Establishment and optimization of PEG-mediated protoplast transformation in the microalga Haematococcus pluvialis

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.