JP2000083679A - Plant Promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new plant promoter which has a specific base sequence, is compact, and has a high transcription activity to express, at a higher level, a desired gene in a specific tissue than other tissue in a host such as a plant. SOLUTION: This is a new plant promoter which has at least base 112-246 in a base sequence shown by formula I, or at least base 186-282 in a base sequence shown by formula II, and a base sequence shown by formula III, and is useful for expressing, at a higher level, a desired gene in a specific tissue than other tissue in a host such as a plant. This promoter is obtained by linking DNA having a base sequence, which is shown by formula I, prepared by PCR using primers complementary to partial sequences of a base sequence shown by formula I and using carrot genomic DNA as a template and so on with DNA having a base sequence shown by formula III so as to be able to function in vivo.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a plant promoter.

[0002]

2. Description of the Related Art In order to express a desired gene in a host cell in order to modify characteristics of a host organism such as a plant, the gene is placed under the control of a promoter operable in the host cell. When it is necessary to express the target gene in a specific tissue of the host organism higher than other tissues depending on the purpose of the trait modification and the nature of the gene to be expressed, the specific tissue is more transcribed than the other tissues. A highly active promoter is used.

[0003]

In general, such a promoter, when its chain length is long, is likely to be affected by various factors in a host cell or to cause recombination or deletion when integrated into a chromosome. And when introduced into host cells, problems such as not achieving the desired trait expression are caused. Therefore, a compact promoter for expressing a desired gene at a higher level in a specific tissue of a host organism than in other tissues has been desired.

[0004]

Under these circumstances, the present inventors have conducted intensive studies and as a result, have found a specific base sequence suitable for the above-mentioned purpose, and have completed the present invention. That is, the present invention provides: 1) a promoter characterized by having the following nucleotide sequence (a) or (b) and the nucleotide sequence represented by SEQ ID NO: 1 (hereinafter referred to as the promoter of the present invention); A) a base sequence having at least the base sequences of base sequences 112 to 246 of the base sequence represented by SEQ ID NO: 2; (b) a nucleotide sequence having at least the nucleotide sequences of nucleotide numbers 186 to 282 of the nucleotide sequence of SEQ ID NO: 3; 2) a promoter having the following nucleotide sequence (a) or (b), and a nucleotide sequence represented by nucleotide sequence 1; (a) a nucleotide sequence 112 to
246, any one of the nucleotide sequences represented by the nucleotide sequences 54 to 246 or the nucleotide sequences 1 to 246. (b) of the nucleotide sequence represented by SEQ ID NO: 3
282, any one of the nucleotide sequences represented by nucleotide sequences 127 to 282 or nucleotide sequences 1 to 282; 3) The promoter according to the above 1) to 2), which has two or more nucleotide sequences represented by SEQ ID NO: 1) 4) The promoter according to the above 3), which has 4 or 8 nucleotide sequences represented by SEQ ID NO: 5) 5) The above 1) ) To 4) and a chimeric gene having a desired gene (hereinafter, referred to as the chimeric gene of the present invention), and 6) a vector having the promoter described in 1) to 4) above (hereinafter, referred to as the vector of the present invention). ), 7) A transformant in which the promoter according to the above 1) to 4), the chimeric gene according to the above 5) or the vector according to the above 6) is introduced into a host cell (hereinafter referred to as the transformant of the present invention). 8) A gene expression method for expressing a desired gene in a host cell under the control of the promoter according to 1) to 4) above, 9) D having the following nucleotide sequence (a) or (b): A
And a DNA having the nucleotide sequence of SEQ ID NO: 1 and a method for preparing a promoter for operably connecting the DNA in a host cell; (a) at least the nucleotide sequence of 112 to 246 of the nucleotide sequence of SEQ ID NO: 2 A base sequence having a base sequence represented by: (b) a nucleotide sequence having at least the nucleotide sequences of nucleotide numbers 186 to 282 of the nucleotide sequence of SEQ ID NO: 3; 10) DNA having the following base sequence (a) or (b)
And a method for preparing a promoter that connects the DNA having the nucleotide sequence of SEQ ID NO: 1 in a form operable in a host cell. (A) At least the nucleotide sequence 112 to 246 of the nucleotide sequence of SEQ ID NO: 2 or Nucleotide sequence 54 to 246 or nucleotide sequence 1
Any of the base sequences represented by the base sequence represented by -246; (b) any one of the nucleotide sequences represented by at least the nucleotide sequences 186 to 282 or the nucleotide sequences 127 to 282 or the nucleotide sequences represented by the nucleotide sequences 1 to 282 in the nucleotide sequence represented by SEQ ID NO: 3. Is provided.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. The genetic engineering technology used here is, for example, J., Sambrook, E., F., Frisch, T., Maniatis,
Molecular Clonin 2nd edition (Molecular Clonin
g 2 ^nd edition), Cold Spring Harbor
Published by Laboratory (Cold Spring Harbor Laboratory pr.)
ess), 1989 and D., M., Glover, DNA Cloni.
ng, IRL issuance, 1985, and the like.

In the promoter of the present invention, “(a) at least the base number of the base sequence represented by SEQ ID NO: 2
A nucleotide sequence having a nucleotide sequence represented by 112 to 246 (hereinafter, referred to as
Described as base sequence A. )) Is, for example, SEQ ID NO: 2
And the base sequences represented by base numbers 112 to 246, the base sequences represented by base numbers 54 to 246, the base sequences represented by base numbers 1 to 246, and the like.
Examples of “(b) a base sequence having a base sequence represented by at least base numbers 186 to 282 of the base sequence represented by SEQ ID NO: 3 (hereinafter referred to as base sequence B)” include, for example, SEQ ID NO: Nucleotide numbers 186 to 2 of the nucleotide sequence represented by 3
The base sequence represented by 82, the base sequence represented by base numbers 127 to 282, the base sequence represented by base numbers 1 to 282, and the like can be given. Usually, the base sequence represented by SEQ ID NO: 1 is preferably located at the 5 ′ upstream side of the base sequence A or the base sequence B, and the base sequence represented by SEQ ID NO: 1 and the base sequence A or the base sequence B The shorter the distance, the shorter the length of the promoter becomes. The promoter of the present invention has at least one nucleotide sequence represented by SEQ ID NO: 1, and preferably has two or more nucleotide sequences.
For example, a promoter having 4 or 8 nucleotide sequences represented by nucleotide sequence number 1 can be mentioned. Although another base sequence may be interposed between the base sequences represented by SEQ ID NO: 1, the base sequence is continuously connected since the intervening other base sequence increases the chain length of the promoter. It is preferred that

The promoter of the present invention is prepared by separately preparing an oligonucleotide having the nucleotide sequence A or the nucleotide sequence B and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 in accordance with a chemical synthesis method. Can be produced by annealing the oligonucleotides in a test tube and binding them by, for example, a method using DNA ligase or the like. Specifically, for example,
An oligonucleotide having a base sequence represented by SEQ ID NO: 6 (+ strand) and its complementary strand having a base sequence represented by SEQ ID NO: 7 (-strand) are chemically synthesized and annealed in a test tube. By doing
A DNA fragment having the nucleotide sequence represented by nucleotide numbers 112 to 246 of the nucleotide sequence represented by SEQ ID NO: 2 can be prepared. On the other hand, for example, an oligonucleotide (+ strand) having the base sequence shown in SEQ ID NO: 4 and an oligonucleotide (-strand) having the base sequence shown in SEQ ID NO: 5, which is a complementary strand thereof, are chemically synthesized and tested. By annealing in a tube, a DNA fragment having a base sequence in which the base sequence of SEQ ID NO: 1 is continuously connected four times can be prepared. The base sequence represented by SEQ ID NO: 1 is 4 times continuous on the 5 ′ upstream side of the DNA fragment having the base sequence represented by base numbers 112 to 246 of the base sequence represented by SEQ ID NO: 2 thus obtained. The promoter of the present invention can be prepared by ligating a DNA fragment having a base sequence that is connected to each other using T4 DNA ligase (Takara Shuzo). Similarly, SEQ ID NO: 1
An oligonucleotide (+ strand) having a base sequence in which eight base sequences are continuously connected and its complementary strand (-
By chemically synthesizing the base sequence and annealing it in a test tube, a DNA fragment having a base sequence in which the base sequence represented by SEQ ID NO: 1 is continuously connected eight times can be prepared. SEQ ID NO: 2 obtained as described above
A DNA having a base sequence in which the base sequence represented by SEQ ID NO: 1 is continuously connected 8 times to the 5 ′ upstream side of the DNA fragment having the base sequence represented by base numbers 112 to 246 of the base sequence represented by The promoter of the present invention can be prepared by ligating the fragments with T4 DNA ligase (Takara Shuzo). As another method for producing the promoter of the present invention, for example, a gene containing a target DNA fragment is cloned from a tissue or a cell of an organism, and a DNA fragment having a base sequence A or a base sequence B is obtained from the obtained gene. Alternatively, a DNA fragment having the nucleotide sequence represented by SEQ ID NO: 1 may be cut out using a restriction enzyme, and the resulting DNA fragment may be bound as necessary, for example, using a method using DNA ligase or the like. it can. Further, the DNA fragment having the base sequence A or the base sequence B or the DNA fragment having the base sequence shown in SEQ ID NO: 1 can be separately or simultaneously amplified using the gene or the like cloned as described above as a template. PCR using primers (polymerase chain
Reaction) to amplify the DNA fragment, and the obtained DNA fragment is, if necessary,
The promoter of the present invention can also be prepared by binding using a method using A ligase or the like. Specifically, for example, an oligonucleotide having a base sequence represented by base numbers 1 to 20 of the base sequence represented by SEQ ID NO: 2, and a base number represented by SEQ ID NO: 2
A DNA fragment comprising the nucleotide sequence represented by SEQ ID NO: 2 by performing PCR using an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by 226 to 246 as a primer and genomic DNA of carrot as a template Can be obtained. Similarly, an oligonucleotide having a base sequence represented by base numbers 1 to 20 of the base sequence represented by SEQ ID NO: 3, and a base represented by base numbers 262 to 282 of the base sequence represented by SEQ ID NO: 3 Using an oligonucleotide having a base sequence complementary to the sequence as a primer and performing PCR using soybean genomic DNA as a template, a DNA consisting of the base sequence represented by SEQ ID NO: 3
Fragments can be obtained. The DN thus obtained
The promoter of the present invention can be prepared by chemically synthesizing the A fragment as described above and binding it to a DNA fragment having the base sequence represented by SEQ ID NO: 1 prepared by annealing in a test tube. Furthermore, the DNA containing the promoter of the present invention produced as described above
Is used as a template, and PCR is performed using a primer containing the nucleotide sequence of SEQ ID NO: 1, whereby the promoter of the present invention having a different number of nucleotide sequences of SEQ ID NO: 1 can be prepared. For example, using a plasmid pGCRG1-1 or pGCRG1-2 containing the promoter of the present invention as a template, a nucleotide sequence comprising a nucleotide sequence in which the nucleotide sequence represented by SEQ ID NO: 1 is connected two or more times, for example, eight times consecutively ( An oligonucleotide having SEQ ID NO: 18 or SEQ ID NO: 19) and a promoter of the present invention in a template plasmid.
Using a primer having a nucleotide sequence complementary to a region located on the 3 ′ downstream side, for example, an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 20 as a primer
By performing PCR, the plasmid pGCRG1-
It is possible to prepare a promoter of the present invention in which the number of base sequences represented by SEQ ID NO: 1 is greater than that of the promoter of the present invention contained in 1 or pGCRG1-2.

By using the promoter of the present invention thus obtained, a desired gene can be expressed in a host cell. In this case, the chimeric gene of the present invention having the promoter of the present invention and the desired gene may be used. Here, the “desired gene” is a gene that is desired to be expressed in a host cell, and includes, for example, a gene encoding a protein such as an enzyme, a storage protein, a receptor, a transcriptional regulator, or a signal transduction factor. These genes may be ligated downstream of the promoter of the present invention in a sense direction or an antisense direction depending on the purpose.
The chimeric gene of the present invention may contain a terminator that can function in a host cell. Examples of a terminator that can function in a host cell include a terminator that exhibits a transcription termination activity in a transformed host cell.
When the host cell is a plant cell, a terminator (NOS) of a nopaline synthase gene derived from Ti-plasmid of Agrobacterium genus bacteria, garlic virus GV1, GV2
And other terminators derived from plant viruses. Usually, the chimeric gene of the present invention is preferably formed by operably linking the promoter of the present invention and a desired gene. Here, "in a operable form" means that when the chimeric gene of the present invention is introduced and a host cell is transformed, the target gene contained in the chimeric gene is
It means that it is in a state linked to the promoter so as to be expressed under the control of the promoter of the present invention.

[0009] In the vector having the promoter of the present invention, the vector is a DNA that can be propagated in a host cell, and is a plasmid, phage, or phage that can be amplified in biological cells such as Escherichia coli, yeast, plant cells, and animal cells. Mid and the like. Specifically, for example, pUC plasmids [pUC118, pUC119 (Takara Shuzo) etc.], pSC101 plasmids, Ti-plasmids [pBI101, pBI121 (CLONTECH) etc.], Bluescript phage mid [pBluescript
SK (+/-) (STRATAGENE), M13 phage [mp10, mp1
1 (Amersham), λ phage [λgt10, gt11 (Amersham
ham)), cosmids [SuperCosI (STRATAGENE)]
And the like, and by incorporating the promoter of the present invention into such a vector using ordinary genetic engineering techniques, a vector having the promoter of the present invention can be constructed.

[0010] If the vector having the promoter of the present invention further has a gene insertion site downstream of the promoter and a terminator operable in the host cell, it is preferable for constructing a vector for expressing a desired gene in the host cell. Available. Here, the “gene insertion site” is a nucleotide sequence that can be specifically recognized and cleaved by a restriction enzyme usually used in genetic engineering techniques, and is preferably a type of restriction enzyme recognition sequence that exists only on a vector.
Such a gene insertion site, the promoter of the present invention, and a terminator operable in a host cell can be obtained by inserting the promoter of the present invention, the desired gene and the host cell into a vector when the desired gene is inserted into the gene insertion site. It is preferred that the terminator be operable in a position such that it is operatively connected. To construct such a vector, for example, a plasmid containing a gene insertion site and a terminator operable in a host cell, specifically, a multicloning site (gene insertion site) such as pBI101.3 (manufactured by CLONTECH) is used. A DNA fragment having the nucleotide sequence represented by SEQ ID NO: 1, and at least a nucleotide sequence represented by nucleotide numbers 112 to 246 or a nucleotide sequence represented by SEQ ID NO: 3 in the nucleotide sequence represented by SEQ ID NO: 2 What is necessary is just to insert the DNA fragment which has the base sequence represented by base number 186-282. Further, a vector having a gene insertion site, specifically, pBIN19 (Nuc.Acid.Res.
12: 8711-8721 (1984)), a promoter of the present invention and a terminator operable in a host cell may be inserted into a multicloning site (gene insertion site).

[0011] The vector having the chimeric gene of the present invention is
Suitable for introducing the chimeric gene into a host cell, for example, cloning the chimeric gene into a vector as described above, or having a promoter of the present invention, a gene insertion site and a terminator operable in the host cell. Such a vector can be prepared by cloning a desired gene into the gene insertion site. For example, a reporter gene (β-glucuronidase gene; hereinafter referred to as GUS) present on pBI101.3 (manufactured by CLONTECH) by cleaving the above-described vector with an appropriate restriction enzyme by using an appropriate restriction enzyme. By removing the gene of interest and inserting the desired gene in place of the reporter gene, a vector having the chimeric gene of the present invention can be prepared.

The vector of the present invention is prepared, for example, by the method described in J., Sambrook,
E., F., Frisch, T., Maniatis, Calcium chloride method described in Molecular Cloning Second Edition (1989) (published by Cold Spring Harbor Laboratory),
It can be introduced into microbial cells such as Escherichia coli and Agrobacterium by electroporation or the like, and such microbial cells into which the vector of the present invention has been introduced can be used for preparing the chimeric gene of the present invention or other hosts of the chimeric gene of the present invention. It is useful for introduction into cells. Further, the promoter of the present invention can be introduced into a plant-derived host cell by a particle gun method or the like. Further, the vector of the present invention can also be introduced into a plant-derived host cell by a known method such as an Agrobacterium infection method, an electric transduction method, or a particle gun method. Among the host organisms capable of introducing the promoter of the present invention, the chimeric gene of the present invention, or the vector of the present invention and expressing the desired gene in the cell under the control of the promoter of the present invention, for example, as a plant, for example, Monocotyledonous plants such as rice, corn, barley, wheat, legumes such as soybean, peas, kidney beans, alfalfa, solanaceous plants such as tobacco, tomato, potato, cruciferous plants such as cabbage, rapeseed and mustard, melon , Pumpkin, cucumber and the like, Cucurbitaceae plants such as carrots and celery, and dicotyledonous plants such as asteraceous plants such as lettuce.

[0013] By introducing the promoter of the present invention, the chimeric gene of the present invention or the vector of the present invention into a host cell, for example, the promoter of the present invention is inserted upstream of a desired gene on genomic DNA, and the gene is replaced with the promoter of the present invention. A transformant expressing under control, a transformant in which the chimeric gene of the present invention is inserted into genomic DNA and expressing a desired gene contained in the chimeric gene under control of the promoter of the present invention, and a vector of the present invention in a host cell. And a transformant expressing the desired gene contained in the vector under the control of the promoter of the present invention. A transformant in which the host cell is a plant cell is, for example, SBGelvi
n, by RASchilperoot and DPSVerma: Plant Molecular Biology Manual (Plant Molecu
lar Biology Manual, Kluwer Academic Publishers pre
ss (1988)), Isao Shimamoto, Kiyotaka Okada: Experimental protocol for model plants (rice, Arabidopsis) (Shujunsha)
(ISBN4-87962-157-9 C3345, 1996) pages 78-143 or by Hirofumi Uchimiya (Plant Gene Manipulation Manual, How to Make Transgenic Plants (Kodansha Scientific)) 19
90, ISBN4-06-153513-7 C3045) By redifferentiating in accordance with the method used in ordinary plant tissue culture techniques described on pages 28 to 33, a plant derived from the plant cell or a part thereof Can be obtained. Further, by cultivating and self-cultivating the plant obtained as described above, progeny of the plant can be obtained. In the present invention,
"Transformants" include such plant cells, plants and progeny thereof.

By linking a desired gene in the sense direction under the control of the promoter of the present invention and expressing it in the cells of the host organism, useful traits can be imparted to the host organism. For example, by expressing a seed storage protein gene such as a soybean glycinin gene or a β-conglycinin gene, the protein content or essential amino acid content in feed crops can be increased, and a Brazil nut 2S albumin gene, a corn γ-zein gene, etc. By expressing a gene encoding a methionine or cysteine-rich storage protein, etc., the methionine or cysteine content of feed crops can be increased, and bioA, bioB, bioC, b derived from microorganisms such as Escherichia coli.
Expression of biotin biosynthesis-related enzyme genes such as the ioD, bioF, and bioH enzyme genes can increase the biotin content in feed crops, and can enhance the α-1.4-glucan branching enzyme of rice. )), The starch component in rice seeds can be modified by expressing an amylopectin synthesis-related enzyme gene or the like,
Stearoyl of soybeans and rapeseed-ACP-desaturase (St
earoyl-ACP-desaturase gene, acyl-ACP-thioesterase (Acyl-ACP-thioesterase) gene, coconut 1-acylglycerol-3-phosphate acyltransferase (1-acylglycerol-3-phosphate acyl trans)
ferase) gene and the like can improve the oxidative stability of lipids in food crops, reduce phospholipids and improve lipid components by increasing oleic acid and linolenic acid, and glycerol-3- such as spinach. Phosphate acyltransferase (glycerol-3-phos
By expressing a phate acyltransferase) gene or the like, the unsaturated fatty acid content can be increased and the low temperature tolerance of the plant can be increased. On the other hand, by linking the desired gene in the antisense direction under the control of the promoter of the present invention and expressing it in the cells of the host organism, useful traits can be imparted to the host organism. For example, by expressing an antisense gene of an amylopectin degrading enzyme gene such as rice isomerase (Isomerase), the starch component in rice seeds can be improved, and pumpkin and the like can be improved.
By expressing an antisense gene of an ethylene synthase gene such as 1-aminocyclopropane-1-carboxylate (ACC) synthase, it is possible to improve the preservability of fruits, flowers and the like. By expressing the antisense gene of the tulonase gene, the preservability of the fruit can be improved. Furthermore, S-locus-type specific RNas is involved in self-incompatibility of plants
The fertility of a plant can also be controlled by expressing a sense or antisense gene of a male sterility-related gene such as the e gene.

[0015]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 (Construction of GUS Expression Plasmid) After digesting 2 μg of plasmid Gbox10 / -90 / GUS (described in JP-A-9-131187) with restriction enzymes XbaI and BamHI, the digested fragment was subjected to 0.8% agarose gel electrophoresis. After separation, a DNA fragment of about 12 kb (hereinafter referred to as G10-Xb-B fragment) was obtained using a DNA purification kit (Prep-A-Gene DNA Purificatio).
n Systems, manufactured by BIO-RAD). Similarly, plasmid-90 / GUS (JP-A-9-131187)
After digesting 2 μg with restriction enzymes XbaI and BamHI, the digestion fragment was separated by 0.8% agarose gel electrophoresis, and a DNA fragment of about 12 kb (hereinafter referred to as Xb-B fragment) was isolated and purified. did. On the other hand, an oligonucleotide having a base sequence represented by any one of SEQ ID NOS:
ied Biosystems). Plasmid pCR16G
Using 1 / Xb (described in Japanese Patent Application No. 8-212680) as a template, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 8 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 9 were used as primers. PCR was performed, and a DNA fragment of 150 nucleotides having a nucleotide sequence represented by nucleotide numbers 112 to 246 of the nucleotide sequence represented by SEQ ID NO: 2 (hereinafter, CR
Described as 16.1 fragment. ) Was prepared. Similarly, PCR was performed using an oligonucleotide having the base sequence of SEQ ID NO: 8 and an oligonucleotide having the base sequence of SEQ ID NO: 10 as primers, and the base of the base sequence of SEQ ID NO: 2 A DNA fragment of 208 bases (hereinafter referred to as CR16.2 fragment) having the base sequence represented by any of SEQ ID NOs: 54 to 246 was prepared, and an oligonucleotide having the base sequence represented by SEQ ID NO: 8 and a base sequence represented by SEQ ID NO: 11 Using oligonucleotides with base sequences
Perform PCR, and have a base sequence represented by SEQ ID NO: 268
A base DNA fragment (hereinafter, referred to as CR16.3 fragment) was prepared. On the other hand, plasmid pGY1 (Iida et al. Plant Cell R
eport 14: 539-544 (1995)) as a template, PCR was performed using, as primers, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 12 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 13, A DNA fragment of 110 bases having a base sequence represented by base numbers 186 to 282 of the base sequence represented by base number 3 (hereinafter referred to as a GY1.1 fragment) was prepared. Similarly, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 12 and SEQ ID NO: 14
PCR was performed using an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 3 as a primer, and a 170-base DNA fragment having the nucleotide sequence represented by nucleotide numbers 127 to 282 of the nucleotide sequence represented by SEQ ID NO: 3 (hereinafter, referred to as GY1.2 fragment) was prepared, and PCR was performed using an oligonucleotide having the nucleotide sequence of SEQ ID NO: 12 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 15 as primers to obtain SEQ ID NO: 3. A DNA fragment of 296 bases (hereinafter, referred to as a GY1.3 fragment) having a base sequence represented by the following formula was prepared. In each of the above PCRs, 10 mM Tris-HCl pH 8.0, 50 mM KCl, 0.2 mM using TAKARA Taq polymerase (Takara Shuzo).
A reaction mixture of dNTP mixed solution (each containing 0.2 mM dATP, dGTP, dCTP, dTTP), each 0.5 μM primer, 0.1 μg template DNA, 2.5 units TAKARA Taq polymerase (Takara Shuzo) at 94 ° C. for 1 minute Then at 55 ° C for 2 minutes, then 7
A total of 30 cycles were performed with one cycle of keeping the temperature at 2 ° C. for 3 minutes. 1 μg of each amplified DNA fragment was mixed with restriction enzymes XbaI and BamHI.
Digested. 0.2 μg of each DNA fragment thus obtained
Was mixed with 2 μg of the above-mentioned G10-Xb-B fragment or 2 μg of the Xb-B fragment, ligated using a ligation kit version 2 (Takara Shuzo), and introduced into Escherichia coli strain HB101 (Takara Shuzo). CR16.1 fragment, CR16.2 fragment, or CR1
Plasmids pGCRG1-1, pGCRG1-2, or pGCRG1-3 were constructed by ligating each of the 6.3 fragments with the G10-Xb-B fragment (FIG. 1). GY1.1 fragment, GY1.2 fragment, or GY1.3
Plasmids pGGY1-1, pGGY1-2, or pGGY1-3 were constructed by ligating each fragment with the G10-Xb-B fragment (FIG. 2). By ligating the CR16.1 fragment, CR16.2 fragment, or CR16.3 fragment with the Xb-B fragment, respectively,
pCRG1-1, pCRG1-2, or pCRG1-3 were constructed (FIG. 3). G
The Y1.1 fragment, the GY1.2 fragment, or the GY1.3 fragment was converted to Xb-B
Plasmids pGY1-1, pGY1-2,
Alternatively, pGY1-3 was constructed (FIG. 4). Furthermore, PCR was performed using the above plasmid pCR16G1-1 as a template, and an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 18 and an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 20 as primers. DNA fragments were prepared. Similarly, using pCR16G1-2 as a template, SEQ ID NO: 19
PCR was performed using an oligonucleotide having a base sequence represented by and a oligonucleotide having a base sequence represented by SEQ ID NO: 20 as primers, and a D
NA fragments were prepared. Each DNA fragment thus obtained was digested with restriction enzymes HindIII and SnaBI. On the other hand, plasmid pBI101.1 (manufactured by CLONTECH) was replaced with restriction enzymes HindIII and SnaBI.
And the PCR-amplified fragment (about 630 bp or about 690 bp) digested with HindIII and SnaBI
By ligating using polymerase (Takara Shuzo), pG8CRG1-1 and pG8CRG1-2 were constructed (FIG. 5).

Example 2 (Plasmid DNA for Gene Transfer)
Preparation of plasmids pGCRG1-1, pGCRG1-2, pGRG obtained in Example 1
CRG1-3, pG8CRG1-1, pG8CRG1-2, pGGY1-1, pGGY1-2, pG
E. coli HB101 strain containing GY1-3, pCRG1-1, pCRG1-2, pCRG1-3, pGY1-1, pGY1-2, or pGY1-3 is inoculated and cultured in 200 ml of L medium containing 50 μg / ml kanamycin, respectively. Then, each plasmid DNA was purified using a commercially available plasmid purification kit (QIAGEN).

Example 3 (Preparation of Transformant by Indirect Transfer Method) Each of the plasmid DNAs purified in Example 2 was
Agrobacterium tumefaciens LBA4 rendered competent by treatment with CaCl ₂
404) (indicating streptomycin resistance and rifampicin resistance) (Hoekma et al. Nature, 303: 179-180 (198
3)) was introduced by heat treatment (37 ° C., 5 minutes). The introduced Agrobacterium is transformed into streptomycin.
300 μg / ml, rifampicin 100 μg / ml, kanamycin 25
By culturing on an L agar medium containing μg / ml, the NPTII gene on the plasmid (Trien-Cuot et al., Gene 2
3: 331-341 (1983)). Transformants were selected using the property of kanamycin resistance conferred by the method. The resulting transformant of Agrobacterium was transformed into streptomycin 30.
0 μg / ml, rifampicin 100 μg / ml, kanamycin 25 μ
g / ml in L medium at 28 ° C for 24 hours, and use the obtained bacterial solution for SBGelvin, RASchilperoort and DPSVer.
Ma; Plant molecular Biology / Manual (1988) (Kluwe
r Academic Publishers), Hirofumi Uchimiya (Plant Gene Manipulation Manual, How to Make Transgenic Plants (Kodansha Scientific)) 1990, ISBN4-06-153513-
7 C3045) by the usual method described on pages 28-33.
A transformant of the above Agrobacterium was infected to a disc piece of tobacco leaf. After culturing the disc pieces of the leaves of the tobacco (SR-1) infected with Agrobacterium on MS-NB agar medium for 4 days, M containing cefotaxime 500 μg / ml
After transferring to an S-NB agar medium, Agrobacterium was removed. On the eleventh day, the cells were transferred to an MS-NB agar medium containing 500 μg / ml of cefotaxime and 100 μg / ml of kanamycin, and the culture was continued. After about 4 weeks, the seedlings that had undergone green foliage differentiation were cut out from the disc pieces, subcultured on an MS-NB agar medium containing 500 μg / ml of cefotaxime and 50 μg / ml of kanamycin, and the rooted seedlings were selected. The selected young tobacco plants were transferred to soil and cultivated in a greenhouse to obtain transformed plants.

Example 4 (Confirmation of Insertion of Transgene in Transformant) (1) Preparation of Genomic DNA from Transformant From leaf pieces of tobacco of the transformed plant obtained in Example 3, Hirofumi Uchimiya (plant Gene manipulation manual, how to make transgenic plants (Kodansha Scientific)) 19
90, ISBN4-06-153513-7 C3045) Genomic DNA was prepared using the CTAB method described on pages 71-74. After about 0.5 g of plant leaf pieces were sufficiently ground in an Eppendorf tube using a homogenizer, a 2xCTAB solution (2%
cetyltrimethyl ammonium bromide, 100mM Tris-HCl,
pH8.0, 20mM EDTA, 1.4M NaCl, 1% polyvinylpyroridon
e (PVP)), and the mixture was kept at 65 ° C for 5 minutes. to this,
0.5 ml of chloroform / isoamyl alcohol (24:
1) The mixture was added and mixed gently for 5 minutes. 12,000 rpm
(10,000xg) for 10 minutes to separate the upper layer, 10% CTAB solution (10% cetyltrimethyl a)
(mmonium bromide, 0.7M NaCl) was added and the mixture was kept at 65 ° C for 3 minutes. An equal volume of a mixed solution of chloroform / isoamyl alcohol (24: 1) was added and mixed well, and the upper layer was separated. 2
Double the volume of CTAB precipitate (1% cetyltrimethyl ammonium bromi
de, 50mM Tris-HCl, pH8.0, 10mM EDTA) and 65 ℃
, And centrifuged at 12,000 rpm (10,000 xg) for 10 minutes to precipitate DNA. The precipitate was washed with high salt TE (10 mM
Tris-HCl pH8.0, 1mM EDTA, 1M NaCl)
100 μl of ethanol was added and mixed. 12,000 rpm (10,
(000xg) for 15 minutes and the resulting precipitate
0 mM Tris-HCl pH 8.0, 1 mM EDTA). RN
aseA was added to a concentration of 10 μg / ml, and the mixture was kept at 37 ° C. for 30 minutes. Then, an equal volume phenol / chloroform / isoamyl alcohol (25: 24: 1) mixed solution was added, mixed well, and the upper layer was separated. Add 1/10 volume of 3M sodium acetate solution (pH 5.
2) and 2.5 volumes of ethanol are added and mixed well, and 12,000 r
Collect the sediment by centrifugation at pm (10,000 xg) for 5 minutes, and
g of genomic DNA was obtained.

(2) Confirmation of Insertion of Transgene by PCR Method Using 50 ng of the genomic DNA obtained in (1) as a template, the oligonucleotide having the nucleotide sequence of SEQ ID NO: 16 and the nucleotide sequence of SEQ ID NO: 17 Using the oligonucleotides as primers, PCR was performed (a total of 30 cycles, one cycle consisting of 94 ° C. for 1 minute, then 55 ° C. for 2 minutes, and further 72 ° C. for 3 minutes). The obtained PCR product was analyzed by 4% agarose gel electrophoresis. As a result, plasmids pGCRG1-1, pGCRG1-2, pGCRG1-3, pG
8CRG1-1, pG8CRG1-2, pGGY1-1, pGGY1-2, pGGY1-3, pCR
G1-1, pCRG1-2, pCRG1-3, pGY1-1, pGY1-2, or pGY1-
Amplification of a DNA fragment of about 500 bp was observed in the genomic DNA of tobacco into which each of 3 was introduced, and the presence of a region from the 3 ′ end of the coding region of the GUS gene to the nopaline synthase gene terminator (NOS) was confirmed.

Example 5 (Self-pollination of Transformant and Breeding of Homoline) Transformed tobacco in which the presence of the transgene was confirmed in Example 4 was transferred to soil and grown in a greenhouse. The flowers were self-pollinated during the flowering stage and seeds were obtained from mature flowers. 1% of the seeds obtained
After sterilization in sodium hypochlorite for 5 minutes, the cells were seeded on an MS agar medium containing 100 μg / ml of kanamycin. A clone in which about 3/4 of the sown seeds germinated and grew was selected. The selected seeds were again inoculated on MS agar medium containing 100 μg / ml of kanamycin, and clones in which all seeds germinated were selected and used in the following experiments.

Example 6 (GUS gene expression in each tissue in a transformant) The transformed tobacco seeds obtained in Example 5 (plasmid pG
CRG1-1, pGCRG1-2, pGCRG1-3, pG8CRG1-1, pG8CRG1-2,
pGGY1-1, pGGY1-2, pGGY1-3, pCRG1-1, pCRG1-2, pCRG1
-3, including the T-DNA region of pGY1-1, pGY1-2, or pGY1-3)
As a control, untransformed tobacco SR-1 seeds were sterilized in 1% sodium hypochlorite for 5 minutes, and then kanamycin was added.
MS agar medium containing 100 μg / ml or MS agar medium (SR-1
Seeds) and grown for about 2 weeks to obtain seedlings.
Approximately 200 mg of leaves and roots of the obtained seedlings (seedlings)
12,000 r in extraction buffer, grind using mortar and pestle
Centrifugation at pm (10,000 × g). The obtained supernatant was used for GUS activity measurement. 5 mM 4-methyl-umbellife in 400 μl of extraction buffer
The reaction solution to which 100 μl of ryl-D-glucronide (MUG) had been added was kept at 37 ° C., and 10 μl of the supernatant was added. The reaction was stopped by taking 100 μl of the reaction solution every 15 minutes and adding it to a 0.2 M calcium carbonate solution. The fluorescence of the sample thus obtained over time was measured using a fluorimeter. The fluorescence analysis was performed at an excitation of 365 nm and an emission of 455 nm.
Thyl-umbelliferone (4-MU) concentrations of 0-50 ng / ml were used.
The protein content of the extract from each plant sample was
n Using an Assay Kit (Bio Rad), specific activity was calculated per protein. Table 1 shows the results of measuring GUS activity in cotyledons, roots, and seeds of 10 transgenic tobacco plants into which the plasmid of the present invention has been introduced and non-transformed tobacco SR-1 used as a control. Each transformed tobacco was transferred to soil, cultured, and grown in a greenhouse. Pollen was obtained from the flowers during the flowering period. GUS gene expression of seeds and pollen of untransformed tobacco SR-1 used as a control and 5 individuals of each transgenic tobacco was written by Hirofumi Uchimiya (Plant Gene Manipulation Manual, How to Make Transgenic Plants (Kodansha Scientific)) 1990, ISBN4-06-153513-7 C3045) pp. 68-70 and Jefferson, Plant Mol. Biol. Rep. 5: 387-405 (1987).
The examination was carried out according to the described staining method. Table 2 shows the results.

[0022]

[Table 1] GUS specific activity in each tissue -(Control): Number of non-transformed tobacco SR-1 +: High level of GUS specific activity.

[0023]

[Table 2] GUS staining degree in seeds and pollen -(Control): Non-transformed tobacco SR-1 Staining intensity: +++ (Tokuno) +++ (Dark), ++ (Medium dark), + (Light),-(No)

The composition of the medium used in the examples is shown. MS agar medium 34.7 g of MURASHIGE AND SKOOG (Flow Laboratories) was dissolved in 1 L of distilled water, adjusted to pH 5.8 with 1 M KOH, added with 8 g of agar, and then sterilized in an autoclave. MS-NB agar medium This is a medium obtained by adding 1 mg / mL of 1-naphthaleneacetic acid (NAA) and 1.0 mg / mL of 6-benzylaminopurine (BA) to an MS agar medium. L medium Bactotryptone (Difco) 10 g, Bacto yeast extract (Difco) 5 g, NaCl 10 g dissolved in distilled water 1 L, 5M Na
The pH was adjusted to 7.0 with OH, and the mixture was autoclaved. Reaction extract for GUS activity measurement 50 mM phosphate buffer (pH 7.0), 10 mM EDTA, 0.1% Triton X-100 (Triton X-100), 0.1% sarkosy
l), a solution containing 10 mM 2-mercaptoethanol.

[0025]

Industrial Applicability According to the present invention, it is possible to provide a compact promoter or the like for expressing a desired gene in a specific tissue of a host organism at a higher level than in other tissues.

[Sequence Listing Free Text] SEQ ID NO: 1 oligonucleotide designed for promoter SEQ ID NO: 4 oligonucleotide designed for promoter SEQ ID NO: 5 oligonucleotide designed for promoter SEQ ID NO: 6 designed for promoter Oligonucleotide SEQ ID NO: 7 Oligonucleotide designed for promoter SEQ ID NO: 8 Oligonucleotide primer designed for PCR SEQ ID NO: 9 Oligonucleotide primer designed for PCR SEQ ID NO: 10 Designed for PCR Oligonucleotide primers SEQ ID NO: 11 Oligonucleotide primers designed for PCR SEQ ID NO: 12 Oligonucleotide primers designed for PCR SEQ ID NO: 13 Designed for PCR Oligonucleotide primers SEQ ID NO: 14 Oligonucleotide primers designed for PCR SEQ ID NO: 15 Oligonucleotide primers designed for PCR SEQ ID NO: 16 Oligonucleotide primers designed for PCR SEQ ID NO: 17 For PCR Designed oligonucleotide primers SEQ ID NO: 18 Oligonucleotide primers designed for PCR SEQ ID NO: 19 Oligonucleotide primers designed for PCR SEQ ID NO: 20 Oligonucleotide primers designed for PCR

[0027]

[Sequence List] SEQUENCE LISTING <110> Sumitomo Chemical Company Limited <120> Plant promoter <130> P150510 <150> JP 10/200372 <151> 1998-07-15 <160> 20 <210> 1 <211> 10 < 212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide for promoter <400> 1 gccacgtgcc 10 <210> 2 <211> 246 <212> DNA <213> Daucus carota L. <400> 2 tctagaatat atcttttgaa atttcaacaa acacagcact aacttttctt ttaacagatt 60 agaatcgttt cctaaacttt taaaattaaa aaatacatta ctataatatt tatcaacacc 120 tcaacattca tgttagcgta ctataaatag gtgctcttgg tgctctacta tcatcacatc 180 aatcttccag cacaaacctt gagcttaatc tttctactaa tttttagcaa aaacattcta 240 aaggtc 246 <210> 3 <211> 282 <212> DNA <213> Glycin max cv. Williams <400> 3 gaaaccatgc atggtcccct cgtcatcacg agtttctgcc atttgcaata gaaacactga 60 aacacctttc tctttgtcac ttaattgaga tgccgaagcc acctcacacc atgaacttca 120 tgaggtgtag cacccaaggc ttccatagcc atgcatactg aagaatgtct caagctcagc 180 accctacttc tgtgacgtgt ccctcattca ccttcctctc ttccctataa ataaccacgc 240 ctcaggttct ccgcttcaca actcaa acat tctctccatt gg 282 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide for promoter <400> 4 agcttgccac gtgccgccac gtgccgccac gtgccgccac gtgcct 46 <210> 5 <211> 46 < 212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide for promoter <400> 5 ctagaggcac gtggcggcac gtggcggcac gtggcggcac gtggca 46 <210> 6 <211> 140 <212> DNA <213> Artificial Sequence <220> <223 > Designed oligonucleotide for promoter <400> 6 ctagatcaac acctcaacat tcatgttagc gtactataaa taggtgctct tggtgctcta 60 ctatcatcac atcaatcttc cagcacaaac cttgagctta atctttctac taatttttag 120 caaaaacatt ctaaaggtcg 140 <210> 7 <211> 220 oligonucleotide for promoter <400> 7 gatccgacct ttagaatgtt tttgctaaaa attagtagaa agattaagct caaggtttgt 60 gctggaagat tgatgtgatg atagtagagc accaagagca cctatttata gtacgctaac 120 atgaatgttg aggtgttgat 140 <210> <123> 8 <1> <2> 2 <1> <1> <2> 2 <1> <1> <2> 2 <1> <2> <8> imer for PCR <400> 8 aaggatccga cctttagaat gtttttgc 28 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 9 tctctagatc aacacctcaa cattc 25 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 10 tctctagaca gattagaatc gtttcc 26 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 11 gccagtgaat gctttctag 19 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 12 aaggatccaa tggagagaat gt 22 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 13 tctctagact tctgtgacgt gtccc 25 <210> 14 <211> 25 <212 > DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 14 tctctagagt agcacccaag gcttc 25 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Designed olig onucleotide primer for PCR <400> 15 tctctagaga aaccatgcat ggtcc 25 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 16 catgcttaac gtaattcaac ag 22 <210 > 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 17 acatgtggag tgaagagtat c 21 <210> 18 <211> 118 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 18 gattacgcca agcttgccac gtgccgccac gtgccgccac gtgccgccac gtgccgccac gtgccg ccac gtgccgccac gtgccgccac gtgcctctag atcaac1111 <223> Designed oligonucleotide primer for PCR <400> 19 gattacgcca agcttgccac gtgccgccac gtgccgccac gtgccgccac gtgccgccac gtgccg ccac gtgccgccac gtgccgccac gtgcctctag ctgcctctag acagattaga atcgtttcc 119 <220> <220> 212 <220> 212 <220> oligonucleotide primer for PCR <400> 20 ctgccagttc agttggttgt tcacac 2 6

[Brief description of the drawings]

FIG. 1. Plasmid pGCR containing the promoter of the present invention.
It is a figure which shows the construction process of G1-1, pGCRG1-2, and pGCRG1-3. In the figure, GUS is the β-glucuronidase gene, T-nos
Indicates a terminator of the nopaline synthase gene derived from the Ti-plasmid.

FIG. 2 Plasmid pGGY containing the promoter of the present invention
BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the construction process of 1-1, pGGY1-2, and pGGY1-3. In the figure, GUS is the β-glucuronidase gene, T-nos
Indicates a terminator of the nopaline synthase gene derived from the Ti-plasmid.

FIG. 3 is a diagram showing the steps of constructing plasmids pCRG1-1, pCRG1-2 and pCRG1-3. In the figure, GUS indicates a β-glucuronidase gene, and T-nos indicates a terminator of a nopaline synthase gene derived from Ti-plasmid.

FIG. 4 is a diagram showing the steps of constructing plasmids pGY1-1, pGY1-2 and pGY1-3. In the figure, GUS indicates a β-glucuronidase gene, and T-nos indicates a terminator of a nopaline synthase gene derived from Ti-plasmid.

FIG. 5 is a diagram showing the steps of constructing plasmids pG8CRG1-1 and pG8CRG1-2. In the figure, GUS indicates a β-glucuronidase gene, and T-nos indicates a terminator of a nopaline synthase gene derived from Ti-plasmid.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:91) (C12N 5/10 C12R 1:91)

Claims (15)

[Claims] 1. A promoter having the following nucleotide sequence (a) or (b) and the nucleotide sequence shown in SEQ ID NO: 1. (A) a base sequence having a base sequence represented by at least base numbers 112 to 246 of the base sequence represented by SEQ ID NO: 2; (B) a base sequence having at least the base sequence represented by base numbers 186 to 282 of the base sequence represented by SEQ ID NO: 3; 2. A promoter having the following nucleotide sequence (a) or (b) and the nucleotide sequence shown in SEQ ID NO: 1. (A) base number 11 of the base sequence represented by SEQ ID NO: 2
Any of the base sequences represented by base numbers 2 to 246, base numbers 54 to 246 or base numbers 1 to 246. (B) base number 18 of the base sequence represented by SEQ ID NO: 3
6 to 282, any one of the nucleotide sequences represented by base numbers 127 to 282 or base numbers 1 to 282. 3. The nucleotide sequence represented by SEQ ID NO: 1 is located 5 ′ upstream of the nucleotide sequence of (a) or (b).
Or the promoter of 2. 4. The promoter according to claim 1, which has two or more nucleotide sequences represented by SEQ ID NO: 1. 5. The promoter according to claim 4, wherein the promoter has 4 or 8 nucleotide sequences represented by SEQ ID NO: 1. 6. A chimeric gene having the promoter according to claim 1 and a desired gene. 7. A vector having the promoter according to claim 1. 8. The vector according to claim 7, having a gene insertion site downstream of the promoter and a terminator operable in a host cell. 9. A vector having the chimeric gene according to claim 6. 10. A transformant obtained by introducing the promoter according to claim 1 to 5, the chimeric gene according to claim 6, or the vector according to claim 7 to 9 into a host cell. 11. The host cell is a microbial cell.
The transformant as described above. 12. The transformant according to claim 10, wherein the host cell is a plant cell. 13. A gene expression method for expressing a desired gene in a host cell under the control of the promoter according to claim 1. 14. A method for preparing a promoter for connecting a DNA having the following nucleotide sequence (a) or (b) and a DNA having the nucleotide sequence of SEQ ID NO: 1 in a form operable in a host cell. (A) a base sequence having a base sequence represented by at least base numbers 112 to 246 of the base sequence represented by SEQ ID NO: 2; (B) a base sequence having at least the base sequence represented by base numbers 186 to 282 of the base sequence represented by SEQ ID NO: 3; 15. A method for preparing a promoter for connecting a DNA having the following nucleotide sequence (a) or (b) and a DNA having the nucleotide sequence of SEQ ID NO: 1 in a form operable in a host cell. (A) base number 11 of the base sequence represented by SEQ ID NO: 2
Any of the base sequences represented by base numbers 2 to 246, base numbers 54 to 246 or base numbers 1 to 246. (B) base number 18 of the base sequence represented by SEQ ID NO: 3
6 to 282, any one of the nucleotide sequences represented by base numbers 127 to 282 or base numbers 1 to 282.