KR101557043B1 - Constitutive expression promoter from chrysanthemum - Google Patents

Abstract

The present invention relates to a constitutive expression promoter separated from an actin gene as a constitutive expression gene of chrysanthemum. It is very useful when improving traits of crops capable of temporarily inducing expression of useful genes regardless of time and tissues of plant growth due to temporarily inducing expression of a target gene using a constitutive expression promoter of the present invention.

Description

Constitutive expression promoter from chrysanthemum < RTI ID = 0.0 >

The present invention relates to a promoter for expression at all times, which is isolated by an actin gene which is an expression gene for chrysanthemums.

The promoter is a genomic region linked to the upper side of the structural gene, and plays a role of regulating transcription of the structural gene linked to the mRNA. Promoters are activated by the binding of several common transcription factors. TATA, which generally controls gene expression,

box, and CAT box. Since proteins necessary for basic metabolism of living organisms must maintain a constant concentration in cells, the promoter linked to these genes is always activated by the action of common transcription factors. On the other hand, proteins that do not have a role in normal times and function only under specific circumstances are connected to inducible promoters that induce the expression of the structural genes. Inducible promoters are activated by the binding of specific transcription factors activated by external stimuli in the development of organisms or from environmental factors from the environment.

In the production of cultivated plants with novel characteristics capable of developing agriculture, the expression of an external gene (i.e., transgene) introduced into the plant may be transcribed, post-transcriptionally, - It is strongly influenced by posttranslational factors. Among the electrical elements, the promoter belonging to the transcriptional element is the most important factor that can directly affect the transcription of the transgene resulting in not only changing the level of expression but also changing the stage of transgene expression, tissue or cell specificity . To date, a number of promoters have been isolated from various plants for the expression of transgene, but only a few of them have been used to transform plants.

It is an object of the present invention to provide a constitutive expression promoter derived from chrysanthemum consisting of the nucleotide sequence shown in SEQ ID NO: 1.

Another object of the present invention is to provide a recombinant plant expression vector containing the promoter.

Yet another object of the present invention is to provide a plant transformed with said recombinant plant expression vector.

It is still another object of the present invention to provide a method of constitutive expression of a foreign gene in a transgenic plant using a recombinant plant expression vector containing the promoter.

In order to solve the above object, the present invention provides a constitutive expression promoter derived from chrysanthemum consisting of the nucleotide sequence shown in SEQ ID NO: 1.

The present invention also provides a recombinant plant expression vector comprising the promoter.

The present invention also provides a plant transformed with the recombinant plant expression vector.

In addition,

1) recombining a foreign gene into the recombinant plant expression vector; And

2) constitutive expression in a transgenic plant of a foreign gene comprising the step of transforming the recombinant plant expression vector into a plant.

In addition,

1) recombining a foreign gene into the recombinant plant expression vector;

2) transforming the recombinant plant expression vector into Agrobacterium;

3) inoculating the transformed Agrobacterium into leaf sections of plants;

4) culturing the inoculated leaves in a co-culture medium;

5) transferring the cultured leaf slices to a selection medium to select a regenerated transgenic plant, and providing a method for constitutive expression of a foreign gene in a plant.

Since the expression of the target gene can be constantly induced by using the expression promoter of the present invention at all times, the expression of the useful gene can be always induced irrespective of the developmental stage and structure of the plant, You can use it very useful.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing the base sequence of an actin promoter derived from chrysanthemum.
Fig. 2 is a view showing the comparison of the types and positions of the action site elements contained in the actin promoter derived from chrysanthemum.
FIG. 3 is a diagram showing the production of a plant transformation vector for the analysis of the activity of an actin promoter derived from chrysanthemum: the reporter gene GUS is regulated by the chrysanthemum actin promoter, and chrysanthemum is selected as an antibiotic screening gene, Resistance gene NPTII and hygromycin resistance gene as an antibiotic screening gene in Arabidopsis thaliana.
4 shows the analysis of the actin promoter activity from chrysanthemum at the stage of developmental development: 1 to 25 days; Transgenic Arabidopsis seeds and seeds 25 days after germination on day 1 after germination.
Fig. 5 is a diagram showing the analysis of GUS expression in the transgenic Arabidopsis thaliana in which chrysanthemum-derived actin promoter is introduced: 35 days; 35 days after germination Transgenic Arabidopsis mature plant, IS; inflorescence stem, F; Flower, Fb; Flower bud, Si: Silique.
FIG. 6 shows GUS expression analysis in transgenic chrysanthemums into which an actin promoter derived from chrysanthemum was introduced: F; Flower, Fl; Floral leaf, S; Stem; R; Root, L; Leaf, C; Cutting region.

Hereinafter, the present invention will be described in detail.

 The present invention provides a constitutive expression promoter derived from chrysanthemum consisting of the nucleotide sequence shown in SEQ ID NO: 1, and more specifically, a chrysanthemum expression gene comprising the nucleotide sequence shown in SEQ ID NO: 1 A recombinant plant expression vector containing an allosteric expression promoter isolated by an actin gene is provided.

The present invention relates to a specific promoter derived from chrysanthemum. Specifically, the promoter is suitable for constitutive expression of a plant gene. Preferably, the promoter may be one that uniformly expresses a gene in the whole plant organs or tissues. Wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a recombinant plant expression vector comprising the promoter. As an example of a recombinant plant expression vector, there may be mentioned, but not limited to, the vector described in Fig. Specifically, the vector is a reporter gene (GUS), a protease inhibitor II terminator gene (TPINII) as a kanamycin resistance gene as an antibiotic selection gene in chrysanthemum, Hygromycin resistance gene The gene and nopaline synthase terminator (TNOS) are operatively linked.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

A preferred example of a plant expression vector is a Ti-plasmid vector that is capable of transferring a so-called T-region into a plant cell when it is present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinotricin, antibiotics such as Kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

In a plant expression vector according to an embodiment of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto. The term "promoter " refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

The terminator can be a conventional terminator such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens octopine And the terminator of the Octopine gene, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

In the recombinant plant expression vector according to an embodiment of the present invention, the plant expression vector may be prepared by operably linking a target gene encoding a target protein downstream of the promoter of the present invention. In the present invention, "operably linked" refers to a component of an expression cassette that functions as a unit for expressing a heterologous protein. For example, a promoter operably linked to a heterologous DNA encoding a protein promotes the production of a functional mRNA corresponding to a heterologous DNA.

The target protein may be any kind of protein. For example, the target protein may be a protein capable of accumulating a large amount of nutrients that can be used for medical purposes such as enzymes, hormones, antibodies, cytokines, However, the present invention is not limited thereto. Examples of the target protein include interleukin, interferon, platelet-derived growth factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor, human growth factor, human serum albumin and erythropoietin.

In addition, the present invention provides a method for producing a target protein constitutively expressed in a plant by transforming a plant with the recombinant plant expression vector. Also provided is a target protein produced by the production method. The target protein produced is as described above.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The method is based on the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202,179-185 (Klein et al., 1987, Nature 327, 70), the infiltration of plants or the transformation of mature pollen or vesicles into Agrobacterium tumefaciens Infection by viruses (non-integrative) in virus-mediated gene transfer (EP 0 301 316), and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell.

"Plant tissue" refers to a tissue of differentiated or undifferentiated plant, including but not limited to roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used for culture, protoplasts, shoots and callus tissue. The plant tissue may be in planta or may be in an organ culture, tissue culture or cell culture.

In addition,

1) recombining a foreign gene into the recombinant plant expression vector; And

2) constitutive expression in a transgenic plant of a foreign gene comprising the step of transforming the recombinant plant expression vector into a plant.

The method of the present invention comprises transforming a plant cell with a recombinant plant expression vector according to the present invention wherein the transformation is mediated by, for example, Agrobacterium tumefaciens GV3101 and LBA4404 .

In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

In addition, the present invention provides a transgenic plant produced by the method for producing a transgenic plant according to the present invention. The plant may be a dicotyledonous plant, but is not limited thereto.

In addition,

1) recombining a foreign gene into the recombinant plant expression vector of claim 2;

2) transforming the recombinant plant expression vector into Agrobacterium;

3) inoculating the transformed Agrobacterium into leaf sections of plants;

4) culturing the inoculated leaves in a co-culture medium;

5) transferring the cultured leaf slices to a selection medium to select a regenerated transgenic plant, and providing a method for constitutive expression of a foreign gene in a plant.

The selection medium in step 5) is preferably, but not limited to, kanamycin and cefotaxim.

The plant of step 3) is preferably a chrysanthemum, but is not limited thereto.

The method of the invention comprises transforming a plant cell with a recombinant plant expression vector according to the invention, said transformation being mediated, for example, by Agrobacterium tumefaciens GV3101 .

The method of the present invention comprises regenerating a transgenic plant from the transgenic plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

"Plant cell" used for transformation of a plant may be any plant cell. Plant cells are cultured cells, cultured tissues, cultivated or whole plants.

"Plant tissue" refers to a tissue of differentiated or undifferentiated plant, including but not limited to roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used for culture, protoplasts, shoots and callus tissue. Preferably, it may be a leaf cut with a spike wound. The plant tissue may be in planta or may be in an organ culture, tissue culture or cell culture.

Transgenic plants in the present invention may include whole plants as well as tissues, cells or seeds obtainable therefrom.

The regeneration selection medium may include naphthalene acetic acid (NAA), benzyladenine (BA), sucrose, agar, cefotaxim, and kanamycin.

In addition, the present invention provides a transgenic plant in which the foreign gene produced by the above method is constantly expressed.

The plant is preferably chrysanthemum, but is not limited thereto.

According to the present invention, the chrysanthemum according to the present invention is a variety of chrysanthemums, including varieties of Santos, Enkali, white line, white horse, autumn lady, japiri, Chinhyang Abcheon, flower, Chrysanthemum, Chrysanthemum, Chrysanthemum, Chrysanthemum, Chrysanthemum, Chrysanthemum, Motherhood, Chrysanthemum, Chrysanthemum, Chrysanthemum, Chrysanthemum, Pusan, It is the most beautiful place in the world. It is the most beautiful place in the world. It is the most beautiful place in Korea. Galaxies, baggies, snow globe, world peace, full moon, crossbows, anti-terrorism, martial law, honorable wife, road metallurgy, peacock, diggings, red lions or new moon.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Chrysanthemum-derived Actin ( actin Separation of promoter region and nucleotide sequence analysis

In order to induce the stable expression of genes in flower crops, we used a promoter derived from chrysanthemum (Dendranthema grandiflorum Kitamura) The promoter was isolated.

Specifically, RT-PCR was performed using a conserved motif primer of actin from total RNA isolated from chrysanthemum to isolate a part of actin gene derived from chrysanthemum. The specific primer sequences are shown in Table 1 below.

SEQ ID NO: Actin-conserved motif primer 2 Forward 5'-TGGCATCACACTTTTCTACAA-3 ' 3 Reverse (reverse) 5'-CAACGGAATCTCTCAGCTCC-3 '

A 1432 bp promoter region containing the first exon of actin was isolated from a part of the chrysanthemum-derived actin gene derived from chrysanthemum using the GenomeWalker technology, and the nucleotide sequence was analyzed. The actin gene specific primer sequences derived from chrysanthemum are shown in Table 2, and the nucleotide sequences of the actin promoter regions derived from chrysanthemums are shown in Fig.

SEQ ID NO:  Actin gene-specific primers 4 GSP1 5'-AGGATCCGATTACTATAGGGCACGCGTGGTC-3 ' 5 GSP2 5'-AACTAGTCTTCACCATTCCGGTACCATTGTC-3 '

< Example  2> Chrysanthemum-derived Actin ( actin ) Analysis of action element of promoter

The actin promoter (1.43 Kb) region derived from chrysanthemum isolated in Example 1 was analyzed.

The cis- acting elements present in the promoter region of actin genes derived from chrysanthemum are called PLACE (Plant Cis-acting), which is a homepage that provides a program to predict the type and location of the action site elements in the promoter Regulatory DNA Elements, http://www.dna.affrc.go.jp/PLACE/) and PlantCARE (Plant Cis-Acting Regulatory Element, http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) Were compared.

As a result, there were a variety of cis- acting elements that respond to plant hormones and various biotic and abiotic stimuli. ARE is the action site element involved in the anaerobic reaction, CAAT-box is the transcription factor binding site, GARE is the action site element involved in the GA reaction, LTR is the action site element involved in the low temperature reaction, MBS is included in the dry induction response TGACG represents the action site element involved in the Me-JA reaction, while SkN-1 is the regulatory element involved in the expression of endosperm, TATA-box is the hogness box, TGAE is the action site element involved in the auxin response, , Especially the Skin-1 element, can explain the expression of GUS in endosperm by the actin promoter of chrysanthemum (Fig. 2).

< Example  3> Chrysanthemum-derived Actin ( actin ) &Lt; / RTI &gt; promoter and transgenic plant &lt; RTI ID = 0.0 &gt;

<3-1> Chrysanthemum-derived Actin ( actin ) Vector for plant transformation including promoter

A plant transformation vector containing the chrysanthemum-derived actin promoter (1.43 Kb) region isolated in Example 2 was prepared.

Specifically, the actin promoter (1.43 Kb) region derived from chrysanthemum isolated in Example 2 was transfected with a vector for transformation through a polymerase chain reaction (PCR) using the primer shown in Table 3 below Respectively.

At this time, the PCR conditions were denatured at 95 ° C for 5 minutes, followed by 35 cycles of 1 cycle at 95 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes, followed by reaction at 72 ° C for 10 minutes. Then, the nucleotide sequence of the DNA obtained in the PCR reaction was confirmed.

SEQ ID NO:  Specific primers for actin gene promoter recombination 6 ChA-Pro-forward 5'-A GGATCC GATTACTATAGGGCACGCGTGGTC-3 ' 7 ChA-Pro- reverse (reverse) 5'-A ACTAGT CTTCACCATTCCGGTACCATTGTC-3 '

The chrysanthemum-derived actin promoter isolated from the above was inserted into pGEM-T-easy, digested with BamHI and SpeI, and inserted into pCAMBIA1381 digested with BamHI and SpeI. An Arabidopsis transforming vector fused with an actin promoter derived from chrysanthemum and a reporter gene GUS was prepared.

The pCAMBIA1390 vector was used to construct a vector for transformation of chrysanthemum. Specifically, the His-tag region present in the pCAMBIA1390 vector was removed by treatment with SpeI and BstEII, and the antibiotic hygromycin resistance gene was replaced with kanamycin for selection of chrysanthemum transformants. The actin promoter derived from chrysanthemums cut with BamH I and Spe I was inserted into pCAMBIA1390 digested with BamH I and Spe I to construct a vector for transformation of chrysanthemum. The map is shown in Fig.

<3-2> Chrysanthemum-derived Actin ( actin ) Transgenic plants containing the promoter

Arabidopsis thaliana fused with GUS and two vectors for transformation of chrysanthemum were introduced into Tumefaciens GV3101 and LBA4404 in the chrysanthemum-derived actin promoter prepared in Example 3-1, and the Agrobacterium GV3101 and LBA4404 Were transformed into Arabidopsis thaliana and Chrysanthemum.

Agrobacterium tumefaciens GV3101 and LBA4404 were transfected with freeze and thaw 2-3 times to introduce the vector into Arabidopsis thaliana and chrysanthemum, then transformed by heat shock method at 37 ℃ and then contained 50 ㎎ / ℓ of antibiotic kanamycin Lt; RTI ID = 0.0 &gt; YEP &lt; / RTI &gt; Colony PCR confirmed the introduction of the carriers in the GV3101 and LBA4404 strains, and the transgenic colonies were used for Arabidopsis and chrysanthemum transformation.

First, in order to transform wild-type Arabidopsis thaliana ecotype Col-0 with transformed GV3101, Arabidopsis thaliana ecotype Col-0 was firstly introduced into Arabidopsis thaliana, Grow for 4 weeks. After removing the primary chiasm of 4 weeks old Arabidopsis plants, it induces 3-4 secondary chests of at least 10 cm. Agrobacterium whose vector introduction was confirmed was inoculated on LB medium containing 50 mg / L of kanamycin, 10 mg / L of rifampicin and 40 mg / L of gentamicin and cultured at 28 DEG C for about 3-4 days. The fully grown Agrobacterium was precipitated by centrifugation and then diluted with a solution containing 5% sucrose and 0.05% silwet77 to an OD of about 0.6 to prepare a suspension. The suspension thus prepared was sprayed on the buds of the Arabidopsis thaliana and cultured for 24 hours in a state of maintaining sufficient humidity and dark conditions, followed by further culturing for 3-4 days on the growth conditions of 22 ° C, 16 hours day and 8 hours night, And the transformation was repeated. Thereafter, the seeds were harvested after growth for about 4-6 weeks until the pods were completely matured on the growth at 22 ° C, 16 hours day and 8 hours night.

The chrysanthemum transformation is carried out by incubating the chrysanthemum leaves in a cabinet for about 2 days in a preculture medium after cutting to a square of about 5 mm. In the meantime, Agrobacterium containing the vector is pre-cultured in a liquid medium (2-3 days), and then about 1.5 ml is inoculated again in 100 ml of medium and harvested to grow to an OD600 of about 1.0. After centrifuging, the pellet is diluted in a co-culture medium, immersed in the pre-cultured plant for about 10 minutes, and then the water is removed and incubated for 2 days in a callus organic medium. Two days later, the co-cultivated leaf tissue was transferred to an antibiotic medium, and Agrobacterium was removed for 7 days, and the transformant shoot was selected by transferring it to a shoot selection medium. Hormone used in the medium was 1 ㎎ / ℓ of IAA and 1 ㎎ / ℓ of BA, and 15 ㎎ / ℓ of kanamycin and 400 ㎎ / ℓ of cytotoxic agent were used as starting antibiotics.

<3-3> Chrysanthemum-derived Actin ( actin ) Selection of transgenic plants containing promoter

In order to select the transgenic Arabidopsis thaliana into which the vector fused with the reporter gene GUS was introduced into the actin promoter derived from the chrysanthemum prepared in Example 3-2, seeds were seeded on MS medium containing 30 / / ml hygromycin the sowing and growth was transferred to the T ₁ transformants of the selected 25 kinds in a greenhouse. Then, seeds were harvested and examined in hygromycin antibiotic medium. As a result of analysis of GUS expression of 8 transformants in which single T-DNA was inserted, which showed separation ratio of about 3 (antibiotic resistance): 1 (antibiotic sensitivity) .

In order to select a chrysanthemum transformant to which a vector fused with the reporter gene GUS was introduced into the actin promoter derived from chrysanthemum prepared in Example 3-2, 15 μg / ml of the antibiotic kanamycin was used to shoot ). Selection of shoots was followed by selection of transformant chrysanthemum using 20 ㎍ / ㎖ of kanamycin as a secondary selection in root medium.

< Experimental Example  1> Transgenic Arabidopsis thaliana Actin ( actin ) Promoter activity assay

The developmental stage of the Arabidopsis thaliana transformant selected in Example 3 was compared and the expression pattern of the promoter was analyzed by GUS staining at T ₃ generation.

In order to confirm whether the actin promoter derived from chrysanthemum constantly induces gene expression in the plant, the growth stage of the Arabidopsis transformed with the recombinant expression vector inserted with the actin promoter derived from chrysanthemum, GUS gene expression patterns were analyzed. That is, three lines isolated from the Arabidopsis thaliana transformed in Example 3-3 were germinated in MS medium to observe GUS (blue coloring gene) expression pattern at the development stage of the seedling, GUS gene expression patterns were observed in various tissues of leaves, stems, and roots at various stages of growth on the 15th and 35th days after planting. The tissues were immersed in GUS-assay buffer (2 mM of cyslohexyl ammonium salt, 50 mM of sodium phosphate, 0.05% of Triton-X100, 1 mM of potassium ferrocyanide, 1 mM of potassium ferricyanide, pH 7.2) After 20 hours of treatment, chlorophyll was completely removed with 70% ethanol and the expression pattern of GUS gene was observed using a stereomicroscope (Olympus, SZX12).

As a result, GUS expression was confirmed in chalazal endosperm and micropylar endosperm in seeds. From 1 to 25 days after seed germination, GUS showed strong systemic expression from seedling development to growth stage. The chrysanthemum actin promoter was found to be a promoter that induces the systemic expression of GUS at all times during the developmental stage of Arabidopsis (Fig. 4).

In the transformed Arabidopsis thaliana, strong GUS expression was observed in the stem, flower, flower, pod, perineum, and mucus. In addition, strong GUS expression was observed at the cut surface of the stem, confirming that it was a promoter inducing expression against wounding stress. Thus, the chrysanthemum actin promoter was identified as a promoter inducing systemic expression constantly in Arabidopsis ( 5).

< Experimental Example  2> Transgenic chrysanthemum is derived from chrysanthemum Actin ( actin ) Promoter activity assay

Expression patterns of promoters were compared and analyzed by GUS staining while advancing the generation of chrysanthemum transformants selected in Example 3 above.

In order to confirm whether or not the actin promoter derived from chrysanthemum constantly induces gene expression in the plant, the expression pattern of GUS gene in each tissue of chrysanthemum transformed with a recombinant expression vector inserted with chrysanthemum-derived actin promoter Respectively. That is, the lines isolated from the chrysanthemum transformed in Example 3-3 were selected to observe GUS (blue coloring gene) gene expression patterns in various tissues of leaves, stems, flowers, and roots in the plants. The tissues were immersed in GUS-assay buffer (2 mM of cyslohexyl ammonium salt, 50 mM of sodium phosphate, 0.05% of Triton-X100, 1 mM of potassium ferrocyanide, 1 mM of potassium ferricyanide, pH 7.2) After 20 hours of treatment, chlorophyll was completely removed with 70% ethanol and the expression pattern of GUS gene was observed using a stereomicroscope (Olympus, SZX12).

As a result, strong GUS expression was observed in flowers, petals, stems, leaves, roots, and the like of the chrysanthemum transformants. In addition, it was confirmed that GUS expression was observed at the cut surface of the stem or leaf, leading to gene expression for wounding stress in chrysanthemum (Fig. 6).

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Constitutive expression promoter from chrysanthemum <130> P14R2D0475 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 1432 <212> DNA <213> Dendranthema grandiflorum Kitamura <400> 1 gattactata gggcacgcgt ggtcgacggc atggaagctg ctggtaaaga cattctatta 60 aacaataaca cctcttttga atcaattttt tcaaacttca acaacccaat taatccacaa 120 aagcaaccaa acaccccgaa atccaacgac ccatgagaac tcatagtttt gatccaacgt 180 ctgtgatttg tcacagctgt caacgacagg tcacgctatc ttttgtccac ctcgtggtct 240 gaatgattct ccccacacat tctccatata actcttttta tctcttcatt ttcccaaaaa 300 aacattccac acacacaaaa cacacacgca aaacacacac agtgacgtgt cataccagct 360 agggtttcat caccgcagat cggaatcttc atcttcttct tctcagatct gatcttataa 420 tcggtatatt tataatcgcc gttactttat ttgactttac gttgactact tcgttgactg 480 atgtgtttta gatctgaatc tgttgatttt gttatgtcta ttgatttggt tattgtagat 540 ctgatgtgtt ttgtttaggt ttgactttat ttgacttaaa attgtgatgt gaagttgcta 600 gatctggtga tttagttgtt aggtagttac gattagttga ttgatgtgtt ttgtagtgtt 660 tggatgattg ttttagctgt tgtttataaa aaaagtgaag atctgagtgt tgttagttat 720 aaatcggtga attcgatgcg tagcgttgag ttgttagatg ttttttcgcc gcctaagttg 780 ttgtatgagc atagtttgat gagtgtaaat attgtacttg ttgatcggta tagctaagtg 840 gaaagttact tcaatagatt attttcgttg tgtaaatgcg gacgatttgt tattttgtca 900 agagttattt gttatgtagc tttgagctgt tgtatcatgt actgatggtt aagttgtcga 960 gttatcggta tgaggtctgg tttgacaaga gtaaatgttg tactcgtttg atcagtgtcg 1020 ttaagcttgc ttgaatagat tcgttgttcg tgtagctgcg gatagtttat tactccgatt 1080 attagttgtt tggtatagtt tggtagttgt attgcgtgta cgattggtat gtttaagcta 1140 aaatatttat attatgcact ttgtttgcag ttgtaactgt tatacttttt tgatcggttt 1200 gttgctgaaa gttactttaa tagatcattt gttcgtgtaa ttgcggattg ggtatgtgtt 1260 gcttagtttg gtttagtagt tgtagtactt gtacgggatg taaatgttga aatccgtatt 1320 ttgcgaatgg tgtagtgact ttaatcttat tttctgttac agtctataaa aaatggccga 1380 taccgaagat attcagcccc ttgtttgtga caatggtacc ggaatggtga ag 1432 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> Actin conserved motif primer (forward) <400> 2 tggcatcaca cttttctaca a 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> Actin conserved motif primer (reverse) <400> 3 caacggaatc tctcagctcc 20 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> GSP1 (forward) <400> 4 aggatccgat tactataggg cacgcgtggt c 31 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> GS21 (reverse) <400> 5 aactagtctt caccattccg gtaccattgt c 31 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> ChA-Pro (forward) <400> 6 aggatccgat tactataggg cacgcgtggt c 31 <210> 7 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> ChA-Pro (reverse) <400> 7 aactagtctt caccattccg gtaccattgt c 31

Claims (12)

A constitutive expression promoter derived from chrysanthemum consisting of the nucleotide sequence shown in SEQ ID NO: 1.
A recombinant plant expression vector comprising the promoter of claim 1.
3. The recombinant plant expression vector according to claim 2, wherein the recombinant plant expression vector is produced by operatively linking a target gene encoding a target protein downstream of the promoter.
A plant transformed with the recombinant plant expression vector of claim 2.
1) recombining a foreign gene into the recombinant plant expression vector of claim 2; And
2) constitutive expression of a foreign gene in a plant, comprising the step of transforming the recombinant plant expression vector into a plant.
A transgenic plant wherein the foreign gene produced by the method of claim 5 is constantly expressed.
7. The transgenic plant according to claim 6, wherein the plant is a dicotyledonous plant.
1) recombining a foreign gene into the recombinant plant expression vector of claim 2;
2) transforming the recombinant plant expression vector into Agrobacterium;
3) inoculating the transformed Agrobacterium into leaf sections of plants;
4) culturing the inoculated leaves in a co-culture medium;
5) A method for constitutive expression of a foreign gene in a plant, comprising the step of transferring the cultured leaf fragment to a selection medium and selecting regenerated transgenic plants.
9. The method according to claim 8, wherein the selection medium of step 5) comprises kanamycin and cefotaxim.
9. The method of constitutionally expressing a foreign gene according to claim 8, wherein the plant of step 3) is chrysanthemum.
A transgenic plant wherein the foreign gene produced by the method of claim 8 is constantly expressed.
12. The transgenic plant according to claim 11, wherein the plant is a chrysanthemum.

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