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
2001-06-22
2003-09-02
Fox, David T. (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
C047S05810R, C424S093700, C435S418000, C435S419000, C800S288000, C800S317000, C800S320000, C800S298000, C800S278000
Reexamination Certificate
active
06613961
ABSTRACT:
TECHNICAL FIELD
The present invention relates to transgenic plants on which capability of metabolizing, for example, drugs is conferred and use thereof.
BACKGROUND ART
Industrial development and advancement has increased kinds of environmental loads which destroy ecosystem. Moreover, numerous substances which disturb reproduction mechanism of organisms with an extremely small amount, such as extrinsic endocrine disruptors, have been reported. Environmental loads, extrinsic endocrine disruptors, and such are concentrated in the body of living organisms and more concentrated through food chain to profoundly affect living organisms and ecosystem. Therefore, it is necessary not only to remove these substances from environment, but also to metabolize and detoxify these in living organisms which have incorporated these substances.
Especially, plants are organisms belonging to the bottom of food chain, and plants in which these substances accumulate can be a source of destroying ecosystem. Thus, metabolizing and detoxifying these substances in plants are very important subjects.
Regarding agricultural crops which are closely related to our diet, agrochemicals such as herbicides, which are environmental loads, are generally used for cultivation. Thus, detoxifying residual agrochemicals in agricultural crops is needed for preventing adverse effect on humans and livestock which ingest these chemicals.
Cytochrome P450 monooxygenase (abbreviated to “P450” hereinafter) is an enzyme which distributes in a broad range of living organisms from microorganisms to plants and mammals and has a role of metabolizing and detoxifying drugs incorporated into living organisms. For P450 to exert its activity, another enzyme which provides p450 with electrons (abbreviated to “P450 reductase” hereinafter), for example, NADPH-P450 reductase, is required. This enzyme is also present in a broad spectrum of living organisms. The present inventors have created artificial enzymes showing extremely high oxidation activity by constructing fusion enzymegenes of various P450 molecular species and P450 reductases (Unexamined Published Japanese Patent Application (JP-A) No. Sho 63-44888, JP-A Hei 2-23870, and JP-A Hei 2-249488).
Recently, P450 in plants has been studied and various P450 molecular species broadly present in plants have been revealed. An activity of many drug-metabolizing P450 molecular species present in plants is, however, low, and capability of metabolizing environmental loads is extremely weak. Therefore, a novel plant with an enhanced capability of metabolizing environmental loads and such has been needed to create.
Specifically, it has been necessary to provide field crops which are used directly for foods and for feed and which play an important role in human life with the capability of metabolizing and detoxifying environmental loads and extrinsic endocrine disruptors represented by agrochemicals.
DISCLOSURE OF THE INVENTION
The present inventors have earnestly studied under these circumstances and successfully created transgenic plants on which capability of metabolizing, for example, drugs is conferred, by expressing drug-metabolizing P450 molecular species of mammals in plants. Transgenic plants of the present invention have the capability of metabolizing various herbicides depending on a molecular species of P450 expressed by them, and thus, show tolerance to these herbicides. Especially, an individual in which multiple P450 molecular species have been expressed can show higher tolerance to a broad spectrum of herbicides by the synergistic effect. Due to such a characteristic, the transgenic plants of the present invention are very useful as herbicide-tolerance plants. Moreover, these can absorb, metabolize, and decompose herbicides dispersed in the soil and, thus, are extremely environment-friendly. In addition, these plants can be used as a phytoremediation plant which can defecate environment by utilizing a plant (by using light energy) to metabolize and decompose not only herbicides but also environmental loads and extrinsic endocrine disruptors.
Therefore, the present invention provides transgenic plants which express a mono-oxygen addition activity derived from P450 or express a fusion enzyme activity comprising both mono-oxygen addition activity derived from P450 and reducing power-providing ability derived from P450 reductases and which can metabolize and decompose environmental loads and extrinsic endocrine disruptors, and use thereof.
Specifically, the present invention provides:
(1) a DNA molecule functionally comprising (a) a promoter capable of functioning in plant cells, (b) a P450 monooxygenase gene, and (c) a terminator capable of functioning in plant cells,
(2) the DNA molecule of (1), wherein the P450 monooxygenase gene is a fusion gene with a P450 reductase gene,
(3) the DNA molecule of (1) or (2), wherein the P450 monooxygenase gene is a gene belonging to CYP2 family,
(4) the DNA molecule of (3), wherein the gene belonging to CYP2 family is a gene selected from the group consisting of CYP2B6, CYP2C9, CYP2C18, and CYP2C19 genes,
(5) the DNA molecule of any one of (1) to (4), wherein the P450 monooxygenase gene is derived from mammals,
(6) a transgenic plant cell into which the DNA molecule of any one of (1) to (5) has been introduced,
(7) the transgenic plant cell of (6), wherein more than one type of P450 monooxygenase genes has been introduced into the transgenic plant cell,
(8) a transgenic plant comprising the plant cell of (6) or (7),
(9) the transgenic plant of (8), wherein the transgenic plant is a gramineous plant or a solanaceous plant,
(10) the transgenic plant of (8) or (9), wherein the transgenic plant is capable of decomposing a foreign compound by the oxidative metabolism,
(11) the transgenic plant of (10), wherein a foreign compound is an environmental load or an extrinsic endocrine disrupter,
(12) the transgenic plant of (10) or (11), wherein the transgenic plant comprises herbicide tolerance,
(13) a propagation material of the plant of any one of (8) to (12),
(14) a method for removing a foreign compound in environment, wherein the transgenic plant of (10) or (11) is used, and
(15) a method for selecting a P450 monooxygenase capable of metabolizing herbicides or a gene thereof, the method comprising:
(a) preparing a microsomal fraction from each transformant expressing one type of P450 monooxygenase gene introduced respectively into each transformant,
(b) reacting the microsomal fraction with an active ingredient of a herbicide,
(c) detecting a parent compound (the active ingredient of the herbicide) and/or a reaction product (a metabolite of the active ingredient of the herbicide), and
(d) selecting the P450 monooxygenase for which the reduction of the amount of the parent compound and/or the production of the reaction product has been detected, or a gene thereof.
In the present invention “environmental loads” mean substances which are released to the environment and provide loads to the ecosystem. “Extrinsic endocrine disruptors” mean substances which are present out of living organisms and cause hormone-like effect when incorporated into living organisms.
Any P450 molecular species can be used herein as long as they have a mono-oxygen addition activity, but those derived from mammals, which comprise higher activity than those derived from plants, are preferable. In this specification, “a mono-oxygen addition activity” means an activity catalyzing a reaction which adds a single oxygen atom to a substrate molecule. P450 molecular species belonging to CYP1, 2, 3, and 4 families are known to be mainly involved in the oxidative metabolism of foreign compounds, such as environmental loads including drugs, toxins, and agrochemicals, and thus, can be preferably used for the present invention (Imai, Y., and Kamataki, T., Protein, Nucleic acid and Enzyme, 1998, 43, 203-215). Specifically a preferable molecular species belonging to CYP1 family is derived from humans, for example, human CYP1A1 and human CYP1A2.
A herbicide metabolized by each P450 molecular speci
Hirose Sakiko
Ohkawa Hideo
Ohkawa Yasunobu
Ozawa Kenjirou
Bio-Oriented Technology Research Advancement Institution
Fox David T.
Kruse David H.
Saliwanchik Lloyd & Saliwanchik
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