WO2005001088A1

WO2005001088A1 - Application of promoter capable of functioning by sugar deficiency in plant cultured cell to secretory production of useful protein

Info

Publication number: WO2005001088A1
Application number: PCT/JP2004/009038
Authority: WO
Inventors: Nozomu Koizumi; Hiroshi Sano; Eun Jeong Lee
Original assignee: National University Corporation NARA Institute of Science and Technology
Priority date: 2003-06-30
Filing date: 2004-06-25
Publication date: 2005-01-06
Also published as: JPWO2005001088A1

Abstract

Nucleic acid molecule comprising a sugar deficiency inducing promoter sequence and, linked in operable fashion thereto, a foreign gene sequence. This sugar deficiency inducing promoter sequence can be selected from the group consisting of a promoter sequence of β-galactosidase, a promoter sequence of β-xylosidase and a promoter sequence of β-glucosidase. The expression of foreign gene sequence can be easily controlled by regulating sugar deficiency by the use of the above nucleic acid molecule.

Description

Specification

Secretion of useful proteins by promoters acting by sugar deficiency in cultured plant cells: Application to production

Technical field

The present invention relates to a novel promoter whose expression is induced by sugar deficiency and a production method using the same.

Background art

[0002] Attempts to mass-produce useful proteins (eg, interferons) by genetic recombination techniques have been made using Escherichia coli and the like for more than 20 years. However, since many proteins require post-translational modification such as glycosylation for activity, use of eukaryotic cells is desired for their production.

[0003] Secretory production of useful human-derived proteins (eg, therapeutic proteins such as interferon) by cultured plant cells has the following advantages:

1. Since plant cells are eukaryotic cells, protein processing and post-translational modifications occur similar to those of mammals;

2. No fear of virus (such as HIV) and prion (BSE) contamination when using animal cells;

3. The plant medium is composed of sugar and inorganic salts, and can be produced at low cost;

4. In the case of secretory production, the target protein is secreted into the medium, which makes recovery and purification relatively easy;

5. Unlike the production method by cultivating genetically modified plants in the field, there is no risk of gene leakage into the environment where the target protein is not mixed into food crops.

[0004] From the above points, it is thought that secretory production of useful proteins by plant cells is effective. However, considering profitability, suppression of purification cost as well as productivity of the target protein is one of the important factors. . Therefore, a control method for specifically secreting the target protein into the medium is desired. [0005] As described above, plants have many advantages in producing useful proteins, and their use has been conventionally attempted. Attempts to mass-produce foreign useful proteins in plants have been made for 10 years, but in many cases the emphasis of research has been on improving gene expression levels. However, high expression of genes does not always lead to high accumulation of proteins, and in many cases, does not lead to practical use. In eukaryotes, protein synthesis occurs in multiple places, and its storage locations vary. In addition, there is an attempt to increase the value of foods by producing nutritious proteins in crops such as rice. In this case, too, there is a problem that the target protein is not highly accumulated. Therefore, despite the fact that many companies or public research institutions have various seeds, the production of useful proteins by plants has not been commercialized or industrialized in most cases.

[0006] For example, regarding the production of foreign proteins using cultured cells, see, for example, Terashima M. et al., Appl. Microbiol. Biotechnol., 52, 1999, 516-523 (Non-patent Document 1).

) Uses human cultured cells to secrete and produce human α_antitrypsin (AAT), enabling production of 24 mg per gram of dry cell weight. However, this is a very efficient case, and there are cases where little production is observed for some proteins.

[0007] Outside of Japan, research on the production of useful proteins using plants is active mainly in the United States. In the United States, because genetically modified plants are easily cultivated in the field, production is performed using transgenic plants instead of using cultured cells. ProdiGene (Texas, USA) already sells beta-dalcuronidase and avidin produced by recombinant corn as test reagents. Ventria Bioscience (California, U.S.A.) produces AAT from transgenic rice and is seeking approval for clinical application by 2004.

[0008] However, in Japan, almost no field-cultivation of such transgenic plants has been observed, and it is therefore preferable to produce foreign useful proteins using cultured cells as much as possible.

[0009] From these facts, it has been desired to establish a method for stably producing large amounts of foreign useful proteins in transgenic plants. [0010] Regarding the sugar deficiency response gene, Non-Patent Document 2 states that-gnorecosidase (denoted as din2) is induced in the dark, and that the sugar-suppressed expression of the din gene is caused by phosphorylation of hexose by hexokinase. It is described to be transmitted and that plants are easily deficient in sugar in the dark. However, this document merely describes the sugar deficiency inducibility of the expression of / 3_darcosidase, and does not disclose or suggest that the promoter of this gene is used for the expression of a useful protein.

[0011] Patent Document 1 discloses that human amylase synthesis and their mRNA levels are greatly induced by sugar deficiency, and that a human amylase-derived vector and a vector containing a gene encoding a desired gene product are used. Methods for producing gene products in angiosperm host cells have been described. However, this document only confirms that expression is possible in rice using the promoter of rice hyperamylase, and it is unclear whether expression is possible in other plants.

Patent Documents 2 and 3 disclose that the promoter of a rice α-amylase-encoding gene is responsive to sugar deficiency, and that human α1-antitrypsin linked to the α-amylase-encoding gene promoter It is described that the transformed rice cells containing (ii) are first maintained in a medium that maintains healthy cell growth, and then replaced or diluted with a sucrose-free ΑΑΤ medium. However, Patent Documents 2 and 3 describe in general descriptions that a plant of the family Poaceae can be used. In the working examples, rice is produced by using a promoter of a gene encoding rice α-amylase. Has been confirmed to be capable of expression in other plants, and it is not known whether expression is possible in other plants.

Patent document 1: U.S. Pat.No. 5,712,112 (column 5, column 9, column 10, column 51) Patent document 2: U.S. Pat.No. 6,048,973 (column 1, (Panel 9-1, Column 12, Column 51) Patent Document 3: International Publication W098Z42853 Pamphlet (Page 1)

Non-Patent Document l: Terashima M. et al., Appl.Microbiol. Biotechnol., 52, 1999,

516-523

Patent Document 2: Fujiki, F. et al., Plant Physiol.Vol. 124, 2000, pp. 1139-1147 Disclosure of the invention

Problems to be solved by the invention

[0013] The present invention is intended to solve the above problems, and it is an object of the present invention to provide a promoter that can be easily controlled in a plant cell and a method for stably and mass-producing a protein using the promoter. And

Means for solving the problem

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that the promoter power of secreted -3-xylosidase gene, / 3-galatatosidase gene and / 3-dal clonidase gene of Arabidopsis thaliana The present inventors have found that the induction is significantly induced by sugar deficiency in cultured cells, and based on this, completed the present invention. In particular, it has been found that the extracellular secretion of the product encoded by the heterologous gene sequence is efficiently performed when the sugar deficiency-inducible promoter and the signal peptide are linked to the heterologous gene sequence.

(1) A nucleic acid molecule comprising a sugar deficiency-inducible promoter sequence and a heterologous gene sequence operably linked to the sugar deficiency-inducible promoter sequence.

(2) The sugar deficiency-inducible promoter sequence is selected from the group consisting of a promoter sequence of a gene encoding galactosidase, a promoter sequence of a gene encoding β-xylosidase, and a promoter sequence of a gene encoding darcosidase. The nucleic acid molecule according to the above item (1).

(3) The sugar deficiency inducible promoter sequence is

(a) the nucleotide sequence shown at positions 643 to 1799 of SEQ ID NO: 1, positions 1 to 1763 of SEQ ID NO: 3, or positions 1 to 2058 of SEQ ID NO: 5;

(b) a nucleotide sequence containing one or more nucleotide substitutions, deletions or additions of nucleotides compared to the nucleotide sequence of (a), and having a sugar deficiency-inducible promoter activity Array;

(c) a nucleotide sequence that hybridizes with the nucleotide sequence of (a) or (b) under stringent conditions and has a sugar deficiency-inducible promoter activity;

(d) nucleotides having at least 70% identity to the nucleotide sequence of (a) A nucleotide sequence having sugar deficiency-inducible promoter activity; and

(e) (a) a truncated sequence of any of the nucleotide sequences of (d), wherein the nucleotide sequence has a sugar deficiency-inducible promoter activity

Selected from the group consisting of

The nucleic acid molecule according to the above item (1), which has an activity of promoting the sugar deficiency-induced expression of the heterologous gene when the heterologous gene sequence is operably linked to the nucleotide sequence.

(4) The nucleic acid molecule according to the above item (1), further comprising a signal peptide coding sequence.

(5) The signal peptide coding sequence is selected from the group consisting of a β-galatatosidase signal peptide coding sequence, a β-xylosidase signal peptide coding sequence, and a β-dalcosidase signal peptide coding sequence. And the nucleic acid molecule according to the above item (4).

(6) The signal peptide coding sequence described above,

(i) a nucleotide sequence encoding the amino acid sequence shown at position 1 to position 27 of SEQ ID NO: 2, position 1 to position 28 of SEQ ID NO: 4, or position 1 to position 28 of SEQ ID NO: 6;

(ii) a nucleotide sequence containing one or several nucleotide substitutions, deletions or additions as compared with the nucleotide sequence of (i), which encodes a peptide having extracellular secretory activity;

(iii) a nucleotide system 1J that hybridizes with the nucleotide system ij of (i) or (ii) under stringent conditions and encodes a peptide having extracellular secretory activity;

(iv) a nucleotide sequence having at least 70% identity to the nucleotide sequence of (i) and is selected from the group consisting of a nucleotide sequence encoding a peptide having extracellular secretory activity;

The nucleic acid molecule according to item (4), wherein the operably linked heterologous gene sequence to the nucleotide sequence results in extracellular secretion of the product of the heterologous gene.

(7) The heterologous gene sequence is a cytoforce or hormone gene sequence. The nucleic acid molecule according to item (1).

(8) The nucleic acid molecule according to the above item (1), wherein the heterologous gene sequence encodes a protein having a desired function when expressed in a plant.

(9) The nucleic acid molecule according to the above item (1), wherein the heterologous gene sequence is a gene sequence of interferon, antibody, human anti-trypsin or green fluorescent protein.

(10) The nucleic acid molecule according to the above item (1), wherein the heterologous gene sequence is a marker gene sequence.

(11) The nucleic acid molecule according to the above item (1), further comprising a regulatory element.

(12) The nucleic acid molecule according to the above item (11), wherein the regulatory element includes at least one element selected from the group consisting of an intron, a terminator, and an enhancer.

(13) A nucleic acid molecule comprising a promoter sequence of a gene encoding an exo-type glycolytic enzyme and a heterologous gene sequence operably linked to one sequence of the promoter.

(14) The above promoter sequence is selected from the group consisting of a promoter sequence of a gene encoding one galactosidase, a promoter sequence of a gene encoding β-xylosidase, and a promoter sequence of a gene encoding β-gnorecosidase. The nucleic acid molecule according to the above item (13).

(15) The above promoter sequence is

(d) a nucleotide sequence having at least 70% identity to the nucleotide sequence of (a) and having a sugar deficiency-inducible promoter activity; and (e) (a) a truncated sequence of any one of the nucleotide sequences of (d), wherein the nucleotide sequence has a sugar deficiency-inducible promoter activity.

Selected from the group consisting of

(13) The nucleic acid molecule according to the above item (13), which has an activity of promoting sugar deficiency-induced expression of the heterologous gene when operably linked to the nucleotide sequence.

(16) The nucleic acid molecule according to the above item (13), further comprising a signal peptide coding sequence.

(17) The signal peptide coding sequence is selected from the group consisting of a β-galatatosidase signal peptide code, a signal peptide coding sequence of β-xylosidase, and a signal peptide coding sequence of β-dalcosidase. The nucleic acid molecule according to item (16) above.

(18) The signal peptide coding sequence described above,

The nucleic acid molecule according to item (16), wherein the operably linked heterologous gene sequence to the nucleotide sequence results in extracellular secretion of the product of the heterologous gene.

(19) The nucleic acid molecule according to the above item (13), wherein the heterologous gene sequence is a gene sequence of cytoforce or hormone. (20) The nucleic acid molecule according to the above item (13), wherein the heterologous gene sequence encodes a protein having a desired function when expressed in a plant.

(21) The nucleic acid molecule according to the above item (13), wherein the heterologous gene sequence is a gene sequence of interferon, antibody, human α-antitrypsin or green fluorescent protein.

(22) The nucleic acid molecule according to the above item (13), wherein the heterologous gene sequence is a marker gene sequence.

(23) The nucleic acid molecule according to the above item (13), further comprising a regulatory element.

(24) The nucleic acid molecule according to the above item (23), wherein the regulatory element includes at least one element selected from the group consisting of an intron, a terminator, and an enhancer.

(25) A nucleic acid molecule comprising a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar, and a heterologous gene sequence operably linked to the promoter sequence.

(26) a promoter sequence of a gene encoding an enzyme capable of degrading the metabolizable sugar, a promoter sequence of a gene encoding galactosidase, a promoter sequence of a gene encoding xylosidase, and; encoding 3-dalcosidase The nucleic acid molecule according to the above item (25), which is selected from the group consisting of a gene's mouth motor sequence.

(27) The promoter sequence of a gene encoding an enzyme capable of degrading the metabolizable sugar,

(d) a nucleotide sequence having at least 70% identity to the nucleotide sequence of (a) and having a sugar deficiency-inducible promoter activity; Bini

Selected from the group consisting of

(25) The nucleic acid molecule according to the above item (25), which has an activity of promoting sugar deficiency-induced expression of the heterologous gene when operably linked to the nucleotide sequence.

(28) The nucleic acid molecule according to the above item (25), further comprising a signal peptide coding sequence.

(29) The signal peptide coding sequence is selected from the group consisting of a signal peptide coding sequence of β-galatatosidase, a signal peptide coding sequence of β-xylosidase and a signal peptide coding sequence of β-dalcosidase. The nucleic acid molecule according to the above item (28).

(30) The signal peptide coding sequence is

The nucleic acid molecule according to item (28), wherein the operably linked heterologous gene sequence to the nucleotide sequence results in extracellular secretion of the product of the heterologous gene.

(31) The above-mentioned heterologous gene sequence is a cytoforce in or hormone gene sequence. The nucleic acid molecule according to item (25).

(32) The nucleic acid molecule according to the above item (25), wherein the heterologous gene sequence encodes a protein having a desired function when expressed in a plant.

(33) The nucleic acid molecule according to the above item (25), wherein the heterologous gene sequence is a gene sequence of interferon, antibody, human anti-trypsin or green fluorescent protein.

(34) The nucleic acid molecule according to the above item (25), wherein the heterologous gene sequence is a marker gene sequence.

(35) The nucleic acid molecule according to the above item (25), further comprising a regulatory element.

(36) The nucleic acid molecule according to (35), wherein the regulatory element includes at least one element selected from the group consisting of an intron, a terminator, and an enhancer.

(37) A nucleic acid molecule comprising a promoter sequence and a heterologous gene sequence operably linked to the promoter sequence, wherein the promoter sequence comprises:

(d) a nucleotide sequence having at least 70% identity to the nucleotide sequence of (a) and having a sugar deficiency-inducible promoter activity; and

Selected from the group consisting of

A nucleic acid molecule having an activity of promoting expression of the heterologous gene when the heterologous gene sequence is operably linked to the nucleotide sequence. (38) The nucleic acid molecule according to the above item (37), further comprising a signal peptide coding sequence.

[0053] (39) The signal peptide coding sequence is selected from the group consisting of a β-galactosidase signal peptide code, a β-xylosidase signal peptide coding sequence, and a β-dalcosidase signal peptide coding sequence. The nucleic acid molecule according to the above item (38).

(40) The signal peptide coding sequence,

(iv) a nucleotide sequence having at least 70% identity to the nucleotide sequence of (i), and is selected from the group consisting of nucleotide sequences encoding a peptide having extracellular secretory activity;

The nucleic acid molecule of claim 38, wherein operatively linking the heterologous gene sequence to the nucleotide sequence results in extracellular secretion of the product of the heterologous gene.

(41) The nucleic acid molecule according to the above item (37), wherein the heterologous gene sequence is a gene sequence of cytoforce or hormone.

(42) The nucleic acid molecule according to the above item (37), wherein the heterologous gene sequence encodes a protein having a desired function when expressed in a plant.

(43) The nucleic acid molecule according to the above item (37), wherein the heterologous gene sequence is a gene sequence of interferon, antibody, human anti-trypsin or green fluorescent protein.

(44) The nucleic acid molecule according to the above item (37), wherein the heterologous gene sequence is a marker gene sequence. (45) The nucleic acid molecule according to the above item (37), further comprising a regulatory element.

(46) The nucleic acid molecule according to the above item (45), wherein the regulatory element includes at least one element selected from the group consisting of an intron, a terminator, and an enhancer.

(47) Sugar deficiency-inducible promoter sequence, promoter sequence of gene encoding exo-type glycolytic enzyme, and promoter sequence of gene encoding enzyme capable of degrading metabolizable sugar A vector comprising at least one promoter sequence and a heterologous gene sequence operably linked to the selected promoter sequence.

(48) Sugar deficiency inducible promoter sequence, promoter sequence of gene encoding exo-type glycolytic enzyme, and promoter sequence of gene encoding enzyme capable of degrading metabolizable sugar A plant cell comprising at least one promoter sequence and a heterologous gene sequence operably linked to the selected promoter sequence.

(49) The plant cell according to the above item (48), which is a dicotyledonous plant cell.

(50) The plant cell according to the above item (48), which is a cultured cell.

(51) The plant cell according to the above item (50), which is a cell capable of growing 50 times or more in one week.

(52) A method for producing a protein, the method comprising the following steps:

At least one promoter sequence selected from the group consisting of a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. When,

Introducing a nucleic acid molecule comprising the protein coding sequence operably linked to the selected promoter sequence into a cell to obtain a transformed cell;

Culturing the transformed cells in the absence of sugar to secrete the protein; and

Step of recovering the protein

A method comprising: (53) The method according to the above item (52), wherein the cell is a plant cell.

(54) The method according to the above item (52), wherein the cell is a dicotyledonous plant cell.

(55) The method according to the above item (52), wherein the cell is a cultured cell.

[0070] (56) The method according to the above item (55), wherein the cells are cells capable of proliferating 50-fold or more in one week.

[0071] (57) The method according to the above item (52), wherein the nucleic acid molecule further comprises a signal peptide coding sequence.

[0072] (58) The signal peptide coding sequence is selected from the group consisting of a β-galatatosidase signal peptide code, a signal peptide coding sequence of β-xylosidase, and a signal peptide coding sequence of β-dalcosidase. The method described in the above item (57).

(59) The signal peptide coding sequence described above,

The method of claim 57, wherein operably linking the heterologous gene sequence to the nucleotide sequence results in extracellular secretion of the product of the heterologous gene.

[0074] (60) The method according to the above item (52), wherein the protein is a cytokinin or a hormone.

(61) A protein having a desired function when the above protein is expressed in a plant The method according to item (52), which is a quality.

(62) The method according to the above item (52), wherein the protein is an interferon, an antibody, human α-antitrypsin or a green fluorescent protein.

[0077] (63) The method according to the above item (52), wherein the sugar is a metabolizable glycolytic sugar or a sugar that can become the glycolytic sugar when metabolized.

[0078] (64) The method according to the above item (52), wherein the sugar is selected from the group consisting of glucose, galactose, fructose, sucrose and xylose.

(65) A method for producing a protein, the method comprising the following steps:

Culturing the transformed cells in the presence of sugar;

Step of recovering the protein

A method comprising:

(66) A protein obtained by the method according to the above item (52).

(67) Use of the nucleic acid molecule according to the above item (1) for sugar-deficiency-induced expression of a heterologous gene.

The invention's effect

[0082] The present invention provides an expression system that can easily control expression by controlling sugar deficiency.

[0083] The promoter sequence of the gene encoding secretory β-galactosidase (Gal), the promoter sequence of the gene encoding β-xylosidase (Xyl), and the promoter sequence of the gene encoding β-darcosidase (Glc) are as follows: The presence of sugars suppresses expression, and sugar deficiency significantly induces expression. Efficient expression of a heterologous gene sequence by culturing cells containing the heterologous gene sequence operably linked to the sequence in the presence of sugar and then reducing the sugar concentration in the medium when the cells have grown to some extent Can be induced.

[0084] In the present invention, particularly, the nucleic acid molecule of the present invention further comprises a signal peptide coding sequence.

Can result in more efficient expression of the product encoded by the heterologous gene sequence.

[0085] By using the present invention, activation of industries related to the production of pharmaceuticals such as interferons, antibodies, vaccines, and the like by plant cells can be expected due to the low productivity and the progress of development research.

Brief Description of Drawings

[0086] [Fig. 1] Figure la is a graph showing the change in signal intensity ratio due to sugar deficiency for about 13,000 Arabidopsis thaliana genes. The horizontal axis indicates the signal intensity in the presence of 2% sucrose, and the vertical axis indicates the signal intensity in the absence of sucrose. Figure lb is a graph showing the change in signal intensity ratio due to sugar deficiency for the 184 Arabidopsis genes initially obtained by screening. The horizontal axis shows the signal intensity in the presence of 1% sucrose, and the vertical axis shows the signal intensity in the absence of sucrose.

[FIG. 2] FIG. 2 is a photograph showing RNA gel plot analysis of nine genes identified in the subarray.

FIG. 3a is a graph showing sucrose or glucose levels when sucrose is supplied to detached leaves. At each time, the white bars on the left show the results under sucrose feeding conditions, the black bars in the middle show the results under sucrose deficient conditions, and the white bars on the left show the results when sucrose was fed again after 72 hours. The results are shown. Each error bar is also shown. FIG. 3b is a photograph showing RNA gel plot analysis showing the time course of induced transcript accumulation of 12 genes during withdrawal leaves.

FIG. 4a is a graph showing the amount of soluble sugar in rosette leaves when the whole plant is shielded from light. White bars indicate the amount of sucrose and black bars indicate the amount of gnorecose. Error bars are also shown for each. FIG. 4b is a photograph of an RNA gel plot showing a transcript of the AtSUG gene when the whole plant is shielded from light. [FIG. 5] FIG. 5a shows yellowing of detached rosette leaves in the upper photograph, and the amount of soluble sugar in detached leaves in the lower graph. White bars indicate the amount of sucrose and black bars indicate the amount of darcos. In each case, an error bar is also shown. FIG. 5b is a photograph of an RNA gel plot showing the transcript of the AtSUG gene in the detached rosette leaves.

[Fig. 6] Fig. 6 is a photograph of an RNA gel plot showing transcripts of AtSUG when sucrose was depleted from a withdrawn leaf for 48 hours, and then sugar or an analog was fed for 24 hours. Each shows the results when the following were added: lane 1, no treatment; lane 2, sugar starvation (48 hours); lane 3, sucrose (24 hours); lane 4, glucose (24 hours); lanes 5, 2 -Deoxyglucose (24 hours); Lane 6, 3_0_ methyl glucose.

FIG. 7 is a photograph of an RNA gel plot showing transcripts of AtSUG due to the presence and absence of sucrose.

FIG. 8 is a schematic diagram showing a phylogenetic tree of a family of β-galatatosidase (also referred to as Gal) and β-xychidoxidase (also referred to as Xyl).

[Fig. 9] Fig. 9 shows Gal-1, Gal-2, Gal-3, Gal_5 and Gal_6, and Xyl-1, Xyl- when Arabidopsis was grown in the absence of sucrose or under conditions containing 1% sucrose. 3 is a photograph of an RNA gel plot showing RNA amounts of 2 and Xyl-3.

[FIG. 10] FIG. 10 is a photograph of an RNA gel blot showing the amount of Gal-1, Xyl-1, and Glc-1 RNA when Arabidopsis is grown in the absence of sucrose or under conditions containing 1% sucrose. It is.

[Figure 11] Figure 11 is a schematic diagram comparing the structures of the gene products of Gal-1 (724 amino acids), Xyl-1 (774 amino acids) and Glc-1 (577 amino acids). is there. Garden 12] FIG. 12 is a schematic diagram showing the advantages of using cultured cells.

[FIG. 13] FIG. 13 is a photograph of an RNA gel plot showing RNA amounts of Gal-1, Xyl-1, and Glc_l when cultured Arabidopsis cells were grown in the absence of sucrose or under conditions containing 1% sucrose. It is.

[FIG. 14] FIG. 14 is a graph showing the enzyme activities of Gal-1, Xyl-1 and Glc-1 when cultured Arabidopsis cells are grown in the absence of sucrose or under conditions containing 1% sucrose. . FIG. 15 is a schematic diagram of regulation by sugar starvation in plant cells.

[Fig. 16] Fig. 16 shows that Gal-1, Xyl_l, and Glc-

3 is a photograph of an RNA gel plot showing the RNA amount of 1.

FIG. 17a is a schematic view showing a part of a method for constructing a plant-introducing construct.

FIG. 17b is a schematic view showing a part of a method for constructing a plant-introducing construct.

FIG. 17c is a schematic view showing a part of a method for constructing a plant-introducing construct.

FIG. 18 is a view showing that deficiency of sugar induces GFP secretion in BY-2 cells. The graph shows the growth of transformed BY-2 cells. The inset in the graph is

Shows the results of Western blotting.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. It is to be understood that throughout this specification, the terms used herein are used in the meaning commonly used in the art, unless otherwise specified.

(1. Nucleic acid molecule)

In one embodiment, a nucleic acid molecule of the invention comprises a sugar deficiency-inducible promoter sequence and a heterologous gene sequence operably linked to the sugar deficiency-inducible promoter sequence.

[0089] In another embodiment, the nucleic acid molecule of the present invention also includes a promoter sequence of a gene encoding an exoglycolytic enzyme, and a heterologous gene sequence operably linked to the promoter sequence.

[0090] In another embodiment, the nucleic acid molecule of the present invention also comprises a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar, and a heterologous gene sequence operably linked to the promoter sequence. including.

[0091] In particular, the nucleic acid molecule of the present invention preferably contains a signal peptide coding sequence. By including the signal peptide coding sequence, extracellular secretion of the product encoded by the heterologous gene sequence can be performed more efficiently.

[0092] As used herein, the term "nucleic acid molecule" is also used interchangeably with nucleic acids, oligonucleotides, and polynucleotides. Examples of nucleic acid molecules include cDNA, mRNA, Nom DNA and the like. As used herein, nucleic acids and nucleic acid molecules are included in the concept of the term "gene", such as when the nucleic acids and nucleic acid molecules encode proteins. Nucleic acid molecules encoding a gene sequence also include "splice variants" and "splice variants." A splice variant and a splice variant are synonymous. Similarly, a particular protein encoded by a nucleic acid includes any protein encoded by a splice variant of the nucleic acid. As the name suggests, "splice variants" are the products of alternative splicing of a gene. After transcription, the initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. The mechanism of production of splice variants varies, but involves alternative splicing of exons. Another polypeptide derived from the same nucleic acid by read-through transcription is also included in this definition. Any product of a splicing reaction, including recombinant forms of the splice product, is included in this definition.

As used herein, the sugar-deficiency-inducible promoter sequence refers to a promoter sequence whose expression is induced by sugar deficiency. Examples of the sugar deficiency-inducible promoter sequence include a promoter sequence of a gene encoding β-galactosidase (Gal), a promoter sequence of a gene encoding β-xylosidase (Xyl), and β-darcosidase (G1c). And the promoter sequences of the sugar-responsive genes listed in Table 1. These promoter sequences were obtained from the Kazusa DNA Research Institute for the gene with the gene ID shown in Table 1, and this gene was deleted little by little from the 5 'end or 3' end to obtain a deleted sequence. It can be readily determined by ascertaining whether the misarrangement has sugar deficiency inducible promoter activity. Whether this deletion sequence has a sugar deficiency-inducible promoter activity can be determined, for example, by ligating the deletion sequence with a reporter sequence, ligating it with a vector, introducing this vector into an appropriate host cell, It can be determined by checking whether expression of the sequence is seen. The j3-galactosidase that can be used herein is preferably Gal_l. Examples of j3-xylosidase that may be used herein include Xyl-1 or Xyl_3, preferably Xyl-1. The j3-gnorecosidase that can be used herein is preferably Glc-1. The sugar deficiency-inducible promoter sequence is not limited to these, and (b) A nucleotide sequence comprising one or more nucleotide substitutions, deletions or additions, compared to the nucleotide sequence of these promoter sequences, wherein the nucleotide sequence has one activity of a sugar deficiency-inducible promoter; (c) a nucleotide sequence IJ that hybridizes under stringent conditions to the nucleotide sequence of (a) or (b) and has a sugar deficiency-inducible promoter activity; (d) the nucleotide sequence of (a) A nucleotide sequence having at least 70% identity and a nucleotide sequence IJ having a sugar deficiency-inducible promoter activity; and (e) a shortened sequence of any one of the nucleotide sequences (a) and (d). In addition, the nucleotide sequence may have a sugar deficiency-inducible promoter activity. The truncated sequence may be a sequence shorter than the full length and longer than the minimum length having a sugar deficiency-inducible promoter activity. The minimum length of the truncated sequence was determined by deleting the original promoter sequence little by little at the 5 'end or 3' end to obtain a truncated sequence, and determining whether this truncated sequence has a sugar deficiency-inducible promoter activity. Can be easily determined. The length of the truncated sequence can be, for example, about 20 nucleotides or more and less than the full length. The length of the truncated sequence can be preferably about 30 nucleotides or more, about 40 nucleotides or more, about 50 nucleotides or more, about 60 nucleotides or more, about 70 nucleotides or more, about 80 nucleotides or more, or about 90 nucleotides or more. The length of the shortened sequence is preferably about 1200 nucleotides or less, more preferably about 1100 nucleotides or less, about 1000 nucleotides or less, about 900 nucleotides or less, about 800 nucleotides, about 700 nucleotides or less, about 600 nucleotides or less, about 500 nucleotides or less. In the following, it can be up to about 400 nucleotides, up to about 300 nucleotides, up to about 200 nucleotides, or up to about 100 nucleotides.

[0094] As used herein, "having a sugar deficiency-inducible promoter activity" means that it has an effect of increasing the expression level under sugar-deficient conditions compared to the expression level under sugar-present conditions. Say. This effect is preferably greater than the expression level when the sugar concentration is about 15 mM or less, and more preferably twice the expression level when the sugar is present at about 50 mM or more. Above, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 50 times or more, 100 times or more. Of course, the sugar deficiency-inducible promoter activity includes a case where it is not expressed under saccharide-existing conditions but has an effect of being expressed under sugar-deficient conditions.

[0095] As used herein, the term "promoter" determines the start point of transcription of a gene, and A region on DNA that directly regulates the frequency. It is a nucleotide sequence at which RNA polymerase starts binding and starts transcription. The promoter region is usually within about 2 kbp upstream of the first exon of the putative protein coding region. One region of the promoter can be estimated. The putative promoter region varies for each structural gene, but is usually, but not limited to, upstream of the structural gene, and may be downstream of the structural gene. Preferably, the putative promoter region is within about 2 kbp upstream of the first exon translation start site. As used herein, “promoter sequence” refers to a sequence having promoter activity. Promoter activity refers to the activity of transcribing DNA RNA. Therefore, "promoter system I" refers to a sequence capable of synthesizing RNA corresponding to DNA when the DNA is ligated downstream (3 'side) and introduced into cells.

[0096] As is well known, the promoters of the Gal_l, Xyl_l, and Glu_l genes can be obtained from the upstream sequence of the coding region. The specification of the promoter region can be performed based on a method well known in the art. Briefly, an expression cassette is constructed in which a candidate sequence for the promoter region and a reporter gene (eg, GUS gene) are operably linked. Using the constructed expression cassette, suitable plant cells are transformed, and the transformed cells are regenerated into plants. The expression of the reporter gene in the transformed plant is detected using an appropriate detection system (eg, dye staining). Based on the detection result, the promoter region and its expression characteristics can be confirmed.

[0097] The sugar deficiency-inducible promoter sequence used in the present invention, the promoter sequence 1J of a gene encoding an exo-type degrading enzyme, or the continuous promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar can be used. Nucleic acid molecules comprising at least 50 (preferably at least 60, more preferably at least 80, even more preferably at least 100, even more preferably at least 150) nucleotide sequences are identified by these promoter sequences. They may have the same or similar activities. Such activity can be confirmed by an assay using the beta-Darc mouth nidase (GUS) gene, luciferase gene, or GFP gene as a reporter gene, biochemical or cytohistological assay. Such atsesses belong to well-known and conventional techniques in the art ( Maliga et al., Methods in Plant Molecular Biology: A laboratory course. Cold Spring Harbor Laboratory Press "995) Jefferson, Plant Molec. Biol. Reporter 5: 387 (1987); 〇w et al., Science 234: 856 (1986); Sheen et al. , Plant J. 8: 777-784 (1995)), without any difficulty for those skilled in the art, the nucleic acid molecule comprising at least 10 contiguous nucleotide sequences of the sequence according to the invention may be used in the sugar deficiency used in the invention. Have the same, similar, or substantially equivalent activity to the promoter sequence of the inducible promoter sequence, the promoter sequence of the gene encoding the exo-type degrading enzyme, or the promoter sequence of the gene encoding the enzyme capable of degrading metabolizable sugars. In the present specification, it is determined that the promoter activity is substantially equal to or greater than the promoter activity when it is determined that the promoter activity is within the range of the detection error in the above-mentioned assay. That.

[0098] The length of the promoter sequence used in the present invention is usually at least 10 nucleotides, but is preferably at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides. , 80 nucleotides or more, 90 nucleotides or more, 100 nucleotides or more, 150 nucleotides or more, 200 nucleotides or more, 300 nucleotides or more.

[0099] The promoter sequence used in the present invention may be a conventional promoter sequence (for example, a minimum promoter (a promoter consisting of about 80 base pairs derived from 35S promoter (Hatton et al., Plant J., 7: 859-876 (1995) Rouster et al., Plant J., 15: 435-440 (1998); Washida et al., Plant Mol. Biol., 40: 1-12 (1999))). In this case, the promoter sequence used in the present invention or a fragment thereof may be added or replaced by a promoter sequence which does not show tissue specificity or shows weak specificity, or a promoter sequence showing another specificity. Thus, a promoter sequence capable of inducing sugar deficiency can be produced (Hatton et al., Plant J., 7: 859-876 (1995); Rouster et al., Plant J., 15: 435-440 (1998) Washida et al., Plant Mol. Biol., 40: 1-12 (1999)).

[0100] The sugar deficiency-inducible promoter preferably has a sugar concentration of about 15 mM or less, more preferably about 10 mM, still more preferably about 5 mM or less, and most preferably a sugar present. A promoter whose expression is induced when not performed. As used herein, “expression is induced” is meant to include both an increase in the expression level and an expression that is not expressed at all under sugar-containing conditions but is expressed under sugar-deficient conditions.

[0101] In the present specification, the sugar acting on the sugar deficiency inducible promoter is preferably a metabolizable sugar. “Metabolizable sugars” refers to sugars that, when taken up and degraded by plant cells, can at least partially enter the glycolysis system and be metabolized. Metabolizable sugars can be used interchangeably with sugars that can be metabolized by plants. Examples of metabolizable sugars include monosaccharides and disaccharides, which are capable of producing ATP via glycolysis via initial phosphorylation of the sugar. Examples of monosaccharides include glucose, fructose, xylose, galactose, maltose and arabinose. An example of a disaccharide is sucrose. Other phosphorylatable sugars may also be metabolizable sugars. Mannitol and sorbitol do not normally fall under the sugars of the present invention because they cannot produce ATP via initial phosphorylation and cannot enter glycolysis. However, the metabolizable sugar varies depending on the organism targeted by the present invention, and when a specific sugar is confirmed to be a metabolizable sugar for the organism by the method described below, the Such sugars are also within the range of metabolizable sugars.

[0102] Metabolizable sugars are well known in the art. Whether a particular sugar is a metabolizable sugar depends on whether the number of plant cells increases when the target plant cells are cultured for one to four weeks using the sugar as the sole carbon source. If the number of plant cells increases, the sugar can be determined to be a metabolizable sugar. The increase in the number of plant cells means that the number of plant cells after culturing for 1 to 4 weeks is preferably about 150% or more, more preferably about 200%, when the cell number before the start of culture is 100%. As mentioned above, more preferably about 250 ° / o or more, more preferably about 300% or more, more preferably about 400% or more, and more preferably about 500% or more.

[0103] As used herein, the term "expression" of a gene, a polynucleotide, a polypeptide, or the like means that the gene or the like undergoes a certain action in vivo to take on another form. Preferably, a force S that means that a gene, a polynucleotide, or the like is transcribed and translated to form a polypeptide, and that mRNA is produced by being transcribed may also be a form of expression. More preferably, such polypeptide forms may be post-translationally processed.

[0104] Therefore, as used herein, "decrease" in "expression" of a gene, polynucleotide, polypeptide, or the like, means that when the factor of the present invention is acted on, the expression is lower than when it is not acted on. Means a significant decrease in the amount of Preferably, the decrease in expression includes a decrease in the expression level of the polypeptide. As used herein, the term "increase" in "expression" of a gene, polynucleotide, polypeptide, or the like means that the amount of expression is significantly increased when the factor of the present invention is acted on as compared to when it is not acted on. Say. Preferably, increasing the expression includes increasing the expression level of the polypeptide.

[0105] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be a natural or non-natural amino acid or a modified amino acid. The term can also be assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of an amino acid (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids) and those skilled in the art. And other modifications known.

[0106] In general, one amino acid of a particular polypeptide sequence may be substituted for another amino acid without any apparent loss or loss of biological activity of the polypeptide. It is the interaction capacity and properties of a polypeptide that define the biological activity of a given polypeptide. Thus, certain amino acid substitutions may be made in the amino acid sequence of the polypeptide, or at the level of the nucleotide sequence encoding the polypeptide, resulting in a polypeptide that retains its original properties after the substitution. obtain. Thus, without obvious loss of biological activity, various modifications may be made in the polypeptide disclosed herein or the nucleotide having the nucleotide sequence encoding this polypeptide. Can be performed.

[0107] As used herein, "biological activity" refers to an activity that a certain factor (eg, a polypeptide or a nucleic acid molecule) may have in a living body, and an activity that exhibits various functions. Included. For example, if a factor is an enzyme, its biological activity includes the enzymatic activity. In another example, where an agent is a ligand, the ligand involves binding to the corresponding receptor. For example, when a factor is an antisense molecule, its biological activity includes binding to the nucleic acid molecule of interest, thereby suppressing expression. Such biological activity can be measured by techniques well known in the art. When a factor is a promoter, its biological activity can be confirmed that the transcription of a target gene is changed (preferably increased) by a stimulus specific to the promoter. Such confirmation can be made using molecular biology techniques well known in the art.

[0108] As used herein, in general, amino acid substitutions, substitutions, deletions, or modifications can be made to produce a polypeptide. Amino acid substitution refers to the replacement of one amino acid with another amino acid. Amino acid addition refers to the addition of one or more, for example, 110, preferably 115, and more preferably 113 amino acids at any position in the original amino acid sequence. Refers to insertion. Amino acid deletion refers to the removal of one or more amino acids from the original amino acid sequence, for example, 110 amino acids, preferably 115 amino acids, more preferably 113 amino acids. Examples of amino acid modifications include forces S that include amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like. Not limited. The amino acid to be substituted or added may be a natural amino acid or an unnatural amino acid or amino acid analog. Natural amino acids are preferred.

[0109] Nucleotides in which some nucleotides have been deleted from full-length nucleotides and polypeptides in which some amino acids have been deleted from full-length polypeptides are also referred to as fragments. As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of up to 11-1 relative to a full-length polypeptide or polynucleotide (length n). The length of the fragment can be appropriately changed depending on its purpose. The lower limit of the length for polypeptides includes 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, where A length represented by an integer not specifically recited in (eg, 11 and the like) may also be appropriate as a lower limit. In the case of polynucleotides, the nucleotides include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. A length represented by a non-integer integer (eg, 11, etc.) may also be appropriate as a lower limit.

[0110] The polypeptide may be a polypeptide analog having a biological activity specific to the polypeptide. The polypeptide may be an enzyme analog, particularly if the polypeptide is an enzyme. As used herein, the term "enzyme analog" refers to an enzyme that is at least one chemical or biological function equivalent to a naturally occurring enzyme that is a different compound from the natural enzyme. Thus, enzyme analogs include those in which one or more amino acid analogs have been added or replaced with the original natural enzyme. Enzyme analogs may have such additions or substitutions such that their function (eg, α-phosphorylase activity or thermostability) is substantially similar to or better than the function of the original native enzyme. I have. Such enzyme analogs can be made using techniques well known in the art. Thus, an enzyme analog can be a polymer comprising an amino acid analog. As used herein, the term “enzyme” includes this enzyme analog unless otherwise specified.

[0111] In the present specification, "amino acid" may be a natural amino acid, an unnatural amino acid, a derivative amino acid, or an amino acid analog. Natural amino acids are preferred.

[0112] The term "natural amino acid" refers to the L-isomer of a natural amino acid. Natural amino acids include glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, fenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, gnoletamic acid, gnoletamine, and 7 — Carboxyglutamic acid, anoreginin, orditin, and lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention. [0113] The term "unnatural amino acid" refers to an amino acid that is not normally found in nature in proteins. Examples of unnatural amino acids include D- or L-forms of nonoleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homoarginine, and D- Phenylalanine.

[0114] "Derivative amino acid" refers to an amino acid obtained by derivatizing an amino acid.

[0115] "Amino acid analog" refers to a molecule that is not an amino acid, but that is similar in physical properties and Z or function of the amino acid. Amino acid analogs include, for example, etyonin, canavanine, 2-methylglutamine and the like.

[0116] Amino acids are represented by their commonly known three-letter symbols or by IUPAC-IUB Biochemical

It may be referred to herein by any of the single letter symbols recommended by the Nomenclature Commission. Nucleotides may also be referred to by the generally accepted single letter code.

[0117] In addition to the desired modification, the modified polypeptide comprising a modification by substitution, addition or deletion of one or more amino acids or more to the amino acid sequence of the natural polypeptide is described in the present invention. Within the scope of the invention. Such modified polypeptides containing one, several or more amino acid substitutions, additions or deletions are described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press "989), Currerrt Protocols. in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), Pro Natl. Acad. Sci., USA, 81, 5662 (1 984) ), Science, 224, 1431 (1984), PCT WO85 / 00817 (1985), Nature, 316, 601 (1985), and the like.

[0118] Amino acid deletion, substitution, or addition of the target polypeptide can be performed by site-specific mutagenesis, which is a well-known technique. Techniques for site-directed mutagenesis are well known in the art. See, for example, Nucl. Acid Research, Vol. 10, pp. 6487-6500 (1982). [0119] In the present specification, "a substitution, addition or deletion of one or several or more amino acids" or "at least one amino acid" when used with respect to a specific polypeptide having a biological activity. "Amino acid substitution, addition or deletion" means that at least one of the biological activities possessed by the specific polypeptide is not lost, and preferably the activity is a reference (for example, , The number of substitutions, additions, or deletions to such an extent as to be equal to or greater than that of the specific polypeptide (natural polypeptide). One skilled in the art can easily select a modified polypeptide having the desired properties.

[0120] The specific modified polypeptide thus produced is preferably about 40%, more preferably about 45%, more preferably about 50%, based on the amino acid sequence of the polypeptide before modification. , more preferably about 55%, more preferably about 60%, more preferably about 65%, more preferably from about 70 ^o / o, more preferably f or about 75 ^_0/0, more preferably ί or about 80 ^o / o , more preferably ί Maitoshaku 85 ^_0/0, more preferably about 90%, more preferably about 95%, and most preferably about 99% identity.

[0121] In designing such modifications, the hydropathic index of amino acids can be considered. The importance of the hydropathic index of amino acids for the biological function of polypeptides is generally recognized in the art (Kyte. J and Doolittle, RFJ Mol. Biol. 157 (1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the resulting polypeptide, which in turn defines the interaction of that polypeptide with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.) . Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge properties. The hydrophobicity index assigned to each amino acid is as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); Cystine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); Tryptophan (-0.9); Tyrosine (_1.3); Proline (_1.6); Histidine (_3.2); Glutamic acid (_3.5); Glutamine (_3.5); Aspartic acid (_3.5) Asparagine (_3.5); lysine (_3.9); and arginine (_4.5)).

[0122] A polypeptide having a certain amino acid in a polypeptide replaced by another amino acid having a similar hydrophobicity index and still having a biological function similar to this polypeptide It is well known in the art that it can produce tides (eg, polypeptides with equivalent enzymatic activity). In such amino acid substitutions, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.

[0123] In the present specification, in studying the design and properties of a protein, the hydrophilicity index may also be considered. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: Arginine (+3.0); Lysine (+3.0); Aspartic acid Glutamic acid (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (+ 3.0 ± 1); -0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (1-1.5); leucine (1-1.8); isoloicin (1-1.8); tyrosine (1-2.3); pheninolealanine (12-5); and tryptophan (13-4). It is understood that certain amino acid forces in certain polypeptides can be substituted for another amino acid that has a similar hydrophilicity index to this amino acid and still confer biological activity. In such amino acid substitutions, the hydrophilic index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0124] In the present invention, the term "conservative substitution" refers to a substitution in which, in an amino acid substitution, the hydrophilicity index and / or the hydrophobicity index of the original amino acid and the amino acid to be substituted are similar as described above. Say. Examples of the conservative substitution include, for example, those having a hydrophilicity index or a hydrophobicity index of two or less within soil 2, preferably within ± 1 and more preferably within ± 0.5. But not limited to them. Thus, examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; daltamic and aspartic acid; serine and threonine; glutamine and asparagine; Leucine, isoleucine, and the like, but are not limited thereto.

[0125] The sugar deficiency-inducible promoter sequence of the present invention is preferably a promoter encoding a gene encoding j3-galactosidase (Ga1), a gene encoding β-xylosidase (Xyl). Selected from the group consisting of a promoter sequence of a gene and a promoter sequence of a gene encoding β-dalcosidase (Glc), more preferably a promoter sequence of a gene encoding β-galactosidase or a promoter sequence of a gene encoding β-xylosidase. It is a data sequence. The sugar deficiency-inducible promoter sequence of the present invention is preferably represented by (a) SEQ ID NO: 1 at positions 643 to 1799, SEQ ID NO: 3 at position 1 to 1763, or SEQ ID NO: 5 at position 1 to position 2058 Nucleotide sequence 1J; (b) a nucleotide sequence containing a substitution, deletion or addition of one or several nucleotides compared to the nucleotide sequence of (a), having a sugar deficiency-inducible promoter activity (C) a nucleotide sequence which hybridizes with the nucleotide sequence of (a) or (b) under stringent conditions and has a sugar deficiency-inducing promoter activity; a nucleotide sequence having at least 70% identity to the nucleotide sequence of a) and having a sugar deficiency-inducible promoter activity; and (e) (a)-(d). Nucleotide sequence Is selected from the group consisting of nucleotide sequences having a sugar deficiency-inducible promoter activity, and operably linked to said heterologous gene sequence, whereby sugar deficiency-induced expression of said heterologous gene is obtained. Has the activity of promoting.

As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes "derivative oligonucleotides" or "derivative polynucleotides." “Derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that contains a derivative of a nucleotide or has an unusual linkage between nucleotides, and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2,1-O-methyl-ribonucleotide, a derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and an oligonucleotide in a oligonucleotide. Derivative oligonucleotide in which phosphodiester bond is converted to N3'_P5, phosphoramidate bond, derivative oligonucleotide in which ribose and phosphoric diester bond in oligonucleotide are converted to peptide nucleic acid bond, peracyl in oligonucleotide Is a derivative oligonucleotide in which is substituted with C-5 propynylperacyl, and peracyl in the oligonucleotide is substituted with C-5 thiazole peracyl Derivative oligonucleotide, a derivative oligonucleotide in which cytosine in the oligonucleotide is replaced with C_5 propynylcytosine, a derivative oligonucleotide in which cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, DNA Derivative oligonucleotides in which ribose is substituted with 2, _0-propylribose and derivative oligonucleotides in which ribose in oligonucleotides are substituted with 2, _methoxyethoxyribose are exemplified. Unless otherwise indicated, a particular nucleic acid sequence also includes conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences thereof, as well as explicitly stated sequences. It is intended to include Specifically, degenerate codon substitutions are those in which the third position of one or several, or a greater number of selected codons) is replaced by a mixed base and a Z or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)).

As used herein, the term "substitution, deletion or addition" of a polypeptide sequence or nucleotide sequence refers to the amino acid or its substitute, or the nucleotide or its substitute for the original polypeptide or polynucleotide, respectively. Replacement, removal or addition. Techniques for such substitution, deletion or addition are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, deletions or additions may be any number as long as it is one or more. The amount can be increased as long as the sugar deficiency inducibility of the promoter and the promoter activity, and in the case of a heterologous gene sequence, the desired protein activity and the like are maintained. For example, such a number can be one or several, and preferably can be no more than 20%, no more than 10% of the total length, or no more than 100, no more than 50, no more than 25, and the like. The sugar deficiency-inducible promoter sequence used in the present invention can be of any length as long as it retains the sugar deficiency-inducing promoter activity. Such a sequence can be easily prepared by, for example, deleting the nucleotide sequence of the original promoter sequence little by little on the 5 ′ side or the 3 ′ side and confirming that the sugar-deficiency-inducing promoter activity is retained. Determined to Can be Methods for shortening such promoter sequences are well known to those skilled in the art and can be easily implemented.

As used herein, the term “variant” refers to a substance in which a substance such as an original polypeptide or polynucleotide is partially changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, glycosylation variants, lipidation variants, variants with complex molecules, etc. Is mentioned. Preferably, the variant retains at least one, and more preferably more than one, property (eg, biological properties) of the substance (eg, enzyme) from which the modification is made. And the like. Alleles refer to genetic variants that belong to the same locus and are distinct from one another. Therefore, “allelic variant” refers to a variant that has an allelic relationship to a certain gene. Such allelic variants usually have the same or very similar composition as their corresponding alleles, and usually have rarely different organisms with almost the same biological activity. May have biological activity. “Species homolog or homolog” refers to homology (preferably 60% or more homology, more preferably 80% or more) of a certain gene at the amino acid or nucleotide level within a certain species. , 85% or more, 90% or more, 95% or more homology). Methods for obtaining such species homologs are well known in the art. The term "ortholog" is also called an orthologous gene and refers to a gene derived from speciation from a common ancestor having two genes. For example, taking the hemoglobin gene family with a multigene structure as an example, human and mouse α-hemoglobin genes are orthologs. is there. In addition, comparing human cystatin 、, a cysteine protease inhibitor, with rice oryzacytin, only three conserved short amino acid motifs, which are considered important for the interaction with the target protease, are conserved. The commonality of amino acids in other parts is very low. However, since both belong to the cystatin gene superfamily and are said to have a common ancestral gene, they share not only amino acid homology overall but also amino acid sequences with locally high homology. Even if it exists, it can be an onoresolog. Thus, orthologs are usually similar to the original species in another species. The ortholog of the sugar deficiency-inducible promoter used in the present invention and the promoter of a gene encoding an enzyme capable of degrading a metabolizable sugar can also be useful in the present invention. Can be

“Conservative (modified) variants” applies to both polypeptide and nucleotide sequences. With respect to a particular nucleotide sequence, a conservatively modified variant is one that encodes the same or essentially the same polypeptide sequence. Where the nucleotide sequence does not encode a polypeptide sequence, such as an antisense coding sequence, a conservatively modified variant refers to essentially identical sequences. Due to the degeneracy of the genetic code, a number of functionally identical nucleotide sequences encode any given polypeptide. For example, the codons GCA, GCC, GCG, and GCT (GCU) all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, that codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. Such variations in the nucleotide sequence are "silent alterations (mutations)," which are one species of conservatively altered mutations. Every nucleotide sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleotide sequence. In the art, each codon in a nucleotide sequence (except for ATG (AUG), which is usually the only codon for methionine, and TGG, which is usually the only codon for tributophan) is used to identify functionally identical molecules. It is understood that it can be modified to produce. Accordingly, each silent variation of a nucleotide sequence that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of the amino acid cysteine, which greatly affects the conformation of the polypeptide. Examples of such a nucleotide sequence modification method include cleavage with a restriction enzyme or the like, ligation treatment with a DNA polymerase, Klenow fragment, DNA ligase, or the like, site-specific base substitution using a synthetic oligonucleotide, or the like. (Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)). Modifications can also be made. [0130] The signal peptide coding sequence and the heterologous gene sequence can be changed according to the codon usage in the organism to be introduced. Codon usage reflects the frequency of use of genes that are highly expressed in the organism. For example, if it is intended to be expressed in Escherichia coli, it may be used for expression in a large recruited bacterium according to a published codon usage table (eg, Sharp et al., Nucleic Acids Research 16, No. 17, page 8207 (1988)). Can be optimized for

[0131] As used herein, the term "identity" of a nucleotide sequence (a polypeptide sequence) refers to the identity of nucleotides in two or more nucleotide sequences (a polypeptide sequence). (When comparing polypeptide sequences, this refers to the degree to which amino acids appear.) Identity is generally determined by comparing the full lengths of the two or more nucleotide sequences or polypeptide acids (IJ) and aligning the two or more sequences in an optimal manner that may include additions or deletions. Determined by comparison. Percent identity determines the number of positions where nucleotides (amino acids when comparing polypeptide sequences) are the same between two or more sequences, and divides the number of identical positions by the total number of positions compared. And to obtain the percent identity between these two sequences, calculated by multiplying the obtained result by 100. When two or more gene sequences are directly compared, the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably less. Genes are identical if they are at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical.

[0132] As used herein, the term "similarity" of a nucleotide sequence or a polypeptide sequence 1J) refers to a conservative substitution defined as positive in the above-mentioned identity. The degree of identity between two or more nucleotide sequences (or polypeptide system 1J) relative to each other. Thus, if there are conservative substitutions, identity and similarity will differ depending on the existence of the conservative substitution. When there is no conservative substitution, identity and similarity show the same numerical value.

[0133] identity ^_0/0, for example, can be force S to determine the NCBI BLAST 2. 2. 9 a (2004. 5.12 Issue) Te use Rere. In the present specification, the value of identity usually refers to a value obtained by aligning the above BLAST using the default conditions. However, if a higher value comes out due to a parameter change, the highest value shall be the value of the identity. Multiple areas are assessed for identity In this case, the highest value is the value of identity.

[0134] The degree of identity or similarity between two or more nucleotide sequences can be confirmed by examining hybridization under stringent conditions other than direct comparison of sequences.

[0135] As used herein, the term "stringent conditions" refers to conditions that hybridize to a specific sequence but do not hybridize to a non-specific sequence. The setting of stringent conditions is well known to those skilled in the art and is described, for example, in Moleculer Cloning (Sambrook et al., Supra). Specifically, for example, using a filter on which DNA derived from colonies or plaques is immobilized, 50% honolemamide, 5 × SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6) 5X Denhardt's solution (0.2% BSA, 0.2% Ficoll 400 and 0.2% polyvinylinolepyrrolidone), 10% dextran sulfate, and 20 μg Zml denatured sheared salmon sperm DNA. After hybridization at 65 ° C for 6 to 24 hours, a 0.1- to 2-fold concentration of SSC (saline-sodium citrate) solution (the composition of the 1-fold concentration SSC solution is 150 mM saline). And 15 mM sodium citrate), and the polynucleotide can be identified by washing the filter at 65 ° C.

[0136] In the present specification, the promoter sequence of a gene encoding an exo-type glycolytic enzyme refers to a promoter sequence of an exo-type glycolytic enzyme gene.

[0137] In the present specification, the "exo-type glycolytic enzyme" refers to an enzyme that acts on a terminal portion of a sugar chain to sequentially release a terminal sugar residue. Examples of exo-type glycolytic enzymes include, but are not limited to: i3-galactosidase, xylosidase, β-darcosidase, xylosidase and / 3-fucosidase. In the present invention, the nucleotide sequence upstream of the nucleotide sequence encoding such an enzyme can be any nucleotide sequence that exhibits promoter activity in the target product. Monkey

[0138] The promoter sequence of the gene encoding the exo-type glycolytic enzyme of the present invention is preferably a promoter sequence of a gene encoding j3-galactosidase, a promoter sequence of a gene encoding β-xylosidase, and / 3-dalcosidase. To code The promoter sequence is selected from the group consisting of gene promoter sequences, and is more preferably a promoter sequence of a gene encoding β-galactosidase, or a promoter sequence of a gene encoding β-xylosidase. The sugar deficiency-inducible promoter sequence of the present invention preferably comprises (a) nucleotides shown at positions 641 to 1799 of SEQ ID NO: 1, positions 1 to 1763 of SEQ ID NO: 3, or positions 1 to 2058 of SEQ ID NO: 5 (B) a nucleotide sequence containing one or several nucleotide substitutions, deletions or additions compared to the nucleotide sequence of (a), wherein the nucleotide sequence has a sugar deficiency-inducible promoter activity; (c) at least the nucleotide sequence of ( _d ) ( _a ), which hybridizes with the nucleotide sequence of (a) or (b) under stringent conditions and has a sugar-deficiency-inducible promoter activity. A nucleotide sequence having 70% identity and having a sugar deficiency-inducing promoter activity; and (e) a nucleotide sequence of (a)-(d), A condensed sequence selected from the group consisting of nucleotide sequences having a sugar deficiency-inducible promoter activity, and operably linked to the nucleotide sequence, the above-described heterologous gene sequence, It has an activity to promote expression.

[0139] In the present specification, the promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar refers to a promoter sequence of a gene encoding an enzyme capable of hydrolyzing a metabolizable sugar. . As used herein, the term "enzyme capable of decomposing metabolizable sugars" refers to an enzyme capable of decomposing metabolizable sugars as described above. Examples of such enzymes include, but are not limited to: _ galactosidase, _ xylosidase, _ darcosidase, <RTIgt; hyxyrosidase </ RTI> and _ fucosidase. In the present invention, any nucleotide sequence upstream of the nucleotide sequence encoding such an enzyme, as long as it exhibits promoter activity in the target organism, can be used.

[0140] The promoter sequence of the gene encoding the enzyme capable of degrading the metabolizable sugar of the present invention is preferably the promoter sequence of the gene encoding j3_galactosidase, the promoter sequence of the gene encoding β-xylosidase, and It is selected from the group consisting of a promoter sequence of a gene encoding β-dalcosidase, and more preferably a promoter sequence of a gene encoding β-galactosidase or encoding a β-xylosidase. This is the promoter sequence of the gene to be expressed. The sugar deficiency-inducible promoter sequence of the present invention

Preferably, (a) the nucleotide sequence IJ shown at positions 643 to 1799 of SEQ ID NO: 1, 1 to 1763 of SEQ ID NO: 3, or 1 to 2058 of SEQ ID NO: 5; (b) (a (C) a nucleotide sequence comprising a substitution, deletion or addition of one or several nucleotides and having a sugar deficiency-inducible promoter activity, as compared to the nucleotide sequence IJ of (c); a nucleotide sequence IJ that hybridizes with the nucleotide sequence of (a) or (b) under stringent conditions and has a sugar-deficiency-inducible promoter activity; (d) at least 70% of the nucleotide sequence of (a). ( _E ) a nucleotide sequence having a sugar deficiency-inducible promoter activity and ( _e ) _a shortened sequence of any one of the nucleotide sequences ( _a ) to (d), Step Is selected from the group consisting of nucleotide sequences that have a motor activity, when operably said heterologous gene sequence to said nucleotide sequence is connected, has an activity of promoting glucose deprivation induced expression of heterologous genes.

[0141] In the present invention, in addition to the promoter sequence of a gene encoding a sugar-deficiency inducible promoter, an exo-type glycolytic enzyme or a gene encoding an enzyme capable of degrading a metabolizable sugar, You can use a promoter together. Examples of such other promoters that can be used include, but are not limited to, the CaMV35S promoter, the nopaline synthase promoter, the ubiquitin promoter, and the like, and their modified promoters. In the present invention, any nucleotide sequence can be used as long as it exhibits promoter activity in the target organism. Such other promoters may be site-specific promoters, stage-specific promoters, constitutive promoters, stress or stimulus responsive promoters, The stress field may be a stimulatory) inducible promoter, or the stress field may be a stimulatory) inducible promoter. Examples of constitutive promoters include the Ca MV35S promoter, the nopaline synthase promoter and the ubiquitin promoter. Examples of specific promoters include tissue-specific and organ-specific promoters.

[0142] In the present invention, the deficiency-inducible promoter encodes an exo-type glycolytic enzyme. The heterologous gene sequence linked to the promoter sequence of the gene or the promoter sequence of the gene encoding the enzyme capable of degrading a metabolizable sugar can be any nucleotide sequence that is heterologous to the linked promoter sequence. It can be a nucleotide sequence. The heterologous gene sequence may be, for example, a protein coding system ij, an antisense coding system ij, a ribozyme coding sequence, a nucleotide sequence for analysis, or the like. The heterologous gene sequence can preferably be a protein coding sequence or an antisense coding sequence. A heterologous gene sequence can be a naturally occurring nucleotide sequence or a modified version of a naturally occurring nucleotide sequence or a complex thereof, whether an artificially synthesized gene or not. (For example, a fusion).

[0143] As used herein, "operably linked" means that a desired nucleotide sequence (eg, a heterologous gene sequence) is a transcription regulatory sequence (eg, a gene) that results in expression (ie, operation). It means that it is placed under the control of a promoter, a terminator, an enhancer, etc.) or a translation control sequence (eg, an intron, a splice donor, a splice acceptor, etc.). For example, in order for a promoter to be operably linked to a gene, the promoter will usually be located immediately upstream of the gene, but need not necessarily be located adjacent thereto.

For DNA synthesis techniques and nucleic acid chemistry for producing artificially synthesized nucleotide sequences, see, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPress; Gait, MJ (1990). Oligonucleotide. Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991) .Oligon ucleotides and Analogues: A Practical Approac, IRL Press; Adams, R. L. etal. (1992) .The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994) .Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996) .Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996) Bioconjugate Techniques, Academic Press, etc., which are incorporated herein by reference, the relevant portions of which are incorporated herein by reference.

[0145] The heterologous gene sequence in the present specification may be any one that can be expressed in a target organism. Anything. For example, if it is intended to be expressed in a specific plant, if it can be expressed in that specific plant, then.

In one embodiment, the heterologous gene sequence is a protein coding sequence. Protein coding sequences include any useful protein that is intended to be expressed in large amounts, including any that are within the scope of the present invention. I will. Examples of protein coding sequences include, but are not limited to: pharmaceutically active peptides (eg, cytokins (interferons, interleukins, chemokines, granulocytes) Macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), multi-CSF (IL-3), erythropoietin (EPII), leukemia suppression Hematopoietic factors such as factor (LIF), c-kit ligand (SCF), tumor necrosis factor, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte proliferation Factors (HGF), vascular endothelial growth factor (VEGF), etc.); hormones (insulin, growth hormone, thyroid hormone, etc.)); vaccine antigens; antigens (eg, human antibodies (preferably fully human antibodies), humanized Body, multispecific antibody, chimeric antibody, single chain antibody, Fab fragment, F (ab ') fragment, fragment produced by Fab expression library, anti-idiotype (anti-Id) antibody, epitope binding fragment); blood products Peptides useful for agricultural production (eg, antibacterial proteins); Various enzymes and hydrolases that synthesize physiologically or pharmacologically active secondary metabolites; Inhibitors that regulate enzymatic reactions; Soybean glycinin; or an artificial protein designed to cleave bioactive peptides by enzymatic degradation in the digestive tract. In addition, examples of nutritionally significant substances include, but are not limited to, casein, albumin and globulin of legumes, synthase of vitamins, saccharide synthase, and lipase. In addition, proteins involved in the kakunje properties as raw materials for various processed foods include, for example, wheat glutenin (bread making), soy globulin group (tofu), and milk casein group (cheese). Proteins that enhance the palatability or functionality of foods (e.g., synthases of special sugars or amino acids such as cyclodextrins, oligosaccharides, and _{diaminoacetic} acid, pigment synthases that improve appearance, and synthesis of taste components) Involved proteins) or gastrointestinal tract These include, but are not limited to, artificial proteins designed to excise physiologically active peptides (eg, angiotensin converting enzyme inhibitory peptides that have a blood pressure effect) by undergoing enzymatic digestion within What? The heterologous gene sequence is preferably a cytodynamic or hormonal gene sequence. The heterologous gene sequence more preferably encodes a protein having a desired function when expressed in a plant. As used herein, "a protein having a desired function when expressed in a plant" means that a gene product expressed in a plant has a desired function in an animal, a plant, or an in vitro mouth. To fulfill. For example, the glycosylation of a gene product expressed in a plant is the same as that expressed in an animal, or performs the intended function in vitro or when administered to an animal, or is harmful. Does not exert any significant effect. The intended function refers to a function that is exhibited when the protein is produced by a conventional method. For example, if the heterologous gene sequence encodes an antibody, the antibody may function as a protective antibody (i.e., as a therapeutic or prophylactic agent) when administered to an animal, or may be specific in the mouth. May bind to a specific antigen (ie, may function as a diagnostic). The heterologous gene sequence is more preferably a gene sequence of interferon ( _α ,; 3 or γ), an antibody, human α-antitrypsin (ΑΑΤ) or green fluorescent protein (GFP). It is a green fluorescent protein, most preferably a green fluorescent protein.

[0147] In one embodiment, the heterologous gene sequence can be a marker gene. The marker one gene is synonymous with the selection gene, and refers to a nucleotide capable of distinguishing between a cell in which the selection marker is present and a cell in which the selection marker is not present, by expression of a product encoded by the selection marker.

[0148] In one embodiment, the heterologous gene sequence can be an antisense coding sequence. An antisense coding sequence encodes an antisense sequence of a specific gene intended to suppress or prevent expression and can have any antisense activity. Such is also included in the scope of the present invention. The antisense sequence refers to a sequence complementary to a coding sequence (also referred to as a sense sequence).

[0149] As used herein, "antisense activity" specifically refers to the expression of a target gene. An activity that can be suppressed or reduced. More specifically, protein expression can be reduced by specifically reducing the mRNA level of a gene having a nucleotide sequence region complementary to a certain nucleotide sequence introduced into a cell, depending on the nucleotide sequence. Activity. Methods include directly introducing RNA molecules complementary to mRNA produced from the target gene into cells, and introducing a construction vector capable of expressing RNA complementary to the target gene into cells. It is roughly divided. In plants, the latter is common.

[0150] Antisense activity is usually achieved by a nucleotide sequence at least about 8 nucleotides in length that is complementary to the coding sequence of the gene intended to suppress or prevent expression. The antisense coding sequence is preferably at least about 9 nucleotides in length, more preferably about 10 nucleotides in length, more preferably about 11 nucleotides in length, even more preferably about 12 nucleotides in length, more preferably About 13 nucleotides in length, more preferably about 14 nucleotides in length, more preferably about 15 nucleotides in length, more preferably about 20 nucleotides in length, more preferably about 25 nucleotides in length, and still more preferably May be about 30 nucleotides in length, more preferably about 40 nucleotides in length, and more preferably about 50 nucleotides in length. Preferably, the sequence complementary to the coding sequence of the gene of interest is present in the antisense coding sequence rather than discretely.

[0151] In the present specification, the length of a nucleotide sequence or a polypeptide sequence is a force that can be represented by the number of nucleotides or amino acids, respectively. The above-mentioned number is intended to include a few above and below (or, for example, 10% above and below) the number. In order to express such an intention, in this specification, “about” may be used before the number. However, in this specification it should be reasoned that the presence or absence of “about” does not affect the interpretation of that number.

[0152] The antisense coding sequence is preferably at least about 70% identical to the sequence of the antisense strand (complementary strand of the coding strand) of the gene intended to suppress or prevent expression, more preferably Are at least about 80% identical, more preferably about 90% identical, and Most preferably contain nucleotide sequences that are about 95% identical. The antisense coding sequence is preferably complementary to the sequence at the 5 'end of the nucleic acid sequence of the gene of interest. Antisense coding sequences having one or several nucleotides, one or more nucleotide substitutions, additions and / or deletions with respect to the antisense coding sequence as described above are also included in the antisense coding sequence. As used herein, “antisense activity” includes, but is not limited to, a decrease in the expression level of a gene.

General antisense technology is described in textbooks (Murray, JAHeds., Antisense RNA and DNA, Wiley-Liss Inc, 1992). The latest research has revealed a phenomenon called RNA interference (RNAi), which has led to the development of antisense technology. In RNAi, when a short-length double-stranded RNA (about 20 bases) having a sequence homologous to the target gene is introduced into cells, the mRNA of the target gene homologous to the RNA sequence is specifically degraded and the expression level is reduced. Is a phenomenon that decreases. This phenomenon found in the first nematode has been found to be a universal phenomenon in living organisms, including plants. It has been elucidated that the mechanism of the molecular level at which target gene expression is suppressed by antisense technology goes through a process similar to that of RNAi. In the conventional antisense technology, a DNA sequence complementary to the nucleotide sequence of a target gene is linked to an appropriate promoter, and an expression vector that expresses artificial mRNA under the control of the promoter is constructed. It was made to be introduced within. In recent antisense technology using RNAi, an expression vector designed to be capable of forming double-stranded RNA in a cell is often used. In RNAi-based antisense technology, the basic structure of an antisense coding sequence is that one DNA sequence complementary to a certain target gene is ligated under a promoter, and the same one is inserted in the opposite direction. It is made by connecting two. In a single-stranded mRNA transcribed from an antisense coding sequence having this basic structure, the one nucleotide sequence portion connected in the reverse direction is in a complementary relationship, and this complementary portion is paired. As a result, a double-stranded RNA with a hairpin-like secondary structure is formed, which causes mRNA degradation of the target gene according to the mechanism of RNAi. In plants, an example used in Arabidopsis has been reported (Smith, NA et al., Nature 407. 319-320, 2000). In addition, RNAi in general (Morita and Yoshida, Protein 'Nucleic Acid' Enzyme 47, 1939–1945, 2002). The contents described in these documents are incorporated herein by reference in their entirety.

[0154] In the present specification, "RNAi" is an abbreviation for RNA interference. Homologous mRNA is specifically expressed by introducing a factor that causes RNAi, such as double-stranded RNA (also referred to as ds RNA), into cells. Refers to the phenomenon in which the synthesis of gene products is suppressed and the technology used for it. As used herein, RNAi may also be used synonymously with factors that cause RNAi in some cases.

[0155] As used herein, the term "factor causing RNAi" refers to any factor capable of causing RNAi. As used herein, the term “factor that causes RNAi to bow I” for “gene” refers to the factor that causes RNAi related to the gene and achieves the effect of RNAi (for example, suppression of expression of the gene). Means Factors that cause such RNAi include, for example, at least a sequence that has at least about 70% homology to a portion of the nucleic acid sequence of the target gene or a sequence that hybridizes under stringent conditions. Examples include, but are not limited to, expression vectors designed to be able to construct RNA containing a double-stranded portion having a length of 10 nucleotides or a variant thereof.

[0156] As the heterologous gene sequence, a sequence which is homologous to any of a naturally occurring protein coding sequence, a naturally occurring antisense coding sequence IJ, a naturally occurring ribozyme coding sequence, and the like can be used. As a nucleotide sequence having such an identity, for example, when compared using the default parameters of Blast, at least about 30%, about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% At least about 30%, about 35%, about 40%, about 45% of the amino acid sequence encoded by the nucleotide sequence 1J having about 99% identity or similarity, or the nucleotide sequence of the control. %, About 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% identity or Examples include, but are not limited to, nucleotide sequences encoding amino acid sequences with similarity.

[0157] The heterologous gene sequence encodes a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and an enzyme capable of degrading a metabolizable sugar. The sequence is heterologous to at least one promoter sequence selected from the group consisting of promoter sequences of genes. The heterologous gene sequence is also preferably heterologous to the signal sequence coding sequence. “Heterologous” with respect to two sequences refers to the fact that these two sequences are derived from different genes or from different species. The term heterologous is selected from the group consisting of a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. Relationship between at least one promoter sequence and a heterologous gene sequence, a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter of a gene encoding an enzyme capable of degrading metabolizable sugars Sequence strength Relationship between at least one selected promoter sequence and other regulatory sequences, sugar deficiency-inducible promoter sequence, promoter sequence of exo-type glycolytic enzyme-encoding gene and degradation of metabolizable sugar Selected from the group consisting of promoter sequences for genes that encode Applied to at least one promoter sequence and a signal coding sequence, a relationship between a plurality of regulatory sequences other than the promoter sequence used in the present invention, and a relationship between a regulatory sequence and a heterologous gene sequence. You. For example, at least one selected from the group consisting of an Arabidopsis sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading metabolizable sugars. One promoter sequence is heterologous to the Arabidopsis actin gene. Similarly, the group consisting of the Arabidopsis sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading metabolizable sugars. At least one promoter sequence selected is heterologous to the human interferon gene. When the heterologous gene sequence is derived from a heterologous organism, it is also called a foreign gene. In this case, "foreign gene" refers to a gene in a certain organism that is not naturally present in the organism.

The nucleic acid of the present invention comprises, in addition to a sugar deficiency-inducible promoter, a promoter sequence of a gene encoding an exo-type glycolytic enzyme or a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar, a signal peptide encoding It may include a sequence. [0159] In the present specification, "signal peptide coding system" refers to a nucleotide sequence that codes for a signal peptide. As used herein, the term "signal peptide" refers to an amino acid sequence mainly containing hydrophobic amino acid residues, which is useful for the attachment of the nascent polypeptide chain to the endoplasmic reticulum membrane and the passage of the membrane. The length of the signal peptide is preferably about 10 to about 50, more preferably about 13 to about 40, and more preferably about 15 to about 30. Whether or not a signal peptide is included in a particular amino acid sequence can be determined according to methods well known in the art, but preferably, Kyte. J and Doolittle, RFJ Mol. Biol. 157 (1) Analysis of hydrophobicity index according to the method described in 105-132, 1982.If about 10 amino acids and about 50 amino acids at the N-terminus are hydrophobic, the hydrophobic amino acid portion is a signal peptide peptide. It can be determined that there is. The signal peptide preferably has a sequence that can be degraded by a signal peptidase.

[0160] The signal peptide coding sequence may be any nucleotide sequence as long as it encodes a signal peptide that binds to the signal peptide and causes extracellular secretion of the polypeptide. Examples of signal peptide coding sequences include the signal / coding peptide sequence of β-galactosidase, the signal sequence of the signal peptide of β-xylosidase, and the signal peptide coding sequence of β-dalcosidase. The signal peptide coding sequence is homologous to the promoter deficiency-inducible promoter, the promoter sequence of the gene encoding the exo-type glycolytic enzyme, or the promoter sequence of the gene encoding the enzyme capable of degrading metabolizable sugar. Or heterogeneous.

[0161] The signal peptide coding sequence is preferably: (i) position 1 to position 27 of SEQ ID NO: 2, SEQ ID NO:

A nucleotide sequence encoding the amino acid sequence shown at position 1 to position 28 of SEQ ID NO: 4 or position 1 to position 28 of SEQ ID NO: 6; (ii) one or several nucleotides compared to the nucleotide sequence of (i). (Iii) a nucleotide sequence containing a substitution, deletion or addition of a nucleotide, which encodes a peptide having extracellular secretory activity; and (iii) the nucleotide sequence of (i) or (ii) under stringent conditions. in and Haiburidizu, and the nucleotide sequence encoding a peptide having an extracellular secretion activity; a nucleotide sequence having at least 70% identity to the nucleotide sequence of and (i _v) (i), and extracellular secretion activity Selected from the group consisting of nucleotide sequences encoding the peptide having When the heterologous gene sequence is operably linked, the extracellular secretion of the product of the heterologous gene is enhanced.

[0162] The nucleic acid of the present invention further comprises a sugar deficiency-inducible promoter, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, or a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. It may include a regulatory element. As used herein, "regulatory element" refers to an element that directly or indirectly affects the expression of a coding sequence. Examples of regulatory elements include, but are not limited to, promoters, introns, terminators, enhancers, silencers, transcription termination sequences, translation termination sequences, transcription origins, intron sequences, and the like. The regulatory element may preferably include at least one element selected from the group consisting of a promoter, an intron, a terminator and an enhancer.

[0163] As used herein, "specifically express" a gene means that the gene is at a different (preferably higher) level at a particular site or stage in a plant than at other sites or stages. It is expressed. To be specifically expressed may be expressed only at a certain site (specific site) or may be expressed at other sites. Preferably, specific expression means expression at a certain site only.

[0164] In the present specification, when used for gene expression, "site specificity" generally refers to a site of an organism (for example, a plant) (for example, in the case of a plant, protein body, root, stem, stem, Leaf, flower, seed, endosperm, germ, embryo, fruit, etc.). “Stage specificity” refers to the stage of development of an organism (eg, a plant) (eg, if it is a plant, the stage of growth (eg, the specific time of formation of the protein body, the number of days of germination after germination)). It refers to the specificity of the expression of that gene. Such specificity can be introduced into a desired organism by selecting an appropriate promoter.

[0165] As described herein, the expression of a promoter is "constitutive" in almost all tissues of an organism, whether in the juvenile or mature stages of growth of the organism. The property expressed in a certain amount. Specifically, when Northern blot analysis is performed under the same conditions as in the examples of the present specification, for example, at any time point (for example, two or more points (for example, in the case of a plant, germination on the 5th and 15th days) )) In either the same or corresponding sites When expression is also observed, expression is constitutive by the definition of the present invention. Constitutive promoters are thought to play a role in maintaining the homeostasis of organisms in their normal habitat.

[0166] The expression of the promoter as "responsive to stress or stimulus" means that the expression level of the promoter changes when at least one stimulus for stress is applied to an organism. U. In particular, the property of increasing the expression level is called "stress (or stimulation) inducibility", and the property of decreasing the expression level is called "stress (or stimulation) reducing property". Expression of “decrease in stress” is a concept that overlaps with “constitutive” expression because it is assumed that the expression is observed in normal times. These properties can be determined by extracting RNA from any part of the organism and analyzing the expression level by Northern blot analysis, or by quantifying the expressed polypeptide by a paste lot. An organism (eg, a plant or plant part (specific cells, tissues, etc.)) transformed with a vector that incorporates a stress-inducible promoter along with a selectable marker and a heterologous gene sequence will be identified by its promoter. By using a stimulating factor having an inducing activity of, the expression of a selectable marker and a heterologous gene sequence can be performed only under certain conditions.

[0167] As used herein, the term "intron" refers to a nucleotide sequence present between any two exons and is not found in RNA after force-maturation that is transcribed into RNA. Say. Introns, when present, may have the effect of increasing the expression level of the polypeptide. The intron may be an intron from any organism. Preferably, the intron is from an organism into which the selectable marker is to be introduced. In the present invention, any nucleotide sequence can be used as long as it exhibits intron activity in the target organism.

[0168] As used herein, "exon" refers to a nucleotide sequence that is transcribed into RNA and translated into a polypeptide.

[0169] In the present specification, the term "terminator" is a sequence located downstream of a protein coding region and involved in termination of transcription when a nucleotide sequence is transcribed into mRNA and addition of a poly A sequence. It is. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene. Terminator is a terminator derived from any organism Force that can be one Preferably, the terminator from the organism into which the selectable marker is to be introduced. Examples of the terminator that can be used in the present invention include CaMV35S terminator, nopaline synthase terminator (Tnos), tobacco PRla gene terminator, Tml terminator, lOKDa prolamin terminator and the like, and modifications thereof. Terminators include, but are not limited to, these. In the present invention, any nucleotide sequence can be used as long as it exhibits the activity of a terminator in a target organism.

[0170] As used herein, "enhancer" can be used to enhance the expression efficiency of a target gene. Such enhancers are well known in the art. A plurality of forces that can be used may be used or one may not be used. The enhancer is a force that can be an enhancer of any organism. Preferably, the enhancer is of an organism from which the selectable marker is to be introduced. In the present invention, any nucleotide sequence can be used as long as it exhibits the activity of the enhancer in the target organism. When used in plants, the enhancer is preferably, for example, an enhancer region containing an upstream sequence in the CaMV 35S promoter.

[0171] As used herein, the term "silencer" refers to a sequence that has the function of suppressing gene expression and quiescing. In the present invention, any type of silencer may be used as long as the silencer has the function.

[0172] The regulatory sequence is preferably operably linked to the promoter and the heterologous gene sequence. As used herein, "operably linked" means that the desired nucleotide sequence is a transcription regulatory sequence (eg, promoter, terminator, silencer, heterozygous gene) that results in expression (ie, operation). Or the like, or under the control of translation control sequences (eg, introns, splice donors, splice acceptors, etc.). For example, in order for a promoter to be operably linked to a gene, the promoter is usually, but not necessarily, positioned immediately upstream of the gene.

[0173] In order to operably link a promoter and a heterologous gene sequence to the above regulatory sequence, the promoter and the heterologous gene sequence may need to be processed. For example, if the distance between the promoter and the coding region is too long and transcription efficiency is expected to decrease, or For example, when the distance between the bososome binding site and the translation initiation codon is not appropriate. Examples of processing methods include digestion with restriction enzymes, digestion with exonucleases such as Bal31 and ExoIII, or introduction of site-directed mutagenesis using single-stranded DNA or PCR such as M13.

[0174] The nucleic acid molecule of the present invention is preferably an isolated nucleotide. As used herein, an `` isolated '' nucleotide is a nucleotide that means that the other nucleotide in the cell of the naturally occurring organism (e.g., a factor other than a nucleotide and a non-nucleotide of interest). Nucleotide) is substantially separated or purified. "Isolated" nucleotides include nucleotides purified by standard purification methods. Thus, an isolated nucleotide includes chemically synthesized nucleotides. In addition, nucleotides mixed with other substances and nucleotides dissolved in a buffer after purification by a standard purification method also correspond to the isolated nucleotides referred to herein.

[0175] As used herein, the term "purified" nucleotide refers to a nucleotide from which at least a part of a factor naturally associated with the nucleotide has been removed. Thus, typically, the purity of the nucleotide in the purified nucleotide is higher (ie, more concentrated) than in the state in which the nucleotide is normally present.

[0176] In the present specification, "nucleotide" may be natural or non-natural. "Derivative nucleotides" or "nucleotide analogs" are those that are different from naturally occurring nucleotides, but that have the same function as the original nucleotides. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioate, phosphonoreamidate, methylphosphonate, chiral methylphosphonate, 2-0-methylribonucleotide, peptide mononucleic acid (PNA). Not done.

The nucleic acid molecule of the present invention comprises a sugar deficiency inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. Group strength, as long as at least one promoter sequence selected has sugar deficiency inducibility and has the necessary promoter activity. As shown in the above, a part of the nucleotide sequence may be deleted, a part of the nucleotide sequence may be replaced by another nucleotide, or another nucleotide sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Also, a group consisting of a natural sugar deficiency-inducible promoter sequence IJ, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. A natural sugar-deficiency-inducible promoter, a promoter sequence of a gene encoding an exo-type glycolytic enzyme and a metabolizable It may be a nucleotide sequence having substantially the same function as at least one promoter sequence selected from the group consisting of a promoter sequence of a gene encoding an enzyme capable of degrading sugar. Such nucleotide sequences are known in the art and can be used in the present invention.

[0178] Such a nucleic acid molecule can be prepared using a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, site-directed mutagenesis, hybridization, and the like.

[0179] (2. Vector)

The vector of the present invention is selected from the group consisting of a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. At least one promoter sequence and a heterologous gene system IJ operably linked to the selected promoter sequence.

[0180] Glucose deficiency-inducible promoter sequence, promoter sequence of a gene encoding exo-type glycolytic enzyme, promoter sequence of gene encoding an enzyme capable of degrading metabolizable sugar, operably linked heterologous gene The sequence and the like are as described in 1 above.

[0181] As used herein, the term "vector" refers to a nucleic acid molecule capable of transferring a heterologous gene sequence to a target cell. Such a vector may be capable of autonomous replication in the target cell, or may be capable of integrating into the chromosome of the target cell and be modified. Those containing a promoter at a position suitable for transcription of the altered base sequence are exemplified. The cells of interest can be host cells such as plant cells and plant individuals. As used herein, a vector can be a plasmid, an expression vector, a recombinant vector, and the like.

[0182] As used herein, the term "expression vector" refers to a sugar deficiency-inducible promoter sequence IJ, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and an enzyme capable of degrading a metabolizable sugar. Refers to a vector capable of expressing a heterologous gene sequence operably linked to at least one promoter sequence selected from the group consisting of the promoter sequence of a target gene in a target cell. The expression vector is selected from the group consisting of one sequence of a sugar deficiency-inducible promoter, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. In addition to at least one promoter sequence and the heterologous gene sequence, various regulatory elements such as gene expression regulators that regulate its expression, and, if necessary, replication and recombinant production in the target cell. Includes factors required for selection (eg, origin of replication (ori), and selectable markers such as drug resistance genes). In an expression vector, the modified base sequence is operably linked so as to be transcribed and translated. Regulatory elements include promoters, terminators and enhancers. The definition of the regulatory element is as described above. If the expressed enzyme is intended to be secreted out of the cell, it is linked in the correct reading frame upstream of the nucleotide sequence whose nucleotide sequence has been modified to encode a secretory signal peptide. The type of expression vector used to introduce into a particular organism (eg, a bacterium), the type of regulatory elements and other factors used in the expression vector, and the ability to vary depending on the cell of interest. Is a matter well known to those skilled in the art.

[0183] In the present specification, examples of the vector include a vector that can be replicated and isolated and purified by common bacteria (typically, an Escherichia coli strain derived from Escherichia coli K12 strain) used in genetic experiments. This is necessary to construct the nucleic acid molecule of interest into the target organism (eg, a plant). Specifically, there are commercially available plasmids such as E. coli PBR322 plasmid, pUC18, pUC19, pBluescript, pGEM-T, and pGEM-T Easy. Elect opening method, polyethylene glycol method, parte When a gene fragment is directly introduced into a plant cell for transformation, such as by the Picklegun method, the gene to be introduced may be constructed using such a commercially available general plasmid. As a special example of a vector, when a plant cell is transformed using a gene transfer method mediated by agrobacterium, the replication origin of both Escherichia coli and agrobacterium, It is necessary to use a plasmid called a "binary vector" having a nucleotide sequence corresponding to the border sequence (Left border and Right Border) derived from T-DNA indicating the border region to be obtained. For example, pBIlOl (commercially available from Clontech), pBIN (Bevan, N., Nucleic Acid Research 12, 8711-8721, 1984), pBINPlus (van Engelen, FA et al., Tranegenic Research 4, 288-290, 1995), pTN Or pTH (Fukuoka H et al., Plant Cell Reports 19, 2000), pPZP (Hajdukiewicz P et al., Plant Molecular Biology 25, 989-994, 1994) and the like, but are not limited thereto. In addition, tobacco mosaic virus vector is also an example of a vector that can be used for plants. Since this type of vector does not introduce the target gene into the plant chromosome, it is necessary to propagate the transgenic plant with seeds. The use is limited in the absence of this, but can be used in the present invention.

[0184] The expression vector may contain the selection marker of the present invention in an expression cassette. An "expression cassette" is defined as comprising a nucleotide sequence to be expressed (for example, a structural gene), a promoter sequence that regulates its expression, a terminator sequence that terminates mRNA transcription, and, if necessary, various other regulatory elements. One unit of an artificially constructed gene, which is operably linked to a nucleotide sequence to be expressed in a target cell. Representative examples of expression cassettes include a selection marker (for example, hygromycin resistance gene) expression cassette for selecting only transformed cells of the target cells, and a useful protein to be expressed in host cells. An expression cassette for the coding sequence is included. The type, structure, and number of expression cassettes to be prepared should be appropriately used depending on the organism, host cell, and purpose, and combinations thereof are well known to those skilled in the art.

[0185] An "expression vector" is also defined as a "vector" that may contain one or more of the above "expression cassettes". Can be justified. Each expression cassette to be introduced into the target cell may be placed on a separate vector, or all expression cassettes may be ligated on one vector. For example, when a selection marker coding sequence is further used in the present invention, it encodes a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and an enzyme capable of degrading a metabolizable sugar. An expression cassette containing at least one promoter sequence selected from the group consisting of a promoter sequence of a gene and a gene gene sequence operably linked to the promoter sequence, and an expression cassette containing a selection marker coding sequence, It may be present on the same expression vector or on another expression vector. Preferably, they are on the same expression vector. More preferably, it is selected from the group consisting of a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. An expression cassette comprising a gene sequence operably linked to at least one promoter sequence and its promoter sequence and an expression cassette comprising a selectable marker coding sequence are included in the same expression cassette. In the present invention, a binary vector-type expression vector can be used as an expression vector for plants.

[0186] (3. Plant cells)

The plant cell of the present invention is selected from the group consisting of a sugar deficiency inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. At least one promoter sequence and a heterologous gene system IJ operably linked to the selected promoter sequence.

[0187] Glucose deficiency-inducible promoter sequence, promoter sequence of gene encoding exo-type glycolytic enzyme, promoter sequence of gene encoding enzyme capable of degrading metabolizable sugar, operably linked heterologous gene The sequence and the like are as described in 1 above.

[0188] The plant cells used in the present invention may be cells derived from any plant (eg, monocotyledonous plants, dicotyledonous plants, etc.). [0189] As the plant cell, preferably, a cell derived from a flowering plant (monocotyledon or dicotyledon) is used, more preferably, a dicotyledonous plant cell is used, and more preferably, Gramineae, Solanaceae, Cells derived from plants of the family Lamiaceae, Brassicaceae, Apiaceae, Rosaceae, Leguminosae, and Musaceae are used. More preferably, wheat, corn, rice, rye, sorghum, tobacco, pepper, eggplant, melon, tomato, strawberry, sweet potato, Arabidopsis, oilseed rape, cabbage, green onion, broccoli, soybean, alfalpha, flax, carrot, Cells derived from cedar, citrus, Chinese cabbage, lettuce, peach, potato, purple, purple, poplar, and apple are used. Even more preferably, cells derived from tobacco or Arabidopsis are used. Most preferably, Arabidopsis-derived cells are used.

[0190] The plant cell may be a part of a plant, an organ, a tissue, a cultured cell, or the like. It is preferably a cultured cell. When aiming at high production of a protein using a plant, it is particularly preferable to use a cultured cell to secrete the target protein into a culture solution since the extraction and purification steps of the protein can be largely omitted. The plant cells are preferably cultured cells having a high growth rate. The fast growth rate means that, when the cultured cells are cultured under arbitrary culture conditions for one week, the number of cells at the start of culture is preferably about 10 times or more, more preferably about 20 times or more, and still more preferably. Is about 30 times or more, more preferably about 40 times or more, further preferably about 50 times or more, more preferably about 60 times or more, further preferably about 70 times or more, further preferably about 80 times or more, and particularly preferably Can grow to a cell number of about 90-fold or more, most preferably about 100-fold or more.

[0191] Examples of such cultured cells having a high proliferation rate include, but are not limited to, cells (for example, tobacco BY-2 cells) that can grow 50 times or more in one week. Tobacco BY-2 Itoda Tsukihachi is a well-established callus force induced by Nicotiana tabacum L. cv. Bright Yellow 2 from the moon-pig, and requires an auxin for growth. Cultured cells having properties similar to BY-2 cells can be prepared according to the method described in Kato, A. et al., "Fermentation Technology Today", 1972, pp. 689-695; It can be distributed by Dr. Seiichiro Touzawa of the Graduate School of Science. Tobacco BY2 Itoda cells have the advantages of an unusually fast growth rate (about 100 times a week) as a plant cell culture, a uniform cell population, and easy transformation. BY—similar to 2 cells In addition, a rapidly proliferating cell population was prepared according to the method described by Kato et al., Above, with 0.2 mg / l 2,4-dichlorophenoxyacetic acid, 0.4 mg / l thiamine hydrochloride and 30 g / l RM containing sucrose—Induced by inducing tobacco hypocotyl force callus in 1964 medium (Linsmaier and Skoog, Physiol. Plant. 18, 1965, p. 100) and further cultivating this callus in the same medium Can be done. Regarding cultured cells derived from Arabidopsis thaliana, cells capable of proliferating 50-fold or more in one week are known. Most preferably, cultured cells derived from Arabidopsis thaliana are used. Arabidopsis thaliana has a small genome and all the genetic information has been elucidated. This is because the operation is easy and various modifications can be made.

[0192] The cells of the present invention can be produced using these cells by a transformation method well known in the art. Alternatively, the cell of the present invention is a cell obtained from a progeny obtained by redifferentiating a cell produced according to a transformation method well known in the art, obtaining a transformed individual, and crossing the individual. possible.

[0193] Methods for transforming cells, tissues, organs or individuals are well known in the art. Such techniques are fully described in the references cited in the present invention and the like. The introduction of the nucleic acid molecule into a biological cell may be transient or permanent. Techniques for transient or homeostatic gene transfer are well known in the art, respectively. Techniques for producing transformed plants by differentiating cells used in the present invention are also known in the art, and such techniques are well described in the literature cited in the present invention and the like. It is understood that. Techniques for obtaining seeds from transformed plants are also well known in the art, and such techniques are described in the literature cited in the present invention and the like.

[0194] "Transformant" refers to all or a part of an organism such as a cell produced by transformation. As the transformant, a plant cell is exemplified. A transformant is also referred to as a transformed cell, a transformed tissue, a transformed host, and the like, depending on the subject, and includes all of these forms in the present specification. Can point.

[0195] In a preferred embodiment, the nucleic acid molecule of the present invention is provided on both sides of the plant of the present invention. Although introduced into the chromosome, those introduced into only one pair may also be useful.

[0196] As a method for arranging a nucleic acid molecule and a target cell under conditions where transformation with the nucleic acid molecule can occur, any method for introducing DNA into a plant cell may be used elsewhere in this specification. As described in detail, any of them can be used. For example, the Agrobacterium method (Agrobacterium) (JP-A-59-140885, JP-A-60-70080, WO94 / 00977), the electoral-portion method (Special 60-251887), a method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813), and the like. Among these methods, examples of the physical method include a polyethylene glycol method (PEG method), an electroporation (electroporation) method, a microinjection method, and a particle gun method. These methods are highly useful in that they can be applied to both monocotyledonous and dicotyledonous plants. However, in the polyethylene glycol method and the electoporation method, since the cell wall becomes an obstacle, protoplasts must be used, and the frequency of integration of the introduced gene into chromosomal DNA of plant cells is low. In addition, the microinjection method using forceless tissue without using protoplasts has many difficulties with regard to the thickness of the needle and the fixation of the tissue. Even with the particle gun method using tissues, there are problems such as the emergence of mutations in the form of chimeras. In addition, these physical methods generally allow the introduced foreign gene to be easily integrated into the nuclear genome as a multicopy gene in an incomplete state. It has been known that when multiple copies of a foreign gene are introduced, the gene is likely to be inactivated.

[0197] On the other hand, methods for introducing an isolated gene using an organism include the agrobacterium terminus method, the Winorespecta method, and a recently developed method using pollen as a vector. . These methods have the advantages of using a plant callus, tissue, or plant without using protoplasts, so that the culture is not extended over a long period of time and is less susceptible to somaclonal mutations and other disorders. I have. Among them, the method using pollen as a vector has many unknowns as a method of transforming plants with few experimental examples. Although the virus vector method has the advantage that the gene to be introduced into the whole plant infected with the virus is spread, it is only amplified and expressed in each cell and there is no guarantee that it will be transmitted to the next generation. The problem is that it cannot introduce long DNA fragments. Agrobacterium method stains DNA larger than about 20 kbp without major rearrangement There are many advantages, such as being able to be introduced into the body, the number of copies of the introduced gene being as small as a few copies, and high reproducibility. For monocotyledonous plants such as grasses, Agrobacterium is out of the host range, and thus, foreign genes have been conventionally introduced into grasses by the physical method as described above. In recent years, however, the agrobacterium method has been applied to monocotyledonous plants, such as rice, for which a culture system has been established. It is used favorably.

[0198] In the introduction of a foreign gene by the agrobacterium method, when a low-molecular-weight phenolic conjugate such as acetociringone synthesized by a plant acts on the Ti plasmid Vir region, a T-DNA region is cut out from the Ti plasmid, It is integrated into the nuclear chromosomal DNA of plant cells through several processes. In dicotyledonous plants, since the plant itself has a mechanism for synthesizing such a phenol compound, a foreign gene can be easily introduced by a leaf disk method or the like, and the reproducibility is high. On the other hand, in monocotyledonous plants, since the plants themselves do not synthesize such a phenolic conjugate, it has been difficult to produce a transformed plant with agrobacterium. However, it is now possible to introduce foreign genes into monocotyledonous plants by adding acetosyringone during infection with agrobacterium.

[0199] When the present invention is used in plants, the method for introducing a plant expression vector into a plant cell includes methods well known to those skilled in the art, for example, a method via agrobacterium and a method for direct introduction into cells. Can be used. As a method via the Agrobacterium terminus, for example, the method of Nagel et al. (Nagel et al. (1990), Microbiol. Lett., 67, 325) can be used. This method involves first transforming an Agrobacterium terminus by, for example, electoporation with an expression vector suitable for a plant, and then transforming the transformed agrobacterium with Gelvin et al. (Ed. 1994), Plant Molecular Biology Manual (Kluwer Ademic Press Publishers)). For a method of directly introducing a plant expression vector into cells, refer to the elect-mouth poration method (Shimamoto et al. (1989), Nature, 338: 274-276; and Rhodes et al. (1989), Science, 240: 204-207). ), Particle gun method (see Christou et al. (1991), BioZ Technology 9: 957-962) and polyethylene glycol (PEG) method. (See Datta et al. (1990), Bio / Technology 8: 736-740). These methods are well known in the art, and a method suitable for a plant to be transformed can be appropriately selected by those skilled in the art.

[0200] In the present invention, in the transformant, the target nucleic acid molecule (transgene) may or may not be introduced into a chromosome. Preferably, the nucleic acid molecule of interest (introduced gene) has been introduced into a chromosome, more preferably both chromosomes.

[0201] When performing the transformation treatment, a selectable marker gene is used, if necessary. By using a selection marker in combination with an appropriate selection factor (eg, an antibiotic, a dye, etc.) for the selection marker, the cells into which the nucleic acid molecule of the present invention has been introduced can be selected from the transformed cells. Can be selected more efficiently. However, this step is not always essential in the present invention. Such selection methods vary depending on the characteristics of the selectable marker possessed by the introduced nucleic acid molecule. For example, when a resistance gene to an antibiotic (eg, hygromycin, kanamycin, etc.) is introduced as a selectable marker, the Target cells can be selected using specific antibiotics. Alternatively, if a marker gene (for example, a green fluorescent gene or the like) is used as a selection marker, a target cell can be selected based on such a marker. Alternatively, when the foreign gene itself causes a discernible difference in phenotype, the transfected cells may be selected based on such a difference. Such identifiable differences include, but are not limited to, for example, the presence or absence of the expression of a dye.

[0202] Next, the thus obtained transformed cells can be redifferentiated into a plant tissue, a plant organ and Z or a plant by a method well known in the art.

[0203] For culturing, differentiating and regenerating plant cells, plant tissues and plants, techniques and media known in the art are used. Such a medium includes, for example, Murashige-Skoog (MS) medium, GaMborg B5 (B) medium, White medium, Nitsch & Nitsch (Nitsch) medium and the like. . These media are usually used after adding an appropriate amount of a plant growth regulator (plant hormone) or the like.

[0204] In the present specification, in the case of a plant, "regenerating" the plant refers to a part of an individual. Means the whole individual is restored. For example, redifferentiation forms an organ or plant from a piece of tissue such as a cell (leaf, root, etc.).

[0205] A method for redifferentiating a transformant into a plant is well known in the art. Such methods include Rogers et al., Methods in Enzymology 118: 627-640 (1986); Tabata III, Plant Cell Physiol., 28: 73-82 (1987); Shaw, Plant Molecular Biology: A practical approach. press (1988); Shimamoto et al., Nature 338: 274 (1989); Maliga III, Methods in Plant Molecular Biology: A laboratory course. Cold Spring Harbor Laboratory Press (1995). Not limited. Therefore, those skilled in the art can use the above-mentioned well-known methods as appropriate according to the transformed plant to be regenerated. The transgenic plant thus obtained has a gene of interest introduced therein, and such gene introduction can be carried out by the methods described herein such as Northern blot, stamp lot analysis, or the like. It can be confirmed using other well-known conventional techniques.

[0206] Further, seeds can be obtained from the obtained transformed plant. The expression of the introduced gene can be detected by Northern blotting or PCR. If necessary, the expression of the protein as a gene product can be confirmed by, for example, Western blotting.

[0207] The present invention can also be used in other organisms that have been shown to be particularly useful in plants. The molecular biology techniques used in the present invention are well known and commonly used in the art and are described, for example, in Ausubel FA et al. (1988), urrent Protocols m Molecular Biology, Wiley, New York ;, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd JX and its 3rd half, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Separate volume experimental medicine "Gene transfer & expression analysis experiment. Law ", Yodosha, 1997.

[0208] (4. Organization)

The tissue of the present invention comprises a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. One promoter A heterologous gene sequence operably linked to the selected promoter sequence.

[0209] Glucose deficiency-inducible promoter sequence, promoter sequence of gene encoding exo-type glycolytic enzyme, promoter sequence of gene encoding enzyme capable of degrading metabolizable sugar, operably linked heterologous gene The sequence and the like are as described in 1 above.

[0210] As used herein, the term "tissue" of an organism refers to a population of cells having a certain similar effect in the population. Thus, tissue can be part of an organ. Organs often have cells with the same function, but sometimes have subtly different functions.Therefore, in this specification, various tissues are used as long as they share certain characteristics. May be present in a mixture. Generally, a "tissue" can be referred to as a tissue, even if the cell populations have the same origin, but different origins, if they have the same function and Z or morphology. Normally, tissues make up part of an organ. Plants are roughly classified into meristems and permanent tissues according to the stage of development of the constituent cells, and are classified into single tissues and composite tissues according to the type of the constituent cells.

[0211] The tissue of the present invention can be obtained by subjecting cells produced according to the following "8. Method for producing transformed cells" to tissue differentiation according to a method known in the art. Alternatively, the tissue of the present invention may be a tissue obtained from offspring obtained by redifferentiating cells produced according to “8. Method for producing transformed cells”, obtaining a transformed individual, and crossing the individual. Can be

[0212] (5. Organs)

The organ of the present invention comprises a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. A promoter sequence and a heterologous gene sequence operably linked to the selected promoter sequence.

[0213] Glucose deficiency-inducible promoter sequence, promoter sequence of gene encoding exo-type glycolytic enzyme, promoter of gene encoding enzyme capable of degrading metabolizable sugar The sequence, operably linked, heterologous gene sequence, etc. are as described in 1 above.

[0214] In the present specification, the term "organ" refers to one that has one independent form and forms a structure that performs a specific function by combining one or more tissues. Plants include, but are not limited to, callus, roots, stems, stems, leaves, flowers, seeds, germs, embryos, fruits, endosperm, and the like.

[0215] The organ targeted by the present invention may be any organ, and the tissue or cell targeted by the present invention may be derived from any organ of an organism. Generally, in a multicellular organism (eg, a plant), an organ is composed of several tissues having a specific spatial arrangement, and a tissue is composed of a large number of cells.

[0216] The organ of the present invention can be obtained by using a cell produced according to a transformation method well known in the art, performing tissue differentiation according to a method known in the art, and forming an organ. Alternatively, the organ of the present invention may be an organ obtained from a progeny obtained by redifferentiating cells produced according to a method for producing transformed cells known in the art, obtaining a transformed individual, and crossing the individual. Can be

[0217] (6. Organisms)

The organism of the present invention is selected from the group consisting of a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. At least one promoter sequence and a heterologous gene sequence operably linked to the selected promoter sequence.

[0218] Glucose deficiency-inducible promoter sequence, promoter sequence of gene encoding exo-type glycolytic enzyme, promoter sequence of gene encoding enzyme capable of degrading metabolizable sugar, operably linked heterologous gene The sequence and the like are as described in 1 above.

[0219] As used herein, the term "organism" (or "plant" in the case of a plant) is used in the broadest sense in the art and refers to a plant that performs a biological phenomenon. Typically, there are various characteristics such as cell structure, proliferation (self-reproduction), growth, regulation, metabolism, and repair ability. It has the basic properties of growth, which involves the inheritance governed by nucleic acids and the metabolism regulated by proteins. Organisms include prokaryotes, eukaryotes (eg, plants, animals). Preferably, in the present invention, the organism can be a plant. As used herein, preferably, such a plant may be fertile. More preferably, such plants are capable of producing seed.

[0220] The organism of the present invention can be obtained by using cells created according to a transformation method well known in the art and redifferentiating according to a method known in the art. Alternatively, the organism of the present invention is obtained from a progeny obtained by redifferentiating cells produced according to a transformation method well known in the art, obtaining a transformed individual, and crossing the individual. It can be an organism.

[0221] As used herein, "plant" is a generic term for organisms belonging to the plant kingdom, and lacks the ability to move, lack the ability to move, have a strong cell wall, abundant and persistent embryonic tissue Characterized by organisms. Typically, plants are flowering plants that have cell wall formation and anabolism by chlorophyll. Plants include both monocots and dicots. Preferred plants include, for example, monocotyledonous plants belonging to the Poaceae family such as wheat, corn, rice, rye, and sorghum. Preferably, the plant is a food crop or a medicinal plant. Other examples of preferred plants include tobacco, peppers, eggplants, melons, tomatoes, strawberries, sweet potatoes, cabbage, leeks, broccoli, carrots and peas.

, Citrus, Chinese cabbage, lettuce, peach, potato, murasaki, olive and apple. More preferably, the plant may be tobacco, rice, tomato, strawberry, purple or olive. Preferred plants are not limited to crops, and include flowers, trees, lawns, weeds and the like. Unless otherwise indicated, a plant refers to any plant, plant organ, plant tissue, plant cell, and seed. Examples of plant organs include roots, leaves, stems, flowers, and the like. Examples of plant cells include callus and suspension culture cells.

[0222] Examples of plants of the Poaceae family include plants belonging to 〇ryza, Hordenum, Secale, Scccharum, Echino chloa, or Zea, and include, for example, rice, oats, rye, hee, sorghum, corn, and the like.

[0223] Plants containing a heterologous nucleotide sequence may express a gene product that is not naturally expressed. The

[0224] In a preferred embodiment, in the organism of the present invention, the nucleic acid molecule of the present invention can be introduced into chromosomes on both sides, but those introduced into only one pair may also be useful.

[0225] (7. Method for producing protein)

The protein production method of the present invention is selected from the group consisting of a sugar deficiency inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. Introducing a nucleic acid molecule comprising at least one promoter sequence to be transformed and the protein coding sequence operably linked to the selected promoter sequence into a cell to obtain a transformed cell; Culturing the transformed cells in the absence of sugar to secrete the protein; and recovering the protein.

[0226] Glucose deficiency-inducible promoter sequence, promoter sequence of a gene encoding exo-type glycolytic enzyme, promoter sequence of gene encoding an enzyme capable of degrading metabolizable sugar, operably linked heterologous gene The sequence and the like are as described in 1 above.

In the method of the present invention, first, a group consisting of a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar can be used. A nucleic acid molecule comprising at least one promoter sequence selected from and a protein coding sequence operably linked to the selected promoter sequence is introduced into a cell to obtain a transformed cell.

[0228] The cells used in the production method of the present invention may be any of plant cells, bacterial cells and animal cells, and are preferably plant cells.

[0229] As the plant cell, preferably, a cell derived from a flowering plant (monocotyledon or dicotyledon) is used, more preferably, a dicotyledonous plant cell is used, and more preferably, Gramineae, Solanaceae, and Cells derived from plants of the family Lamiaceae, Brassicaceae, Apiaceae, Rosaceae, Legumes, and Musaceae are used. More preferably, wheat, corn, rice, corn, sorghum, tobacco, pepper, eggplant, melon, tomato, strawberry, sweet potato, rape, cabbage, green onion, broccoli, soybean, alfalfa, ama, carrot, cricket, citrus, Chinese cabbage, lettuce, peach, ja Cells derived from potato, purple, ogle, poplar and apple are used. Most preferably, cells from tobacco are used.

[0230] The plant cell may be a part of a plant body, an organ, a tissue, a cultured cell, or the like. It is preferably a cultured cell. The plant cells are preferably cultured cells having a high growth rate. The fast growth rate means that the number of cells at the start of culture is preferably about 10 times or more, more preferably about 20 times or more, and still more preferably, when the cultured cells are cultured under arbitrary culture conditions for one week. Is about 30 times or more, more preferably about 40 times or more, more preferably about 50 times or more, more preferably about 60 times or more, more preferably about 70 times or more, more preferably about 80 times or more, and particularly preferably about 80 times or more. It means that it can grow to a cell number of 90 times or more, most preferably about 100 times or more.

[0231] Examples of such cultured cells having a high growth rate include tobacco BY-2 cells. Even in cultured cells derived from Arabidopsis thaliana, a cell line having a high growth rate has been established and is known. Examples of such cell lines, James AH M urray Dr., Institute oi Biotechnology, University of Cambridge , Te nnis Court Road, Cambridge CB2 1QT, include United Kingdom force ^s established cell lines. Of course, a cell line having a high growth rate can also be obtained by culturing an Arabidopsis thaliana plant, growing the cultured cells, and repeatedly selecting cells at a high growth rate from the grown cells. These cells are as described above.

[0232] The cells of the present invention can be transformed using these cells as described in "3. Cells" above, using a well-known transformation method in the relevant field. Alternatively, the transformed cell may be a cell obtained from a progeny obtained by redifferentiating a cell produced according to a transformation method well known in the art, obtaining a transformed individual, and crossing the individual. .

[0233] The nucleic acid molecule used to transform a cell may further include a signal peptide coding sequence. The signal peptide coding sequence is as described in 1 above.

[0234] Next, the transformed cell may be cultured as it is in the absence of sugar to secrete the protein. Alternatively, the transformed cells are first grown in the presence of sugar. Thereafter, the cells may be transferred and cultured in the absence of sugar.

[0235] As used herein, the "sugar" may be any sugar, but is preferably a metabolizable glycolytic sugar or a sugar that can be metabolized into this glycolytic sugar. Glycolytic sugars are sugars that can be metabolized. The metabolizable sugar is as described in “1. Nucleic acid molecule” above. The metabolizable sugar is preferably glucose, fructose, xylose, galactose, sucrose, maltose and arabinose, more preferably sucrose. This is because sucrose is cheaper and easier to obtain than other monosaccharides.

[0236] The concentration of sugar at the time of culturing the transformant can be easily determined by a method known in the art. Appropriate media compositions for the cells of interest are known in the art. The medium may be liquid or solid, but is preferably liquid. Shaking culture in a liquid medium is preferred. Shaking culture in liquids results in faster cell growth

[0237] When the transformed cells are cultured in the presence of sugar before the transformed cells are cultured in the absence of sugar, the sugar concentration under these conditions is preferably about 10 mM or more. It is preferably about 15 mM or more, and more preferably about 20 mM or more. There is no particular upper limit to the sugar concentration, but there is no particular lower limit if necessary. However, the concentration can be about 200 mM or less, about 150 mM or less, about 100 mM or less, or about 90 mM or less as necessary. When transformed cells are cultured in the presence of sugar, the sugar-deficient responsive promoter does not respond, resulting in the normal metabolism that cells normally have. In the presence of sugar, transformed cells preferably grow rapidly.

[0238] After culturing the transformed cells in the presence of sugar and then transferring them in the absence of sugar, for example, the transformed cells are cultured in the presence of a certain concentration of sugar, and cultured for a certain period of time. By feeding, the cells may be transferred by consuming sugar from the medium due to the growth of the transformed cells. Preferably, however, after culturing the transformed cells in the presence of sugar for a period of time, recovering the transformed cells according to methods known in the art, and placing the transformed cells in a fresh medium without sugar Can be achieved by: When cells are cultured in liquid media, it is preferable to recover them, for example, by passage through a mesh of appropriate mesh size that does not allow the cells to pass through (eg, a 20 zm mesh). Les ,. The mesh can be manufactured from any material. For example, a nylon mesh can be used. Cultured cells can also be harvested by centrifugation at 1, OOOg x 5 minutes. When cells are cultured on a solid medium, the clumps of cells can be picked up with tweezers or the like and replaced with fresh medium.

[0239] "In the absence of sugar" mainly refers to conditions in which sugar is substantially absent. The condition in which a sugar is substantially absent refers to a condition in which a sugar deficiency-responsive promoter promotes expression. The concentration of sugar under conditions substantially free of sugar is preferably less than 15 mM, more preferably less than about 10 mM, more preferably less than about 5 mM, and even more preferably less than about 3 mM. Yes, even more preferably less than about ImM, and particularly preferably less than about 0.1 ImM. Conditions that are substantially free of sugar include conditions that are completely free of sugar.

[0240] Methods for preparing a medium containing an appropriate concentration of sugar are well known to those skilled in the art.

[0241] When a transformed cell is cultured in the absence of sugar, the sugar-deficient inducible promoter in the transformed cell responds, and the expression of a heterologous gene sequence operably linked to the sugar-deficient inducible promoter is expressed. Occurs. Furthermore, operably linked to the heterologous gene sequence, the signal peptide coding sequence results in secretion of the gene product encoded by the heterologous gene sequence.

[0242] Next, the secreted protein is recovered. The protein can be recovered using methods well known in the art.

[0243] Techniques for separating proteins are well known in the art, and any technique that can separate proteins may be used. In order to isolate or purify a protein (e.g., a useful protein) encoded by a protein coding sequence from a culture of the transformed cell of the present invention, a conventional protein known and commonly used in the art can be used. Alternatively, a purification method can be used. For example, when the polypeptide of the present invention is secreted extracellularly from the transformant of the present invention, the culture is treated by a method such as centrifugation to obtain a soluble fraction. . From the soluble fraction, solvents such as solvent extraction, salting out with ammonium sulfate, etc., precipitation with organic solvents, resins such as getylaminoethylene (DEAE) -Sepharose and DIAION HPA-75 (Mitsubishi Chemical) were used. Shade On-exchange chromatography, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose, phenyl sepharose, and molecular sieve A purified sample can be obtained using a method such as gel filtration, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric focusing.

[0244] As used herein, an "isolated" polypeptide is defined as another biological factor (eg, a factor other than a polypeptide) within a cell of an organism in which the polypeptide naturally exists. And polypeptides other than the polypeptide of interest). An “isolated” polypeptide includes a polypeptide that has been purified by standard purification methods. Thus, an isolated polypeptide includes chemically synthesized polypeptides. In addition, polypeptides mixed with other substances and dissolved in a buffer after purification by a standard purification method also correspond to the isolated polypeptides referred to in the present specification.

[0245] As used herein, the term "purified" refers to a polypeptide in which at least a part of a factor naturally associated with the polypeptide has been removed. Thus, typically, the purity of the polypeptide in the purified polypeptide will be higher (ie, more concentrated) than in the state in which the polypeptide is normally present.

[0246] When the target protein accumulates in a state of lysis in the cells of the transformant of the present invention, the cells in the culture are collected by centrifuging the culture, and the cells are washed. The cells are crushed by an ultrasonic crusher, French press, Mantongaulin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, getylaminoethyl (DEAE) — Anion exchange chromatography using a resin such as Sepharose, DIAION HP A-75 (Mitsubishi Chemical), cation exchange chromatography using a resin such as S-Sepharo se FF (Pharmacia), butyl sepharose, Hydrophobic chromatography using resin such as phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing By using The ability to gain S.

[0247] When the protein of interest is expressed by forming an insoluble substance in the cells, the cells are similarly collected, crushed, and centrifuged to obtain a precipitate fraction obtained by a conventional method. After recovering the protein, the insoluble form of the protein is solubilized with a protein denaturant. This lysate is diluted or dialyzed to contain no protein denaturant, or diluted so that the protein denaturant concentration is low enough not to denature the protein. After being formed into a three-dimensional structure, a purified sample can be obtained by the same isolation and purification method as described above. In addition, when it can be accumulated in a specific organelle in a cell, for example, in a protein body, the target protein can be purified after separating the organelle.

[0248] The protein can be purified according to a conventional protein purification method (J. Evan. Sadler et al .: Methods in Enzymology, 83, 458). For example, the target protein can be produced as a fusion protein with other proteins, and purified using affinity chromatography using a substance having affinity for the fused protein [Yama J 11 Akio, Experimental Medicine, 13, 469-474 (1995)] ₀ For example, the method of Lowe et al. (Larsen et al., Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Kukowska —Latallo JF, Genes Dev., 4, 1288 (1990)), producing a target protein as a fusion protein with protein A, and affinity chromatography using immunoglobulin G. Can be further purified.

[0249] For example, a protein of interest and a tag for purification (for example, His tag (for example, 6 His residual 6X His tags), HA tag, protein A, IgG domain, or maltose binding protein) If a fusion protein designed to be fused to add a cleavage site by a protease, for example, between the target protein and the tag for purification, is produced, affinity chromatography for the tag for purification can be performed by using affinity chromatography. By purifying the fusion protein and thereafter cleaving the protein of interest from the fusion protein using, for example, a protease, the protein of interest can be easily purified.

[0250] For example, when using a 6X His tag, cells expressing the fusion protein are collected by centrifugation (eg, at about 6000 X g for 20 minutes) and the cell pellet is treated with a chaotropic agent. In 6M guanidine HC1 by stirring at 4 ° C for 3-4 hours. The cell debris is then removed by centrifugation, and the supernatant containing the polypeptide is applied to a Nickel nitrite triacetic acid (“Ni_NTA”) affinity resin column (available from QIAGEN, Inc. supra). To load. Proteins with a 6 X His tag bind to Ni—NTA resin with high affinity and can be purified by a simple one-step procedure (see The QIA expressionist (1995) QIAGEN, Inc., supra). Out). Briefly, the supernatant was loaded onto a 6Μ guanidine-HC1, pH 8 column, the column was first washed with 10 volumes of 6M guanidine-HC1, pH 8, then 10 volumes of 6M guanidine-HC1, pH 6 And finally elute the polypeptide with 6M guanidine-HCl, pH5. The purified protein is then regenerated by dialysis against phosphate buffered saline (PBS) or 50 mM sodium acetate, pH 6 buffer and 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on a Ni-NTA column. The recommended conditions are as follows: regeneration using a linear gradient of 6M-1M urea in 500mM NaCl, 20% glycerolone, 20mM Tris / HCl pH 7.4, including protease inhibitors. Regeneration is preferably performed over a period of at least 1.5 hours. After regeneration, the protein is eluted by adding 250 mM imidazole. The imidazole is removed by a final dialysis step against PBS or 50 mM sodium acetate pH 6 buffer and 200 mM NaCl. Thus, a purified protein is obtained.

[0251] Alternatively, the target protein can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody (Larsen et al., Proc. Natl. Acad. Sci., USA , 86, 8227 (1989), Kukowska-Latallo JF, Genes Dev., 4, 1288 (1990)).

[0252] Further, the polypeptide can be purified by affinity mouth chromatography using an antibody against the target polypeptide itself. The protein of the present invention can be transcribed in vitro according to a known method ϋ. Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, 』. Biochem., 110, 166-168 (1991)]. It can be produced using a translation system.

[0253] The present invention provides a composition produced by the method of the present invention, comprising a translation product of the nucleotide of interest. The translation product contained in such a composition is Force that varies depending on the peptide Preferably it can be a protein.

[0254] Hereinafter, the present invention will be described based on examples which are preferable embodiments of the present invention. However, the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the detailed description of the invention nor the examples below, but is limited only by the appended claims. Patents, patent applications, and references cited in this specification should be incorporated by reference into this specification as if the content itself were specifically described in this specification. Understood.

Example

(Example 1: Isolation of sugar deficiency responsive gene)

(Outline of Example 1)

To identify sugar-responsive genes, Arabidopsis thaliana cDNA macroarrays were hybridized with probes prepared from seedlings cultured in the presence or absence of sucrose. Initial screening identified 36 affected cDNAs. Twelve of these cDNAs were identified as being induced under sugar-deficient conditions. Nine of these were new with a known sugar response. Based on the properties of the encoded proteins, they were divided into three groups involved in amino acid metabolism, carbohydrate metabolism and unknowns. Subsequently, the correlation between these expression profiles and sugar levels was analyzed using plants. Subjecting withdrawn leaves to sucrose deficiency for 36 hours resulted in a significant decrease in glucose, but not sucrose, and concomitantly induced all 12 genes. However, these transcripts declined rapidly within 7 hours when the sample was resupplied with sucrose. A similar pattern of induction and suppression was observed by in-situ treatment of whole plants, but not during natural senescence of mature leaves. Inhibition analysis using glucose analogs showed that nine of these genes could be involved in the hexokinase signaling pathway. This observation suggests that plants activate a set of genes involved in substance recycling during sugar deficiency, and that these genes are simultaneously and coordinated, despite their functional diversity. Implies that it is regulated.

[0256] Details of this method and the results are described below. [0257] (1.1 Materials and methods)

(1.1.1 Plant material and growth conditions)

Arabidopsis thaliana (Ecotype Columbia) in a 0.5 × MS liquid medium containing 2% sucrose (w / v) (Murashige and Skoog basic salt, pH 5.8), 16 hours light period / 8 hours photoperiod And grown at 21 ° C. Three weeks after sowing, the sprouts were depleted of sugar by transplanting to a sucrose-free medium and further cultured in place for up to 79 hours. After 72 hours of culture in sucrose-free medium, the seedlings were resupplied with sucrose by transplanting the seedlings into medium supplemented with 2% sucrose (wZv) for 7 hours.

[0258] (1.1.2 Preparation of cDNA macroarray)

Arabidopsis thaliana cDNA macroarray was supplied by Kazusa DNA Research Institute. One filter (8 X I 2 cm) contained approximately 13,000 PCR-amplified cDNA fragments prepared from four different tissues (aboveground organs, flower buds, roots and green siliques). Amplicon production and array production were performed as described in Asamizu et al., DNA Research 7, 2000, pp. 175-180 and Sasaki et al., DNA Research 8, 2001, pp. 153-161.

[1259] RNA extraction and macroarray hybridization

Total RNA was isolated from seedlings using the AGPC (acidic guanidinium-phenol-chloroform) method (Suzuki et al., Plant Cell and Environment 24, 2001, pp. 1177-1188). Poly (A) ⁺ RNA was purified using a PolyATract mRNA Isolation System (Promega) from 600 / ig total RNA. 12 μl solution of 0.3 μg / ig poly (A) + RNA and 1 μg oligo (dT) at night. Denature for 10 min in C, chill on ice for 3 min, and add 28 μl total volume (3 μl buffer, 3 μl 25 mM MgCl, 1.5 μl

2

. Each 10mM of dNTP, 1 μ 1 (7) 0 1Μ DTT, of 6 mu 1 [shed - ³³ P] with dCTP and 1. 5 mu 1 reverse transcriptase), subjected to 90 minutes reverse transcription at 37 ° C for did. The resulting ³³ P-labeled cDNA probe, Quant G-50 Micro Kahum, Amersham Pharmacia Biotech) (purified. Church-phosphate buffer (pH 7.2) supplemented with 10 μΐ oligo (dA) (pH 7.2) 65 hours in 5M NaHP〇, ImM EDTA and 7% SDS). After hybridization, the microarray filters were subjected to hybridization using probes in a total volume of 10 ml at 65 ° C for 16-20 hours. The filters were washed once with 2 × SSC containing 0.1% SDS and then twice in 0.5 × SSC containing 0.1% SDS for 30 minutes at 65 ° C. Each microarray filter was used sequentially for alternative hybridization with a ³³ P-labeled cDNA probe prepared from treated and untreated plant material.

[0260] (1.1.4 Data analysis)

Filters were scanned using a BAS 5000 and signal intensities were knocked down and analyzed by Array Vision (Amersham Pharmacia Biotech). Spots showing values below the knock ground were excluded from further analysis. To improve the comparison between the different hybridizations, the quantified signal intensities were normalized with respect to the integral per filter. Induced or repressed sugar response genes were identified using criteria of more than 3 times untreated levels or less than 0.3 times untreated levels.

[0261] (1.1.5 Subarray preparation, hybridization and data analysis)

To eliminate conflicting clones between the genes identified by the initial array screening, a subarray was constructed using 184 genes and 8 controls, and these adjacent overlapping genes totaled 384 Generates a cDNA subarray of elements (Gene-Lab Co. Ltd.). Hybridization was performed according to the protocol used for cDNA macroarray analysis. The reproducibility of the hybridization signal was evaluated from the mean of triplicate experiments.

[0262] (1.1.6 RNA gel plot analysis)

Total RNA was extracted from total sprouting or detached leaf tissue as described in Suzuki et al., Plant Cell and Environment 24, 2001, pp. 1177-1188. 10 μg of each RNA sample was size fractionated by 1.2% formaldehyde-agarose gel electrophoresis, transferred to a nylon membrane, and cross-linked by UV irradiation. 42 of this membrane. C for 16 hours of hybridization, 0.1%

Wash twice at 65 ° C. in 0.5 × SSC containing SDS, and use Suzuki et al., Plant Cell a Contact with @finolene as described in nd Environment 24, 2001, pp. 1177-1188.

[0263] (1.1.7 Determination of soluble sugar)

Soluble sugars were extracted sequentially with 4 mL of 80% (vZv) ethanol and 1 mL of 50% ethanol. The two layers were pooled and incubated at 80 ° C for 30 minutes. The solution was centrifuged at 9, OOO rpm for 10 minutes and the supernatant was dried in a Speed Vac Concentrator. The dried residue was resuspended in 400 μl of 80% ethanol, mixed with 400 μl of chloroform and centrifuged at 7,500 rpm for 10 minutes. Supernatants were analyzed for soluble sugars by the enzymatic method (Sigma Diagnostics Glucose reagent, USA).

[0264] (1.2 results)

(1.2.1 Identification of sugar-responsive genes)

Using a cDNA macroarray for approximately 13.000 Arabidopsis genes, we screened cDNAs whose transcript levels changed in response to sugar deficiency. Probe cDNA was prepared from hydroponic shoots that had been subjected to 79 hours of sugar deficiency or returned to a sucrose-rich medium after 72 hours of sucrose depletion for 7 hours. Hybridization was performed in duplicate for each sample to obtain high reproducibility. After scaling with Array Vision, the signal intensities were normalized to calculate the intensity ratio between the two probes. Initial screening yielded 184 genes whose signal ratios changed more than 3-fold or changed less than 0.3-fold (Fig. La). Subsequently, duplicate subarrays (each containing 192 genes) were prepared. Of these, 184 were the first identified genes and 8 were positive control genes. Always in place 1 Differential hybridization of subarrays with probes prepared from seedlings 3 weeks after seeding, cultured in the absence or presence of / ₀ sucrose, resulted in control of 73 gene transcript levels. It showed a 1.5-fold increase or decrease in comparison (Figure lb).

[0265] (1.2.2 accumulation of transcript)

1. The seedlings grown in the culture medium containing / ₀ sucrose are transferred to fresh medium without sucrose and incubated for 12, 36 and 79 hours, Then, RNA gel plot analysis was performed on the 73 genes identified in the subarray (Fig. 2). The expression patterns of the nine genes induced by sugar deficiency were analyzed by RNA blot hybridization (Figure 2). For all 9 genes, the RNA gel plot data was consistent with the subarray profiles, and we concluded that the quantitative results for these arrays were valid, and Atsug (Arabidopsis thaliana sugar up-regulated) 1-9 was named.

[0266] (1.2.3 Response to leaving leaf)

When sucrose was fed to the developed leaves of Sampnole, detached from the plant 25 days after sowing, it was found that the levels of sucrose and gnorecose remained relatively constant (Figure 3a). In contrast, when sucrose was depleted from the sample, the sucrose level decreased gradually, and the level of gnorecose declined rapidly (Figure 3a). In particular, gnorecoc decreased to less than 10% of the starting level after 79 hours (Figure 3a). When sucrose was depleted from the sample for 72 hours, and then fed 30 mM sucrose for 7 hours, both sucrose and gnorecose showed levels equal to those in the sugar-fed sample (Figure 3a).

[0267] The transcript accumulation profile was determined for the 73 genes identified to respond to glycosylation by RNA blot hybridization using detached leaf samples.

In each case, 12 genes were induced. The time course of the induced transcript accumulation of the 12 genes was then examined in detached leaves supplied with sucrose and detached leaves lacking sucrose (FIG. 3b). All of these transcripts were absent in the sucrose-fed sample, and they were rapidly accumulated when sucrose was depleted. Remarkably, sucrose-induced transcripts decreased within 7 hours of resupplying sucrose (Fig. 3b). These patterns seem to be commonly shared. The two forces (At SUG4 and AtSUGlO) showed some differences. Based on the predicted function of the encoded protein, the twelve induced genes were classified into three groups (amino acid metabolism, carbohydrate metabolism and unknown) (Table 1). [0268] [Table 1]

Table 1. Sugar-responsive genes

Gene filed Gene ID Description Estimation function

AtSUGl 11.72 At3g30775

AtSUG2 4.44 At3g47340

AISUG3 6.24 At2gl3360 Amino acid metabolism

AtSUG4 2.66 At5g07440

AtSUGS 2.33 At5g43060

AtSUG6 2.27 At5g56870 β-galactosidase-

AtSUG7 7.7 At5g49360 ~ pheasant

Carbohydrate metabolism AtSUG8 3.55 At5g20250 Seed assimilation protein

AtSUG9 3.70 At3g57520 Anabolic protein homolog 1

AtSUGl 0 2.49 At4g20260 Inner membrane binding protein of vomerula ―

AtSUGl 1 10.32 At5g48180 myrosinase binding protein S-like protein unknown AtSUGl 2 2.07 At2g33830 putative auxin regulatory protein

(1.2.4 Effect of location)

When the whole plant was shaded, both sucrose and glucose content in rosette leaves were significantly reduced after 3 days to 25% of the original level (Figure 4a). This reduction was more pronounced for glucose than for sucrose, showing a 70% reduction after one day of treatment (FIG. 4a). All AtSUG transcripts in detached leaves were highly induced within 1 day of transfer to location and this level was maintained for up to 3 days (FIG. 4b).

[0270] (1.2.5 Effect of aging)

The detached leaves are known to age and visibly yellow (Fig. 5a). The detached rosette leaves (which were aged) were used to examine the variation in sugar content and the transcription level of AtSUG. The experimental system was set up with a Columbia Ecotype. In this experimental system, rosette leaves are fully spread approximately 25 days after germination, and inflorescences and flowers appear at 35 days. On day 50, rosette leaves lose chlorophyll, and on day 70 plants mature and reach an senescent state. Sucrose and glucose, which accumulated until day 25, declined rapidly during the next 10 days (Figure 5a). Again, the levels of both of these sugars then increased, reaching about the same level on day 70 as on day 25. This may indicate degradation of accumulated starch or cellular compounds (FIG. 5a). [0271] Surprisingly, the response of AtSUG was variable (Figure 5b). It is the first time that not all transcripts have been induced, but that induction becomes evident after day 50. AtSUG3 and AtSUG5 transcripts were present in non-senescent leaves at day 25 and persisted at relatively constant levels during the senescence period (FIG. 5b). These results suggest that the expression system of AtSUG is different from artificial aging induced by location and sugar deficiency and natural aging associated with aging.

[0272] (1.2.6 Effect of glucose analog)

The ability to be phosphorylated by hexokinase The glucose is not metabolized to 16-phosphate, and the effects of 2-dexoxyglucose are thought to indicate the involvement of the hexokinase signaling pathway. 3_〇_methylglucose is not a substrate for hexokinase, and its effects indicate hexokinase-dependent signaling. When sucrose was depleted from the withdrawn leaves for 48 hours and then sugar or analogs were fed for 24 hours, all AtSUGs were first derepressed by the deficiency (FIG. 6). Resupply of sucrose and glucose was not sufficient to be effective with respect to the forces S that suppressed their expression, the latter with respect to some genes. 2-Deoxyglucose also did not suppress AtSUG5, AtSUG7, and AtSUGlO, which did not suppress all of the forces that suppressed the expression of AtSUG. 3-O-methylglucose had no effect on AtSUG. These results indicate that nine of the twelve AtSUGs are under the control of the suspicious hexokinase signaling pathway, while three are clearly independent.

(Example 2: Confirmation of sugar deficiency-responsive expression of galactosidase, xylosidase and dalcosidase in plants)

The expression of AtSUGl isolated in Example 1 in the presence and absence of sucrose from AtSUGl was 12.2. In the same manner as described above, RNA gel plot analysis was used. Fig. 7 shows the results. As a result, it was found that AtSUG6 and AtSUG7 were significantly induced in response to sugar deficiency. Therefore, the sequences of AtSUG6 and AtSUG7 were converted to the DNA sequencer (model 373, PE Biosystems, Foster City, (CA). Searches of the determined sequences for homology to other sequences indicated that AtSUG6 encodes β-galactosidase and AtSUG7 encodes β-xylotsidase.

[0274] Using the obtained sequence, perform sequence analysis using the ClustalW and DNA analysis software on the website of the Japan DNA Data Bank (http: www.ddbj.nig.ac.jp/Welcome-j.html). This created a phylogenetic tree of the family of 3 / 3-galatatosidase (also described as Gal) and β-xychididase (also described as Xyl). Fig. 8 shows the results. Gal_l Gal_6 is present in the j3-galactosidase family. Xyl_l—Xyl—3 exists in the / 3—xylosidase family.

[0275] In order to observe the expression patterns of these similar genes, the expression of Gal-1-Gato 6 and Xyl-1-Xyl-3 in the presence and absence of sucrose was determined using the respective probes as probes. RNA gel blot analysis was performed in the same manner as described in 1.2.2 above, except that the gene was used and that the cells were grown under conditions containing 1% sucrose in the presence of sucrose. The results are shown in FIG. As a result, it was found that Gal-1 and Xyl-1 responded sensitively to sugar deficiency, and expression was significantly induced by sugar deficiency. Xyl-3 expression was enhanced by sugar deficiency.

[0276] Since these glycolysis-related genes respond sensitively to sugar deficiency, we suspect that other glycolysis-related genes might respond similarly. —Using Glc_l as a probe for dalcosidase (Glc-1) RNA gel plot analysis was performed in the same manner as described in 1.2.2 above, except that the cells were used and grown under conditions containing 1% sucrose in the presence of sucrose. The results are shown in FIG. As a result, it was found that the expression of β-dalcosidase was also significantly induced in response to sugar deficiency.

[0277] The structures of the Gal-1 gene product (724 amino acids), the Xyl_l gene product (774 amino acids), and the Glc-1 gene product (531 amino acids) were compared. (FIG. 11). Therefore, these gene products are expected to be secreted out of the cell. In addition, all of them had a sugar chain addition signal and were considered to be glycoproteins. In other parts, the homology among these three gene products was low. (Example 3: Confirmation of glucose deficiency-responsive expression of -galactosidase, -xylosidase and -dalcosidase in cultured cells)

Cultured cells are characterized in that the sugar source can be easily adjusted and that cell wall proteins are secreted into the culture solution (see FIG. 12). If expression of j3-galactosidase, j3-xylosidase and / 3-darcosidase is similarly induced in cultured cells by sugar deficiency, construct a sugar deficiency-inducible expression system using the promoters of these genes. Is possible. Therefore, Arabidopsis thaliana (ecotype Columbia) was placed in a 0.5X MS liquid medium (Murashige Chobi Skoog basic salt, pH 5.8) containing 2% sucrose (wZv) for 16 hours during the light period and 8 hours during the Z8 period. The photoperiod was grown at 21 ° C. One week after seeding, a portion of the hypocotyl of Arabidopsis thaliana was cut out, and this portion was cut out with 3% sucrose and lmg / L 2,4-dichlorophenoxyacetic acid, 1 X B5 vitamin (0.4 mg / L myo-inositol, 0. 1 month on 1X MS solid medium containing 004 mg / L nicotinamide, 0.004 mg / L pyridoxine HC1, 0.04 mg / L thiamine HC1) and 0.8% agarose for 1 month at 21 ° C Callus was induced by culturing over a period of time. One month later, the induced callus was replaced with agarose-free, 3% sucrose and lmg / L 2,4-dichlorophenoxyacetic acid, 1 X B5 vitamin (0.4 mg / L myo-inositol, 0.004 mg / L Planted in 1 XMS medium containing nicotinamide, 0.004 mg / L pyridoxine HC1, and 0.04 mg / L thiamine HC1) and cultivated in suspension in the dark with shaking at 100 rpm at 21 for 1 week. . After one week of suspension culture, the Arabidopsis cultured cells were treated with 0, 10, 30 or 90 mM sucrose and lmg / L 2,4-dichlorophenoxyacetic acid, 1 X B5 vitamin (0.4 mg / L myo-inositol The cells were subcultured at 21 ° C. for 36 hours in a 1 × MS medium containing 0.0004 mg / L nicotinamide, 0.004 mg / L pyridoxine HC1, and 0.004 mg / L thiamine HC1). At the start of the subculture (0 hours), 12 hours, 24 hours, and 36 hours after the subculture, a portion of the cultured cells is removed, and Gal-1, Xyl-1, and Glc-1 in the cells are removed. Expression was examined by RNA gel plot analysis in the same manner as described in 1.2.2 above, except that each gene was used as a probe for each gene. The results are shown in FIG. As a result, Gal-1, Xyl_l and Glc_l were remarkably induced in mRNA expression in cultured cells due to sugar deficiency, and in the presence of sucrose, It was found that the expression of gene mRNA was almost completely suppressed.

[0279] To determine whether the actual enzymatic activity of Gal-1, Xyl-1, and Glc-1 in the culture was also increased in a glucose-deficient manner, 3% sucrose and lmg / L 2 , 4-dichloro mouth phenoxyacetic acid, 1 X B5 arsenic, Tamine (0.4 mg / L genuine inositol), 0.004 mg /: L nicotinamide, 0.004 mg / L pyridoxine HC1, 0.04 mg / L thiamine HC1 Precultured in 1X MS medium containing, and then subcultured by transplanting the cultured cells to the same medium containing 3% sucrose or without sucrose, and after 10 hours and 20 hours, After 30 hours or 40 hours, collect 0.1 ml of the culture supernatant and use artificial substrates (Methyl-Bennveriferon-Gal, Methyl-Bembelliferone-Xyl and Methyl-Bembelliferone-Glu, respectively), 365 nm Using a fluorescence microscope at an excitation wavelength of 455 nm and an absorption wavelength of 455 nm. By measuring the fluorescence of 4-methyl © Nbe Rif We Ron (4-MU), beta one-galactosidase activity of this supernatant was measured β- xylosidase activity and β- Darco oxidase activity. The results are shown in FIG. As a result, it was confirmed that the expression of all of galactosidase, β-xylosidase and _darcosidase was significantly induced at the protein level due to sugar deficiency and was secreted into the culture medium.

[0280] From these results, during sugar deficiency (sugar starvation), sugar starvation induces the expression of cell wall degrading enzyme genes, secretes cell wall degrading enzymes, and causes the sugar chains present in the cell wall by the cell wall degrading enzymes. Is decomposed to release monosaccharides such as galactose, xylose, and gnorecose, and the released sugars are considered to be used as carbon sources by plants (Fig. 15). In other words, it is considered that when a plant becomes sugar-starved due to inhibition of photosynthesis or the like, it degrades cell wall polysaccharide and uses it as an energy source.

[0281] In order to confirm what kind of sugar controls the sugar-deficiency-induced expression by Gal-1, Xyl-1 and Glc-1, the cultured Arabidopsis thaliana cells were used. / ₀ sucrose and lmg / L 2,4-dichlorophenoxyacetic acid, 1 X B5 vitamin (0.4 mg ZL myo-inosito-nore, 0.004 mg / L nicotinamide, 0.004 mg / L pyridoxine HC1, 0.0 4 mg / L Preculture in IX MS medium containing thiamine HC1), then 2-doxy, instead of 3% sucrose, each of which is sucrose, glucose, galactose, fructose, xylose, mannose, mannitol, or a glucose analog as the carbon source —Cultured in the same medium containing 3% glucose (2_d_gluc) or 3_-methylglucose (3_oM_gluc) at 21 ° C in the dark for 24 hours. After culturing for 24 hours, cultured cells were collected, mRNA was isolated, and examined by RNA gel plot analysis. The results are shown in FIG. As a result, mRNA expression of Gal-1, Xyl_l and Glc_l was suppressed by sucrose, glucose, galactose, fructose and xylose, and metabolizable sugars, while 2-deoxy-glucose, mannose and 3_0_methyl It was found that the expression of Gal-1, Xyl-1, and Glc_l mRNAs was not suppressed by glucose, mannitol, and sugars that were not metabolized by plants and that did not enter glycolysis. These results indicate that mRNA expression of Gal-1, Xyl_1 and Glc_l is controlled by the presence or absence of metabolizable sugars (Fig. 15). Thus, it is expected that the expression control by sugar is controlled by substances in and after the glycolysis system.

(Example 4: Expression of heterologous gene sequence by β-galactosidase promoter or β-xylosidase promoter in tobacco BY-2 cells)

Controlling the expression level and timing of gene expression of the target protein is an important factor when aiming for efficient protein production. Proliferation of cultured cells is also important for high protein production. For example, tobacco BY-2 cells (Kato, A. et al., Fermentation Technology Today, 1972, pp. 689-695, can be prepared according to the method described in myself; Dr. Seiichiro Tosawa of the Graduate School of Frontier Sciences, The University of Tokyo ) Are very fast growing cell lines that grow about 100-fold in one week. Use of such fast-growing cultured cells would allow more efficient production of useful proteins using the sugar deficiency-inducible promoter. Therefore, it was examined whether the promoter of the sugar deficiency-inducible gene isolated from Arabidopsis thaliana could be expressed in a sugar deficiency-inducing manner even in a heterologous plant, Tabaco.

(4.1 Preparation of Construct in Which Heterologous Gene Sequence Is Linked to Promoter of j3_galactosidase Gene)

A region containing a 1418 bp promoter (GaP) derived from the β-galactosidase (Gal-1) gene (MIP ID: At5g56870) and a signal peptide (SP) coding sequence of 27 amino acid sequence (Gal_P_SP) as a promoter and a signal peptide ) The GFP coding sequence is used as the gene sequence. As the promoter region of Gal-1, a DNA sequence 1418 bp upstream from the start codon of Ga1 was used. Of course, as is known to those skilled in the art, a similar result can be obtained even if a longer region is not necessarily used as the promoter sequence and the system J 'is used.

[0284] In detail, genomic DNA of Arabidopsis thaliana (ecotype: Colombia) was prepared by cetyltrimethylammonium bromide precipitation method (Murray and Thompson, Nucleic Acids Res 8, 1980, pp. 4321-4325). Using the prepared genomic DNA as a template, TAKARA BIO PCR was performed using pi-mouth best polymerase to amplify the sequence of the / 3_galactosidase promoter and the signal peptide coding sequence (Gal_P_SP) behind it. Primer 2 is an amino acid 5 amino acids downstream from the putative cleavage site of the gal signal peptide. Having a sequence consisting of the sequence of the BamHI site to the PCR reaction conditions at this time, the reaction conditions are in accordance with the instructions attached, were as follows.:

PCR reaction conditions: 30 cycles at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute, 30 cycles, and 72 ° C for 5 minutes.

[0285] After confirming the amplification of the PCR product by electrophoresis, the amplified DNA fragment 1 was ligated to pGEM-T (registered trademark) Easy vector (obtained from Promega) and cloned into E. coli. The cloned nucleotide sequence was determined, and it was confirmed that the target DNA fragment had been cloned. After culturing this E. coli in large quantities, vectors are extracted from this E. coli, the extracted vector is cut with restriction enzymes Hindlll and BamHI, electrophoresed, recovered from the gel, and purified. A DNA fragment 1 (Gal_P—SP; also described as Ga to P—Gal_SP) containing the promoter sequence of the gene encoding j3-galactosidase and the signal peptide coding sequence following it was obtained (see FIG. 17a).

) o

[0286] On the other hand, pBI121 (obtained from Clontech) was cut with restriction enzymes EcoRI and Hindlll, Fragment 2, a vector without the CaMV 35S promoter, GUS gene and Nos terminator, was obtained. Also, CaMV35S_sGFP (S65T) _N〇S3 '(Hiroho Fukuda, Mikio Nishimura, and Kenzo Nakamura, "Experimental protocol for observing plant cells: From gene expression to intracellular structure'function", Shujunsha, 1997, pp. Described in 196-199; donated by Yasuo Niwa), digested with the restriction enzymes EcoRI and Hindlll, electrophoresed, recovered from the gel and purified to obtain the CaMV35S promoter, sGFP gene and NOS gene. Fragment 2 containing the minator was obtained. The vector fragment 2 and the fragment 2 were ligated to obtain a vector 4 containing the CaMV35S promoter, the sGFP gene and the NOS terminator downstream of the kanamycin resistance gene. After cloning this vector into E. coli and culturing this E. coli in large quantities, extract vector 4 from this E. coli, cut the extracted vector 4 with Hindlll and BamHI, A vector fragment 5 containing the promoter insertion site, the sGFP gene and the NOS terminator was obtained downstream of the sex gene (see FIG. 17b).

[0287] Next, the DNA fragment 1 prepared above and the vector fragment 5 were ligated, and the Gal promoter, the Gal signal peptide coding system IJ, and the first of the Gal mature polypeptide sequence were placed downstream of the force namycin resistance gene. A plasmid 6 (construct for plant introduction; pBI-Gal-P-SP-GFP) was obtained, in which the system IJ encoding 5 amino acids, the GFP gene sequence and the Nos terminator sequence were linked.

(4.2 Establishment of Transformed BY2 Cells)

This construct for plant introduction (pBI-Gal-P-SP-GFP) was supervised by Hiroho Fukuda, Mikio Nishimura and Kenzo Nakamura, "Experimental protocol for observing plant cells: From gene expression to intracellular structure 'function," Junsha, 1997, pp. 125-129, introduced into Agrobacterium tumefaciens (EHA105 strain) according to the agrobacterium terminus method described in p. 125-129. The infected BY-2 cells were transformed into a BY-2 solid medium containing 200 μg ZmL kanamycin and 500 μg / mL carbenicillin (Yuho Fukuda, Mikio Nishimura, Kenzo Nakamura, “ Experimental Protocol: From gene expression to intracellular structure * function ”, described in Shujunsha, 1997, pp. 187-191) to obtain transformed BY-2 cells. Transformation B obtained Y-2 cells in the presence of kanamycin (50 mg / L) in 3% sucrose in liquid BY-2 medium (Hiroki Fukuda, Mikio Nishimura, Kenzo Nakamura, "Experimental protocol for observing plant cells. Liquid culture was performed at 27 ° C with shaking in “internal structure to function” (described in Shujunsha, 1997, pp. 187-191). Every week, 0.5 ml of cells were subcultured into 50 ml of fresh medium to maintain the cell line. The sucrose concentration in the medium used for subculture was 3%, and the sugar was sufficiently present.

(4.3 Confirmation of secretion induction and secretion of GFP)

The transformed BY-2 cells were removed on day 4 by removing the medium through a nylon mesh. The removed transformed BY-2 cells were transferred to a liquid BY-2 medium having the same composition except that it did not contain sucrose, and cultured with shaking at 27 ° C. Protein was extracted from the medium on days 3 and 4 after transplantation to a medium containing no sucrose, and the protein was fractionated by SDS-PAGE. Then, using an anti-GFP antibody (Roche's Diagnostics), an antibody specific to GFP, supervised by Kenzo Nakamura et al., Shujunsha, Cell Engineering Separate Volume, “Plant Protein Experiment Protocol Genes and Tissues” Western Blotting was performed according to the method described in “Functions and Structures of Proteins Approaching”, pp. 164-172. The results are shown in FIG. In this example, the growth of transformed BY-2 cells was measured by optical density (OD) at 600 nm. In the graph of Fig. 18, OD

600 600 is shown on the vertical axis and the number of days of culture with the passage time in the sucrose-containing medium as day 0 is shown on the horizontal axis. When culturing in sucrose-containing medium was continued, the cells continued to grow until day 7. When passaged to culture medium without sucrose on day 4 of culture, the cells hardly grew, but secreted GFP protein. The inset in the graph of FIG. 18 shows the detection of GFP protein by Western blotting. The GFP protein detected from the medium three days after transplantation to the medium without sucrose is indicated by the arrow on the left. The GFP protein detected in the medium 4 days after transplantation to the medium without sucrose is indicated by the right arrow. As a result, it was confirmed that the GFP protein was secreted into the medium due to sugar deficiency, and the secreted GFP was accumulated in the medium.

[0290] Therefore, even in tobacco cells heterologous to Arabidopsis thaliana, Gal-1 is induced in a sugar-deficient manner, and GFP is secreted into the culture medium. From this, sugar deficiency It can be seen that by using a poorly inducible promoter and a signal peptide sequence, a heterologous gene sequence can be controllably and efficiently expressed and secreted.

Example 5 Expression of Heterologous Gene Sequence Using Xylosidase Gene Promoter

The promoter of the secretory β-xylosidase gene was isolated, and a vector linked to / 3-glucuronidase (GUS) as a reporter gene was prepared. This vector was introduced into the Arabidopsis cultured cells prepared above and the tobacco BY-12 cultured cells distributed by Prof. Kazuyuki Hiratsuka by the Agrobacterium-mediated transformation method in the same manner as 3.1 above. did.

[0292] As a result, the GUS protein was remarkably induced by sugar deficiency, like the original / 3-xylosidase. In other words, by using the xylosidase promoter, it was possible to induce the expression of the target protein in cultured plant cells by sugar deficiency.

[0293] By using this expression system, efficient secretory production of useful proteins in plant cells becomes possible.

(Example 6: Experiment in which GFP is secreted extracellularly in Arabidopsis thaliana cells (medium) by controlling Arabidopsis β-galactosidase promoter (Gal-II) and signal peptide (SP))

According to the construct prepared in Example 4 above (construct for plant introduction; pBI-Gal-P-SP-GFP), according to Shujunsha, Cell Engineering Supplement, "Model Plant Experimental Protocol", pp. 109-113) Transgenic Arabidopsis seeds were obtained by introducing the transgenic Arabidopsis thaliana. The seeds were allowed to germinate and grow, and the leaves of the fully grown transformed plant were cut off, floated in water (sugar-deficient conditions) or a 1% sucrose aqueous solution (sugar-existing conditions), and placed in a place overnight. The leaves were ground to extract proteins and subjected to Western blotting in the same manner as in Example 4. As a result, expression of the GFP gene and accumulation of the GFP protein were confirmed only in the leaves floating on the water. It was confirmed that in the leaves of the transgenic Arabidopsis thaliana plants, the expression of the GFP gene and the accumulation of the GFP protein occurred specifically in sugar deficiency.

(Example 7: Arabidopsis thaliana β-galactosidase promoter (Gal-Ρ) and signa Of secretion of mouse antibodies to the extracellular (medium) of Arabidopsis thaliana cells by the control of peptide (SP)

A mouse antibody expression construct is prepared in the same manner as in Example 4 except that a mouse antibody gene is used instead of the GFP gene. This expression construct is introduced into Arabidopsis cultured cells in the same manner as in Example 6 to obtain transformed Arabidopsis cells. By confirming by stamp lotting as in Example 4, it is confirmed that in this transformed Arabidopsis thaliana, expression of mouse antibody gene and accumulation of protein occur specifically in sugar deficiency. Further, by purifying the mouse antibody expressed in the medium and conducting an antibody specificity test, it can be confirmed that the expressed mouse antibody functions as an antibody.

(Example 8: Experiment of secreting the interfacial protein out of the Arabidopsis thaliana cells (medium) by controlling the Arabidopsis β-galactosidase promoter (Gal-G) and the signal peptide (SP))

An expression construct for interferon α is prepared in the same manner as in Example 4 except that the interferon α gene is used instead of the GFP gene. This expression construct is introduced into Arabidopsis cultured cells in the same manner as in Example 6 to obtain transformed Arabidopsis cells. By confirming by Western blotting in the same manner as in Example 4, it is confirmed that in the transformed Arabidopsis thaliana, the expression of interferon _α gene and the accumulation of protein occur specifically in sugar deficiency. Further, by purifying the interferon α expressed in the medium and administering it to mice, it can be confirmed that the protein (interferon) expressed in the plant has the desired function.

[0297] As described above, the present invention has been described using the preferred embodiment of the present invention. However, the present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the appended claims. It is understood that those skilled in the art can implement equivalent ranges based on the description of the present invention and common general knowledge from the description of the specific preferred embodiments of the present invention. Patents, patent applications, and references cited herein should be incorporated by reference in their entirety, as though the content itself was specifically described herein. Is understood.

Industrial applicability

[0298] The present invention provides an expression system that can easily control expression by controlling sugar deficiency.

[0299] The use of the present invention can be expected to activate industries related to the production of pharmaceuticals such as interferons, antibodies, and vaccines using plant cells, which have not been developed and developed due to low productivity.

[0300] (Description of Sequence Listing)

SEQ ID NO: 1: nucleotide sequence encoding Arabidopsis β-galactosidase (including the open motor region and the signal coding region);

SEQ ID NO: 2: amino acid sequence of Arabidopsis β-galactosidase;

SEQ ID NO: 3: Nucleotide sequence encoding Arabidopsis β-xylosidase (including the open motor region and the signal coding region);

SEQ ID NO 4: Amino acid sequence of Arabidopsis β-xylosidase;

SEQ ID NO: 5: nucleotide sequence encoding Arabidopsis β-dalcosidase (including the open motor region and the signal coding region);

SEQ ID NO: 6: amino acid sequence of Arabidopsis β-darcosidase;

SEQ ID NO: 7: nucleotide sequence of primer 1;

SEQ ID NO: 8: nucleotide sequence of primer 2.

Claims

The scope of the claims

[1] A nucleic acid molecule comprising a sugar deficiency-inducible promoter sequence and a heterologous gene sequence operably linked to the sugar deficiency-inducible promoter sequence.

[2] The sugar deficiency-inducible promoter sequence is selected from the group consisting of a promoter sequence of a gene encoding j3 galactosidase, a promoter sequence of a gene encoding β-xylosidase, and a promoter sequence of a gene encoding / 3-Darcosidase. The nucleic acid molecule of claim 1, wherein

[3] the sugar deficiency inducible promoter sequence is

Selected from the group consisting of

The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule has an activity of promoting sugar deficiency-induced expression of the heterologous gene when the heterologous gene sequence is operably linked to the nucleotide sequence.

[4] The nucleic acid molecule according to claim 1, further comprising a signal peptide coding sequence.

[5] The signal peptide coding sequence is a signal peptide coding sequence of a gene encoding galactosidase, a signal peptide coding sequence of a gene encoding β-xylosidase, and a signal peptide coding sequence of a gene encoding β-darcosidase. 5. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule is selected from the group consisting of a sequence. [6] the signal peptide coding sequence,

(iii) a nucleotide sequence that hybridizes with the nucleotide sequence of (i) or (ii) under stringent conditions and encodes a peptide having extracellular secretory activity;

5. The nucleic acid molecule of claim 4, wherein operably linking said heterologous gene sequence to said nucleotide sequence results in extracellular secretion of said heterologous gene product.

[7] The nucleic acid molecule according to claim 1, wherein the heterologous gene sequence is a cytoforce or hormone gene sequence.

[8] The nucleic acid molecule according to claim 1, wherein the heterologous gene sequence encodes a protein having a desired function when expressed in a plant.

[9] The nucleic acid molecule according to claim 1, wherein the heterologous gene sequence is a gene sequence of interferon, antibody, human α-antitrypsin or green fluorescent protein.

[10] The nucleic acid molecule according to claim 1, wherein the heterologous gene sequence is a marker gene sequence.

[11] The nucleic acid molecule according to claim 1, further comprising a regulatory element.

[12] The nucleic acid molecule according to claim 11, wherein the regulatory element includes at least one element selected from the group consisting of an intron, a terminator, and an enhancer.

[13] A nucleic acid molecule comprising a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a heterologous gene sequence operably linked to the promoter sequence.

[14] The promoter sequence is a promoter of a gene encoding j3-galactosidase. 14. The nucleic acid molecule according to claim 13, wherein the nucleic acid molecule is selected from the group consisting of a sequence, a promoter sequence of a gene encoding β-xylosidase, and a promoter sequence of a gene encoding β-darcosidase.

[15] the promoter sequence is

Selected from the group consisting of

14. The nucleic acid molecule according to claim 13, wherein the nucleic acid molecule has an activity of promoting sugar deficiency-induced expression of the heterologous gene when the heterologous gene sequence is operably linked to the nucleotide sequence.

[16] The nucleic acid molecule according to claim 13, further comprising a signal peptide coding sequence.

[17] The signal peptide coding sequence is a signal peptide coding sequence of a gene encoding galactosidase, a signal peptide coding sequence of a gene encoding β-xylosidase, and a signal peptide coding sequence of a gene encoding β-darcosidase. 17. The nucleic acid molecule of claim 16, wherein the nucleic acid molecule is selected from the group consisting of:

[18] the signal peptide coding sequence,

(ii) Compared to the nucleotide sequence of (i), substitution or deletion of one or several nucleotides Or a nucleotide sequence containing an addition, which codes for a peptide having extracellular secretory activity;

(iii) a nucleotide sequence that hybridizes with the nucleotide system ij of (i) or (ii) under stringent conditions and encodes a peptide having extracellular secretory activity;

17. The nucleic acid molecule of claim 16, wherein operably linking said heterologous gene sequence to said nucleotide sequence results in extracellular secretion of said heterologous gene product.

[19] The heterologous gene sequence is a cytoforce or hormone gene sequence.

14. The nucleic acid molecule according to 13.

[20] The nucleic acid molecule according to claim 13, wherein the heterologous gene sequence encodes a protein having a desired function when expressed in a plant.

[21] The nucleic acid molecule according to claim 13, wherein the heterologous gene sequence is a gene sequence of interferon, antibody, human α-antitrypsin or green fluorescent protein.

[22] The nucleic acid molecule according to claim 13, wherein the heterologous gene sequence is a marker gene sequence.

[23] The nucleic acid molecule according to claim 13, further comprising a regulatory element.

[24] The nucleic acid molecule according to claim 23, wherein the regulatory element includes at least one element selected from the group consisting of an intron, a terminator, and an enhancer.

[25] A nucleic acid molecule comprising a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar, and a heterologous gene sequence operably linked to the promoter sequence.

[26] The promoter sequence of a gene encoding an enzyme capable of degrading the metabolizable sugar is a promoter sequence of a gene encoding β-galactosidase, a promoter sequence of a gene encoding β-xylosidase, and encoding a / 3-dalcosidase. 26. The nucleic acid molecule according to claim 25, wherein the nucleic acid molecule is selected from the group consisting of a promoter sequence of a gene to be expressed.

[27] The promoter sequence of a gene encoding an enzyme capable of degrading the metabolizable sugar, (a) the nucleotide sequence shown at positions 641 to 1799 of SEQ ID NO: 1, positions 1 to 1763 of SEQ ID NO: 3, or positions 1 to 2058 of SEQ ID NO: 5;

Selected from the group consisting of

26. The nucleic acid molecule of claim 25, wherein said nucleic acid molecule has an activity to promote sugar deficiency-induced expression of said heterologous gene when operably linked to said nucleotide sequence.

[28] The nucleic acid molecule according to claim 25, further comprising a signal peptide coding sequence.

[29] The signal peptide coding sequence is a signal peptide coding sequence of a gene encoding galactosidase, a signal peptide coding sequence of a gene encoding β-xylosidase, and a signal peptide coding sequence of a gene encoding β-darcosidase. 29. The nucleic acid molecule according to claim 28, which is selected from the group consisting of sequences.

[30] the signal peptide coding sequence,

(iii) a nucleotide sequence that hybridizes with the nucleotide sequence of (i) or (ii) under stringent conditions and encodes a peptide having extracellular secretory activity; (iv) a nucleotide sequence having at least 70% identity to the nucleotide sequence of (i), and is selected from the group consisting of a nucleotide sequence encoding a peptide having extracellular secretory activity;

29. The nucleic acid molecule of claim 28, wherein operably linking said heterologous gene sequence to said nucleotide sequence results in extracellular secretion of said heterologous gene product.

[31] The claim, wherein the heterologous gene sequence is a cytoforce or hormone gene sequence.

26. The nucleic acid molecule according to 25.

32. The nucleic acid molecule according to claim 25, wherein the heterologous gene sequence encodes a protein having a desired function when expressed in a plant.

[33] The nucleic acid molecule according to claim 25, wherein the heterologous gene sequence is a gene sequence of interferon, antibody, human anti-trypsin or green fluorescent protein.

[34] The nucleic acid molecule according to claim 25, wherein the heterologous gene sequence is a marker gene sequence.

[35] The nucleic acid molecule of claim 25, further comprising a regulatory element.

[36] The nucleic acid molecule according to claim 35, wherein the regulatory element includes at least one element selected from the group consisting of an intron, a terminator, and an enhancer.

[37] A nucleic acid molecule comprising a promoter sequence and a heterologous gene system IJ operably linked to the promoter sequence, wherein the promoter sequence comprises

Selected from the group consisting of

A nucleic acid molecule having an activity of promoting expression of the heterologous gene when the heterologous gene sequence is operably linked to the nucleotide sequence.

[38] The nucleic acid molecule of claim 37, further comprising a signal peptide coding sequence.

[39] The signal peptide coding sequence is a signal peptide coding sequence of a gene encoding / 3_galactosidase, a signal peptide coding sequence of a gene encoding β-xylosidase, and a signal of a gene encoding β-dalcosidase. 39. The nucleic acid molecule of claim 38, wherein the nucleic acid molecule is selected from the group consisting of a peptide code sequence.

[40] the signal peptide coding sequence,

39. The nucleic acid molecule of claim 38, wherein operably linking said heterologous gene sequence to said nucleotide sequence results in extracellular secretion of a product of said heterologous gene.

[41] The heterologous gene sequence is a cytoforce or hormone gene sequence.

38. The nucleic acid molecule according to 37.

[42] a protein having a desired function when the heterologous gene sequence is expressed in a plant; 38. The nucleic acid molecule of claim 37, encoding a quality.

[43] The nucleic acid molecule according to claim 37, wherein the heterologous gene sequence is a gene sequence of interferon, antibody, human α-antitrypsin or green fluorescent protein.

[44] The nucleic acid molecule according to claim 37, wherein the heterologous gene sequence is a marker gene sequence.

[45] The nucleic acid molecule of claim 37, further comprising a regulatory element.

[46] The nucleic acid molecule according to claim 45, wherein the regulatory element includes at least one element selected from the group consisting of an intron, a terminator, and an enhancer.

[47] At least one selected from the group consisting of a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. Two promoter sequences,

A heterologous gene sequence operably linked to the selected promoter sequence.

[48] At least one selected from the group consisting of a sugar deficiency-inducible promoter sequence, a promoter sequence of a gene encoding an exo-type glycolytic enzyme, and a promoter sequence of a gene encoding an enzyme capable of degrading a metabolizable sugar. Two promoter sequences,

A heterologous gene sequence operably linked to the selected promoter sequence.

[49] The plant cell according to claim 48, which is a dicotyledonous plant cell.

[50] The plant cell according to claim 48, which is a cultured cell.

[51] The plant cell according to claim 50, which is a cell that can grow 50 times or more in one week.

[52] A method for producing a protein, comprising the following steps:

Culturing the transformed cells in the absence of sugar to secrete the protein; and And

Step of recovering the protein

A method comprising:

53. The method of claim 52, wherein said cells are plant cells.

53. The method of claim 52, wherein said cells are dicot cells.

53. The method of claim 52, wherein said cells are cultured cells.

56. The method of claim 55, wherein said cells are cells capable of growing 50-fold or more in one week. 53. The method of claim 52, wherein said nucleic acid molecule further comprises a signal peptide coding sequence.

The signal peptide coding sequence is a signal peptide coding sequence of a gene coding for / 3-galatatosidase, a signal peptide coding sequence of a gene coding for β-xylosidase, and a signal peptide coding sequence of a gene coding for β-dalcosidase. 58. The method of claim 57, wherein the method is selected from the group consisting of:

The signal peptide coding sequence,

58. The method of claim 57, wherein operably linking said heterologous gene sequence to said nucleotide sequence results in extracellular secretion of a product of said heterologous gene.

53. The method of claim 52, wherein the protein is a cytoforce or a hormone. [61] The method according to claim 52, wherein the protein is a protein having a desired function when expressed in a plant.

[62] The method according to claim 52, wherein the protein is an interferon, an antibody, human α-antitrypsin or a green fluorescent protein.

63. The method according to claim 52, wherein the sugar is a metabolizable glycolytic sugar or a sugar that can become the glycolytic sugar when metabolized.

[64] The method according to claim 52, wherein the sugar is selected from the group consisting of glucose, galactose, funolectose, sucrose, and xylose.

[65] A method for producing a protein, comprising the following steps:

Culturing the transformed cells in the presence of sugar;

Step of recovering the protein

A method comprising:

[66] A protein obtained by the method according to claim 52.

[67] Use of the nucleic acid molecule according to claim 1, for sugar deficiency-induced expression of a heterologous gene.