JP2003199582A - Promoter derived from plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a promoter of S-adenosylmethionine decarboxylase gene in Cucurbita ficifolia Bouche polyamine synthetases and promoters of a spermidine synthetase gene and an arginine decarboxylase gene capable of expressing a chimeric gene. <P>SOLUTION: The 5' upstream sequence of the S-adenosylmethionine decarboxylase gene and the 5' upstream sequence of the spermidine synthetase gene are obtained by treating a genomic DNA of the Cucurbita ficifolia Bouche through the inverse PCR method using DNA sequence information of the S- adenosylmethionine decarboxylase gene of the Cucurbita ficifolia Bouche, the permidine synthetase gene of the Cucurbita ficifolia Bouche and the arginine decarboxylase gene of the Cucurbita ficifolia Bouche. These upstream sequences are subcloned and the base sequences thereof are determined by the cycle sequence method. GUS activity is tested using a recombinant plant and the activity of the promoter is examined. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a promoter for S-adenosylmethionine decarboxylase gene, a promoter for spermidine synthase gene and a promoter for arginine decarboxylase gene of Scutellaria scutella, which is a gene. It is used to develop new plant varieties using recombination and to modify plants that produce useful substances.

[0002]

2. Description of the Related Art Plant genetic engineering is a very useful technique for producing a plant having desired characteristics without using a time-consuming and costly crossing. Therefore, the development of a method for introducing a gene into plant cells has enabled the production of various transformed plants. Currently, various transgenic plants are being actively developed using such genetic engineering, and have been put to practical use not only in Europe and America but also in Japan.

Breeding using plant genetic engineering is
Mainly in the field of crops, there are long-lasting tomatoes using antisense technology, herbicide-resistant soybeans and rape, insect-resistant potatoes and corn. , Play an immeasurable role in solving food problems. Further, in recent years, it has been actively attempted to cause plants other than agricultural crops to carry out substance production and environmental restoration by using genetic engineering techniques. It is expected that they will greatly contribute to energy problems and environmental problems in the future.

An important problem in producing a transformant plant is efficient expression of a gene introduced into plant cells. The promoter sequence is a major factor that determines the transcription level of a gene in plant cells, and generally, by using a promoter sequence having a strong transcription activity, it is possible to improve the expression level of the target gene. However, promoter sequences are known to have specificity based on differences in RNA polymerases existing in plant species, organs, tissues, cells, or environmental conditions. When trying to do so, it is necessary to use a promoter sequence that functions under the desired conditions. Currently, the promoters that can be used at a practical level are limited, and generally, the 35S promoter derived from cauliflower mosaic virus is often used.

By the way, it is known that Kurodane squash expresses genes of the polyamine synthase group and accumulates polyamines in its body when subjected to stress such as low temperature, high salt concentration and drought. Polyamine is a general term for aliphatic hydrocarbons having two or more primary amino groups and is a natural product that is universally present in the body, and 20 or more types of polyamines have been found. Representative polyamines include putrescine, spermidine, and spermine. The main physiological actions of polyamines are stabilization and structural changes of nucleic acids due to interaction with nucleic acids, promotion of various nucleic acid synthesis systems, activation of protein synthesis systems, stabilization of cell membranes and membrane permeability of substances. It is known to enhance resistance.

[0006] As a role of polyamines in plants, it has been reported that nucleic acid and protein biosynthesis are promoted and cells are protected during cell growth and division, and salt stress and acid stress (Plant cell physiol., 38) against environmental stress. (Ten),
156-1166, 1997), low temperature stress (Plant Science, 12
2, 111-117, 1997, Plant Science, 126, 1-10, 199
7, Japan Jour. Crop Sci., 50 (3), 411-412, 1981) are mainly reported.

Arginine decarboxylase (AD) is a polyamine metabolism-related enzyme involved in biosynthesis of polyamines in plants.
C), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), spermidine synthase (SPDS), spermine synthase (SPM)
S) etc. are known. Some genes encoding these polyamine metabolism-related enzymes have already been isolated from plants.

The ADC gene is an oat (Mol. Gen. Gene
t., 224, 431-436, 1990), tomato (Plant Physiol.,
103, 829-834, 1993), Arabidopsis (plant physio)
l., 111, 1077-1083, 1996), Pea (Plant Mol. Bi
ol., 28, 997-1009, 1995), and the ODC gene is Datura (Biocem. J., 314, 241-248, 199).
6), SAMDC gene is potato (Plant Mol. Bio
l., 26, 327-338, 1994), spinach (Plant Physi
ol., 107, 1461-1462, 1995), tobacco, and SPDS genes are Arabidopsis thaliana (Plant cell Physiol., 39 (1), 73
-79, 1998) and the like. However, none of these plant-derived polyamine metabolism-related enzyme gene promoters have been reported.

[0009]

One of the objects of the present invention is to
It is an object of the present invention to provide a promoter capable of controlling a gene required for breed improvement and the like by using a genetic engineering technique in plants. Furthermore, it is to provide a promoter capable of controlling a target gene by controlling temperature.

[0010]

[Means for Solving the Problems] In order to obtain the plant promoter of the present invention, the present inventors have already conducted earnest research with the aim of elucidating at the gene level based on the knowledge of the physiology of Kurodane squash, and Some cd more than pumpkin
NA was isolated (JP 2001-46079A). These isolated cds
NA was investigated, and further, the upstream sequences of S-adenosylmethionine decarboxylase gene, spermidine synthase gene and arginine decarboxylase gene were cloned and analyzed. As a result, a promoter expressed in tobacco of a model plant was found, The present invention has been completed.

The present invention relates to items 1 to 22 below. Item 1, a DNA fragment containing a promoter region of a plant-derived S-adenosylmethionine decarboxylase gene. Item 2, a DNA fragment containing the DNA of (a) or (b) below. (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 1 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
The DNA fragment according to claim 1, which further comprises DNA having the ability to act as a plant promoter, and the DNA of (a) or (b) below. (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 1 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
A DNA fragment capable of acting as a plant promoter and hybridizing with a DNA comprising the nucleotide sequence of claim 4, claim 2 or 3 under stringent conditions and having the ability of acting as a plant promoter. Item 5. The DNA fragment according to any one of claims 1 to 4, which can be controlled by temperature. A vector comprising the DNA fragment according to claim 6 or any one of claims 1 to 5. 7. The vector according to claim 6, wherein the heterologous gene is under the transcriptional control of the DNA fragment. A transformed plant obtained by introducing the vector according to claim 8 or claim 6 or 7 into a host plant cell. Item 9. A seed obtained from the transformed plant according to claim 9 or its progeny. Item 10, a DNA fragment having a promoter activity of a plant-derived spermidine synthase gene. Item 11, a DNA fragment containing the following DNA (a) or (b): (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 13 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
And a DNA having the ability to act as a plant promoter, and the DNA of (a) or (b) below:
The DNA fragment according to 0. (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 13 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
DNA having the ability to act as a plant promoter and comprising the nucleotide sequence of claim 13 or 11 or 12.
And a DNA fragment capable of hybridizing to the plant under stringent conditions and acting as a plant promoter. The DN according to claim 14 or any one of claims 10 to 13.
A vector containing the A fragment. 15. The vector according to claim 14, wherein the heterologous gene is under the transcriptional control of the DNA fragment. A transformed plant obtained by introducing the vector according to claim 16 or 14 or 15 into a host plant cell. Item 17. A seed obtained from the transformed plant according to claim 17 or its progeny. Item 18, a DNA fragment having a promoter activity of a plant-derived arginine decarboxylase gene. Item 19, a DNA fragment containing the following DNA (a) or (b): (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 25 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
A DNA having the ability to act as a plant promoter and a DNA having the following (a) or (b):
The DNA fragment according to item 8. (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 25 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
And DNA having the ability to act as a plant promoter
fragment. It has the ability to hybridize with the DNA comprising the nucleotide sequence according to claim 21, claim 19 or 20 under stringent conditions and to act as a plant promoter.
DNA. Item 22. The DNA fragment according to any one of items 18 to 21, which is controllable by temperature. The DN according to claim 23 or any one of claims 18 to 22.
A vector containing the A fragment. 24. The vector according to claim 23, wherein the heterologous gene is under the transcriptional control of the DNA fragment. A transformed plant obtained by introducing the vector according to claim 25 or 23 or 24 into a host plant cell. Item 26. A seed and its progeny obtained from the transformed plant according to item 26 or item 25.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The “plant promoter” of the present invention is a DNA fragment having the ability to act as a promoter (promoter function) in plants.
“Promoter” means a specific base sequence on DNA that starts mRNA synthase (transcription) using DNA as a template, has a common base sequence, and recognizes this enzyme to synthesize mRNA (RNA polymerase ) Synthesizes mRNA. here,
The "promoter function" refers to the action of RNA polymerase binding to a specific region on DNA and initiating transcription. Specifically, it is a promoter of S-adenosylmethionine decarboxylase gene, spermidine synthase gene or arginine decarboxylase gene, or (a), (b) below.
Alternatively, it includes a DNA fragment containing the DNA of (c). (A) D consisting of the nucleotide sequence of SEQ ID NO: 1, 13 or 25
NA (b) (a) consists of a base sequence in which one or more base sequences are deleted, substituted or added,
And DNA capable of acting as a plant promoter (c) DNA capable of hybridizing with a DNA comprising the nucleotide sequence of (a) or (b) under stringent conditions and having the ability of acting as a plant promoter.

The plant promoter of the present invention comprises a DNA sequence consisting of the nucleotide sequence of SEQ ID NO: 1, 13 or 25 in which one or more DNAs have been deleted, substituted or added, and which serves as a plant promoter. It is DNA that has the ability to act. The plant promoter of the present invention has a nucleotide sequence of SEQ ID NO: 1, 13 or 25 to which a nucleotide sequence for improving translation efficiency is added to the 3'or 5'end, or the 3'or 5'end has promoter activity. As long as there is, the deletion is included.

The "vector" of the present invention is obtained by introducing the above promoter into a vector. Examples of the vector include a vector derived from Escherichia coli, for example, pBluescript vector, a plasmid containing β-glucuronidase gene (GUS), pBI101-. HmB, shuttle vector,
Helper plasmid, pRK2013 and the like can be mentioned. Alternatively, a plant virus such as cauliflower mosaic virus can be used. The vector is selected according to each host cell. The method for introducing the plant promoter into the vector follows the method for introducing a normal gene into the vector.

The "transformed plant" of the present invention is a transformed plant obtained by introducing the above vector into a host plant cell. As a host plant cell, Arabidopsis Wassilewskija
Strains, tomatoes, tobacco, petunias, wheat, rice, corn, pumpkins, cucumbers, cotton and the like can be mentioned. Examples of the transformation method for introducing a plant expression vector into a host plant cell include an electroporation method, a protoplast fusion method, a microinjection method, a polyethylene glycol method and a particle gun method. Furthermore, it is a transformed plant regenerated from cells into which the gene has been introduced. As a method for regeneration, there is a method in which callus-like transformed cells are transferred to a medium in which the type and concentration of hormone are changed and cultured to form adventitious embryos to obtain a complete plant body. The medium used for cotton is M
SIC medium (MS salt, 0.75% MgCl2, 1.9g / l KNO3, 30g / l
Examples of glucose, 2.0 g / l gellan gum, pH 5.8) and carrot include Kamada & Harada medium.

The following steps are exemplified as specific steps of the present invention. (1) S-adenosylmethionine decarboxylase gene (SAMD
C), cloning of upstream region of spermidine synthase gene (SPDS) or arginine decarboxylase gene (ADC) (2) fusion with β-glucuronidase gene (GUS gene) (3) introduction of plasmid into Agrobacterium ( 4) Aseptic tobacco cultivation (5) Agrobacterium infection (6) Sterilization (7) Selection of transformed plants (8) Regeneration of transformed plants (9) Confirmation of transformants (10) Measurement of GUS activity <1> Isolation of promoter regions of SAMDC gene, SPDS gene and ADC gene S-adenosylmethionine decarboxylase gene SAMDC (Japanese Patent Laid-Open No. 2001-4607)
9), spermidine synthase gene SAMDC (JP 2001-46
079), arginine decarboxylase gene ADC (JP 2001-460A).
A synthetic oligonucleotide is prepared from the nucleotide sequence of 79) and the inverse PCR method is performed. Genomic DNA is extracted from Kurodane squash, cut with several restriction enzymes such as EcoRI, self-ligated, and then used as a template for PCR. PCR using the first primer group
After that, the reaction product is further subjected to PCR using the second primer group to clone the upstream region. After obtaining a clone of the desired length by agarose gel electrophoresis,
Subcloned into A cloning vector and sequenced using an autosequencer. Obtained P
Since a part of the sequence of the CR fragment completely matched with the sequences of SAMDC, SPDS and ADC, it was judged that they were upstream (plant promoter) of SAMDC, SPDS and ADC, respectively. <2> Utilization of promoter region of SAMDC gene, SPDS gene, ADC gene The plant promoter obtained by the above method can be used for controlling protein expression by producing a chimeric gene construct in a model plant tobacco. Furthermore, since this promoter can be regulated by temperature, it can be applied to breeding of a new plant having stress resistance such as low temperature resistance when connected to a stress resistance gene such as low temperature. <3> Introduction of plant promoter into vector and transformation of host plant cell As a transformation method of a plant cell, an electroporation method in which protoplasts are subjected to electric pulse treatment to introduce a plasmid into plant cells, small cells, cells Examples include a fusion method of lysosome or the like and protoplast, a microinjection method, a polyethylene glycol method, or a particle gun method. Moreover, the target gene can be introduced into a plant by using a plant virus as a vector. As the plant virus to be used, for example, cauliflower mosaic virus (CaMV) can be used. That is, first, the viral genome is first inserted into a vector derived from E. coli or the like to prepare a recombinant, and then these target genes are inserted into the viral genome. These target genes can be inserted into a plant by excising the thus modified viral genome from the recombinant with a restriction enzyme and inoculating the plant [Horn (Ho
hn) et al., Molecular Biology of Plant Tumor
s), Academic Press, New York (Academic
Press, New York), pages 549-560 (1982), U.S. Patent No.
4,407,956].

Further, there is a method of using a Ti plasmid of Agrobacterium, which has the property that when a bacterium belonging to the genus Agrobacterium infects a plant, a part of the plasmid DNA carried by the plant is transferred into the plant genome. Can also be used to introduce these target genes into plants. Among bacteria belonging to the genus Agrobacterium, Agrobacte tumefaciens (Agrobacte
rium tumefaciens) infects a plant with a tumor called crown gall, which causes Agrobacterium rhizogenes (Agr
obacterium rhizogenes) infect plants to cause hairy roots, which are called T-DNA regions (Transferred DNA) on the plasmids present in each bacterium, called Ti plasmid or Ri plasmid during infection. Due to the region translocating into the plant and integrating into the plant genome. Furthermore, on the Ti or Ri plasmid, there is a region called a vir region which is essential for the T-DNA region to be transferred into the plant and to be integrated into the plant genome. The vir region itself is not transferred into plants, and this vir region can function even on a plasmid different from that in which the T-DNA region is present [Nature, 303, 179, (1983)].

T-DNA on Ti or Ri plasmid
By inserting a DNA to be incorporated into the plant genome into the region, the target DNA can be incorporated into the plant genome when a bacterium of the genus Agrobacterium infects the plant. Here, T-DN of Ti or Ri plasmid
The crown gall in A or the part that causes hairy roots can be removed without impairing the target migration function, and the obtained one can be used as a vector. In the present invention, such various vectors can be used. For example, pB called a binary vector
These can be introduced into plant cells by inserting into the vector such as I101 (Clontech) the promoter of the present invention to which the gene involved in fiber formation and elongation is connected in the sense or antisense direction. In addition,
These vectors do not have the vir region described above,
The bacterium of the genus Agrobacterium used by introducing the vector needs to contain another plasmid having a vir region.

Further, these vectors are shuttle vectors that can be amplified not only in bacteria of the genus Agrobacterium but also in Escherichia coli. Therefore, the recombinant operation of Ti plasmid can be performed using Escherichia coli. Furthermore, these vectors contain an antibiotic resistance gene, and when transforming Escherichia coli, bacteria of the genus Agrobacterium, plant cells or the like, transformants can be easily selected. In addition, the 35S promoters of cauliflower mosaic virus and CaMV are present in these vectors, and the genes inserted into these vectors can be expressed unregulated after being integrated into the plant genome. <4> Regeneration of Plant from Transformed Plant Cell Hereinafter, a method for introducing a target gene by Agrobacterium in tobacco and a method for regenerating a transformed cell into a plant will be described in detail. Tobacco seeds are sown on an MS plate and cultivated aseptically according to a conventional method. Tobacco leaves cut to 1 cm square by culturing Agrobacterium transformed with a plasmid having a kanamycin and hygromycin resistance gene by connecting a target gene to the plant promoter of the present invention , And co-culture in liquid MS medium for several days. When Agrobacterium has grown sufficiently to be visible to the naked eye, the bacteria are sterilized and cultured on an MS plate for several days. These leaf pieces are finally cultured on an MS plate containing a hormone necessary for germination, and transplantation is repeated every week on a new plate. The transformed leaf pieces continue to grow and shoots appear. When it shows a perfect shoot shape, cut it with a scalpel so that it does not contain the callus part at the root of the shoot, transfer it to an MS plate containing the hormones necessary for rooting, and sterilize the culture until rooting is confirmed. Then, a transformant is obtained. From this transformant, DNA was prepared according to a conventional method.
It is possible to confirm the presence or absence of transformation by extracting the protein, designing primers for the promoter portion and the GUS gene portion, and performing PCR.

RNA is extracted from the transformant or non-transformant according to a conventional method to prepare a probe having a sense or antisense sequence of a chimeric gene, and Northern hybridization is carried out using these probes. , It is possible to examine the expression status of the target gene. Method for determining element As a method for determining the element site on the promoter of the present invention, for example, Joseph Sambrook, David W. Rus
sell Molecular Cloning 3rd edition Chapter 13 Create a promoter whose length is regulated by using the deletion method or PCR, and transform it into plant cells. There is a method to investigate using. For more detailed element sequencing, see Joseph Sambrook, David W. Russell Mo.
As described above, by introducing a DNA mutation on the promoter as described in Chapter 13 of lecular Cloning 3rd edition, or by introducing a mutation mutation using the PCR method, after transforming into a plant cell or the like, There is a way to investigate promoter activity and determine the most important element sequences. As a specific example, the determination of the Arabidopsis DRE element (T
he Plant Cell 6: 251-264 (1994)) is helpful.

The plant journal (1996) 9 (2) 14-158
Reported that a transformant could not be formed by connecting the SAMDC gene to a known 35S promoter, and that a transformant could be formed by changing to an inducible promoter. Is advantageous to obtain transformants over the 35S promoter. It was

The promoter of the present invention can be modified to obtain a promoter whose activity is changed by various methods such as connecting the elements of the promoter in tandem or connecting the Ω sequence of tobacco mosaic virus (Pl.
ant cell physiol 37 (1): 49-59 (1996), Efficient Pro
moter Cassettes for Enhanced Expression of Foreign
Genes in Dicotyledonous and Monocotyledonous Plan
t, JP 2000-166577).

The following are examples of genes that are preferably connected to the promoter of the present invention. (i) Genes encoding enzymes that synthesize osmoprot
ectants

[0024]

[Table 1]

(Ii) Late embryogenesis abundant (LEA)-
related genes

[0026]

[Table 2]

(Iii) Regulatory genes

[0028]

[Table 3]

(Iv) Oxidative stress related genes

[0030]

[Table 4]

(V) Genes encoding for molecular chape
rones

[0032]

[Table 5]

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[0034]

INDUSTRIAL APPLICABILITY In the present invention, when the GUS gene widely used in plants as a reporter gene is linked to the 3'end of the promoter of the present invention and used, the strength of the promoter can be easily determined by examining the GUS activity. Can be evaluated.
As the reporter gene, not only the GUS gene but also luciferase and green fluorescein protein can be used. SAMDC gene, SPDS gene and ADC gene are one of the genes of the polyamine synthesizing gene enzyme group, and the timing of expression of polyamine having an unknown function, the tissue of expression, etc. are utilized by the promoter of the present invention. It is also useful for identifying and elucidating the function of polyamines.

The promoter of the present invention is SAMDC gene, S
PDS gene and ADC gene, that is, promoters involved in the polyamine synthase group, which can be used to isolate cis and trans factors to elucidate plant polyamine synthesis signal pathways, and gene regulation by temperature Therefore, it is extremely useful in the technical field of studying the stress response system of plants by conducting a comparative study with a promoter capable of gene regulation by a known temperature.

Furthermore, a chimeric gene in which a nucleotide sequence encoding a protein involved in stress resistance such as low temperature is bound to the plant promoter of the present invention is prepared, and a chimeric gene of the target gene is produced only under specific environmental conditions. It is possible to produce a stress-resistant transformed plant that can be expressed and has a low burden on the plant. Further, generally, by using the 35S promoter of cauliflower mosaic virus, it is possible to bring about a morphological change in the entire organ of a plant through the whole process of the life cycle. However, when the promoter of the present invention is used, the morphology is changed depending on the growth environment. Variable plants can be created.

[0037]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 Construction of transgenic tobacco having upstream region of SAMDC (1) S-adenosylmethionine decarboxylase gene, SAMD
Cloning of the upstream region of C (SAMDCpro) Using seedlings that had been grown for 1 month after sowing Cucurbita ficifolia Bouche, Plant DNA ZOL (G
Genomic DNA was extracted using IBCO BRL, and the genomic DNA was cloned by the inverse PCR method. That is, the restriction enzymes NcoI, BamHI, SphI, Dra
After cutting 1 μg of genomic DNA with I and AIW44I,
Self-ligation was performed at tion high (Toyobo Co., Ltd.). PC using 300 ng of ligated DNA as template
Using R reagent kit (LA-PCR kit, Takara Shuzo), SAMDC
5'upstream region was amplified. The reaction was carried out using the first primer group having the nucleic acid sequences shown in SEQ ID NOs: 2 and 3 for 28 cycles of 94 ° C. for 15 seconds and 65 ° C. for 12 minutes. Next, using this PCR product, PCR was performed under the same conditions using the second primer group having the nucleotide sequences shown in SEQ ID NOS: 4 and 5. This PC
The amount and length of the R product was confirmed by electrophoresis, and the obtained D
The NA fragment was introduced into the vector pTAdv using AdvanTAge PCR Cloning Kit (Clontech), and Escherichia coli JM109 was introduced.
Was transformed and cloned. Then ABI PRIS
SAMDC 5 using M 310 Genetic Analyzer (ABI)
'The upstream nucleotide sequence was determined. It was confirmed from the primer sequence that the base sequence was entirely in agreement with the cDNA and was a genomic region corresponding to the cDNA. Furthermore, the primers of SEQ ID NOs: 6 and 7 with restriction enzyme sites (XbaI, BamHI) added to both ends were designed to be approximately 1000 bp from the transcription initiation point, and inserted into the MCS of pBluescript.
I named it 00pro. This base sequence is shown in SEQ ID NO: 1.
Similarly, a gene having a transcription start point of 500 bp was also prepared from SEQ ID NOs: 8 and 9 and named pSAMDC500pro. This base sequence is shown in SEQ ID NO: 10. (2) Fusion with β-glucuronidase gene (GUS gene) Plasmid containing β-glucuronidase gene (GUS), pBI221 (Clontech), restriction enzymes, BamHI and Eco
After cutting with RI, agarose gel electrophoresis was performed to take out the GUS gene in the plasmid. This GUS
The gene is a plasmid, the vector pSAMDC1000pro and p
BamHI and E, restriction enzymes on pBluescript of SAMDC500pro
Inserted into the coRI site, plasmid pSAMDCpro1000:
GUS and pSAMDCpro500: GUS were obtained.

Further, pSAMDC1000pro and pSAMDC500pro were cleaved with restriction enzymes, XbaI and BamHI, and after electrophoresis, XbaI
-The BamHI fragment was inserted into the restriction enzyme XbaI and BamHI sites of the plasmid pBI101-HmB (obtained from Prof. Atsuhiko Shinna, Nara Institute of Technology) to obtain a new plasmid pB.
I101-SAMDCpro1000: GUS and pBI101-SAMDCpro500: GUS
Got Then, the plasmid, pBI101-1000: GUS
And pBI101-500: GUS transformed E. coli JM109,
Escherichia coli JM109 / pBI101-SAMpro1000: GUS and
Escherichia coli JM109 / pBI101-SAMpro500: GUS was obtained. This process is shown in FIG. (3) Introduction of plasmid into Agrobacterium Escherichia coli JM109 / pBI1 obtained in (2)
01-SAMDCpro1000: GUS and Escherichia coli JM109 / pB
I101-SAMDCpro500: GUS was cultured in LB medium containing 59 mg / L of kanamycin at 37 ° C. overnight, and a plasmid was extracted from each E. coli. 2 μl of extracted plasmid and ELECTOROMAX A. tumefaciens LBA4404ce
ll (GIBCO BRL) 50 μl are mixed, and Cuv is applied by Gene Pulser II electroporation system (BIO-RAD).
ett Gap 0.2cm, Voltage 2.5KV, Field strength 12.5K
Electroporation was performed under the conditions of V / cm, Capacity 25 μF, and Resistor 200Ω. Then, the cells were cultivated in SOC medium for 2 hours at 28 ° C, and 50 mg / l kanamycin YEP
It was spread on the medium. After culturing at 28 ° C for 2 days, a single colony was selected. The transformant obtained was designated as LBA4404 / pBI1.
01-SAMDCpro1000: GUS, LBA4404 / pBI101-SAMDCpro500: GU
I named it S. (4) Aseptic tobacco cultivation Tobacco seeds (obtained from Prof. Yoshihiko Shinna, Nara Institute of Technology)
Several dozen particles were placed in a 1.5 ml tube, 1 ml of 70% ethanol was added, and the mixture was left for 3 minutes. Then sterilized liquid (5%
Soaked in sodium hypochlorite, 0.02% TritonX-100) for 3 minutes and washed 5 times with sterilized water, then MS plate (Murashige-Skoog inorganic salts 4.3 g, sucrose 30 g per liter)
, Myo-inositol 0.1g, gellan gum 2.5g, pH 5.7)
It was placed on the floor. This plate was cultured in a plant incubator (manufactured by Sanyo, MLR-350HT) at 25 ° C for about 2 weeks. After germination, the plants were transplanted to a plant culture pot containing the same MS medium as above, and grown for about 1 month. (5) Infection with Agrobacterium Tobacco leaves cultured for 1 month in the above (4) were treated with a female for about 1.
Cut to about 0 cm, float on liquid MS medium,
The transformant obtained in (3) above, LBA4404 / pBI101-SAMDCpr
o1000: GUS and LBA4404 / pBI101-SAMDCpro500: GUS at 28 ℃,
50 μL of the culture solution that had been cultivated for 2 days was added, and coculture was carried out for 2 days at 25 ° C. in the dark in each Petri dish. (6) Leaf pieces after sterilization co-culture were washed several times with sterilized water. (7) Selectively eradicated tobacco leaf pieces of transformed plants are kanamycin (100 mg / L), hygromycin (20 mg / L), carbenicillin (250 mg / L)
L), naphthalene acetic acid (0.02mg / L), benzylaminopurine (1mg / L)
Cultured in lux. Thereafter, it was transplanted to a new MS plate every week. The transformed leaf pieces continued to proliferate, and shoots were formed from the callus-shaped dome-shaped rises after about 1 month. (8) The root of the plant that became a regenerated shoot of the transformed plant is cut off with a shaving blade or a scalpel so that it does not contain the callus part, and it is lightly placed in a plant culture pot containing MS medium at 25 ° C. It was grown under the conditions of 16 hours of light irradiation and 8 hours of darkening. (9) Confirmation of transformant One leaf was cut out from the plant grown in the above (8), and genomic DNA was obtained using Plant DNA ZOL (GIBCO BRL).
A was extracted using the primers of SEQ ID NOs: 11 and 12
It was confirmed by PCR that the target DNA fragment was contained. Example 2: Preparation of transformed tobacco having CaMV35S promoter (1) Plasmid containing fusion CaMV35S promoter (35S) with β-glucuronidase gene (GUS gene), pBI22
1 (Clontech) was digested with restriction enzymes, XbaI and HindIII, and then subjected to agarose gel electrophoresis to take out the 35S promoter in the plasmid. This 35S promoter is a plasmid, restriction enzymes XBI and Hi of pBI101-HmB (obtained from Nara Institute of Technology, Professor Akihiko Shinna).
A new plasmid, pBI35, was inserted into the ndIII site.
S: GUS was obtained. Then the plasmid, pBI35S:
Escherichia coli JM109 was transformed with GUS, and Escherichi
A coli JM109 / pBI101-35S: GUS was obtained. This process is shown in Figure 1.
Shown in. (2) Introduction of plasmid into Agrobacterium Escherichia coli JM109 / pBI obtained by (1)
101-35S: GUS was cultured in LB medium containing 59 mg / L of kanamycin at 37 ° C. overnight, and a plasmid was extracted from each E. coli. Extracted plasmid 2
μl and ELECTOROMAX A. tumefaciens LBA4404cell (GIBCO
BRL) 50 μl, and mixed with Gene Pulser II electroporation system (BIO-RAD) by Cuvett Gap
0.2cm, Voltage 2.5KV, Field strength 12.5KV / cm, Ca
Electroporation was performed under the conditions of pacity 25 μF and Resistor 200Ω. Then, the cells were cultured in SOC medium for 2 hours at 28 ° C. and spread on 50 mg / l kanamycin YEP medium. After culturing at 28 ° C for 2 days, a single colony was selected. The resulting transformant was transformed into LBA4404 / pBI101-35S: GUS
I named it. (3) Aseptic tobacco cultivation Tobacco seeds (obtained from Prof. Yoshihiko Shinna, Nara Institute of Technology)
Several dozen particles were placed in a 1.5 ml tube, 1 ml of 70% ethanol was added, and the mixture was left for 3 minutes. Then sterilized liquid (5%
Soaked in sodium hypochlorite, 0.02% TritonX-100) for 3 minutes and washed 5 times with sterilized water, then MS plate (Murashige-Skoog inorganic salts 4.3 g, sucrose 30 g per liter)
, Myo-inositol 0.1g, gellan gum 2.5g, pH 5.7)
It was placed on the floor. This plate was cultured in a plant incubator (manufactured by Sanyo, MLR-350HT) at 25 ° C for about 2 weeks. After germination, the plants were transplanted to a plant culture pot containing the same MS medium as above, and grown for about 1 month. (4) Infection with Agrobacterium Tobacco leaves cultured for 1 month in (4) above were treated with a female for about 1.
Cut to about 0 cm, float on liquid MS medium,
The transformant obtained in (3) above, LBA4404 / pBI35S: GUS
50 μL of the culture solution that had been cultivated at 2 ° C. for 2 days was added, and cocultured at 25 ° C. in the dark for 2 days in separate Petri dishes. (5) Leaf pieces after sterilization co-culture were washed several times with sterilized water. (6) Selectively eradicated tobacco leaf pieces of transformed plants are kanamycin (100 mg / L), hygromycin (20 mg / L), carbenicillin (250 mg / L)
L), naphthalene acetic acid (0.02mg / L), benzylaminopurine (1mg / L)
Cultured in lux. Thereafter, it was transplanted to a new MS plate every week. The transformed leaf pieces continued to proliferate, and shoots were formed from the callus-shaped dome-shaped rises after about 1 month. (7) The root of the plant that has become the regenerated shoot of the transformed plant is cut with a shaving blade or a scalpel so that it does not contain the callus part, and it is lightly placed in a plant culture pot containing MS medium, and the temperature is 25 ° C. It was grown under the conditions of 16 hours of light irradiation and 8 hours of darkening. (8) Confirmation of transformant One leaf was cut out from the plant grown in the above (8) and genomic DNA was used using Plant DNA ZOL (GIBCO BRL).
A was extracted using the primers of SEQ ID NOs: 11 and 12
It was confirmed by PCR that the target DNA fragment was contained. Example 3: Measurement of GUS activity (1) Evaluation of promoter activity by particle gun pSAMDCpro1000: GUS obtained in the above Example 1 (2)
Using wild type tobacco leaves, pBI221 was observed for GUS activity in transient expression by particle gun (Bio-Rad). Tobacco leaves, 0.25%
They were arranged in a medium containing gellan gum. The particles to shoot are
60 mg of gold particles were weighed, 1 ml of 70% ethanol was added, and the mixture was vortexed for 5 minutes. After allowing to stand for 15 minutes, centrifuge at 10,000 rpm for 5 seconds, remove the supernatant, and repeat the following operations 3 times.
Add 1 ml of sterilized water, vortex for 1 minute, leave for 1 minute, centrifuge at 10,000 rpm for 2 seconds, and remove the supernatant. 1m after repeating
l sterilized water was added, dispensed in 50 μl aliquots and stored at -20 ° C.
The gold particles were vortexed for 5 minutes. While vortexing, 5μl DNA (1mg / 1ml), 50μl 2.5M CaCl2
, 20 μl 0.1M spermidine was added, and the mixture was vortexed for another 3 minutes and allowed to stand for 1 minute to precipitate fine particles. Ten
After centrifuging at 000 rpm for 10 seconds, the supernatant was removed, 250 μl of 100% ethanol was added without disturbing the pellet, and the mixture was vortexed to firmly stir, and then centrifuged to remove the supernatant. Add 30 μl 100% ethanol and vortex thoroughly to add pSAMDCpro1000: GUS to the gold particles.
And PBI221 were adsorbed. Then wash the carrier discs one by one in 70% ethanol and dry thoroughly,
Add 5 gold particles prepared above to the center of the carrier.
A particle gun was prepared by adding microliters of each and drying.

The condition of the particle gun is that the gold particles are 0.1 μm.
The pressure was 1350 psi, and there were 3 shots from the launch port. For staining, soak in GUS staining solution (50 mM phosphate buffer, 0.5 M potassium ferricyanide, 0.5 M potassium ferrocyanide, 1 mM X-Gluc) and suck with a vacuum device to thoroughly infiltrate the solution, and then overnight at 37 ° C. After incubation, the spot of GUS was confirmed. (Fig. 2,
3) (2) Confirmation of promoter activity by staining of transformed tobacco LB obtained in the above Example 1 (9) and Example 2 (8)
A4404 / pBI101-SAMDCpro1000: GUS and LBA4404 / pBI101-SAMD
Transformed tobacco with Cpro500: GUS and LBA4404 / pBI101-35S: GUS was subjected to low temperature treatment at 4 ° C. for 4 days, followed by low temperature treatment for 4 days.
After darkening and darkening for 1 day, GUS stain
(50mM phosphate buffer, 0.5M potassium ferricyanide, 0.5M potassium ferrocyanide, 1mM X-Gluc), suck the solution with a vacuum device to infiltrate the solution well, and incubate at 37 ℃ overnight to remove chlorophyll. For removal, it was immersed in 50% ethanol for 10 minutes and 100% ethanol for 10 hours. Especially LBA4404 / pBISAMpro100
Staining by GUS was observed in 0: GUS and LBA4404 / pBI35S: GUS (FIGS. 4 and 6). In addition, LBA4404 / pBISAMpro500: GUS
, No staining with GUS was seen. (Fig. 5). (3) Quantitative measurement of GUS activity of transformed tobacco LB obtained in Example 1 (9) and Example 2 (8)
A4404 / pBI101-SAMDCpro1000: GUS and LBA4404 / pBI101-SAMD
Transformed tobacco with Cpro500: GUS and LBA4404 / pBI35S: GUS was subjected to low temperature treatment at 4 ° C for 4 days in the dark, followed by low temperature treatment at 25 ° C.
Darkening, treated for 1 day, at 25 ℃, darkening,
The one that was processed during the same period was prepared. GUS activity is 4
-Methylumbelliferyl glucuronide (4-MUG) was decomposed to measure 4-methylumbelliferone (4-MU) reaction as an index. Treated leaves, about 1
00 mg to 500 μl of buffer solution (50 mM sodium phosphate
(pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 0.1% sarcosyl) was ground using a mortar and pestle, and the ground solution was centrifuged to obtain a supernatant. Of this, 10 μl to 90 μl of 1 m
M 4-methylumbelliferyl glucuronide (4-MU
G) was added and the reaction was carried out at 37 ° C, and after 1 hour, 900μ
The reaction was stopped by adding 1 of 0.2 M sodium carbonate. 4-Methylambelliferone (4-M
U) fluorescence (365 nm excitation, 455 nm emission) is measured, and G
US-activity was calculated. In addition, the protein concentration of the obtained extract was quantified using the Protein assay reagent (BIO-RAD
Company). The results of measuring the GUS activity of each transformed tobacco are shown in Table 1 below.
The numbers are shown as the amount of 4-MU produced per minute per 1 mg of protein in the extract. Especially LBA4404 / pBI1
01-35S: GUS always showed strong promoter activity regardless of temperature treatment. LBA4404 / pBI101-SAMDC1000: G
In the US, a particularly strong promoter activity was found in those given low temperature treatment. Also, LBA4404 / pBI101-SAMDCpro500: G
In the US, almost no promoter activity was found.
(FIGS. 7 and 8) From these results, it was found that the DNA of the present invention can be controlled so as to express the chimeric gene in about 5 to 10 times more depending on the temperature. Example 4 Construction of transgenic tobacco having upstream region of SPDS (1) Spermidine synthase gene, upstream region of SPDS (SP
(DS pro) cloning Using seedlings that had been grown for 1 month after sowing Cucurbita ficifolia Bouche, Plant DNA ZOL (G
Genomic DNA was extracted by using IBCO BRL, and the genomic DNA was cloned by the inverse PCR method. That is, the restriction enzymes NcoI, BamHI, SphI, Dra
After cutting 1 μg of genomic DNA with I and AIW44I,
Self-ligation was performed at tion high (Toyobo Co., Ltd.). PC using 300 ng of ligated DNA as template
SPDS using R reagent kit (LA-PCR kit, Takara Shuzo)
5'upstream region was amplified. The reaction was performed at 94 ° C. for 15 seconds and at 65 ° C. for 12 minutes using the first primer group having the nucleic acid sequences shown in SEQ ID NOS: 14 and 15 for 28 minutes.
Cycled. Next, using this PCR product, PCR was performed under the same conditions using the second primer group having the nucleotide sequences shown in SEQ ID NOs: 16 and 17. The amount and length of this PCR product were confirmed by electrophoresis, and the resulting DNA fragment was used for the AdvanTAge PCR Cloning Kit (Clontech
E. coli JM
109 was transformed and cloned. Then AB
SPD using I PRISM 310 Genetic Analyzer (ABI)
The 5'upstream region base sequence of S was determined. It was confirmed from the primer sequence that the base sequence was entirely in agreement with the cDNA and was a genomic region corresponding to the cDNA. Furthermore, the primers of SEQ ID NOs: 18 and 19 with restriction enzyme sites (XbaI, BamHI) added to both ends were designed to be approximately 1000 bp from the transcription initiation point, inserted into the MCS of pBluescript, and named p SPDS 1000pro. It was This base sequence is shown in SEQ ID NO: 13. Similarly, a sequence of 500 bp from the transcription start point was prepared using SEQ ID NOS: 20 and 21, and pSPD
I named it S500pro. This base sequence is shown in SEQ ID NO: 22. (2) Fusion with β-glucuronidase gene (GUS gene) Plasmid containing β-glucuronidase gene (GUS), pBI221 (Clontech), restriction enzymes, BamHI and Eco
After cutting with RI, agarose gel electrophoresis was performed to take out the GUS gene in the plasmid. This GUS
The gene is a plasmid and the vector pSPDS 1000pro described above
The plasmid pSPDSpro100 was inserted into the restriction enzymes, BamHI and EcoRI sites on pBluescript of pSPDS500pro.
0: GUS and pSPDSCpro500: GUS were obtained. This step is shown in FIG. In addition, p SPDS 1000pro and p SPDS 500pro were digested with restriction enzymes, XbaI and BamHI, and after electrophoresis, XbaI
-The BamHI fragment was inserted into the restriction enzyme XbaI and BamHI sites of the plasmid pBI101-HmB (obtained from Prof. Atsuhiko Shinna, Nara Institute of Technology) to obtain a new plasmid pB.
I101- SPDS pro1000: GUS and pBI101- SPDS pro500: G
I got US. Then the plasmid, pBI101-SPDS1000:
E. coli JM109 was transformed with GUS and pBI101-SPDS500: GUS to obtain Escherichia coli JM109 / pBI101-SPpro1000:
GUS and Escherichia coli JM109 / pBI101-SPpro500: GU
Got s. This process is shown in FIG. (3) Introduction of plasmid into Agrobacterium Escherichia coli JM109 / pBI1 obtained in (2)
01-SPDSpro1000: GUS and Escherichia coli JM109 / pBI
101-SPDSpro500: GUS was cultured in LB medium containing 59 mg / L of kanamycin at 37 ° C. overnight, and a plasmid was extracted from each E. coli. 2 μl of extracted plasmid and ELECTOROMAX A. tumefaciens LBA4404 cell
(GIBCO BRL) 50 μl are mixed, and Cuvet is applied by Gene Pulser II electroporation system (BIO-RAD).
t Gap 0.2cm, Voltage 2.5KV, Field strength 12.5KV /
Electroporation was performed under the conditions of cm, Capacity 25 μF and Resistor 200Ω. Then, in SOC medium for 2 hours, 2
The cells were cultured at 8 ° C. and spread on 50 mg / l kanamycin YEP medium. After culturing at 28 ° C for 2 days, a single colony was selected. The transformant obtained was designated as LBA4404 / pBI101-SP.
They were named DS pro1000: GUS and LBA4404 / pBI101- SPDS pro500: GUS. (4) Aseptic tobacco cultivation Tobacco seeds (obtained from Prof. Yoshihiko Shinna, Nara Institute of Technology)
Several dozen particles were placed in a 1.5 ml tube, 1 ml of 70% ethanol was added, and the mixture was left for 3 minutes. Then sterilized liquid (5%
Soaked in sodium hypochlorite, 0.02% TritonX-100) for 3 minutes and washed 5 times with sterilized water, then MS plate (Murashige-Skoog inorganic salts 4.3 g, sucrose 30 g per liter)
, Myo-inositol 0.1g, gellan gum 2.5g, pH 5.7)
It was placed on the floor. This plate was cultured in a plant incubator (manufactured by Sanyo, MLR-350HT) at 25 ° C for about 2 weeks. After germination, the plants were transplanted to a plant culture pot containing the same MS medium as above, and grown for about 1 month. (5) Infection with Agrobacterium Tobacco leaves cultured for 1 month in the above (4) were treated with a female for about 1.
Cut to about 0 cm, float on liquid MS medium,
The transformant obtained in (3) above, LBA4404 / pBI101- SPDS p
ro1000: GUS and LBA4404 / pBI101- SPDS pro500: GUS 28
50 μL of the culture solution that had been cultivated at 2 ° C. for 2 days was added, and cocultured at 25 ° C. in the dark for 2 days in separate Petri dishes. (6) Leaf pieces after sterilization co-culture were washed several times with sterilized water. (7) Selectively eradicated tobacco leaf pieces of transformed plants are kanamycin (100 mg / L), hygromycin (20 mg / L), carbenicillin (250 mg / L)
L), naphthalene acetic acid (0.02mg / L), benzylaminopurine (1mg / L)
Cultured in lux. Thereafter, it was transplanted to a new MS plate every week. The transformed leaf pieces continued to proliferate, and shoots were formed from the callus-shaped dome-shaped rises after about 1 month. (8) The root of the plant that became a regenerated shoot of the transformed plant is cut off with a shaving blade or a scalpel so that it does not contain the callus part, and it is lightly placed in a plant culture pot containing MS medium at 25 ° C. It was grown under the conditions of 16 hours of light irradiation and 8 hours of darkening. (9) Confirmation of transformant One leaf was cut out from the plant grown in the above (8), and genomic DNA was obtained using Plant DNA ZOL (GIBCO BRL).
By extracting A and performing PCR using the primers of SEQ ID NOS: 23 and 24, it was confirmed that the target DNA fragment was contained. Example 5: Measurement of GUS activity (1) Evaluation of promoter activity by particle gun pSPDS pro1000: GUS obtained in the above Example 4 (2)
Using wild type tobacco leaves, pBI221 was observed for GUS activity in transient expression by particle gun (Bio-Rad). Tobacco leaves, 0.25%
They were arranged in a medium containing gellan gum. The particles to shoot are
60 mg of gold particles were weighed, 1 ml of 70% ethanol was added, and the mixture was vortexed for 5 minutes. After allowing to stand for 15 minutes, centrifuge at 10,000 rpm for 5 seconds, remove the supernatant, and repeat the following operations 3 times.
Add 1 ml of sterilized water, vortex for 1 minute, leave for 1 minute, centrifuge at 10,000 rpm for 2 seconds, and remove the supernatant. 1m after repeating
l sterilized water was added, dispensed in 50 μl aliquots and stored at -20 ° C.
The gold particles were vortexed for 5 minutes. While vortexing, 5μl DNA (1mg / 1ml), 50μl 2.5M CaCl2
, 20 μl 0.1M spermidine was added, and the mixture was vortexed for another 3 minutes and allowed to stand for 1 minute to precipitate fine particles. Ten
After centrifuging at 000 rpm for 10 seconds, the supernatant was removed, 250 μl of 100% ethanol was added without disturbing the pellet, and the mixture was vortexed to firmly stir, and then centrifuged to remove the supernatant. Add 30 μl 100% ethanol and vortex thoroughly to mix the gold particles with pSPDSpro1000: GUS.
PBI221 was adsorbed. After that, 1 carrier disk
Each particle was washed in 70% ethanol, dried sufficiently, and 5 μl each of the gold particles prepared above was added to the center of the carrier and dried to prepare a particle gun.

The condition of the particle gun is that the gold particles are 0.1 μm.
The pressure was 1350 psi, and there were 3 shots from the launch port. For staining, soak in GUS staining solution (50 mM phosphate buffer, 0.5 M potassium ferricyanide, 0.5 M potassium ferrocyanide, 1 mM X-Gluc) and suck with a vacuum device to thoroughly infiltrate the solution into the sample, and then overnight at 37 ° C. After incubation, the spot of GUS was confirmed. (Fig. 1
0, 11) (2) Confirmation of promoter activity by staining of transformed tobacco LBA4404 / pBI101-SPDSpro obtained in Example 4 (9) above.
1000: GUS-transformed tobacco was subjected to low temperature treatment at 4 ° C for 4 days, low temperature treatment for 4 days, followed by treatment at 25 ° C for 1 day in the dark, and then GUS staining solution (50 mM phosphate buffer, 0.5 M
Soaked in potassium ferricyanide, 0.5M potassium ferrocyanide, 1mM X-Gluc), sucked with a vacuum device to thoroughly infiltrate the solution into the sample, and then incubated overnight at 37 ° C, 10% in 50% ethanol to remove chlorophyll. It was soaked in 100% ethanol for 10 minutes. LBA4
Staining with GUS was observed in 404 / pBISPDSpro1000: GUS (FIG. 12). Example 6 Construction of transgenic tobacco having upstream region of arginine decarboxylase (1) Arginine decarboxylase gene, upstream region of ADC (ADC
Cpro) cloning Seedlings of Cucurbita ficifolia Bouche were planted for 1 month after seeding, and plant DNAZOL (G
Genomic DNA was extracted by using IBCO BRL, and the genomic DNA was cloned by the inverse PCR method. That is, after cutting 1 μg of genomic DNA with restriction enzymes EcoRV, SacI, and SphI, Ligation high
(Toyobo Co., Ltd.) was used for self-ligation. PCR reagent kit using 300 ng of ligated DNA as template
(LA-PCR kit, Takara Shuzo) was used to amplify the 5'upstream region of ADC. The reaction was carried out at 94 ° C. for 15 hours using the first primer group having the nucleic acid sequences shown in SEQ ID NOS: 27 and 28.
28 cycles were performed for 12 seconds at 65 ° C. for seconds.
Next, using this PCR product, PCR was performed under the same conditions using the second primer group having the nucleotide sequences shown in SEQ ID NOs: 26 and 29. The amount and length of this PCR product were confirmed by electrophoresis, and the resulting DNA fragment was used as AdvanTA
ge PCR Cloning Kit (Clontech) was used to introduce into vector pTAdv and transform E. coli JM109,
Cloned. After that, ABI PRISM 310 Genetic An
The alyzer (ABI) was used to determine the 5'upstream region base sequence of the ADC. The base sequence is cDNA based on the sequence of the primer.
It was confirmed that the genomic region corresponds to the cDNA and corresponds to the cDNA. Furthermore, we designed the primers of SEQ ID NOs: 30 and 31 with restriction enzyme sites (XbaI, BamHI) added to both ends so that it would be approximately 1000 bp from the transcription start point.
It was amplified by R, inserted into MCS of PUC118 using TAKARA BKL Kit, and named pADC1000pro. This base sequence is shown in SEQ ID NO: 25. (2) Fusion with β-glucuronidase gene (GUS gene) A plasmid containing the β-glucuronidase gene (GUS), pBI221 (Clontech), was cleaved with a restriction enzyme,
Agarose gel electrophoresis was carried out, and G in the plasmid was analyzed.
The US gene was removed. This GUS gene was inserted into the plasmid, the restriction enzymes on pBluescript of the above vector pADC1000pro, BamHI and EcoRI sites to obtain the plasmid, pADCpro1000: GUS. This process is shown in FIG. In addition, pADC1000pro was digested with restriction enzymes XbaI and BamHI.
Cleavage and digestion with XbaI-BamHI fragment,
A new plasmid, pBI101-ADCpro1000: GUS, was inserted into the restriction enzyme XbaI and BamHI sites of pBI101-HmB (obtained from Nara Institute of Technology, Professor Akihiko Shinna).
Got Then, the plasmid, pBI101-ADC1000: GU
E. coli JM109 was transformed with S and Escherichia co
li JM109 / pBI101-ADCpro1000: GUS was obtained. This process is illustrated
Shown in 14. (3) Introduction of plasmid into Agrobacterium Escherichia coli JM109 / pBI1 obtained in (2)
01-ADCpro1000: GUS was cultured in LB medium containing 59 mg / L of kanamycin at 37 ° C. overnight, and a plasmid was extracted from each E. coli. 2 μl of extracted plasmid and ELECTOROMAX A. tumefaciens LBA4404 cell
(GIBCO BRL) 50 μl are mixed, and Cuvet is applied by Gene Pulser II electroporation system (BIO-RAD).
t Gap 0.2cm, Voltage 2.5KV, Field strength 12.5KV /
Electroporation was performed under the conditions of cm, Capacity 25 μF and Resistor 200Ω. Then, in SOC medium for 2 hours, 2
The cells were cultured at 8 ° C. and spread on 50 mg / l kanamycin YEP medium. After culturing at 28 ° C for 2 days, a single colony was selected. The transformants obtained were LBA4404 / pBI101-ADC.
It was named pro1000: GUS. (4) Aseptic tobacco cultivation Tobacco seeds (obtained from Prof. Yoshihiko Shinna, Nara Institute of Technology)
Several dozen particles were placed in a 1.5 ml tube, 1 ml of 70% ethanol was added, and the mixture was left for 3 minutes. Then sterilized liquid (5%
Soaked in sodium hypochlorite, 0.02% TritonX-100) for 3 minutes and washed 5 times with sterilized water, then MS plate (Murashige-Skoog inorganic salts 4.3 g, sucrose 30 g per liter)
, Myo-inositol 0.1g, gellan gum 2.5g, pH 5.7)
It was placed on the floor. This plate was cultured in a plant incubator (manufactured by Sanyo, MLR-350HT) at 25 ° C for about 2 weeks. After germination, the plants were transplanted to a plant culture pot containing the same MS medium as above, and grown for about 1 month. (5) Infection with Agrobacterium Tobacco leaves cultured for 1 month in the above (4) were treated with a female for about 1.
Cut to about 0 cm, float on liquid MS medium,
The transformant obtained in (3) above, LBA4404 / pBI101-ADCpro1
000: Add 50 μL of the culture solution obtained by culturing GUS at 28 ° C. for 2 days,
Cocultivation was carried out in a separate petri dish for 2 days at 25 ° C. in the dark. (6) Leaf pieces after sterilization co-culture were washed several times with sterilized water. (7) Selectively eradicated tobacco leaf pieces of transformed plants are kanamycin (100 mg / L), hygromycin (20 mg / L), carbenicillin (250 mg / L)
L), naphthalene acetic acid (0.02mg / L), benzylaminopurine (1mg / L)
Cultured in lux. Thereafter, it was transplanted to a new MS plate every week. The transformed leaf pieces continued to proliferate, and shoots were formed from the callus-shaped dome-shaped rises after about 1 month. (8) The root of the plant that became a regenerated shoot of the transformed plant is cut off with a shaving blade or a scalpel so that it does not contain the callus part, and it is lightly placed in a plant culture pot containing MS medium at 25 ° C. It was grown under the conditions of 16 hours of light irradiation and 8 hours of darkening. (9) Confirmation of transformant One leaf was cut out from the plant grown in the above (8), and genomic DNA was obtained using Plant DNA ZOL (GIBCO BRL).
A was extracted using the primers of SEQ ID NOs: 23 and 24
It was confirmed by PCR that the target DNA fragment was contained. Example 7 Measurement of GUS activity (1) Evaluation of promoter activity by particle gun pADCpro1000: GUS and pB obtained in the above Example 6 (2)
Using I221 wild-type tobacco leaves, GUS activity was observed with transient expression by particle gun (Bio-Rad). Tobacco leaves, 0.25%
They were arranged in a medium containing gellan gum. The particles to shoot are
60 mg of gold particles were weighed, 1 ml of 70% ethanol was added, and the mixture was vortexed for 5 minutes. After allowing to stand for 15 minutes, centrifuge at 10,000 rpm for 5 seconds, remove the supernatant, and repeat the following operations 3 times.
Add 1 ml of sterilized water, vortex for 1 minute, leave for 1 minute, centrifuge at 10,000 rpm for 2 seconds, and remove the supernatant. 1m after repeating
l sterilized water was added, dispensed in 50 μl aliquots and stored at -20 ° C.
The gold particles were vortexed for 5 minutes. While vortexing, 5μl DNA (1mg / 1ml), 50μl 2.5M CaCl2,
20 μl 0.1M spermidine was added, vortexed for 3 minutes, and allowed to stand for 1 minute to precipitate fine particles. 1000
After centrifuging at 0 rpm for 10 seconds, the supernatant was removed, 250 μl of 100% ethanol was added without disturbing the pellet, and the mixture was vortexed firmly, and then centrifuged to remove the supernatant. Add 30 μl 100% ethanol and vortex thoroughly to add pSAMDCpro1000: GUS to the gold particles.
And PBI221 were adsorbed. Then wash the carrier discs one by one in 70% ethanol and dry thoroughly,
Add 5 gold particles prepared above to the center of the carrier.
A particle gun was prepared by adding microliters of each and drying.

The conditions of the particle gun are 0.1 μm of gold particles.
The pressure was 1350 psi, and there were 3 shots from the launch port. For staining, soak in GUS staining solution (50 mM phosphate buffer, 0.5 M potassium ferricyanide, 0.5 M potassium ferrocyanide, 1 mM X-Gluc) and suck with a vacuum device to thoroughly infiltrate the solution into the sample, and then overnight at 37 ° C. After incubation, the spot of GUS was confirmed. (2) Quantitative measurement of GUS activity of transformed tobacco LB obtained in Example 6 (9) and Example 2 (8)
Transformed tobacco with A4404 / pBI101-ADCpro1000: GUS and LBA4404 / pBI35S: GUS was subjected to low temperature treatment at 4 ° C in the dark for 5 days, followed by treatment at 25 ° C in the dark for 1 day. ℃, in the dark, after the processing during the same period, 4
℃, with light, low temperature treatment for 5 days, followed by 25 ℃, with light,
One that was treated for 1 day and one that was treated at 25 ° C with light and during the same period were prepared. The GUS activity was measured using the reaction of degrading 4-methylumbelliferyl glucuronide (4-MUG) to produce 4-methylumbelliferone (4-MU) as an index. Treated leaves, about 100m
500 μl of buffer solution (50 mM sodium phosphate (pH 7.
(0), 10 mM EDTA, 0.1% Triton X-100, 0.1% sarcosyl) was ground using a mortar and pestle, and the ground solution was centrifuged to obtain a supernatant. 90 μl of 1 mM 4-methylumbelliferyl glucuronide (4-MUG) was added to 10 μl
Was added and the reaction was carried out at 37 ° C. After 1 hour, 900 μl of 0.2 M sodium carbonate was added to stop the reaction. Fluorescence (excitation at 365 nm, emission at 455 nm) of 4-methylambelliferone (4-MU) generated in the reaction solution was measured, and GUS-activity was calculated. The protein concentration of the obtained extract was quantified by the method using Protein assay reagent (BIO-RAD). The results of measuring the GUS activity of each transformed tobacco are shown in Table 1 below. The numbers are shown as the amount of 4-MU produced per minute per 1 mg of protein in the extract. Especially LBA4404 / pBI101-3
In 5S: GUS, a strong promoter activity was always observed regardless of temperature treatment. Regarding LBA4404 / pBI101-ADC1000: GUS, particularly strong promoter activity was observed in those given low temperature treatment. (FIGS. 15 and 16) From these results, the DNA of the present invention showed about 2 to about 2 chimera genes depending on the temperature without being influenced by light.
It was found that the expression can be controlled so as to be expressed four times more.

[0042]

[Sequence list]                                SEQUENCE LISTING <110> TOYOBO RESEARCH CENTER CO., LTD <120> A PROMOTER DERIVED FROM PLANT <130> 16802JP <140> <141> <160> 31 <170> PatentIn Ver. 2.1 Sequence number 1 Array length: 1212 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence Type: Genomic DNA Array ggtccggtac ttggtctctc caatctattt gccatgtgta aagtcgaaaa taaccctatt 60 aggcagcggt gcgtgttgag tcgcacgtga gaaactaaat taaaaaaatg caacgctgat 120 agagttggaa tcacgtgata agaatttctt ccggctttga cactaagcca cctacgttta 180 taaagaataa aaattacttt ttatgtctaa taaatattta aaatttaaaa tattaaatta 240 cggatattta taatgggatg attttggaat agtttaaaaa tgttttttta aaaaagattt 300 tcaataattt ttttatataa tttaccattt atttaaaaat actctaaata acattttttt 360 tccctttatc ttcaaaagat attttattaa ttaagcggct caaatttatt ggttttttca 420 agacaatact cttgatttgc aacttcccat aaaattatta tttattatta ttattattaa 490 ttaagtataa atctttttcc ctatccctct cattattttc tgtttttttc ccttaaaaat 540 cccttcttct tagtttaatt ttgaaactcg ttttttatgg acccaattgg ataaaaaagt 600 taaatatact taattaataa ataataaaaa tatattggtt tggatttgat aacaattcta 660 tgataattaa aagaaaaaac ttttacccat atttttatgt ttaatttttc aagttaaaaa 720 atggttataa tctctctaag acagattatc tctataatat ttatttaata aattattttt 780 taaaaagtgt agaaacaaaa aagagatata tttttaaagc ttcgagaata aaattaaaat 840 ttaaaataat aatatttttt taaatccgga gaaaaggaat ggcagttatt ttattatgta 900 ataatttggc accccgcgta gggaaagccg ataagggcta cgctgtcatt tcacatagct 960 aagttacgga atttagggat gagagaaaat agggcaaaac cgtaattaaa cccaaaccgc 1020 gtggtttttt cgctatataa gttcctcttc gctattgaag acctcacaga aacgcaaacc 1080 gtctgcggcc tcttgaattc tcatcgtttc tctctctttc gaattttgtt ttctttcgct 1140 ctcagccctt tcttgcaagt tttattttag gcattctccg ggtttccttc tcctccgcta 1200 attcttttca tc1212 Sequence number 2 Array length: 30 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array tacgaacgca gttggagtag gcagcagatc 30 Sequence number 3 Array length: 30 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array atgcggagtc taccatccat gtcactccag 30 Sequence number 4 Array length: 30 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array ccaccgtgtg gtctaacgtc ttcaatgctg 30 Sequence number 5 Array length: 30 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array gaattggacg acctggacct gtgtaaggtg 30 Sequence number 6 Array length: 33 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array aatctagagg tccggtactt ggtctctcca atc 33 Sequence number 7 Array length: 35 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array ccggatccga tgaaaagaat tagcggagga gaagg 35 Sequence number 8 Array length: 36 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array cctctagaga aactcgtttt ttatggaccc aattgg 36 SEQ ID NO: 9 Array length: 35 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array ccggatccga tgaaaagaat tagcggagga gaagg 35 SEQ ID NO: 10 Array length: 650 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: genomic DNA Array gaaactcgtt ttttatggac ccaattggat aaaaaagtta aatatactta attaataaat 60 aataaaaata tattggtttg gatttgataa caattctatg ataattaaaa gaaaaaactt 120 ttacccatat ttttatgttt aatttttcaa gttaaaaaat ggttataatc tctctaagac 180 agattatctc tataatattt atttaataaa ttatttttta aaaagtgtag aaacaaaaaa 240 gagatatatt tttaaagctt cgagaataaa attaaaattt aaaataataa tattttttta 300 aatccggaga aaaggaatgg cagttatttt attatgtaat aatttggcac cccgcgtagg 360 gaaagccgat aagggctacg ctgtcatttc acatagctaa gttacggaat ttagggatga 420 gagaaaatag ggcaaaaccg taattaaacc caaaccgcgt ggttttttcg ctatataagt 480 tcctcttcgc tattgaagac ctcacagaaa cgcaaaccgt ctgcggcctc ttgaattctc 540 catcgtttct tctctttcga attttgtttt ctttcgctct cagccctttc ttgcaagttt 600 tattttaggc attctccggg tttccttctc ctccgctaat tcttttcatc 650 SEQ ID NO: 11 Array length: 20 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array ttgtgtggaa ttgtgagcgg 20 SEQ ID NO: 12 Array length: 19 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array cgcgatccag actgaatgc 19 SEQ ID NO: 13 Array length: 1212 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: genomic DNA Array aaacctaaat gggaacgcat atgttttaaa attttaattt ctaaatagtt aaaagttatt 60 ttcgagagaa taaataataa ctaaataatt atactataaa caaaatataa attcaaatat 120 atatctgtta agtaaatcaa gcatcaccta atttggatta taaattcaaa tcgtcattat 180 catccatcat aatatactct ctaccaaatt taaggcaaaa aaaatttgtt taattacatt 240 actttttgag tcaatttcat gtcaagatta gtctcgtagc cctacgtctt ttagtctaaa 300 ttctctctgt tcacgtcata aattttgttt catataacta tctaacgtta ttgcactttg 360 caccgttttt attgatatct tcaactgtac gaggtgaaga aaagccacga aaaaccctat 420 ttagtacacg tgttggtgcc tctgtgctta atgcattaat catacgaccc tttaaggcaa 480 aaattcgaac atttgtcatt ttagttgaac atataatata taatcatgta ttgttaagat 640 tctaaccgta acgtttcatg gccccgatat actcttgcga tatggtcatg ttatttcgac 600 agaggtccgt ttaacatatt ttgtcctctt tcacatgcct ctcaatttta gatattatag 660 atttatttat gtatttctgg cgaaaaataa aattatattt taataaaata ttatattaca 720 ctaaaaaaaa tatacctttc atggtcaaaa ctacactata gtatttggaa gtttatggaa 780 aaatcgaacc tatacaaaaa catgaaagaa cgagatgaaa aaaaaaacat aaatcgaggg 840 cagtaagaaa aagaaaaaag atcgaggaaa ccgtaatttg attccttcaa aagcgaagcc 900 tttcacggac cctttgcgat tatataaatg gaggaagaac tgggtcagtt tgctcagtgc 960 tcatatccaa cgggtcatac agaagcactc cccactgtat tgggatttgg gattttagcg 1020 agtcgatagt agaggga 1037 SEQ ID NO: 14 Array length: 30 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array acagaggaaa tagaatcggg ctgtgaggcg 30 SEQ ID NO: 15 Array length: 26 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array ggttctcgtt atcggtggag gagacg 26 SEQ ID NO: 16 Array length: 30 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array attctcaatc tcaggtttct tcgccgccgc 30 SEQ ID NO: 17 Array length: 30 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array gaggtggctc gccattcatc tgttgagcag 30 SEQ ID NO: 18 Array length: 30 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array cctctagaaa cctaaatggg aacgcatatg 30 SEQ ID NO: 19 Array length: 28 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array ccggatcctc cctctactat cgactcgc 28 SEQ ID NO: 20 Array length: 29 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array cctctagaca cgtgttggtg cctctgtgc 29 SEQ ID NO: 21 Array length: 28 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array ccggatcctc cctctactat cgactcgc 28 SEQ ID NO: 22 Array length: 618 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: genomic DNA Array acacgtgttg gtgcctctgt ggctaatgca ttaatcaata cgacctttaa ggcaaaaatt 60 cgaacatttg tcatttttag ttgaacatat aatatataat catgtattgt taagattcta 120 accgtaacgt ttcatggccc cgatatactc ttgcgatatg gtcatgttat ttcgacagag 180 gtccgtttaa catattttgt cctctttcac atgcctctca attttagata ttatagattt 240 atttatgtat ttctggcgaa aataaaatta tattttaata aaatattata ttacactaaa 300 aaaaatatac ctttcatggt caaaactaca ctatagtatt tggaagttta tggaaaaatc 360 gaacctatac aaaaacatga aagaacgaga tgaaaaaaaa aacataaatc gagggcagta 420 agaaaaagaa aaaagatcga ggaaaccgta atttgattcc ttcaaaagcg aagccttttc 480 acggaccctt tgcgattata taaatggagg aagaactggg tcagtttgct caagtgctca 540 tatccaacgg gtcatacaga agcactcccc actgtattgg ggattttggg attttaagcg 600 agtcgatagt agaaggga 618 SEQ ID NO: 23 Array length: 20 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array ttgtgtggaa ttgtgagcgg 20 SEQ ID NO: 24 Array length: 19 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array cgcgatccag actgaatgc 19 SEQ ID NO: 25 Array length: 919 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: genomic DNA Array aataagatta ttttttaaaa ttctaaattt tcatctttac tgtactaaaa tattaaaata 60 tctcagaatt atctggctcg acaattgaga tattaatgag tttacatata ttcaatttgt 120 taaaacataa tcttttttac taaaaattag tcgtttaaat ttctatattt acatattgta 180 aaaaatttag gtatttatga tggtgacacg cccaatacta gggctggatg agtgtcactc 240 gagagtagcc gatttgcgag tcacaaccta ctcattagtt acgatatttg catatctcac 300 cgtctttcat attttcattt cgacttgctg aagaatcttt ctcgagcttg tttcttaatg 360 gagagttcga agacaattca cgagtcgaca atgctgataa aaaaaagata catgaatgaa 420 ttgagatcat atttattctc acaaatttta agtcggagag agattcaaat gagggggcaa 480 gatccagatg gtccacctca gcaacatctt aacaccacga aacttcccgt taccttcgtc 540 ttcttgctaa taatatcgta tttcattata ccgcgatatt tttattttta aaaatcgcca 600 cgtaagcaaa gttaacctga aatcaaagcc tcgggagacg taattgccaa tggagggtcc 660 cgcatggaag tcggccacgc ggcaacccga cgctgattag tccacgatag aatctcagtt 720 ttttttaaaa aaattattat tattaattac gacaaaaata attaatcgtc cttttttaaa 780 aaccgcgata aaattaaatc gggtcatcat aataaactac cgggttgatg gagggctgtt 840 aagtccgacc cgacccgaga catattctcc tataaaatgg cacttggttg ttcatgcaaa 900 tctcgctcat tctaacctc 919 SEQ ID NO: 26 Array length: 31 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array aatctagacg ctcttcacca tgcagatctg g 31 SEQ ID NO: 27 Array length: 19 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array tgtccgacga cggatcaag 19 SEQ ID NO: 28 Array length: 20 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array cctcttggtc tccttctctg 20 SEQ ID NO: 29 Array length: 31 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array aaggatcccg tttggagtct ctccaatcgg c 31 SEQ ID NO: 30 Array length: 28 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array aatctagacg gaacaaagcg gtatatcc 28 SEQ ID NO: 31 Array length: 28 Sequence type: Nucleic acid Sequence type: single-stranded Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array aaggatccat tggagagccc tcggagac 28

[Brief description of drawings]

FIG. 1 pSAMDCpro1000 containing the promoter of the present invention:
GUS, pSAMDCpro500: GUS and pBI101-SAMDCpro1000: GU
It is a figure showing the construction process of S, pBI101-SAMDCpro1000: GUS, pBI101-35S: GUS.

FIG. 2 is a diagram in which transient expression was confirmed in tobacco leaves using pSAMDCpro1000: GUS.

FIG. 3 is a diagram in which transient expression was confirmed in tobacco leaves using pBI221.

FIG. 4 is a diagram showing GUS staining of LBA4404 / pBI101-SAMDCpro1000: GUS-transformed tobacco.

FIG. 5 is a diagram showing GUS staining of LBA4404 / pBI101-SAMDCpro500: GUS-transformed tobacco.

FIG. 6 shows GUS staining of tobacco transformed with LBA4404 / pBI101-SAMDCpro1000: GUS.

FIG. 7: The expression level of GUS enzyme in transformed tobacco containing the promoter of the present invention was quantitatively measured, and the activity at 25 ° C. was 1.0.
FIG. 4 is a diagram relatively showing the activity at 4 ° C. LBA4404 / pBI101-SAMDCpro1000: GUS and S described in the previous examples
Compatible with AMDCpro1000, LBA4404 / pBI101-SAMDCpro500: GU
Compatible with S and SAMDCpro500, LBA4404 / pBI101-35S: GUS and 35
S corresponds. Two independent lines were repeated twice and the average value is shown.

FIG. 8: The expression level of GUS enzyme in transgenic tobacco containing the promoter of the present invention was quantitatively measured. LBA4404 / pBI101-SAMDCpro1000: GUS and SAMDCpro100 as described in the previous examples.
0 supports, LBA4404 / pBI101-SAMDCpro500: GUS and SAMDCpr
o500 supports, LBA4404 / pBI101-35S: GUS and 35S support. Two independent lines were repeated twice and the average value is shown.

FIG. 9: pSPDSpro1000: G containing the promoter of the present invention
It is a figure showing the construction process of US and pSPDSpro500: GUS.

FIG. 10 is a diagram in which transient expression was confirmed in tobacco leaves using pSPDS pro1000: GUS.

FIG. 11 is a diagram in which transient expression was confirmed in tobacco leaves using pBI221.

FIG. 12 is a GUS-stained image of tobacco transformed with LBA4404 / pBI101-SPDSpro1000: GUS.

FIG. 13: pBI101-SPDSpro1000: GUS and pBI101-ADCpro10
It is a figure showing the construction process of 00: GUS.

FIG. 14: pBI101-SPDSpro1000: GUS, pBI101-SPDSpro50
It is a figure showing the construction process of 0 and pBI101-ADCpro1000: GUS.

FIG. 15: The expression level of GUS enzyme in transgenic tobacco containing the promoter of the present invention was quantitatively measured to show its activity at 25 ° C.
It is the figure which showed the activity of 4 degreeC when set to 1.0 relatively. LBA4404 / pBI101-ADCpro1000: GU described in the above example
Compatible with S and SAMDCpro1000, LBA4404 / pBI101-35S: GUS and 3
5S corresponds. dark is a sample processed in the dark,
light is a sample processed in the presence of light.
Two independent lines were repeated twice and the average value is shown.

FIG. 16: The expression level of GUS enzyme in transformed tobacco containing the promoter of the present invention was quantitatively measured. LBA4404 / pBI101-ADCpro1000: GUS and SAMDCpro100 described in the above example.
0 corresponds, and LBA4404 / pBI101-35S: GUS and 35S correspond.
dark is a sample processed in the dark, light is a sample processed in the presence of light. Two independent lines were repeated twice and the average value is shown.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Izumi Inohara             2-1-1 Katata, Otsu City, Shiga Prefecture Stock Association             Company Toyobo Research Institute F term (reference) 2B030 AA02 AB03 AD08 CA16 CA17                       CA19 CB02 CD03 CD07 CD09                       CD10 CD13 CD17                 4B024 AA08 CA01 DA01 DA06 EA04                       FA02 GA11 GA17 GA19 HA14                 4B065 AA88Y AA89X AB01 CA53

Claims (26)

[Claims] 1. A DNA fragment containing a promoter region of a plant-derived S-adenosylmethionine decarboxylase gene. 2. A DNA fragment containing the following DNA (a) or (b): (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 1 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
And DNA having the ability to act as a plant promoter 3. The DNA fragment according to claim 1, which comprises the following DNA (a) or (b): (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 1 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
And DNA having the ability to act as a plant promoter 4. A DNA comprising the nucleotide sequence according to claim 2 or 3.
And a DNA fragment capable of hybridizing to the plant under stringent conditions and acting as a plant promoter. 5. The DNA fragment according to claim 1, which is controllable by temperature. 6. The DN according to any one of claims 1 to 5.
A vector containing the A fragment. 7. The vector according to claim 6, wherein the heterologous gene is under the transcriptional control of the DNA fragment. 8. A transformed plant in which the vector according to claim 6 or 7 is introduced into a host plant cell. 9. A seed and its progeny obtained from the transformed plant according to claim 8. 10. A DNA fragment having a promoter activity for a plant-derived spermidine synthase gene. 11. A DNA containing the following DNA (a) or (b):
fragment. (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 13 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
And DNA having the ability to act as a plant promoter 12. The DNA fragment according to claim 10, which comprises the following DNA (a) or (b): (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 13 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
And DNA having the ability to act as a plant promoter 13. A hybridizing with a DNA consisting of the nucleotide sequence of claim 11 or 12 under stringent conditions and having the ability to act as a plant promoter.
DNA fragment. 14. A vector containing the DNA fragment according to any one of claims 10 to 13. 15. The vector according to claim 14, wherein the heterologous gene is under the transcriptional control of the DNA fragment. 16. A transformed plant obtained by introducing the vector according to claim 14 or 15 into a host plant cell. 17. A seed and its progeny obtained from the transformed plant according to claim 16. 18. A DNA fragment having a promoter activity of a plant-derived arginine decarboxylase gene. 19. A DNA containing the following DNA (a) or (b):
fragment. (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 25 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
And DNA having the ability to act as a plant promoter 20. The DNA fragment according to claim 18, which comprises the following DNA (a) or (b): (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 25 (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted or added in the nucleotide sequence of (a),
And DNA having the ability to act as a plant promoter
fragment. 21. A DNA capable of hybridizing with a DNA comprising the nucleotide sequence according to claim 19 or 20 under stringent conditions and having the ability to act as a plant promoter. 22. The DNA fragment according to claim 18, which is controllable by temperature. 23. A vector comprising the DNA fragment according to any one of claims 18 to 22. 24. The vector according to claim 23, wherein the heterologous gene is under the transcriptional control of the DNA fragment. 25. A transformed plant, which is prepared by introducing the vector according to claim 23 or 24 into a host plant cell. 26. A seed and its progeny obtained from the transformed plant according to claim 25.