JP2001136978A - Plant promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a plant promoter capable of being used for plant breeding techniques by introducing a gene. SOLUTION: This plant promoter contains a DNA expressed by the following (a) or (b); (a) a DNA consisting of a base sequence having base numbers from 1743 to 2301 out of the base sequence expressed by the sequence number 1, (b) a DNA capable of hybridizing to the DNA described in (a) under a stringent condition and having promoter function in plant cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a plant promoter.

[0002]

2. Description of the Related Art Production of transgenic plants to which useful traits have been imparted by introducing and expressing genes into plants has been attempted, and some of the produced plants have been put to practical use. In order to express a gene introduced into a plant, the gene is usually placed under the control of a promoter operable in plant cells. In the above-described plant breeding, in order to produce a transformed plant having a desired trait, it is necessary to select a promoter having properties suitable for the purpose of breeding and to express a gene under the control.

[0003]

However, the types of plant promoters that can be used for such a plant breeding technique by gene transfer are not yet sufficient, and the development of a new plant promoter has been strongly desired.

[0004]

Under these circumstances, the present inventors have conducted intensive studies, and as a result, have found a new DNA having a promoter function in plant cells, and have reached the present invention. That is, the present invention provides: 1) a promoter containing the following DNA (a) or (b): (a) a DNA comprising a base sequence from base numbers 1743 to 2301 of the base sequence shown in SEQ ID NO: 1 b) DNA that hybridizes to the DNA of (a) under stringent conditions and has a promoter function in plant cells 2) DN of the following (c), (d), (e) or (f)
(C) a DNA consisting of the base sequence from base numbers 1020 to 2301 out of the base sequence shown in SEQ ID NO: 1; and (d) a base out of the base sequence shown in SEQ ID NO: 1 (E) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 (f) DNA comprising the nucleotide sequence of SEQ ID NO: 1 (f) Under conditions of stringency with the DNA of (c), (d) or (e) above DNA that hybridizes and has a promoter function in plant cells 3) A chimeric gene comprising the promoter of the preceding clause 1, a desired gene and a terminator operably linked, 4) a vector containing the promoter of the preceding clause 1 5) The vector according to 4 above, which comprises a gene insertion site and a terminator downstream of the promoter. A vector containing the chimeric gene according to item 3; 7) a transformant having the promoter according to item 1 introduced into a host cell; 8) a transformant having the chimeric gene according to item 3 introduced into a host cell; 9) a transformant obtained by introducing the vector according to 4 above into a host cell; 10) a transformant according to 7 above wherein the host cell is a microbial cell; 11) a transformant according to 7 above wherein the host cell is a plant cell. A transformant, 12) a method for producing a chimeric gene, comprising a step of operably linking the promoter, the desired gene and the terminator according to the above 1; And a method for producing a vector, which comprises the step of operably linking the promoter to a plant cell. The method of producing a transformed plant, which comprises the step of expressing the desired gene under the control of the motor, there is provided a.

[0005]

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

In the present invention, “promoter function” means the ability to act as a promoter, that is, the ability to initiate gene transcription, and “plant promoter” means a DN having a promoter function in a plant.
A. The promoter of the present invention includes the following DNA (a) or (b) (hereinafter referred to as the promoter DNA of the present invention). (A) a DNA consisting of the nucleotide sequence of base numbers 1743 to 2301 in the base sequence represented by SEQ ID NO: 1 (b) hybridizing to the above DNA under stringent conditions, and having a promoter function in plant cells DNA having herein Here, the DNA that “hybridizes under stringent conditions” refers to the following DNA. That is, (1) under high ion concentration [for example, 6XSSC (900mM
Sodium chloride, 90 mM sodium citrate) and the like. ], Hybridized under the temperature condition of 65 ° C., and D consisting of the base sequence from base numbers 1743 to 2301 in the base sequence represented by SEQ ID NO: 1
A DNA-DNA hybrid is formed with NA, and (2) low ion concentration [for example, 0.1 × SSC (15 mM sodium chloride, 1.5 mM sodium citrate) or the like is used. ]
And DNA that can maintain the hybrid even after washing at 65 ° C. for 30 minutes. Specifically, for example, in the base sequence represented by SEQ ID NO: 1, a DNA consisting of a base sequence in which a part of the base sequence has been deleted, substituted or added, and the like, more specifically, for example, A DNA consisting of the nucleotide sequence represented by SEQ ID NO: 26 can be mentioned. Such a DNA may be a DNA cloned from the natural world, a DNA obtained by artificially introducing a deletion, substitution or addition of a base into a DNA cloned from the natural world, or an artificial DNA. DNA may be synthesized. The promoter of the present invention may be upstream and / or of the promoter DNA of the present invention as long as it has the ability to act as a promoter.
Alternatively, other DNA may be included downstream. In particular,
For example, of the base sequence represented by SEQ ID NO: 1, DN consisting of the base sequence from base numbers 1020 to 2301
A, of the nucleotide sequence represented by SEQ ID NO: 1,
From the base sequence of SEQ ID NO: 1 to 2301, the DNA of the base sequence of SEQ ID NO: 1, the DNA of the base sequence of SEQ ID NO: 27, and the like.

The promoter DNA of the present invention is, for example, Gl
It can be isolated from soybean genomic DNA such as ycin max using the PCR method. Specifically, Glycin m
Genomic DNA is extracted from a piece of tissue such as soybean leaves such as ax. For the extraction method, Hirofumi Uchimiya, "A Manual for Plant Gene Manipulation, How to Make Transgenic Plants (Kodansha Scientific)" 1990 (ISBN4-06-15351)
3-7 C3045), CTAB method described on pages 71-74 and SORogers and A.
The urea-phenol method described in J. Bendich, PlantMol. Biol., 5:69 (1985) and the like can be mentioned. Using the obtained genomic DNA as a template, for example, an oligonucleotide having a nucleotide sequence represented by nucleotide numbers 1743 to 1782 of SEQ ID NO: 1 and a nucleotide number 227 of SEQ ID NO: 1
An oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by 0 to 2301,
By performing the CR, of the base sequence represented by SEQ ID NO: 1, a DNA consisting of the base sequence from base numbers 1743 to 2301, or one or more bases in the base sequence of the DNA is deleted, substituted, or added. DNA consisting of the base sequence shown in SEQ ID NO: 26, for example, DNA consisting of the base sequence of base numbers 1742 to 2300 in the base sequence shown in SEQ ID NO: 26, Or about 0.6 kbp DNA such as a DNA consisting of a base sequence from base numbers 1742 to 2300 in the base sequence represented by SEQ ID NO: 29
Can be amplified. In this case, if necessary, a gapped duplex (described later)
It is also possible to combine DNA site-specific mutagenesis methods such as the x) method or the Kunkel method. Take
PCR conditions include, for example, 10 × Ex
5 μl of Taq buffer (Takara Shuzo), 2.5 mM dNTP mixture (each
Includes 2.5 mM dATP, dGTP, dCTP, dTTP. ) 4μl (dATP,
dGTP, dCTP, and dTTP each have a final concentration of 0.2 mM), 20 μM
Primer 0.25-1.25μl (final concentration 0.1-0.5μl)
M), template genomic DNA 0.1-0.5 μg, ExTaq polymerase
(Takara Shuzo Co., Ltd.) 94
The conditions include, for example, keeping the temperature at 1 ° C. for 1 minute, then at 55 ° C. for 2 minutes, and further at 72 ° C. for 5 minutes as one cycle, and performing this for 30 cycles in total. Further, by appropriately designing the oligonucleotide used for the primer as described above based on the base sequence shown in SEQ ID NO: 1,
Or a DNA consisting of a base sequence in which one or more bases have been deleted, substituted or added in the base sequence of the DNA can also be obtained by PCR. In addition, a restriction enzyme recognition sequence or the like may be added to the 5 'end of the primer used for PCR. The DNA amplified as described above is referred to as “M
olecular Cloning: A Laboratory Manual 2nd editio
n "(1989), Cold Spring Harbor Laboratory Press,
`` Current Protocols in Molecular Biology '' (1987),
It can be cloned into a vector according to the usual method described in John Wiley & Sons, Inc. ISBNO-471-50338-X and the like. Specifically, for example, T
Plasmid vectors and S included in A cloning kit
It can be cloned using a plasmid vector such as pBluescriptII from tratagene.

Further, in the above method, for example, an oligonucleotide having a base sequence represented by base numbers 1743 to 1782 of SEQ ID NO: 1 and a base complementary to the base sequence represented by base numbers 2270 to 2301 of SEQ ID NO: 1 Instead of using an oligonucleotide having a sequence as a primer, base numbers 1020 to 1 of SEQ ID NO: 1
When PCR is performed using, as primers, an oligonucleotide having a base sequence represented by base sequence 050 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 2270 to 2301 of SEQ ID NO: 1, the promoter of the present invention can be obtained. Of the base sequence represented by SEQ ID NO: 1, a DNA consisting of the base sequence from base numbers 1020 to 2301 or a base sequence in which one or more bases are deleted, substituted or added in the base sequence of the DNA DNA, for example, a DNA consisting of the base sequence of base numbers 1020 to 2300 in the base sequence represented by SEQ ID NO: 26, or the base sequence represented by base numbers 1022 to 2302 of the base sequence represented by SEQ ID NO: 28 Or a base sequence represented by SEQ ID NO: 29, It is possible to amplify DNA of approximately 1.3kbp of DNA such that the nucleotide sequence from base No. 1019 to 2300. In this case, if necessary, gapped duplex (described later)
Or a DNA site-specific mutagenesis method such as the Kunkel method. The amplified DNA can be cloned into a vector by the same method as described above.

[0009] For example, base number 17 of SEQ ID NO: 1
An oligonucleotide having a nucleotide sequence represented by any one of SEQ ID NOs: 43 to 1782;
Instead of using an oligonucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO: 01 as a primer, an oligonucleotide having a base sequence represented by base numbers 2 to 32 of SEQ ID NO: 1 and a base number 227 of SEQ ID NO: 1
An oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by 0 to 2301,
By performing CR, the promoter of the present invention, SEQ ID NO: 1
In the base sequence represented by the following, a DNA consisting of a base sequence from base numbers 2 to 2301 or a DNA consisting of a base sequence in which one or more bases are deleted, substituted or added in the base sequence of the DNA, for example, the sequence Number 26
DNA consisting of the base sequence from base number 2 to 2300 in the base sequence represented by the above, or DNA consisting of the base sequence from base number 2 to 2302 in the base sequence shown by the SEQ ID NO: 28, or SEQ ID NO: Of the base sequence represented by 29, a DNA of about 2.3 kbp such as a DNA consisting of a base sequence of base numbers 2 to 2300 can be amplified. In this case, if necessary, a gapped duplex method described later,
Alternatively, a DNA site-specific mutagenesis method such as the Kunkel method can be combined. Amplified DN
A can be cloned into a vector in the same manner as described above.

When a long chain DNA is amplified by using the PCR method as described above, DNA containing an unexpected deletion, substitution or addition mutation is amplified due to the nature of Ex Tap polymerase. Methods for isolating a DNA having a specific sequence from the obtained DNA include, for example,
Isao Shimamoto, Takuji Sasaki “Cell Engineering Separate Volume, Plant Cell Engineering Series 7, PCR Experiment Protocol for New Plants (Shujunsha)” 1997, ISBN4-87962-173-0 C3345 A PCR method using a plurality of characteristic primer sets can be given. That is, to isolate a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1, for example, using the obtained genomic DNA as a template, an oligonucleotide having the nucleotide sequence represented by nucleotide numbers 2027 to 2063 of SEQ ID NO: 1 And an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by nucleotide numbers 2270 to 2301 of SEQ ID NO: 1 as a primer, to thereby obtain a P of about 280 bp.
A CR product can be obtained. Next, an oligonucleotide having a base sequence represented by base numbers 1743 to 1782 of SEQ ID NO: 1 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 2027 to 2063 of SEQ ID NO: 1 as primers PCR using
, A PCR product of about 320 bp can be obtained. The above about 280 bp and about 320 bp DN
A, the 3′-end was blunted using a Klenow fragment (manufactured by Takara Shuzo Co., Ltd.), and an oligonucleotide having a nucleotide sequence represented by nucleotide numbers 1743 to 1782 of SEQ ID NO: 1 and a nucleotide of SEQ ID NO: 1 By performing PCR using an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by any of SEQ ID NOs: 2270 to 2301 as a primer, the nucleotide sequence represented by SEQ ID NO: 1
About 0.6 kb consisting of the base sequence from 743 to 2301
p DNA can be amplified, and the amplified DNA can be cloned into a vector by the same method as described above. Further, using the obtained genomic DNA as a template, for example, nucleotide numbers 1020 to 1058 of SEQ ID NO: 1
By using an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by nucleotide numbers 1197 to 1239 of SEQ ID NO: 1 as primers, to obtain about 2
A 20 bp PCR product can be obtained. SEQ ID NO: 1
An oligonucleotide having a base sequence represented by base numbers 1320 to 1361 and base number 15 of SEQ ID NO: 1
A PCR product of about 300 bp can be obtained by performing PCR using an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by 76 to 1618 as a primer. Nucleotide number 1576 to 1 of SEQ ID NO: 1
Approximately 210 bp of PCR is performed by using as a primer an oligonucleotide having a nucleotide sequence represented by base number 618 and an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by base numbers 1747 to 1782 of SEQ ID NO: 1. The product can be obtained. An oligonucleotide having a base sequence represented by base numbers 1197 to 1239 of SEQ ID NO: 1 and base number 1 of SEQ ID NO: 1
A PCR product of about 170 bp can be obtained by performing PCR using an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by 320 to 1361 as a primer. About 220 bp, about 300 bp,
In addition to about 210 bp and about 170 bp, SEQ ID NO: 1
In the base sequence represented by, base numbers 1743 to 2343
Said DNA of about 0.6 kbp consisting of a base sequence up to 01
And the 3′-end is blunted using a Klenow fragment (manufactured by Takara Shuzo Co., Ltd.).
By performing PCR using, as primers, an oligonucleotide having a base sequence represented by base sequence No. 058 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 2270 to 2301 of SEQ ID NO: 1, thereby obtaining SEQ ID NO: In the base sequence represented by No. 1, base number 1
About 1.3 kb consisting of the nucleotide sequence from 020 to 2301
p DNA can be amplified, and the amplified DNA can be cloned into a vector in the same manner as described above. Alternatively, using the obtained genomic DNA as a template, for example, an oligonucleotide having a base sequence represented by base numbers 2 to 40 of SEQ ID NO: 1;
About 1.1 kbp by performing PCR using an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 1081 to 1117 as primers.
PCR product can be obtained. The PCR product of about 1.1 kbp and the above-mentioned DNA of about 1.3 kbp consisting of the base sequence of base numbers 1020 to 2301 of the base sequence shown in SEQ ID NO: 1 were mixed, and a Klenow fragment (Takara Shuzo) was mixed. After blunting the 3'-end using
PCR is performed using, as primers, an oligonucleotide having a base sequence represented by base numbers 2 to 40 of SEQ ID NO: 1 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 2270 to 2301 of SEQ ID NO: 1. As a result, a PCR product of about 2.3 kbp consisting of the base sequence from base numbers 2 to 2301 in the base sequence shown in SEQ ID NO: 1 can be obtained, and the amplified DNA can be obtained by the same method as described above. Can be cloned.

In order to isolate a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 28, for example, the obtained genomic DNA
Is used as a template, and from base number 1744 to 1
By performing PCR using, as primers, an oligonucleotide having a nucleotide sequence represented by nucleotide sequence No. 783 and an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by nucleotide numbers 2271 to 2302 of SEQ ID NO: 28, whereby SEQ ID NO: Of the base sequence represented by 28, about 0.6 consisting of the base sequence from base numbers 1744 to 2302
kbp PCR product can be obtained and the amplified DNA
Can be cloned into a vector in the same manner as described above. In addition, using the obtained genomic DNA as a template, an oligonucleotide having a base sequence represented by base numbers 1022 to 1067 of SEQ ID NO: 28 and a base sequence complementary to the base sequence represented by base numbers 2271 to 2302 of SEQ ID NO: 28 By performing PCR using an oligonucleotide having a base sequence as a primer, about 1.3 kbp of PC consisting of the base sequence from base numbers 1022 to 2302 in the base sequence represented by SEQ ID NO: 28
An R product can be obtained, and the amplified DNA can be cloned into a vector in the same manner as described above. Further, using the obtained genomic DNA as a template, an oligonucleotide having a base sequence represented by base numbers 2 to 40 of SEQ ID NO: 28, and an oligonucleotide having base number 5 of SEQ ID NO: 28
An oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence shown by 44 to 583 was used as a primer
By performing the CR, a PCR product of about 580 bp can be obtained. Nucleotide numbers 589 to 6 of SEQ ID NO: 28
By performing PCR using, as primers, an oligonucleotide having a base sequence represented by base sequence 31 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 1037 to 1073 of SEQ ID NO: 28, about 480 bp PCR product can be obtained. Further, an oligonucleotide having a base sequence represented by base numbers 544 to 583 of SEQ ID NO: 28 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 589 to 631 of SEQ ID NO: 28 as primers By performing PCR using the PCR, a PCR product of about 90 bp can be obtained. In addition to the above about 580 bp, about 480 bp, and about 90 bp, of the base sequence represented by SEQ ID NO: 28,
After mixing the above approximately 1.3 kbp DNAs consisting of up to 2 nucleotide sequences and blunting the 3′-end using a Klenow fragment (manufactured by Takara Shuzo Co., Ltd.), it is represented by nucleotide numbers 2 to 40 of SEQ ID NO: 28. PCR using a base sequence and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 2271 to 2302 of SEQ ID NO: 28 as primers
Of the base sequence represented by SEQ ID NO: 28, a PCR product of about 2.3 kbp consisting of the base sequence from base numbers 2 to 2302 can be obtained, and the amplified DNA can be obtained by the same method as described above. Can be cloned into a vector.

In order to isolate a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 29, for example, the obtained genomic DNA
Is used as a template, and base numbers 1742 to 1
By performing PCR using, as primers, an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 781 and an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by nucleotide numbers 2269 to 2300 in SEQ ID NO: 29, whereby SEQ ID NO: Of the base sequence represented by 29, about 0.6 consisting of the base sequence from base numbers 1742 to 2300
kbp PCR product can be obtained and the amplified DNA
Can be cloned into a vector in the same manner as described above. In addition, using the obtained genomic DNA as a template, an oligonucleotide having a nucleotide sequence represented by nucleotide numbers 1019 to 1057 of SEQ ID NO: 29 and a nucleotide sequence complementary to the nucleotide sequence represented by nucleotide numbers 1196 to 1238 of SEQ ID NO: 29 By performing PCR using an oligonucleotide having a base sequence as a primer, about 22
A 0 bp PCR product can be obtained. SEQ ID NO: 29
PCR is performed using, as primers, an oligonucleotide having a base sequence represented by base numbers 1491 to 1531 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 2269 to 2300 in SEQ ID NO: 29 Thereby, a PCR product of about 810 bp can be obtained. Furthermore, an oligonucleotide having a base sequence represented by base numbers 1196 to 1238 of SEQ ID NO: 29 and a base number 1487 of SEQ ID NO: 29
And an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by
By performing R, a PCR product of about 340 bp can be obtained. After mixing the above DNAs of about 220 bp, about 810 bp, and about 340 bp and blunting the 3′-end using a Klenow fragment (manufactured by Takara Shuzo), SEQ ID NO: 29
By performing PCR using, as primers, a base sequence represented by base numbers 1019 to 1057 and a oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 2269 to 2300 in SEQ ID NO: 29, Of the base sequence represented by No. 29, about 1.3 consisting of the base sequence from base numbers 1019 to 2300
kbp PCR product can be obtained and the amplified DNA
Can be cloned into a vector in the same manner as described above. Further, using the obtained genomic DNA as a template, an oligonucleotide having a base sequence represented by base numbers 2 to 40 of SEQ ID NO: 29 and a base sequence complementary to the base sequence represented by base numbers 369 to 409 of SEQ ID NO: 29 By performing PCR using an oligonucleotide having a base sequence as a primer, a PC of about 410 bp is obtained.
The R product can be obtained. Base number 4 in SEQ ID NO: 29
Oligonucleotides having the nucleotide sequences of 96 to 530, and nucleotides 588 to 628 of SEQ ID NO: 29
By performing PCR using an oligonucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by as a primer, a PCR product of about 130 bp can be obtained. An oligonucleotide having a base sequence represented by base numbers 657 to 699 of SEQ ID NO: 29;
By performing PCR using an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 752 to 793 of No. 9 as a primer, a PCR product of about 140 bp can be obtained. Using, as primers, an oligonucleotide having a base sequence represented by base numbers 752 to 793 of SEQ ID NO: 29 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 1034 to 1070 of SEQ ID NO: 29 By performing PCR, a PCR product of about 320 bp can be obtained. Using, as primers, an oligonucleotide having a base sequence represented by base numbers 369 to 409 of SEQ ID NO: 29 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 496 to 530 of SEQ ID NO: 29 By performing PCR, about 160
bp PCR product can be obtained. Further, an oligonucleotide having a base sequence represented by base numbers 588 to 628 of SEQ ID NO: 29 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 657 to 699 of SEQ ID NO: 29 as primers By performing PCR using the PCR, a PCR product of about 110 bp can be obtained. About 410 bp, about 130 b
p, about 140 bp, about 320 bp, about 160 bp, and about 110 bp, and in addition to the above-mentioned about 1.3 kbp DNA consisting of the base sequence from base numbers 1019 to 2300 of the base sequence shown in SEQ ID NO: 29, Klenow
After blunting the 3'-end using a fragment (Takara Shuzo),
Performing PCR using, as primers, a base sequence represented by base numbers 2 to 40 of SEQ ID NO: 29 and an oligonucleotide having a base sequence complementary to the base sequence represented by base numbers 2269 to 2300 of SEQ ID NO: 29 As a result, a PCR product of about 2.3 kbp consisting of the base sequence of base numbers 2 to 2300 in the base sequence represented by SEQ ID NO: 29 can be obtained, and the amplified DNA is converted into a vector in the same manner as described above. Can be cloned.

Further, the promoter DNA of the present invention may be, for example, at least a part of the base sequence represented by SEQ ID NO: 1,
Preferably, the DNA is obtained by labeling a DNA consisting of about 250 bases or more, using this as a probe to hybridize to DNA derived from a plant or the like, and detecting and isolating DNA specifically bound to the probe. it can. By using the above method, it is possible to obtain a DNA having a further reduced length of the promoter DNA of the present invention. Such a DNA can be used as a more compact promoter as long as it has the ability to act as a promoter, and is included in the scope of the present invention as one method of using the promoter DNA of the present invention. As the DNA for hybridizing the probe as described above, for example, a genomic DNA library derived from plants such as soybean and the like can be used. The DNA library includes "Molecular Cloning: A Laboratory Ma
nual 2nd edition "(1989), Cold Spring Harbor Lab
oratory Press, `` Current Protocols in Molecular Bio
logy "(1987), John Wiley & Sons, Inc. ISBNO-471-50
338-X and the like, according to the usual library production method, for example, STRATAGENE λFixII, λEMBL3, λ
Using vectors such as EMBL4 and λDASH II, STRATAGENE
A genomic DNA library can be prepared using Gigapack packaging Extracts or the like for in vitro packaging and used. Alternatively, a commercially available genomic library can be used. Examples of a method for hybridizing a probe to such DNA include a colony hybridization method and a plaque hybridization method, and a method may be selected depending on the type of a vector used for preparing a library. When the library to be used is constructed using a plasmid vector, colony hybridization is performed. Specifically, first, a transformant is obtained by introducing the DNA of the library into host microbial cells, the resulting transformant is diluted and plated on an agar medium, and cultured at 37 ° C. until colonies appear. . If the library has been prepared using a phage vector, a plaque hybridization method is performed. Specifically, first, the host microbial cells and the phage of the library are mixed under conditions capable of infecting, and then mixed with a soft agar medium and spread on an agar medium. Thereafter, the cells are cultured at 37 ° C. until plaques appear.
More specifically, for example, "Molecular Cloning: A Labo
ratory Manual 2nd edition (1989) "Cold Spring Har
bor Laboratory Press, according to the method described in 2.60 to 2.65 p. etc., agar 1 mm <sup>2</sup> per 0.1 to NZY agar
A phage library of approximately 9.0 × 10 <sup>5</sup> pfu is spread at a density of 1.01.0 pfu and cultured at 37 ° C. for 6-10 hours. Next, in any of the above-mentioned hybridization methods, a membrane is placed on the surface of the agar medium on which the above-mentioned culture has been performed, and the transformant or phage is transferred to the membrane. After the membrane is subjected to an alkali treatment, a neutralization treatment is performed, and then a treatment for immobilizing DNA on the membrane is performed. More specifically, for example, in the case of plaque hybridization, cloning and sequencing: Plant Biotechnology Experiment Manual (edited by Watanabe, Sugiura, Rural Bunkasha)
1989), etc., on the agar medium, such as nitrocellulose membrane or nylon membrane, for example, Hybond-N + (registered trademark of Amersham)
And allowed to stand for about 1 minute to allow the phage particles to adsorb to the membrane. Next, the membrane was washed with an alkaline solution (1.5 M
The phage particles were dissolved by soaking in sodium chloride and 0.5N sodium hydroxide for about 3 minutes to elute the phage DNA on the membrane, and then neutralized (1.5 M sodium chloride, 0.5 M Tris-HCl buffer pH7. Perform the treatment of dipping in 5) for about 5 minutes. Further, the membrane is washed with a washing solution (0.3 M sodium chloride, 30 mM sodium citrate, 0.2 M Tris-HCl buffer pH 7.5) for about 5 minutes, and then baked at about 80 ° C. for about 90 minutes, for example. Immobilize the phage DNA on the membrane. Using the thus prepared membrane, hybridization is performed using the DNA as a probe. Hybridization, for example,
DMGlover `` DNAcloning, A practical Approach '' IRL
Press (1985) ISBN 0-947946-18-7, Cloning and sequencing: Plant biotechnology experiment manual (edited by Watanabe and Sugiura, Rural Culture Co., 1989), or J. Samb
rook, EFFrisch, T. Maniatis, Molecular Cloning: A
Laboratory Manual 2nd edition (1989) "Cold Sprin
It can be performed according to the description of gHarbor Laboratory Press and the like. To label with a radioactive isotope of DNA used as a probe, for example, it can be used Boehringer Co. and Takara Shuzo Co. Random Labeling Kit or the like, a dCTP normal PCR reaction solution composition ^(alpha-32 P) Labeling can also be performed by performing a PCR reaction using DNA used as a probe as a template instead of dCTP. When the DNA used for the probe is labeled with a fluorescent dye, for example, ECL Direct Nucleic Acid Labeling and Detectio
n System or the like can be used. There are various types of reagents and temperature conditions for performing hybridization, for example, including 450 to 900 mM sodium chloride and 45 to 90 mM sodium citrate, and sodium dodecyl sulfate (SD
S) at a concentration of 0.1-1.0% and denatured non-specific DNA
200200 μg / ml, optionally containing albumin, ficoll, polyvinylpyrrolidone, etc.
Prehybridization solution, which may contain a concentration of ~ 0.2%, preferably 900 mM sodium chloride, 90 mM
Of sodium citrate, 1.0% SDS, and 100 μg / ml denatured Calf-thymus DNA was added to the pre-hybridization solution per cm ^{2 of} the membrane prepared as described above.
The membrane is immersed in the solution and kept at 42 to 65 ° C for 1 to 4 hours, preferably at 65 ° C for 2 hours. It then contains, for example, 450-900 mM sodium chloride and 45-90 mM sodium citrate and has an SDS of 0.1
0-200 μg of denatured non-specific DNA at a concentration of ~ 1.0%
/ ml, optionally containing albumin, ficoll, polyvinylpyrrolidone, etc. each at a concentration of 0-0.2%, preferably a hybridization solution, preferably 900 mM sodium chloride, 90 mM sodium citrate, 1.0 mM % SDS and 100 μg / ml denatured Calf-thymu
hybridization solution containing s DNA, probes (membrane 1 cm ² per obtained by preparing the above-mentioned method
1.0 × 10 ^{4 to} 2.0 × 10 ⁶ cpm) is prepared at a rate of 50 to 200 μl per 1 cm ² of the membrane, and the membrane is immersed in the solution for 12 to 20 hours at 42 to 65 ° C., preferably
Incubate at 65 ° C for 16 hours to perform hybridization reaction. After the hybridization reaction, the membrane is taken out, and containing 42 to 65 ° C. containing 15 to 300 mM sodium chloride, 1.5 to 30 mM sodium citrate, and 0.1 to 1.0% SDS.
Washing is preferably performed twice with a washing solution containing 15 mM sodium chloride, 1.5 mM sodium citrate and 1.0% SDS at 65 ° C. for 15 minutes. Further, the membrane is lightly rinsed with a 2 × SSC solution (300 mM sodium chloride, 30 mM sodium citrate) and then dried. This membrane is subjected to, for example, autoradiography to detect the position of the probe on the membrane, thereby detecting the position of the DNA that hybridizes with the used probe on the membrane. A clone having the DNA can be isolated by specifying a clone corresponding to the position of the detected DNA on the membrane on the original agar medium and picking it. Specifically, for example, the membrane is attached to an imaging plate (Fuji Film Co., Ltd.).
Expose for 4 hours, then transfer the imaging plate to BAS2
Analyze using 000 (manufactured by Fuji Film) and detect the signal. In the agar medium used for preparing the membrane, a portion corresponding to the position where the signal was detected (having a diameter of about
5mm), and then add about 0.5ml of SM buffer (50mM
The phage particles are eluted by soaking in a Tris-HCl solution (pH 7.5, 0.1 M sodium chloride, 7 mM magnesium sulfate, 0.01% gelatin) for 2 to 16 hours, preferably 3 hours. The obtained phage particle eluate was used in J. Sambrook, EFFrisch, T. Mania.
tis, Molecular Cloning: A Laboratory Manual 2nd e
dition (1989) "Cold Spring Harbor Laboratory Pres
s Spread on an agar medium according to the method described in (2.60 to 2.65) and culture at 37 ° C for 6 to 10 hours. Using this agar medium, phage DNA is immobilized on a membrane in the same manner as described above, and hybridization is performed using this membrane and the aforementioned probe. The phage particles were eluted from the portion of the agar medium used for preparing the membrane corresponding to the position where the signal was detected, spread on an agar medium, prepared a membrane in the same manner as described above, and hybridized. I do. By repeatedly specifying and purifying such a phage clone, a phage clone containing a DNA having a nucleotide sequence that hybridizes with the probe used can be obtained. DNA contained in a clone obtained by performing screening by hybridization as described above is a plasmid vector that is easy to prepare and analyze, such as commercially available pUC118, pUC119, and pBLU.
It can be subcloned into ESCRIPT KS +, pBLUESCRIPT KS- and the like. Plasmid DNA was prepared from the clone isolated by the method described above, and F. Sanger, S. Nicklen,
ARCoulson, Proceeding of Natural Academy of Sc
The nucleotide sequence can be analyzed by the dideoxy terminator method described in ience USA (1977) 74: 5463-5467 and the like. For sample preparation for nucleotide sequence analysis,
For example, ABI PRISM Dye Terminat from PerkinElmer
A commercially available reagent such as or Cycle Sequencing Ready Reaction Kit may be used. Alternatively, sample preparation for nucleotide sequence analysis is described in Molecul, J. Sambrook, EFFrisch, T. Maniatis.
ar Cloning: A Laboratory Manual 2nd edition (198
9) "can also be carried out according to the primer extension method described in Cold Spring Harbor Laboratory Press, page 13.15.

[0014] A reporter gene, for example, a β-glucuronidase gene is ligated downstream of the DNA obtained as described above, and this is cloned into a vector as necessary, and then the particle gun method and Agrobacterium infection described below are used. By introducing the DNA into a plant cell using a method such as a method and examining the expression of a reporter gene, the promoter activity of the DNA in the plant cell, that is, the promoter function can be examined. For example, a cell extract of plant cells such as tobacco cultured cells BY-2 into which the DNA has been introduced is prepared and its β-glucuronidase activity is measured by an enzymatic method, and the activity value is used as an index, or The cells were immersed in a staining solution to which 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid had been added to observe the deposition of a blue pigment, and the degree of pigment deposition was used as an index. Check if there is a promoter function,
The promoter DNA of the present invention can be obtained. Also,
Similarly, the above D linked to the reporter gene
For example, NA is introduced into a plant cell such as a tobacco wild-type cell to regenerate a plant, and β-glucuronidase activity in each tissue of the plant or its progeny is measured, or the plant or Each tissue of the progeny or a section thereof was immersed in a staining solution to which 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid was added, and the deposition of the blue pigment was observed. By examining the promoter function, the promoter DNA of the present invention can also be obtained. The reporter gene is β
-Not only the glucuronidase gene but also a luciferase gene, a chloramphenicol acetyltransferase gene, a green fluorescein protein gene and the like can be used.

The promoter DNA of the present invention has SEQ ID NO: 1.
Can also be obtained by introducing a mutation into the nucleotide sequence of DNA consisting of the nucleotide sequence represented by Specifically, for example, A. Greener, M. Callahan, Strategies, 1994, 7
Vol., Pages 32-34, etc., mutations may be randomly introduced into the DNA consisting of the nucleotide sequence of SEQ ID NO: 1. Also, W. Kramer, et al., Nucleic Acids Res
earch, 1984, vol. 12, p. 9441 or W. Kramer, HJF
rits, Methods in Enzymology, 1987, vol. 154, p. 350, etc.
lex) method or TAKunkel, Proc. of Natl. Acad.
Sci. USA, 1985, 82, 488 or TAKunke
l, et al., Methods in Enzymology, 1987, 154, 36
According to the Kunkel method described on page 7, etc., site-specific mutations may be introduced into the DNA comprising the nucleotide sequence of SEQ ID NO: 1. Using a DNA consisting of the base sequence represented by SEQ ID NO: 1 as a template, an oligonucleotide having a base sequence in which one or more bases have been deleted, substituted or added in a part of the base sequence represented by SEQ ID NO: 1 as a primer PCR was performed using SEQ ID NO: 1.
Can also be used to amplify a DNA consisting of a base sequence in which one or more bases have been deleted, substituted or added in the base sequence represented by. Further, a chimeric DNA in which one to several nucleotide sequences of the nucleotide sequence represented by SEQ ID NO: 1 are replaced with a part of the nucleotide sequence of another promoter.
May be produced. Further, for example, S. Henikoff, et a
l., Gene, 1984, 28, 351, C. Yanisch-Perron, et.
al., Gene, 1985, vol. 33, p. 103, etc., may be used to prepare a DNA having a nucleotide sequence in which a part of the nucleotide sequence shown in SEQ ID NO: 1 has been deleted. By examining the promoter function of the DNA thus obtained as described above, the promoter DNA of the present invention can be obtained.

The promoter of the present invention is the promoter DN of the present invention of (a) or (b) obtained as described above.
A may contain any one of A, (a),
The DNA of (b) may be contained together, or may be contained repeatedly. Further, the promoter of the present invention may include a nucleotide sequence having an effect of increasing the transcription efficiency of a gene in a plant. As the base sequence, for example, a sequence constitutively exhibiting a transcription efficiency increasing effect in a plant, a species-specific, tissue-specific or time-specific sequence exhibiting the transcription efficiency increasing effect in a plant, or a pathogenic microorganism in a plant Infection, light, heat, dryness,
Sequences whose transcription efficiency increasing effect is induced by stress such as salt or injury can be given. Specific examples of the nucleotide sequence having the effect of increasing the efficiency of gene transcription in plants include, for example, upstream of the transcription start site of the octopine synthase gene of Agrobacterium.
Downstream of the region from base 333 to base 116, upstream of the transcription start site of the mannopine synthase gene
A transcription translation activation sequence in which the region from the eighth base to the 138th base is also linked, downstream of the region from the 318th base to the 213th base upstream of the transcription start point of the mannopine synthase gene , A transcription / translation activation sequence consisting of a region from the 333rd base upstream to the 116th base upstream of the transcription start point of the octopine synthase gene (The Plant Journal, 7 (4): 661-676 (1995) ), A nucleotide sequence comprising the 343rd base to the 91st base upstream of the transcription start site of the cauliflower mosaic virus 35S promoter (Nature, 313: 810-812 (1985)), tomato ribulose-1,5-diphosphate Nucleotide sequence (Plant Cell, 1: 217-227 (1990)) containing from the 1099th base to the 205th base upstream of the transcription start site of the acid carboxylase oxidase small subunit gene (rbc-3A). PR1a gene Nucleotide sequence containing bases 902 to 287 from the upstream of the transcription start site (Plant Cell,
2: 357-366 (1990)), a nucleotide sequence comprising from the 1300th base upstream to the 195th base upstream of the transcription start site of the potato protease inhibitor gene (PI-II) (Pla
nt Cell, 2: 61-70 (1990)).

Further, the promoter of the present invention has SEQ ID NO: 1.
And a base sequence having a transcription efficiency increasing effect included in the base sequence represented by Furthermore, by identifying such a base sequence, for example, the promoter of the present invention containing the base sequence repeatedly can be prepared, or by linking the base sequence to another DNA having a promoter function in a plant cell. Alternatively, a new plant promoter can be created. In order to identify the nucleotide sequence, for example, a reporter gene is expressed in plant cells under the control of various promoter DNAs of the present invention, and the expression levels are compared to analyze the nucleotide sequence contributing to the effect of increasing the transcription efficiency. Good to do. More specifically, for example, a plurality of DNAs each consisting of a base sequence in which one or more bases are deleted or substituted in the base sequence represented by SEQ ID NO: 1 are prepared, and a reporter gene such as β-
The glucuronidase genes are ligated and introduced into a plant cell such as a cultured tobacco cell BY-2 using a method such as a particle gun method or an Agrobacterium infection method described below. Next, a cell extract of the cells is prepared and its β-glucuronidase activity is measured by an enzymatic method and the activity value is used as an index, or the cells are treated with 5-bromo-4-chloro-3-indolyl-β- The cells were immersed in a staining solution to which D-glucuronic acid was added, and the deposition of blue pigment was observed. Using the degree of pigment deposition as an index, the expression levels of reporter genes in each cell were compared, and the results were linked to the reporter gene. By comparing with the base sequence of the DNA, a base sequence contributing to the effect of increasing the transcription efficiency can be clarified. Similarly, the DNA linked to a reporter gene is introduced into a plant cell such as a tobacco wild-type cell to regenerate a plant from the cell, and the β in the tissue of the plant or its progeny is determined. -Glucuronidase activity is measured, or each tissue of the plant or its progeny or a section thereof is subjected to 5-
By immersing in a staining solution to which bromo-4-chloro-3-indolyl-β-D-glucuronic acid was added and observing the deposition of a blue pigment, the expression levels of reporter genes controlled by the above DNAs were compared, and the transcription efficiency was determined. The nucleotide sequence that contributes to the enhancement effect can also be clarified.

To express a desired gene in a plant cell using the promoter of the present invention, the promoter of the present invention
A gene in which a desired gene and a terminator are operably linked (hereinafter, referred to as the chimeric gene of the present invention) can be used. Here, the desired gene is a gene to be expressed in a plant, 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 are ligated downstream of the promoter of the present invention in a sense direction or an antisense direction depending on the purpose. The terminator is a DNA having a transcription terminating action in a plant to be introduced, and is, for example, a terminator (NOS) of a nopaline synthase gene derived from a Ti-plasmid of Agrobacterium sp. A virus-derived terminator can be used. Further, the chimeric gene of the present invention may contain the nucleotide sequence of the promoter of the present invention or a part thereof in a repeated form. The term "in a operable form" means that when a chimeric gene of the present invention is introduced and a plant cell is transformed, the target gene contained in the chimeric gene is expressed under the control of the promoter and the terminator of the present invention. As such, it is in a state linked to these promoters and terminators.

In the vector having the promoter of the present invention, the vector is a DNA which can be propagated in a cell, such as a plasmid, a phage, a phagemid or the like which can be amplified in cells such as Escherichia coli, yeast, plant cells and animal cells. And the selection may be made according to the host cell and application. Specifically, for example, pUC-based plasmids [pUC118, pUC119 (Takara Shuzo) etc.], pSC101-based plasmids, Ti-plasmid [p
BI101, pBI121 (CLONTECH), Bluescript phagemid [pBluescript SK (+/-) (STRATAGENE)
M13 phage [mp10, mp11 (Amersham) etc.],
λ phage [λgt10, gt11 (Amersham) etc.], cosmids [SuperCosI (STRATAGENE) etc.] and the like,
By incorporating the promoter of the present invention into such a vector, a vector having the promoter of the present invention can be constructed. In the vector having the promoter of the present invention as described above, if a gene insertion site and a terminator are further provided downstream of the promoter, it is convenient for constructing a vector for expressing a desired gene in plant cells. Here, the gene insertion site is, for example,
Restriction enzymes commonly used in genetic engineering techniques are nucleotide sequences that can be specifically recognized and cleaved, and the only type of restriction enzyme recognition sequence that is present only on the vector having the promoter and terminator of the present invention is preferable. Such a gene insertion site, the promoter and the terminator of the present invention are ligated on a vector so that the promoter of the present invention, the desired gene and the terminator can function when the desired gene is inserted into the gene insertion site. It is preferable to be in such a position. Such a vector can be obtained, for example, by inserting the DNA of the promoter of the present invention into a plasmid containing a gene insertion site and a terminator, specifically, a multicloning site (gene insertion site) such as pBI101.3 (manufactured by CLONTECH). Can be built. Further, a vector having a gene insertion site, specifically, pBIN19 (Nucl.Acid Res. 12: 8711-8721 (1984))
Can also be constructed by inserting the promoter and terminator of the present invention into a multiple cloning site (gene insertion site). The vector having the chimeric gene of the present invention can be used for introducing the chimeric gene into host cells. In the vector, for example, the chimeric gene of the present invention is cloned into the vector as described above, or a desired gene is cloned into the gene insertion site of the vector having the promoter, gene insertion site and terminator of the present invention as described above. Can be prepared. Specifically, for example, pBI101.3
The vector prepared by inserting the DNA of the promoter of the present invention into a multicloning site (gene insertion site) of (CLONTECH) is cleaved with a restriction enzyme, and a reporter gene (β- By removing the glucuronidase gene) 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 as described above is a promoter of the present invention,
In addition to the desired gene or terminator to be expressed, a marker gene (for example, a kanamycin resistance gene, a hygromycin resistance gene, a neomycin resistance gene,
A gene capable of conferring herbicide resistance on a plant). The vector may contain the nucleotide sequence of the promoter of the present invention in a repeated form.

The vector of the present invention is described, for example, in J., Sambrook,
E., F., Frisch, T., Maniatis, Molecular Cloning 2nd Edition (1989) (published by Cold Spring Harbor Laboratory), etc. by the calcium chloride method, electroporation method, etc. Such a microbial cell (transformant) that can be introduced into a bacterium and has the vector of the present invention introduced therein is useful for preparing DNA of the chimeric gene of the present invention, introducing the chimeric gene into plant cells, and the like. . DNA encoding the promoter of the present invention or DNA of the chimeric gene of the present invention
Can be introduced into plant cells by a particle gun method (a method of direct introduction into tissue cells or cultured cells using a particle gun) or the like. Further, the vector of the present invention,
For example, known methods such as Agrobacterium infection method (method of infecting plant tissue with Agrobacterium fungi), electric introduction method (electroporation method and electric introduction method to protoplast), or particle gun method Can be introduced into plant cells.

The types of plant cells into which the promoter of the present invention, the chimeric gene of the present invention or the vector of the present invention can be introduced to express the gene under the control of the promoter of the present invention include, for example, rice, maize, barley and wheat. Legumes such as soybeans, peas, kidney beans or alfalfa,
Solanaceous plants such as tobacco, tomato or potato, cruciferous plants such as cabbage, rapeseed or mustard,
Cells derived from Cucurbitaceae plants such as melon, pumpkin or cucumber, Umbelliferae plants such as carrot or celery, or cells derived from dicotyledonous plants such as Asteraceous plants such as lettuce can be mentioned. By introducing a promoter of the present invention, a chimeric gene of the present invention or a vector of the present invention into plant cells as described above to obtain a transformant, for example, the promoter of the present invention is inserted upstream of a desired gene on genomic DNA. A plant cell expressing the gene under the control of the promoter of the present invention; a plant cell expressing the desired gene contained in the chimeric gene by inserting the chimeric gene of the present invention into genomic DNA under the control of the promoter of the present invention; A plant cell or the like which has a vector having a chimeric gene in a cell and expresses the gene contained in the chimeric gene under the control of the promoter of the present invention can be obtained. Plant cells transformed as described above are, for example, SBGelvin, RASchilperoot
and DPSVerma: Plant Molecular Biology Manual (Plant Molecular Biology Manua)
l, Kluwer Academic Publishers press (1988)), Isao Shimamoto, Kiyotaka Okada: Experimental protocol for model plants (rice, Arabidopsis) (Hidejunsha) (ISBN4-87962-157-9)
C3345, 1996) pp. 78-143 or by Hirofumi Uchimiya (Plant Gene Manipulation Manual, How to Make Transgenic Plants (Kodansha Scientific)) 1990, ISBN4-06-153
513-7 C3045) A transformed plant derived from the plant cell or a part thereof is obtained by redifferentiation in accordance with the method used in ordinary plant tissue culture techniques described on pages 28-33. be able to. Further, by cultivating and self-cultivating the plant obtained as described above, progeny of the plant can be obtained. From the transformed plant cells or plants as described above, DNA is prepared according to a conventional method.
Is extracted, the DNA is cleaved with a restriction enzyme, and the introduced gene can be confirmed by performing Southern hybridization using the DNA used for transforming the host cell or a part thereof as a probe. In addition, RNA is extracted from such a plant cell or plant according to a conventional method, and Northern hybridization is performed using, as a probe, an oligonucleotide or DNA derived from the gene of interest to be expressed under the control of the promoter of the present invention. Thus, the state of expression of the target gene can be examined.

Under the control of the promoter of the present invention, a specific gene can be ligated in a sense direction or an antisense direction and expressed in a plant. As a gene to be expressed, for example, soybean glycinin gene, β-conglycinin gene, 2N albumin gene of Brazil nut,
Alternatively, storage protein genes such as 10 kDa protein gene or 15 kDa protein gene of corn or rice, bioA gene derived from microorganisms such as Escherichia coli, bioB gene,
biotin biosynthesis-related enzyme genes such as bioC gene, bioD gene, bioF gene or bioH gene, stearoyl-A
A gene involved in lipid metabolism such as a CP-desaturase gene, an acyl-ACP-thioesterase gene, a 3-phosphate acyltransferase gene or an acyltransferase gene, or an amylopectin degrading enzyme gene such as rice isomerase (Isomerase);
Raffinoses and oligosaccharide synthase genes can be mentioned.

[0023]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples. Example 1 (Preparation of Genomic DNA) From the leaf piece of soybean (Glycin max cv. Williams), Hirofumi Uchimiya (Manual for Plant Gene Manipulation, How to Make Transgenic Plants (Kodansha Scientific)) 199
0, ISBN4-06-153513-7 C3045) Genomic DNA was prepared using the CTAB method described on pages 71-74. After thoroughly grinding about 0.5 g of plant leaf pieces using a homogenizer in an Eppendorf tube,
2xCTAB solution [2% cety
ltrimethylammonium bromide, 100mM Tris-HCl, pH8.0,
20mM EDTA, 1.4M NaCl, 1% polyvinylpyroridone (PV
P)], and the mixture was kept at 65 ° C for 5 minutes. Add 0.5ml
Of chloroform / isoamyl alcohol (24: 1) was added and mixed gently for 5 minutes. 12,000 rpm (10,000
xg) for 10 minutes to separate the upper layer.
10% CTAB solution (10% cetyltrimethyl ammoniu
mBromide, 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 thereto, mixed well, and the upper layer was separated.
Double the amount of CTAB precipitate (1% cetyltrimethyl am
monium bromide, 50 mM Tris-HCl, pH 8.0, 10 mM EDTA), and incubated at 65 ° C for 1 minute, then 12,000 rpm (10,000xg)
For 10 minutes to precipitate DNA. The obtained precipitate was subjected to high salt concentration TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1M Na
Cl) was dissolved in 50 μl, and 100 μl of ethanol was added and mixed. The precipitate obtained by centrifugation at 12,000 rpm (10,000 × g) for 15 minutes was subjected to 50 μl TE (10 mM Tris-HCl pH 8.0, 1 mM EDT
A). To this, RNaseA was added to a concentration of 10 μg / ml, and the mixture was incubated at 37 ° C. for 30 minutes.
A mixed solution of chloroform / isoamyl alcohol (25: 24: 1) was added and 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, mix well, centrifuge at 12,000 rpm (10,000 xg) for 5 minutes to collect the sediment, and DNA was obtained.

Example 2 (Acquisition of the Promoter of the Present Invention by Inverse PCR) 450 ng of the genomic DNA obtained in Example 1 was digested with the restriction enzyme EcoT22I.
After complete digestion with T4 DNA ligase (Takara Shuzo)
A ligation reaction (15 ° C., 10 hours) was performed using The DNA was then recovered by ethanol precipitation and dissolved in 20 μl sterile water. Using 5 μl of the lysate, an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 3 (SRS-5R) and an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 4 (SRS-6) were used as primers, PCR using Ex Taq polymerase (Takara Shuzo) at 94 ° C for 1 minute, then 55 ° C
C. for 2 minutes, and further at 72.degree. C. for 3 minutes as one cycle for a total of 30 cycles). The obtained PCR reaction solution 1/1
Using 0 volumes, an oligonucleotide (SRS-6R) consisting of the base sequence shown in SEQ ID NO: 5 and an oligonucleotide (SRS-5) consisting of the base sequence shown in SEQ ID NO: 2 were used as primers, and Ex Taq Polymerase (Takara Shuzo)
PCR (using 94 ° C for 1 minute, then 55 ° C for 2 minutes, and 72 ° C for 3 minutes as one cycle).
30 cycles). As a result of analyzing the DNA contained in the PCR reaction solution thus obtained by 0.8% agarose gel electrophoresis, amplification of a DNA of about 3 kbp was confirmed. This approximately 3 kbp DNA was recovered from the gel, digested with EcoT22I,
Furthermore, blunt-end treatment was performed using T4 DNA polymerase. Next, the DNA and a HincII digest of plasmid pUC119 (Takara Shuzo) were mixed, and a ligation reaction was performed using T4 DNA ligase (Takara Shuzo). The DNA after the reaction was introduced into E. coli JM109 (competent cell manufactured by Takara Shuzo Co., Ltd.),
Three types of clones having a plasmid containing a kbp DNA were selected, and the nucleotide sequence of the DNA contained in each clone was determined. The nucleotide sequence was analyzed by performing a PCR reaction by a dye terminator method using fluorescent dideoxynucleotide (manufactured by Applied Biosystems) and analyzing using a fluorescent DNA sequencer (manufactured by Applied Biosystems, model 373A). In addition, an M13 primer (manufactured by Takara Shuzo Co., Ltd.) and an oligonucleotide having a base sequence represented by any of SEQ ID NOS: 6 to 18 were used as primers for determining a base sequence. The nucleotide sequence of the region of about 0.6 kbp in the DNA of about 3 kbp contained in the three types of clones is all the raffinose synthase gene derived from soybean (European Patent Publication No. 08
No. 49359) and the nucleotide sequence after the start codon of the coding region. About the upstream region of the coding region
The nucleotide sequence read from the DNA of one clone is shown as SEQ ID NO: 1 or 27 as the 2.4 kbp nucleotide sequence. In the base sequence in the upstream region, differences were observed in 13 bases (in the case of SEQ ID NO: 1) and 16 bases (in the case of SEQ ID NO: 27) among the three types of clones. Thus, using the genomic DNA obtained in Example 1 as a template, SEQ ID NO: 9
An oligonucleotide consisting of the nucleotide sequence represented by (SRS-1
6) and an oligonucleotide (SRS-17) consisting of the base sequence represented by SEQ ID NO: 10, an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 11 (SRS-18) and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12 Nucleotide (SRS-1
9), an oligonucleotide (SRS-15) consisting of the base sequence represented by SEQ ID NO: 8 and an oligonucleotide (SRS-31) consisting of the base sequence represented by SEQ ID NO: 17, and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12 An oligonucleotide (SRS-26) consisting of the nucleotide sequence represented by nucleotide (SRS-19) and SEQ ID NO: 30 or an oligonucleotide (SRS-20) consisting of the nucleotide sequence represented by SEQ ID NO: 13 and SEQ ID NO: 25
Oligonucleotide consisting of the nucleotide sequence represented by (SRS-2
1) was used for each primer, and PCR was performed using Ex Taq polymerase (Takara Shuzo) at 94 ° C. for 1 minute, then 55 ° C.
C. for 2 minutes, and further at 72.degree. C. for 3 minutes as one cycle for a total of 30 cycles). T4 DNA
The DNA was blunt-ended using a polymerase, the DNA was mixed with a HincII digest of plasmid pUC119 (manufactured by Takara Shuzo), and a ligation reaction was performed using T4 DNA ligase (manufactured by Takara Shuzo). The DNA after the reaction was introduced into Escherichia coli JM109 (competent cell manufactured by Takara Shuzo Co., Ltd.), and plasmids containing the DNA obtained by PCR were selected. Then, the nucleotide sequence of the DNA contained in each plasmid was determined. . As described above, the analysis of the base sequence was carried out using fluorescent dideoxynucleotide (Applied Bi
osystems Co., Ltd.)
Perform the R reaction and perform a fluorescent DNA sequencer (Applied Biosys
The analysis was performed using a tems company (model 373A). In addition, an M13-based primer (Takara Shuzo) was used as a primer for nucleotide sequence determination. In this manner, the above-mentioned three clones (the nucleotide sequence of the DNA of each clone has nucleotide numbers 27 and 2)
(Base sequences represented by 8 or 29) were identified. The base sequence in the upstream region of the coding region based on the identification result was set to SEQ ID NO: 26
Shown in The 16 locations where differences in bases were observed are described below. A corresponding to base number 389 in SEQ ID NO: 26 corresponds to G corresponding to base number 389 in SEQ ID NO: 29. 2. A corresponding to base number 515 in SEQ ID NO: 26 This corresponds to G corresponding to base number 515 in SEQ ID NO: 29. 3. Position between T at base number 563 and A at base number 564 in SEQ ID NO: 26 T at base number 564 in SEQ ID NO: 28 (insertion)
Corresponding to 4. A which is base number 609 in SEQ ID NO: 26 corresponds to the deletion at the position of base number 609 in SEQ ID NO: 29. 5. Position between A of base number 609 and C of base number 610 in SEQ ID NO: 26 A (insertion) of base number 611 in SEQ ID NO: 28
Corresponding to 6. T corresponding to base number 679 in SEQ ID NO: 26 corresponds to C corresponding to base number 678 in SEQ ID NO: 29. 7. C corresponding to base number 774 in SEQ ID NO: 26 corresponds to T corresponding to base number 773 in SEQ ID NO: 29. 8. T corresponding to base number 1052 in SEQ ID NO: 26 Corresponds to A corresponding to base number 1054 in SEQ ID NO: 28. 9. Position between T at base number 1215 and A at base number 1216 in SEQ ID NO: 26 This corresponds to T (insertion) at base number 1216 in SEQ ID NO: 27. Base number 121 in SEQ ID NO: 29
5 corresponds to T (insertion). 10. T corresponding to base number 1340 in SEQ ID NO: 26 corresponds to C corresponding to base number 1341 in SEQ ID NO: 27. 11. T corresponding to base number 1511 in SEQ ID NO: 26 This corresponds to C corresponding to base number 1511 in SEQ ID NO: 29. 12. A corresponding to base number 1596 in SEQ ID NO: 26 corresponding to G corresponding to base number 1597 in SEQ ID NO: 27. 13. T corresponding to base number 2045 in SEQ ID NO: 26 This corresponds to C corresponding to base number 2046 in SEQ ID NO: 27. 14. C corresponding to base number 2332 in SEQ ID NO: 26 corresponds to T corresponding to base number 2333 in SEQ ID NO: 27. 15. T corresponding to base number 2333 in SEQ ID NO: 26 This corresponds to C corresponding to base number 2333 in SEQ ID NO: 29. 16. G corresponding to base number 2358 in SEQ ID NO: 26 corresponding to A corresponding to base number 2358 in SEQ ID NO: 27.

Example 3 (Construction of the Vector of the Present Invention for Expression of Luciferase) Of the three types of clones cloned in Example 2, the plasmid DNA containing the DNA having the nucleotide sequence of SEQ ID NO: 27 was replaced with NdeI, BglII, or EcoT22
After digestion with I, each end was blunted using T4 DNA polymerase. These DNAs were further digested with NcoI to obtain approximately 0.6 kbp, 1.4 kbp, and 2.4 kbp of DNA, respectively.
(Hereinafter, referred to as promoter DNAs 1 to 3 of the present invention). That is, the promoter DNA 1 of the present invention is a DNA of about 0.6 kbp consisting of the base sequence from base numbers 1743 to 2362 in the base sequence shown in SEQ ID NO: 27.
In the promoter DNA 2 of the present invention, nucleotides 1020 to 236 of the nucleotide sequence represented by SEQ ID NO: 27
The promoter DNA 3 of the present invention is a DNA of about 2.4 kbp consisting of the base sequence of base numbers 2 to 2362 among the base numbers shown in SEQ ID NO: 27. is there. A HincII digest of plasmid pUC118 (Takara Shuzo) and an NcoI linker were ligated using T4 DNA ligase (the resulting plasmid is referred to as pUC118 / NcoI). Then, pUC118 / NcoI
Was digested with NcoI and HindIII, and plasmid pSUM-GY1-LUC (Iida et al. (1995) Plant Cell Report
s 14: 539-544) from NcoI and HindIII digestion
Plasmid pUC118-LUC was constructed by ligating A (including the luciferase gene and the soybean glycinin gene terminator region). Then, the plasmid
The DNA of pUC118-LUC was digested with SmaI and NcoI, and the DNA was digested with the above-mentioned promoter DNAs 1 to 3 of the present invention (0.6 kbp, 1.4 kbp, 2.
4kbp) and ligated to each other using T4 DNA ligase (Takara Shuzo Co., Ltd.).
3 of the present invention (pRS1 / LUC, pRS2
/ LUC, pRS3 / LUC) (see FIG. 1).

Example 4 (Introduction of the Vector of the Present Invention into Soybean Seeds) The vector of the present invention for expressing luciferase prepared in Example 3 was separately treated with a gene gun (CM Particle Gu) of Morikawa et al.
n System, Rehbock Co., Tokyo, Japan), direct introduction method (Yang NS, Christou P edition, Particle Bombardme
nt Technology for Gene Transfer, WH Freeman and C
o. Publishers, New York, pp. 52-59). 0.5-1μm gold particles (Tokuriki Honten
Co., Ltd, Japan) was washed several times with ethanol and adjusted to 60 mg / ml with sterile water. The vector of the present invention for luciferase expression prepared in Example 3 and pBI221 (C
lonetech) at a molar ratio of 3: 1 and a final DNA concentration of 500 μg / ml with sterile water. 20 μl (DNA 1
0 μg) and 50 μl of gold particle liquid (3 mg of gold particles),
Add 50 μl of M calcium chloride, stir quickly, and add 0.1 M
20 μl of spermidine was added and quickly stirred. After leaving it at room temperature for at least 30 minutes, it was lightly centrifuged (9000 rpm, 5 seconds) to precipitate gold particles. The supernatant was removed, and the gold particles were washed with 70% ethanol and suspended in 50 μl of ethanol. This gold particle suspension was placed on a projectile by 10 μl and dried. This projector was set on a gene gun, and 335 m / sec under 110 mmHg vacuum.
Soybeans immature seeds placed on MS agar medium were bombarded. As a control, only pBI221 (manufactured by Clonetech) was similarly shot on soybean immature seeds. As described above, the vector of the present invention for luciferase expression and pBI221 (Clonete
ch.) or pBI221 (Clonetech) alone is ground in an extraction buffer (0.1 M potassium phosphate, 1 mM DTT, pH 7.5) using a mortar and pestle. Then, the obtained triturated liquid was centrifuged at 12,000 rpm (10,000 × g). The supernatant was collected and used for luciferase activity measurement. Luciferase activity was measured using a Pica Gene luminescence kit (manufactured by Toyo Ink Mfg. Co., Ltd.) and a luminometer. The supernatant was mixed with 100 μl of the luminescent substrate solution, and the amount of luminescence was measured for 30 seconds with a luminometer. The β-glucuronidase activity was measured using a GUS-light kit (TROPIX) and a luminometer. After mixing 20 μl of the supernatant and 180 μl of the GUS reaction reagent, and allowed to stand at room temperature for 60 minutes,
300 µl of a luminescent reagent was added and mixed, and the amount of luminescence was measured for 5 seconds with a luminometer. The value obtained by dividing the luciferase activity value by the β-glucuronidase activity value was calculated, and this was defined as the luciferase relative activity (Table 1). In all of the soybean seeds into which each of the three types of luciferase expression vectors of the present invention were introduced, higher luciferase relative activity was detected than in the soybean seeds (control) into which only pBI221 (manufactured by Clonetech) was introduced.

[0027]

[Table 1] * Luciferase relative activity when only pBI221 (internal standard) was introduced (control) is shown as 1.

Example 5 (Construction of Luciferase Expression Binary Vector of the Present Invention) Binary plasmid pBI101 (manufactured by Clonetech) is digested with restriction enzyme EcoRI, and then blunt-ended using T4 DNA polymerase (manufactured by Takara Shuzo). . This is further digested with a restriction enzyme SmaI, and the vector DNA from which the GUS gene and the nopaline synthase gene-derived terminator have been removed is recovered by agarose gel electrophoresis. On the other hand, plasmid pSUM
-GY1 (Iida et al. (1995) Plant Cell Reports 14: 539-
544) was digested with HindIII, blunt-ended using T4 DNA polymerase (Takara Shuzo), and
EcoRI linker (Takara Shuzo) is ligated using DNA ligase (Takara Shuzo). The DNA obtained in this way is PstI
(Takara Shuzo), blunt-ended using T4 DNA polymerase (Takara Shuzo), and restriction enzyme EcoRI
(Manufactured by Takara Shuzo Co., Ltd.), and a DNA of about 0.7 kbp containing a soybean glycinin gene terminator region is recovered by agarose gel electrophoresis. The pBI101-derived vector DNA (Eco
RI / SmaI) and about 0.7 kbp of DNA containing the soybean glycinin gene terminator region were ligated using DNA ligase (Takara Shuzo) to give plasmid pBI101-GY1te.
get r. Plasmid pBI101-GY1ter is digested with restriction enzyme XbaI (Takara Shuzo) and blunt-ended using T4 DNA polymerase (Takara Shuzo). A ligation reaction between this DNA and KpnI linker (Takara Shuzo) using DNA ligase (Takara Shuzo) is performed, followed by digestion with a restriction enzyme KpnI.
On the other hand, the three luciferase expression vectors of the present invention prepared in Example 3 are digested with a restriction enzyme KpnI (Takara Shuzo), and a DNA containing the promoter DNA of the present invention and a luciferase gene is recovered by agarose gel electrophoresis. . The thus obtained DNA and plasmid pBI101-GY1ter
By linking the digest of KpnI with T4 DNA ligase (Takara Shuzo Co., Ltd.), three types of binary luciferase expression binary including a chimeric gene to which the promoter DNA of the present invention, the luciferase gene, and the soybean glycinin gene terminator are linked. Vector (pTiRS1 / LUC,
pTiRS2 / LUC, pTiRS3 / LUC) can be produced (see FIG. 2).

Example 6 (Construction of the Vector of the Present Invention for Expressing Raffinose Synthase) An oligonucleotide (SRS-N) having the nucleotide sequence of SEQ ID NO: 19 and an oligonucleotide (4RV) having the nucleotide sequence of SEQ ID NO: 20 ) Or an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 21 (seq1) and an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 22 (C
PCR (94 ° C for 1 minute, then soybean cDNA library as template)
A total of 30 cycles are performed with one cycle of keeping the temperature at 55 ° C. for 2 minutes and 72 ° C. for 3 minutes. Then the products of each PCR 30
ng was used as a template, and an oligonucleotide (SRS-N) consisting of the base sequence represented by SEQ ID NO: 19 and SEQ ID NO: 2
An oligonucleotide consisting of the nucleotide sequence represented by 2 (C2ps
By performing PCR again using the combination of t) as a primer, a DNA consisting of the base sequence represented by base numbers 61 to 2451 encoding raffinose synthase among the base sequence represented by SEQ ID NO: 23 is obtained. Mung Bea
n Blunt-end treatment using Nuclease (Takara Shuzo)
Furthermore, digestion is performed using restriction enzyme PstI (Takara Shuzo). On the other hand, the three types of luciferase expression vectors of the present invention prepared in Example 3 were each digested with the restriction enzyme NcoI, and
After blunt-end treatment using ng Bean Nuclease (Takara Shuzo), digest with restriction enzyme PstI (Takara Shuzo). The digested solution is subjected to agarose electrophoresis to recover the DNA of the vector of the present invention from which the luciferase gene has been removed.
By ligating the DNA derived from the vector of the present invention thus obtained and the DNA encoding the raffinose synthase using T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.), the vector pRS4 for expressing raffinose synthase, pRS5,
And pRS6 can be produced (see FIG. 3). Also,
The vector pRS4, pRS5, and pRS6 of the present invention were digested with restriction enzymes SacI and PstI (Takara Shuzo Co., Ltd.), and then T4 D
The protruding ends are blunt-ended using NA polymerase (Takara Shuzo). This DNA is ligated with a BamHI linker (Takara Shuzo) using T4 DNA ligase (Takara Shuzo), and then digested with a restriction enzyme BamHI (Takara Shuzo). on the other hand,
The plasmid pBI101-GY1ter prepared in Example 5 was digested with a restriction enzyme BamHI (manufactured by Takara Shuzo Co., Ltd.), and the DNA and each of the DNAs derived from the plasmids pRS4, pRS5, or pRS6 were digested with T
4 By performing a ligation reaction using DNA ligase (manufactured by Takara Shuzo Co., Ltd.), three types of binary vectors (pTiRS4, pTiRS5, pTiRS6) in which the raffinose synthase gene is ligated under the control of the promoter of the present invention can be produced. (See FIG. 4).

Example 7 (Preparation of Transformed Tobacco by Indirect Transfer Method) Agrobacterium made competent by treating the DNA of the binary vector of the present invention prepared in Examples 5 and 6 with 20 mM CaCl ₂ Agrobact
erium tumefaciens LBA4404) (showing streptomycin resistance and rifampicin resistance) (Hoekma et al. Na
ture, 303: 179-180 (1983)) and heat treatment (37 ° C, 5 minutes)
Introduced by The introduced Agrobacterium was treated with 300 μg / ml of streptomycin and 100 μm of rifampicin.
g / ml and kanamycin 25 μg / ml by culturing on an L agar medium, the NPTII gene on the plasmid (Trien
-Cuotet al., Gene 23: 331-341 (1983)) to select transformants imparted with kanamycin resistance. The obtained transformant of Agrobacterium was transformed with streptomycin 300 μg / ml, rifampicin 100 μg / ml, kanamycin
Cultured in an L medium containing 25 μg / ml overnight at 28 ° C., and using the obtained bacterial solution, SBGelvin, RASchilperoort and DP
S.Verma; Plant molecular Biology / Manual (1988)
(Published by Kluwer Academic Publishers), written by Hirofumi Uchimiya (plant gene manipulation manual, how to make transgenic plants (Kodansha Scientific)) 1990, ISBN4-06-1
53513-7 C3045) by the method described on pages 28-33,
A transformant of the above Agrobacterium is 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
Transfer to S-NB agar medium and culture. On the eleventh day, the cells are transferred to an MS-NB agar medium containing 500 μg / ml of cefotaxime and 100 μg / ml of kanamycin, and the culture is continued. Approximately 4 weeks later, the green shoot-and-leaf-differentiated seedlings were cut off from the disc pieces, and MS-NB containing cefotaxime 500 μg / ml and kanamycin 50 μg / ml.
The rooted seedlings are transferred to an agar medium and selected. The selected tobacco seedlings are transferred to soil and cultivated in a greenhouse,
A transformed plant can be obtained.

Example 8 (Confirmation of Insertion of Transgene in Transformed Plant) (1) Preparation of Genomic DNA from Transformed Plant Leaf pieces of the tobacco transformed plant of the present invention obtained in Example 7 From Hirofumi Uchimiya (Manual for Plant Gene Manipulation, How to Make Transgenic Plants (Kodansha Scientific)) 1990, ISBN4-06-153513-7 C3045) C described on pages 71-74
Prepare genomic DNA using TAB method. 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, 100 mM Tris-H
Cl, pH8.0, 20mM EDTA, 1.4M NaCl, 1% polyvinylpyror
Add 0.5 ml of idone (PVP) and keep at 65 ° C for 5 minutes. 0.5 ml of chloroform / isoamyl alcohol (2
4: 1) Add the mixture and mix gently for 5 minutes. 12,000 r
Centrifuge at pm (10,000 xg) for 10 minutes to separate the upper layer, and
10% CTAB solution (10% cetyltrimethyl)
ammonium bromide, 0.7M NaCl) and keep at 65 ° C for 3 minutes. Add an equal volume of a mixture of chloroform / isoamyl alcohol (24: 1), mix well, and separate the upper layer.
2 volumes of CTAB precipitate (1% cetyltrimethyl ammonium bro
mide, 50 mM Tris-HCl, pH 8.0, 10 mM EDTA) and add 65
After incubating at 1 ° C. for 1 minute, the mixture is centrifuged at 12,000 rpm (10,000 × g) for 10 minutes to precipitate DNA. Precipitate with high salt concentration TE (10m
M Tris-HCl (pH 8.0, 1 mM EDTA, 1 M NaCl) is dissolved in 50 μl, and 100 μl of ethanol is added and mixed. 12,000 rpm
The precipitate obtained by centrifugation at (10,000 xg) for 15 minutes was
Dissolve in E (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Add RNaseA to this at a concentration of 10 μg / ml, incubate at 37 ° C for 30 minutes, add an equal volume of a phenol / chloroform / isoamyl alcohol (25: 24: 1) mixture, mix well, and separate the upper layer. I do. To this, add 1/10 volume of 3M sodium acetate solution (pH 5.2) and 2.5 volume of ethanol and mix well,
The precipitate is collected by centrifugation at 12,000 rpm (10,000 × g) for 5 minutes to obtain about 5 μg of genomic DNA.

(2) Confirmation of Insertion of Transgene by PCR Method The binary vector of the present invention (pT
iRS4, pTiRS5, pTiRS6), using as a template 50 ng of tobacco genomic DNA into which an oligonucleotide (SRS-27) consisting of the nucleotide sequence shown in SEQ ID NO: 16 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 (SR
PCR using S-6R) as primer (94 ° C for 1 minute, then
A total of 30 cycles (one cycle of keeping the temperature at 55 ° C. for 2 minutes and 72 ° C. for 3 minutes) is performed. 4% of the resulting PCR product
By analyzing by agarose gel electrophoresis,
Amplification of the 800 bp DNA fragment was detected, confirming that the raffinose synthase gene had been integrated into the chromosome. In the case of the tobacco into which each of the binary vectors (pTiRS1 / LUC, pTiRS2 / LUC, pTiRS3 / LUC) prepared in Example 5 was introduced, an oligonucleotide (SRS-27) having the base sequence represented by SEQ ID NO: 16 and Amplification of a DNA fragment of about 700 bp was detected by PCR using an oligonucleotide (luc1) consisting of the nucleotide sequence of SEQ ID NO: 24 as a primer, and it was confirmed that the luciferase gene had been integrated into the chromosome.

The composition of the medium used in the examples is shown below. 1. MS agar medium Dissolve 34.7 g of MURASHIGE AND SKOOG (manufactured by Flow Laboratories) in 1 L of distilled water, adjust to pH 5.8 with 1 M KOH, and agar 8 g
After the addition, the solution was autoclaved. 2. MS-NB agar medium 1-naphthaleneacetic acid (NAA) 0.1 mg / mL and 6
-A medium supplemented with 1.0 mg / mL of benzylaminopurine (BA). 3. L medium 10 g of bactotryptone (manufactured by Difco), 5 g of bactoeast extract (manufactured by Difco) and 10 g of NaCl were dissolved in 1 L of distilled water, adjusted to pH 7.0 with 5M NaOH, and sterilized in an autoclave. In the case of an agar medium, 15 g of Bacto agar (manufactured by Difco) was added, followed by autoclaving. 4. NZY medium 10 g of NZ amine (TypeA) (manufactured by Wako Pure Chemical Industries), 5 g of Bacto yeast extract (manufactured by Difco) and 5 g of NaCl were dissolved in 1 L of distilled water, adjusted to pH 7.0 with 5 M NaOH, and sterilized in an autoclave. . In the case of an agar medium, 15 g of Bacto agar (manufactured by Difco) was added, followed by autoclaving.

[0034]

According to the present invention, a new plant promoter or the like which can be used for plant breeding technology by gene transfer can be provided.

[Sequence List Free Text] SEQ ID NO: 2 Oligonucleotide primer designed for PCR SEQ ID NO: 3 Oligonucleotide primer designed for PCR SEQ ID NO: 4 Oligonucleotide primer designed for PCR SEQ ID NO: 5 Oligonucleotide primer designed for PCR SEQ ID NO: 6 Oligonucleotide primer designed for PCR SEQ ID NO: 7 Oligonucleotide primer designed for PCR SEQ ID NO: 8 Oligonucleotide primer designed for PCR SEQ ID NO: 9 oligonucleotide primer designed for PCR SEQ ID NO: 10 oligonucleotide primer designed for PCR SEQ ID NO: 11 oligonucleotide primer designed for PCR SEQ ID NO: 12 oligonucleotide primer designed for PCR SEQ ID NO: 13 oligonucleotide primer designed for PCR SEQ ID NO: 14 oligonucleotide primer designed for PCR SEQ ID NO: 15 designed for PCR Oligonucleotide primers SEQ ID NO: 16 oligonucleotide primers designed for PCR SEQ ID NO: 17 oligonucleotide primers designed for PCR SEQ ID NO: 18 oligonucleotide primers designed for PCR SEQ ID NO: 19 designed for PCR Oligonucleotide primers designed for PCR SEQ ID NO: 20 Oligonucleotide primers designed for PCR SEQ ID NO: 21 Oligonucleotide primers designed for PCR SEQ ID NO: Designed oligonucleotide primer for oligonucleotide primers SEQ ID NO: 30 PCR designed for oligonucleotide primers SEQ ID NO: 25 PCR designed for oligonucleotide primers SEQ ID NO: 24 PCR designed for 2 PCR

[0036]

[Sequence List] <110> Sumitomo Chemical Co. Ltd. <120> Plant promoter <130> P151481 <150> JP 11/124527 <151> 1999-04-30 <150> JP 11/247211 <151> 1999-09 -01 <160> 30 <210> 1 <211> 2301 <212> DNA <213> Glycin max cv. Williams 82 <220> <221> promoter <222> (1) ... (2301) <400> 1 atgcatctat aaaaattgta taataacaaa atgaattttg ttatgtattt tatttaataa 60 aattatgatt atgtgaaaag aataatttaa aaataaacaa aaattcattt tcttaatagg 120 aggccagaat aaattgcatg atgccaatta ggccaatagt aactcccaac cgttaaactt 180 gggcaaactg ttttccaatt cagtttggtt taagggagaa ttgtttttca attggcttta 240 attcgatttt ttgtgttgaa ctgaattgtg aacaagccta accaaaggta atgacccttt 300 tctcattcct agttcggttc aatttagaga ggaaaaattg aattcaattg tcccaaacta 360 atttgattcg aattttagaa tatagtgcat cgagccaaaa aaataattca attcggttga 420 actattttct aattcagttt ggttcgatct tctctactga atcaaaacca tgtacatact 480 tatttacaag tagtagcccc ccacatctaa acacaaagga tacatcaagc ttaattttaa 540 ccacacatga agaatcattt cctaaattaa tgcacactat tgtcagagaa atcacaaag acaaaaaac gttaga tatataaaat tatcgtttta gatgagacta 660 tatacagaga gaaaatattt ttttaaaacc tgaagaatcc tctaataaag agcaatgtct 720 ctgagtattg aatttattta atggtccaaa ctcatttaac atatgttttt tttcatataa 780 atagatcatt tatgaaatca tatttatctt taaaaataat aatatttatc ttaaattttc 840 aatagaaaat gttctgataa atattaaatt aaaagcaaaa tacattttac tttttattta 900 agaatagagc tactttaaaa agattctttt ccttttacga ataaatcatt cgatatgact 960 acacttttgt gttttgtttt ctttctttct ttttactatg aagtttttct ggaactagag 1020 atctctagag ttttctagtg ggacaaaaca gtgctgccgt gcgggttatc cattataaaa 1080 ctattgttcg atgaaccaaa aagactatta aaatatgttt tagtattcat tttaatatgc 1140 tttacattta aatttataaa caaactcttg tataatagtt ttttcatgaa ataaatgttt 1200 aattaacttt ttttttacat tttttttacg gaataatact tatctcattt attataaatt 1260 aaatattcaa taaaaaaaat tataggatcg atagaataga gactaatgaa gaatgaaatc 1320 acccttacta acatatttat catttttctt catcacttta cttattgcat cttttatttt 1380 ccccgtttct ctatatatat atatgcagga aatgagtgta acaaactaag aatcgtcctt 1440 gaagaaaaca tacttagcat atctaaacac gacaa acggt gccttctggc ttctacgtag 1500 atagataagg cttgattgcc gagggataaa gcaataagtc tcctctcgaa tgtgtccacc 1560 gaaaccatat tagtactaat taaatataag accccaggca aggccaaaaa gagctacccc 1620 ttgaaaacaa aacaaactcc atattatcaa gcatttaaga aataagattt gtttatatca 1680 tgtctaagtt tttaattaaa taaattcaac acatgaaatt tactctaaat ctttatattt 1740 acatatgatg caaacaaatc ttatctgaat atttaattta actaatgcct attattttta 1800 gtcagattat acaatctagc tagtacttta ttttttctat cacgtacatc caatatcatt 1860 tttttcatag cagttcatga aatattttat taaatattaa ttaaagcata atttgacctc 1920 taaggtgtga cacgtgtaac taatatagct ttgatttttt tttattatgg tttattttat 1980 attcaaaatc tatcctgatt taaattctaa ataatggatt aaaaaacata aattactaca 2040 ctaatcgagt caacaattat tactgtcgtc ggttcaactt tgagctttta agtatatgag 2100 agtggttgaa ttgctgccta tatgaataaa acaatattta tgggggataa aaatgagtct 2160 catattgtac atggtagttt gactttgaca catataccct ttgctctggc tgtaactaga 2220 atgcactagg cacaattaaa caaaaataaa ttctccttct ctatataaac ccaccatgtc 2280 accacaccct acccagcaaa a 2301 < 210> 2 <211> 3 5 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 2 actaatgcat acccgtatca gttgcttaga gaagc 35 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 3 catgcaaacg tccacgtaat catcc 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 4 agctgcttat acgtccacgt tggcc 25 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 5 actaatgcat gttgcaggga ggctcggaac gaggc 35 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 6 atccttgaat tcagtcccat ggctccaagc 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 7 cttggagcca tgggactgaa ttcaaggatc 30 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 8 ggaaa tgatt cttcatgtgt gg 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 9 taaccacaca tgaagaatca tttc 24 <210> 10 <211> 24 <212 > DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 10 tttataatgg ataacccgca cggc 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 11 gactaatgaa gaatgaaatc accc 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 12 tcttaaatgc ttgataatat ggag 24 <210 > 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 13 ttgacctcta aggtgtgaca cgtg 24 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 14 aaataaattc aatactcaga gacattgctc 30 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for P CR <400> 15 tttttctgga actagagatc tctagag 27 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 16 gtcaacaatt attactgtcg tcggttc 27 <210> 17 < 211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 17 tgtgaacaag cctaaccaaa ggtaatgacc 30 <210> 18 <211> 29 <212> DNA <213> Artificial Sequence <220 > <223> Designed oligonucleotide primer for PCR <400> 18 ttacatatga tgcaaacaaa tcttatctg 29 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 19 catggctcca agcataagca aaactgtgga 30 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 20 gacacccttg gagccaatac gtgccatttg 30 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 21 cgggtggcaa gccatttgtc acgacgagga 30 <210> 22 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 22 taactgcaga aagagagtca aacatcatag tatccc 36 <210> 23 <211> 2498 <212> DNA <213> Glycin max cv. Williams 82 <220> <221> CDS <222 > (62) ... (2407) <400> 23 ccaaaccata gcaaacctaa gcaccaaacc tctttctttc aagatccttg aattcagtcc 60 c atg gct cca agc ata agc aaa act gtg gaa cta aat tca ttt ggt ctt 109 Met Ala Pro Ser Ile Leu Lyr Thr Val Asn Ser Phe Gly Leu 1 5 10 15 gtc aac ggt aat ttg cct ttg tcc ata acc cta gaa gga tca aat ttc 157 Val Asn Gly Asn Leu Pro Leu Ser Ile Thr Leu Glu Gly Ser Asn Phe 20 25 30 ctc gcc aac ggc cac cct ttt ctc acg gaa gtt ccc gaa aac ata ata 205 Leu Ala Asn Gly His Pro Phe Leu Thr Glu Val Pro Glu Asn Ile Ile 35 40 45 gtc acc cct tca ccc atc gac gcc aag agt agt aag aac aac gag gac 253 Val Thr Pro Ser Pro Ile Asp Ala Lys Ser Ser Lys Asn Asn Glu Asp 50 55 60 gac gac gtc gta ggt tgc ttc gtg ggc ttc cac gcg gac gag ccc aga 301 Asp Asp Val Val Gly Cys Phe Val Gly Phe His Ala Asp Glu Pro Arg 65 70 75 80 agc cga cac gtg gct tcc ctg ggg aag ctc aga gga ata aaa ttc atg 349 Ser Arg His Val Ala Ser Leu Gly Lys Leu Arg Gly Ile Lys Phe Met 85 90 95 agc ata ttc cgg ttt aag gtg tgg tgg acc act cac tgg gtc ggt 397 Ser Ile Phe Arg Phe Lys Val Trp Trp Thr Thr His Trp Val Gly Ser 100 105 110 aac gga cac gaa ctg gag cac gag aca cag atg atg ctt ctc gac aaa 445 Asn Gly His Glu Leu Glu His Glu Thr Gln Met Met Leu Leu Asp Lys 115 120 125 aac gac cag ctc gga cgc ccc ttt gtg ttg att ctc ccg atc ctc caa 493 Asn Asp Gln Leu Gly Arg Pro Phe Val Leu Ile Leu Pro Ile Leu Gln 130 135 140 gcc tcg ttc cga gcc tcc ccc ggt ttg gat gat tac gtg gac 541 Ala Ser Phe Arg Ala Ser Leu Gln Pro Gly Leu Asp Asp Tyr Val Asp 145 150 155 160 gtt tgc atg gag agc ggg tcg aca cgt gtc tgt ggc tcc agc ttc ggg G 589 Val Cy Ser Gly Ser Thr Arg Val Cys Gly Ser Ser Phe Gly 165 170 175 agc tgc tta tac gtc cac gtt ggc cat gac ccg tat cag ttg ctt aga 637 Ser Cys Leu Tyr Val His Val Gly His Asp Pro Tyr Gln Leu Leu Arg 180 185 19 0 gaa gca act aaa gtc gtt agg atg cat ttg ggg acg ttc aag ctt ctc 685 Glu Ala Thr Lys Val Val Arg Met His Leu Gly Thr Phe Lys Leu Leu 195 200 205 gag gag aaa acc gcg cca gtg atc ata gac aag ttt ggt tgg tgt aca 733 Glu Glu Lys Thr Ala Pro Val Ile Ile Asp Lys Phe Gly Trp Cys Thr 210 215 220 tgg gac gcg ttt tac ttg aag gtg cat ccc tca ggt gtg tgg gaa ggg 781 Trp Asp Ala Phe Tyr Leu Lys Val His Pro Ser Gly Val Trp Glu Gly 225 230 235 240 gtg aaa ggg ttg gtg gag gga ggg tgc cct cca ggg atg gtc cta atc 829 Val Lys Gly Leu Val Glu Gly Gly Cys Pro Pro Gly Met Val Leu Ile 245 250 255 gac gac ggg tgg caa gcc att tgt cac gac gag gac ccc ata acg gac 877 Asp Asp Gly Trp Gln Ala Ile Cys His Asp Glu Asp Pro Ile Thr Asp 260 265 270 caa gag ggt atg aag cga acc tcc gca ggg gag caa atg cca tgc agg 925 Gln Glu Gly Met Lys Arg Thr Ser Ala Gly Glu Gln Met Pro Cys Arg 275 280 285 ttg gtg aag ttg gag gaa aat tac aag ttc aga cag tat tgt agt gga 973 Leu Val Lys Leu Glu Glu Asn Tyr Lys Phe Arg Gln Tyr Cy Gl y 290 295 300 aag gat tct gag aag ggt atg ggt gcc ttt gtt agg gac ttg aag gaa 1021 Lys Asp Ser Glu Lys Gly Met Gly Ala Phe Val Arg Asp Leu Lys Glu 305 310 315 320 cag ttt agg agc gtg gag cag gtg gtg tgg cac gcg ctt tgt ggg 1069 Gln Phe Arg Ser Val Glu Gln Val Tyr Val Trp His Ala Leu Cys Gly 325 330 335 tat tgg ggt ggg gtc aga ccc aag gtt ccg ggc atg ccc cag gct aag 1117 Tyr Trp Gly Val Lyg Pro Lys Val Pro Gly Met Pro Gln Ala Lys 340 345 350 gtt gtc act ccg aag ctg tcc aat gga cta aaa ttg aca atg aag gat 1165 Val Val Thr Pro Lys Leu Ser Asn Gly Leu Lys Leu Thr Met Lys Asp 355 360 365 tta gcg gtg gat aag atc gtc agt aac gga gtt gga ctg gtg cca cca 1213 Leu Ala Val Asp Lys Ile Val Ser Asn Gly Val Gly Leu Val Pro Pro 370 375 380 cac ctg gct cac ctt ttg tac gag ggg ctc cac tcc cgt tct 1261 His Leu Ala His Leu Leu Tyr Glu Gly Leu His Ser Arg Leu Glu Ser 385 390 395 400 gcg ggt att gac ggt gtt aag gtt gac gtt ata cac ttg ctc gag atg 1309 Ala Gly Ile Asp Gly Val Lys Val Asp Val Ile His Leu Leu Glu Met 405 410 415 cta tcc gag gaa tac ggt ggc cgt gtt gag cta gcc aaa gct tat tac 1357 Leu Ser Glu Glu Tyr Gly Gly Arg Val Glu Leu Ala Lys Ala Tyr Tyr 420 425 430 430 aaa gcg ctc act tcg gtg aag aag cat ttc aaa ggc aat ggg gtc 1405 Lys Ala Leu Thr Ala Ser Val Lys Lys His Phe Lys Gly Asn Gly Val 435 440 445 att gcg agc atg gag cat tgt aat gac ttc ttt ctc ctt ggt acc gaa 1453 Ser Met Glu His Cys Asn Asp Phe Phe Leu Leu Gly Thr Glu 450 455 460 gcc ata gcc ctt ggg cgc gta gga gat gat ttt tgg tgc act gat ccc 1501 Ala Ile Ala Leu Gly Arg Val Gly Asp Asp Phe Trp Cys Thr Asp Pro 465 470 475 480 tct gga gat cca aat ggc acg tat tgg ctc caa ggg tgt cac atg gtg 1549 Ser Gly Asp Pro Asn Gly Thr Tyr Trp Leu Gln Gly Cys His Met Val 485 490 495 cac tgt gcc tac aac agc ttg tgg atg aat ttt att cag ccg gat 1597 His Cys Ala Tyr Asn Ser Leu Trp Met Gly Asn Phe Ile Gln Pro Asp 500 505 510 tgg gac atg ttc cag tcc act cac cct tgt gcc gaa ttc cat gca gcc 1645 Trp Asp Met Phe Gln Ser Thr His Pro Cys Ala Glu Phe His Ala Ala 515 520 525 tct agg gcc atc tct ggt gga cca gtt tac gtt agt gat tgt gtt gga 1693 Ser Arg Ala Ile Ser Gly Gly Pro Val Tyr Val Ser Asp Cys Val Gly 530 535 540 aag cac aac ttc aag ttg ctc aag agc ctc gct ttg cct gat ggg acg 1741 Lys His Asn Phe Lys Leu Leu Lys Ser Leu Ala Leu Pro Asp Gly Thr 545 550 555 560 att ttg cgt tgt caa cac tat gca ccc gcc tgt ttg ttt 1789 Ile Leu Arg Cys Gln His Tyr Ala Leu Pro Thr Arg Asp Cys Leu Phe 565 570 575 gaa gac ccc ttg cat gat ggg aag aca atg ctc aaa att tgg aat ctc 1837 Glu Asp Pro Leu His Asp Gly Lys Thr M Leu Lys Ile Trp Asn Leu 580 585 590 aac aaa tat aca ggt gtt ttg ggt cta ttt aat tgc caa gga ggt ggg 1885 Asn Lys Tyr Thr Gly Val Leu Gly Leu Phe Asn Cys Gln Gly Gly Gly 595 600 605 tgg tgt ccc act agg aga aac aag agt gcc tct gaa ttt tca caa 1933 Trp Cys Pro Val Thr Arg Arg Asn Lys Ser Ala Ser Glu Phe Ser Gln 610 615 620 act gtg aca tgc tta gcg agt cct caa gac att gaa tgg agc aat ggg 1981 T hr Val Thr Cys Leu Ala Ser Pro Gln Asp Ile Glu Trp Ser Asn Gly 625 630 635 640 aaa agc cca ata tgc ata aaa ggg atg aat gtg ttt gct gta tat ttg 2029 Lys Ser Pro Ile Cys Ile Lys Gly Met Asn Val Phe Ala Val Tyr Leu 645 650 655 ttc aag gac cac aaa cta aag ctc atg aag gca tca gag aaa ttg gaa 2077 Phe Lys Asp His Lys Leu Lys Leu Met Lys Ala Ser Glu Lys Leu Glu 660 665 670 gtt tca ctt gag ccat act gag cta ttg aca gtg tct cca gtg 2125 Val Ser Leu Glu Pro Phe Thr Phe Glu Leu Leu Thr Val Ser Pro Val 675 680 685 att gtg ctg tca aaa aag tta att caa ttt gct cca att gga tta gtg 2173 Ile Val Leu Ser Lys Lys Leu Ile Gln Phe Ala Pro Ile Gly Leu Val 690 695 700 aac atg ctt aac act ggt ggt gcc att cag tcc atg gag ttt gac aac 2221 Asn Met Leu Asn Thr Gly Gly Ala Ile Gln Ser Met Glu Phe Asp Asn 705 710 715 720 cac ata gat gtg gtc aaa att ggg gtt agg ggt tgt ggg gag atg aag 2269 His Ile Asp Val Val Lys Ile Gly Val Arg Gly Cys Gly Glu Met Lys 725 730 735 gtg ttt gca tca gag aaa cca gtt agt tgc aaa cta gat ggg gta gtt 2317 Val Phe Ala Ser Glu Lys Pro Val Ser Cys Lys Leu Asp Gly Val Val 740 745 750 gta aaa ttt gat tat gag gat aaa atg ctg aga gtg caa gtt ccc tgg 2365 Val Lys Phe Asp Tyr Glu Asp Lys Met Leu Arg Val Gln Val Pro Trp 755 760 765 cct agt gct tca aaa ttg tca atg gtt gag ttt tta ttt tga tccctga 2414 Pro Ser Ala Ser Lys Leu Ser Met Val Glu Phe Leu Phe 770 775 780 aggtgaattt gggatacatt gatgtttg tctagtattgatt aaaaaaaaaa aaaa 2498 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 24 aatgttcata ctgttgagca attcacg 27 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 25 aaacgtccac gtaatcatcc aaacc 25 <210> 26 <211> 2361 <212> DNA <213> Glycin max cv. Williams 82 <220> < 221> promoter <222> (1) ... (2361) <400> 26 atgcatctat aaaaattgta taataacaaa atgaattttg ttatgtattt tatttaataa 60 aattatgatt atgtgaaaag aataatttaa aaataaacaa aa attcattt tcttaatagg 120 aggccagaat aaattgcatg atgccaatta ggccaatagt aactcccaac cgttaaactt 180 gggcaaactg ttttccaatt cagtttggtt taagggagaa ttgtttttca attggcttta 240 attcgatttt ttgtgttgaa ctgaattgtg aacaagccta accaaaggta atgacccttt 300 tctcattcct agttcggttc aatttagaga ggaaaaattg aattcaattg tcccaaacta 360 atttgattcg aattttagaa tatagtgcat cgagccaaaa aaataattca attcggttga 420 actattttct aattcagttt ggttcgatct tctctactga atcaaaacca tgtacatact 480 tatttacaag tagtagcccc ccacatctaa acacaaagga tacatcaagc ttaattttaa 540 ccacacatga agaatcattt cctaaattaa tgcacactat tgtcagagaa atcacaaaaa 600 aaaaaaaaac accattgcag aggtgttaga tatataaaat tatcgtttta gatgagacta 660 tatacagaga gaaaatattt ttttaaaacc tgaagaatcc tctaataaag agcaatgtct 720 ctgagtattg aatttattta atggtccaaa ctcatttaac atatgttttt tttcatataa 780 atagatcatt tatgaaatca tatttatctt taaaaataat aatatttatc ttaaattttc 840 aatagaaaat gttctgataa atattaaatt aaaagcaaaa tacattttac tttttattta 900 agaatagagc tactttaaaa agattctttt ccttttacga ataaatcatt cgatatgact960 acacttttgt gttttgtttt ctttctttct ttttactatg aagtttttct ggaactagag 1020 atctctagag ttttctagtg ggacaaaaca gtgctgccgt gcgggttatc cattataaaa 1080 ctattgttcg atgaaccaaa aagactatta aaatatgttt tagtattcat tttaatatgc 1140 tttacattta aatttataaa caaactcttg tataatagtt ttttcatgaa ataaatgttt 1200 aattaacttt tttttacatt ttttttacgg aataatactt atctcattta ttataaatta 1260 aatattcaat aaaaaaaatt ataggatcga tagaatagag actaatgaag aatgaaatca 1320 cccttactaa catatttatt atttttcttc atcactttac ttattgcatc ttttattttc 1380 cccgtttctc tatatatata tatgcaggaa atgagtgtaa caaactaaga atcgtccttg 1440 aagaaaacat acttagcata tctaaacacg acaaacggtg ccttctggct tctacgtaga 1500 tagataaggc ttgattgccg agggataaag caataagtct cctctcgaat gtgtccaccg 1560 aaaccatatt agtactaatt aaatataaga ccccaagcaa ggccaaaaag agctacccct 1620 tgaaaacaaa acaaactcca tattatcaag catttaagaa ataagatttg tttatatcat 1680 gtctaagttt ttaattaaat aaattcaaca catgaaattt actctaaatc tttatattta 1740 catatgatgc aaacaaatct tatctgaata tttaatttaa ctaatgccta ttatttttag 1800 tc agattata caatctagct agtactttat tttttctatc acgtacatcc aatatcattt 1860 ttttcatagc agttcatgaa atattttatt aaatattaat taaagcataa tttgacctct 1920 aaggtgtgac acgtgtaact aatatagctt tgattttttt ttattatggt ttattttata 1980 ttcaaaatct atcctgattt aaattctaaa taatggatta aaaaacataa attactacac 2040 taattgagtc aacaattatt actgtcgtcg gttcaacttt gagcttttaa gtatatgaga 2100 gtggttgaat tgctgcctat atgaataaaa caatatttat gggggataaa aatgagtctc 2160 atattgtaca tggtagtttg actttgacac atataccctt tgctctggct gtaactagaa 2220 tgcactaggc acaattaaac aaaaataaat tctccttctc tatataaacc caccatgtca 2280 ccacacccta cccagcaaaa ccaaaccata gcaaacctaa gcaccaaacc tctttctttc 2340 aagatccttg aattcagtcc c 2361 <210> 27 <211> 2362 <212> DNA <213> 222 <220> Glycin ) ... (2362) <400> 27 atgcatctat aaaaattgta taataacaaa atgaattttg ttatgtattt tatttaataa 60 aattatgatt atgtgaaaag aataatttaa aaataaacaa aaattcattt tcttaatagg 120 aggccagaat aaattgcatg atgccaagattact ggccaacgattactg ttccaatt cagtttggtt taagggagaa ttgtttttca attggcttta 240 attcgatttt ttgtgttgaa ctgaattgtg aacaagccta accaaaggta atgacccttt 300 tctcattcct agttcggttc aatttagaga ggaaaaattg aattcaattg tcccaaacta 360 atttgattcg aattttagaa tatagtgcat cgagccaaaa aaataattca attcggttga 420 actattttct aattcagttt ggttcgatct tctctactga atcaaaacca tgtacatact 480 tatttacaag tagtagcccc ccacatctaa acacaaagga tacatcaagc ttaattttaa 540 ccacacatga agaatcattt cctaaattaa tgcacactat tgtcagagaa atcacaaaaa 600 aaaaaaaaac accattgcag aggtgttaga tatataaaat tatcgtttta gatgagacta 660 tatacagaga gaaaatattt ttttaaaacc tgaagaatcc tctaataaag agcaatgtct 720 ctgagtattg aatttattta atggtccaaa ctcatttaac atatgttttt tttcatataa 780 atagatcatt tatgaaatca tatttatctt taaaaataat aatatttatc ttaaattttc 840 aatagaaaat gttctgataa atattaaatt aaaagcaaaa tacattttac tttttattta 900 agaatagagc tactttaaaa agattctttt ccttttacga ataaatcatt cgatatgact 960 acacttttgt gttttgtttt ctttctttct ttttactatg aagtttttct ggaactagag 1020 atctctagag ttttctagtg ggacaaaaca gtgctgccgt gcgggttatc cattataaaa 1080 ctattgttcg atgaaccaaa aagactatta aaatatgttt tagtattcat tttaatatgc 1140 tttacattta aatttataaa caaactcttg tataatagtt ttttcatgaa ataaatgttt 1200 aattaacttt ttttttacat tttttttacg gaataatact tatctcattt attataaatt 1260 aaatattcaa taaaaaaaat tataggatcg atagaataga gactaatgaa gaatgaaatc 1320 acccttacta acatatttat catttttctt catcacttta cttattgcat cttttatttt 1380 ccccgtttct ctatatatat atatgcagga aatgagtgta acaaactaag aatcgtcctt 1440 gaagaaaaca tacttagcat atctaaacac gacaaacggt gccttctggc ttctacgtag 1500 atagataagg cttgattgcc gagggataaa gcaataagtc tcctctcgaa tgtgtccacc 1560 gaaaccatat tagtactaat taaatataag accccaggca aggccaaaaa gagctacccc 1620 ttgaaaacaa aacaaactcc atattatcaa gcatttaaga aataagattt gtttatatca 1680 tgtctaagtt tttaattaaa taaattcaac acatgaaatt tactctaaat ctttatattt 1740 acatatgatg caaacaaatc ttatctgaat atttaattta actaatgcct attattttta 1800 gtcagattat acaatctagc tagtacttta ttttttctat cacgtacatc caatatcatt 1860 tttttcatag cagttcatga aatattttat taaat attaa ttaaagcata atttgacctc 1920 taaggtgtga cacgtgtaac taatatagct ttgatttttt tttattatgg tttattttat 1980 attcaaaatc tatcctgatt taaattctaa ataatggatt aaaaaacata aattactaca 2040 ctaatcgagt caacaattat tactgtcgtc ggttcaactt tgagctttta agtatatgag 2100 agtggttgaa ttgctgccta tatgaataaa acaatattta tgggggataa aaatgagtct 2160 catattgtac atggtagttt gactttgaca catataccct ttgctctggc tgtaactaga 2220 atgcactagg cacaattaaa caaaaataaa ttctccttct ctatataaac ccaccatgtc 2280 accacaccct acccagcaaa accaaaccat agcaaaccta agcaccaaac ctttttcttt 2340 caagatcctt gaattcaatc cc 2362 <210> 28 <211> 2363 <212> DNA <213> Glycin max cv. Williams 82 <220> <221> promoter <222> (1) ... (2363) <400> 28 atgcatctat aaaaattgta taataacaaa atgaattttg ttatgtattt tatttaataa 60 aattatgatt atgtgaaaag aataatttaa aaataaacaa aaattcattt tcttaatagg 120 aggccagaat aaattgcatg atgccaatta ggccaatagt aactcccaac cgttaaactt 180 gggcaaactg ttttccaatt cagtttggtt taagggagaa ttgtttttca attggcttta 240 attcgatttt ttgtgttgaa ctgaattgtg aacaagccta accaa aggta atgacccttt 300 tctcattcct agttcggttc aatttagaga ggaaaaattg aattcaattg tcccaaacta 360 atttgattcg aattttagaa tatagtgcat cgagccaaaa aaataattca attcggttga 420 actattttct aattcagttt ggttcgatct tctctactga atcaaaacca tgtacatact 480 tatttacaag tagtagcccc ccacatctaa acacaaagga tacatcaagc ttaattttaa 540 ccacacatga agaatcattt ccttaaatta atgcacacta ttgtcagaga aatcacaaaa 600 aaaaaaaaaa acaccattgc agaggtgtta gatatataaa attatcgttt tagatgagac 660 tatatacaga gagaaaatat ttttttaaaa cctgaagaat cctctaataa agagcaatgt 720 ctctgagtat tgaatttatt taatggtcca aactcattta acatatgttt tttttcatat 780 aaatagatca tttatgaaat catatttatc tttaaaaata ataatattta tcttaaattt 840 tcaatagaaa atgttctgat aaatattaaa ttaaaagcaa aatacatttt actttttatt 900 taagaataga gctactttaa aaagattctt ttccttttac gaataaatca ttcgatatga 960 ctacactttt gtgttttgtt ttctttcttt ctttttacta tgaagttttt ctggaactag 1020 agatctctag agttttctag tgggacaaaa cagagctgcc gtgcgggtta tccattataa 1080 aactattgtt cgatgaacca aaaagactat taaaatatgt tttagtattc attttaatat 1 140 gctttacatt taaatttata aacaaactct tgtataatag ttttttcatg aaataaatgt 1200 ttaattaact tttttttaca ttttttttac ggaataatac ttatctcatt tattataaat 1260 taaatattca ataaaaaaaa ttataggatc gatagaatag agactaatga agaatgaaat 1320 cacccttact aacatattta ttatttttct tcatcacttt acttattgca tcttttattt 1380 tccccgtttc tctatatata tatatgcagg aaatgagtgt aacaaactaa gaatcgtcct 1440 tgaagaaaac atacttagca tatctaaaca cgacaaacgg tgccttctgg cttctacgta 1500 gatagataag gcttgattgc cgagggataa agcaataagt ctcctctcga atgtgtccac 1560 cgaaaccata ttagtactaa ttaaatataa gaccccaagc aaggccaaaa agagctaccc 1620 cttgaaaaca aaacaaactc catattatca agcatttaag aaataagatt tgtttatatc 1680 atgtctaagt ttttaattaa ataaattcaa cacatgaaat ttactctaaa tctttatatt 1740 tacatatgat gcaaacaaat cttatctgaa tatttaattt aactaatgcc tattattttt 1800 agtcagatta tacaatctag ctagtacttt attttttcta tcacgtacat ccaatatcat 1860 ttttttcata gcagttcatg aaatatttta ttaaatatta attaaagcat aatttgacct 1920 ctaaggtgtg acacgtgtaa ctaatatagc tttgattttt ttttattatg gtttatttta 1980 ta ttcaaaat ctatcctgat ttaaattcta aataatggat taaaaaacat aaattactac 2040 actaattgag tcaacaatta ttactgtcgt cggttcaact ttgagctttt aagtatatga 2100 gagtggttga attgctgcct atatgaataa aacaatattt atgggggata aaaatgagtc 2160 tcatattgta catggtagtt tgactttgac acatataccc tttgctctgg ctgtaactag 2220 aatgcactag gcacaattaa acaaaaataa attctccttc tctatataaa cccaccatgt 2280 caccacaccc tacccagcaa aaccaaacca tagcaaacct aagcaccaaa cctctttctt 2340 tcaagatcct tgaattcagt ccc 2363 <210> 29 <211> 2361 <212> DNA <213> Glycin max cv. Williams 82 <220> <221> promoter <222> (1) ... (2361) <400> 29 atgcatctat aaaaattgta taataacaaa atgaattttg ttatgtattt tatttaataa 60 aattatgatt atgtgaaaag aataatttaa aaataaacaa aaattcattt tcttaatagg 120 aggccagaat aaattgcatg atgccaatta ggccaatagt aactcccaac cgttaaactt 180 gggcaaactg ttttccaatt cagtttggtt taagggagaa ttgtttttca attggcttta 240 attcgatttt ttgtgttgaa ctgaattgtg aacaagccta accaaaggta atgacccttt 300 tctcattcct agttcggttc aatttagaga ggaaaaattg aattcaattg tcccaaacta 360 atttgattcg aat tttagaa tatagtgcgt cgagccaaaa aaataattca attcggttga 420 actattttct aattcagttt ggttcgatct tctctactga atcaaaacca tgtacatact 480 tatttacaag tagtagcccc ccacatctaa acacgaagga tacatcaagc ttaattttaa 540 ccacacatga agaatcattt cctaaattaa tgcacactat tgtcagagaa atcacaaaaa 600 aaaaaaaaca ccattgcaga ggtgttagat atataaaatt atcgttttag atgagactat 660 atacagagag aaaatatctt tttaaaacct gaagaatcct ctaataaaga gcaatgtctc 720 tgagtattga atttatttaa tggtccaaac tcatttaaca tatgtttttt tttatataaa 780 tagatcattt atgaaatcat atttatcttt aaaaataata atatttatct taaattttca 840 atagaaaatg ttctgataaa tattaaatta aaagcaaaat acattttact ttttatttaa 900 gaatagagct actttaaaaa gattcttttc cttttacgaa taaatcattc gatatgacta 960 cacttttgtg ttttgttttc tttctttctt tttactatga agtttttctg gaactagaga 1020 tctctagagt tttctagtgg gacaaaacag tgctgccgtg cgggttatcc attataaaac 1080 tattgttcga tgaaccaaaa agactattaa aatatgtttt agtattcatt ttaatatgct 1140 ttacatttaa atttataaac aaactcttgt ataatagttt tttcatgaaa taaatgttta 1200 attaactttt tttttacatt ttttttac gg aataatactt atctcattta ttataaatta 1260 aatattcaat aaaaaaaatt ataggatcga tagaatagag actaatgaag aatgaaatca 1320 cccttactaa catatttatt atttttcttc atcactttac ttattgcatc ttttattttc 1380 cccgtttctc tatatatata tatgcaggaa atgagtgtaa caaactaaga atcgtccttg 1440 aagaaaacat acttagcata tctaaacacg acaaacggtg ccttctggct tctacgtaga 1500 tagataaggc ctgattgccg agggataaag caataagtct cctctcgaat gtgtccaccg 1560 aaaccatatt agtactaatt aaatataaga ccccaagcaa ggccaaaaag agctacccct 1620 tgaaaacaaa acaaactcca tattatcaag catttaagaa ataagatttg tttatatcat 1680 gtctaagttt ttaattaaat aaattcaaca catgaaattt actctaaatc tttatattta 1740 catatgatgc aaacaaatct tatctgaata tttaatttaa ctaatgccta ttatttttag 1800 tcagattata caatctagct agtactttat tttttctatc acgtacatcc aatatcattt 1860 ttttcatagc agttcatgaa atattttatt aaatattaat taaagcataa tttgacctct 1920 aaggtgtgac acgtgtaact aatatagctt tgattttttt ttattatggt ttattttata 1980 ttcaaaatct atcctgattt aaattctaaa taatggatta aaaaacataa attactacac 2040 taattgagtc aacaattatt actgtcgtcg gtt caacttt gagcttttaa gtatatgaga 2100 gtggttgaat tgctgcctat atgaataaaa caatatttat gggggataaa aatgagtctc 2160 atattgtaca tggtagtttg actttgacac atataccctt tgctctggct gtaactagaa 2220 tgcactaggc acaattaaac aaaaataaat tctccttctc tatataaacc caccatgtca 2280 ccacacccta cccagcaaaa ccaaaccata gcaaacctaa gcaccaaacc tccttctttc 2340 aagatccttg aattcagtcc c 2361 <210> 30 <211> 27 <212> DNA <213 > Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 30 tttttctgga actagagatc tctagag 27

[Brief description of the drawings]

FIG. 1 shows a vector of the present invention containing the promoter of the present invention.
It is a figure which shows the construction process of pRS1 / LUC, pRS2 / LUC, and pRS3 / LUC. In the figure, LUC indicates a luciferase gene derived from firefly, and T-gy indicates a terminator of the glycinin gene derived from soybean. Restriction enzyme sites described in parentheses indicate that the restriction enzyme recognition sequence described has been modified, and means that the restriction enzyme site is not digested by the restriction enzyme described.

FIG. 2 is a diagram showing the steps of constructing the luciferase-expressing binary vectors pTiRS1 / LUC, pTiRS2 / LUC, and pTiRS3 / LUC containing the promoter of the present invention. In the figure, LUC
Indicates a luciferase gene derived from fireflies, GUS indicates a β-glucuronidase gene derived from Escherichia coli, nptII indicates a neomycin phosphotransferase gene derived from Escherichia coli, and P-nos indicates a promoter of a nopaline synthase gene derived from a Ti plasmid, P-gy indicates the promoter of the soybean-derived glycinin gene, T-nos indicates the terminator of the nopaline synthase gene derived from the Ti plasmid, and T-gy indicates the terminator of the soybean-derived glycinin gene. Restriction enzyme sites described in parentheses indicate that the described restriction enzyme recognition sequence has been modified,
It means that it is not digested with the restriction enzymes described.

FIG. 3 is a diagram showing the steps of constructing the raffinose synthase expression vectors pRS4, pRS5, and pRS6 containing the promoter of the present invention. In the figure, LUC represents a firefly-derived luciferase gene, T-gy represents a terminator of the soybean-derived glycinin gene, and RS represents a soybean-derived raffinose synthase gene. Restriction enzyme sites described in parentheses indicate that the restriction enzyme recognition sequence described has been modified, and means that the restriction enzyme site is not digested by the restriction enzyme described.

FIG. 4 is a diagram showing the steps of constructing the binary vectors pTiRS4, pTiRS5, and pTiRS6 for expressing raffinose synthase containing the promoter of the present invention. In the figure, RS indicates the raffinose synthase gene from soybean, nptII indicates the neomycin phosphotransferase gene from Escherichia coli, P-nos indicates the promoter of the nopaline synthase gene derived from the Ti plasmid, and T-nos indicates the Ti plasmid. Indicates the terminator of the nopaline synthase gene derived from soybean, and T-gy indicates the terminator of the glycinin gene derived from soybean. Restriction enzyme sites described in parentheses indicate that the restriction enzyme recognition sequence described has been modified, and means that the restriction enzyme site is not digested by the restriction enzyme described.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 5/00 A (72) Inventor Kenji Oeda 4-2-1 Takashi Takarazuka City, Hyogo Prefecture No. Sumitomo Chemical Co., Ltd. F term (reference) 2B030 CA15 CA16 CA17 CA19 4B024 AA08 CA01 DA01 EA04 FA02 FA07 4B065 AA01X AA57X AA88X AA88Y AA90X AB01 BA01 CA53

Claims (14)

[Claims] 1. A promoter containing the following DNA (a) or (b): (A) a DNA consisting of the nucleotide sequence of base numbers 1743 to 2301 in the base sequence represented by SEQ ID NO: 1 (b) hybridizing to the DNA of (a) under stringent conditions, and DNA having promoter function 2. The promoter according to claim 1, comprising the following DNA of (c), (d), (e) or (f). (C) DNA consisting of the base sequence of base numbers 1020 to 2301 in the base sequence shown in SEQ ID NO: 1 (d) From the base sequence of base numbers 2 to 2301 in the base sequence shown in SEQ ID NO: 1 (E) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 (f) hybridizing to the DNA of the above (c), (d) or (e) under stringent conditions, and a promoter in a plant cell Functional DNA 3. A chimeric gene comprising the promoter according to claim 1, a desired gene and a terminator operably linked. 4. A vector containing the promoter according to claim 1. 5. The vector according to claim 4, which comprises a gene insertion site and a terminator downstream of the promoter. 6. A vector containing the chimeric gene according to claim 3. 7. A transformant obtained by introducing the promoter according to claim 1 into a host cell. 8. A transformant obtained by introducing the chimeric gene according to claim 3 into a host cell. 9. A transformant obtained by introducing the vector according to claim 4 into a host cell. 10. The transformant according to claim 7, wherein the host cell is a microbial cell. 11. The transformant according to claim 7, wherein the host cell is a plant cell. 12. A method for producing a chimeric gene, comprising a step of operably linking the promoter according to claim 1, a desired gene and a terminator. 13. A method for producing a vector, comprising a step of operably linking the promoter according to claim 1 and a desired gene. 14. A method for producing a transformed plant, comprising a step of introducing the promoter according to claim 1 into plant cells and expressing a desired gene under the control of the promoter.