Gene Research Center, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan b RIKEN Plant Science Center, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama-shi, Kanagawa, Japan
A taste-modifying protein, miraculin, is highly accumulated in ripe fruit of miracle fruit (Richadella dulcifica) and the content can reach up to 10% of the total soluble protein in these fruits.
Miracle fruit (Richadella dulcifica), a shrub native to tropical West Africa, yields red berries that have the unusual ability to modify a sour taste into a sweet taste. For example, lemons taste like oranges when the pulp of these berries is chewed. The active ingredient in miracle fruit is a taste-modifying protein, miraculin. Miraculin contents can reach up to 10% of the total soluble protein
in the miracle fruits. Miraculin is obviously an attractive alternative to more traditional sweeteners such as sucrose, but miracle fruit is a tropical plant that is difficult to cultivate outside its natural environment.
Miraculin is most successfully produced in transgenic tomato plants, although the miraculin content of these plants was at least twice lower than in miracle fruit. Because of the unique feature, taste-modifying activity, of miraculin protein, studies on the practical application have been carried out.
METHODS AND MATERIALS
I. Plant materials for immunoelectron microscopy
Miracle fruit (Richardella dulcifica (Schumach. & Thonn.) Baehni) plants were grown in a greenhouse at Inplanta Innovations, Inc. in Hayama town, Kanagawa, Japan. transgenic tomato (Solanum lycopersicum L.) highly expressing miraculin was produced.
II. Immunoelectron microscopy analysis
In this analysis, the pulp and leaves of the miracle fruit was experimented. There were methods done in order to extract valuable results to help figure how miraculin progresses in electron microscopy sciences.
III. Imaging miraculin–GFP fusion protein in tomato hypocotyls
The experiment here was conducted and executed by the doctors of Japan where in to construct the miraculin–GFP fusion gene, the GFP plasmid, a pUC18-based vector containing CaMV-35S-sGFP (S65 T)-NOS3 (Chiu et al., 1996), which was kindly provided by Dr. Yasuo Niwa (University of Shizuoka, Japan)
IV. Detection of miraculin protein in culture medium from liquid cultures of transgenic tomato.
Seeds of transgenic tomato were subjected to sterilization, germination & pH adjustments before it was transferred to a room for culture maintenance. The resulting supernatant was used for protein gel blot analysis.
The results demonstrate that recombinant miraculin is also specifically accumulated in intercellular layers and junctions of transgenic tomato as in miracle fruit.
Protein secretion is important for protein production in biotechnology. The miraculin precursor has a cleavable N-terminal signal sequence rich in hydrophobic amino acids and the mature protein is an N-glycosylated glycoprotein. Therefore, miraculin is expected to be secreted from cells.
In this study, we confirmed the localization of miraculin in miracle fruit and in transgenic tomato plants. Miraculin is located in the intercellular layers of both plant tissues, indicating that it is a secreted protein.
Transgenic tomato plants expressing the natural miraculin gene without modification unexpectedly accumulated a high level of miraculin protein.
In this study, we demonstrated that miraculin protein was secreted into the culture medium from the roots of transgenic tomato plants expressing miraculin, although the functions of secreted miraculin into the culture media remain to be elucidated. This suggests the possibility of producing miraculin using a root culture system.
The experiments showed that miraculin is secreted into the intercellular spaces, and when expressed in tomato, into the surrounding environment. Taken together, there was a speculation that miraculin is involved in plant defense. Transgenic tomato constitutively expressing miraculin will provide a powerful tool for elucidating the biological roles of miraculin in plants.
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