JP6427329B2 - Wheat-derived promoter - Google Patents

Description

  The present invention relates to a wheat-derived promoter that is activated under drought stress conditions, salt stress conditions, and low temperature stress conditions. Furthermore, the present invention provides a method of producing a plant having tolerance to vector, microorganism or virus, transformant, drought stress, salt stress and low temperature stress, and drought stress, salt stress and low temperature stress using the promoter of the present invention. It relates to a method of conferring resistance on plants.

  Plants have tolerance mechanisms to cope with various environmental stresses such as drought stress conditions, salt stress conditions, and low temperature stress. In recent years, plants having resistance to such stress have been produced. For example, Patent Document 1 describes a trehalose-producing transgenic plant for the purpose of protecting agricultural products from drought, high salinity or extreme temperatures. In addition, Patent Document 2 describes a method of enhancing tolerance to environmental stress in plants, which is characterized by promoting the production of photosynthesis-related proteins of higher plants. Furthermore, in Patent Document 3, at least one polyamine synthetase enzyme gene is stably maintained under the control of an inducible promoter that can function in plants, and does not have the exogenous polyamine synthetase enzyme gene. Plants and their progeny have been described in which at least one stress tolerance has been improved compared to a control plant.

  Also, it has been reported to impart environmental stress tolerance to plants using a stress inducible promoter. For example, Patent Documents 4 and 5 describe a stress-inducible promoter derived from rice having a predetermined base sequence. In addition, Patent Document 6 describes an environmental stress responsive promoter located upstream of a choline monooxygenase gene in Schizophora (Chenopodium album L.) known as a salt-tolerant plant. Furthermore, Patent Documents 7 and 8 describe environmental stress responsive promoters derived from Arabidopsis thaliana.

Japanese Patent Publication H10-501978 International Publication WO2004 / 058975 Japanese Patent Application Publication No. 2007-21 JP 2004-248638 A International Publication WO2004 / 085641 JP 2004-33026 A Japanese Patent Application Publication No. 2005-80613 JP, 2007-202461, A

  As described above, there have been reports of environmental stress responsive promoters derived from rice, shiroza and Arabidopsis thaliana, but there are few reports of environmental stress responsive promoters for wheat. In addition, there is no report on a promoter having the ability to be activated under any of drought stress conditions, salt stress conditions and low temperature stress conditions to increase the expression level of downstream linked genes. An object of the present invention is to provide a novel promoter having an ability to activate under drought stress conditions, salt stress conditions, and low temperature stress conditions.

  As a result of intensive studies to solve the above problems, the present inventors have developed a novel wheat-derived promoter having the ability to activate under any of drought stress conditions, salt stress conditions and low temperature stress conditions. It succeeded in cloning and came to complete this invention.

According to the present invention, the following inventions are provided.
(1) A wheat-derived promoter having the ability to be activated under drought stress conditions, salt stress conditions, and low temperature stress conditions to increase the expression level of downstream linked genes.
(2) The promoter according to (1), which is derived from Triticum monococcum.
(3) The ability to increase the expression level of downstream linked genes under drought stress conditions, salt stress conditions and low temperature stress conditions is 1 to 150 times that of the cauliflower mosaic virus 35S promoter, The promoter as described in (1) or (2).
(4) The ability to increase the expression level of downstream linked genes under drought stress conditions is 3.0 times or more in leaves and 0.7 times or more in roots as compared to the start of drought stress treatment Yes,
The ability to increase the expression level of downstream linked genes under salt stress conditions is 1.2-fold or more in leaves and 20-fold or more in roots as compared to the beginning of salt stress treatment,
The ability to increase the expression level of downstream linked genes under cold stress conditions is 3.0 times or more in leaves and 10 times or more in roots as compared to the start of cold stress treatment, (1) The promoter according to any one of (3) to (3).
(5) The ability to increase the expression level of downstream linked genes under drought stress conditions is 4.0 to 63.1 times in leaves and 10.9 in roots as compared to that at the start of drought stress treatment ~ 41.8 times,
The ability to increase the expression level of downstream linked genes under salt stress conditions is 1.5 to 33.5-fold in leaves compared to that at the start of salt stress treatment, and in the roots from 3 to 190. 5 times or less,
The ability to increase the expression level of downstream linked genes under cold stress conditions is 3.8 to 43.3 times in leaves compared to that at the start of cold stress treatment and 14.1 to 881 in roots. The promoter according to any one of (1) to (4), which is 5 times or less.
(6) MYCATRD22 (base sequence is CACATG), MYCATERD1 (base sequence is CATGTG), MYBCORE (base sequence is CNGTTR), MYB2AT (base sequence is TAACTG), CBFHV (base sequence is RYCGAC), ABRELATERD1 (base sequence is ACGTG) , ABREATRD 22 (base sequence is RYACGTGGYR), GT1 GMSCAM 4 (base sequence is GAAAAA), LTRECOREATCOR15 (base sequence is CCGAC), and CCAATBOX1 (base sequence is CCAAT) at least one or more motifs selected from the group consisting of The promoter according to any one of (1) to (5), which has the above.
(7) The promoter according to any one of (1) to (6), which is any of the following (a) to (d).
(A) a promoter comprising the nucleotide sequence set forth in SEQ ID NO: 17, 18 or 21;
(B) A base sequence having a sequence identity of 90% or more with the base sequence set forth in SEQ ID NO: 17, 18 or 21, activated under dry stress conditions, salt stress conditions and low temperature stress conditions, A promoter having the ability to increase the expression level of downstream linked genes;
(C) In the base sequence shown in SEQ ID NO: 17, 18 or 21, it contains a base sequence in which one or more bases are deleted, inserted, substituted and / or added, under dry stress conditions, salt stress conditions, And a promoter having the ability to activate under cold stress conditions to increase the expression level of the downstream linked gene; or (d) a base sequence and a string complementary to the base sequence set forth in SEQ ID NO: 17, 18 or 21 A promoter comprising a base sequence that hybridizes under a gen condition, and having the ability to be activated under drought stress conditions, salt stress conditions and low temperature stress conditions to increase the expression level of downstream linked genes.
(8) A vector comprising the promoter according to any one of (1) to (7) and a gene linked downstream of the promoter.
(9) A microorganism or a virus comprising the vector according to (8).
(10) A transformant obtained by transforming a host cell with the vector according to (8) and / or the microorganism or virus according to (9).
(11) The transformant according to (10), wherein the host cell is a monocotyledon-derived cell.
(12) The transformant according to (10) or (11), wherein the host cell is a cell derived from wheat (Triticum aestivum).
(13) A method for producing a plant having tolerance to drought stress, salt stress and low temperature stress, comprising the step of introducing the vector of (8) and / or the microorganism or virus of (9) into a plant cell.
(14) A method for imparting tolerance to drought stress, salt stress and low temperature stress to a plant, comprising the step of introducing the vector according to (8) and / or the microorganism or virus according to (9) into a plant cell.

  The promoter of the present invention has the ability to be activated under drought stress conditions, salt stress conditions, and low temperature stress conditions to increase the expression level of downstream linked genes. By using the promoter of the present invention, it is possible to produce plants having tolerance to drought stress, salt stress and low temperature stress.

FIG. 1 is an electrophoresis photograph of RT-PCR showing the expression level of GUS gene of leaves in drought stress treatment. The upper row of each cDNA shows the amplification result of the GUS gene, and the lower row shows the amplification result of the endogenous gene EF-1α. FIG. 2 is an RT-PCR electrophoresis photograph showing the expression levels of leaf and root GUS genes in salt stress treatment. The upper row of each cDNA shows the amplification result of the GUS gene, and the lower row shows the amplification result of the endogenous gene EF-1α. FIG. 3 is an electrophoresis photograph of RT-PCR showing the expression levels of leaf and root GUS genes in low temperature stress treatment. The upper row of each cDNA shows the amplification result of the GUS gene, and the lower row shows the amplification result of the endogenous gene EF-1α. FIG. 4 shows photographs of GUS staining during drying, salt and cold stress treatment. FIG. 5 shows the results of quantitative PCR of the expression level of GUS gene during drying, salt and low temperature stress treatment as compared with cauliflower mosaic virus 35S promoter. In the leaf A of FIG. 5, from the left side in each column of tplb0004111, Contig1515, tplb0015p04, 0 hours, 1, 3, and 5 hours after stress application to normal cultivation conditions. Return to 2 days later. In the root of A of FIG. 5, from the left side in each column of tplb0004111, Contig1515, tplb0015p04, 0 hour, 1 hour, 3 hours, and 5 hours after stress application are shown. In the leaves and roots of B and C in FIG. 5, from the left side in each column of tplb0004111, Contig1515, tplb0015p04, 0 hour, 1 hour, 1 hour, 5 hours, 10 hours and 24 hours after stress application, respectively 2 days after returning to normal cultivation conditions from stress application. FIG. 6 shows the results of quantitative PCR of the expression level of GUS gene during dry, salt and low temperature stress treatment as compared with the treatment start time. FIG. 7 shows the position of the type of motif present in the promoter of the present invention.

Hereinafter, embodiments of the present invention will be described.
(1) Promoter The promoter of the present invention is characterized by having the ability to be activated under drought stress conditions, salt stress conditions, and low temperature stress conditions to increase the expression level of downstream linked genes. When the promoter is activated to increase the expression level of the gene linked downstream, RNA polymerase binds to the promoter when the promoter is exposed under dry stress conditions, salt stress conditions, or low temperature stress conditions, It refers to activating the transcription of a gene linked downstream of a promoter to increase the expression level of the gene.

  The dry stress condition means a state of lack of water, for example, placing the plant body in a state where it can not absorb water, such as putting it on a towel. The salt stress condition means a state where NaCl at a concentration of 50 mM to 600 mM, preferably 100 mM to 400 mM, is continuously treated for 0.5 hours to several weeks. The low temperature stress condition means a condition exposed to a temperature lower than the optimum temperature for living species, for example, in the case of wheat, at a temperature of -20 ° C to + 10 ° C, preferably -15 ° C to + 5 ° C. It says to treat continuously for one hour to several weeks.

  Whether it has the ability to be activated under drought stress conditions, salt stress conditions, and low temperature stress conditions to increase the expression level of downstream linked genes can be confirmed by the following procedure. . That is, a vector is prepared by linking various reporter genes (eg, β-glucuronidase gene (GUS), luciferase gene (LUC), green fluorescent protein gene (GFP), etc.) downstream of the promoter, and the vector is It can be confirmed by measuring the expression of the reporter gene under drought stress conditions, salt stress conditions, and low temperature stress conditions after introduction into the genome of the host. The promoter of the present invention is characterized in that it is activated under drought stress conditions, salt stress conditions, and low temperature stress conditions.

  The organism from which the promoter of the present invention is derived is wheat, and as wheat, T. agilopoides, T. thaoudar, T. monococcum, 2 grains T. dicoccoides, T. dicoccum (2 grains, Emmer wheat), T Pyromidale, T. orientale (cola wheat), T. durum (durum wheat, macaroni wheat), T. turgidum (rivet wheat), T. polonicum (Polish wheat), T. persicum (Persia wheat), ordinary strain T . aestivum (normal wheat, bread wheat), T. spelta (spelled wheat (dinkel wheat, farlo wheat)), T. compactum (club wheat, thick eared wheat) T. sphaerococcum (Indian dwarf wheat), T. maha (maca wheat) , T. vavilovii (Babilovi wheat), Tymofevi strain T. timopheevi etc., but not particularly limited. Among the above-mentioned, a promoter derived from Triticum monococcum is particularly preferable.

  In a preferred embodiment, the promoter of the present invention has the ability to increase the expression level of downstream linked genes under drought stress conditions, salt stress conditions and low temperature stress conditions compared to the cauliflower mosaic virus 35S promoter It is 1 to 150 times (preferably 1.02 to 140 times, more preferably 1.04 to 138.7 times).

In a preferred embodiment, the promoter of the present invention is a promoter satisfying any of the following (condition i) to (condition iii):
(Condition i)
The ability to increase the expression level of downstream linked genes under drought stress conditions is 3.0 times or more (preferably 3.5 times or more, more preferably 4.) or more in leaves as compared to that at the start of drought stress treatment. 0 times or more), and 0.7 times or more (preferably 0.75 times or more, more preferably 0.8 times or more) in the root,
The ability to increase the expression level of downstream linked genes under salt stress conditions is at least 1.2 times (preferably 1.4 times or more, more preferably 1. times or more) in the leaves as compared to the start of salt stress treatment. 5 times or more), 20 times or more (preferably 25 times or more, more preferably 30.1 times or more) in the root,
The ability to increase the expression level of downstream linked genes under low temperature stress conditions is 3.0 times or more (preferably 3.5 times or more, more preferably 3. or more) in the leaves as compared to the start of low temperature stress treatment. 8 times or more) and 10 times or more (preferably 12 times or more, more preferably 14.1 times or more) in the root.

(Condition ii)
The ability to increase the expression level of downstream linked genes under drought stress conditions is at least 40 times (preferably 50 times or more, more preferably 55.6 times or more) in the leaves as compared to the start of drought stress treatment And 30 times or more (preferably 35 times or more, more preferably 41.8 times or more) in the root,
The ability to increase the expression level of downstream linked genes under salt stress conditions is at least 20 times (preferably 30 times or more, more preferably 33.5 times or more) in leaves as compared to when salt stress treatment is initiated And 100 times or more (preferably 150 times or more, more preferably 190.5 times or more) in the root,
30-fold or more (preferably 40-fold or more, more preferably 41.8-fold or more) in leaves as compared with the initiation of low temperature stress treatment, the ability to increase the expression level of downstream linked genes under low temperature stress conditions And 300 times or more (preferably 500 times or more, more preferably 881.5 times or more) in the root.

(Condition iii)
The ability to increase the expression level of downstream linked genes under drought stress conditions is at least 40-fold (preferably 50-fold or more, more preferably 63.1-fold or more) in leaves as compared to the start of drought stress treatment And 5 times or more (preferably 8 times or more, more preferably 10.9 times or more) in the root,
The ability to increase the expression level of downstream linked genes under salt stress conditions is 3 times or more (preferably 4.0 times or more, more preferably 5.3 times or more) in the leaves as compared to the beginning of salt stress treatment Or more, and 100 times or more (preferably 150 times or more, more preferably 182.6 times or more) in the root,
30-fold or more (preferably 40-fold or more, more preferably 43.3-fold or more) in leaves compared to the start of low temperature stress treatment, the ability to increase the expression level of downstream linked genes under low temperature stress conditions In the root, it is 200 times or more (preferably 300 times or more, more preferably 447.4 times or more).

In a more preferred embodiment, the promoter of the present invention is a promoter satisfying the following.
The ability to increase the expression level of downstream linked genes under drought stress conditions is 4.0-fold or more and 63.1-fold or less, more preferably 55. 6 times or more and 63.1 times or less, and 0.8 times or more and 41.8 times or less, more preferably 10.9 times or more and 41.8 times or less in the root,
The ability to increase the expression level of downstream linked genes under salt stress conditions is 1.5 times or more and 33.5 times or less in the leaves as compared to the start of salt stress treatment, and more preferably 5. 3 times or more and 33.5 times or less, and 30.1 times or more and 190.5 times or less, more preferably 182.6 times or more and 190.5 times or less, in the root,
41. The ability to increase the expression level of downstream linked genes under cold stress conditions is 3.8 times or more and 43.3 times or less in the leaves as compared to the start of cold stress treatment, more preferably 41. It is 8 times or more and 43.3 times or less, and in the root, 14.1 times or more and 881.5 times or less, more preferably 447.4 times or more and 881.5 times or less.

Specific examples of the promoter of the present invention include a promoter comprising the nucleotide sequence set forth in SEQ ID NO: 17, 18 or 21 obtained in the following Examples.
The position of the type of motif present in the promoter comprising the nucleotide sequence set forth in SEQ ID NO: 17 (RFL_Contig 1515), SEQ ID NO: 18 (tplb0004111) and SEQ ID NO: 21 (tplb0015p04) is shown in FIG. The promoter of the present invention is MYCATRD22 (base sequence is CACATG), MYCATERD1 (base sequence is CATGTG), MYBCORE (base sequence is CNGTTR), MYB2AT (base sequence is TAACTG), CBFHV (base sequence is RYCGAC), ABRELATERD1 (base sequence Is ACGTG, ABREATRD 22 (base sequence RYACGTGGYR), GT1GMSCAM4 (base sequence is GAAAAA), LTRECOREATCOR15 (base sequence is CCGAC), and at least one selected from the group consisting of CCAATBOX1 (base sequence is CCAAT) (preferably) At least one (preferably 3 or more, more preferably 5 or more, particularly preferably 8 or more, preferably 2 or more, more preferably 3 or more, further preferably 4 or more, more preferably 5 or more motifs. Or more) is preferable.

The following can be mentioned as specific examples of the promoter of the present invention.
-One MYCATRD22 (base sequence is CACATG), three MYBCORE (base sequence is CNGTTR), one MYB2AT (base sequence is TAACTG), ABRELATERD1 (base sequence is ACGTG) or ABREATRD22 (base sequence is RYACGTGGYR) A promoter having two GT1GMSCAM4 (base sequence is GAAAAA) and three CCAATBOX1 (base sequence is CCAAT) (for example, RFL_Contig 1515 of SEQ ID NO: 17)
・ 3 MYCATRD22 (base sequence is CACATG), 1 MYCATERD1 (base sequence is CATGTG), 5 MYBCORE (base sequence is CNGTTR), 1 MYB2AT (base sequence is TAACTG), 1 GT1 GMSCAM4 (base sequence is A promoter having one GAAAAA) and one CCAATBOX1 (the base sequence is CCAAT) (for example, tplb0004111 of SEQ ID NO: 18)
・ 1 MYCATRD22 (base sequence is CACATG), 2 MYBCORE (base sequence is CNGTTR), 4 CBFHV (base sequence is RYCGAC), ABRELATERD1 (base sequence is ACGTG) or ABREATRD22 (base sequence is RYACGTGGYR) One promoter, one GT1 GMS CAM4 (base sequence is GAAAAA), 3 LTRECOREATCOR15 (base sequence is CCGAC), and 3 CCAATBOX1 (base sequence is CCAAT) (for example, tplb0015p04 of SEQ ID NO: 21)

  Furthermore, as long as it has the ability to be activated under drought stress conditions, salt stress conditions and low temperature stress conditions to increase the expression level of downstream linked genes, the bases described in SEQ ID NO: 17, 18 or 21 The promoter may be a promoter containing a nucleotide sequence having a sequence identity of 90% or more (preferably 95% or more, more preferably 97% or more, more preferably 98% or more, particularly preferably 99% or more).

  Furthermore, as long as it has the ability to be activated under drought stress conditions, salt stress conditions and low temperature stress conditions to increase the expression level of downstream linked genes, the bases described in SEQ ID NO: 17, 18 or 21 The sequence may be, for example, a promoter containing a nucleotide sequence in which one or more bases are deleted, inserted, substituted and / or added, and is 1 to 20, preferably 1 to 10, more preferably 1 to 5 It is also possible to use a promoter comprising a nucleotide sequence in which 1 to 3, particularly preferably 1 to 3 bases have been deleted, inserted, substituted and / or added.

Furthermore, the promoter of the present invention may have one or more bases deleted, inserted, substituted and / or added in a base sequence other than the motif of the promoter.
For example, the following promoters can be mentioned. In addition, 1-200 bp means the 1st-200th base, and the description of those other than the above is also the same.
(I) deletion of one or more bases from the transcription start point (ATG) in the base sequence of SEQ ID NO: 17, 18 or 21 to the nearest TATA box and / or in the base sequence of 1 to 200 bp , Inserted, substituted and / or added promoters.
(Ii) 201 to 362 bp, 369 to 471 bp, 477 to 487 bp, 493 to 759 bp, 765 to 880 bp, 887 to 1061 bp, 1068 to 1090 bp, 1097 to 1211 bp, 1218 to 1374 bp, 1380 to 1437 bp in the base sequence described in SEQ ID NO: 17 , A promoter in which one or more bases are deleted, inserted, substituted and / or added in the base sequence of 1444-1509 bp.
(Iii) 201 to 205 bp, 213 to 583 bp, 590 to 689 bp, 703 to 721 bp, 1163 to 1365 bp, 1371 to 1407 bp, 1414 to 1540 bp, 1549 to 15188 bp, 1595 to 1619 bp of the nucleotide sequence of SEQ ID NO: 18 , A promoter in which one or more bases are deleted, inserted, substituted and / or added in the base sequence of 1626 to 1752 bp.
(Iv) 201-427 bp, 434-525 bp, 532-538 bp, 544-612 bp, 618-763 bp, 769-1190 bp, 1197-1462 bp, 1481-1531 bp, 1538-1607 bp, 1614-1634 bp of the nucleotide sequence set forth in SEQ ID NO: 21 A promoter in which one or more bases are deleted, inserted, substituted and / or added in the base sequence of

  Furthermore, as long as it has the ability to be activated under drought stress conditions, salt stress conditions and low temperature stress conditions to increase the expression level of downstream linked genes, the bases described in SEQ ID NO: 17, 18 or 21 It may be a promoter containing a base sequence that hybridizes under stringent conditions with a base sequence complementary to the sequence. Here, under stringent conditions, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, the temperature is 42 to 68 ° C., preferably 42 to 65 ° C., more preferably 50 to 65 ° C., particularly preferably 55-65 ° C. More specifically, 5 × SSC (83 mM NaCl, 83 mM sodium citrate) at a temperature of 42 ° C., 50 ° C., 55 ° C., 60 ° C. or 65 ° C.

  The promoter of the present invention may have a base sequence to improve translation efficiency added to the 3 'end of the above-mentioned base sequence, or may have the 5' end deleted as long as promoter activity is not lost. .

  After the base sequence of the promoter of the present invention is determined, the present invention is subsequently carried out by chemical synthesis, by PCR using a cloned probe as a template, or by hybridizing a DNA fragment having the base sequence as a probe. Promoters can be obtained. Furthermore, it is possible to synthesize a mutant of the promoter of the present invention which has the same function as the promoter before mutation by site-directed mutagenesis and the like. The site-directed mutagenesis can be carried out using a kit for mutagenesis (for example, Mutant-K (manufactured by TAKARA) or Mutant-G (manufactured by TAKARA)), and LA manufactured by TAKARA. Mutation introduction can also be performed using the PCR in vitro Mutagenesis series kit.

2. Vector The vector of the present invention can be produced by ligating the promoter of the present invention to a suitable vector. The vector for inserting the promoter of the present invention is not particularly limited as long as it can replicate in the host, and examples include plasmids, shuttle vectors, helper plasmids and the like.

  Examples of plasmid DNA include plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YCp50, etc.) And the like. Examples of the phage DNA include λ phage (Charon 4A, Charon 21A, EMBL 3, EMBL 4, λgt 10, λgt 11, λ ZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.

  In order to insert the promoter of the present invention into a vector, first, purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or multiple cloning site of an appropriate vector DNA, and ligated to the vector, etc. Will be adopted.

  In the present invention, a gene can be further inserted into the above-described vector in order to express a desired gene. The method of inserting a gene is the same as the method of inserting a promoter into a vector. Although the type of gene is not particularly limited, for example, a gene encoding a protein capable of conferring tolerance to drought stress, salt stress and / or low temperature stress to a plant is preferable.

  In the vector of the present invention, the promoter of the present invention needs to be incorporated into the vector such that the gene can be expressed so that the function of the desired gene described above can be exerted. The state capable of expression means that a gene and a promoter are linked and integrated into a vector such that the gene is expressed under the control of the promoter in a host organism into which the gene is introduced. That is, in addition to the promoter of the present invention and a desired gene, a terminator, an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence) and the like can be ligated to the vector of the present invention . Examples of selection markers include antibiotic resistance genes such as ampicillin resistance gene and kanamycin resistance gene, and herbicide resistance genes such as bialaphos resistance gene.

  Furthermore, a promoter of the present invention in which an arbitrary gene of interest is connected in a sense or antisense direction can be prepared and inserted into a vector such as pBI101 (Clonetech) called a binary vector.

3. Transformant The transformant of the present invention can be obtained by introducing the expression vector of the present invention into a host. Here, the host is not particularly limited as long as it can express a promoter or a target gene, an environmental stress responsive transcription factor, but a plant is preferable. When the host is a plant, a transformed plant (transgenic plant) can be obtained as follows.

  Plants to be transformed in the present invention include whole plants, plant organs (eg leaves, petals, stems, roots, seeds etc.), plant tissues (eg epidermis, phloem, soft tissue, xylem, vascular bundles etc.) Or plant cultured cells. Plants used for transformation include Brassicaceae (eg, Arabidopsis thaliana etc.), Gramineae (eg, Wheat (Triticum), maize (Zea mays), rice (Oryza sativa) etc.), solanaceous plants (eg, And tobacco (Nicotiana tabacum etc.), plants belonging to the leguminous family (eg, soybean (Glycine max) etc.), etc., but it is not limited to these plants.

  The vector of the present invention can be introduced into a host by a conventional transformation method such as electroporation, Agrobacterium method, particle gun method, PEG method and the like.

  For example, in the case of using electroporation, a gene can be introduced into a host by treatment under conditions of voltages of 500 to 1600 V, 25 to 1000 μF, and 20 to 30 msec by an electroporation apparatus provided with a pulse controller.

  When the particle gun method is used, a plant, a plant organ, or a plant tissue itself may be used as it is, may be used after preparing a section, or may be used after preparing a protoplast. The sample thus prepared can be processed using a gene transfer device (eg, PDS-1000 / He etc. from Bio-Rad). The treatment conditions vary depending on the plant or sample, but usually the pressure is about 1000 to 1800 psi and the distance is about 5 to 6 cm.

  In addition, by using a plant virus as a vector, a target gene can be introduced into a plant. Available plant viruses include, for example, cauliflower mosaic virus. That is, first, a viral genome is inserted into a vector derived from E. coli, etc. to prepare a recombinant, and then these target genes are inserted into the viral genome. The target gene can be introduced into a plant host by excising the thus modified virus genome from the recombinant with restriction enzymes and inoculating the plant host.

  In the method of using the Agrobacterium Ti plasmid, when a bacterium belonging to the genus Agrobacterium infects a plant, it utilizes the property of transferring a part of the plasmid DNA it has into the plant genome. , The target gene is introduced into a plant host. Among bacteria belonging to the Agrobacterium genus, Agrobacterium tumefaciens (Agrobacterium tumefaciens) infects plants to form a tumor called crown gall, and Agrobacterium rhizogenes (Agrobacterium rhizogenes) infects plants. To generate hairy roots. In these, the region called T-DNA region (Transferred DNA) on the plasmid present in each bacterium called Ti plasmid or Ri plasmid during infection is transferred into the plant and integrated into the plant genome. It is due to

  If DNA to be integrated into the plant genome is inserted into the T-DNA region on the Ti or Ri plasmid, the target DNA can be incorporated into the plant genome when Agrobacterium bacteria infect the plant host. It can be incorporated.

  Tumor tissue, shoots, hairy roots and the like obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture, and can be used at appropriate concentrations using conventionally known plant tissue culture methods. The plant body can be regenerated by administration of a plant hormone (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.)

  The vector of the present invention can be introduced not only into the above-mentioned plant host but also from the genus Escherichia, such as Escherichia coli, Bacillus, such as Bacillus subtilis, or Pseudomonas, such as Pseudomonas putida. (Saccharomyces cerevisiae), yeast such as Schizosaccharomyces pombe (Shizosaccharomyces pombe), animal cells such as COS cells, CHO cells, or insect cells such as Sf9, and transformants are transformed. You can also get it. When using a bacterium such as E. coli or yeast as a host, the recombinant vector of the present invention is autonomously replicable in the bacterium, and simultaneously comprises the promoter of the present invention, a ribosome binding sequence, a target gene, and a transcription termination sequence Is preferred. In addition, a gene that controls a promoter may be included.

  The method for introducing the recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using calcium ion, an electroporation method and the like can be mentioned.

  When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc. are used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, a lithium acetate method and the like.

  When an animal cell is used as a host, monkey cell COS-7, Vero, Chinese hamster ovary cell (CHO cell), mouse L cell or the like is used. Examples of methods for introducing the recombinant vector into animal cells include electroporation, calcium phosphate method, lipofection method and the like.

  When an insect cell is used as a host, Sf9 cells or the like are used. Examples of methods for introducing a recombinant vector into insect cells include calcium phosphate method, lipofection method, electroporation method and the like.

  Whether or not the gene has been incorporated into the host can be confirmed by PCR method, Southern hybridization method, Northern hybridization method or the like. For example, DNA is prepared from transformants, DNA-specific primers are designed, and PCR is performed. The PCR is performed under the same conditions as those used to prepare the plasmid. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis or the like, stained with ethidium bromide, SYBR Green solution or the like, and the amplified product is detected as a single band, Confirm that it has been transformed. Alternatively, amplification products can be detected by conducting PCR using primers previously labeled with a fluorescent dye or the like. Furthermore, a method may be employed in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or an enzyme reaction.

4. Production of Plant In the present invention, the above-mentioned transformed plant cells can be regenerated into transformed plants. As a regeneration method, a method is employed in which callus-like transformed cells are transferred to a culture medium in which the type and concentration of hormone are changed, and cultured to form somatic embryos to obtain a complete plant. As the medium to be used, LS medium, MS medium and the like are exemplified.

  In the present invention, an expression vector into which the promoter of the present invention has been inserted is introduced into a host cell to obtain a transformed plant cell, and the transformed plant is regenerated from the transformed plant cell, and the transformed plant obtained. Plant seeds can be obtained, and plants can be produced from the plant seeds.

  To obtain plant seeds from transformed plants, for example, transformed plants are harvested from a rooting medium, transplanted to a pot containing water-containing soil, and grown under constant temperature to form flowers. And finally form seeds. Also, in order to produce a plant from the seed, for example, when the seed formed on the transformed plant matures, it is isolated, sowed in soil containing water, and grown under constant temperature and illuminance. Produce a plant body. In plants thus bred, in particular, expression of a gene located downstream of the promoter of the present invention can be induced.

  Transformed plants into which the promoter of the present invention has been introduced have tolerance to drought stress, salt stress and low temperature stress by increasing the expression level of the gene linked downstream.

  The present invention will be specifically described by the following examples, but the present invention is not limited by the examples.

Example 1 Isolation of Stress-Responsive Genes In the present invention, genes that are specifically expressed at the time of environmental stress were comprehensively analyzed using Wheat Oligonucleotide Microarray (manufactured by Agilent Technologies). The microarray contains 43,000 oligo DNA based on the latest public databases such as RefSeq (Release 31), Unigene (Build 53), TIGR Plant Transcript Assemblies (Release 5), and TIGR Gene Indicators (Release 11). It covers the above transcripts.

  The samples used for the experiment were prepared as follows. Wheat seeds (Triticum aestivam Chinese Spring) were absorbed on filter paper on a petri dish for 2 days at 4 ° C., and then transferred to a growth chamber under illumination at 22 ° C. for 20 hours for germination. After that, they were transferred onto glass beads and subjected to hydroponic culture for 10 days to grow wheat leaves and roots.

  Genes that respond to drought stress as environmental stress were analyzed. In the drying stress treatment, a plant under hydroponic culture was put on a Kim towel to bring it into a drying stress state, and samples were taken over time at 1, 5, 10 and 24 hours after treatment. The samples that had been subjected to dry stress treatment each time were cut into leaves and roots, and the samples were cryopreserved with liquid nitrogen in situ. Further, as a comparative control, samples were also taken over time at 1, 5, 10 and 24 hours from those continuing hydroponic culture.

  The RNeasy Plant mini kit (manufactured by QIAGEN) was used for extraction of total RNA from the cryopreserved sample, and concentration measurement was performed using a NanoDrop ND-2000 spectrophotometer (manufactured by NanoDrop Technologies). The degree of degradation of the total RNA was measured using an Agilent 2100 bioanalyzer, and the degree of degradation of RNA was checked using RNA 6000 Nano kit (manufactured by Agilent Technologies) as an analysis kit.

  For labeling of RNA, Low Input Quick Amp Labeling Kit (manufactured by Agilent Technologies) was used to synthesize Cy3-labeled cDNA.

  The cDNA was hybridized with Wheat Oligonucleotide Microarray using Gene Expression Hybridization Kit (Agilent Technologies). Thereafter, using a Gene Expression Wash Pack (containing Wash buffer 1 and Wash buffer 2) (manufactured by Agilent Technologies), non-specific labeled targets and non-specific adsorption were washed away to dry the slide glass of the microarray.

The slide glass was read into a DNA Microarray Scanner (manufactured by Agilent Technologies), and the fluorescence intensity of each spot was quantified. Using the GeneSpring (Agilent Technologies) software for microarray data analysis, the fluorescence intensity (numerical value) of each spot, the number of a 20-mer probe mounted on each spot (ID), the original CDS number (contig or tplb The analysis was carried out by collating the information of the proteins encoded by the gene, and other plants (rice, arabi, barley, etc.) having homology with CDS. As a result, seven genes were selected as stress response genes, whose expression amount specifically increases during drying.

[Example 2] Isolation of Promoter Sequence As a result of microarray analysis, each selected gene (CDS) is searched by searching the wheat CDS database (URL: http://trifldb.psc.riken.jp/index.pl) Sequence information of stress response gene was acquired. In addition, wheat sequences registered in DDBJ were downloaded and assembled to obtain sequence information of response genes. The promoter sequence is presumed to be about 1 to 2 kb upstream of the position assumed to be the start codon (ATG) of the gene as a promoter region.

Since the sequence information of the wheat genome has not been completely decoded yet, it is possible to use the 5 'RACE method, TAIL-PCR method, etc. which are conventional methods for isolation of the promoter region present upstream of each gene Plant cell engineering series 7 New version PCR experiment protocol of plant Shujunsha). In addition, DNA Walking Speedup Premix kit (manufactured by Seegene) specialized for the sequence of the unknown region can also be used.

  The present inventors first attempted to isolate a promoter sequence from wheat (Triticum aestivam Chinese Spring), but it was difficult to isolate a single promoter sequence because it is a heteroploid (AABBDD). Therefore, isolation of a promoter sequence was performed from single grain wheat (Triticum monococcum A'A ') which is an ancestor of wheat.

Seeds of single grain wheat (Triticum monococcum DV 92) were hydroponically cultured according to the method described in Example 1, and genomic DNA was extracted from grown leaves (first to second leaves) by CTAB method (Shimamoto et al. Plant cells Engineering series 7 New version PCR experiment protocol of plant Shujunsha). Using this as a template, DNA Walking Speedup Premix kit was used to isolate upstream sequences of each stress response gene. The obtained sequence was incorporated into pGET vector to identify a promoter region. The sequence analysis was carried out by the dye terminator method, using an Applied Biosystems 3130xl genetic analyzer.

Based on the decoded genomic information, primers for amplifying the upstream sequence of each cDNA were redesigned (Table 1). The 4-base sequence of CACC is added to the 5 'end side of the forward primer, and the DNA is amplified by PCR and can be cloned into the gateway entry vector pENTR / D-TOPO (manufactured by Invitrogen).

  Genomic PCR was performed using forward and reverse primers of each cDNA under the following conditions. Using PrimeSTAR GXL polymerase (TaKaRa BIO) with very high accuracy as PCR enzyme, 50 μl reaction system (1 × PrimeSTAR GXL buffer, 0.2 mM dNTP solution, 0.2 μM PCR primer, template (Triticum monococcum as described above) DV92 genome) 100 ng, 1.25 U PrimeSTAR GXL polymerase). The PCR conditions are as follows: first react at 94 ° C for 5 minutes, then 30 cycles of 98 ° C for 10 seconds, 55 ° C for 15 seconds and 68 ° C for 2 minutes, and finally at 68 ° C for 5 minutes went. The PCR product was confirmed by 1.0% agarose gel electrophoresis to confirm the fragment size and amplification efficiency, and purified using NucleoSpin Gel and PCR Clean-up (TaKaRa BIO).

  The purified PCR product was incorporated into pENTR / D-TOPO, and sequence analysis of the promoter region was performed to confirm that there was no mutation. The respective promoter sequences are shown in SEQ ID NOs: 15-21.

Sequence of CDS promoter region
7AL.Contig 16213: SEQ ID NO: 15
Scaffold 2239: SEQ ID NO: 16
RFL_Contig1515: SEQ ID NO: 17
tplb0004l11: SEQ ID NO: 18
tplb0009k09: SEQ ID NO: 19
tplb0010o09: SEQ ID NO: 20
tplb0015p04: SEQ ID NO: 21

Example 3 Preparation of Agrobacterium for Transformation pENTR / D-TOPO into which a promoter having a confirmed sequence was introduced is a gateway vector pMDC164 (Curtis et. Al. (2003) Plant Physiol. Oct; 133 (2) It was recombined by using 462-9) and LR Clonase Enzyme mix (manufactured by invitrogen) to obtain an expression vector in which a β-glucuronidase gene (GUS) and a Nos terminator (Tnos) were linked downstream of the promoter sequence. The pMDC164 vector also contains a hygromycin resistant gene in the T-DNA region.

  For the expression vector into which the above promoter sequence has been introduced, freeze-thaw method (Hofgen et. Al. (1988) Nucleic Acids Res., Oct 25; 16 (20) 9877) in Agrobacterium (Agrobacterium tumefacience EHA 105) It was introduced and transformed. In order to select for transformed Agrobacterium, it was applied to an LB agar medium containing kanamycin (50 μg / L) and subjected to stationary culture at 28 ° C. for 2 days. The single colony thus obtained was inoculated in LB liquid medium, shake cultured at 28 ° C. for 2 days, and stored at -80 ° C. as a glycerol stock with a final concentration of 20%.

[Example 4] Transformation of a Poaceae model plant In the present invention, promoter activity is evaluated using Brachypodium distachyon, which is an annual herbaceous plant of Poaceae. Minato-kadoshi is a plant classified in the same family as the wheat and barley, and is similar in taxonomically to wheat than sorghum and rice, and is smaller in genome size, and the plant is relatively small and transformation is possible. From the point of view, it is considered to be suitable for model plants.

  Agrobacterium infection of callus was carried out according to the method of Alves et. Al. (Nature Protocol 2009; 4 (5): 638-49) for transformation of C. versicolor. Callus was formed from immature embryos by removing immature embryos of self-pollinated Minatokamodigusa Bd21 and culturing in BDD medium containing 2,4-D (2.5 μg / ml) in the dark at 25 ° C. for about 3 weeks. .

  The glycerol stock Agrobacterium was inoculated into LB liquid medium containing kanamycin (50 μg / ml), and shake culture was carried out at 28 ° C. overnight. The resultant was applied to BDA medium containing kanamycin (50 μg / ml) and statically cultured at 28 ° C. for 2 days. The grown cells were scraped, suspended in an agrobacterium infection solution containing acetosyringone (40 μg / ml), and adjusted to OD 600 nm = 1.0 to obtain an inoculation suspension.

Embryo calli were immersed in the inoculum suspension for 5 minutes, transferred to a medium containing hygromycin and carbenicillin and statically cultured at 25 ° C. in the dark for about 6 weeks. The callus which showed hygromycin resistance and continued to grow was transferred to shoot formation medium containing kinetin and cultured for 2 to 5 weeks under illumination at 25 ° C. for 16 hours. Individuals in which shoot formation was observed were transferred to MS medium in agripot to promote root formation. Plants sufficiently rooted in an agripot were raised, and grown for 3 to 4 months at 22 ° C., 50% humidity, and 20 hours of light to collect seeds (T1). As a result of transformation of C. elegans using Agrobacterium expressing each of the expression vectors, transformed plants were not generated only for the callus infected with Agrobacterium in which the promoter sequence of tplb0009k09 was incorporated.

[Example 5] Evaluation of environmental stress response Drying stress response experiment T1 seeds of the transformed plants collected in Example 4 were allowed to absorb water at 4 ° C. for 2 days on filter paper on a petri dish, and then transferred to a growth chamber under illumination at 22 ° C. for 20 hours for germination. . After that, it was transferred onto glass beads and subjected to hydroponic culture for 10 days to grow leaves and roots. In the drying stress treatment, a plant under hydroponic culture was put on a Kim towel to bring it into a drying stress state and treated for 5 hours.

After drying, the leaves and roots were separated and lyophilized. Similarly, the leaves and roots of transformants that continued to be subjected to hydroponic culture as no treatment (control) were also collected. RNA was extracted from each sample using RNeasy Plant mini kit and then using PrimeScript II 1st strand cDNA synthesis Kit (TaKaRa BIO) to synthesize cDNA. The drought stress response was confirmed by performing semiquantitative RT-PCR of the GUS gene linked downstream of the promoter sequence. RT-PCR conditions are shown below.

  The polymerase used was Quick Taq HS DyeMix (manufactured by TOYOBO), and was performed in 50 μl of a reaction system (1 × Premix, 0.2 μM PCR primer, 25 ng of the template (cDNA above)). The PCR conditions are: first react at 94 ° C. for 2 minutes, then 30 cycles of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds and 68 ° C. for 1 minute, and finally at 68 ° C. for 2 minutes went. The PCR products were checked for fragment size and amplification efficiency by 1.0% agarose gel electrophoresis. The PCR primers used here are shown in Table 2 below.

  The results of electrophoresis of the PCR product are shown in FIG. Expression was strongly responded by four promoters by drying treatment (Scaffold 2239, Contig 1515, tplb0004l11, tplb0015p04). One kind (7AL.Contig 16213) showed a response even though it was weak. For tplb0010o09, no expression response of GUS gene due to drought stress was observed.

2. Salt Stress Response Experiment T1 seeds of the transformed plants collected in Example 4 were allowed to absorb water at 4 ° C. for 2 days on filter paper on a petri dish, and then transferred to a growth chamber under illumination at 22 ° C. for 20 hours for germination. . After that, it was transferred onto glass beads and subjected to hydroponic culture for 10 days to grow leaves and roots. In the salt stress treatment, the plants under hydroponic culture were brought into a salt stress state by transferring the plants to a water tank at 22 ° C. to which 200 mM NaCl was added, and the treatment was carried out for 24 hours.

  Similar to the above-described method, RNA was extracted from the sample, cDNA synthesis and RT-PCR were performed, and the expression of the GUS gene was confirmed by electrophoresis. The results of electrophoresis of the PCR product are shown in FIG. By salt treatment, expression was observed in the three promoters (Contig1515, tplb0004111, tplb0015p04), and a stronger response was seen particularly in roots than in leaves. On the other hand, no salt stress response was observed for Scaffold 2239.

3. Low temperature stress response experiment T1 seeds of the transformed plants collected in Example 4 were allowed to absorb water at 4 ° C. for 2 days on filter paper on a petri dish, and then transferred to a growth chamber under illumination at 22 ° C. for 20 hours for germination. . After that, it was transferred onto glass beads and subjected to hydroponic culture for 10 days to grow leaves and roots. The low temperature stress treatment was carried out for 10 hours as it was in a low temperature stress state by transferring the plants under hydroponic cultivation to a 4 ° C. water tank in a growth chamber set at 4 ° C.

  Similar to the above-described method, RNA was extracted from the sample, cDNA synthesis and RT-PCR were performed, and the expression of the GUS gene was confirmed by electrophoresis. The results of electrophoresis of the PCR product are shown in FIG. It has been shown that the three promoters that have shown dry and salt stress responses up to now also respond to cold stress.

4. Observation of expression of transgene by GUS staining In order to observe the activity of the promoter introduced into C. sativa L., GUS staining was performed. A reaction solution for GUS staining (100 mM sodium phosphate buffer (pH 7.0), 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (D) for GUS staining of dried, salted and low temperature stress-treated plants. It was immersed in X-Gluc), 0.5 mM K ₃ [Fe (CN) ₆ ], 0.5 mM K ₄ [Fe (CN) ₆ ], 0.1% Triton-X), and degassed for 30 minutes. Then, the reaction was allowed to proceed at 37 ° C. overnight, the reaction solution was discarded, and the reaction solution was replaced with 70% ethanol to stop the reaction. The chlorophyll was removed by exchanging 70% ethanol several times.

The result observed with a stereomicroscope is shown in FIG. The samples of each stress treatment were stained in leaves, particularly in young leaves.

[Example 6] Expression amount analysis of reporter gene (GUS) With respect to promoters confirmed to respond at dryness, salt and low temperature, temporally under each stress condition (stressing to analyze expression pattern and expression intensity) RNA was extracted at 0 hour, 1 hour, 3 hours, 5 hours, 10 hours, and 24 hours), and quantitative PCR was performed. After each time of stressing, the culture conditions (hydroponic culture, fresh water, 22 ° C.) were returned to normal conditions, and the response of the promoter and changes in the expression amount of downstream genes when cultivated for 2 days were also confirmed. Fast SYBR Green Mster Mix (manufactured by life technologies) and StepOnePlus (manufactured by life technologies) were used for quantitative PCR. As a control to be compared, a transformed plant was used to which a plasmid in which a cauliflower mosaic virus (CaMV) 35S promoter, a GUS gene and a Nos terminator (Tnos), which are widely used for genetically modified plants, were introduced was introduced. The UBC18 gene was used as an internal control gene (Hong et. Al. (2008) BMC Plant Biol., Nov 7; 8: 112), and the sequences of the primers are shown in Table 3 below.

  FIG. 5 shows the results of quantitative PCR of relative expression to the control cauliflower mosaic virus 35S promoter, and FIG. 6 shows the results of quantitative PCR of relative expression to the start of stress treatment. The expression patterns were different in all three types, and there were those that responded earlier and those that continued. In addition, in any promoter, when stress conditions were returned to normal cultivation conditions, the activity of the promoter was reduced to the same level as in the case of no treatment. Regarding the expression intensity, one of the three types of promoters showed an expression intensity similar to that of the control 35S promoter, and the other two types were promoters that induced expression approximately 10 to 150 times. It has become clear that the three types of promoter sequences have an expression level equal to or higher than general-purpose promoters in plants.

  In tplb0004111, the ability to increase the expression level of downstream linked genes under drought stress conditions is up to 4.0 times in leaves and up to 0.8 in roots compared to the beginning of drought stress treatment And the ability to increase the expression level of downstream linked genes under salt stress conditions is at most 1.5 times in leaves and at most 30 in roots compared to when salt stress treatment is initiated. 1 times, the ability to increase the expression level of downstream linked genes under cold stress conditions is at most 3.8 times in leaves and at most 14 in roots compared to the start of cold stress treatment .1 times.

  In Contig 1515, the ability to increase the expression level of downstream linked genes under drought stress conditions is up to 55.6 times in leaves and up to 41.8 in roots compared to the beginning of drought stress treatment And the ability to increase the expression level of downstream linked genes under salt stress conditions is at most 33.5 times in leaves and at most 190 in roots compared to when salt stress treatment is initiated. The ability to increase the expression level of downstream linked genes under cold stress conditions is up to 41.8 times in leaves and up to 881 in roots compared to that at the start of cold stress treatment. .5 times.

  In tplb0015p04, the ability to increase the expression level of downstream linked genes under drought stress conditions is at most 63.1 times in leaves compared to the beginning of drought stress treatment and at most 10.9 in roots And the ability to increase the expression of downstream linked genes under salt stress conditions is up to 5.3 times in leaves and up to 182. 2 in roots compared to when salt stress treatment is initiated. 6 times, the ability to increase the expression level of downstream linked genes under cold stress conditions is at most 43.3 times in leaves compared to the start of cold stress treatment, at most 447 in roots .4 times.

Claims (13)

A wheat-derived promoter having the ability to be activated under drought stress conditions, salt stress conditions and low temperature stress conditions to increase the expression level of downstream linked genes, comprising the following (a) to (c) Promoter which is any of).
(A) a promoter comprising the nucleotide sequence set forth in SEQ ID NO: 17 or 21;
(B) A base sequence having a sequence identity of 90% or more with the base sequence set forth in SEQ ID NO: 17 or 21 and activated downstream under dry stress conditions, salt stress conditions and low temperature stress conditions; A promoter having the ability to increase the expression level of linked genes;
(C) The nucleotide sequence set forth in SEQ ID NO: 17 or 21, containing a nucleotide sequence in which one or more bases are deleted, inserted, substituted and / or added, under dry stress conditions, salt stress conditions, and low temperature A promoter that has the ability to be activated under stress conditions to increase the expression level of downstream linked genes. The promoter according to claim 1, which is derived from Triticum monococcum. The ability to increase the expression level of a downstream linked gene under drought stress conditions, salt stress conditions and low temperature stress conditions is 1 to 150 times that of the cauliflower mosaic virus 35S promoter. Or the promoter as described in 2. The ability to increase the expression level of downstream linked genes under drought stress conditions is 3.0 times or more in leaves and 0.7 times or more in roots as compared to the beginning of drought stress treatment,
The ability to increase the expression level of downstream linked genes under salt stress conditions is 1.2-fold or more in leaves and 20-fold or more in roots as compared to the beginning of salt stress treatment,
The ability to increase the expression level of downstream linked genes under cold stress conditions is 3.0 times or more in leaves and 10 times or more in roots as compared to the start of cold stress treatment. The promoter according to any one of 3. The ability to increase the expression level of downstream linked genes under drought stress conditions is 4.0 to 63.1 times in leaves compared to the beginning of drought stress treatment and 10.9 to 41. in roots. 8 times,
The ability to increase the expression level of downstream linked genes under salt stress conditions is 1.5 to 33.5-fold in leaves compared to that at the start of salt stress treatment, and in the roots from 3 to 190. 5 times or less,
The ability to increase the expression level of downstream linked genes under cold stress conditions is 3.8 to 43.3 times in leaves compared to that at the start of cold stress treatment and 14.1 to 881 in roots. The promoter according to any one of claims 1 to 4, which is 5 times or less. MYCATRD22 (base sequence is CACATG), MYCATERD1 (base sequence is CATGTG), MYBCORE (base sequence is CNGTTR), MYB2AT (base sequence is TAACTG), CBFHV (base sequence is RYCGAC), ABRELATERD1 (base sequence is ACGTG), ABREATRD22 (base sequence is ACGTG) The base sequence has at least one or more motifs selected from the group consisting of RYACGTGGYR, GT1 GMSCAM4 (base sequence is GAAAAA), LTRECOREATCOR15 (base sequence is CCGAC), and CCAATBOX1 (base sequence is CCAAT), The promoter according to any one of claims 1 to 5. A vector comprising the promoter according to any one of claims 1 to 6 and a gene linked downstream of the promoter. A microorganism or a virus comprising the vector according to claim 7. A transformant obtained by transforming a host cell with the vector according to claim 7 and / or the microorganism or virus according to claim 8. The transformant according to claim 9, wherein the host cell is a monocot plant-derived cell. The transformant according to claim 9 or 10, wherein the host cell is a cell derived from wheat (Triticum aestivum). A vector according to claim 7 and / or a microorganism or virus according to claim 8 (wherein the gene linked downstream of said promoter confers resistance to drought stress, salt stress and cold stress on plants) A method of producing a plant having tolerance to drought stress, salt stress and low temperature stress, which comprises the step of introducing into the plant cells a gene encoding a protein capable of A vector according to claim 7 and / or a microorganism or virus according to claim 8 (wherein the gene linked downstream of said promoter confers resistance to drought stress, salt stress and cold stress on plants) A method for imparting tolerance to drought stress, salt stress and low temperature stress to a plant, which comprises the step of introducing into the plant cell a gene encoding a protein capable of

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