WO2011010485A1

WO2011010485A1 - Process for production of eicosanoid, eicosanoid biosynthesis gene derived from marchantia polymorpha, and use of the gene

Info

Publication number: WO2011010485A1
Application number: PCT/JP2010/053533
Authority: WO
Inventors: 莞爾大山; 美保小林
Original assignee: 石川県
Priority date: 2009-07-22
Filing date: 2010-03-04
Publication date: 2011-01-27

Abstract

A process for producing an eicosanoid, which is characterized by comprising a step of proliferating or growing a transgenic plant or a tissue thereof, wherein the transgenic plant is produced by introducing at least one gene selected from a phospholipase gene, a lipoxygenase gene and a cyclooxygenase gene into a plant in such a manner that the gene can be expressed in the plant.

Description

Eicosanoid production method, eicosanoid biosynthetic gene derived from genus genus and use thereof

The present invention relates to the genes involved in biosynthesis of eicosanoids (Prostaglandin (PG), thromboxane (TX), leukotriene (LT), and lipoxin (LX)) derived from Marchantia polymorpha, ie, phospholipase, lipoxygenase and cyclooxygenase. It relates to genes and their use. The present invention also relates to a method for producing eicosanoids by plants.

Eicosanoid prostaglandins have peripheral vasodilatory action, platelet aggregation inhibitory action, cytoprotective action, angiogenic action, etc., and are used as these therapeutic agents as so-called lipoPG agents. Leukotrienes, a kind of eicosanoid, are leukocyte activators, chemotaxis to inflammatory sites, lysosomal enzyme secretion from neutrophils, active oxygen production promoting action, neutrophil adhesion to vascular endothelial cells It shows the action. Phospholipase (PL) is an enzyme that hydrolyzes the ester bonds of glycerophospholipids that make up biological membranes, and is an information exchange enzyme that is activated by various stimuli through the receptor and transmits its information through the production of lipid mediators. Function.

Polyunsaturated fatty acids such as arachidonic acid (hereinafter sometimes abbreviated as “AA”) and eicosapentaenoic acid (hereinafter sometimes abbreviated as “EPA”) are lipids in the cell membrane mainly in the nervous system in humans and the like. Is included in many. From these highly unsaturated fatty acids, pharmacologically important physiologically active substances such as prostaglandins and leukotrienes are biosynthesized by the reaction of phospholipase, lipoxygenase, and cyclooxygenase. These biosynthetic metabolic pathways are called arachidonic acid cascades.

Thus, it has been reported that eicosanoids such as prostaglandins (PG), thromboxane (TX), leukotriene (LT), and lipoxin (LX), which are physiologically active substances, are biosynthesized in animal cells. Yes. Prostaglandins in blood are prepared for quantitative analysis (Patent Document 1).

Currently, eicosanoids are produced by total chemical synthesis from lactones by chemical synthesis methods. This raises the problem of high production costs. Eicosanoids are considered to be biosynthesized by reaction of phospholipase, lipoxygenase, and cyclooxygenase from AA or EPA, for example, in the human body. In these reactions, AA or EPA is first released from cell membrane lipids into cells by phospholipase, and the AA or EPA is oxidized by two molecules of oxygen by cyclooxygenase and converted into prostaglandins and thromboxanes. On the other hand, AA or EPA released from the cell membrane is oxidized by one molecule of oxygen by lipoxygenase and converted into leukotriene and lipoxin.

The phospholipase A1 gene has been cloned in several plant species. For example, phospholipase genes derived from Arabidopsis (Non-patent Document 1) and pepper (Non-patent Document 2) have been reported. A plant-derived phospholipase A2 involved in the release of AA or EPA from the cell membrane has not been reported.

The lipoxygenase gene has been cloned from several plant species. For example, a lipoxygenase gene derived from Arabidopsis thaliana (Non-patent Document 3), Physcomitrella patens (Non-patent Document 4), and potato (Non-patent Document 5) has been reported. The cyclooxygenase gene has not been cloned from plants.

Land plants are composed of moss plants (Bryophyte), fern plants, gymnosperms and angiosperms. Moss plants are the oldest branched group of terrestrial plants, and are composed of three groups: sens, Thais, and hornworts. Zenigoke is said to have first inhabited the land among the above-mentioned organisms (Non-patent Document 6). The presence of prostaglandins or prostaglandin-like substances has been reported in onions, poplars, larch, evening primroses, morning glory, bean diatoms and certain Thais, but details are unknown (Non-patent Document 7). It has been reported that AA and EPA are already biosynthesized and accumulated by Amanita (non-patent document 8). The genes involved in these have been cloned. However, there has been no report that phospholipase, lipoxygenase and cyclooxygenase genes have been identified from Xenopus. Furthermore, there are no reports of producing eicosanoid (prostaglandin (PG), thromboxane (TX), leukotriene (LT), lipoxin (LX)) compounds by Zenigo.

JP 2007-278750 A

The present invention has been made in view of the above-mentioned conventional problems, and the object thereof is to use plant species and produce eicosanoids, which are derived from plants such as the genus Coleoptera, a lipoxygenase gene and a cyclooxygenase gene, and It is to provide usage.
Another object of the present invention is to provide a technique for producing eicosanoids by plants using animal-derived phospholipase genes, lipoxygenase genes and cyclooxygenase genes.

As a result of intensive studies to solve the above problems, the present inventors have identified each gene encoding the above phospholipase, lipoxygenase, and cyclooxygenase from a cDNA clone derived from Marchantia polymorpha, and a strong promoter for the gene. It has been found that the ability to produce eicosanoids can be improved by introducing and expressing it in the moss. Furthermore, the present inventors have found that the ability to produce eicosanoids can be improved also by introducing and expressing each gene encoding phospholipase, lipoxygenase and cyclooxygenase derived from an animal in the moss. The present inventors have further studied based on the above findings and have completed the present invention. That is, the present invention includes the following inventions [1] to [33].

[1] An eicosanoid production comprising a step of growing or growing a plant transformant or a tissue thereof into which one or more of a phospholipase gene, a lipoxygenase gene, and a cyclooxygenase gene are introduced so as to be expressed in a plant Method.
[2] The method according to the above [1], wherein the plant is a genus Pleurotus.
[3] A plant transformant in which one or more of a phospholipase gene, a lipoxygenase gene, and a cyclooxygenase gene are introduced into a plant so that the plant transformant can be expressed, or the plant transformant having a modified eicosanoid composition, or the plant transformant The method according to [1] or [2] above, which is a plant transformant that is a descendant of the plant transformant having the same properties as the body.

[4] The phospholipase gene is any one of the following genes (a1) to (a8), the lipoxygenase gene is any one of the following genes (b1) to (b8), and the cyclooxygenase gene is: The method according to any one of [1] to [3], wherein the gene is any one of (c1) to (c8).
(A1) a gene comprising DNA consisting of the base sequence shown in SEQ ID NO: 1 (a2) consisting of a part of the base sequence shown in SEQ ID NO: 1, and arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into the cell A gene containing DNA derived from the genus Coleoptera encoding a protein having a phospholipase activity to be released (a3) A DNA comprising all or part of the base sequence complementary to the base sequence shown in SEQ ID NO: 1 under stringent conditions And a gene containing a DNA derived from the genus Coleoptera that encodes a protein having a phospholipase activity that liberates arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into the cell (a4) a base represented by SEQ ID NO: 1 DNA comprising the 394th to 2091th nucleotide sequences in the sequence The gene (a5) is hybridized under stringent conditions with either DNA consisting of the 394th to 2091th base sequences of the base sequence shown in SEQ ID NO: 1 or DNA consisting of base sequences complementary to the DNA, And a gene comprising a DNA encoding a protein derived from the genus Coleoptera having a phospholipase activity that releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into the cell (a6) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 The encoding gene (a7) consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 2, and from cell membrane phospholipid to arachidonic acid or Phospholipa that releases eicosapentaenoic acid into cells A gene encoding a protein derived from the genus Coleoptera having ze activity (a8) a phospholipase A2 gene derived from a mouse (b1) a gene comprising a DNA consisting of the base sequence shown in SEQ ID NO: 3 (b2) a base shown in SEQ ID NO: 3 A gene comprising a part of the sequence and containing a DNA derived from the genus Coleoptera encoding a protein having lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate (b3) complementary to the base sequence shown in SEQ ID NO: 3 Including DNA derived from the genus Lepidoptera that hybridizes under stringent conditions with DNA consisting of all or part of a simple nucleotide sequence and encodes a protein having lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate Gene (b4) nucleotide sequence shown in SEQ ID NO: 3 A gene comprising DNA having the 127th to 2994th base sequence in the sequence (b5) consisting of DNA consisting of the 127th to 2994th base sequence of the base sequence shown in SEQ ID NO: 3 or a base sequence complementary to the DNA A gene (b6) comprising a DNA that hybridizes with any one of the DNAs under stringent conditions and encodes a protein derived from the genus Coleoptera having lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate. A gene encoding a protein comprising the amino acid sequence (b7) comprising the amino acid sequence shown in SEQ ID NO: 4, wherein one or several amino acids are substituted, deleted, inserted and / or added, and arachidone Lipoxy containing acid or eicosapentaenoic acid as substrate A gene encoding a protein derived from the genus Coleoptera having a genease activity (b8) A 5-lipoxygenase gene derived from a mouse (c1) a gene comprising a DNA consisting of the base sequence shown in SEQ ID NO: 5 (c2) shown in SEQ ID NO: 5 A gene comprising a part of the base sequence and containing a DNA derived from the genus Coleoptera encoding a protein having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate (c3) complementary to the base sequence shown in SEQ ID NO: 5 A DNA derived from the genus Amanita which encodes a protein having a cyclooxygenase activity that hybridizes under stringent conditions with all or part of a DNA having a basic nucleotide sequence and that uses arachidonic acid or eicosapentaenoic acid as a substrate. Containing gene (c4) SEQ ID NO: A gene comprising DNA having the 321st to 2153th base sequences in the base sequence shown in (c5) DNA consisting of the 321st to 2153th base sequences in the base sequence shown in SEQ ID NO: 5 or complementary to the DNA A gene (c6) comprising a DNA that hybridizes with any one of DNAs comprising a base sequence under a stringent condition and encodes a protein derived from a genus Coriolis having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate (c6) A gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 6 (c7) in the amino acid sequence shown in SEQ ID NO: 6, wherein one or several amino acids are substituted, deleted, inserted and / or added Arachidonic acid or eicosapentae A gene encoding a protein derived from the genus Coleoptera having cyclooxygenase activity using acid as a substrate (c8) a cyclooxygenase gene derived from zebrafish

[5] The phospholipase gene is any one of the genes (a1) to (a7), the lipoxygenase gene is any one of the genes (b1) to (b7), and the cyclooxygenase gene is the gene (c1). ) To (c7), the method according to the above [4].

[6] A gene comprising the DNA of any one of (a) to (c) below:
(A) DNA comprising the base sequence shown in SEQ ID NO: 1
(B) a DNA derived from the genus Pleurotus that encodes a protein having a phospholipase activity, which comprises a part of the base sequence represented by SEQ ID NO: 1 and releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into the cell.
(C) Hybridizing under stringent conditions with DNA consisting of all or part of the base sequence complementary to the base sequence shown in SEQ ID NO: 1, and arachidonic acid or eicosapentaenoic acid from the cell membrane phospholipid, DNA derived from the genus Pleurotus that encodes a protein having phospholipase activity to be released in the body
[7] A gene comprising the following DNA (a) or (b):
(A) DNA consisting of the 394th to 2091th base sequences in the base sequence shown in SEQ ID NO: 1
(B) a cell membrane that hybridizes under stringent conditions with either DNA comprising the 394th to 2091th nucleotide sequences of the nucleotide sequence represented by SEQ ID NO: 1 or DNA comprising a nucleotide sequence complementary to the DNA. DNA that encodes a protein derived from the genus Lepidoptera having phospholipase activity that releases arachidonic acid or eicosapentaenoic acid from phospholipids into cells.
[8] A gene encoding the following protein (A) or (B).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (B) an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 2 And a protein derived from the genus Pleurotus having a phospholipase activity that releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into the cell.

[9] A gene containing the DNA of any one of (a) to (c) below.
(A) DNA consisting of the base sequence shown in SEQ ID NO: 3
(B) a DNA derived from the genus Pleurotus that encodes a protein having a lipoxygenase activity comprising a part of the base sequence shown in SEQ ID NO: 3 and having arachidonic acid or eicosapentaenoic acid as a substrate;
(C) Lipoxygenase activity that hybridizes under stringent conditions with DNA consisting of all or part of the base sequence complementary to the base sequence shown in SEQ ID NO: 3 and that uses arachidonic acid or eicosapentaenoic acid as a substrate DNA derived from the genus Pleurotus that encodes a protein having
[10] A gene comprising the following DNA (a) or (b):
(A) DNA consisting of the 127th to 2994th nucleotide sequences in the nucleotide sequence shown in SEQ ID NO: 3
(B) hybridizing under stringent conditions with either DNA comprising the 127th to 2994th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 3 or DNA comprising a complementary nucleotide sequence to the DNA, and arachidon DNA encoding a protein derived from the genus Pleurotus having lipoxygenase activity using acid or eicosapentaenoic acid as a substrate
[11] A gene encoding the following protein (A) or (B).
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (B) from the amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 4 And a protein derived from the genus Pleurotus having lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate

[12] A gene comprising the DNA of any one of (a) to (c) below:
(A) DNA consisting of the base sequence shown in SEQ ID NO: 5
(B) a DNA derived from the genus Pleurotus that encodes a protein having a cyclooxygenase activity comprising a part of the base sequence shown in SEQ ID NO: 5 and having arachidonic acid or eicosapentaenoic acid as a substrate;
(C) Cyclooxygenase activity that hybridizes under stringent conditions with all or part of DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 5 and uses arachidonic acid or eicosapentaenoic acid as a substrate DNA derived from the genus Pleurotus that encodes proteins
[13] A gene comprising the following DNA (a) or (b):
(A) DNA consisting of nucleotide sequences 321 to 2153 among the nucleotide sequence shown in SEQ ID NO: 5
(B) hybridizing under stringent conditions with either DNA comprising the 321st to 2153rd base sequences of the base sequence shown in SEQ ID NO: 5 or DNA comprising a base sequence complementary to the DNA; and arachidone DNA encoding a protein derived from the genus Pleurotus having cyclooxygenase activity using acid or eicosapentaenoic acid as a substrate
[14] A gene encoding the following protein (A) or (B).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (B) From the amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 6 And a protein derived from the genus Pleurotus having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate

[15] A protein encoded by the gene according to any one of [6] to [14].
[16] An antibody that recognizes the protein encoded by the gene according to any one of [6] to [14].
[17] A recombinant expression vector comprising the gene according to any one of [6] to [14].

[18] A plant transformant obtained by introducing at least the gene according to any one of [6] to [14] above into a plant, or the plant transformant having the same properties as the plant transformant A plant transformant to be a progeny or a tissue of the plant transformant.
[19] The plant transformant or the tissue of the plant transformant according to the above [18], wherein the plant is a genus Coleoptera.
[20] A plant transformant having a modified eicosanoid composition, wherein at least the gene according to any one of [6] to [14] is introduced into a plant so that the gene can be expressed, or the plant transformant A plant transformant that is a descendant of the plant transformant having the same properties, or a tissue of the plant transformant.
[21] A propagation material obtained from the plant transformant according to any one of [18] to [20] or a tissue of the plant transformant.

[22] A method for modifying the eicosanoid composition of a plant or a tissue thereof, comprising the step of introducing the gene according to any one of [6] to [14] above into the plant.

[23] A gene detection instrument using, as a probe, at least a part of the base sequence of the gene according to any one of [6] to [14] or a complementary sequence thereof.
[24] A method for screening a gene that regulates the protein or a substance that regulates the protein, using the protein encoded by the gene according to any one of [6] to [14].
[25] A gene or substance obtained by the screening method according to [24].

[26] A recombinant expression vector obtained by inserting a mouse-derived phospholipase A2 gene, a mouse-derived 5-lipoxygenase gene or a zebrafish-derived cyclooxygenase gene into a plant cell vector.
[27] A plant transformant obtained by introducing at least one or more of a mouse-derived phospholipase A2 gene, a mouse-derived 5-lipoxygenase gene and a zebrafish-derived cyclooxygenase gene, or the plant transformant, A plant transformant that is a descendant of the plant transformant having the same properties, or a tissue of the plant transformant.
[28] The plant transformant or the tissue of the plant transformant according to the above [27], wherein the plant is a genus Coleoptera.

[29] A plant trait having an altered eicosanoid composition, wherein at least one or more of a mouse-derived phospholipase A2 gene, a mouse-derived 5-lipoxygenase gene, and a zebrafish-derived cyclooxygenase gene are introduced so as to be expressed in the plant A transformant, a plant transformant that is a descendant of the plant transformant having the same properties as the plant transformant, or a tissue of the plant transformant.
[30] A propagation material obtained from the plant transformant according to any one of [27] to [29] or a tissue of the plant transformant.

[31] A method for modifying the eicosanoid composition of a plant or a tissue thereof, comprising introducing one or more of a mouse-derived phospholipase A2 gene, a mouse-derived 5-lipoxygenase gene, and a zebrafish-derived cyclooxygenase gene into the plant.
[32] A method for screening a gene that regulates the protein or a substance that regulates the protein, using the protein encoded by the recombinant expression vector according to [26].
[33] A gene or substance obtained by the screening method according to [32].

One of the preferred embodiments of the gene according to the present invention encodes a phospholipase gene, a lipoxygenase gene and a cyclooxygenase gene isolated from the same species of genus moss, and a protein having an activity equivalent to the protein encoded by these genes. It is a gene. For example, when a phospholipase gene, a lipoxygenase gene and a cyclooxygenase gene derived from the same species of genus moss are expressed in a plant species, genes related to eicosanoid biosynthesis are expressed in the plant rather than expressing three genes derived from different species. It has the effect of functioning well. Furthermore, the genus moss is considered to be a model system of plants, so that these genes have the effect that they can function better in plants than genes derived from biological species other than plants.

Also, the plant transformant according to the present invention has an effect that it can produce eicosanoids. For this reason, when the plant transformant according to the present invention is used, the production cost of an eicosanoid useful as an active ingredient of a pharmaceutical can be reduced, and at the same time, a more environmentally friendly production process can be realized. That is, according to the present invention, an eicosanoid can be produced at a low cost by a low-cost and environmentally friendly production process. Furthermore, since a genetically modified plant can be mass-produced by a plant factory system, it is possible to supply a cheaper eicosanoid. The eicosanoid thus obtained has an effect that it can be used as a pharmacologically active substance, a raw material for health foods, and the like.

FIG. 1 is a diagram showing only a leukotriene biosynthetic system and a prostaglandin biosynthetic system from a reaction in which AA or EPA is released into cells from a lipid conjugate according to the present invention in the arachidonic acid cascade. 2 (A) to 2 (C) respectively show (A) a procedure for preparing an expression construct for introducing a phospholipase gene derived from a genus genus, (B) a procedure for preparing an expression construct for introducing a lipoxygenase gene derived from a genus genus, And (C) shows a procedure for producing an expression construct for introducing a cyclooxygenase gene derived from genus Amanita. FIG. 3 (A) is a schematic diagram of a vector for introducing a phospholipase gene derived from Xenopus, and FIG. 3 (B) is a schematic diagram of a vector for introducing a lipoxygenase gene derived from Xenopus. (C) is a schematic diagram of a vector for introducing a gene for cyclooxygenase derived from genus Amanita. 4 (A) to 4 (C) respectively show (A) a procedure for preparing an expression construct for introducing a mouse-derived phospholipase A2 gene, and (B) an expression construct for introducing a mouse-derived 5-lipoxygenase gene. It is a figure which shows the preparation procedure and the preparation procedure of the expression construct for introduce | transducing the cyclooxygenase gene derived from (C) zebrafish. FIG. 5 (A) is a schematic diagram of a vector for introducing a mouse-derived phospholipase A2 gene, and FIG. 5 (B) is a schematic diagram of a vector for introducing a mouse-derived 5-lipoxygenase gene. FIG. 5 (C) is a schematic diagram of a vector for introducing a zebrafish-derived cyclooxygenase gene.

An embodiment of the present invention will be described as follows. Note that the present invention is not limited to this.
The eicosanoid production method of the present invention includes a step of proliferating or growing a plant transformant or a tissue thereof in which one or more of a phospholipase gene, a lipoxygenase gene, and a cyclooxygenase gene are introduced so as to be expressed in a plant.
According to the present invention, an eicosanoid can be produced using a plant. The plant is preferably a genus Coleoptera which will be described later. The plant transformant is preferably a plant transformant having a modified eicosanoid composition, wherein one or more of a phospholipase gene, a lipoxygenase gene, and a cyclooxygenase gene are introduced so as to be expressed in the plant, or the plant A plant transformant that is a descendant of the plant transformant having the same properties as the transformant. As the phospholipase gene, lipoxygenase gene, and cyclooxygenase gene, the genes described later are suitable, and any of plant-derived and animal-derived genes can be used.

Hereinafter, the biosynthetic pathway of eicosanoids (prostaglandins, leukotrienes, etc.), the gene according to the present invention, the protein according to the present invention, the method for obtaining the protein and gene according to the present invention, and the method for using the gene and protein according to the present invention The present invention will be described in detail in the order of (usefulness). Further, a method for producing eicosanoids in plants using an eicosanoid biosynthetic gene derived from animals will be described.

(1) Biosynthetic pathway of eicosanoids (prostaglandins and leukotrienes) (FIG. 1).
In cells, eicosanoids (prostaglandins, leukotrienes, etc.) originate from arachidonic acid and eicosapentaenoic acid (substrate), respectively, and the substrate is oxidized by one molecule of oxygen, and the substrate is oxidized by two molecules of oxygen. And biosynthesized into prostaglandins. These reactions are catalyzed by intracellular lipoxygenase (LOX) and cyclooxygenase (COX), respectively, as shown in FIG. These reaction pathways are called arachidonic acid cascades.

(2) Gene according to the present invention The genes of the present invention are the phospholipase gene, lipoxygenase gene, and cyclooxygenase gene described below. The gene of the present invention can be suitably used for, for example, preparation of a recombinant protein, production of a plant transformant having a modified eicosanoid composition, and the like.

(Phospholipase gene according to the present invention)
The phospholipase gene according to the present invention is a gene encoding a protein having phospholipase activity that releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into cells by hydrolysis of ester bonds. ~ 7. It is a gene described in. These genes are preferably used in the eicosanoid production method of the present invention.

1. A gene comprising DNA consisting of the base sequence shown in SEQ ID NO: 1.
2. A gene comprising a part of the base sequence shown in SEQ ID NO: 1 and containing a DNA derived from a genus Coleoptera that encodes a protein having a phospholipase activity that releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into cells.
3. Hybridizes under stringent conditions with DNA consisting of all or part of the base sequence complementary to the base sequence shown in SEQ ID NO: 1 and releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into the cell A gene comprising DNA from the genus Pleurotus that encodes a protein having phospholipase activity.

4). A gene comprising DNA consisting of the 394th to 2091th base sequences of the base sequence shown in SEQ ID NO: 1.
5. It hybridizes under stringent conditions with either DNA consisting of the 394th to 2091th base sequences in the base sequence shown in SEQ ID NO: 1 or DNA consisting of base sequences complementary to the DNA, and from cell membrane phospholipids A gene comprising a DNA encoding a protein derived from the genus Coleoptera having phospholipase activity that releases arachidonic acid or eicosapentaenoic acid into cells.

6). A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2.
7). The amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and arachidonic acid or eicosapentaenoic acid is converted from cell membrane phospholipids into cells. A gene that encodes a protein derived from the genus Pleurotus having a phospholipase activity that is released inside.
The phospholipase gene according to the present invention is preferably derived from Marchantia polymorpha.

The DNA consisting of the base sequence shown in SEQ ID NO: 1 is a gene derived from Marchantia polymorpha. Of the base sequence shown in SEQ ID NO: 1, the 394th to 2091st base sequence is a region translated into a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is a phospholipase that releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into cells by hydrolysis of ester bonds.

As a DNA derived from the genus Coleoptera which consists of a part of the base sequence shown in SEQ ID NO: 1 and encodes a protein having phospholipase activity that releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into cells, For example, a DNA having the 394th to 2091th base sequences of the base sequence of SEQ ID NO: 1 is preferred. The DNA comprising a part of the base sequence complementary to the base sequence shown in SEQ ID NO: 1 is, for example, a base complementary to the DNA having the 394th to 2091th base sequences in the base sequence of SEQ ID NO: 1. Sequence DNA is preferred.

(Lipoxygenase gene according to the present invention)
The lipoxygenase gene according to the present invention is a gene encoding a protein having lipoxygenase activity that uses arachidonic acid or eicosapentaenoic acid as a substrate and oxidizes the substrate with one molecule of oxygen. ~ 7. It is a gene described in. These genes are preferably used in the eicosanoid production method of the present invention.
1. A gene comprising DNA consisting of the base sequence shown in SEQ ID NO: 3.
2. A gene comprising a part of the base sequence shown in SEQ ID NO: 3 and containing a DNA derived from the genus Coleoptera which encodes a protein having lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate.
3. A protein that hybridizes under stringent conditions with DNA consisting of all or part of the base sequence complementary to the base sequence shown in SEQ ID NO: 3 and has lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate A gene containing DNA derived from the genus Pleurotus that encodes.

4). A gene comprising DNA having the 127th to 2994th base sequence in the base sequence shown in SEQ ID NO: 3.
5. It hybridizes under stringent conditions with either the DNA consisting of the 127th to 2994th base sequences of the base sequence shown in SEQ ID NO: 3 or the DNA complementary to the DNA, and arachidonic acid or e A gene comprising a DNA encoding a protein derived from a genus Cryptococcus having lipoxygenase activity using icosapentaenoic acid as a substrate.

6). A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4.
7). The amino acid sequence shown in SEQ ID NO: 4 consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and has lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate. A gene that encodes a protein derived from the genus Pleurotus.
The lipoxygenase gene according to the present invention is preferably derived from Marchantia polymorpha.

DNA consisting of the base sequence shown in SEQ ID NO: 3 is a gene derived from Marchantia polymorpha. Of the nucleotide sequence shown in SEQ ID NO: 3, the 127th to 2994th nucleotide sequence is a region translated into a protein consisting of the amino acid sequence shown in SEQ ID NO: 4. The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is a lipoxygenase that oxidizes arachidonic acid or eicosapentaenoic acid with one molecule of oxygen.

Examples of DNA encoding a protein having a part of the base sequence shown in SEQ ID NO: 3 and having lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate include, for example, the base sequence shown in SEQ ID NO: 3 Of these, DNA having the 127th to 2994th nucleotide sequence is preferred. The DNA comprising a part of the base sequence complementary to the base sequence shown in SEQ ID NO: 3 is, for example, a base complementary to the DNA having the 127th to 2994th base sequences in the base sequence of SEQ ID NO: 3. Sequence DNA is preferred.

(Cyclooxygenase gene according to the present invention)
The cyclooxygenase gene according to the present invention is a gene encoding a protein having cyclooxygenase activity that uses arachidonic acid or eicosapentaenoic acid as a substrate and oxidizes the substrate with two molecules of oxygen. ~ 7. It is a gene described in. These genes are preferably used in the eicosanoid production method of the present invention.
1. A gene comprising DNA consisting of the base sequence shown in SEQ ID NO: 5.
2. A gene comprising a part of the base sequence shown in SEQ ID NO: 5 and containing a DNA derived from a genus Coleoptera that encodes a protein having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate.
3. A protein having a cyclooxygenase activity that hybridizes under stringent conditions with all or part of a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 5 and uses arachidonic acid or eicosapentaenoic acid as a substrate A gene containing DNA derived from the genus Pleurotus that encodes.

4). A gene comprising DNA having a nucleotide sequence of positions 321 to 2153 of the nucleotide sequence represented by SEQ ID NO: 5.
5. It hybridizes under stringent conditions with either DNA consisting of the 321st to 2153th base sequences of the base sequence shown in SEQ ID NO: 5 or DNA complementary to the DNA, and arachidonic acid or A gene comprising a DNA encoding a protein derived from a genus Coriolis having cyclooxygenase activity using eicosapentaenoic acid as a substrate.

6). A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 6.
7). The amino acid sequence shown in SEQ ID NO: 6 consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and has cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate A gene that encodes a protein derived from the genus Pleurotus.
The cyclooxygenase gene according to the present invention is preferably derived from Marchantia polymorpha.

The DNA consisting of the base sequence shown in SEQ ID NO: 5 is a gene derived from Marchantia polymorpha. Of the base sequence shown in SEQ ID NO: 5, the 321st to 2153rd base sequence is a region translated into a protein consisting of the amino acid sequence shown in SEQ ID NO: 6. The protein consisting of the amino acid sequence shown in SEQ ID NO: 6 is a cyclooxygenase that oxidizes arachidonic acid or eicosapentaenoic acid with two molecules of oxygen.

Examples of the DNA derived from the genus Coleoptera which consists of a part of the base sequence shown in SEQ ID NO: 5 and encodes a protein having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate include, for example, SEQ ID NO: 5 Of the nucleotide sequences shown, DNA having the 321st to 2153rd nucleotide sequences is preferred. The DNA comprising a part of the base sequence complementary to the base sequence shown in SEQ ID NO: 5 is, for example, a base complementary to the DNA having the 321st to 2153rd base sequences in the base sequence of SEQ ID NO: 5. Sequence DNA is preferred.

The above “stringent conditions” means that hybridization occurs only when at least 90% identity, preferably at least 95% identity, most preferably 97% identity exists between sequences. means.

The hybridization, J. Sambrook et al. Molecular Cloning, A lLaboratory Manual. 2 nd Ed., Like the method described in Cold Spring Harbor Laboratory (1989), can be performed by a conventionally known method. Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous gene can be obtained. As hybridization conditions, conventionally known conditions can be preferably used, and are not particularly limited. For example, a commercially available DIG (digoxigenin) hybridization buffer (Roche Diagnostics) is used. And so on.

The above-mentioned “Amanitaceae organisms” are not limited to (Marchantia polymorpha), but include organisms belonging to the subgenus Amanitaceae (Marchantiales). Examples of organisms belonging to the order of the genus Mantiantiaceae (Marchantiales) include plants such as the clam sphagnum family, sphagnum moss family, jagoke department, azuma genus department, zingagoceae, gentian moss family, sphagnum department, sphagnum department, Can be mentioned. Prostaglandins or prostaglandin-like substances have been reported to exist in the moss genus Ambrystechium serpens, raBrachythecium implicantum, and moss marchantia parviloba (Groenewald, EG, Kruger, G. H. J., de Wet , H., Botes, P. J. and van der Westhuizen, A. J., South African J. Sci. 86: 152-153 (1990)).

It is easy to obtain phospholipase gene, lipoxygenase gene and cyclooxygenase gene from these organisms with the current state of the art. For example, it is generally known that genes encoding enzymes with similar functions in related organisms cross-hybridize.

The gene of the present invention includes not only double-stranded DNA but also single-stranded DNA and RNA such as sense strand and antisense strand constituting the DNA. The antisense strand can be used as a probe or as an antisense compound. DNA includes, for example, cDNA and genomic DNA obtained by cloning or chemical synthesis techniques, or a combination thereof. Furthermore, the gene of the present invention may include sequences such as untranslated region (UTR) sequences and vector sequences (including expression vector sequences).

(3) Protein according to the present invention The protein according to the present invention is the following phospholipase protein, lipoxygenase protein, and cyclooxygenase protein.

(Phospholipase protein according to the present invention)
The phospholipase protein according to the present invention may be a protein having the above-described phospholipase activity, that is, a function of releasing AA or EPA into cells from phospholipids bound to the cell membrane. More specifically, the following proteins are preferred. More preferably, it is a protein having phospholipase activity derived from the genus Pleurotus.
1. 1. a protein encoded by the phospholipase gene according to the present invention described in (2) above; 2. A protein consisting of the amino acid sequence shown in SEQ ID NO: 2. It consists of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 2 are substituted, deleted, inserted, and / or added, and arachidonic acid or eicosapentaenoic acid is intracellularly converted from cell membrane phospholipids. A protein having a phospholipase activity that is released into the protein The protein comprising the amino acid sequence shown in SEQ ID NO: 2 is a phospholipase derived from Marchantia polymorpha.

(Lipoxygenase protein according to the present invention)
The lipoxygenase protein according to the present invention may be any protein as long as it has the above-described lipoxygenase activity, that is, substrate specificity for AA or EPA, and has an effect of converting these into leukotrienes. More specifically, the following proteins are preferred. More preferably, it is a protein having lipoxygenase activity derived from the genus Pleurotus.
1. 1. a protein encoded by the lipoxygenase gene according to the present invention described in (2) above; 2. A protein consisting of the amino acid sequence shown in SEQ ID NO: 4. It consists of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 4 are substituted, deleted, inserted, and / or added, and has lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate Protein The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is a lipoxygenase derived from Marchantia polymorpha.

(Cyclooxygenase protein according to the present invention)
The cyclooxygenase protein according to the present invention may be any protein as long as it exhibits substrate specificity for the above-described cyclooxygenase activity, that is, AA or EPA, and has an action of converting these into prostaglandins. More specifically, the following proteins are preferred. More preferably, it is a protein having cyclooxygenase activity derived from the genus Pleurotus.
1. 1. a protein encoded by the cyclooxygenase gene according to the present invention described in (2) above; 2. A protein consisting of the amino acid sequence shown in SEQ ID NO: 6. Cyclooxygenase activity comprising an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 6 are substituted, deleted, inserted and / or added, and using arachidonic acid or eicosapentaenoic acid as a substrate Protein with Protein The protein consisting of the amino acid sequence shown in SEQ ID NO: 6 is a cyclooxygenase derived from Marchantia polymorpha.

The above “one or several amino acids are substituted, deleted, inserted, and / or added” means substitution, deletion, insertion by a known mutant protein production method such as site-directed mutagenesis. And / or the number of amino acids that can be added (usually 20 or less, preferably 10 or less, more preferably 7 or less, and even more preferably 5 or less) are substituted, deleted, inserted, and / or added. Means that.

(4) Protein and gene acquisition method according to the present invention The protein and gene acquisition method (production method) according to the present invention is not particularly limited, but the following methods are listed as representative methods. Can do.

(Protein acquisition method)
The method (production method) for obtaining the protein of the present invention is not particularly limited as described above. First, a method for simply purifying the protein from cells, tissues, etc. that express the protein of the present invention is mentioned. be able to. The purification method is not particularly limited, and a cell extract may be prepared from cells or tissues by a known method, and the cell extract may be purified using a known method such as a column. Examples of the cell that expresses the protein of the present invention include the above-mentioned cells of the genus Coleoptera.

In addition, as a method for obtaining the protein of the present invention, a method using a gene recombination technique or the like can also be mentioned. In this case, for example, after the gene of the present invention is incorporated into a vector or the like, it is introduced into a host cell so that it can be expressed by a known method, and the resulting transformed cell is cultured and translated into the cell. For example, a method of purifying a protein can be employed. The host cell may be a plant, microorganism, or animal cell.

In addition, when a foreign gene is introduced into a host cell for the purpose of obtaining the protein of the present invention, there are various expression vectors and hosts incorporating a promoter that functions in the host for the expression of the foreign gene. Since it exists, what is necessary is just to select the thing according to the objective. The method for purifying the protein produced in the host cell differs depending on the host used and the nature of the protein, but the protein is recovered from the host cell or its culture supernatant by a method known to those skilled in the art. Is possible. For example, the target protein can be purified relatively easily by using tags or the like.

The method for producing the mutant protein is not particularly limited. For example, site-directed mutagenesis (Hashimoto et al., Gene 152, 271-275 (1995)), a method of introducing a point mutation into a base sequence using PCR, or a transposon insertion A well-known method for producing a mutant protein, such as a method for producing a mutant strain by the above method, can be used. A commercially available kit may be used for the production of the mutant protein.

The method for obtaining the protein of the present invention is not limited to the above-described method, and may be, for example, chemically synthesized. For example, the protein of the present invention may be synthesized from the gene of the present invention using a cell-free protein synthesis solution. PeptideeptSynthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; Peptide Synthesis Fundamentals and Experiments, Maruzen Co., Ltd. 1985 Development of pharmaceuticals, Vol. 14, peptide synthesis, Hirokawa Shoten, 1991, etc., can also be synthesized by peptide synthesis methods.

(Gene acquisition method)
The method (production method) for obtaining the gene of the present invention is not particularly limited, and examples thereof include a method using differential screening (subtraction cloning). In this method, according to a known technique, direct hybridization in a test tube is repeated to concentrate the target cDNA (the gene of the present invention).

Each step in the above differential screening may be performed under conditions that are normally used. The clone thus obtained can be analyzed in more detail by preparing a restriction enzyme map and determining its nucleotide sequence (sequencing). From these analyses, it can be easily confirmed whether a DNA fragment containing the gene sequence of the present invention has been obtained.

Further, as a method for obtaining the gene of the present invention, a method of isolating and cloning a DNA fragment containing the gene of the present invention by a known technique can be mentioned. For example, a probe that specifically hybridizes with a part of the base sequence of the gene of the present invention may be prepared and a genomic DNA library or cDNA library may be screened. As such a probe, any probe of any sequence / length may be used as long as it specifically hybridizes to at least a part of the base sequence of the gene of the present invention or its complementary sequence.

In addition, for example, if the sequence of the probe is selected from the regions that are well conserved among the above genus, and the genomic DNA (or cDNA) library of other genus genus is screened, the above protein and Genes encoding homologous or related molecules with similar functions can be isolated and cloned.

Alternatively, examples of a method for obtaining the gene of the present invention include a method using amplification means such as PCR. For example, among the cDNA sequences of the gene of the present invention, primers are prepared from the 5 ′ side and 3 ′ side sequences (or their complementary sequences), and genomic DNA (or cDNA) is used as a template using these primers. By performing PCR or the like and amplifying the DNA region sandwiched between both primers, a large amount of DNA fragments containing the gene of the present invention can be obtained. Operations such as PCR and primer preparation can be performed by a known genetic engineering technique (gene manipulation technique). The gene of the present invention can also be synthesized by a DNA synthesizer.

The protein encoded by the gene obtained by the above method has phospholipase activity, lipoxygenase activity or cyclooxygenase. For example, the protein is produced by introducing the gene into Escherichia coli, yeast, etc., and the prepared protein fraction is used. Thus, it can be confirmed by analyzing each enzyme activity in vitro.

(5) Gene and protein utilization method (usefulness) according to the present invention
(5-1) Recombinant Expression Vector The recombinant expression vector of the present invention is used for, for example, producing the above-described proteins (phospholipase protein, lipoxygenase protein and cyclooxygenase protein) according to the present invention in a host cell, or an eicosanoid described later. It can be used for the production of plant transformants having a modified composition. The recombinant expression vector of the present invention can be suitably used for the production of a transformant used in the eicosanoid production method of the present invention.

The recombinant expression vector according to the present invention is not particularly limited as long as it contains the phospholipase gene, lipoxygenase gene or cyclooxygenase gene according to the present invention described in (2) above. For example, a recombinant expression vector into which the cDNA of the gene according to the present invention is inserted can be mentioned. In the phospholipase gene, lipoxygenase gene and cyclooxygenase gene of the present invention, these two or more genes may be contained in the same recombinant expression vector, or each may be contained in a separate recombinant expression vector. The recombinant expression vector preferably further contains regulatory sequences such as a promoter necessary for the expression of the gene in order to express the gene according to the present invention in the host. The recombinant expression vector may be produced using a known method.

The recombinant expression vector containing the gene according to the present invention usually incorporates the gene, promoter, terminator and the like according to the present invention into the multicloning site of the basic vector (in the following description, referred to as the basic vector for convenience). You can build with.

The specific type of basic vector is not particularly limited, and a vector that can be expressed in a host cell may be appropriately selected. Here, plasmids, phages, cosmids, and the like can be used as the basic vector, but are not particularly limited. That is, according to the type of host cell, a promoter sequence or the like is appropriately selected in order to reliably express the gene, and a gene obtained by incorporating this and the gene of the present invention into a basic vector such as various plasmids can be used as a recombinant expression vector. That's fine.
When the recombinant expression vector of the present invention is introduced into a plant to produce a plant transformant or the eicosanoid composition of a plant is modified, a plant cell vector described later is preferably used as a basic vector.

In order to confirm whether or not the gene of the present invention has been introduced into the host cell, and whether or not it is reliably expressed in the host cell, various markers may be used. The marker is not particularly limited, and a marker known per se may be used. For example, a gene deleted in the host cell is used as a marker, and a plasmid or the like containing this marker and the gene of the present invention is introduced into the host cell as a recombinant expression vector. Thus, the introduction of the gene of the present invention can be confirmed from the expression of the marker gene. Examples of such marker genes include various drug resistance genes and genes that complement plant auxotrophy. More specifically, examples include hygromycin, neomycin resistance gene (G418 resistance), chloramphenicol resistance gene, kanamycin resistance gene, tetracycline resistance gene or herbicide chlorsulfuron resistance gene, spectinomycin resistance gene and the like. It is done. Alternatively, the protein of the present invention may be expressed as a fusion protein. For example, a green fluorescent protein GFP (Green Fluorescent Protein) derived from Aequorea jellyfish may be used as a marker, and the protein of the present invention may be expressed as a GFP fusion protein. When the host cell is a plant cell, the marker is preferably a hygromycin resistance gene. Moreover, it is preferable to have a promoter and a terminator for recognizing the gene upstream and downstream of the marker gene.

The host cell is not particularly limited, and various conventionally known cells can be suitably used. Specific examples of host cells used for preparing the protein of the present invention include plants, animals other than humans, insects, bacteria such as Escherichia coli, yeasts (budding yeast Saccharomyces cerevisiae, fission yeast). Examples of such cells include, but are not limited to, oocytes of Schizosaccharomycesbepombe, Caenorhabditis elegans, and Xenopus laevis. As bacteria, Escherichia coli is preferable. As the plant, for example, white crocodile, algae, Physcomitrella patens, and the above-mentioned genus Coleoptera are preferable, and the genus Asteraceae is more preferable. Of these, Escherichia coli and yeast are preferred as host cells for preparing the protein of the present invention.

In the case where the recombinant expression vector of the present invention is introduced into a plant to produce a plant transformant, preferably a plant transformant having a modified eicosanoid composition, the plant is preferably a genus Coleoptera. That is, a preferred host cell is a cell of the order genus Coleoptera.

The method for introducing the above recombinant expression vector into a host cell, that is, the transformation method is not particularly limited, and may be appropriately selected depending on the host. For example, conventionally known methods such as an electroporation method, a calcium phosphate method, a liposome method, a DEAE dextran method, an Agrobacterium method, and a particle gun method can be suitably used. In addition, for example, when the protein of the present invention is transferred and expressed in insects, an expression system using baculovirus can be employed.

(5-2) Transformant A transformant according to the present invention is a trait into which one or more of the phospholipase gene, lipoxygenase gene and cyclooxygenase gene according to the present invention described in (2) above is introduced. If it is a converter, it will not specifically limit. Such a transformant is capable of producing (expressing) the protein according to the present invention (phospholipase protein, lipoxygenase protein and cyclooxygenase protein). Moreover, the plant transformant obtained by introducing the gene of the present invention into a plant can be used for the production of eicosanoids as described later. Here, the “plant transformant” means not only plant cells, tissues and organs but also individual plants (plants). Preferably, it is a transformant in which the phospholipase gene, lipoxygenase gene and cyclooxygenase gene according to the present invention are introduced.

The plant transformant according to the present invention is a plant trait having a modified eicosanoid composition, wherein one or more of the phospholipase gene, lipoxygenase gene and cyclooxygenase gene according to the present invention are introduced so as to be expressed in a plant. A converter is more preferable. Such a plant transformant can be suitably used for the production of eicosanoids. Preferably, it is a plant transformant in which the phospholipase gene, lipoxygenase gene and cyclooxygenase gene according to the present invention are introduced into a plant so that they can be expressed. Such a plant transformant having a modified eicosanoid composition can be produced, for example, by introducing a phospholipase gene, a lipoxygenase gene and a cyclooxygenase gene according to the present invention into a plant. In addition, a plant transformant that is a descendant of the plant transformant having the same properties as the plant transformant, or a tissue of the plant transformant is also a preferred embodiment of the present invention.

Here, “a gene has been introduced so that it can be expressed” means that the gene is introduced into a target cell (for example, a plant host cell) so that it can be expressed by a known genetic engineering technique (gene manipulation technique). Means. In the present invention, the plant may be, for example, the whole plant body or a part of the plant body, or may be a plant cell such as protoplast or callus.

Eicosanoids such as leukotrienes and prostaglandins can be produced by a plant transformant (transformed plant) obtained by introducing such a gene according to the present invention into a plant by a low-cost and environment-friendly production process.

The method for producing the transformant (production method) of the present invention is not particularly limited, and examples thereof include a method for transformation by introducing the above-described recombinant expression vector into a host cell. As a method for introducing the recombinant expression vector into the host cell, a known method may be used, and it may be appropriately selected depending on the host. In addition, the organism to be transformed is not particularly limited, and examples thereof include various microorganisms, plants, animals and the like exemplified in the host cell. As described above, when the gene of the present invention is introduced into a host cell and the protein according to the present invention is produced using the transformed cell, the host cell includes plants, animals other than humans, insects, bacteria, Yeast and the like are preferable. As bacteria, Escherichia coli is preferable. As the plant, for example, white crocodile, algae, Physcomitrella patens, the above-mentioned genus Coleoptera, and the like are more preferable, and C. elegans are more preferable. Among them, E. coli and yeast are preferable as the host cell used for obtaining the protein according to the present invention.
When eicosanoids are produced by the plant transformant of the present invention, the host plant is preferably a genus Genus. Among these, Zenigoke (Marchantia polymorpha) is preferable.

The recombinant expression vector used for plant transformation is not particularly limited as long as it can express the inserted gene in the plant cell. For example, as a basic vector used for the production of a recombinant expression vector, for example, pUC plasmids such as pUC18 and pUC19; plasmid DNA for plant host cells such as pBI221, or pWTT23132 (manufactured by DNAP), Gateway (manufactured by Invitrogen) A vector for plant cells such as a binary vector such as PCS31 vector (for chloroplast) is preferred. Further, as described above, a recombinant expression vector having a promoter that constantly expresses a gene in a plant cell (for example, the 35S promoter of the cauliflower mosaic virus described above) or an inducibly activated by an external stimulus. A recombinant expression vector having a promoter can be used. The plant cells into which the recombinant expression vector is introduced include various types of plant cells, such as suspension culture cells, protoplasts, leaf sections, and callus. The gene of the present invention may be introduced into intracellular organelles other than the nucleus such as chloroplasts in plant cells by a recombinant expression vector.

Any promoter may be used as long as it can be expressed in plant cells as a promoter for expressing the gene according to the present invention contained in the recombinant expression vector in plant cells. For example, a plant-derived promoter such as the cauliflower mosaic virus 35S promoter, rd29A gene promoter, or rbcS promoter; or a cauliflower mosaic virus 35S promoter enhancer sequence added to the 5 ′ side of the Agrobacterium-derived mannopine synthase promoter sequence. A constitutive promoter such as -1 promoter is preferred. Furthermore, an artificially designed and modified promoter such as a tac promoter may be used. Various promoters derived from plant genes can also be used. Among them, a constitutive promoter is preferably used because the gene involved in biosynthesis of the introduced eicosanoid is preferably constitutively expressed in plants, and the cauliflower mosaic virus 35S promoter and the like are more preferable.
The terminator can be appropriately selected depending on the gene to be introduced, and examples thereof include AG7 terminator, NOS terminator, 35S terminator, rps16 terminator, CaMV35S terminator and the like.

For introduction of a recombinant expression vector into a plant cell, a polyethylene glycol method, an electroporation method (electroporation method), an Agrobacterium-mediated method (for example, Heiei, Y. et al., Plant J., 6, 271-282, 1994, Takaiwa, F., et al., Plant Sci. 111, 39-49, 1995), and various methods known to those skilled in the art, such as a particle gun method. Regeneration of a plant body from a transformed cell can be performed by a method known to those skilled in the art depending on the type of plant cell.

For example, there are already several techniques for creating plant transformants of the genus Amanita, such as a method of directly regenerating plants by introducing a gene directly into cells by the particle gun method, and a method using Agrobacterium. Has been established. In the present invention, these methods can be suitably used. A technique for producing such a transformant of the genus Coleoptera is described in, for example, Plant Cell Physiol., 49, p1048 2008.

Once a plant transformant in which the gene of the present invention has been introduced into the genome is obtained, offspring can be obtained from the plant body of the plant transformant by sexual reproduction or asexual reproduction. Further, from the plant transformant, its progeny, or clone, a propagation material such as a leaf, embryo, spore, temporary root, strain, callus, protoplast, etc. is obtained and based on them It is possible to mass-produce plant transformants. Therefore, the present invention includes a plant transformant into which the gene of the present invention has been introduced, a plant transformant that is a descendant of the plant transformant having the same properties as the plant transformant, and the plant trait This includes the organization of converters. Furthermore, the present invention also includes a propagation material obtained from the plant transformant or the tissue of the plant transformant. As a method for obtaining a propagation material from the plant transformant or the tissue of the plant transformant, a known method can be used. There are no particular restrictions on the method for growing or growing a plant or plant tissue into which a gene has been introduced by the above recombinant expression vector, the method for regenerating a plant from plant cells, the method for cultivating, etc. The conditions according to the above can be used as appropriate.

A plant transformant having a modified eicosanoid composition, the plant transformant having the same properties as the plant transformant, wherein the gene according to the present invention is introduced so that it can be expressed in the plant, and the plant transformant serving as a descendant of the plant transformant The plant, the tissue of the plant transformant, and the propagation material obtained from the plant transformant or the tissue of the plant transformant are also included in the present invention. “The eicosanoid composition has been altered” means that the eicosanoid composition in the plant before transformation is different from the eicosanoid composition in the plant transformant or its tissue after transformation. For example, (1) a plant that originally did not contain leukotriene, prostaglandin, etc. in the eicosanoid composition was transformed with the gene according to the present invention, so that the eicosanoid composition of the plant transformant produced was leukotriene, prostaglandin. In the case where gin or the like is included, (2) the case where the content of leukotriene, prostaglandin, etc. in the eicosanoid composition is increased as compared with that before transformation, and the like can be mentioned.

(5-3) Eicosanoid Production Method In the present invention, a plant transformant transformed with the gene of the present invention (preferably a plant transformant having a modified eicosanoid composition) or a tissue of the plant transformant is used. Methods for producing eicosanoids. For example, as described above, the plant transformant according to the present invention having an increased content of leukotrienes, prostaglandins and the like has a high content of leukotrienes and prostaglandins, and is highly valuable as a raw material having high pharmaceutical and pharmacological activity. . Such plant transformants are also useful as raw materials for animal drugs, functional foods, feeds, and the like.

In the production of an eicosanoid using a plant body of a plant transformant or a tissue of the plant transformant, the eicosanoid is efficiently produced in the cells of the plant body or the tissue as the plant body or the tissue grows or grows. Produced. The produced eicosanoid accumulates in the cells of the plant transformant or its tissue. The method for producing an eicosanoid using a plant transformant or a tissue of the plant transformant may include a step of growing or growing the plant transformant or the tissue (preferably by asexual reproduction (vegetative reproduction)). preferable. In order to proliferate or grow a plant transformant or tissue thereof, the plant transformant or tissue thereof may be appropriately cultivated or cultured. The conditions and time for growing or growing (cultivating or cultivating) the plant transformant or its tissue may be appropriately selected depending on the type of plant. For example, when using the genus Coleoptera transformed with the gene according to the present invention, it is preferable to use asexual reproduction (vegetative reproduction) as the breeding form of the genus Coleoptera. That is, for the genus Coleoptera, it is preferable to grow or grow the transformant or the tissue thereof by asexual reproduction (vegetative reproduction) because the growth speed is high and mass production is possible even as a plant factory system. A well-known method can be used about the plant cultivation floor and the plant cultivation method. Further, for example, the method described in Japanese Patent Application No. 2008-259372 (cultivation apparatus using a mat type system using a vertical multi-stage cultivation shelf) can be used. For example, a transformant (transformed cell) in which the gene of the present invention has been introduced into a cell of the genus Coleoptera is usually cultured for 20 to 49 days, preferably 31 to 49 days to proliferate the eicosanoid into the cell. accumulate. In the case of the genus Coleoptera, the culture or cultivation temperature is usually about 20 to 30 ° C., preferably about 25 to 30 ° C.

The plant transformant produced by producing the eicosanoid according to the present invention or the tissue thereof can be used as it is as a material containing eicosanoid as it is for a pharmaceutical, food, veterinary medicine, feed, etc. A step of purifying the produced eicosanoid from the tissue may be included. The method for purifying eicosanoid is not particularly limited, and can usually be performed by a high-pressure high-temperature steam distillation method.

(5-4) Material The present invention includes a material obtained by the above-described eicosanoid production method, that is, a material containing at least one of leukotriene and prostaglandin. This “raw material” means all materials that can be used for the above-mentioned pharmaceutical raw materials, food raw materials, veterinary pharmaceutical raw materials, feeds, and the like.

The eicosanoids of the above materials have unique physical properties that have multiple double bonds in the molecule. Therefore, it is currently synthesized by total chemical synthesis using lactone as a raw material. For this reason, for example, the production cost of these eicosanoids can be reduced by producing leukotrienes and prostaglandins with the transformed plant of the present invention. In addition, the present invention can realize an eco-friendly eicosanoid production process.

Examples of the eicosanoids produced by the present invention include eicosanoids produced from arachidonic acid, such as leukotrienes (LT) such as LTA4; prostaglandin G ₂ (PGG ₂ ), prostaglandin H ₂ (PGH ₂ ), prosta Examples include prostaglandins (PG) such as glandin F (PGF2α). Examples of eicosanoids produced from eicosapentaenoic acid include leukotrienes such as LTA5; prostaglandins such as prostaglandin G ₃ (PGG ₃ ) and prostaglandin H ₃ (PGH ₃ ). Among them, the method of the present invention is applicable to leukotrienes (LT) such as leukotriene LTA5; prostaglandin H ₂ (PGH ₂ ), prostaglandin G ₃ (PGG ₃ ), prostaglandin H ₃ (PGH ₃ ), and prostaglandin F. Suitable for the production of prostaglandins (PG) such as (PGF2α).

(5-5) Eicosanoid Composition Modification Method The present invention includes a method of modifying the eicosanoid composition of a host using the gene according to the present invention. For example, as described above, the eicosanoid composition of the host cell can be altered by preparing a transformant into which the gene according to the present invention has been introduced. The target for modifying the eicosanoid composition is not particularly limited, and it is possible to target any organism other than plants, such as animals, bacteria, and yeasts.

A method for modifying the eicosanoid composition of a plant or its tissue including the step of introducing the gene according to the present invention described above into the plant is also one aspect of the present invention. Preferably, the phospholipase gene, lipoxygenase gene and cyclooxygenase gene according to the present invention are introduced into a plant. As the plant, the above-mentioned genus Coleoptera is preferable, and among them, the genus Marchantia polymorpha is more preferable.

(5-6) Gene Detection Instrument The gene detection instrument according to the present invention uses at least a partial base sequence of the gene according to the present invention or a complementary sequence thereof as a probe. The gene detection instrument can be used for detection and measurement of the expression pattern of the gene of the present invention under various conditions.
Examples of the gene detection instrument of the present invention include a DNA chip in which the probe that specifically hybridizes with the gene of the present invention is immobilized on a substrate (carrier). Here, the “DNA chip” mainly means a synthetic DNA chip using a synthesized oligonucleotide as a probe, but also includes an affixed DNA microarray using a cDNA such as a PCR product as a probe.

The sequence used as a probe can be determined by a conventionally known method for specifying a characteristic sequence from a cDNA sequence. Specifically, for example, SAGE: Serial Analysis of （Gene Expression method (Science 276: 1268, 1997; Cel1 88: 243, 1997; Science 270: 484,1995; Nature 389: 300,1997; US Patent No. 5,695,937) Etc.

In addition, what is necessary is just to employ | adopt a well-known method for manufacture of a DNA chip. For example, when a synthetic oligonucleotide is used as an oligonucleotide, the oligonucleotide may be synthesized on a substrate by a combination of a photolithography technique and a solid phase DNA synthesis technique. On the other hand, when cDNA is used as the oligonucleotide, it may be attached to the substrate using an array machine.

Also, as in a general DNA chip, a perfect match probe (oligonucleotide) and a mismatch probe in which one base is substituted in the perfect match probe may be arranged to further improve gene detection accuracy. Furthermore, in order to detect different genes in parallel, a plurality of types of oligonucleotides may be fixed on the same substrate to constitute a DNA chip.

The gene detection instrument according to the present invention is not limited to the above-described DNA chip, and may be any one that uses at least a partial base sequence of the gene according to the present invention or a complementary sequence thereof as a probe. For example, a kit containing at least a partial base sequence of the gene according to the present invention or a complementary sequence thereof is also included.

(5-7) Antibody An antibody that recognizes the protein encoded by the gene of the present invention is also encompassed by the present invention. The antibody according to the present invention is preferably an antibody obtained as a polyclonal antibody or a monoclonal antibody by a known method using the protein according to the present invention, or a partial protein or partial peptide thereof as an antigen. Known methods include, for example, literature (Harlow et al. “Antibodies: A laboratory manual” (Cold Spring Harbor Laboratory, New York (1988)), Iwasaki et al. “Monoclonal antibody hybridoma and ELISA, Kodansha (1991)”). The method of description is mentioned. The antibody thus obtained can be used for detection and measurement of the protein of the present invention.

(5-8) Screening Method The screening method according to the present invention is a method for screening a gene that regulates the protein or a substance that regulates the protein, using the protein according to the present invention. As the screening method of the present invention, various conventionally known methods for examining the presence or absence of binding or dissociation between substances can be applied and are not particularly limited. For example, screening for substances that promote or inhibit the activity of the protein according to the present invention (the above-mentioned phospholipase activity, lipoxygenase activity, or cyclooxygenase activity) can be mentioned.
The present invention also includes genes or substances obtained by the above screening methods.

(6) Use of animal-derived eicosanoid biosynthetic genes One or more of the above-described phospholipase gene, lipoxygenase gene and cyclooxygenase gene according to the present invention, or instead of these genes, an animal-derived phospholipase A2 gene Plant transformants produced by introducing one or more of animal-derived lipoxygenase genes and animal-derived cyclooxygenase genes into plants can also be suitably used for the production of eicosanoids. Hereinafter, the animal-derived phospholipase A2 gene, lipoxygenase gene and cyclooxygenase gene are also simply referred to as “animal-derived eicosanoid biosynthesis genes”.

When an animal-derived eicosanoid biosynthetic gene is introduced into a plant, an animal-derived eicosanoid biosynthetic system is used instead of the phospholipase gene, lipoxygenase gene and cyclooxygenase gene of the present invention in the above-described method for producing a plant transformant. A gene may be used, and the transformation method and preferred embodiments thereof are the same as those described above.

(6-1) Recombinant Expression Vector Animal-derived eicosanoid biosynthetic genes are usually introduced into plants using a recombinant expression vector obtained by inserting the gene into a plant cell vector.
A recombinant expression vector obtained by inserting an animal-derived phospholipase A2 gene, lipoxygenase gene, or cyclooxygenase gene into a plant cell vector is one of the preferred embodiments of the present invention. A method for producing a recombinant expression vector containing such an animal-derived eicosanoid biosynthetic gene is not particularly limited. Similarly to the above-described method for producing a recombinant expression vector containing a gene according to the present invention, a known method can be used. It can be carried out. Usually, a plant cell vector is used as a basic vector, and a recombinant expression vector can be constructed by incorporating an animal-derived eicosanoid biosynthesis gene, promoter, terminator, and the like into the multicloning site of the basic vector. In the construction of a recombinant expression vector containing an eicosanoid biosynthetic gene derived from animals, plant cell vectors used as basic vectors, promoters and terminators that can be expressed in plant cells are the same as those described above. Two or more of these animal-derived phospholipase A2 gene, lipoxygenase gene and cyclooxygenase gene may be contained in the same vector, or may be contained in different vectors.

The animal-derived phospholipase A2 gene may be any gene that is an animal-derived gene and encodes the above-described protein having phospholipase activity. As such an animal-derived phospholipase A2 gene, a mouse-derived phospholipase A2 gene (Cell 65, p1043, 1991) is preferable.

The animal-derived lipoxygenase gene may be any gene that is an animal-derived gene and encodes the above-described protein having lipoxygenase activity. As such a gene, a mouse-derived 5-lipoxygenase gene (J. Biol. Chem. 270, p17993, 1995) is preferable.

The animal-derived cyclooxygenase gene may be any gene that is an animal-derived gene and encodes the above-described protein having cyclooxygenase activity. As such a gene, a zebrafish-derived cyclooxygenase gene (Biochem. Biophys. Res. Commun. 352, p181, 2007) is preferable.

A recombinant expression vector obtained by inserting the above mouse-derived phospholipase A2 gene, mouse-derived 5-lipoxygenase gene, or zebrafish-derived cyclooxygenase gene into a plant cell vector is also one aspect of the present invention.

(6-2) Transformant A plant transformant can be obtained by introducing one or more of phospholipase A2 gene, 5-lipoxygenase gene and cyclooxygenase gene derived from animals into a plant. A plant transformant obtained by introducing at least one or more of the mouse-derived phospholipase A2 gene, mouse-derived 5-lipoxygenase gene or zebrafish-derived cyclooxygenase gene into a plant is a preferred embodiment of the present invention. One. More preferably, it is a plant transformant obtained by introducing a mouse-derived phospholipase A2 gene, a mouse-derived 5-lipoxygenase gene, and a zebrafish-derived cyclooxygenase gene into a plant. Since such a plant transformant also produces eicosanoid and accumulates it in the cell, it can be suitably used for the production of eicosanoid. As the plant, the above-mentioned genus Coleoptera is preferable. The plant transformation method and the like are the same as those described above.

A plant transformant having a modified eicosanoid composition, in which one or more of a mouse-derived phospholipase A2 gene, a mouse-derived 5-lipoxygenase gene, and a zebrafish-derived cyclooxygenase gene are introduced so as to be expressed in a plant, This is one of the preferred embodiments of the invention. Moreover, the plant transformant which becomes the descendant of this plant transformant which has the same property as such a plant transformant, or the structure | tissue of this plant transformant is also preferable as a plant transformant of this invention.

Producing eicosanoids such as leukotrienes and prostaglandins by a low-cost and environmentally friendly production process by using plant transformants (transformed plants) obtained by introducing such an eicosanoid biosynthetic gene derived from animals into plants. Can do.

Once a plant transformant in which the above-described animal-derived eicosanoid biosynthesis gene is introduced into the genome is obtained, offspring can be obtained from the plant body of the plant transformant by sexual reproduction or asexual reproduction. . Moreover, it is possible to obtain the above-mentioned propagation material from the plant transformant, its progeny, or clone, and mass-produce the plant transformant based on them. Therefore, the present invention includes a plant transformant obtained by introducing the above-described animal-derived eicosanoid biosynthesis gene into a plant, and a plant that is a descendant of the plant transformant having the same properties as the plant transformant. The transformant and the tissue of the plant transformant are also included. Furthermore, the present invention also includes a propagation material obtained from the plant transformant or the tissue of the plant transformant. As a method for obtaining a propagation material from the plant transformant or the tissue of the plant transformant, a known method can be used in the same manner as described above. The method for growing or growing a plant or plant tissue introduced with the eicosanoid biosynthetic gene derived from the animal, the method for regenerating a plant from plant cells, the method for cultivating, etc. is not particularly limited. Conditions according to the type and the like can be used as appropriate.

(6-3) Eicosanoid Production Method The present invention also includes a method for producing eicosanoid using a plant transformant transformed with an eicosanoid biosynthesis gene derived from an animal or a tissue thereof. For example, as described above, a plant transformant according to the present invention having an increased leukotriene or prostaglandin content has a high leukotriene or prostaglandin content, and is highly valuable as a raw material having high pharmaceutical and pharmacological activity. Such plant transformants are also useful as raw materials for animal drugs, functional foods, feeds, and the like.

In the production of an eicosanoid using a plant transformant or a tissue thereof, eicosanoid is efficiently produced in the cells of the plant body or the tissue as the plant body or the tissue grows or grows. The produced eicosanoid accumulates in the cells of the plant transformant or its tissue. The method for producing an eicosanoid using a plant transformant or a tissue of the plant transformant may include a step of growing or growing the plant transformant or the tissue (preferably by asexual reproduction (vegetative reproduction)). preferable. In order to proliferate or grow a plant transformant or tissue thereof, the plant transformant or tissue thereof may be appropriately cultivated or cultured. The conditions and time for growing or growing (cultivating or cultivating) the plant transformant or its tissue may be appropriately selected depending on the type of plant. For example, in the case of using a genus Coleoptera transformed with an eicosanoid biosynthetic gene derived from an animal, as described above, it is preferable to use asexual reproduction (vegetative reproduction) as a breeding form of the genus Coleoptera.

The plant transformant produced by producing the eicosanoid according to the present invention or the tissue thereof can be used as it is as a material containing eicosanoid as it is for a pharmaceutical, food, veterinary medicine, feed, etc. A step of purifying the produced eicosanoid from the tissue may be included. The eicosanoid purification method is not particularly limited, and can be performed by the above-described known methods.

(6-4) Material The present invention includes a material obtained by the above-described eicosanoid production method, that is, a material containing at least one of leukotriene and prostaglandin. This “raw material” means the same general materials as those described above, which can be used for the above-mentioned pharmaceutical raw material use, food raw material use, veterinary pharmaceutical raw material use, feed use and the like.

(6-5) Eicosanoid Composition Modification Method In the present invention, an eicosanoid biosynthetic gene derived from an animal (preferably a phospholipase A2 gene derived from a mouse, a 5-lipoxygenase gene derived from a mouse and a cyclooxygenase gene derived from a zebrafish or 2 or more) is used to modify the eicosanoid composition of the host. For example, it is possible to modify the eicosanoid composition of the host cell by preparing a transformant into which an animal-derived eicosanoid biosynthesis gene is introduced as described above. The target for modifying the eicosanoid composition is not particularly limited, and it is possible to target any organism other than plants, such as animals, bacteria, and yeasts.

A method for modifying the eicosanoid composition of a plant or tissue thereof comprising the step of introducing one or more of the mouse-derived phospholipase A2 gene, mouse-derived 5-lipoxygenase gene and zebrafish-derived cyclooxygenase gene into the plant, It is one of the inventions. As the plant, the above-mentioned genus Coleoptera is preferable.

(6-6) Screening method The screening method according to the present invention uses a protein encoded by the above-described animal-derived eicosanoid biosynthesis gene to screen for a gene that regulates the protein or a substance that regulates the protein. The method is also one aspect of the present invention. The screening method of the present invention is the same as the screening method described above, except that a protein encoded by an eicosanoid biosynthesis gene derived from an animal is used instead of the protein according to the present invention described above.
The present invention also includes genes or substances obtained by the above screening methods.

Hereinafter, some embodiments and drawings will be shown to describe the embodiment of the present invention in more detail. The present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Furthermore, the present invention is not limited to the technical embodiments, and various modifications shown in the claims can be made, and the embodiments of the present invention also apply to the embodiments obtained by appropriately combining the disclosed technical means. Included in the scope.

In this example, unless otherwise specified, the experimental method was in accordance with the method described in Molecular Cloning (Sambrook et al. Cold Spring Harbor Laboratory Press, 1989).

(Example 1) Isolation of phospholipase gene derived from genus Amanita As a result of intensive comparison and study of the amino acid sequence of the cloned phospholipase gene of other organisms, a clone showing homology with an animal phospholipase was found from the genus moss EST library. It was found that the amino acid sequences Leu-Gly-Arg-Arg-Asp (SEQ ID NO: 7) and Thr-Gly-His-Ser-Leu-Gly (SEQ ID NO: 8) were conserved. Therefore, in order to isolate the phospholipase gene derived from genus Amanita, the following primers encoding the above amino acid sequences were used.
PLA1F: 5'-CTCGGCCGACGAGAC-3 '(SEQ ID NO: 9)
PLA1R: 5'-GCCCAAGCTGTGCCCCGT-3 '(SEQ ID NO: 10)

As a sample, a leaf-like body of genus moss (see Transgenic Res. 9, P179, 2000) was used. Isolation of total RNA from the frond was carried out according to the method recommended by the manufacturer using an RNA extraction kit “RNeasy Plant Mini Mini kit” (manufactured by QIAGEN). The isolated total RNA 5 μg was reverse transcribed into cDNA using ReverTra Ace (TOYOBO). Using 1 μL of cDNA as a template, PCR was performed by the method recommended by the manufacturer using the above primers (PLA1F and PLA1R) and 0.5 U of enzyme (Takara Ex Taq, Takara). The reaction volume was 20 μL, and the reaction was repeated 25 times using a PCR-Thermal-Cycler-Dice (Takara), held at 95 ° C. for 2 minutes, then 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute. Then, it was cooled to 4 ° C.

The obtained PCR product was electrophoresed on a 0.7% (w / v) agarose gel, and the amplified fragment having the size expected from the base sequence of phospholipase was recovered from the gel using Gel Extraction kit (QIAGEN). did. The recovered amplified fragment was ligated to pGEM-Teasy (Promega) and transformed into competent E. coli DH5α.

Using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems) and automated sequencer ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), the base sequence of all the obtained clones was determined, and the partial sequence of the phospholipase gene was obtained. . Therefore, in order to obtain a full-length cDNA sequence, the following primers were used.
3 'RACE (PLA1): 5'-TGACAGCGGGCTTCTGGCAC-3' (SEQ ID NO: 11)
5 'RACE (PLA1): 5'-CCTGGCGCAATACGGAGTCC-3' (SEQ ID NO: 12)

By the above method, total RNA was isolated from the genus Amanita, and 5 μg of total RNA was reverse-transcribed into cDNA using SMART® RACE® cDNA® kit (BD Bioscience). 5 ′ and 3′-RACE® PCR uses 1 μL of the above cDNA as a template, the above primers (3′RACE (PLA1) and 5′RACE (PLA1)) and enzyme (Takara® Ex® Taq, manufactured by Takara) 0.5 U. And performed as recommended by the manufacturer. The reaction volume is 20 μL, and the PCR-Thermal-Cycler-Dice (manufactured by Takara) is used and kept at 95 ° C. for 2 minutes. Then, it was cooled to 4 ° C. The obtained DNA fragment was subcloned, the base sequence was determined, and the full-length cDNA sequence was obtained.

As a result, one type of clone (SEQ ID NO: 1) was isolated as a phospholipase gene of Xenopus. This clone encoded 565 amino acids as shown in SEQ ID NO: 2. The coding region of the base sequence shown in SEQ ID NO: 1 was the 394th to 2091th base sequence. A clone having a length encoding the amino acid sequence represented by SEQ ID NO: 2 was used in the following examples.

As a result of comparing the deduced amino acid sequence encoded by the phospholipase gene derived from Xenopus obtained above with the amino acid sequence of phospholipase reported in Arabidopsis thaliana, only 21% identity was shown.

(Example 2) Isolation of Lipoxygenase Gene Derived from Agaricus As a result of extensive comparison and study of the amino acid sequence of the lipoxygenase gene cloned from other organisms, a clone showing homology with the lipoxygenase of animals was found from the Agaricus EST library. It was found that the amino acid sequences Pro-Ile-Ala-Ile-Glu-Leu (SEQ ID NO: 13) and Asn-Phe-Gly-Gln-Tyr (SEQ ID NO: 14) were conserved. Therefore, the following primers encoding the above amino acid sequences were used in order to isolate the genus Lipoxygenase gene.
LOX-F1: 5'-CCCATCGCCA TTGAGCTC-3 '(SEQ ID NO: 15)
LOX-R1: 5'-GTACTGGCCA AAGTT-3 '(SEQ ID NO: 16)

PCR was performed using the above primers, and the resulting DNA fragment was subcloned. The base sequence of the obtained clone was determined, and the partial sequence of the target lipoxygenase gene was obtained. Therefore, in order to obtain a full-length cDNA sequence, the following primers were used. The experimental materials and methods are the same as in Example 1.
3 'RACE (LOX): 5'-GAGACAGAAATCCCTACTGG-3' (SEQ ID NO: 17)
5 'RACE (LOX): 5'-ATTCCAGTCGCATTGATGAG-3' (SEQ ID NO: 18)

As a result, one type of homologous gene candidate was isolated, and this gene was designated as the MpLOX gene. The MpLOX gene is DNA consisting of the 127th to 2994th base sequences in the base sequence shown in SEQ ID NO: 3. The length of the isolated MpLOX gene cDNA (excluding the poly A portion) was 3,224 bp, and the deduced amino acid sequence encoded was 955 residues. The nucleotide sequence of the MpLOX gene cDNA is shown in SEQ ID NO: 3, and the deduced amino acid sequence is shown in SEQ ID NO: 4, respectively.

As a result of comparing the deduced amino acid sequence of MpLOX cDNA with the amino acid sequence of the lipoxygenase of Arabidopsis thaliana, it showed only 36.5% identity.

(Example 3) Cyoxygenase-derived cyclooxygenase gene As a result of intensive comparison and study of the amino acid sequence of the cyclooxygenase gene cloned from other organisms, a clone showing homology with animal cyclooxygenase was found from the genus Zenigo EST library. It was found that the amino acid sequences Glu-His-Asn-Leu-Ile-Cys-Asp (SEQ ID NO: 19) and His-Pro-Leu-Leu-Pro-Glu (SEQ ID NO: 20) are conserved. Therefore, the following primers encoding the above amino acid sequences were used in order to isolate the genus Lipoxygenase gene.
COX-F 5'-GAGCACAACCTTATCTGTGAT-3 '(SEQ ID NO: 21)
COX-R 5'-CTCGGGCAGAAGAGGGTG-3 '(SEQ ID NO: 22)

PCR was performed using the above primers (COX-F and COX-R), and the resulting DNA fragment was subcloned. The base sequence of the obtained clone was determined, and the partial sequence of the target cyclooxygenase gene was obtained. Therefore, in order to obtain a full-length cDNA sequence, the following primers were used. The experimental materials and methods are the same as in Example 1.
3'RACE (COX): 5'-GGATCGTTGGGCACTCGAAG-3 '(SEQ ID NO: 23)
5'RACE (COX): 5'-TAACCTTGGCGTGCTTGTAG-3, (SEQ ID NO: 24)

As a result, one type of homologous gene candidate was isolated, and this gene was designated as the MpCOX gene. The length of the MpCOX gene cDNA (excluding the poly A portion) was 2464 bp, and the deduced amino acid sequence was 610 residues. The nucleotide sequence is shown in SEQ ID NO: 5, and the deduced amino acid sequence is shown in SEQ ID NO: 6.

As a result of comparing the deduced amino acid sequence of MpCOX cDNA with the amino acid sequences of zebrafish and human cyclooxygenase, they were found to show 13.2% and 12.1% identity, respectively.

(Example 4) Construction of a vector to be introduced into the genus Amanita and introduction of the vector into the genus Amanita, a phospholipase gene derived from the genus Amanita, obtained in Example 1, In order to express the lipoxygenase gene and / or the cyclooxygenase gene derived from the genus Coleoptera obtained in Example 3, an expression construct was prepared by the following procedure. Further, this production procedure is shown in FIG.

FIG. 2 (A) shows a method for preparing a vector (plasmid pBin-Hyg-TX-MpPLA) for expressing the phospholipase gene (MpPLA) derived from the genus Coleoptera in Amanita.
FIG. 2 (B) shows a method for producing a vector (plasmid pBin-Hyg-TX-MpLOX) for expressing the repolicigenase gene (MpLOX) derived from the genus Coleoptera in Amanita.
FIG. 2 (C) shows a method for preparing a vector (plasmid pBin-Hyg-TX-MpCOX) for expressing the cyclooxygenase gene (MpCOX) derived from the genus Coleoptera in Amanita.

As shown in FIGS. 2 (A) to (C), the SmaI restriction enzyme between the cauliflower mosaic virus (CaMV) 35S promoter of pBIN-HYG-TX (Plant J., 2, p397, 1992) and the OCS terminator Cleavage site, XbaI restriction enzyme cleavage site (SmaI-Xbal cleavage site) contains SmaI-XbaI fragment of cDNA containing ORF of phospholipase gene derived from genus Lepidoptera, ORF of lipoxygenase gene (MpLOX gene) derived from genus Lepidoptera The SmaI-XbaI fragment of the cDNA and the SmaI-XbaI fragment of the cDNA containing the ORF of the cyclooxygenase gene (MpCOX gene) derived from the genus Coleoptera were ligated.

For each ORF amplification, the following primers containing the recognition sequence indicated by the underline were used.
Primers used for ORF amplification of phospholipase genes derived from the genus Pleurotus
PLA-F5: 5'-AA CCCGGG ATGCAGTATCCATCAGTG-3 '(SEQ ID NO: 25)
PLA-R4: 5'-GC TCTAGA TAATCGTAATCATCAGAG-3 '(SEQ ID NO: 26)

Primers used for ORF amplification of the lipoxygenase gene derived from the genus Pleurotus
LOX-F3: 5'-TA CCCGGG ATGCTTAGTGTCCGAGCC-3 '(SEQ ID NO: 27)
LOX-R9: 5'-AC TCTAGA TCAGATGGATATGCTGTT-3 '(SEQ ID NO: 28)

Primers used for ORF amplification of cyclooxygenase genes derived from the genus Pleurotus
COX-F5: 5'- CCCGGG ATGGGGCTCCTATCGTTTG-3 '(SEQ ID NO: 29)
COX-R7: 5'- TCTAGA TAGGGAAAGCGAATTAGAG-3 '(SEQ ID NO: 30)

In PCR for amplifying each gene, PrimeSTAR DNA polymerase (manufactured by Takara) 0.5U was used and maintained at 95 ° C for 2 minutes, then at 95 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 2 minutes. The reaction was repeated 30 times and then cooled to 4 ° C. The obtained DNA fragment was subcloned, the base sequence was determined, and it was confirmed that there was no mistake. The obtained plasmid was treated with the above restriction enzymes and ligated to the pBIN-HYG-TX vector cut with the same restriction enzymes. This was transformed into E. coli DH5α. Plasmid DNA (transformation vector) was isolated from the obtained colonies. Agrobacterium C58rifR was transformed.

The structure of the transformation vector obtained above is schematically shown in FIGS. 3 (A) to 3 (C). FIG. 3A is a schematic diagram showing the structure of the plasmid pBin-Hyg-TX-MpPLA. FIG. 3B is a schematic diagram showing the structure of the plasmid pBin-Hyg-TX-MpLOX. FIG. 3C is a schematic diagram showing the structure of the plasmid pBin-Hyg-TX-MpCOX.
In FIGS. 3A to 3C, LB represents a left boundary array. RB represents the right boundary array. CaMV p35S represents the cauliflower mosaic virus (CaMV) 35S promoter. tOCS represents the OCS gene terminator. pNOS represents the NOS gene promoter. HPT II represents the hygromycin resistance gene. tAG7 represents an Ag7 gene terminator.
The above-mentioned three kinds of constructs (transformation vectors) thus obtained are introduced into Xenoke by the Agrobacterium method by a known method (Plant Cell Rep. 27, p1467, 2008), and transformed Xenoke Acquired.
Eicosanoids can be produced by proliferating or growing the transformed genus moss.

(Example 5)
Construction of a vector for introducing a mouse-derived phospholipase gene, a mouse-derived 5-lipoxygenase gene, and a zebrafish-derived cyclooxygenase gene into the moss and introduction of the vector into the moss. In the moss, the phospholipase A2 gene derived from the mouse (Cell 65 , p1043, 1991), expressing 5-lipoxygenase gene from mouse (J. Biol. Chem. 270, p17993, 1995) and cyclooxygenase gene from zebrafish (Biochem. Biophys. Res. Commun. 352, p181, 2007) Therefore, an expression construct was prepared according to the procedure shown below. In addition, the manufacturing procedure is shown in FIGS.

FIG. 4 (A) shows a method for preparing a vector (plasmid pGWB2-mPLA2) for expressing mouse-derived phospholipase A2 gene (mPLA2) in the moss. FIG. 4 (B) shows a method for preparing a vector (plasmid pGWB2-mLOX) for expressing the mouse-derived 5-lipoxygenase gene (mLOX) in Amanita. FIG. 4 (C) shows a method for producing a vector (plasmid pGWB2-zCOX) for expressing the zebrafish-derived cyclooxygenase gene (zCOX) in the mushroom.

As shown in FIGS. 4 (A) to (C), a mouse-derived phospholipase A2 gene is placed at the EcoRI restriction enzyme cleavage site and the XhoI cleavage site (EcoRI-XhoI cleavage site) between homologous recombination sequences of pENTR (manufactured by Invitrogen). The EcoRI-XhoI fragment of the mouse 5-lipoxygenase gene and the zebrafish cyclooxygenase gene cDNA (ORF) were ligated. For ORF amplification, the following primers containing the recognition sequence shown underlined were used.

Primer used for amplification of mouse-derived phospholipase A2 gene (mPLA2)
mPLA-1FE: 5'-GGGCCCGAATTCAATGTCTTTCATAGATCCTTATCAG-3 '(SEQ ID NO: 31)
mPLA-1RX: 5'-AACGGG CTCGAG TACACAGTGGGTTTACTTAGAA-3 '(SEQ ID NO : 32)

Primers used to amplify mouse-derived 5-lipoxygenase gene (mLOX)
mLOX-1FE: 5'-AAATCC GAATTC CATGCCCTCCTACACGGTCAC-3 '(SEQ ID NO: 33)
mLOX-1RX: 5'-AAGGAA CTCGAG ATGGCTACGCTGTTGGGAA-3 '(SEQ ID NO : 34)

Primers used for amplification of zebrafish-derived cyclooxygenase gene (zCOX)
zCOX-1FE: 5'-ACACGC GAATTC AATGAAAAGTTCAGTATTATTTAT-3 '(SEQ ID NO: 35)
zCOX-1RX: 5'-AGTGTA CTCGAG AGCTCAGATGTCCTTTC-3 '(SEQ ID NO : 36)

In PCR for amplifying each gene, PrimeSTAR DNA polymerase (manufactured by Takara) 0.5U was used and maintained at 95 ° C for 2 minutes, then at 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 2 minutes. The reaction was repeated 30 times and then cooled to 4 ° C. The obtained fragment was treated with restriction enzymes (EcoRI and XhoI) and ligated to the pENTR vector cut with the same enzymes. The obtained plasmid was transformed into E. coli DB3.1, plasmid DNA was isolated from the obtained colony, the base sequence was determined, and it was confirmed that there was no mistake. These plasmids and pGWB2 (manufactured by Invitrogen) are mixed, recombination reaction (LR recombination reaction) is performed by LR recombinase (manufactured by Invitrogen), and cauliflower mosaic virus (CaMV) promoter of pGWB2 and NOS terminator The ORF of each gene was ligated to.

The structure of the transformation vector obtained above is schematically shown in FIGS. 5 (A) to (C).
FIG. 5 (A) is a schematic diagram showing the structure of plasmid pGWB2-mPLA2. FIG. 5 (B) is a schematic diagram showing the structure of plasmid pGWB2-mLOX. FIG. 5 (C) is a schematic diagram showing the structure of plasmid pGWB2-zCOX.
In FIGS. 5A to 5C, LB represents a left boundary array. RB represents the right boundary array. CaMV p35S represents the cauliflower mosaic virus (CaMV) 35S promoter. tNOS represents the NOS gene terminator. p35S represents the cauliflower mosaic virus (CaMV) 35S promoter. HPT II represents the hygromycin resistance gene.
The above-described construct thus obtained was introduced into Xenoke by the Agrobacterium method by a known method (Plant Cell Rep. 27, p1467, 2008), and transformed Xenoke was obtained.
Eicosanoids can be produced by proliferating or growing the transformed genus moss.

As described above, the gene and protein of the present invention are useful for eicosanoid production. Moreover, the plant transformant introduced so that the gene of the present invention can be expressed is extremely useful for supplying raw materials such as drugs and reagents in producing eicosanoids in the pharmaceutical industry and various material industries. In addition, in a plant transformant in which the gene of the present invention has been introduced so that it can be expressed, the content of eicosanoids in the plant body increases, so such a plant transformant is very useful in the pharmaceutical field and the like.

Claims

An eicosanoid production method comprising a step of growing or growing a plant transformant or a tissue thereof into which one or more of a phospholipase gene, a lipoxygenase gene, and a cyclooxygenase gene are introduced so as to be expressed in a plant.
The method according to claim 1, wherein the plant is a genus Pleurotus.
A plant transformant having one or more of a phospholipase gene, a lipoxygenase gene, and a cyclooxygenase gene introduced into a plant so that the plant transformant can be expressed, or a plant transformant having a modified eicosanoid composition, or the same as the plant transformant The method according to claim 1 or 2, which is a plant transformant that is a descendant of the plant transformant having the following properties.
The phospholipase gene is any one of the following genes (a1) to (a8), the lipoxygenase gene is any one of the following genes (b1) to (b8), and the cyclooxygenase gene is any of the following (c1 The method according to any one of claims 1 to 3, wherein the gene is any one of (1) to (c8).
(A1) a gene comprising DNA consisting of the base sequence shown in SEQ ID NO: 1 (a2) consisting of a part of the base sequence shown in SEQ ID NO: 1, and arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into the cell A gene containing DNA derived from the genus Coleoptera encoding a protein having a phospholipase activity to be released (a3) A DNA comprising all or part of the base sequence complementary to the base sequence shown in SEQ ID NO: 1 under stringent conditions And a gene containing a DNA derived from the genus Coleoptera that encodes a protein having a phospholipase activity that liberates arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into the cell (a4) a base represented by SEQ ID NO: 1 DNA comprising the 394th to 2091th nucleotide sequences in the sequence The gene (a5) is hybridized under stringent conditions with either DNA consisting of the 394th to 2091th base sequences of the base sequence shown in SEQ ID NO: 1 or DNA consisting of base sequences complementary to the DNA, And a gene comprising a DNA encoding a protein derived from the genus Coleoptera having a phospholipase activity that releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into the cell (a6) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 The encoding gene (a7) consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 2, and from the cell membrane phospholipid to arachidonic acid or Phospholipa that releases eicosapentaenoic acid into cells A gene encoding a protein derived from the genus Coleoptera having ze activity (a8) a phospholipase A2 gene derived from a mouse (b1) a gene comprising a DNA consisting of the base sequence shown in SEQ ID NO: 3 (b2) a base shown in SEQ ID NO: 3 A gene comprising a part of the sequence and containing a DNA derived from the genus Coleoptera encoding a protein having lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate (b3) complementary to the base sequence shown in SEQ ID NO: 3 Including DNA derived from the genus Lepidoptera that hybridizes under stringent conditions with DNA consisting of all or part of a simple nucleotide sequence and encodes a protein having lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate Gene (b4) nucleotide sequence shown in SEQ ID NO: 3 A gene comprising DNA having the 127th to 2994th base sequence in the sequence (b5) consisting of DNA consisting of the 127th to 2994th base sequence of the base sequence shown in SEQ ID NO: 3 or a base sequence complementary to the DNA A gene (b6) comprising a DNA that hybridizes with any one of the DNAs under stringent conditions and encodes a protein derived from the genus Coleoptera having lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate. A gene encoding a protein comprising the amino acid sequence (b7) comprising the amino acid sequence shown in SEQ ID NO: 4, wherein one or several amino acids are substituted, deleted, inserted and / or added, and arachidone Lipoxy containing acid or eicosapentaenoic acid as substrate A gene encoding a protein derived from the genus Coleoptera having a genease activity (b8) A 5-lipoxygenase gene derived from a mouse (c1) a gene comprising a DNA consisting of the base sequence shown in SEQ ID NO: 5 (c2) shown in SEQ ID NO: 5 A gene comprising a part of the base sequence and containing a DNA derived from the genus Coleoptera encoding a protein having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate (c3) complementary to the base sequence shown in SEQ ID NO: 5 A DNA derived from the genus Amanita which encodes a protein having a cyclooxygenase activity that hybridizes under stringent conditions with all or part of a DNA having a basic nucleotide sequence and that uses arachidonic acid or eicosapentaenoic acid as a substrate. Containing gene (c4) SEQ ID NO: A gene comprising DNA having the 321st to 2153th base sequences in the base sequence shown in (c5) DNA consisting of the 321st to 2153th base sequences in the base sequence shown in SEQ ID NO: 5 or complementary to the DNA A gene (c6) comprising a DNA that hybridizes with any one of DNAs comprising a base sequence under a stringent condition and encodes a protein derived from a genus Coriolis having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate (c6) A gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 6 (c7) in the amino acid sequence shown in SEQ ID NO: 6, wherein one or several amino acids are substituted, deleted, inserted, and / or added Arachidonic acid or eicosapentae A gene encoding a protein derived from the genus Coleoptera having cyclooxygenase activity using acid as a substrate (c8) a cyclooxygenase gene derived from zebrafish
The phospholipase gene is any one of the genes (a1) to (a7), the lipoxygenase gene is any one of the genes (b1) to (b7), and the cyclooxygenase gene is any of the above (c1) to (c The method according to claim 4, wherein the gene is any one of c7).
A gene comprising any of the following DNAs (a) to (c):
(A) DNA comprising the base sequence shown in SEQ ID NO: 1
(B) a DNA derived from the genus Pleurotus that encodes a protein having a phospholipase activity, which comprises a part of the base sequence represented by SEQ ID NO: 1 and releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into cells.
(C) Hybridizing under stringent conditions with DNA consisting of all or part of the base sequence complementary to the base sequence shown in SEQ ID NO: 1, and arachidonic acid or eicosapentaenoic acid from the cell membrane phospholipid, DNA derived from the genus Pleurotus that encodes a protein having phospholipase activity to be released in the body
A gene comprising the following DNA (a) or (b):
(A) DNA consisting of the 394th to 2091th base sequences in the base sequence shown in SEQ ID NO: 1
(B) a cell membrane that hybridizes under stringent conditions with either DNA comprising the 394th to 2091th nucleotide sequences of the nucleotide sequence represented by SEQ ID NO: 1 or DNA comprising a nucleotide sequence complementary to the DNA. DNA that encodes a protein derived from the genus Lepidoptera having phospholipase activity that releases arachidonic acid or eicosapentaenoic acid from phospholipids into cells.
A gene encoding the following protein (A) or (B).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (B) an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 2 And a protein derived from the genus Pleurotus having a phospholipase activity that releases arachidonic acid or eicosapentaenoic acid from cell membrane phospholipids into the cell.
A gene comprising any of the following DNAs (a) to (c):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 3
(B) a DNA derived from the genus Pleurotus that encodes a protein having a lipoxygenase activity comprising a part of the base sequence shown in SEQ ID NO: 3 and having arachidonic acid or eicosapentaenoic acid as a substrate;
(C) Lipoxygenase activity that hybridizes under stringent conditions with DNA consisting of all or part of the base sequence complementary to the base sequence shown in SEQ ID NO: 3 and that uses arachidonic acid or eicosapentaenoic acid as a substrate DNA derived from the genus Pleurotus that encodes a protein having
A gene comprising the following DNA (a) or (b):
(A) DNA consisting of the 127th to 2994th nucleotide sequences in the nucleotide sequence shown in SEQ ID NO: 3
(B) hybridizing under stringent conditions with either DNA comprising the 127th to 2994th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 3 or DNA comprising a complementary nucleotide sequence to the DNA, and arachidon DNA encoding a protein derived from the genus Pleurotus having lipoxygenase activity using acid or eicosapentaenoic acid as a substrate
A gene encoding the following protein (A) or (B).
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (B) from the amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 4 And a protein derived from the genus Pleurotus having lipoxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate
A gene comprising any of the following DNAs (a) to (c):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 5
(B) a DNA derived from the genus Pleurotus that encodes a protein having a cyclooxygenase activity comprising a part of the base sequence shown in SEQ ID NO: 5 and having arachidonic acid or eicosapentaenoic acid as a substrate;
(C) Cyclooxygenase activity that hybridizes under stringent conditions with all or part of DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 5 and uses arachidonic acid or eicosapentaenoic acid as a substrate DNA derived from the genus Pleurotus that encodes proteins
A gene comprising the following DNA (a) or (b):
(A) DNA consisting of nucleotide sequences 321 to 2153 among the nucleotide sequence shown in SEQ ID NO: 5
(B) hybridizing under stringent conditions with either DNA comprising the 321st to 2153rd base sequences of the base sequence shown in SEQ ID NO: 5 or DNA comprising a base sequence complementary to the DNA; and arachidone DNA encoding a protein derived from the genus Pleurotus having cyclooxygenase activity using acid or eicosapentaenoic acid as a substrate
A gene encoding the following protein (A) or (B).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (B) From the amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 6 And a protein derived from the genus Pleurotus having cyclooxygenase activity using arachidonic acid or eicosapentaenoic acid as a substrate
A protein encoded by the gene according to any one of claims 6 to 14.
An antibody that recognizes the protein encoded by the gene according to any one of claims 6 to 14.
A recombinant expression vector comprising the gene according to any one of claims 6 to 14.
A plant transformant obtained by introducing at least the gene according to any one of claims 6 to 14 into a plant, or a plant transformant that is a descendant of the plant transformant having the same properties as the plant transformant Body or tissue of the plant transformant.
The plant transformant or the tissue of the plant transformant according to claim 18, wherein the plant is a genus Coleoptera.
A plant transformant having a modified eicosanoid composition, wherein the gene according to any one of claims 6 to 14 is introduced into a plant so that the gene can be expressed, or the plant transformant having the same properties as the plant transformant A plant transformant that is a descendant of the plant transformant, or a tissue of the plant transformant.
21. A propagation material obtained from the plant transformant according to any one of claims 18 to 20 or a tissue of the plant transformant.
A method for modifying the eicosanoid composition of a plant or a tissue thereof, comprising a step of introducing the gene according to any one of claims 6 to 14 into the plant.
A gene detection instrument using, as a probe, at least a partial base sequence of the gene according to any one of claims 6 to 14 or a complementary sequence thereof.
A method for screening a gene that regulates the protein or a substance that regulates the protein, using the protein encoded by the gene according to any one of claims 6 to 14.
A gene or substance obtained by the screening method according to claim 24.
A recombinant expression vector obtained by inserting a mouse-derived phospholipase A2 gene, a mouse-derived 5-lipoxygenase gene or a zebrafish-derived cyclooxygenase gene into a plant cell vector.
A plant transformant obtained by introducing at least one or more of a mouse-derived phospholipase A2 gene, a mouse-derived 5-lipoxygenase gene and a zebrafish-derived cyclooxygenase gene, or the same property as the plant transformant A plant transformant that is a descendant of the plant transformant, or a tissue of the plant transformant.
28. The plant transformant or the tissue of the plant transformant according to claim 27, wherein the plant is a genus Coleoptera.
A plant transformant having a modified eicosanoid composition, wherein at least one or more of a mouse-derived phospholipase A2 gene, a mouse-derived 5-lipoxygenase gene, and a zebrafish-derived cyclooxygenase gene are introduced so as to be expressed in the plant, Or the plant transformant used as the descendant of this plant transformant which has the same property as this plant transformant, or the structure | tissue of this plant transformant.
A propagation material obtained from the plant transformant according to any one of claims 27 to 29 or a tissue of the plant transformant.
A method for modifying the eicosanoid composition of a plant or its tissue, comprising introducing one or more of a mouse-derived phospholipase A2 gene, a mouse-derived 5-lipoxygenase gene and a zebrafish-derived cyclooxygenase gene into the plant.
A method for screening a gene that regulates the protein or a substance that regulates the protein, wherein the protein encoded by the recombinant expression vector according to claim 26 is used.
A gene or substance obtained by the screening method according to claim 32.