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.
A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps. We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient PCR-based detection of transgenes from a variety of transgenic plant and algal species. Our protocol consists of homogenizing plant tissue with a pestle, boiling the homogenized tissue in a microfuge tube with 5% Chelex 100 for 5 min, and centrifuging the boiled mixture. The supernatant, which is used for PCR analysis, was able to successfully amplify transgenes in transgenic tobacco, tomato, potato, Arabidopsis, rice, strawberry, Spirodela polyrhiza, Chlamydomonas, and Porphyra tenera. The entire DNA extraction procedure requires \15 min and is therefore comparable to that used for bacteria, Chlamydomonas, and animal cell lines.
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