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

Abstract

Porphyra is a commercially valuable source of food and drugs, and represents an important model organism for algal research. However, genetic research on P. tenera has been limited due to lack of a heterologous gene expression system. In the present study, we isolated a native promoter, the PtHSP70 promoter, for efficient expression of foreign genes in this organism. This promoter lies approximately 1 kb upstream of the coding sequence for Heat Shock Protein 70 (HSP70) and was isolated using adapter-mediated genomic PCR. Promoter activity was evaluated using the synthetic GUS gene (PyGUS) with optimized codons for Porphyra yezoensis. Interestingly, the PtHSP70 promoter allowed equivalent expression of PyGUS in both P. tenera and P. yezoensis, whereas the GAPDH promoter from P. yezoensis was not fully functional in P. tenera. These data suggest that the PtHSP70 promoter has a more conserved regulatory mechanism than the PyGAPDH promoter between these species.We also established an efficient transient transformation system for P. tenera by evaluating various transformation parameters such as quantity of gold particles, pressure of helium and vacuum, developmental stages of leafy gametophytes, and target distance. Under the optimal conditions of transient transformation, the frequency of GUS expression was determined by histochemical staining to be 30-50 cells per bombardment. 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.

Transient Transformation of Red Algal Cells: Breakthrough Toward Genetic Transformation of Marine Crop Porphyra Species

“…Thus, it is possible that the PyGUS gene could be commonly functional in red algae as a reporter of the gene expression. Indeed, applicability of the PyGUS gene in P. tenera has recently been confirmed by another laboratory (Son et al, 2011). The applicability of the P. yezoensis transient gene expression system was examined using the PyGUS and sGFP(S65T) reporter genes under the direction of the PyAct1 promoter in the red macroalgae listed in Table 2.…”

Heterologous activation of thePorphyra tenera HSP70promoter in Bangiophycean algal cells

“…7 In addition, the promoter region of the HSP70 gene derived from P. tenera (PtHSP70 promoter) was shown to be active in P. yezoensis cells. 8 However, it remains to be determined whether the PtHSP70 promoter is available as a heterologous promoter in other Bangiophycean algal cells.…”

“…In addition to our previous report on some heterologously employable promoters in Bangiophycean algae, 7,8 here we evaluate the P. tenera HSP70 promoter as an additional candidate for the heterologous promoter in red algal cells.…”

Current Advances in Seaweed Transformation

“…Taken together, PyGUS and sGFP(S65T) genes act synergistically with the PyAct1 promoter as a heterologous promoter for transient transformation in Bangiophycean algae. Recently, the same synergistic effect was found in P. tenera; that is, Son et al [85] clearly indicated that the heat shock protein 70 (PtHSP70) promoter from P. tenera can activate the PyGUS gene in gametophytic cells of this alga. Moreover, the PtHSP70-PyGUS gene was expressed in P. yezoensis, P. okamurae, P. psedolinearis and B. fuscopurpurea [85,87].…”

“…EST analysis of P. haitanensis revealed that the GC content of the ORFs in this alga was as high as that in P. yezoensis, and analysis of the GAPDH gene from a Florideophycean alga Chondrus crispus showed a high GC content (approximately 60%) in the coding region [83,84], which is consistent with the codon preference in P. yezoensis. Since efficient expression of the GAPDH-PyGUS gene has recently been confirmed in P. tenera [85], the applicability of the P. yezoensis transient gene expression system in other red seaweeds is expected. Indeed, using the PyGUS and sGFP(S65T) reporter genes under the direction of the PyAct1 promoter, efficient expression of PyGUS and sGFP(S65T) genes was observed in Bangiophyceae including P. tenera, P. okamurae, P. psedolinearis and Bangia fuscopurpurea, although the expression efficiency varied among species [86].…”