WO2007145267A1

WO2007145267A1 - Promoter capable of being expressed in root of plant

Info

Publication number: WO2007145267A1
Application number: PCT/JP2007/061968
Authority: WO
Inventors: Hironori Mano; Kazuhito Sato; Hiromi Higo; Yuzo Minobe
Original assignee: Plant Genome Center Co., Ltd.
Priority date: 2006-06-15
Filing date: 2007-06-14
Publication date: 2007-12-21
Also published as: JP2009201357A

Abstract

A cDNA library is produced from a tuberous root of a sweet potato plant, and cDNA expressed at high frequency (IT394) is obtained by cluster analysis, and a region upstream to the cDNA is obtained. When GFP is linked to the region upstream to IT394 and the linked product is introduced into a plant Arabidopsis thaliana, the emission of fluorescence is observed in a root and an embryonic axis of the plant. When the linked product is introduced into a sweet potato plant, the strong emission of fluorescent is observed in a tuberous root of the plant. These results reveal that the region upstream to IT394 can be used as a promoter acting in a tuberous root of a sweet potato plant.

Description

Promoters expressed in plant roots

Technical field

[0001] The present invention relates to a gene having promoter activity that is specifically expressed in plant roots. More specifically, the present invention relates to a method for specifically expressing a foreign gene in a plant body using a promoter region of a gene (IT394) expressed in tuberous root of sweet potato or a part thereof.

Background art

[0002] Genetically modified plants can produce useful substances such as insulin, growth hormone, erythrocyte pouchin (EPO), granulocyte stimulating factor (G-CSF), interferons, vaccines, antibodies, etc. Expected as a place. Advantages of using genetically modified plants include production at a much lower cost than using animal cells, etc., no concern about contamination with viruses and BSE that infect humans, and low environmental impact It can be used as an eating medicine.

Research on tobacco, potato, corn, etc. has progressed as recombinant plants that produce useful substances, and some have already been put into practical use.

[0003] Sweet potatoes are about five times as productive as rice, and can be grown loosely, grown under poor conditions, with low fertilizers, and with low pesticides. Therefore, it is considered promising as a place for material production. So far, raw materials for biodegradable plastics using sweet potato starch and pigment materials have been produced. However, there have been few reports on the use of recombinant sweet potato as a place for producing useful substances as described above.

[0004] Sweet potato is an important crop, but its production area is biased toward developing countries in Asia, and molecular genetics research is particularly delayed compared to other crops (for example, the number of ESTs registered in GenBank). As of February 2006, rice is 1.02 million, corn power is 680,000, and potato is 220,000, whereas sweet potato is less than 8,000.

[0005] In addition, there is little information about the promoter derived from sweet potato. There are limited reports on promoters that are known to be expressed in roots.

[0006] The promoters listed below are isolated from sweet potato, but it has not been experimentally confirmed to be expressed in a sweet potato transformant. is there.

[0007] 'Sporamine promoter

Since sporamine accounts for 60-80% of the soluble protein of sweet potato tuberous root and is rarely detected in other tissues, much research has been conducted on promoters, and it is a sucrose and injury-inducible promoter. It has been reported. Kenzo Nakamura et al. (See Patent Document 1) claim that the gene can be expressed specifically in potato tubers by the sporamine promoter. We have not conducted any force experiments that claim to be expressed even in the roots of sweet potato.

Yu, SM. (See Patent Document 2) reports that the sporamine promoter allowed high expression of recombinant protein in potato leaves, stems and tubers. No experiments have been conducted on plants that form tuberous roots or tubers, such as sweet potatoes, carrots, and onions. Wang, SJ. Et al. (See Non-Patent Document 1) confirm that the sporamin promoter responds to injury in transformed tobacco.

[0008] · β-amylase promoter

β-Amylase is a protein with abundant abundance of sporamine among sweet potato tuber proteins. When a transformant of tobacco was produced by connecting the 13 amylase promoter to GUS, it was reported that sucrose induction was observed as in the case of the sporamine promoter (see Non-Patent Document 2).

[0009] 'Peroxidase promoter

It has been reported that the promoter of sweet potato peroxidase was strongly expressed in tobacco protoplasts. (See Non-Patent Document 3)

[0010] -MADS-box gene promoter

Myung BJ. Et al. Showed that the MADS-box gene of sweet potato is expressed at a high level in the root of sweet potato, and a transient expression analysis using carrot and radish (small radishes) tuber. It has been confirmed that GUS is expressed by connecting the promoter of the MADS-box gene. From these facts, it is claimed that the promoter of the MADS-box gene in sweet potato can be expressed at a high level in the storage root of plants (see Patent Document 3). However, we did not perform expression analysis in sweet potato, and the results of transient expression analysis and analysis using recombinants do not necessarily match.

[0011] When a recombinant sweet potato was created and a foreign gene was actually expressed in its tuberous root, a report using a CaMV35S promoter derived from a virus is commonly used. I haven't found anything else. However, since the CaMV35S promoter may adversely affect plant growth depending on the gene to be expressed, it is desirable to use a tissue-specific promoter.

[0012] Patent Document 1: Patent No. 2833789

Patent Document 2: US20030177517

Patent Document 3: WO2006022467

Tokubori 1: Wang, SJ. Et al., “Wouna—response regulation of the sweet potato spo ramin gene promoter region”, Plant Mol. Biol., 2002, Vol. 48, p.223-231 Non-patent document 2 : Maeo, K. et al., “Sugar- responsible elements in the promoter of a gene for β-amylase of sweet potato”, Plant Mol. Biol., 2001, Vol. 46, p.627-637 Patent Document 3: Kim, KY. Et al., “A novel oxidative stress—inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco piants and cultured cells”, Plant Mol. Biol., 2003, Vol.51, p.831. — 838

Disclosure of the invention

Problems to be solved by the invention

[0013] The present invention provides a promoter that specifically controls gene expression in the roots of a plant, an expression vector containing the promoter, a transformed plant or plant containing the expression vector, a method for producing the same, and the promoter It is an object of the present invention to provide a method for causing a desired gene to be expressed in a plant body and to provide a drug that induces the expression of a foreign gene containing the promoter as an active ingredient.

Means for solving the problem [0014] The present inventors have conducted intensive research to solve the above problems. That is, the present inventors have isolated a gene and its promoter that are expressed in sweet potato tuberous roots, and have demonstrated that the promoter actually expresses a foreign gene in sweet potato tuberous root.

[0015] Specifically, the present inventors first tuberous force Sammaimo also create a cDNA library to obtain the _C DNA (IT394) with high expression frequency by cluster analysis to obtain its upstream region. When GFP was connected to the upstream region of IT394 and introduced into Arabidopsis thaliana, fluorescence was observed at the root and hypocotyl. When introduced into sweet potato, strong fluorescence was observed in tuberous roots. This indicates that the upstream region of IT394 can be used as a promoter that functions in the roots of sweet potato.

[0016] It should be noted that IT394 cDNA has a homology in metamouthonein. When the expression of IT394 was analyzed by RT-PCR, it was confirmed that it was expressed in roots, leaves and calli of sweet potato. Forces using sequences derived from high system No. 14 There is almost the same sequence in Ayamurasaki.

[0017] Further, the present inventors have found that the promoter of the present invention has a much stronger expression ability than the CaMV 35S promoter that is usually used as a strong promoter.

[0018] Furthermore, the inventors of the present invention, when a gene is injected into a sweet potato leaf with a particle gun to cause transient expression, the gene of the present invention is expressed more continuously than the SCaMV 35S promoter. I found out that I can make it.

[0019] As described above, the present inventors have succeeded in isolating a promoter expressed in the tuberous root of sweet potato and completed the present invention. So far it has been shown that it functions in tubers of sweet potatoes, and most promoters have been reported! ,.

[0020] The present invention relates to a promoter that specifically controls gene expression in the roots of sweet potato, an expression vector containing the promoter, a transformed plant or plant containing the expression vector, a method for producing the same, and a method using the promoter More specifically, a method for expressing a desired gene in a plant body V, and provision of a drug that induces the expression of a foreign gene containing the promoter as an active ingredient, more specifically,

[1] DNA having promoter activity according to any of the following (a) to (c), (a) DNA having the nucleotide sequence of SEQ ID NO: 8

(b) DNA comprising one or more bases deleted, substituted or added in the base sequence set forth in SEQ ID NO: 8

(c) DNA that hybridizes under stringent conditions with the DNA having the nucleotide sequence of SEQ ID NO: 8

[2] The DNA according to [1], wherein the DNA has promoter activity in plant roots,

[3] The DNA according to [2], wherein the root is a tuber of sweet potato,

[4] DNA having a structure in which a foreign gene is operably linked under the control of the DNA according to any one of [1] to [3],

[5] [1] to [4] !, a vector containing the DNA of any one of the above,

[6] Transformed cells comprising the DNA of any one of [1] to [4] or the vector of [5],

[7] The transformed cell according to [6], which is a microorganism,

[8] The transformed cell according to [6], which is a plant cell,

[9] A transformed plant comprising the cell according to [8],

[10] A transformed plant that is a descendant or clone of the transformed plant according to [9]

[11] A propagation material for the transformed plant according to [9] or [10],

[12] A method for producing a transformed plant comprising the step of introducing the DNA according to [4] or the vector according to [5] into a plant cell and regenerating the plant from the plant cell,

[13] A method for expressing a foreign gene in a plant, the method comprising introducing the DNA according to [4] or the vector according to [5] into a cell of the plant,

[14] A method for producing a plant containing a foreign protein, the method comprising the step of introducing the DNA according to [4] or the vector according to [5] into a plant cell,

[15] A method for producing a foreign protein-producing plant, wherein a foreign gene is expressed in the plant, wherein the DNA of [4] or the vector of [5] is used as a plant cell A method comprising a step of introducing into [16] The transformed cell according to any one of [6] to [8] is cultured, and the cell or a culture supernatant thereof is encoded by a foreign gene expressed by the vector according to [5]. A method for producing a foreign protein, characterized by recovering the protein,

[17] A method for producing a foreign protein comprising the following steps (a) and (b):

(a) A step of producing a plant containing a foreign protein by the method according to [14] or [15]

(b) recovering foreign protein from the plant

[18] The method according to any one of [13] to [15], [17], which is the plant physical strength potato or root vegetable

[19] The method according to any one of [13] to [15], [17], wherein the plant is a plant having a tuberous root or a tuber.

[20] The tuber root sweet potato tuber, the method according to [19],

[21] A plant obtained by the method according to [14] or [15],

[22] An artificially produced plant comprising the DNA according to [4], wherein a protein encoded by a foreign gene is expressed,

[23] The plant according to [21] or [22], wherein the plant is sweet potato,

[24] A screening method for a compound that modulates the promoter activity of DNA according to any one of [1] to [3], comprising the following steps (a) to (c):

(a) Under the control of the DNA according to any one of [1] to [3], a test compound is brought into contact with a cell or a cell extract containing DNA having a structure in which a reporter gene is functionally bound. Process

(b) measuring the expression level of the reporter gene

(c) selecting a compound that changes the expression level of the reporter gene

[25] A DNA drug used as a promoter for expressing a foreign gene in a plant, wherein the DNA according to any one of [1] to [4] or the vector according to [5] is effective. The present invention provides a gene expression inducer as a component.

Brief Description of Drawings

FIG. 1 is a diagram showing the procedure for creating a promoter activity test vector upstream of IT394. . The procedure for constructing a vector PHM161 capable of verifying the promoter activity in the plant body upstream of IT394 (SEQ ID NO: 8) using the GFP gene as a reporter is shown.

[Fig. 2] A photograph showing a recombinant Arabidopsis plant expressing GFP with the IT394 promoter. Arabidopsis thaliana on the 6th day after sowing was used. The top photo is taken with visible light, and the bottom photo is a fluorescent image of GFP. The roots and hypocotyls are strong and fluoresce.

FIG. 3 is a photograph showing a cross section of a tuberous root of a recombinant sweet potato expressing GFP with ΙΤ394 and CaMV35S promoter. The right end shows high line 14 of non-transformant. A: Visible light

, B: Fluorescent image. When GFP was expressed with the IT394 promoter, much stronger fluorescence was observed than when expressed with the CaMV35S promoter.

[Fig. 4] A photograph of a plasmid shot with a particle gun on a sweet potato leaf. A and B are photographs of the 3rd day when a plasmid in which GFP or IbMYBl is linked to the IT394 promoter (A) or 35S promoter (B) is simultaneously inserted. Visible light on the left and fluorescent image on the right. For C and D, the vertical axis indicates the percentage of cells in which GFP fluorescence (NIBA) or anthocyanin accumulation (Light) is observed, and the horizontal axis indicates the number of days (days) when the plasmid is injected. C is the result of using IT394 promoter and D is the result of using 35S promoter.

BEST MODE FOR CARRYING OUT THE INVENTION

[0022] The present invention is described in detail below. The present inventors have identified DNA having promoter activity in the roots of plants such as sweet potato. Preferred examples of the above-described DNA V ヽ of the present invention include DNA having a high expression frequency in the root of sweet potato and upstream of cDNA (IT394). The nucleotide sequence of 730 bp upstream of IT394 is shown in SEQ ID NO: 8.

[0023] The present invention provides a DNA having promoter activity expressed in the roots of a plant. A preferred embodiment of the DNA of the present invention is a DNA having the promoter activity described in any of the following (a) to (c).

(a) DNA having the nucleotide sequence of SEQ ID NO: 8

(c) DNA having the nucleotide sequence described in SEQ ID NO: 8 Soy DNA

[0024] The "DNA having promoter activity" (in this specification, promoter DNA and V, in some cases) is necessary for initiation of mRNA synthesis (transcription) using DNA as a cage. This means DNA containing a specific nucleotide sequence, including DNA created by human modification such as recombination using only DNA existing in nature.

[0025] Promoter activity can be appropriately evaluated by those skilled in the art using a known method. As a known method, as described later, a method of measuring by using a reporter gene and using the expression of the gene as an index can be exemplified.

[0026] The DNA having the promoter activity of the present invention is composed of only the DNA consisting of the base sequence set forth in SEQ ID NO: 8. The base sequence set forth in SEQ ID NO: 8 lacks one or more bases. DNA having the ability to act as a promoter in plants that has lost, substituted or added base sequence, or the base sequence described in SEQ ID NO: 8, such as a base sequence that increases translation efficiency at the 3 ′ end Also included are those that have been added and those that have been deleted at the 5 'end without losing promoter activity.

[0027] In order to prepare the above DNA, methods well known by those skilled in the art include the hybridization technique (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR). In addition to technology (Saiki, RK. Et al, Science, 1985, 230, 1350., Saiki, RK. Et al, Science, 1988, 239, 487.), for example, site-directed mutagenesis (Kramer, W. & Fritz, HJ "Methods Enzymol, 1987, 154, 350.).

[0028] In the present invention, the number of bases into which mutations such as deletions and substitutions are introduced is not particularly limited as long as the DNA into which mutations are introduced has promoter activity, but is usually within 20 base pairs. Preferably, it is within 10 base pairs, more preferably within 5 base pairs, and most preferably within 3 base pairs.

[0029] Furthermore, the DNA having promoter activity of the present invention includes DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 8. Here, the stringent conditions are not particularly limited. For example, 42 ° C, 2 X SSC (3 00 mM NaCl, 30 mM citrate), 0.1% SDS, preferably 50 ° C, 2 X SSC, 0.1% SDS, more preferably 65 ° C, 0.1 X SSC and 0.1% It is a condition of SDS. Under these conditions, it can be expected that DNA having high homology can be efficiently obtained as the temperature is increased. Multiple factors such as temperature and salt concentration can be considered as factors affecting the stringency of a hybridization, and those skilled in the art will realize the same stringency by appropriately selecting these factors. It is possible.

[0030] In addition, endogenous DNA of other plants corresponding to the DNA consisting of the base sequence described in SEQ ID NO: 8 generally has high homology with the DNA described in SEQ ID NO: 8. High homology is preferably 70% or higher, more preferably 80% or higher, more preferably 90% or higher (e.g. 95% or higher, even 96%, 97%, 98% or 99% or higher). Means sex. This homology was determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. U SA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873- 7) can be determined by.

[0031] The DNA having promoter activity of the present invention is characterized by having promoter activity in the roots of plants.

[0032] The "plant" in which the DNA of the present invention exhibits promoter activity is not particularly limited, but is preferably a plant of the genus Convolvulaceae, preferably sweet potato.

[0033] The ability of various varieties to be known for sweet potatoes The DNA having the promoter activity of the present invention may be DNA inherent in any cultivar. For example, varieties of Satsumaimo include high line 14 (Koukei), Ayamurasaki, Naruto Kintoki, Tosa Beni, Kotobuki, Murasaki Sakimasari, Purple Sweet Road, Koganesengan, Beni Tatsuma, Beni Ryo, Ben-Satsuma, Benino, , Beniotome, Benimasari, Benimase, Benikomachi, Satsuma Hikari, Satsuma Hikari, Sunny Red, Giei Red, Joy White, Aya Komachi, Hamakomachi, Yamakawa Murasaki, Tanegashima Gold, Tanegashima Roma, Annoimo, Anno Red, Anno Koney , Tama Yutaka, Shiro Yutaka, Minami Yutaka, White Satsuma, Kona Homare, Tamaotome, Daiichi Nome, Oki Kogane, Akemura Saki, Satsuma Musti, Agricultural Forest No. 1, Quick Sweet, etc. Other varieties may be used.

[0034] It is to be noted that, as a preferred embodiment of the "plant root" of the present invention, mention is made of sweet potato tuberous root. Can do.

[0035] Furthermore, the present invention provides a DNA having a structure in which a foreign gene is functionally linked under the control of the DNA having the promoter activity of the present invention. In the present invention, the foreign gene is not particularly limited, and a desired gene can be used.

[0036] Examples of the "foreign gene" in the present invention include genes such as erythropoietin (EPO), granulocyte stimulating factor (G-CSF), insulin, interferon, growth hormone, antibody, or vaccine. . By producing these genes in plants, it is possible to produce cheap and large quantities of safe pharmaceutical raw materials.

[0037] Furthermore, examples of the foreign gene include genes involved in the nutritional value and functionality of plants such as anthocyanin activity factor, phytase, and sporamine.

[0038] Anthocyanins of sweet potato have been reported to have antioxidant, antimutagenic properties, liver dysfunction reducing effects, blood pressure lowering effects and the like. By expressing the anthocyanin activity factor, these functionalities can be imparted to the plant body.

[0039] By expressing phytase, the absorption efficiency of phosphate present in the plant can be increased.

[0040] Further, by suppressing the expression of sporamine, the trypsin inhibitor activity is suppressed and the nutritional value of the feed is improved.

[0041] In the "suppression" of the present invention, the expression of a target gene (for example, a sporamine gene) is completely suppressed, and the expression level of the gene is significantly reduced. Etc. are included.

Further, examples of the foreign gene include a stress tolerance gene, a nutrient transporter gene, and a heavy metal chelator gene. By expressing these genes in plants, the plants can grow even in poor soil environments (under conditions such as dryness, flooding, poor nutrition, alkalinity, acidity, and heavy metals).

[0043] Further, examples of the foreign gene include genes resistant to pests such as nematodes and viruses. By expressing a resistance gene to nematodes, the plant body can be protected from pests and the amount of pesticide used can be reduced.

[0044] In addition to the foreign gene, the above DNA of the present invention is further linked to a terminator. It may be a structure. The terminator generally refers to a plant-derived terminator (sometimes referred to as a plant terminator), which is a DNA sequence arranged in the vicinity of the promoter of the present invention. For example, a terminator derived from cauliflower mosaic virus, Alternatively, a terminator derived from a non-synthesizing enzyme gene can be exemplified, but it is not particularly limited as long as it has a function as a terminator.

In the present invention, “functionally linked” means an exogenous gene force under the control of the DNA having the promoter activity of the present invention, so that the promoter activity is received so as to be transcribed from the DNA having the promoter activity of the present invention. Refers to the state of binding to DNA having “Functionally linking” a DNA having a promoter activity and a foreign gene is easily performed and obtained by those skilled in the art using general genetic engineering techniques.

[0046] The DNA of the present invention includes natural or isolated / purified genomic DNA, and chemically synthesized DNA. Preparation of genomic DNA can be performed by those skilled in the art using conventional means.

[0047] The DNA of the present invention can be isolated by PCR by extracting and purifying genomic DNA from a target plant, for example, sweet potato tissue, and using the obtained DNA as a cocoon.

[0048] In the present invention, in order to isolate DNA having the nucleotide sequence ability described in SEQ ID NO: 8 and DNA having a promoter activity that is hybridized under stringent conditions therewith, for example, SEQ ID NO: The primer set for amplifying the DNA having the nucleotide sequence ability according to 8 and having the promoter activity of the present invention can be used. Using this primer set, PCR can be performed using plant genomic DNA as a saddle, and then the obtained amplified DNA fragment can be used as a probe to screen the genomic library of the same plant.

[0049] PCR may be performed based on the manufacturer's guidelines for commercially available kits and devices, or may be performed by techniques known to those skilled in the art. Methods for preparing gene libraries and gene cloning methods are well known to those skilled in the art. For example, experiments such as “Cloning and Sequence” (supervised by Watanabe Katsu, edited by Masahiro Sugiura, Rural Bunkasha (1989)) and “Molecular Cloning (Sambrook et al. (1989), Cold Spring Harbor Laboratory Press)” You can refer to the book. The base sequence of the obtained gene is a nucleotide sequence analysis method known in the art or is commercially available. It can be determined using an automatic sequencer. The DNA of the present invention also includes a DNA that hybridizes with the DNA having the nucleotide sequence shown in SEQ ID NO: 8, which can be isolated by a PCR technique or a hybridization technique.

[0050] Promoter DNA isolated and identified by screening as described above (that is, DNA having the promoter activity shown in SEQ ID NO: 8, or a homolog thereof) Force Gene expression specific to plant roots The inductivity can be analyzed as follows.

[0051] The above sequence is ligated upstream of a reporter gene such as, for example, β-darc mouth-dase (GUS). As the reporter gene, in addition to the GUS gene, chloramphie-coal acetyltransferase (CAT) gene, luciferase (LUC) gene, GFP gene, sGFP (S65T) gene and the like can be used.

[0052] The chimeric gene construct prepared as described above can be introduced into plants such as sweet potato through, for example, agrobacterium, and the function thereof can be analyzed. When pHM 161 is used as a vector, a recombinant plasmid containing a chimeric gene is introduced into, for example, EHA101 strain of Agrobacterium tumefaciens using the freeze-thaw method, and the resulting transformed bacterium is For example, plants such as Arabidopsis thaliana are infected by vacuum infiltration. Seeds obtained from the infected plants are sown in a medium containing a drug suitable for the vector used, such as rhodium and idaromomycin, and analyzed for GFP using the obtained drug-resistant individuals. By attaching a GFP filter to a stereomicroscope or digital camera for observation, it is expected that GFP will be specifically detected in the roots.

[0053] The gene (foreign gene) that can be controlled by the DNA having promoter activity of the present invention is not limited to the genes described above. It is possible to use a desired gene to be specifically expressed in a plant body (for example, a plant root).

[0054] It is also possible to modify the function of the DNA having promoter activity of the present invention by linking other expression control sequences to the DNA having promoter activity of the present invention. Examples of such expression control sequences include enhancer sequences, repressor sequences, and insulator sequences. For example, a chimeric promoter in which a repressor sequence that is derepressed in response to a drug is linked to the promoter DNA of the present invention is prepared, and the target promoter is downstream of it. When a gene-linked construct is introduced into a plant, the resulting transformant suppresses the expression of the target gene under conditions where the drug is not present, but the suppression is released by administering the drug, It is expected that the target gene will be expressed in plant bodies (eg plant roots).

[0055] The present invention also provides a vector comprising the DNA having the promoter activity of the present invention, a vector comprising a DNA having a structure in which a foreign gene is functionally linked under the control of the DNA of the present invention, and Provided are a vector containing DNA having a structure having a gene insertion site downstream of the promoter, and a vector carrying a terminator on the vector.

[0056] The vector of the present invention is usually one in which the DNA having the promoter activity of the present invention is inserted into a vector capable of replicating in various cells. As this replicable vector, various known vectors can be used. Examples include vectors that can be amplified in E. coli such as pUC derivatives, shuttle vectors that can be amplified in both E. coli and agrobacterium such as pPZP2H-lac. Plant viruses such as cauliflower mosaic virus can also be used. A person skilled in the art can appropriately select a vector capable of replicating in a plant cell according to each host cell. The method of inserting the DNA having the promoter activity of the present invention into a vector follows the usual method of inserting a normal gene into a vector.

[0057] The DNA having promoter activity of the present invention or an expression vector containing the same can be used as follows. An expression vector is constructed in which a chimeric gene in which the gene of interest is linked downstream of the DNA having promoter activity of the present invention is inserted. This vector is introduced into plants such as sweet potato. The obtained transformant is expected to express the target gene specifically in the root and introduce the target trait by the action of the DNA having the promoter activity of the present invention. . In this case, it is not expressed even in an unnecessary tissue such as a 35S plug motor.

It is expected that V が traits will not appear.

[0058] The present invention also provides a transformed cell comprising the promoter DNA of the present invention or a vector having the DNA. The cells of the present invention are not particularly limited, but are preferred. Or microbial cells are plant cells.

[0059] The transformed plant cell of the present invention is a plant cell transformed by introducing the DNA or vector of the present invention into a host cell. Examples of host cells include plant cells such as scutellum in leaves, roots, stems, flowers and seeds, callus, suspension culture cells and the like. The plant species from which the cells originate is not particularly limited, and examples thereof include convolvulaceae plants. Examples of the convolvulaceae plant include sweet potato, morning glory, turkey, yuugao, and American potato. A most preferred example in the present invention is sweet potato.

[0060] The present invention also provides a method for producing a transformed plant comprising the steps of introducing the DNA or vector of the present invention into a plant cell and regenerating the plant from the plant cell. A variety of techniques can be used to introduce the DNA or vector of the present invention into host plant cells. These methods include a method for transforming plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transforming factor, An electoresis method that introduces a plasmid into plant cells by applying an electric pulse to protoplasts, a fusion method of protoplasts with small cells, cells, lysosomes, microinjection method, polyethylene glycol method, particle gun method, etc. Other known methods are included.

[0061] The plant cell into which the DNA or vector of the present invention is introduced may be an explant cell, and a cultured cell is prepared from these plant cells and introduced into the obtained cultured cell. May be. For example, plant cells such as scutellum in leaves, roots, stems, flowers and seeds, protoplasts, callus, suspension culture cells and the like can be mentioned.

[0062] For direct introduction into protoplasts, there is usually no specially required vector. For example, a simple plasmid such as a pUC derivative can be used. Depending on how the gene of interest is introduced into the plant cell, other DNA sequences may be required. For example, when Ή or Ri plasmids are used for transformation of plant cells, the sequences of at least the right end of the T-DNA region of the Ή and Ri plasmids, usually the sequences at both ends, are inserted into the gene to be introduced. It must be connected so that it is an adjacent area. [0063] When Agrobacterium is used for transformation, it is necessary to clone the gene to be introduced into a special plasmid, that is, an intermediate vector or a binary vector. Intermediate vectors are not replicated in Agrobacterium. The intermediate vector is transferred into the genus Agrobataterium by a helper plasmid or electoral position. The intermediate vector has a region that is homologous to the T-DNA sequence, and is incorporated into the Agrobacterium sputum or Ri plasmid by homologous recombination. Agrobacterium used as a host must contain the vir region. Usually, vir or Ri plasmid contains vir region, and T-DNA can be transferred to plant cells by its function.

[0064] On the other hand, the binary vector can be replicated and maintained in the genus Agrobacterium. Therefore, when the binary vector is incorporated into the genus Agrobacterium by the helper plasmid, the electroporation method or the freeze lysis method, By virtue of the vir region, T-DNA on a binary vector can be transferred to plant cells.

[0065] It should be noted that the intermediate vector or binary vector thus obtained and microorganisms such as Escherichia coli containing the same are also the subject of the present invention.

[0066] In order to efficiently select plant cells transformed by introducing the DNA or vector of the present invention, the vector contains a suitable selection marker gene or a plasmid containing a selection marker gene. It is preferable to introduce it into plant cells together with the vector. Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene resistant to the antibiotic hygromycin, the neomycin phosphotransferase resistant to kanamycin or gentamicin, and the acetyl transferase gene resistant to the herbicide phosphinothricin. Can be mentioned.

[0067] Plant cells into which the above-described vector has been introduced are placed on a selection medium containing a selection agent according to the introduced selection marker and cultured. As a result, transformed plant cells can be obtained.

[0068] The transformed plant of the present invention is a transformed plant regenerated (re-differentiated) from the transformed plant cell of the present invention. Those skilled in the art can appropriately carry out the redifferentiation method using a known technique according to the type of plant cell. For example, in potatoes Visser et al. (Visser et al. (1989), Theor. Appl. Genet., Vol. 78: p589—). In Arabidopsis, Akama et al. (Akama et al. (1992), Plant Cell Rep., Vol. 12: p7- ), In rice, Fujimura et al. (Fujimura et al. (1995), PlantTissue Culture Lett., Vol. 2: p74-), in maize, Shillito et al. (Shillito et al. (1989), Bio / Technology, vol. 7: p581). -) Redifferentiation method.

[0069] A transformed object created by these methods is a subject of the present invention. Further, obtained from the above-mentioned transformation, body, or propagation material (for example, seed, fruit, tuber, cutting ear, tuberous root, strain, callus, protoplast, etc.) obtained (consisting) The transformed plant body is also an object of the present invention.

[0070]-If a transformed plant into which the promoter (DNA) of the present invention has been introduced into the chromosome is obtained, progeny can be obtained by vigorous or asexual reproduction of the plant. In addition, the plant body, its progeny, or clonal power can also be obtained as a propagation material, and the plant body can be mass-produced based on them.

[0071] In a preferred embodiment of the method for producing a plant of the present invention, the DNA or vector of the present invention is introduced into a plant cell (host cell) to obtain a transformed plant cell, and the transformation is performed. It includes the process of transforming and regenerating objects from plant cells. Further, the silkworm may include a step of obtaining plant seeds from the thus obtained transformation and body, and producing a plant body from the plant seeds.

[0072] Plant regeneration can be performed by methods known to those skilled in the art depending on the type of plant cells (Toki. Et al., Plant Physiol, 1995, 100, 1503-1507.). For example, as a method for producing transformed plants, gene transfer to protoplasts using polyethylene glycol and regeneration of plants (Datta, S K. et al., In Gene Transfer To Plants (Potr ykus I and Spangenberg Eds.), 1995, 66-74.), A method of regenerating plants by introducing genes into protoplasts by electric pulses (Toki. Et al, Plant Physiol, 1992, 100, 1503-15 07.), particle gun The gene is directly introduced into cells by the method to regenerate plants (Christou, et al., Bio / technology, 1991, 9, 957-962.) And the gene is introduced via agrobacterium. , A method of regenerating a plant body (Hiei. Et al, Plant J, 1994, 6, 271 -282.) Etc. have already been established and widely used in the technical field of the present invention. In the present invention, these methods can be suitably used. Transformation Plants that have also been regenerated with cell strength are then cultivated in normal medium. Thereafter, when the ordered regenerated plant body is cultivated under normal cultivation conditions, the plant body can be obtained, and matured and fruited to obtain a seed.

[0073] The process of obtaining plant seeds from the transformation and the body is, for example, collecting the transformation and the body from the rooting medium, transplanting it to a pot containing soil containing water, and growing it at a constant temperature. The process of forming flowers and finally forming seeds. In addition, the process of producing a plant from a seed is, for example, when a seed formed on a transformed plant matures, it is isolated and sown in water-containing soil, under a constant temperature and illuminance. This refers to the process of producing a plant by growing it.

[0074] It should be noted that the presence of foreign DNA or nucleic acid introduced into a transformed plant that has been regenerated and cultivated in this manner is determined by a known PCR method or Southern hybridization method, or the plant body. It can be confirmed by analyzing the base sequence of the nucleic acid therein. In this case, the DNA or nucleic acid having a transformation strength can be extracted in accordance with the method of J. Sambrook et al. (Molecular and loning, ¾f2¾x, Cold bpnngHarbor laboratory Press, 1989).

[0075] The present invention also provides a method for expressing a foreign gene in a plant. In a preferred embodiment of the present invention, a plant comprising a DNA having a structure in which an exogenous gene is operably linked under the control of a DNA having the promoter activity of the present invention, or a vector comprising the DNA having the promoter activity of the present invention. It is a method including the step of introducing into the cell. This method can also be carried out by introducing the DNA into plant cells and regenerating the cells into plants. The DNA can be introduced into a plant or plant cell by the method described above.

[0076] By using a nucleic acid having a function of suppressing gene expression (for example, antisense RNA or siRNA as shown below) as a foreign gene, it is also possible to suppress the expression of an endogenous gene. Included in the method of the invention. The “suppression” in the present invention includes the case where the expression of the endogenous gene is completely suppressed, and the above-described endogenous expression in the plant body. This includes cases where the expression level of the sex gene is significantly reduced compared to the expression level of the gene in other plants.

[0077] As a method for inhibiting the expression of a specific endogenous gene, a method using an antisense technique is well known to those skilled in the art. There are a number of factors that cause the antisense nucleic acid to inhibit the expression of the target gene. Inhibition of transcription initiation due to triplex formation, transcription inhibition due to hybridization with a site where an open loop structure was locally created by RNA polymerase, transcription inhibition due to hybridization with RNA undergoing synthesis, intron Inhibition of splicing by hybridization at the junction of Etason and etason, inhibition of splicing by spliceosome formation site, inhibition of splicing by hybrid formation, inhibition of nuclear force by hybridization with mRNA, inhibition of migration to cytoplasm, capping site and poly (A) Splicing inhibition by hybridization with an additional site, translation initiation inhibition by hybridization with a translation initiation factor binding site, translation inhibition by hybridization with a ribosome binding site near the initiation codon, translation region of mRNA and polysome binding site By hybrid formation Outgrowth inhibitory peptide chain, and gene expression inhibition by hybrid formation at sites of interaction between nucleic acids and proteins, and the like. In this way, antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kenkyusho 2 Nucleic acid IV gene replication and expression, Japan Biochemical Society, Tokyo Chemical Doujin, 1993, 319-347.).

[0078] The antisense nucleic acid may inhibit the expression of the endogenous gene by the above-described action. In one embodiment, if a complementary antisense sequence is designed in the untranslated region near the 5 ′ end of the mRNA of the endogenous gene, it is considered effective for inhibiting translation of the gene. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, the nucleic acid containing the antisense sequence of the sequence of the untranslated region as well as the translated region of the endogenous gene is also included in the antisense nucleic acid. Such an antisense nucleic acid is linked downstream of the DNA having promoter activity of the present invention, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The nucleic acid thus prepared can be transformed into a desired plant by using a known method. The sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous gene or a part of the plant to be transformed. Preferably, it may not be completely complementary as long as gene expression can be effectively suppressed. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcript of the target gene. In order to effectively suppress the expression of the target gene using an antisense nucleic acid, it is preferred that the length of the antisense nucleic acid is at least 15 bases and less than 25 bases! Not limited!

[0079] Furthermore, inhibition of endogenous gene expression can also be performed by RNA interference (RNAi) using double-stranded RNA having the same or similar sequence as the target gene sequence. A nucleic acid having an inhibitory action due to the RNAi effect is generally also referred to as siRNA. RNAi introduces double-stranded RNA consisting of a sense RNA that has a sequence sequence homologous to the mRNA of the target gene and an antisense RNA that has a complementary sequence capacity into the cell, etc. It is a phenomenon that can induce destruction and suppress the expression of target genes. Since RNAi can suppress the expression of target genes in this way, it can be used as a simple gene knockout method instead of the conventional complicated and low-efficiency gene disruption method by homologous recombination, or a method applicable to gene therapy. I have attracted attention as ^^. The RNA used for RNAi need not be completely identical to the target gene or a partial region of the gene, but preferably has perfect homology.

[0080] As one embodiment of the nucleic acid having an inhibitory action by the RNAi effect, for example, double-stranded RNA (RNAi) having an RNAi effect on a target gene can be mentioned. The RNA molecule also includes a molecule having one end closed structure, for example, a siRNA (shRNA) having a hairpin structure. That is, a single-stranded RNA molecule that can form a double-stranded RNA structure in the molecule is also included.

The above-mentioned “double-stranded RNA having RNAi effect” can be appropriately prepared by those skilled in the art based on the base sequence of the gene targeted by the double-stranded RNA. That is, it is normal for those skilled in the art to select an arbitrary continuous RNA region of mRNA that is a transcription product of an arbitrary base sequence and prepare a double-stranded RNA corresponding to this region. This can be done as appropriate within the scope of the trial. Moreover, those skilled in the art can appropriately select siRNA sequences having a stronger RNAi effect from mRNA sequences that are transcripts of the sequences by known methods. In addition, if one strand (one base sequence) is known, those skilled in the art It is easy to know the base sequence of the other strand (complementary strand). The siRNA as described above can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer. In addition, a general synthesis contract service can be used for synthesis of a desired RNA.

[0082] "Nucleic acid" in the present invention means RNA or DNA. Further, chemically synthesized nucleic acid analogs such as so-called PNA (peptide nucleic acid) are also included in the nucleic acid of the present invention. PNA has a three-dimensional structure very similar to nucleic acid, in which the pentasaccharide 'phosphate skeleton, which is the basic skeleton structure of nucleic acids, is replaced with a polyamide skeleton with glycine units. So-called LNA (Locked Nucleic Acid) is also included in the nucleic acid of the present invention.

[0083] The present invention also provides a method for producing a plant containing a foreign protein. In a preferred embodiment of the present invention, a plant having a DNA having a structure in which an exogenous gene is operably linked under the control of a DNA having a promoter activity of the present invention, or a vector containing a DNA having a promoter activity of the present invention is used. It is a method including the step of introducing into cells.

[0084] The present invention also provides a method for producing a foreign protein-producing plant characterized in that a foreign gene is expressed in a plant. In a preferred embodiment of the present invention, a plant cell comprising a DNA having a structure in which a foreign gene is operably linked under the control of a DNA having the promoter activity of the present invention, or a vector comprising the DNA having the promoter activity of the present invention. It is a method including the process of introducing into.

[0085] The present invention also provides a method for producing a foreign protein. In a preferred embodiment of the present invention, the transformed cell of the present invention is cultured, and encoded by a foreign gene expressed from the cell or its culture supernatant by a vector containing a DNA having the promoter activity of the present invention. The production method is characterized in that the protein to be collected is recovered.

[0086] A preferred embodiment of the method for producing a foreign protein of the present invention is, for example, a method comprising the following steps (a) and (b).

(a) A step of producing a plant containing a foreign protein by the method for producing a plant containing the foreign protein of the present invention or the method for producing a foreign protein producing plant.

(b) recovering foreign protein from the plant

[0087] The plant to be subjected to the method of the present invention is not particularly limited. A plant or a root vegetable plant is mentioned.

[0088] Examples of the "imo" include sweet potato, potato, cyssano, taro, yam, taro, nagaimo, yam, konnyakumo, and kikumo, but these are not limiting.

[0089] Examples of root vegetables include Japanese radish, Japanese radish, sugar beet (sugar beet), carrots, power, onions, shallots, burdock, lotus root, kwai, ginger, chorogi, garlic, Japanese sea bream, coral rust, and urine. Yes, but these are only examples and are not limited.

[0090] Examples of the plant in the present invention include plants having seeds, fruits, leaves, stems, tubers, roots, tuberous roots and the like. In the present invention, a plant having a tuberous root or tuber is preferred, a plant having a tuberous root is more preferred, and a sweet potato having a tuberous root is most preferred.

[0091] The DNA having promoter activity of the present invention controls gene expression in the roots of sweet potato, but exhibits the same structure as the above-mentioned plant body, preferably the root of sweet potato tubers. Even in a plant body, the DNA having promoter activity of the present invention is considered to have a function as a promoter.

[0092] The present invention also provides a plant obtained by the method of the present invention. Also provided is an artificially produced plant characterized by having a DNA having the promoter activity of the present invention and expressing a protein encoded by a foreign gene.

[0093] Such a plant is not particularly limited, but is preferably sweet potato.

[0094] Furthermore, the present invention provides a method for screening a compound that modulates the promoter activity of a DNA having the promoter activity of the present invention, comprising the following steps (a) to (c).

(a) a step of bringing a test compound into contact with a cell or cell extract containing DNA having a structure in which a reporter gene is functionally linked under the control of the DNA of the present invention

(b) measuring the expression level of the reporter gene

(c) selecting a compound that changes the expression level of the reporter gene

[0095] The test compound used in the screening method of the present invention is not particularly limited. For example, natural compounds, organic compounds, inorganic compounds, proteins, peptides, etc. are unified. Compound, and compound library, gene library expression product, cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract, prokaryotic cell extract, eukaryotic single cell extract Or an animal cell extract etc. can be mentioned.

[0096] In this screening method, first, a test compound is brought into contact with a cell or a cell extract containing DNA having a structure in which a reporter gene is functionally linked under the control of the promoter DNA of the present invention.

[0097] In the present invention, "functionally linked" means that the promoter of the present invention is such that expression of a reporter gene is induced by binding of a transcription factor to DNA having promoter activity of the present invention. It means that the active DNA and the reporter gene are bound. In the present screening method, “cells containing DNA having a structure in which a reporter gene is functionally linked under the control of DNA having promoter activity of the present invention”, for example, a cell into which a vector containing the above DNA has been introduced. Can be mentioned. The vector can be prepared by methods well known to those skilled in the art. Introduction of the vector into the cells can be carried out by a general method, for example, calcium phosphate precipitation method, electric pulse perforation method, ribofactoramine method, microinjection method and the like.

[0098] In addition, "a cell containing DNA having a structure in which a reporter gene is functionally linked under the control of DNA having promoter activity of the present invention" includes a cell into which the DNA is inserted into a chromosome. . DNA can be inserted into a chromosome by a method generally used by those skilled in the art, for example, a gene introduction method using homologous recombination.

[0099] The "cell extract containing DNA having a structure in which a reporter gene is functionally linked under the control of DNA having promoter activity of the present invention" in this method means, for example, commercially available in vitro transcription translation The cell extract contained in the kit may include those containing a DNA having a structure in which the DNA having the promoter activity of the present invention and a reporter gene are functionally linked.

[0100] In the present screening method, “contact” is performed by adding a test compound to a culture solution of cells containing DNA having a structure in which a reporter gene is functionally linked under the control of DNA having promoter activity of the present invention. Or by adding a test compound to the above-mentioned commercially available cell extract containing the DNA. The test compound is protein In some cases, for example, a vector containing DNA encoding the protein can be introduced into the cell, or the vector can be added to the cell extract.

[0101] In this screening method, the expression level of the reporter gene is then measured as follows. The expression level of a reporter gene can be measured by those skilled in the art in consideration of the type of the reporter gene.

[0102] In this screening method, when the test compound changes the expression level of the reporter gene as compared with the case where it is measured in the absence of the test compound (control), the test compound is the present invention. It is determined that the compound regulates the promoter activity of DNA.

[0103] Furthermore, in the present invention, the above screening method is used to evaluate whether or not the promoter activity of the DNA of the present invention is regulated for a plurality of test compounds, thereby regulating the promoter activity. By selecting a compound, a compound that efficiently modulates promoter activity can be screened. The compound obtained by the screening method is very useful because it can control the expression specific to the plant of the gene.

[0104] Furthermore, by introducing the DNA or vector of the present invention into a desired plant body, it is possible to induce the expression of a foreign gene in the plant body.

[0105] The "gene expression inducer" in the present invention is a substance containing the DNA or vector of the present invention as an active ingredient for the purpose of expressing a desired foreign gene in a plant. Refers to a composition (mixture).

[0106] In addition to the active ingredient DNA or vector, the drug of the present invention requires, for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, preservative, and the like. May be mixed accordingly.

[0107] The compound obtained by the screening method of the present invention is useful as a compound that modulates the promoter activity of a plant. By adding the compound to the plant, it is considered that the character and function of the plant body can be regulated at the cultivation site.

In addition, all prior art documents cited in this specification are incorporated herein by reference. Be incorporated.

Example

[0108] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Unless otherwise stated, methods required for general gene recombination, such as genomic DNA preparation, mRNA adjustment, DNA cleavage, ligation, E. coli transformation, and gene sequencing, are described in each operation. Instructions attached to the commercially available reagents and equipment used, and experimental documents (for example, “Molecular Cloning (Sambrook et al. (1989), Cold Spring Harbor Laboratory Press”) J .

[Example 1] Isolation of genes expressed in tuberous roots of sweet potato

The tuberous strength of two varieties of sweet potato (high line 14 and Ayamurasaki) was also used to create a cDNA library.

[0110] RNA extraction using phenol from sweet potato tubers was performed in The NSF potato genome project (〃RNA isolation using phenol protocol in the URL of http://www.tigr.org/tdb/potato/microarray_SO Ps. shtml) method was basically followed.

[0111] cDNA synthesis was performed using Stratagene's cDNA Synthesis Kit !, and cDNA size fractionation was performed using Amersham Bioscience's Size Sep400 Spun Columns. This was incorporated into a vector pBluescriptll-SK + (Stratagene) cleaved with restriction enzymes Xhol and EcoRI using Toyobo's Ligation High to complete a cDNA library.

[0112] Clones were randomly isolated from the cDNA library, and the nucleotide sequences of 2804 clones derived from High Line 14 and 3783 clones derived from Ayamurasaki were determined.

[0113] The nucleotide sequence was determined using PCR product Pre-Sequencing Kit (USB) and DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBA, and E (amersham pharmacia biotech), and using MegaBACElOOO.

[0114] Cluster analysis of these clones was performed, and clone IT394 that appeared relatively frequently was selected. IT394 appeared 4 times from the library derived from the high line 14 and 7 times from the library derived from Ayamurasaki. In the case of IT948, which is homologous to actin, it appeared 3 times from the library derived from high line 14 and 6 times from the library derived from Ayamurasaki.

[0115] SEQ ID NO: 1 shows the cDNA sequence of IT394. When a homology search of IT394 was performed, There was homology with mouth thionein.

[012] [Example 2] Acquisition of upstream region of gene IT394 expressed in tuberous root of sweet potato

In order to obtain the promoter region of IT394, the upstream region of IT394 genomic DNA was isolated by Clontech's BD GenomeWalker Kits. In this case, Takashi 14 and Ayamurasaki genomic DNA are used as the cage type, and two primers (394-R3; based on IT394 cDNA sequence (SEQ ID NO: 1)) are used as gene-specific primers. SEQ ID NO: 2, 394-R4; SEQ ID NO: 3) was synthesized. The amplified DNA fragment of about 0.9 kb was subjected to TA cloning with TOPO TA CIoning kit of invitrogen, and the base sequence of the isolated clone was determined.

[0117] As a result, a base sequence of about 0.8 kb upstream from the IT394 cDNA sequence (SEQ ID NO: 1) was successfully obtained. SEQ ID NO: 4 shows the sequence obtained by using the No. 14 genomic DNA as a cocoon type, and SEQ ID NO: 5 shows the sequence obtained as a genomic DNA cocoon from Ayamura Saki.

[Example 3] Preparation of promoter activity assay vector upstream of IT394

From the IT394 cDNA sequence (SEQ ID NO: 1), a long ORF is not detected, but the metagene of thioneine homologous to the IT394 cDNA sequence (eg RCMIT; accession number: L02306; IT394 and RCMIT (The ORF region has 43% homology) The translation start point of SEQ ID NO: 1 matches with position 54 of the sequence, so this is predicted as the translation start point, and the sequence upstream from this is a candidate for the IT394 promoter region As isolated.

[0119] Based on the upstream sequence (SEQ ID NO: 4) of IT394 cDNA obtained by using the genome DNA of high system No. 14 as a cage type! / And a pair of primers (394-F07s; SEQ ID NO: 6 , 394-Rb; SEQ ID NO: 7) was synthesized.

[0120] 394-F07S is a forward primer, has a restriction enzyme Sadl site (ccgcgg) on the 5 'end side, and introduces a mutation (gaCctc) into the Sad site (gaGctc) on the genome sequence. 394-Rb is a reverse primer and has a restriction enzyme BamHI site (ggatcc) on the 5 ′ end side. Using this pair of primers, PCR was carried out using the type 14 genomic DNA as a saddle and an amplified DNA fragment of approximately 730 bp was obtained. The amplified DNA fragment was subjected to TA cloning with the in vitro TOPO TA Cloning kit (pSP53), and the base sequence of the isolated clone was determined (SEQ ID NO: 8).

[0121] Next, the vector pBluescript SK (Stratagene) emits strong green light in plants. Fluorescent protein mutant sGFP (S65T) (Yasuo Niwa: Plant Cell Engineering Series 4, Model Plant Experiment Protocol, PPH7-121, Shujunsha, 1996) and pblue-sGFP, a plasmid incorporating Nos terminator ( S65T) -NOS SK and plasmid pSP53 were digested with BamHI and SacII, respectively, and ligated using Toyobo's Ligation High to produce plasmid pHM160.

[0122] This plasmid was further cleaved with Sad and Kpnl, and a sequence of one upstream of IT394-GFP-Nos terminator was inserted into the multicloning site of binary vector pPZP2H-lac. A vector PHM161 that can verify the promoter activity in the plant upstream of the GFP gene as a reporter was completed (Fig. 1).

[Example 4] Preparation of transformed plant and detection of GFP

The prepared vector PHM161 was introduced into Agrobacterium tumefaciens EHA101 by freeze-thawing. Freezing and thawing refers to a method in which a plasmid solution is placed in a frozen EHA101 competent cell and incubated at 37 ° C for 5 minutes.

[0124] Genes were introduced into Arabidopsis thaliana (variety: Columbia) and sweet potato (breed: high line 14) using a strain into which the vector pHM161 was introduced.

[0125] Arabidopsis thaliana was introduced by the method described in “Transformation by reduced pressure infiltration method (Takashi Araki)” (Plant cytology series 4, Experimental protocol for model plants, ppl09-113, Shujunsha, 1996). Followed the basics. However, the described decompression process was omitted.

[0126] Introduction into sweet potato was performed according to the method described in Otani et al. Plant Biotechnology, 15: 11-16, 1998.

[Example 5] Detection of GFP in transformed plants

A blue LED spotlight (MeCan Imaging, MC-L12B) is equipped with an Ex filter for GFP (Asahi halo) and irradiated to the plant as excitation light. The fluorescence emitted is stereo microscope (OLYMPU)

S, SZX12), or a digital camera (OLYMPUS, E-10) with a GFP Ba filter (Asahi spectroscopy) was used for observation.

[0128] In sweet potato tuberous roots, a much stronger expression was detected than the CaMV35S promoter, which is normally used as a strong promoter. The base sequence of 730 bp upstream of IT394 was shown to function as a promoter to express a large amount of foreign genes in sweet potato tuberous roots. Moreover, since it was expressed in Arabidopsis thaliana, it could be used as a root promoter in other plant species.

[Example 6] Transient expression analysis in leaves

A plasmid in which GFP or IbMYBl was linked to the IT394 promoter or 35S promoter was simultaneously injected into the leaves of a sweet potato with a particle gun. Plasmid construction and particle gun method (Mano H, Ogasawara F, Sato K, Higo H, Minobe Y (2 007) Isolation of a regulatory gene of anthocyanin biosynthesis in tuberous roots of purple-fleshed sweet potato.Plant Physiol. 143: 1252-1268).

[0130] As a result, the percentage of cells in which GFP fluorescence was observed over a long period of time was greater when the IT394 promoter was used than when the 35S promoter was used. For example, in the IT394 promoter, fluorescence was observed in 80% of cells after 3 days and in half of the cells after 1 week, but in the 35S promoter, fluorescence was observed in only about 10% of cells after 3 days and after 1 week. . In addition, when the transcription factor IbMYBl that controls anthocyanin synthesis is expressed, anthocyanin accumulation is observed in about 80% of cells when the IT3 94 promoter is used. About half of the cells are observed when the 35S promoter is used. Only anthocyanin accumulation was observed.

[0131] From this, it was shown in the transient expression analysis that the IT394 promoter has the ability to continuously express the introduced gene over a longer period than the 35S promoter.

[0132] The need for long-term expression is not necessarily high when the introduced gene can be directly detected, such as reporter genes such as GFP, GUS, and LUC. However, for example, since a transcription factor manifests itself by inducing the expression of another gene, the need to continuously express the transgene for a longer period of time becomes higher. In fact, in an experiment in which anthocyanin synthesis system genes were introduced by introducing anthocyanin transcription factors and as a result the accumulation of anthocyanins was observed (Fig. 4) Coloring was observed in more than 20% of the transgenic cells, whereas in the case of 35S, about half of the cells were force-colored. In addition to this, for example, when analyzing genes that induce morphological changes of cells such as guard cells and root hair formation, it is considered that the need for continuous expression of the transgene is further increased. Therefore, if IT394 probe motor is used for transient expression analysis, it will be possible to analyze genes that could not be analyzed with 35S promoter.

Industrial applicability

[0133] The present inventors have succeeded in isolating a promoter expressed in the roots of plants such as sweet potato tuberous roots. By using the promoter, it is possible to efficiently produce useful substances such as pharmaceutical raw materials and industrial raw materials in plants containing sweet potato tuberous roots. In particular, by expressing appropriate genes in the roots of plants, they can grow on nematode-resistant, disease-resistant, water-resistant, drought-tolerant plants, soils lacking specific nutrients, and excessive soils. New plants may be created. It can also be used for phytoremediation and soil purification by creating plants that can absorb harmful substances efficiently. Alternatively, the range of applications by expressing foreign genes in plants, such as nutritional modification of the plants and production of raw materials such as pharmaceuticals and biodegradable plastics, is wide and useful.

[0134] Further, by modifying the components of the plant body, it is possible to produce a plant body having a good taste and a plant body containing a component good for the body. For example, in sweet potatoes, it is possible to produce delicious sweet potatoes and healthy sweet potatoes by modifying the components of tuberous roots.

Claims

The scope of the claims

[I] A DNA having promoter activity described in any one of (a) to (c) below.

(a) DNA having the nucleotide sequence of SEQ ID NO: 8

(c) DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence of SEQ ID NO: 8

[2] The DNA _{0 according} to claim 1, which has promoter activity in the roots of the plant.

3. The DNA according to claim 2, wherein the root is a tuber root of sweet potato.

[4] A DNA having a structure in which a foreign gene is operably linked under the control of the DNA according to any one of claims 1 to 3.

[5] A vector comprising the DNA according to any one of claims 1 to 4!

[6] A transformed cell comprising the DNA according to any one of claims 1 to 4 or the vector according to claim 5.

[7] The transformed cell according to claim 6, which is a microorganism.

[8] The transformed cell according to claim 6, which is a plant cell.

[9] A transformed plant comprising the cell according to claim 8.

[10] The transformed ^ ¾ object which is a descendant or clone of the transformed ^ ¾ object according to claim 9.

[II] The propagation material for the transformed plant according to claim 9 or 10.

[12] A method for producing a transformed plant comprising the steps of introducing the DNA according to claim 4 or the vector according to claim 5 into a plant cell and regenerating the plant from the plant cell.

[13] A method for expressing a foreign gene in a plant, comprising the step of introducing the DNA according to claim 4 or the vector according to claim 5 into cells of the plant.

[14] A method for producing a plant containing a foreign protein, comprising the step of introducing the DNA of claim 4 or the vector of claim 5 into a plant cell.

[15] A foreign protein-producing plant characterized in that a foreign gene is expressed in a plant. A method for producing an object, the method comprising introducing the DNA according to claim 4 or the vector according to claim 5 into a plant cell.

[16] The transformed cell according to any one of claims 6 to 8 is cultured, and the protein encoded by the foreign gene expressed by the vector according to claim 5 is recovered from the cell or a culture supernatant thereof. A method for producing a foreign protein, characterized by comprising:

[17] A method for producing a foreign protein, comprising the following steps (a) and (b):

(a) A step of producing a plant containing a foreign protein by the method according to claim 14 or 15.

(b) recovering foreign protein from the plant

[18] The method according to any one of [13] to [15] and [17], which is the plant strength potato or root vegetable.

[19] The method according to any one of claims 13 to 15 and 17, wherein the plant is a plant having tuberous roots or tubers.

20. The method according to claim 19, wherein the tuberous root is a sweet potato tuber.

[21] A plant obtained by the method according to claim 14 or 15.

[22] An artificially produced plant comprising the DNA of claim 4 and expressing a protein encoded by a foreign gene.

[23] The plant according to claim 21 or 22, wherein the plant is sweet potato.

[24] The method for screening a compound that modulates one activity of the promoter of DNA according to any one of claims 1 to 3, comprising the following steps (a) to (c):

(a) a step of bringing a test compound into contact with a cell or cell extract containing DNA having a structure in which a reporter gene is functionally bound under the control of the DNA according to any one of claims 1 to 3;

(b) measuring the expression level of the reporter gene

(c) selecting a compound that changes the expression level of the reporter gene

[25] A DNA agent used as a promoter for expressing a foreign gene in a plant, wherein the DNA according to any one of claims 1 to 4 or the vector according to claim 5 is effective. Gene expression inducer as a component.