KR102390866B1 - Constitutive promoter derived from chrysanthemum and use thereof - Google Patents

Abstract

The present invention relates to a systemic expression promoter derived from chrysanthemum and its use, and more specifically, a constitutive promoter (ConP) 10 gene derived from chrysanthemum for inducing expression of a target gene in the entire plant tissue promoter, including the promoter It relates to a recombinant expression vector, a method for producing a transgenic plant using the recombinant expression vector, and a plant transformed by the method.

Description

Systemic expression promoter derived from chrysanthemum and its use {Constitutive promoter derived from chrysanthemum and use thereof}

The present invention relates to a systemic expression promoter derived from chrysanthemum and its use, and more specifically, a constitutive promoter (ConP) 10 gene derived from chrysanthemum for inducing expression of a target gene in the entire plant tissue promoter, including the promoter It relates to a recombinant expression vector, a method for producing a transgenic plant using the recombinant expression vector, and a plant transformed by the method.

Known promoters used to achieve transformation purposes capable of systemic-expression or tissue-specific expression of foreign genes in plants can be classified according to their functions as follows.

First, there is a promoter that drives systemic expression. As such a plant systemic-expression induction promoter, the promoter of the 35S RNA gene of cauliflower mosaic virus (CaMV) is used as a representative promoter for dicotyledonous plants. In addition, as a systemic expression inducing promoter for monocotyledonous plants, the rice actin gene promoter and the corn ubiquithin gene promoter have been mainly used. Recently, the rice cytochrome C gene (OsOc1) promoter has been developed and used by a domestic research team. Yes (Korean Patent No. 10-0429335). They are inherent to induce the expression of an antibiotic or herbicide resistance gene and a reporter gene, which are mainly used as selection markers in plant transformation basic vehicles. Therefore, in terms of research, promoters are preferentially considered when attempting to elucidate the function of a target gene in a plant.

Second, there is a promoter specific to the seed. As this seed-specific promoter, the promoters of major storage protein genes in rice are a representative example, and the rice glutelin promoter used in the development of golden rice is currently used a lot to induce seed-specific expression of monocotyledonous plants. is being used In addition, promoters mainly used to induce seed-specific expression of dicotyledonous plants include soybean-derived lectin promoter, Chinese cabbage-derived napin promoter, and gamma-tocopherol methyl transferase (γ-tocopherol methyl transferase) from seeds of Arabidopsis thaliana: Examples include the carrot-derived DC-3 promoter used in a study to promote vitamin E production by inducing γ-TMT) gene expression. In particular, induction of seed-specific expression of the perilla-derived oleosin promoter has already been A patent application has been filed (Korean Patent No. 10-0685520). The seed-specific promoters are mainly used for the purpose of accumulating useful proteins and generating useful substances in major crops in which seeds themselves are used as food or as raw materials for food or food.

Third, there is a promoter specifically expressed in the root. Although this root-specific promoter has not yet been commercialized, Arabidopsis peroxidase (prxEa) has been isolated and root-specific expression has been confirmed. It was reported that the glucose pyrophosphatase (ADP-glucose pyrophosphatase, AGPase) gene was isolated and the promoter induces specific expression in the root. In addition, these promoters were confirmed to induce root-specific transient expression in carrots and radishes, and patents were registered (Korean Patent Nos. 10-0604186 and 10-0604191). These promoters can be expected to be mainly used for the purpose of improving agricultural traits, accumulating useful proteins, and generating useful substances in food or main root crops used as raw materials for food.

Fourth, there may be mentioned promoters specific to other tissues such as leaves. This tissue-specific promoter includes the ribulose bisphosphate carboxylase/oxygenase small subunit (rbcS) promoter derived from rice and corn that induces strong gene expression only in photosynthetic tissues such as leaves, and RolD that induces the expression of plant roots derived from Agrobacterium. A promoter, a tuber-derived tuber-derived patatin promoter, a tomato-derived phytoene synthase (PDS) promoter, and a cabbage-derived oleosin whose pollen-specific expression was confirmed in Chinese cabbage flowers. ) promoters and the like.

As described above, various plant-derived promoters for various purposes have been discovered and reported in each tissue of the plant, and development is still ongoing. However, most of the types of promoters that have already been commercialized or widely used among plant researchers belong to the above-mentioned cases, and there is a need to continue to develop new promoters that can more precisely control the site of gene expression according to the intention of the developer. .

Under this background, the present inventors made intensive efforts to develop a new promoter capable of systemically expressing a target gene in a plant, specifically, a useful promoter capable of specifically expressing the target gene in all tissues of a plant such as chrysanthemum. As a result, the present invention was completed by isolating the constitutive promoter (ConP) 10 gene promoter that can express the target gene in chrysanthemum in all tissues of the plant.

KR 10-1163051 B1 (2004.04.29. Announcement) KR 10-0685520 B1 (2007.03.09. Announcement) KR 10-0604186 B1 (2006.07.25. Announcement) KR 10-0604191 B1 (2006.07.25. Announcement)

It is an object of the present invention to use a promoter stably expressed in all tissues of a plant, an expression vector containing the promoter, a transformant transformed with the expression vector, a method for producing the transformant, and the promoter and vector An object of the present invention is to provide a method for inducing expression of a target protein.

As one aspect for achieving the above object, the present invention provides a promoter that specifically expresses a target protein in all tissues of a plant, an expression vector including the promoter, a transformant transformed with the expression vector, and the transformation A method for producing a transformant and a method for inducing expression of a target protein using the promoter and vector are provided.

By replacing the CaMV35S promoter, which is often used as a representative dicotyledonous systemic expression promoter, the promoter of the chrysanthemum-derived Constitutiven promoter (ConP) 10 gene according to the present invention can be effectively used for dicotyledonous crops such as chrysanthemum. In addition, using the promoter according to the present invention, there is an advantage that can be usefully used to improve the plant by systemically expressing the target gene in the plant.

1 shows the expression pattern of the ConP 10 gene, which is a systemic expression gene of the present invention, for each chrysanthemum organ.
2 shows the promoter sequence (554 bp) of the ConP 10 gene, which is a systemic expression gene of the present invention.
3A schematically shows an expression vector for plant transformation including a promoter of the ConP 10 gene, which is a systemic expression gene of the present invention, and FIG. 3B is a systemic expression gene of the present invention, comprising a promoter of the ConP 10 gene Transformed Arabidopsis thaliana is shown.
Figure 4 shows the GUS staining analysis results for each tissue of the transgenic Arabidopsis thaliana into which the promoter of the ConP10 gene, a systemic expression gene of the present invention, is inserted (1-9: Arabidopsis transformants introduced with the ConP10 promoter; Col- 0: control (non-transformed) Arabidopsis thaliana; CaMV35S: Arabidopsis transformant introduced with a systemic expression promoter).
5 shows the GUS gene expression results for each tissue of the transgenic Arabidopsis thaliana into which the promoter of the ConP10 gene, a systemic expression gene of the present invention, is inserted (1 to 9: Arabidopsis transformants introduced with the ConP10 promoter; WT: Control (non-transformed) Col-0 Arabidopsis thaliana; GUS: Arabidopsis transformant introduced with the systemic expression promoter CaMV35S).

The present invention relates to a systemic expression promoter derived from chrysanthemum and its use, and more particularly, a promoter of the chrysanthemum-derived Constitutive promoter (ConP) 10 that induces expression of a target gene in the entire tissue of a plant, recombinant comprising the promoter It relates to an expression vector, a method for producing a transgenic plant using the recombinant expression vector, and a plant transformed by the method.

In one aspect, the present invention provides a systemic expression promoter derived from chrysanthemum, comprising the nucleotide sequence of SEQ ID NO: 1.

As used herein, the term “promoter” refers to a region of a genome linked to the upper side of a structural gene, and serves to regulate the transcription of the structural gene linked thereto into mRNA. The promoter is activated by binding of several general transcription factors. TATA box, CAAT box region that generally regulates gene expression, region, position and direction that affects gene expression in response to external stimuli It consists of enhancers, etc. that promote the expression of almost all genes regardless of Since proteins necessary for the basic metabolism of the living body must be maintained at a certain concentration in the cell, promoters linked to these genes are always activated only by the action of general transcription factors. On the other hand, proteins that do not have a role in normal times and whose function is required only under special circumstances are linked to a tissue specific promoter or an inducible promoter that induces the expression of the corresponding structural gene. That is, the tissue-specific promoter is activated during the development of an organism, and the inducible promoter is activated by binding of a specific transcription factor activated by an external stimulus from an environmental factor.

The promoter of the present invention is a promoter for systemically inducing expression in a plant, and has the nucleotide sequence of SEQ ID NO: 1, but a sequence having 80% or more homology with the sequence, preferably 90% or more homology, more preferably A sequence exhibiting 95% or more homology, more preferably 98% or more homology, and most preferably 99% or more homology, and may include a base having substantially the same or corresponding biological activity. In addition, as a sequence having such homology, as long as it is a nucleotide sequence having substantially the same or corresponding biological activity, it is apparent that promoters having nucleotide sequences in which some sequences are deleted, modified, substituted or added are also included in the scope of the present invention.

In the present invention, "homology" is intended to indicate a degree of similarity to a wild type nucleotide sequence, and may include a sequence having the same percentage or more as the nucleotide sequence of the present invention as described above. Comparison of such homology can be performed visually or using an easily purchased comparison program. A commercially available computer program can calculate the homology between two or more sequences as a percentage (%), and the homology (%) can be calculated for adjacent sequences.

The systemic expression promoter of the present invention can express a target protein or target gene RNA in all tissues of a plant, and more preferably includes leaves, leaves, stems, roots, and seeds (by flower development period) of the plant. The target protein can be expressed equally in all tissues or organs.

Specifically, in one embodiment of the present invention, as a result of transforming Arabidopsis with a recombinant expression vector containing a promoter of the chrysanthemum-derived Constitutive promoter (ConP) 10 of the present invention, the target protein operably linked to the promoter is Arabidopsis thaliana It was confirmed that it was strongly expressed in all tissues of the plant including the leaves and the stem.

The systemic expression promoter of the present invention can act as a promoter for conventional recombinant expression vectors.

As another aspect, the present invention is a recombinant expression vector comprising a foreign gene encoding a target protein, operably linked to the promoter of the chrysanthemum-derived Constitutive promoter (ConP) 10 gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 provides

As used herein, the term “recombinant expression vector” refers to a vector capable of expressing a target protein or target RNA in a host cell, and refers to a gene construct including essential regulatory elements operably linked to express a gene insert.

The recombinant expression vector of the present invention is a plasmid known in the art, into which the systemic expression promoter of the present invention and a gene sequence encoding a target protein operatively linked to the promoter can be inserted or introduced. , virus or other vector. The systemic expression promoter of the plant according to the present invention and the nucleotide sequence encoding the target protein operably linked to the promoter may be operably linked to the expression control sequence, and the operably linked gene sequence and the expression control sequence are It may be included in one expression vector including a selection marker and a replication origin.

The "operatively linked" may be a gene and an expression control sequence linked in a manner that enables gene expression when an appropriate molecule is bound to the expression control sequence. By "expression control sequence" is meant a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a particular host cell. Such regulatory sequences may include promoters to effect transcription, optional operator sequences to control transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation.

The expression vector according to the present invention is prepared by inserting the systemic expression promoter of the present invention using an existing vector used for protein expression as a basic skeleton, and inserting a nucleotide sequence encoding a target protein downstream of the promoter. can do. Therefore, as a vector that can be used in the present invention, E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119) having various cloning sites, Bacillus subtilus-derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50) ) and Ti plasmid, plant virus vectors, and binary vectors such as pCHF3, pPZP, pGA and pCAMBIA families may be used. However, those skilled in the art may use any vector as long as it is capable of introducing the systemic expression promoter of the present invention and the nucleotide sequence encoding the target protein operably linked to the promoter into a host cell.

The target protein that can be used in the present invention may be any external target protein desired to be systemically expressed in plants by the promoter of the present invention, ie, an exogenous protein, or an endogenous protein and a reporter protein. The exogenous protein refers to a protein not naturally present in a specific tissue or cell, and the endogenous protein refers to a protein expressed by a gene naturally present in a specific tissue or cell. In addition, the reporter protein is a protein expressed by a reporter gene, and refers to a label protein that informs its activity in a cell by its presence. Target proteins include antibiotic resistance protein, herbicide resistance protein, virus resistance protein, pest resistance protein, metabolic protein, luminescent protein, GFP (green fluorescence protein), GUS (β-glucuronidase), GAL (β-galactosidase), etc. However, the present invention is not limited thereto.

In addition, the expression vector of the present invention may preferably contain one or more selectable markers. The marker is a nucleic acid sequence having a characteristic selectable by a conventional chemical method, and may include any gene capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinotricin, G418, kanamycin, Bleomycin, hygromycin, antibiotics such as cholramphenicol There is a resistance gene, but is not limited thereto.

Specifically, in one embodiment of the present invention, the promoter of chrysanthemum-derived ConP10 of the present invention having the nucleotide sequence of SEQ ID NO: 1 in a known vector having a reporter system linked to Gus, a blue color gene, is inserted upstream of the Gus gene. A recombinant expression vector, pBGWFS7-ConP10, operably linked to the moiety was constructed.

As another aspect, the present invention provides a transformant transformed by a recombinant expression vector including the promoter of ConP10 derived from chrysanthemum of the present invention.

In the present invention, the term "transformation" refers to genetically changing the trait of an individual or cell by DNA, which is a genetic material given from the outside. In the present invention, by introducing the promoter represented by SEQ ID NO: 1 It means that the target gene is uniformly expressed in all tissues or organs of the plant, including systemically, specifically, leaves, stems, roots, seeds, and the like in the plant.

Transformation with the recombinant expression vector of the present invention in the present invention can be performed by transformation techniques known to those skilled in the art. For example, microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion ( PEG-mediated fusion, microinjection, liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method), or a method mediated by Agrobacterium sp, etc. may be used, but is not limited thereto.

As used herein, the term "transformant" refers to a host cell transformed with a recombinant expression vector comprising the systemic expression promoter of the present invention and a nucleotide sequence encoding a target protein operably linked to the promoter.

The transformant is preferably a microorganism, but is not limited thereto, Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis ), Staphylococcus and Agrobacterium tumefaciens may be, and more preferably, Agrobacterium tumefaciens.

Specifically, in an embodiment of the present invention, Agrobacterium GV3101 was used.

As another aspect, the present invention provides a plant transformed by introducing a recombinant expression vector comprising a nucleotide sequence encoding a target protein operably linked to the promoter of ConP10 derived from chrysanthemum of the present invention into the plant. provides

The transformed plant according to the present invention can be obtained through a sexual reproduction method or asexual reproduction method, which is a conventional method in the art. More specifically, the plant of the present invention can be obtained through sexual reproduction, which is a process of producing seeds through the pollination process of flowers and propagating from the seeds. In addition, it can be obtained through the asexual reproduction method, which is a process of induction, rooting, and soil acclimatization of callus according to a conventional method after transforming a plant with the recombinant expression vector according to the present invention. That is, the explants of plants transformed with the recombinant expression vector of the present invention are plated in a suitable medium known in the art, and then cultured under appropriate conditions to induce the formation of callus, and when shoots are formed, they are transferred to a hormone-free medium. incubate After about 2 weeks, the shoots are transferred to a rooting medium to induce roots. After the roots are induced, the transformed plant according to the present invention can be obtained by transplanting them into soil and acclimatizing them. In the present invention, the transgenic plant may include not only the whole plant but also tissues, cells or seeds obtained therefrom.

Specifically, in one embodiment of the present invention, Arabidopsis thaliana transformed plants by inoculating Arabidopsis thaliana with Agrobacterium GV3101, a microorganism transformant transformed by a recombinant expression vector including a promoter of chrysanthemum-derived ConP10 was produced.

As another aspect, the present invention comprises the step of transforming a plant with a recombinant vector comprising a promoter consisting of the nucleotide sequence of SEQ ID NO: 1 and a foreign gene encoding the target protein operably linked thereto, the target protein It provides a method for producing a transgenic plant for systemically expressing in the plant.

In the present invention, the transgenic plant may be a dicotyledonous plant, but is not limited thereto, but preferably chrysanthemum, soybean, potato, red bean, Arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, Melon, Pumpkin, Carrot, Ginseng, Tobacco, Cotton, Sesame, Beet, Perilla, Peanut, Rapeseed, Apple Tree, Pear Tree, Jujube Tree, Peach, Amphibian, Grape, Tangerine, Persimmon, Plum, Apricot, Rose, Gerbera, It may be carnation or red clover, and more preferably chrysanthemum.

In one embodiment of the present invention, the whole tissue of Arabidopsis thaliana into which the promoter of chrysanthemum-derived ConP10 was inserted was collected and analyzed by GUS staining method. As a result, GUS expression was strongly induced in the entire plant tissue including the leaves and stems of Arabidopsis Therefore, it was confirmed that the promoter of chrysanthemum-derived ConP10 can systemically express the target protein in all tissues of the plant.

As another aspect, the present invention provides a method of inducing expression of a target protein in whole tissues of a plant.

Prepared by operatively linking the ConP10 promoter derived from chrysanthemum for systemic expression of the target protein in all tissues of a plant comprising the nucleotide sequence represented by SEQ ID NO: 1 according to the present invention and the structural gene encoding the target protein It is possible to provide a method of inducing expression of a target protein in all tissues or organs including leaves, stems, roots, seeds, etc. of plants by transforming plants with recombinant expression vectors.

The target protein may be any type of protein, for example, a protein that has medical utility such as enzymes, hormones, antibodies, cytokines, etc., or a protein capable of accumulating a large amount of nutrients that can improve the health of animals including humans , but is not limited thereto. Examples of the target protein include interleukin, interferon, platelet-induced growth factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor, human serum albumin, erythropoietin, and the like.

If the promoter of the present invention is used, in the case of a crop that consumes all parts of a plant, for example, seeds, leaves, roots or stems, the target protein is expressed together with the seeds, leaves or stems by expressing a useful target protein in all tissues of the plant. Crops that can be consumed together can be developed.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Isolation and nucleotide sequence analysis of ConP10 derived from Chrysanthemum

In order to induce stable expression of genes in flower crops, the promoter of ConP10 was isolated from chrysanthemum to use a promoter derived from chrysanthemum.

First, as a result of examining the expression pattern of ConP10 by organs from standard chrysanthemum varieties, it was confirmed that systemic expression was carried out in leaves, roots, stems, and flowers (by flower development period), as shown in FIG. 1 .

Thereafter, the promoter of the ConP10 gene, which is the systemic expression gene, was isolated.

Specifically, PCR was performed using the gene specific primer (GSP) of Table 1 below.

SEQ ID NO: Primer name Primer direction Sequence (5'→3') 2 ConP10-F forward 5'-GGTACAGCCGCATGTGAGGTGGG-3' 3 ConP10-R reverse 5'-GTGAATGAAGAAGCCATTTCGATTG-3'

The size of the amplified product is 554 bp, and the promoter sequence was analyzed using new PLACE (http://www.dna.affrc.go.jp/PLACE). One enhancer element (-446 to -441) that promotes gene expression in the previously reported promoter region, one SV40 core enhancer (-337 to -329), one conserved enhancer elements EEC (-317 to -311, - 244 to -238, -154 to -148), and 2 GATA boxes (-185 to -182, -39 to -36) showing high gene expression were confirmed to exist.

The promoter nucleotide sequence of ConP10 derived from chrysanthemum confirmed above is shown as SEQ ID NO: 1 (FIG. 2).

Example 2: Construction of vectors and transformants for plant transformation

To construct a vector for plant transformation, the ConP10 promoter region was amplified and introduced into the pUC57mini carrier through infusion. For infusion, the ConP10 promoter region was PCR amplified using ConP10-F (pUC57mini) (5'-AAAGCAGGCTGAATTCGGTACAGCCGCATGT-3') and ConP10-R (pUC57mini) (5'-AAAGCTGGGTGGATCCGTGAATGAAGAAGCCATT-3') and then the QIAgen gel extraction kit was used for elution. The pUC57mini vector was subjected to EcoRI and BamHI enzyme treatment, followed by electrophoresis, and then elution was performed using the QIAgen gel extraction kit. After infusion of the pUC57mini vector and the PCR product, E. coli DH5α was transformed to select colonies in which the ConP10 promoter was introduced into the pUC57mini carrier. After the plasmid DNA was isolated, nucleotide sequence analysis was performed.

After the LR reaction was performed to introduce the ConP10 promoter into pBGWFS7, a plant expression vehicle, a carrier into which the ConP10 promoter was introduced was selected by transformation into E. coli DH5α, and the base sequence was analyzed. Finally, the vehicle for plant expression into which the ConP10 promoter was introduced was named pBGWFS7_ConP10 (FIG. 3). Agrobacterium GV3101 was transformed with pBGWFS7_ConP10 carrier by freeze-thaw method. The selected pBGWFS7_ConP10 Agrobacterium culture medium was transformed into Arabidopsis thaliana by the floral-dip method. Selected transformants were treated with 0.3% basta to select transformants (FIG. 3B).

Experimental Example 1: GUS staining results for each tissue of Arabidopsis transformation

GUS staining for each tissue of the transformed Arabidopsis thaliana into which the ConP10 promoter was introduced was comparatively analyzed. As a negative control, Arabidopsis untransformed was used, and as a positive control, Arabidopsis thaliana introduced with the CaMV35S promoter, a systemic expression promoter, was used.

Histochemical staining was performed by immersion in GUS staining solution (X-gluc (cyclohexyl ammonium salt) 20 mM, NaH ₂ PO ₄ H 2 _O 100 mM, NaEDTA 10 mM, Triton X-100 0.1%, pH 7.5) at 37 °C for 24 hours. After time immersion, the GUS staining solution was removed and chlorophyll was removed by treatment with 70% ethanol and observed under a stereoscopic microscope.

Referring to FIG. 4 , as a result of confirming GUS staining in the Arabidopsis transformant into which the ConP10 promoter was introduced, strong GUS staining was confirmed in the leaves of all transformants. In addition, as expected, GUS staining was not observed in the leaves of the negative control, and GUS staining was confirmed in the leaves of the positive control. In the Arabidopsis transformant into which the ConP10 promoter was introduced, GUS staining was confirmed in the flower peduncle (stem) and flower. Since the ConP10 promoter shows GUS staining in all tissues of leaves and flowers, it was confirmed that it can be used to express the target gene in various tissues for crop improvement.

Experimental Example 2: GUS gene expression assay for each tissue of Arabidopsis transformation

To check the expression of GUS gene in leaf tissue, samples of Arabidopsis thaliana were collected and total RNA was extracted using FavorPrep™ Plant Total RNA Mini Kit (Favorgen, Pingtung, Taiwan). Using 1 μg of total RNA, cDNA was synthesized using amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Barker, TX, USA) and used for real-time gene expression analysis (qPCR). qPCR was performed using GUS-specific primers (GUS-Forward, 5'-TGGAGTGAAGTATCAGTGTGC-3' (SEQ ID NO: 4); GUS-Reverse, 5'-CCATACCTGTTCACCGACG -3' (SEQ ID NO: 5)), and gene expression To standardize the Arabidopsis EF1α (elongation factor 1 alpha) specific primer (EF1α-Forward, 5'-GCCACACCTCTCACATTG-3' (SEQ ID NO: 6); EF1α Reverse 5'-TACCAGCGTCACCATTCT-3' (SEQ ID NO: 7)) qPCR was performed using For qPCR, Bio-Rad CFX96 Detection System (Bio-Rad Laboratories) was used, and after denaturation at 95°C for 10 minutes, 15 seconds at 95°C and 30 seconds at 55°C were performed once, a total of 40 times.

Referring to FIG. 5 , as expected, almost no GUS gene was detected in the negative control (WT), and high expression of the GUS gene was confirmed in the positive control (GUS).

In the transgenic plants introduced with the ConP10 promoter, the expression of the GUS gene in the leaf tissue showed high expression at a level similar to that of the positive control.

These results indicate that the ConP10 promoter secured in the present invention maintains the expression of the transgene at a high level in the leaf tissue, which has the advantage that it can be usefully used to induce gene expression in crops such as chrysanthemum. .

<110> REPUBLIC OF KOREA (RURAL DEVELOPMENT ADMINISTRATION) <120> Constitutive promoter derived from chrysanthemum and use thereof <130> DP20200044 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 554 <212> DNA <213> Artificial Sequence <220> <223> ConP10 promoter <400> 1 ggtacagccg catgtgaggt gggggaccca cagctgggag agagagggag gacaaaaggg 60 gacaaaagcc tgacagtcta aggggctagg gggtagagcc cgtattaata gtatgacctg 120 gctaaaaata gagcgaagat cccttctttg aattgggatc tttggtgtaa atttgtaatt 180 atgtaattgg ccttgtggtg ttgtgccgtc ccgttttgtg gattggcggt ttctaatgaa 240 attcatcttt caaaaaaaaa acttagttag tcatttataa gtcattaaat tgagtcaagt 300 tgatcttctc gaattccatg taaaaaaagt gagatggtgt gccgattaat tttagtaaaa 360 agtgagttag atagtcgggc ccctgattga ctgatgggcg gagttcccaa agtaagttga 420 acctaattcc acctacaatc tcaaataaaa agagcacaca cctataacta aatacacaca 480 ctctccttat ttcacattca tcgcccccga ttatttatca aatcaaatcc aatcgaaatg 540 gcttcttcat tcac 554 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ConP10-forward primer <400> 2 ggtacagccg catgtgaggt ggg 23 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ConP10-reverse primer <400> 3 gtgaatgaag aagccatttc gattg 25 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> gus-forward primer <400> 4 tggagtgaag agtatcagtg tgc 23 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> gus-reverse primer <400> 5 ccatacctgt tcaccgacg 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> EF1alpha-forward primer <400> 6 gccacacctc tcacattg 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> EF1alpha-Reverse primer <400> 7 taccagcgtc accattct 18

Claims (9)

A systemic expression promoter derived from Chrysanthemum consisting of the nucleotide sequence of SEQ ID NO: 1. A recombinant expression vector comprising a foreign gene encoding a target protein, operably linked to the promoter of claim 1 . A transformant transformed with the vector of claim 2 . 4. The method of claim 3,
The transformant is a transformant that is Agrobacterium (Agrobacterium). A plant transformed with the vector according to claim 2 or the transformant according to claim 3 . 6. The method of claim 5,
The transgenic plant is a transgenic plant that is a dicotyledonous plant. 7. The method of claim 6,
The transgenic plant, wherein the dicotyledonous plant is a chrysanthemum. A transgenic plant for systemically expressing the target protein in a plant, comprising transforming the plant with a recombinant vector comprising a promoter consisting of the nucleotide sequence of SEQ ID NO: 1 and a foreign gene encoding the target protein operably linked thereto manufacturing method. A method of inducing the expression of a target protein in all tissues of a plant by transforming the plant with the vector of claim 2 .

Images