; Wang et al 2000; Gross and Lockwood 2005; Izumi-Nagai et al 2008; Chang et al 2010; Curek et al 2010). Haematococcus pluvialis has been proven to be the most effective producer of astaxanthin among microalgae, bacteria, and fungi (Hagen et al 2002). Haematococcus pluvialis is highly adaptable to environmental changes, such as temperature, light, and salinity, when compared to other algae. H. pluvialis is in the motile flagellar stage and has a gelatinous extracellular matrix. When exposed to various stress conditions, such as nitrogen and phosphate limitations, as well as salt stress and high light intensities, the cells change into the aplanospore stage in which astaxanthin accumulates significantly. The cell wall is thickened which greatly hinders the extraction of astaxanthin and the transformation of H. pluvialis (Damiani et al 2006)
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