KR101080288B1 - Constitutive promoter UK1 - Google Patents

Abstract

The present invention relates to a novel plant constitutive promoter, and more particularly to promoter AtUK1 derived from Arabidopsis , a method for preparing the same, a recombinant expression vector including the same, and a method for transforming an edible crop. It is about the use to use. The whole-expression promoter of the present invention can express foreign genes regardless of the place of expression to induce systemic-expression of various herbicide resistance genes and reporter genes or to develop transgenic plants that improve useful traits in food crops. It can be widely used.

Transformation of constitutive promoter UK1, Arabidopsis, edible crops

Description

Whole-expression promoter UK1 {Constitutive promoter UK1}

The present invention relates to a novel plant constitutive promoter, and more particularly, promoter UK1 derived from Arabidopsis and a method for preparing the same, a recombinant expression vector including the same, and a method for transforming an edible crop. It is about the use to use. The whole-expression promoter of the present invention can express foreign genes regardless of the place of expression to induce systemic-expression of various herbicide resistance genes and reporter genes or to develop transgenic plants that improve useful traits in food crops. It can be widely used.

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

First, there is a promoter that induces systemic-expression. As such a plant whole-expression induction promoter, a promoter of 35S RNA gene of CaMV (cauliflower mosaic virus) is used as a representative dicotyledonous promoter. In addition, rice actin gene promoter and corn ubiquithin gene promoter have been mainly used as a systemic expression induction promoter for monocotyledonous plants. Recently, a promoter of rice cytochrome C gene (OsOc1) has been developed and used by domestic researchers. (Korean Patent Registration No. 10-0429335). They are primarily induced to induce the expression of antibiotic or herbicide resistance genes and reporter genes used as selection markers in the plant transformation basic carrier. Therefore, in terms of research, it is the promoters that are considered first when attempting to reveal the function of the target gene in the plant.

Second, there are promoters specific to seed. These seed-specific promoters are representative of the promoters of the major storage protein genes of rice, and the rice gluterin promoter used for the development of golden rice is currently used to induce seed-specific expression of monocotyledonous plants. It is used. In addition, promoters mainly used to induce seed-specific expression of dicotyledonous plants include a lectin promoter derived from soybean, a napin promoter derived from cabbage, and gamma-tocopherol methyl transferase (γ-tocopherol methyl transferase) in seeds of Arabidopsis. Examples include carrot-derived DC-3 promoters used in studies that induce γ-TMT) gene expression to promote vitamin E production, and in particular, seed specific expression induction of perilla-derived oleosin (oleosin) promoter is already patented. Application (Korean Patent Application No. 10-2006-0000783). The seed specific promoters are mainly used for the purpose of accumulating useful proteins and generating useful substances in major crops in which the seeds themselves are used as food, food or food ingredients.

Third, there may be mentioned a promoter specifically expressed in the root. This root-specific promoter has not been commercialized yet, but root-specific expression has been confirmed by the separation of Arabidopsis peroxidase (peroxidase, prxEa). Recently, the sweet potato-derived Maz gene ( ibMADS ) and sugar-induced APD Glucose pyrophosphatase (ADP-glucose pyrophosphatase, AGPase) gene has been isolated and reported that the promoter induces specific expression in the roots. In addition, these promoters have been confirmed to induce root-specific transient expression in carrots and radishes (patent registration number 10-0604186, 10-0604191). These promoters can be expected to be used mainly for the purpose of agricultural transformation, accumulation of useful proteins and production of useful substances in food, food or major root crops used as raw materials of food.

Fourth, promoters specific to other tissues such as leaves may be mentioned. This tissue-specific promoter includes rice and corn-derived albics (rbcS) promoters, which induce potent gene expression only in photosynthetic tissues such as leaves, and RolD, which induces plant root expression from Agrobacterium. Promoter, potato-induced tuber-specific expression patatin promoter, tomato-derived fruit maturation-specific expression-induced phytoene synthase (PDS) promoter, and cabbage-derived oleosin identified pollen-specific expression in Chinese cabbage flowers ) Promoters and the like. In addition, the research team of the present invention has used the male vocal cord tissue-specific BcA9 promoter of flowers to induce expression of Bt protein genes toxic to cells, and has developed and registered a patent for male sterile crops (Korea Patent Registration No. 10- 0435143).

As described above, various plant-derived promoters have been found and reported in each tissue of a plant according to various purposes, and development is still underway. However, most of the types of promoters that are already commercialized or are widely used among plant researchers belong to the cases mentioned above, and there is a need for further development of new promoters that can precisely control the place of gene expression according to the developer's intention. .

Accordingly, the present inventors continued efforts to develop a new plant constitutive promoter, confirming that the promoter UK1 derived from Arabidopsis is systemically expressed and that SEQ ID NO: 6 and SEQ ID NO: Using oligonucleotides as primers to amplify the promoter UK1 site, and then insert expression vector for plant transformation into which it is inserted, and transform it into Arabidopsis from which GUS (β-glucuronidase) blue coloration and GUS transcripts were measured. The present invention was successfully completed by checking the degree of whole body-expression by confirming that the whole body-expressing promoter UK1 can express a foreign gene regardless of the place of expression.

It is an object of the present invention to provide a novel plant whole-expressing promoter, a method for producing the same, and a use thereof for improving plant traits.

In order to achieve the above object, the present invention provides a plant whole-expressing promoter UK1 consisting of SEQ ID NO: 1.

The present invention also provides a recombinant expression vector pBGWFS7-PAtUK1 (Accession No .: KACC 95084P) comprising the plant whole-expressing promoter.

The present invention also provides a method for producing the plant whole-expressing promoter UK1 by carrying out a polymerase chain reaction using oligonucleotides of SEQ ID NO: 6 and SEQ ID NO: 7 using a primer.

In addition, the present invention is a system for producing a plant expression vector expressing a target gene using the systemic-expressing promoter UK1 and transforming the plant carrier and then introducing it into the plant systemically (constitutively) ) Provides a method of expression.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel plant whole-expressing promoter.

Specifically, the present invention provides a plant promoter UK1 consisting of SEQ ID NO: 1. The plant body-expression promoter is preferably derived from can include both that derived from the genus Arabidopsis, but Arabidopsis Italia or (Arabidopsis thaliana) (Arabidopsis sp. ).

The whole-expressing promoter is a promoter of a new gene whose function is not known in Arabidopsis, and the whole-expressing promoter preferably corresponds to 1,897 bp upstream of the coding region of the gene. The base sequence of SEQ ID NO: 1 may include a modified sequence in which some bases are substituted, deleted or added, but it is preferable to have sequence homology of 90% or more.

The present invention also provides a pair of primers consisting of oligonucleotides of SEQ ID NO: 6 and SEQ ID NO: 7 used to amplify the systemic-expressing promoter. The primers consist of a forward primer and a reverse primer each consisting of 32bp that specifically amplify the systemic-expressing promoter UK1, which primers can be prepared according to the synthesis of oligonucleotides known to those skilled in the art and commercially synthesized. It can also be used. The primer may include a modification sequence in which a mutation occurs in part of an individual nucleotide sequence. The modified sequence is a result of performing PCR using a mutated primer and a result of performing PCR using primers of SEQ ID NO: 6 and SEQ ID NO: 7 consisting of an unmodified sequence. Or added sequences.

The present invention also provides a method for producing the plant whole-expressing promoter UK1 by carrying out a polymerase chain reaction using oligonucleotides of SEQ ID NO: 6 and SEQ ID NO: 7 using a primer.

The present invention also provides a recombinant expression vector comprising the plant whole-expressing promoter. Specifically, the present invention provides an expression vector pBGWFS7-PAtUK1 for plant transformation comprising the plant whole-expressing promoter having the nucleotide sequence of SEQ ID NO: 1.

The expression vector pBGWFS7-PAtUK1 for plant transformation was deposited on July 04, 2008 to the Gene Bank of Agricultural Biotechnology Research Institute (Accession Number: KACC 95084P).

The expression vector may include a herbicide resistance gene and / or a reporter gene in addition to the systemic-expressing promoter of SEQ ID NO: 1, and the herbicide resistance gene may include a Bar gene and the like, and the reporter gene may include Egfp: gus gene and the like are preferable.

The present invention also provides a plant transformed with a recombinant expression vector comprising the plant whole-expressing promoter. Specifically, the present invention provides a plant transformed with the plant expression vector pBGWFS7-P At UK1 comprising the systemic-expressing promoter sequence of SEQ ID NO: 1, wherein the plant is a carrot, sweet potato, radish, etc. It may include root crops, medicinal plants such as ginseng and deodeok, feed crops such as corn and main grain crops such as rice. In addition, the plant may include various dicotyledonous plants.

The present invention also provides a seed of a plant transformed with a recombinant expression vector comprising the plant whole-expressing promoter. Seeds of the plant may include seeds such as carrots, sweet potatoes and radish crops, medicinal plants such as ginseng and deodeok, feed crops such as corn and main grain crops such as rice. In addition, the seeds of the plant may include seeds of various dicotyledonous plants.

In addition, the present invention is to produce a plant expression vector expressing a target gene using the systemic-expression promoter, transforming the plant carrier and then introducing it into the plant systemically (constitutively) the target protein in the plant It provides a method of expression.

Genes that can be expressed using the systemic-expressing promoter of the present invention may include all plant genes, but in particular, herbicide resistance genes, marker genes and reporter genes, etc. are preferably included. It is more preferable to include useful genes that can be used. In addition, it can be used in various ways for research purposes, such as comparing the performance of the promoter.

As described above, the present invention provides a promoter UK1 derived from Arabidopsis as a new plant constitutive promoter, a preparation method thereof, a recombinant expression vector including the same, and the use of the same for the transformation of food crops. It is about use. The whole-expression promoter of the present invention can express foreign genes regardless of the expression site, and induces systemic-expression of various herbicide resistance genes, other marker genes, and reporter genes or transforms useful traits in food crops. It can be widely used to develop plants.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

General experimental methods not specifically mentioned in the examples below refer to the molecular genetic cloning book of Sambrook et al . ("Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, second edition, 1989"). .

Example 1 Analysis of Expression Pattern of UK1 Gene from Arabidopsis

In the present invention, in order to investigate the expression pattern of gene UK1 whose function is unknown from Arabidopsis , total RNA (total) from pods including Arabidopsis root, leaf, stem and flower and seeds during development RNA) was isolated and analyzed by performing reverse transcriptase-polymerase chain reaction (RT-PCR). Detailed description of the entire RNA separation process is as follows. Approximately 1 g of each sample was ground in a mortar with liquid nitrogen and transferred to a 1.5 ml tube, then 500 μl of RNA extraction buffer (50 mM sodium acetate pH 5.5), 150 mM LiCl, 5 mM EDTA, 0.5% in each tube. SDS) and 500 μl phenol were added and mixed. The mixture was treated at 65 ° C. for 10 minutes and mixed at room temperature for 15 minutes using a rotary shaker, followed by centrifugation (10,000 rpm, 10 minutes) at 4 ° C. FIG. The supernatant layer obtained from this was carefully transferred to a new tube, mixed well by adding 500 µl of chloroform, and centrifuged again to obtain the supernatant. To this was added 0.6 times the volume of 8M lithium chloride (LiCl) and stored at -20 ° C for 2 hours or more, followed by centrifugation at 4 ° C and 12,000rpm for 20 minutes, and RNA precipitates were added once with 4M LiCl and 80% ethanol. After washing, the final RNA precipitate obtained was dissolved in 50 μl of distilled water.

The RNA eluate obtained here was quantified by measuring A ₂₆₀ / A ₂₈₀ using a UV spectrophotometer, and then 50 μM oligo dT primer was used as the template for 5 μg total RNA at 50 ° C. for 50 minutes at 85 ° C. Treated for 5 minutes and cooled on ice. 1 μl of RNase H was added thereto and treated for 20 minutes at 37 ° C. to synthesize cDNA (10 × RT buffer, 25 mM MgCl _2, 0.1 M DTT, RNase OUT, SuperScript ^™ RT). The synthesized cDNA thus obtained was used as a template, and a polymerase chain reaction was performed using a specific primer set for the UK1 gene, that is, the UK1-F primer of SEQ ID NO: 2 and the UK1-R primer of SEQ ID NO: 3. In addition, in order to uniformly adjust the relative amount of RNA used, EF1α-F primer of SEQ ID NO: 4 and EF1α-R of SEQ ID NO: 5, which are primer sets for Arabidopsis elongation factor 1a, were simultaneously used at 95 ° C. Denatured for 7 minutes, reacted for 30 seconds at 95 ° C, annealing for 30 seconds at 60 ° C, and polymerized series of 40 reactions for 30 seconds at 72 ° C, followed by PCR reactions for 7 minutes at 72 ° C. Was performed.

As a result of the reverse transcription PCR reaction, it was confirmed that the transcript of the UK1 gene derived from Arabidopsis was all systemically expressed in pod tissue including leaves, stems, flowers, roots and seeds. At this time, it was proved that the relative amount of RNA used was uniform by using primers specific for Arabidopsis elongation element 1α.

1 shows the AtUK1 promoter gene of the present invention amplified using total RNA and specific primers isolated from roots, leaves, stems, flowers, and seed tissues of Arabidopsis spp., Respectively, compared with a positive control. At this time, M is a 1Kb plus DNA ladder marker, AtUK1 is a Arabidopsis promoter gene and EF1α is an Arabidopsis elongation factor 1α gene.

Example 2 Construction of Plant Expression Vectors Including Plant Whole-Expressing Promoter UK1

In the present invention, the expression pattern was examined according to various tissues of the Arabidopsis as described in Example 1, and the UK1 gene corresponding to the systemic-expressing protein was searched for genes from the already disclosed Arabidopsis nucleotide sequence. . In addition, about 1.89 kb region (SEQ ID NO: 1) containing a TATA box and the like above the start codon of the UK1 gene was isolated, and a carrier for P At UK1 promoter function analysis was prepared using the gateway carrier pBGWFS7 purchased from the University of Ghent in Belgium. . A small amount of leaf tissue of the transformed Arabidopsis plants was placed in an Eppendorf tube and separated and purified using a Genomic DNA Purification Kit (IJBIO DNA System). DNA eluate was quantified by measuring A ₂₆₀ / A ₂₈₀ using an ultraviolet spectrophotometer. SEQ ID NO: 6, as shown in Table 1 below, designed to specifically amplify P At UK1, including some recombinant sequence regions specific for bacteria when the bacteria were infected from bacteriophages from genomic DNA isolated from Arabidopsis; A primer set of SEQ ID NO: 7 (PUK1-B1 primer and PUK2-B2 primer) was prepared. Using this primer set, the promoter site of about 1.89 kb was isolated again through a gene amplification reaction (PCR: polymerase chain reaction).

primer Sequence PUK1-B1 5'-AAAAAGCAGGCTTCCTTCAAAGCAACTCTCAT-3 ' PUK1-B2 5'-AGAAAGCTGGGTAAAACGATCAACCACGTACT-3 '

In addition, in order to compare the activity of the P At UK1 promoter region with the activity of the P35S promoter mainly used for transformation of dicotyledonous plants, SEQ ID NO: 8 designed to amplify the P35S promoter while including a part of a nucleic acid sequence specific for bacteria Primer 35S-F and primer 35S-R of SEQ ID NO: 9 were prepared to separate the P35S site. The amplified PCR products were then amplified again by PCR using adapter primer B1 of SEQ ID NO: 10 and adapter primer B2 of SEQ ID NO: 11, which contain the entire nucleic acid sequence site specific for the bacteria when the bacteriophage was infected. I was.

In addition, the secondary amplified PCR products are recombinant cloning vectors pDONR-P At UK1 and pDONR-P35S by BP recombination using a cloning vector pDONR221 containing a nucleic acid sequence site specific for bacteriophage when bacteria are infected with bacteriophage. Was made. These two subcloned carriers were used in the plant expression vector pBGWFS7 incorporating the reporter system in which Egfp and Gus genes were linked to produce a plant transformation carrier. Complete recombinant expression vectors pBGWFS7-P At UK1 and pBGWFS7-P35S. At this time, the plant expression vector pBGWFS7 used as a binary vector has a spectinomycin resistance gene for microbial selection and includes herbicide resistance Bar gene as a plant selection factor. The transforming carriers thus prepared were transformed into the Agrobacteria tuberculosis GV3101 strain according to the direct Agrobacteria transformation method (Holster et al., Freeze-thaw method, 1978) using liquid nitrogen, followed by the alkaline method of Ahn et al. Agrobacteria were introduced into Agrobacteria using the quick-screen method based on alkaline lysis.

Fig. 2 schematically shows a genetic map of the plant expression vector pBGWFS7-P At UK1 of the present invention. At this time, LB is the left side, Tnos is the nos terminator, BAR is the herbicide resistance gene, Pnos is the nos promoter, PAtUK1 is the promoter of the present invention, P35S is the cauliflower virus 35S promoter, EGfp is the green fluorescent protein gene, GUS represents the blue chromosome β-glucuronidase gene, T35S represents the cauliflower virus 35S terminator, and RB represents the right side. 3 schematically shows a genetic map of the control plant expression vector pBGWFS7-P35S.

Example 3. Transformation and Culture of Arabidopsis

In the present invention, Arabidopsis was transformed as a plant using the expression vector for plant transformation prepared in Example 2 as follows. Before sowing to 22 ℃ in an incubator (16 hours day / 8 hour night) removing approximately four weeks the growth of Arabidopsis plants Italia or bolting of the primary (Arabidopsis thaliana ecotype Columbia), which was induced at least three to four secondary bolting. Agrobacteria cultures containing plant transformation carriers were then diluted to a solution containing 5% sucrose and 2,500 ppm Tween 20 at an OD of 0.8 and the suspension obtained was sprayed onto the buds to infect Agrobacteria. . The infected Agrobacterium was incubated overnight with sufficient humidity and cancer conditions, followed by incubation for 4 to 5 days in a 22 ° C. incubator (16 hours day and 8 hours night) and incubated and diluted in the same manner as above. Was sprayed on the fire tissue again. Then incubate overnight with sufficient humidity and cancer conditions, and then incubate for 3 to 5 weeks in a 22 ° C incubator (16 hours day / 8 hours night) until the mature silique matures to brown. After seeding. The seeded seeds were sown again and sprayed with 0.3% batha on the 10th day and the second day of 17th day, and the transformants of T1 generation were selected therefrom. The T1 transgenic plants were grown for about 2 months in a 22 ° C. incubator (16 hours day / 8 hours night) until the pods silique matured completely to brown and the T2 seeds were seeded.

Example 4 Analysis of GUS Blue Color Response in Transgenic Arabidopsis

In the present invention, whether the whole body-expression in the transgenic Arabidopsis was investigated by GUS blue color reaction as follows. First, P At UK1 of the present invention T2 seeds transformed with the promoter and P35S promoter were sown and sprayed with 0.3% batha on the 1st day of the 10th day and the 2nd day of the 17th day, and various tissues such as leaves, stems, roots, flowers, and pods of the transgenic Arabidopsis plant were selected. The blue color reaction was observed at each growth stage. As a control, non-transgenic Arabidopsis plants were germinated without treatment with batha. 7, 21 and 42 days after sowing, transgenic Arabidopsis plants were GUS assay buffer [GUS assay buffer; Sol. I: X-gluc (cyslohexyl ammonium salt) 20 mM, Sol. II: NaH ₂ PO ₄ H ₂ O 100mM, NaEDTA 10mM, Triton-X-100 0.1%, pH7.5] and treated at 37 ° C. for 24 hours, followed by 70% ethanol to remove chlorophyll It was. Plants were observed using a light microscope or visually.

As a result, P35S and P At UK1 The transformants showed GUS blue coloration throughout the body, whereas no GUS blue coloration was observed in the nontransgenic Arabidopsis. In addition, the plants at days 21 and 42 had no GUS blue coloration at all in the transgenic Arabidopsis, whereas P35S and P At UK1 The GUS blue color was observed in the whole body of the transformant, and it was confirmed that the P At UK1 promoter was a systemic-expressing promoter as well as the P35S promoter.

Figure 4 shows the observation of the blue color reaction during seedling and leaf development process of Arabidopsis transformed with plant expression vector pBGWFS7-P At UK1 of the present invention. Col0 is a non-transgenic Arabidopsis, P35S :: GUS is a control transgenic Arabidopsis and P At UK1 :: GUS is a Arabidopsis transformed with the expression vector pBGWFS7-P At UK1, 7D after seeding 7 It is the first seedling seedling, the 21D is the 21st day after planting, the 42D is the 42nd day after planting.

Example 5 Quantitative Analysis of GUS Blue Color in Transgenic Arabidopsis

In the present invention, the degree of whole-expression in the transgenic Arabidopsis was investigated by GUS blue color quantitative analysis as follows. First, in order to measure the GUS blue color expression level of the promoter of the present invention, the Arabidopsis T2 generation 2 transformed using the PAtUK1 promoter and the control Arabidopsis and nontransgenic Arabidopsis leaves transformed with the P35S promoter, Whole proteins were isolated from roots, stems, pods and flowers. To isolate the whole protein, transfer 50 mL of tissue-specific samples ground in a mortar with liquid nitrogen to a 1.5 ml tube, then place 100 μl of protein extraction buffer (50 mM sodium phosphate, pH 7.0, 10 mM dithio) in each tube. Dithiothreitol (DTT), 1 mM disodium EDTA, 0.1% SDS, 0.1% Triton X-100) was added and mixed vigorously. The mixed solution was centrifuged at 4 ° C. and 15,000 rpm for 5 minutes, the supernatant was transferred to a new tube, and 50 μl of this was taken. 50 μl of analytical solution set at 37 ° C. [1.2 mM MUGluc, 10.0 mM β-mercaptoethanol (β -mercaptoethanol in 1X reaction buffer solution), FluorAce β-glucuronidase reporter assay kit (β-glucuronidase reporter assay kit, Bio-Rad)] and reacted for 30 minutes in a 37 ℃ water bath. 100 μl of 1 × Stop buffer was added to each sample, and GUS activity was quantified using a fluorophotometer (excitation filter 355 nm, emission filter 460 nm). Figure 5 shows the quantitative blue chromogenic protein expressed by the PAtUK1 promoter of the present invention compared to the positive control. As a result, it was confirmed that the P At UK1 promoter of the present invention was a whole-expressing promoter capable of expressing a foreign gene to the same or more increased level than the P35S promoter.

Example 6 Systemic-Expression Investigation in Transgenic Arabidopsis by RT-PCR Reaction

In the present invention, whether or not the gene expression in the transgenic Arabidopsis was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) as follows. First, sowing transformed T2 seed, spraying 0.3% batha on the 1st day of the 10th day and the 2nd day of the 17th day, and leaves, stems, roots, flowers and pods of the transgenic Arabidopsis plants selected from the seed (3rd week after flowering). Several organizations were collected. Then, the total RNA extracted in the same manner as in Example 1 was treated with 50 μM oligo dT primer for 50 minutes at 50 ° C. and 5 minutes at 85 ° C., cooled on ice, and then 1 μl of RNase H was added thereto. CDNA was synthesized by addition and treatment at 37 ° C. for 20 minutes (10 × RT buffer, 25 mM MgCl _2, 0.1M DTT, RNase OUT, SuperScript ^™ RT). Using the synthesized cDNA as a template, the primer GUS-F primer of SEQ ID NO: 12, the primer GUS-F primer of SEQ ID NO: 12, and the GUS-R primer of SEQ ID NO: 13, which is a blue chromosome gene, were used as a template. Denatured at 95 ° C. for 7 minutes, at 95 ° C. for 30 seconds, at 60 ° C. for 30 seconds, using the primer EF1α-F of SEQ ID NO: 4 and EF1α-R of SEQ ID NO. After performing 40 series of polymerization reactions for 21 seconds at 21 ° C, PCR reactions were performed at 72 ° C. for 7 minutes.

As a result, it was confirmed that the transcript of the blue chromogenic protein gene GUS was clearly detected in the leaves, stems, flowers, roots, and seed pods of the Arabidopsis transformed with the P At UK1 promoter of the present invention. Using the Arabidopsis elongation element EF1α it was proved that the relative amount of RNA used is uniform.

FIG. 6 is an extension factor 1α of the Arabidopsis endogenous gene, which is a chromogenic color gene GUS and a positive control after reverse transcription using total RNA extracted from each tissue of Arabidopsis transformed with the plant expression vector pBGWFS7-P At UK1 of the present invention. An electrophoretic photograph showing gene bands amplified with primers that specifically amplify each gene. Where M is a 1Kb plus DNA ladder marker, GUS is a blue-colored beta-glucuronidase gene, EF1a is a Arabidopsis elongation factor 1α gene, and 1: non-transgenic Arabidopsis leaf , 2: flower of the untransformed Arabidopsis, 3: stem of the untransformed Arabidopsis, 4: seed of the untransformed Arabidopsis, 5: the root of the untransformed Arabidopsis, 6: the Arabidopsis transformed with PAtUK1 Leaves, 7: flowers of Arabidopsis transformed with PAtUK1, 8: stems of Arabidopsis transformed with PAtUK1, 9: seeds of Arabidopsis transformed with PAtUK1, 10: roots of Arabidopsis transformed with PAtUK1 , 11: leaves of Arabidopsis transformed with P35S, 12: Arabidopsis flowers transformed with P35S, 13: stems of Arabidopsis transformed with P35S, 14: seeds of Arabidopsis transformed with P35S, 15 : Rooted Arabidopsis transformed with P35S.

[Sequence list]

SEQ ID NO: 1

TCCTTCAAAG CAACTCTCAT ATCTTCAAAC TTGACACAAT CACCATACAA AAACTCACCA AGTTTCTTCA ACTCAACATC TTCCAAATCA TCAGAAAACG

GTTTAATCGG AAACGCATTC TCCGGTTGAA GCGCGTACGA GTTCGGATTA TCATCAACAA TCACCACGCG CCTCAAATCT CTCATCACAA ACCCTAAATC

CTTCACTAAC CTCCCATCAA TTTCGCTACA CGCGTCTCTG TAAAAGCTAC GCGATATCAC GCGCCGCTCT GGATCCAATT TATCGAGCAC TAACGACGCG

TACTCTCTCA ATCCCGCCGT GAAAACAACG ATCTGGTACT TTTCTCCGAT CTTCTTCAAA AACTCGTCGA CTCCTGGCCG TTTGATCACG AAAAACGTTA

AGATCTGACC GTCGATTTTG GGATTCACGA CGAAATCATA CGGTACTTCT GGTTTCTCCA TTGATGAATG AACAAGAGTT TCGTCTAGAT CTAACACGAT

CGTCTTCTTC GTCTCGTCGA ATGATTTACC AGATCTTTCG TGAAACAATG AATACGGAGA ATTCTGAAAA TCTTCGCGAG GTTTGAGGAT TTTGAATCCT

TTTGTTGCGT GACGAGAAAA CAAACAGAGA AAAGTTCGAT GACATCTGTA GATCGATTTG TTGATGGTGG CGATTACGGT GGTTGTGGCC GGAGACGGTG

AGCATCCGAT CTGAGATTTC CGGTGACGGC GGAGGTAGTT ACGTTGGTGA CGGCTGATGG ATTTTGTCGT TTTCTTGAGT ATGAGTTTCG TCGCCATTGA

AGAAGAAAGA GAGAGATTAG AGTTTGCGAG ATTTTTGGGA GAGAGAGGAG AGTGATGATG TTGTATTTAA ATTAGGATCG GTGTAACGGT CATGTGTTTA

TTTTAGGTTC GGAATCCGAA GCAAAGCTTG ATTCGGAAGC AGTTTTTTTTTT TTTTTTTAAA GTTTGGACCT ACACGGAGTT TACGTTGTAT TATTACCGTT

GGGAATTCTA TATTAATCTC GAGATTCATT TCCTAAATTA AGAAACAAAA TTCTATATAT AACACAGAAT AGACAAATAT TATATATATA TATATATTTA

TCTTATTATA TAAATTGTGA ATCATAAAAG GTGTTCAAAG AACAGACAAA ATGATCTAAT ATATGATATG AGTTCTTAAT AGTTTCTAAT TTTTAATTAC

ATAAATTCTA TTTGTAGTTG TAAATTTAAT GTGACAAAAT ATTGTCAAAG TGTTCATAAT TCATATTCAA AACTATTGTA TTGGAGTGAA ACGACTTGAA

ACAAAACAAA ACAAAGTCCT ACATAACATC TATAAAACTT TATCTAAAAA ACAATAAATA AAGTTCACAA AAATTTAATT TATATCAAAG CGAACCGCTT

TAATTATGTT GCTTGGATGT CATAACTTAA CACATTTTGA TGTGGAGAAG GAAAAAAAAA TAGATTTTGA TAGTTCAATT AATATTTTAA TAATGAAATA

AAACTTGACT TGACATGAAA AAAATCGAAA CTTAGATAAA TCATAAAACC CGGCCCGTAT ATAGATTTTT TTTGAAAAAG ACTATAACGA TCTTTTTTTT

TTCTATGAAG ATTATACAGA GTCCAACCAA ATTCAAGAAT CTCAATTATA AGTGCCAAGT GGCCAAAGGT AATCACCATC ATTAACATAT CCACGTGTCA

TCCACAAAGC TCAATCTCAA TATTTCTCGC CACATAACCA CAAACAACGC AATGACGTGT CCTTCTCAAA TCATCCACGT CACATTTTGC TCATGCAGCA

ATGACACCAA ACACAATCAC ATCCCATATT TATAATAACA CACAAAAGAA AGAAAAGATA CATAAAATTT GTATAATAGT ACGTGGTTGA TCGTTTT

SEQ ID NO: 2

5'-AGTATGGAATCTCGACGGAATCTT-3 '

SEQ ID NO: 3

5'-ATTTGTTTACGTTGAAGGTGGTTAT-3 '

SEQ ID NO: 4

5'-GTTTCACATTAACATTGTGGTCATT-3 '

SEQ ID NO: 5

5'-GAGGTACCAGTAATCATGTTCTTG-3 '

SEQ ID NO: 6

5'-AAAAAGCAGGCTTCCTTCAAAGCAACTCTCAT-3 '

SEQ ID NO: 7

5'-AGAAAGCTGGGTAAAACGATCAACCACGTACT-3 '

SEQ ID NO: 8

5'-AAAAAGCAGGCTGGTCCCCAGATTAGCCT-3 '

SEQ ID NO: 9

5'-AGAAAGCTGGGTCCCGGGGATCCTCTAGA-3 '

SEQ ID NO: 10

5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3 '

SEQ ID NO: 11

5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3 '

SEQ ID NO: 12

5'-ACCTGCGTCAATGTAATGTTCTGC-3 '

SEQ ID NO: 13

5'-CTCCCTGCTGCGGTTTTTCA-3 '

1 shows the AtUK1 promoter gene of the present invention amplified using total RNA and specific primers isolated from roots, leaves, stems, flowers, and seed tissues of Arabidopsis spp., Respectively, compared with a positive control.

M: 1 Kb plus DNA ladder marker; AtUK1: Arabidopsis promoter gene; And EF1α: Arabidopsis elongation element 1α gene.

Fig. 2 schematically shows a genetic map of the plant expression vector pBGWFS7-P At UK1 of the present invention.

LB: left side; Tnos: nos terminator; BAR: herbicide resistance gene; Pnos: nos promoter; PAtUK1: promoter of the present invention; P35S: cauliflower virus 35S promoter; EGfp: green fluorescent protein gene; GUS: blue chromosome β-glucuronidase gene; And T35S: cauliflower virus 35S terminator; RB: Right side.

3 schematically shows a genetic map of the control plant expression vector pBGWFS7-P35S.

Figure 4 shows the observation of the blue color reaction during seedling and leaf development process of Arabidopsis transformed with plant expression vector pBGWFS7-P At UK1 of the present invention.

Col0: untransformed Arabidopsis; P35S :: GUS: control transgenic Arabidopsis; P At UK1 :: GUS: Arabidopsis transformed with pBGWFS7-P At UK1; 7D: Arabidopsis seedlings 7 days after sowing; 21D: Arabidopsis 21 days after sowing; 42D: Baby pole on day 42 after sowing.

Figure 5 shows the quantitative blue chromogenic protein expressed by the PAtUK1 promoter of the present invention compared to the positive control.

Figure 6 is compared with the positive control and GUS gene promoter gene of the present invention amplified using a specific primer and the total RNA isolated from each tissue of Arabidopsis transformed with the plant expression vector pBGWFS7-P At UK1 of the present invention It is shown.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION <120> Constitutive promoter UK1 <130> PA080072 <160> 13 <170> KopatentIn 1.71 <210> 1 <211> 1897 <212> DNA <213> Arabidopsis thaliana <400> 1 tccttcaaag caactctcat atcttcaaac ttgacacaat caccatacaa aaactcacca 60 agtttcttca actcaacatc ttccaaatca tcagaaaacg gtttaatcgg aaacgcattc 120 tccggttgaa gcgcgtacga gttcggatta tcatcaacaa tcaccacgcg cctcaaatct 180 ctcatcacaa accctaaatc cttcactaac ctcccatcaa tttcgctaca cgcgtctctg 240 taaaagctac gcgatatcac gcgccgctct ggatccaatt tatcgagcac taacgacgcg 300 tactctctca atcccgccgt gaaaacaacg atctggtact tttctccgat cttcttcaaa 360 aactcgtcga ctcctggccg tttgatcacg aaaaacgtta agatctgacc gtcgattttg 420 ggattcacga cgaaatcata cggtacttct ggtttctcca ttgatgaatg aacaagagtt 480 tcgtctagat ctaacacgat cgtcttcttc gtctcgtcga atgatttacc agatctttcg 540 tgaaacaatg aatacggaga attctgaaaa tcttcgcgag gtttgaggat tttgaatcct 600 tttgttgcgt gacgagaaaa caaacagaga aaagttcgat gacatctgta gatcgatttg 660 ttgatggtgg cgattacggt ggttgtggcc ggagacggtg agcatccgat ctgagatttc 720 cggtgacggc ggaggtagtt acgttggtga cggctgatgg attttgtcgt tttcttgagt 780 atgagtttcg tcgccattga agaagaaaga gagagattag agtttgcgag atttttggga 840 gagagaggag agtgatgatg ttgtatttaa attaggatcg gtgtaacggt catgtgttta 900 ttttaggttc ggaatccgaa gcaaagcttg attcggaagc agtttttttt tttttttaaa 960 gtttggacct acacggagtt tacgttgtat tattaccgtt gggaattcta tattaatctc 1020 gagattcatt tcctaaatta agaaacaaaa ttctatatat aacacagaat agacaaatat 1080 tatatatata tatatattta tcttattata taaattgtga atcataaaag gtgttcaaag 1140 aacagacaaa atgatctaat atatgatatg agttcttaat agtttctaat ttttaattac 1200 ataaattcta tttgtagttg taaatttaat gtgacaaaat attgtcaaag tgttcataat 1260 tcatattcaa aactattgta ttggagtgaa acgacttgaa acaaaacaaa acaaagtcct 1320 acataacatc tataaaactt tatctaaaaa acaataaata aagttcacaa aaatttaatt 1380 tatatcaaag cgaaccgctt taattatgtt gcttggatgt cataacttaa cacattttga 1440 tgtggagaag gaaaaaaaaa tagattttga tagttcaatt aatattttaa taatgaaata 1500 aaacttgact tgacatgaaa aaaatcgaaa cttagataaa tcataaaacc cggcccgtat 1560 atagattttt tttgaaaaag actataacga tctttttttt ttctatgaag attatacaga 1620 gtccaaccaa attcaagaat ctcaattata agtgccaagt ggccaaaggt aatcaccatc 1680 attaacatat ccacgtgtca tccacaaagc tcaatctcaa tatttctcgc cacataacca 1740 caaacaacgc aatgacgtgt ccttctcaaa tcatccacgt cacattttgc tcatgcagca 1800 atgacaccaa acacaatcac atcccatatt tataataaca cacaaaagaa agaaaagata 1860 cataaaattt gtataatagt acgtggttga tcgtttt 1897 <210> 2 <211> 24 <212> DNA <213> Arabidopsis thaliana <400> 2 agtatggaat ctcgacggaa tctt 24 <210> 3 <211> 25 <212> DNA <213> Arabidopsis thaliana <400> 3 atttgtttac gttgaaggtg gttat 25 <210> 4 <211> 25 <212> DNA <213> Arabidopsis thaliana <400> 4 gtttcacatt aacattgtgg tcatt 25 <210> 5 <211> 24 <212> DNA <213> Arabidopsis thaliana <400> 5 gaggtaccag taatcatgtt cttg 24 <210> 6 <211> 32 <212> DNA <213> Arabidopsis thaliana <400> 6 aaaaagcagg cttccttcaa agcaactctc at 32 <210> 7 <211> 32 <212> DNA <213> Arabidopsis thaliana <400> 7 agaaagctgg gtaaaacgat caaccacgta ct 32 <210> 8 <211> 29 <212> DNA <213> Arabidopsis thaliana <400> 8 aaaaagcagg ctggtcccca gattagcct 29 <210> 9 <211> 29 <212> DNA <213> Arabidopsis thaliana <400> 9 agaaagctgg gtcccgggga tcctctaga 29 <210> 10 <211> 29 <212> DNA <213> Arabidopsis thaliana <400> 10 ggggacaagt ttgtacaaaa aagcaggct 29 <210> 11 <211> 29 <212> DNA <213> Arabidopsis thaliana <400> 11 ggggaccact ttgtacaaga aagctgggt 29 <210> 12 <211> 24 <212> DNA <213> Arabidopsis thaliana <400> 12 acctgcgtca atgtaatgtt ctgc 24 <210> 13 <211> 20 <212> DNA <213> Arabidopsis thaliana <400> 13 ctccctgctg cggtttttca 20  

Claims (7)

Plant whole-expressing promoter UK1 consisting of SEQ ID NO: 1. The method of claim 1, The systemic-expressing promoter UK1., Characterized in that it is derived from Arabidopsis thaliana . A method for producing a plant whole-expressing promoter UK1 according to claim 1, wherein the oligonucleotides of SEQ ID NO: 6 and SEQ ID NO: 7 are subjected to polymerase chain reaction using a primer. A recombinant expression vector comprising the plant whole-expressing promoter of claim 1. The method of claim 4, wherein The recombinant expression vector is pBGWFS7-PAtUK1 (Accession Number: KACC 95084P), characterized in that the recombinant expression vector. Transgenic plant transformed with the recombinant expression vector of claim 4. A method of expressing a target protein systemically in a plant by preparing a plant expression vector expressing a gene of interest using the systemic-expression promoter of claim 1, transforming the plant carrier, and introducing the same into a plant .

Images