Development of an expression system using the heat shock protein 70 promoter in the red macroalga, Porphyra tenera

Porphyra is a commercially valuable source of food and drugs and an important model organism for algal research. However, genetic research on Porphyra tenera has been limited by a lack of a heterologous gene expression system. In this study, we isolated native promoter PtHSP70 for the efficient expression of foreign genes in this organism. This promoter lies approximately 1 kb upstream of the heat shock protein 70 coding sequence and was isolated using adapter ligation-mediated genomic polymerase chain reaction. Promoter activity was evaluated using the synthetic GUS gene (PyGUS) with optimized codons for Porphyra yezoensis. Interestingly, the PtHSP70 promoter allowed the efficient expression of PyGUS in P. tenera and P. yezoensis, whereas the PyGAPDH promoter from P. yezoensis was not fully functional in P. tenera. The PtHSP70 promoter may have a more conserved regulatory mechanism than the PyGAPDH promoter between these species, suggesting that PtHSP70 could serve as a universal promoter for Porphyra species. We also established an efficient transient transformation system for P. tenera by evaluating transformation parameters including gold particle quantity, helium and vacuum pressure, developmental stages of leafy gametophytes, and target distance. Under optimal conditions of transient transformation, the frequency of GUS expression was determined by histochemical staining as 30-50 cells per bombardment. In addition, PyGUS expression was detected during the regeneration of monospores in P. tenera, indicating successful genetic transformation. Therefore, the new transient transformation system using the PtHSP70 promoter can be used for foreign gene expression in P. tenera, which may advance the development of P. tenera as a model organism.