KR20130033056A - Brref promoter specific for cotyledon and hypocotyl - Google Patents

Abstract

PURPOSE: A BrREF promoter which is specific to cotyledon and hypocotyls is provided to specifically induce the expression of a gene in a certain tissue, and to effectively produce a gene product under a predetermined condition. CONSTITUTION: A promoter contains a nucleic acid sequence in sequence number 1. An expression vector contains the promoter, and is pPBrREF. A cell is transformed with the expression vector. The cell is an Agrobacterium sp. microorganism or a plant cell. A transgenic plant contains the expression vector. The plant is dicotyledones.

Description

Cotyledon and hypocotyl {BrREF promoter specific for cotyledon and hypocotyl}

The present invention relates to novel promoters capable of specifically inducing the expression of genes in specific tissues at certain plant developmental stages. More specifically, the present invention specifically induces the expression of genes in cotyledon and hypocotyl from seed germination to seedling stages, which are the early stages of plant growth, and pistils, pistil bulkheads, and pollen during flowering and seed maturation. A novel BrREF promoter capable of specifically inducing the expression of a gene in.

It is very important to develop promoters with tissue specific expression inducing activity. For example, if a heterologous gene is desired to be tissue-specific, a recombinant expression vector in which the heterologous gene of interest is operably linked to a promoter having expression activity specific for the target tissue is produced and introduced into the plant. Specific expression of the heterologous gene of interest is possible within. Plant tissue specific expression of heterologous genes is of significant value in the research and horticultural industries because they can allow them to exhibit altered properties in plants.

As a promoter having such tissue-specific expression-inducing activity, the Arabidopsis-derived AtPFP α2 gene, which specifically induces expression in trichome, cotyledon vein, root and leaf surgery, and pistils Promoter (Korean Patent No. 10-0827553), a novel promoter derived from ZPT2-10, ZPT2-11 and ZPT3-3 genes of petunia with expression activity specific to pistil tissue (Korean Patent No. 10-0443487) Has been developed.

The present invention specifically induces the expression of genes in cotyledon and hypocotyl during the specific growth and development period of plants, ie, seed germination and seedling development, and genes in the pistil, pistil and pollen of flower buds during flowering and seed maturation. It is intended to provide a novel promoter that can specifically induce the expression of.

In order to solve the above problems, the present invention provides a promoter consisting of the nucleic acid sequence of SEQ ID NO: 1.

In another aspect, the present invention provides an expression vector comprising the promoter.

The present invention also provides a transformed cell transformed with the expression vector and a transformed plant into which the expression vector is introduced.

In another aspect, the present invention comprises the steps of transforming plant cells with the expression vector; And regenerating the transformed plant from the transformed plant cell.

According to the present invention, a transgenic plant capable of specifically inducing gene expression in cotyledon and hypocotyls from seed germination to seedling stages and flowering and seed maturation stages in pistils, pistil septum and pollen of flowering plants can do. Therefore, it can be usefully utilized for the efficient production of gene products under the specific conditions during the plant growth stage.

1 is a diagram showing the results of BrREF gene expression analysis using Semi-quantitative RT-PCR in the growth and development of Chinese cabbage. (0d ~ 7d: Seedlings of Chinese cabbage before seed germination ~ 7 days after germination, R: Root, l: Leaf, Sa: Shoot apical region, Fb: Flower bud , F: Flower, St: Stem, Si: Silique
2 shows a cleavage map of the expression vector pPBrREF.
3 is BrREF through GUS expression in the growth and development stage of transgenic Arabidopsis It is a figure which shows the result of promoter activity analysis. (0d ~ 35d: seed germination pole before seed germination ~ 35 days after germination)
Figure 4 BrREF through GUS expression in the hypocotyl of the transgenic Arabidopsis It is a figure which shows the result of promoter activity analysis. (15d: 15 days after seed germination of transformed Arabidopsis, 35d: 35 days after seed germination of transgenic Arabidopsis, Hc: Hypocotyl)
Figure 5 BrREF through GUS expression in the reproductive growth of transgenic Arabidopsis It is a figure which shows the result of promoter activity analysis. (35d: 35 days after seed germination of transformed Arabidopsis, F: Flower, Fb: Flower bud, At: Pollen, Styt: Style, St: Pistil Sept. )

The present invention provides a promoter consisting of the nucleic acid sequence of SEQ ID NO: 1.

The nucleic acid sequence of SEQ ID NO: 1 is Chinese cabbage (Brassica rapa, Brassica rapa ) BrREF ( Brassica) rapa Rubber Elongation Factor A promoter isolated from the gene, which is germinated in the cotyledon and hypocotyl from seed germination, seedling to seedling, during the early stages of plant growth, and during flowering and seed maturation. (Style), pistil septum (Peptum) and pollen (Anther) of the (flowr bud) is characterized by inducing the expression of the gene in a time and tissue specific.

In the present invention, in addition to the promoter consisting of the nucleic acid sequence of SEQ ID NO: 1 is considered to be included within the scope of the invention variants of the promoter. The promoter variant refers to a promoter consisting of a nucleic acid sequence having a functional property similar to that of the nucleic acid sequence of SEQ ID NO: 1, although the nucleic acid sequence is changed. Specifically, the promoter according to the present invention may include a nucleic acid sequence having a sequence homology of at least 70%, at least 80%, at least 90%, or at least 95% with the nucleic acid sequence of SEQ ID NO: 1.

In the present invention, "promoter" means a DNA upstream region from a structural gene, and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.

In another aspect, the present invention provides an expression vector comprising the promoter.

In one embodiment of the invention, the expression vector may be a pPBrREF vector having a cleavage map disclosed in FIG. However, the expression vector of the present invention is not limited to the vector described in FIG. 2, and any known vector useful for transforming plants can be used.

Preferred examples of plant expression vectors may be appropriately selected by those skilled in the art, and a general binary vector or cointegration vector may be used. There are many types of binary vectors widely used in plant transformation. For example, they are obtained from international centers such as CAMBIA (Center for the Application of Molecular Biology to International Agriculture, GPO Box 3200, Canberra ACT 2601, Australia) It is possible. The skeleton of the basic binary vector can be transformed into a transformant selection marker, a promoter, a transcription termination site gene, and the like at the left and right boundaries where a gene is transferred using a Ti plasmid as a host.

As used herein, the term "vector" means a DNA product that retains a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in the appropriate host. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself.

As used herein, the term “expression vector” refers to a recombinant DNA molecule comprising a DNA sequence of interest and a suitable nucleic acid sequence necessary to express a DNA sequence operably linked in a particular host organism. The appropriate nucleic acid sequence necessary for expressing the DNA sequence may include, for example, a promoter, an enhancer, a termination signal, and a polyadenylation signal.

In addition, the expression vector may comprise one or more selection markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. There are resistance genes, but are not limited thereto, and may be appropriately selected by those skilled in the art.

In another embodiment of the present invention, the expression vector may include a promoter consisting of the nucleic acid sequence of SEQ ID NO: 1 and a homologous or heterologous gene operably linked thereto. "Operably linked" means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. Therefore, the expression vector of the present invention, due to the influence of the promoter consisting of the nucleic acid sequence of SEQ ID NO: 1, the expression of the homologous or heterologous gene in the transgenic plant into which the expression vector is introduced, the seed germination from seed germination, the early stage of plant growth. Until cotyledons and hypocotyls can be induced specifically in time and tissue.

By “homologous gene” in the present invention is meant that the gene is endogenous to the host plant species. That is, the homologous genes are inherent to the host plant species and the untransformed host plant species can express the homologous genes to produce gene products.

In the present invention, "heterologous gene" means that the gene is exogenous to the host plant species. In other words, the heterologous gene is not native to the host plant species and the untransformed host plant species does not express the heterologous gene to produce a gene product.

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

In one embodiment of the invention, the transformed cells are Agrobacterium microorganisms ( Agrobacterium) spp.), Escherichia coli ), Bacillus subtilis ( Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteusmirabilis) or Staphylococcus (prokaryotes, fungi such as Staphylococcus) (e. g., Aspergillus (Aspergillus)), yeast (For example, Pichia pastoris), saccharide as MY processes Sergio non jiae (Saccharomyces cerevisiae), investigating car break in Rome Seth (Schizosaccharomyces), Castello La Neuro Chrysler Corporation (Neurospora crassa ) may be a cell derived from higher eukaryotes, including but not limited to lower eukaryotic cells, insect cells, plant cells, animal cells, and the like. According to an embodiment of the present invention, the recombinant vector pPBrREF of the present invention was transformed into Agrobacterium tumefaciens, and then the expression vector of the present invention was introduced into plant cells by the transformed Agrobacterium. Thus, the transformed cells may preferably be Agrobacterium microorganisms or plant cells.

Preparation of the transformed cells can use any transforming method known in the art for introducing the vector into a host cell. For example, but not by way of limitation, Agrobacterium spp.) microbial transformation, particle gun bombardment, silicon carbide whiskers, sonication, electroporation and precipitation by PEG (Polyethylenglycol) Can be used. Preferably, Agrobacterium-mediated transformation can be used, and the methods exemplified in the literature can be used (Horsch et al., Science 227: 1229-1231, 1985).

The transformed plant cells in the transformed cells may be cultured according to a conventional method to be liquid cultures, callus, plasma cultures, and differentiated to be plant tissues or plants. That is, the transgenic plant cells into which the recombinant vector of the present invention is introduced can be re-differentiated into plants through processes such as callus induction, rooting, and soil purification using standard techniques known in the art, such as folding, grafting, and the like. It can be produced by voice breeding method using asexual breeding method and seed. The cultivation of plant cells is carried out by culturing a part of the plant isolated from the mother, cultivating it aseptically under appropriate conditions, and cultivating the plant cell by any method known to those skilled in the art such as liquid culture of tissue slice, callus culture of tissue slice, protoplast culture, And the culture conditions and methods can be carried out by conditions and methods known to those skilled in the art. Differentiating the cultured plant cells into plants is to induce differentiation of cultured plant cells in callus, protoplast form under appropriate conditions to differentiate into plant tissues or plants, and the differentiation conditions and methods are known to those skilled in the art, and It may be carried out by the method.

Accordingly, the present invention also provides a transgenic plant into which the expression vector is introduced.

Specifically, the method of introducing the expression vector into the transgenic plant may apply the same various transformation methods for introducing the vector described above into the host cell. Alternatively, the transgenic plant may be prepared by re-differentiating from the transgenic plant cell as described above.

In one embodiment of the invention, the plant is for example a food crop selected from the group consisting of rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum; Vegetable crops selected from the group consisting of Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops selected from the group consisting of ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And it may be a feed crop selected from the group consisting of lysis, red clover, orchardgrass, alpha alpha, tolsque cue and perennial lysegrass, but is not limited thereto. Preferably it may be Arabidopsis, cruciferous, and more broadly dicotyledonous plants.

In another aspect, the present invention comprises the steps of transforming plant cells with the expression vector according to the present invention; And regenerating the transformed plant from the transformed plant cell.

In one embodiment of the invention, the transformation of the plant cell refers to any method of transferring DNA to the plant cell, which transformation does not necessarily have a period of regeneration and tissue culture. Transformation of plant species is now common for plant species, including both dicotyledonous plants as well as monocotyledonous plants. “Plant cells” used for transformation of plant cells may be any plant cell, and may be, for example, cultured cells, cultured tissues, cultured organs or whole plants. Specific transformation may use any transformation method known in the art as described above.

In another embodiment of the present invention, the method of regenerating the transformed plant from the transformed plant cell is subjected to a process such as callus induction, rooting and soil purification using standard techniques known in the art as described above. It may be to re-differentiate into a plant, it may be produced by an oily breeding method using a seedless breeding method such as folding, grafting and the like, in addition to any method known in the art can be used.

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: from cabbage BrREF ( B rassica r apa R ubber E longation F actor ) Gene expression analysis

From the cabbage microarray results, BrREF genes were obtained from the gene group whose expression was greatly increased in the seedling development stage, and the expression was analyzed by RT-PCR in the cabbage growth stage.

RNA was isolated from each tissue of cabbage (seedling stage, root, stem apical meristem, bud, flower, stem, pod) and then brREF through semi-quantitative RT-PCR using the following gene specific primers. Gene expression patterns were analyzed. At this time, the RT-PCR conditions are 5 cycles at 95 ℃, 1 cycle, 95 seconds 30 seconds, 55 ℃ 30 seconds, 72 ℃ 45 cycles 35 cycles, 72 ℃ 10 minutes 1 cycle gene in the cabbage plant tissue gene Expression was confirmed.

Specific primer nucleic acid sequences for gene expression analysis:

BrREF - Forward : 5'-ATGGCTGAAGATGAGATTAT-3 '(SEQ ID NO: 2)

BrREF - Reverse : 5'-CTAGTGGGCTCGACAGTGAT-3 '(SEQ ID NO: 3)

As a result, as shown in Figure 1, BrREF Genes increased during seedling development, including seed, and were weakly expressed in roots and buds. BrREF It can be seen that the gene is expressed at a specific growth stage early in plant growth and exhibits a tissue-specific expression pattern.

Example  2: BrREF  Promoter region separation and Nucleic acid sequence  analysis

Using the nucleotide sequence of the Chinese cabbage branch BAC (KBrB042E01) clone containing the Chinese cabbage BrREF , we searched for the region of the -2.2 kb promoter including the TATA box above the BrREF gene initiation codon. The promoter region was separated from the BAC clone by performing a polymerase chain reaction (PCR). At this time, the PCR conditions were denatured at 95 ° C. for 5 minutes, followed by 35 cycles of 1 minute at 95 ° C., 1 minute at 55 ° C., and 2 minutes at 72 ° C., followed by reaction at 72 ° C. for 10 minutes. Since the base sequence of the DNA obtained in the PCR reaction was confirmed.

Specific primer nucleic acid sequences for promoter isolation:

BrREF - Pro - Forward : 5'-GGATCCTTGGATAAACAGAGTAGAGATAGC-3 '(SEQ ID NO: 4)

BrREF - Pro - Reverse : 5'-GAATTCGACTATAATCTCATCTTCAGCCAT-3 '(SEQ ID NO: 5)

As a result, the sequence of the promoter region of the cabbage BrREF gene of the BAC clone was 100% identical, which is represented by SEQ ID NO: 1.

Example  3: Preparation and transformation of expression vector for plant transformation

1) Plant transformation vector production

BrREF isolated in Example 2 The promoter was inserted into pGEM-T-easy, cleaved with Hind III and EcoR I, and then inserted into pCAMBIA1391 cleaved with Hind III and EcoR I. A plant transformation vector pPBrREF in which the reporter gene GUS was fused to the BrREF promoter was prepared and a map is shown in FIG. 2.

2) Arabidopsis transformant production

Agrobacterium was introduced to introduce the produced vector pPBrREF into the Arabidopsis. After repeating the freeze and thaw 2-3 times in tumefaciens GV3101, transformed by heat shock method at 37 ℃ colony was confirmed in YEP medium containing the antibiotic kanamycin 50 ㎎ / ℓ. The introduction of the carrier in the GV3101 strain was confirmed by colony PCR, and the colonies in which the gene introduction was confirmed were used for Arabidopsis transformation. Wild type Arabidopsis Col-o ( Arabidopsis) using transformed GV3101 strain To transform the thaliana ecotype Col-o), the Arabidopsis cultivar was first seeded and grown for about 4 weeks at 22 ° C (16 hours day, 8 hours night). After removal of the primary stem of the 4 week Arabidopsis plant, 3-4 secondary shafts of at least 10 cm were induced. Agrobacterium confirmed the introduction of the vector was inoculated in LB medium containing kanamycin 50 mg / L, rifampicin 10 mg / L, gentamycin 40 mg / L and incubated at 28 ℃ for 3-4 days. A suspension was prepared by diluting the fully grown Agrobacterium by centrifugation and OD 0.8 in a solution containing 5% sucrose and 0.05% silwet77. The suspension was sprayed onto the Arabidopsis bud and then incubated for 24 hours under sufficient humidity and dark conditions, followed by further incubation for 3-4 days at 22 ° C., 16 hours day and 8 hours night. Transformation was repeated in the same manner as. Thereafter, the seeds were harvested after growing for about 4-6 weeks until the silique matured completely to brown at 22 ° C., 16 hours day and 8 hours night. Harvested seeds were seeded in MS medium containing 30 μg / ml hygromycin and selected for transformants, and then analyzed for expression of BrREF gene promoter through GUS staining from the selected transformants.

Example  4: Transgenic Arabidopsis Growth Stages BrREF  Promoter Activity Assay

Growth stage of development of the promoter of the cabbage BrREF gene transformed with a recombinant expression vector, the promoter of the cabbage BrREF gene is inserted in order to confirm that specific lead to gene expression in the plant Arabidopsis thaliana with the GUS gene expression in each tissue Analyzed. That is, some of the seeds harvested by selecting three lines independent from the transformed Arabidopsis in Example 3 germinated in MS medium to observe the appearance of GUS (blue color gene) expression in the development stage of seedlings, and some GUS gene expression was observed in various tissues of leaves, stems, and roots in plants at 15 and 35 days of growth. The germinated seedlings were immersed in GUS-assay buffer (X-gluc (cyslohexyl ammonium salt) 20 mM, NaH ₂ PO 4 H ₂ O 100 mM, NaEDTA 10 mM, Triton-X-100 0.1%, pH7.5) and at 37 ℃ After 24 hours of treatment, chlorophyll was completely removed with 70% ethanol, and GUS gene expression was observed using stereomicroscope (Olympus, SZX12).

As a result, Strong GUS expression began to appear from day 1 after seed germination and GUS expression was observed in hypocotyls and cotyledons up to 10 days (FIG. 3), and GUS expression was decreased in 15-day hypocotyls and cotyledons, but not on day 35 (FIG. 4). At the root, they were not expressed at all stages of development.

This suggests that the BrREF promoter induces cotyledon and hypocotyl specific expression during seedling development.

Example  5: In developmental stages such as transgenic Arabidopsis flowering and seed maturation BrREF  Promoter Activity Assay

Flower bud, flower, and pod tissues in transgenic Arabidopsis cultured at day 35 to examine whether the promoter of the cabbage BrREF gene specifically induces gene expression during developmental stages such as plant flowering and seed maturation GUS expression in each tissue, including (silique), was analyzed.

As a result, GUS expression was confirmed in the style, pistil septum, and pollen of the BrREF promoter-transformed Arabidopsis bud (Flowr bud) (FIG. 5). However, GUS expression was not observed in mature flowers.

<110> REPUBLIC OF KOREA <120> BrREF promoter specific for cotyledon and hypocotyl <130> P110743 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 2200 <212> DNA <213> Brassica rapa <400> 1 ttggataaac agagtagaga tagcgcccac catggcacct ataaggagag aaatgtcata 60 tagtagtagt actcctcctc atgttgttat atcattaaga aaggctttga tagttgaata 120 ccataccaca tcaggagtct tgagatagat gaacaagacg atggacagat tggcaatgta 180 gagaacacca aaaagaatct gcacacaaca tcaaacgaac gatttgttaa aatgcagagg 240 ccgagaaaga gagagtcaat ttgaaggtac ctgagcaggg tagactgctc ccccagtcaa 300 attctggaca gcagagtaaa catagaggaa accagccgga taaaccaaag gccctgtgtc 360 gcctttcaag ctcccgtagt ctctctctcc cccgaggaac ccactaacct gcccccattt 420 tgtaaaattt caccaagaga gttgactttg tatattctac tggatgtaat gcaaacctgt 480 gacatgtacg catcccaatc gatcttcgtg tctgttcagt aaatttaaaa aagagagtga 540 gcttaacacc cccatgtcca attgcacatt tcgaaattaa aaaaaaacgt aggaaattag 600 atctgacatg gaacataggc gatgataagt gcgacgagga ttgcatcagc cacgattaag 660 cccaaggcga atgcaatttt aggcacatta cggctacgag aagctcttga tgagctcgcc 720 tccattttcg ccggtgattg aacgaacaat cgccccagtc gtcgtctgtc gtaaagaatc 780 ctgcgtacgc cgcgaaacag acaactaact cttagtttta gcccatagta aagcccaatt 840 cactcctaac tattgggctt atcatcttta cttagtgaca cgtgcgtgcc tacgctttat 900 tacagagtcg agtcattatc caaaacaaaa caattttatt tccacgtgta ccacgtctgt 960 gtcaactacc cgccgcgtgt ctctccaaca ctcttcttct tttataaact tgagctcctc 1020 ctcattattc tctttgacca agcttcttct ctcatttctc cgtctcaaat atcttaaact 1080 cgttatggat tctcaggaca tcaggtaccg tgccggagac gccgctatgg ctgagacgga 1140 gaggaaacaa gccgacgaca acaacaacaa caaaggcaaa cgcgatcaaa agagggcgat 1200 ggctaaacgc ggtctcaaat ccctgacgtt agctgttgca gctcctgtgc tcctgactct 1260 cttcacatcc tacttcctcg ggaatcaggc tcggtcctcg tcgtgggtcc ttcacctcat 1320 gcgtctcgcc tcgagcggtc tgatgggctt ggctgcgtgg ctcgtatggg tcgacgctgg 1380 gttccacaag aagcccaacg ctctgtatct ttacttggct cagtttgtgc tttgtttgac 1440 tacgtgcatg gtcgggtcgg gactagcagg gcttgcagtg tgcttgtgtc agtctgcggc 1500 cttgttccga tgctacaagg cctttaatga gatcagtccg gtcgctggta atatggtaaa 1560 gccgtgtttg gcttttgctg cgtttgtagc agccgttaat gtaaagctcg caatcgcgtg 1620 agatctataa acagtataaa gccaacgata gatgtgatag tctctgagaa tgttgtggag 1680 tcgtaattaa tcgttttttt ttacaagtgt tttgtgacaa tatacgtaat taattatctg 1740 ttagtatgtg tccattgggc tttaatatat atcaagattg acaaaataaa agtggctaca 1800 tttgagttta gggtcctgat tattgcagac ttattggccg ttcactcgga actttttacc 1860 cccaactgat atatttaggc ttttcaaggt gaaaataacg gtataataaa ttaggcccaa 1920 taagttcgaa gacgtgatgg aaaagttagg gctattcttt ttgcattttc cctcgagact 1980 cgtgccagac ggaatgggag tgcttatctt gcaacaatct cgacccatgc cctgacacgt 2040 gctgtgagcg gtccattagc atcagtacac gtgtcccagc ggtcactctt cgcttacaat 2100 ccttgtcgtc tgcagatctt gtattcagtc cttctattta aagagtaaat tcaatcggaa 2160 cgagtaggat ttgagaatgg ctgaagatga gattatagtc 2200 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BrREF-Forward <400> 2 atggctgaag atgagattat 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BrREF-Reverse <400> 3 ctagtgggct cgacagtgat 20 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> BrREF-Pro-Forward <400> 4 ggatccttgg ataaacagag tagagatagc 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> BrREF-Pro-Reverse <400> 5 gaattcgact ataatctcat cttcagccat 30

Claims (8)

A promoter consisting of the nucleic acid sequence of SEQ ID NO: 1. An expression vector comprising the promoter of claim 1. The method of claim 2,
The vector is an expression vector which is a pPBrREF vector having a cleavage map disclosed in FIG. A transformed cell transformed with the expression vector of claim 2. 5. The method of claim 4,
The cell is a transformed cell Agrobacterium microorganism or plant cell. The transgenic plant into which the expression vector of Claim 2 or 3 was introduce | transduced. The method according to claim 6,
The plant is a dicotyledonous transgenic plant. Transforming the plant cell with the expression vector of claim 2; And
Regenerating a transformed plant from the transformed plant cells.

Images