Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2001-08-13
2003-06-10
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S298000, C800S278000, C800S287000, C536S023100, C536S023600, C435S468000
Reexamination Certificate
active
06576820
ABSTRACT:
This application is a national stage filing of PCT/JP99/01057, filed on Mar. 4, 1999, under 35 U.S.C. §371. This application claims priority under 35 U.S.C. §119 and 365 to application number 10/223897 filed in Japan on Jul. 8, 1998.
TECHNICAL FIELD
The present invention relates to a plant expressing soybean glycinin, which is useful as an ingredient in food, and to a propagation material of the plant, and the invention belongs to the fields of agriculture, food, and the like.
BACKGROUND ART
Soybean-extract protein (which has soybean globulin for its main ingredients), which is obtained by extracting proteins from soybeans and acid-precipitating the proteins (pH 4.5), is useful for preventing and treating arteriosclerosis and coronary heart diseases because it lowers the serum cholesterol level in human; particularly, it has the remarkable effection lowering a total cholesterol level, an LDL-cholesterol level, and a triglyceride level in patients with hypercholesterolemia (Mercer, N. J. H., Carroll, K. K., Giovannetti, P. M., Steinke, F. H. and Wolfe, B. M., Nutr. Rep. Int., 35, 279-287 (1987); T. Imura, M. Tanaka, T. Watanabe, S. Kudo, T. Uchida and T. Kanazawa, Ther. Res., 17, 2451-2456 (1996); M. Kanno, Food Industry, 39(18), 59-68 (1996)). In addition, the protein extract significantly reduces a total cholesterol level in healthy persons whose cholesterol levels are within normal limits (Kito, M., Moriyama, T., Kimura, Y. and Kambara, H., Biosci. Biotech. Biochem., 57, 354-355 (1993); Kambara, H., Nohara T., Kito, M., Ther. Res., 14, 3197-3204 (1993)).
By inhibiting the absorption of cholesterol and bile acid, soybean proteins play a pivotal role in lowering the cholesterol level (M. Kanno, Food Industry, 39 (18), 59-68 (1996)). Namely, the inhibition of cholesterol absorption results in an increase in cholesterol synthesis in the liver. However, the re-absorption of bile acid is inhibited at the same time and, as a result, cholesterol in the liver is oxidized into bile acid. This reduces the hepatic cholesterol concentration: and enhances the uptake of serum cholesterol into the liver. Thus, the serum cholesterol level is lowered (M. Kanno, Food Industry, 39(18), 59-68 (1996)).
It has been known that, an indigestible fraction obtained by intensive digestion of soybean protein with a protease markedly lowers the serum cholesterol level (M. Kanno, Food Industry, 39(18), 59-68 (1996)). The primary structure of the peptide present in the indigestible fraction still remains to be clarified (M. Kanno, Food Industry, 39(18), 59-68 (1996)). On the other hand, it has been reported that a peptide derived from A1aB1b subunit of glycinin, which is a major constituent of soybean globulins, has the bile acid-binding activity (Y. Makino, Food Industry, 39(24), 77-87 (1996)), and thus, the above-mentioned indigestible fraction has been believed to be derived from the bile acid-binding peptide originating from the A1aB1b subunit (M. Kanno, Food Industry, 39(18), 59-68 (1996)). Furthermore, a hydrophobic peptide derived from soybean protein is known to have bile acid-binding activity (Iwami, K., Sakakibara, K. and Ibuki, F., Agric. Biol. Chem., 50, 1217-1222 (1986)). Glycinin is the most hydrophobic protein among soybean globulins and there are two hydrophobic regions in the bile acid-binding peptide of A1aB1b subunit (S. Utsumi, Food Industry, 40(10), 68-79 (1997)). Consequently, it can be considered that, among soybean proteins, glycinin chiefly has the activity lowering the serum cholesterol level and, particularly, A1aB1b subunit is highly responsible for the activity.
Examples of industrial application of soybean-extract protein in the field of food include the following commercial products: Soybean Kara-age (fried soybean) and Protein Ganmo (fried tofu, where tofu is soybean curd) from Fuji Oil Co., Ltd.; G-9 and G-9 100 from Kanesa Co.; and Teriyaki Meatball (grilled meatball in teriyaki style), Hamburger, Pork Frankfurt from Nippon Meat Packers, Inc. However, there has been no report on functional food to which glycinin itself is added.
Furthermore, in the field of agriculture, there is no report on an attempt of giving the properties provided by soybean glycinin to other plants.
DISCLOSURE OF THE INVENTION
The present inventors focused on the above-mentioned properties of soybean glycinin and then predicted that the properties of soybean such as an activity of lowering the cholesterol level should be transplantable to plant species other than soybean by expressing soybean glycinin in these plants. The present inventors further focused on not only the above-mentioned activity of lowering the cholesterol level but also the facts that soybean glycinin protein is water soluble and has a wide variety of uses for processing food like tofu and that it excels nutritionally because it contains an essential amino acid “lysine” abundantly, which is less abundant in rice. Thus, the inventors considered that it could be possible to produce new-type processed foods and new agricultural products having higher nutritive values by expressing soybean glycinin protein in other agricultural plants. Thus, an objective of the present invention is to provide a plant expressing soybean glycinin, particularly agricultural products useful as food.
The rice plant, of which seed, rice, is indispensable to Japanese eating habits, is one of the most important agricultural products. A major storage protein, glutelin, accounts for 80% of total protein in rice. Glutelin exhibits 32 to 37% homology to soybean glycinin at the amino acid sequence level, and the two proteins share a fundamental structure. Specifically, both glycinin and glutelin are synthesized as prepro-forms consisting of a signal peptide, A chain, and B chain, converted to pro-forms, and then processed to mature forms by being processed at specific sites of which sequences are highly conserved in each other. There are some differences between them; glycinin forms a trimer and then a hexamer by molecular association and is soluble in a salt solution; glutelin is insoluble in a salt solution because of its own giant molecule formed by disulfide bonding and hydrophobic interaction. Nonetheless, based on the similarity of their fundamental structures, the two proteins are considered to have evolved from the common ancestral gene (FIG.
1
). Thus, just as with glutelin, it may be possible, by using glutelin gene promoter, to express and accumulate soybean glycinin, which is excellent in nutrition and processing suitability and has the effect on maintaining and promoting health by lowering the serum cholesterol level in human, in rice seeds, and then new values are thought to be added to rice while the wild-type characteristics of rice are maintained. Furthermore, the accumulation of a hybrid protein of glycinin and glutelin is also promising. Hence,the present inventors selected especially the rice plant as a plant to be used for the expression of soybean glycinin and attempted to express and accumulate soybean glycinin in rice, which is seed of rice plant and is one of useful agricultural products.
Specifically, a region containing the glutelin gene promoter, which expresses a gene specifically in the endosperm of rice plant seed, was isolated; a vector was constructed to have the natural or modified soybean glycinin gene ligated downstream of the promoter; the chimera gene construct was introduced into a cultured cell of rice plant; and thus, a transgenic rice plant was obtained. Subsequently, the inventors studied the tissue specificity and form of soybean glycinin expressed in the created transgenic rice plant. Then, they found that the expressed soybean glycinin was accumulated in rice plant seeds in the transgenic rice plant and the accumulated soybean glycinin was present as a matured protein formed by protein processing. Namely, the present inventors succeeded in expressing and accumulating soybean glycinin as a functional form in rice plant seeds and thus completed the present invention.
The present invention relates to a plant cell and a pla
Katsube-Tanaka Tomoyuki
Takaiwa Fumio
Utsumi Shigeru
Baum Stuart F
McElwain Elizabeth F.
National Institute of Agrobiological Sciences
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