KR100987009B1 - A root-specific promoter r - Google Patents

Abstract

The present invention relates to a root specific expression promoter for limiting and expressing the expression site of a foreign useful gene in a plant, and more particularly, consisting of a root specific expression promoter consisting of SEQ ID NO: 1. A primer for amplifying the root specific promoter consisting of an oligonucleotide and an oligonucleotide consisting of SEQ ID NO: 3, an expression vector pBGWFS7-PAtRA166 and the expression vector pBGWFS7- comprising the promoter sequence of SEQ ID NO: 1 It relates to a plant cell transformed with PAtRA166.

Root-specific, promoter, Arabidopsis, tissue-specific, transformant

Description

Root specific expression promoter R {A ROOT-SPECIFIC PROMOTER R}

Crop molecular breeding is the key to lead the next generation of agriculture in that it is possible to use all kinds of genes as a material and to fine-tune the effects of infinite breeding by pulling down the breeding technology that was possible only in the existing genome. Technology.

In order to maximize the effect of the technology, such crop molecular breeding must be preceded by the accumulation of a large amount of genetic data that can represent genes of various plants, a transformation system of various crops must be established, and the expression of externally inserted genes. The development of various promoters that can control this should be preceded.

The present invention relates to a root specific expression promoter for limiting and expressing the expression site of a foreign useful gene in a plant, and to provide an AtRA166 promoter useful when expressing a foreign gene only in root tissues when developing a plant transformant. .

Known promoters used to achieve transformation purposes by confining the expression of foreign genes to specific tissues of plants can be classified as follows according to their function.

First is a root specific expression promoter. There is no commercialized case yet, but the sweet potato-derived Maz gene (ibMADS) and the sugar-induced APD-glucose pyrophosphatase (ADGase) gene are isolated and the promoter induces specific expression in the roots. It was confirmed that it induces specific transient expression (patent registration number 10-0604186, 10-0604191), and recently, the promoter of Arabidopsis-derived extensin gene expresses root-specifically Confirmed and received a patent registration. (Korean Registered Number 10-0790809). 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.

Second, seed specific promoters can be mentioned. As a representative example, the rice gluten promoter used to develop golden rice as a promoter of the major storage protein gene of rice has been widely used to induce seed-specific expression of monocotyledonous plants. Promoters mainly used to induce seed specific expression include soybean-derived lectin promoters, cabbage-derived napin promoters, and gamma-tocopherol methyl transferase (γ-TMT) in seedlings of Arabidopsis. The carrot-derived DC-3 promoter used in studies that enhanced the production of vitamin E by inducing gene expression, and the patent was confirmed by the team confirmed the induction of seed specific expression of perilla-derived oleosin promoter There is an example. (Korean Register No. 10-0685520) The seed specific promoters are mainly seed. Is mainly used for the purpose of useful proteins accumulate and produce useful materials in major crop used as a food or a body material of a food or food.

Third, systemic expression induction promoters can be cited. The promoter of 35S RNA gene of CaMV (caaflower mosaic virus) is used as a representative dicotyledonous promoter as a plant whole-body induction promoter, and rice actin (actin) as a whole-body induction promoter for a monocotyledonous plant such as rice. ) And corn ubiquithin gene promoters have been mainly used, and recently, a promoter of rice cytochrome C gene (OsOc1) has been developed and used by domestic researchers (Korean Register No. 10-0429335). These are already inherent to induce the expression of antibiotic or herbicide resistance genes and reporter genes used as selection markers in the plant transformation basic carriers. Promoters are considered.

Fourth, other tissue specific promoters, such as a leaf, can be mentioned. Rice and corn-derived albics (rbcS) promoters that induce potent gene expression only in photosynthetic tissues such as leaves, RolD promoters that induce plant root expression from Agrobacterium, and tubers from potato Specific expression-induced patatin promoter, tomato-derived fruit maturation specific expression-induced phytoene synthase (PDS) promoter, and cabbage-derived oleosin promoter whose function has been identified as pollen-specific of cabbage flower, The research team of the Institute developed and registered a patent for male sterile crops by inducing the expression of the BtA9 promoter (flower registration No. 10-0435143) of the male endophytic drug of flowers to induce the expression of Bt protein genes toxic to cells. There is an example.

In addition, a large number of promoters have been reported for various tissues of various plant-derived tissues for various purposes, and are still under development, but most of the types of promoters that have been commercialized or widely used among plant researchers are mentioned above. New promoters need to continue to be developed that can more precisely control the location of gene expression according to the developer's intention.

The present invention has been carried out by the need for the development of a new promoter that can more precisely control the place of gene expression according to the intention of the developer, the present invention induces the expression of useful genes and markers or reporter genes or improve agricultural characteristics It is to provide a novel root expression promoter, primers for amplifying the promoter, and an expression vector comprising the promoter. In addition, the present invention is to provide a plant cell transformed with the expression vector.

The present invention relates to a root specific expression promoter for restricting and expressing the expression site of a foreign useful gene in the root portion in a plant, and more specifically,

The novel root specific promoter (PAtRA166) of the present invention is characterized by consisting of the nucleotide sequence of SEQ ID NO: 1.

In addition, the present invention is characterized by a primer for amplifying the root-specific promoter comprising an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 3.

In addition, the present invention, the expression vector pBGWFS7-PAtRA166 And the plant cell transformed with the expression vector pBGWFS7-PAtRA166 comprises the promoter sequence of SEQ ID NO: 1.

The expression vector comprising the root-specific expression promoter according to the present invention and the plant cells transformed with the expression vector induced limited expression of foreign genes introduced by genetic engineering methods into the root tissues. It has the effect of limiting the expression to roots without affecting the growth of plants, so it is necessary to produce a large amount of useful proteins in transformed storage root tissues or to control metabolism and functional substances of storage root tissues using transformants. It can be effectively used for production.

The novel root specific promoter (PAtRA166) of the present invention is characterized by consisting of the nucleotide sequence of SEQ ID NO: 1.

The root-specific promoter includes a nucleotide sequence showing root-specific promoter activity as a modified sequence in which some bases of SEQ ID NO: 1 as well as some bases of SEQ ID NO: 1 are substituted, deleted or added.

The novel root specific promoters of the invention are promoters involved in the transcription of unidentified genes isolated from Arabidopsis. Although the gene has not been tested for biological function, it appears to be located in the cell membrane and further studies are needed for root-specific expression, function, and encoding of the gene.

The RA166 promoter region of the Arabidopsis corresponds to 1,926 bp upstream of the gene coding region.

In addition, the present invention is composed of an oligonucleotide consisting of a base sequence of SEQ ID NO: 2 and an oligonucleotide consisting of a base sequence of SEQ ID NO: 2 and 32 bp forward and reverse primers for amplifying the root-specific expression promoter It provides a pair of primers, characterized in that made. The primer may be prepared by a method for synthesizing oligonucleotides known to those skilled in the art, and may be used by commercially synthesizing.

The primer of the present invention may include a modification sequence in which a mutation occurs in a part of the individual nucleotide sequences constituting the primer. The modified sequence is a result of performing a polymerase chain reaction (Polymerase Chain Reaction, PCR) using a primer having a mutation and PCR using a primer (SEQ ID NO: 2 and SEQ ID NO: 3) consisting of an unmodified sequence One result refers to a sequence that has been deleted, substituted or added within substantially the same range.

The present invention also provides an expression vector PAtRA166 (hereinafter referred to as pBGWFS7-PAtRA166) comprising a promoter sequence consisting of SEQ ID NO: 1.

The expression vector pBGWFS7-PAtRA166 was deposited on April 2, 2008 with the gene accession number KACC 95080P to the Institute of Agricultural Biotechnology.

The expression vector pBGWFS7-PAtRA166 may include a promoter consisting of the nucleotide sequence of SEQ ID NO: 1 of the present invention, Bar, a herbicide resistance gene, and Egfp: gus, a reporter gene.

The present invention also provides a plant cell, characterized in that transformed with the expression vector pBGWFS7-PAtRA166 comprising a promoter sequence of SEQ ID NO: 1.

Plant cells that can be transformed using the promoter include Arabidopsis and similar dicotyledonous plants, but not limited thereto, and any plant cells can be used as long as they can be used as roots.

When using the root-specific promoter of the present invention can be expected to reduce the chemical control through the root expression of the pathogenic gene for the control of root disease, while the root of a specific useful protein in root crops, root crops, radish, sweet potato, ginseng, carrot, etc. Expression can be induced.

The invention can be understood in more detail by the following examples. However, the following examples are intended to illustrate the invention and are not intended to limit the protection scope of the invention. On the other hand, for commonly used DNA test methods not specifically mentioned in the following examples, see Sambrook et al., “Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, second edition, 1989”.

[ Example ]

1. Baby Pole Promoter Acquisition of PAtRA166

To obtain the Arabidopsis promoter PAtRA166, bacteria were isolated from bacteriophages from genomic DNA based on the Arabidopsis genome sequence (http://www.tigr.org/tdb/e2k1/ath1/, At1g56320). Forward primers (RA166 B1: 5'-AAAAAGCAGGCT AATGCTGGCAAAACTACATT-3 ', SEQ ID NO: 2) to specifically amplify the PAtRA166 promoter, including some of the recombinant nucleic acid sequence sites that the bacterial side specifically possesses when infected. A reverse primer (RA166 B2: 5'-AGAAAGCTGGGT GGGAGAGAAATAAGCAGAGA-3 ', SEQ ID NO: 3) was prepared, genomic DNA was extracted from the leaves of Arabidopsis Ecotype Columbia-0, and then polymerase chain reaction (PCR) was performed. About 1.9 kb of the RA166 promoter (SEQ ID NO: 1) was isolated. At this time, after 10 minutes of denaturation at 95 ° C, 1 minute of reaction at 94 ° C, annealing reaction at 55 ° C for 1 minute, and 30 minutes of polymerization reaction at 68 ° C for 2 minutes, followed by polymerization reaction at 68 ° C for 10 minutes. PCR was performed under the same PCR conditions.

2. Transformation plasmid preparation

In order to transform the RA166 promoter isolated above into the Arabidopsis, a carrier for RA166 promoter function analysis was constructed using a gateway carrier pBGWFS7 purchased from the University of Ghent in Belgium. The amplified PCR products contained adapter primers B1 (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3 ', SEQ ID NO: 4) and B2 (5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3') containing the entire bacterial-specific recombinant nucleic acid sequence site when the bacteria were infected with bacteriophage. , SEQ ID NO: 5) was respectively re-PCR. The secondary PCR products were produced by the pDONR-PAtRA166 intermediate cloning vector by BP recombination with the pDONR221 vector containing the bacteriophage-specific recombinant nucleic acid sequence site when the bacteria were infected with the bacteriophage. The subcloned carrier was then subjected to LR recombination with the pBGWFS7 vector incorporating a reporter system in which the Egfp and Gus genes were linked to produce the final plant transformation carrier, resulting in the final pBGWFS7-PAtRA166 carrier. At this time, pBGWFS7 used as a binary vector has a spectinomycin resistance gene for microbial selection, and includes a herbicide resistant Bar gene as a plant selection factor. 1 is a schematic diagram of a genetic map of the plasmid pBGWFS7-PAtRA166.

The transformant carrier was transformed into Agrobacterium tumefaciens strain GV3101 according to the direct Agrobacterium transformation method (Holster et al., Freeze-thaw method, 1978) using liquid nitrogen, followed by an alkaline method such as An (alkaline). Agrobacterium quick selection based on lysis was used to confirm the introduction into Agrobacteria.

3. Plant material and Arabidopsis transformation

After seeding, the primary rods of Arabidopsis thaliana ecotype Columbia-0 plants grown for about 4 weeks in a 22 ° C incubator (16 hours day / 8 hours night) were removed to induce at least 3 to 4 secondary rods. Agrobacteria cultures containing plant transformation carriers are diluted to contain 5% sucrose and 250 ppm tween 20 and then the suspension is sprayed onto the bud to infect Agrobacteria. Incubate overnight with sufficient humidity and dark conditions, then incubate for 4-5 days in a 22 ℃ incubator (16 hours day / 8 hours night), repeat in the same manner as above, and then incubate at 22 ℃ (16 hours day / 8 hours night) the pod tissue (silique) was grown for about 3 to 5 weeks until full maturation of brown and then harvested. The seeded seeds were sown again and the transformants of T1 generation were selected by spraying 0.3% batha on the 10th day and the 17th day. T1 transgenic plants were grown for about 2 months in a 22 ° C. incubator (16 hours day / 8 hours night) until the pod tissue (silique) had fully matured to brown and then T2 seeded.

4. GUS Blue color  Reaction analysis

PAtRA166 transformed T2 seeds were partially germinated in water, and some were sown on the 7th day, and then the leaves, stems, and roots of the transgenic Arabidopsis plants survived by spraying 0.3% batha on the first and 17th days. Expression of blue color gene (GUS) was observed at various stages of growth from tissues such as, flowers and pods. As a control, nontransgenic Arabidopsis Columbia-0 and P35S promoter transformed Arabidopsis germinated and used. In order to observe the expression of blue color gene, seedlings and various tissues were treated with GUS-assay buffer [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], overnight treatment at 37 ° C, discarded the buffer solution and wiped with water, then immersed in 70% by weight ethanol and repeatedly changed until chlorophyll (chlorophyll) is removed once every hour at 37 ℃. Plants were observed under a stereoscopic microscope, Figure 2 is to observe the expression of the blue color gene. From the seedling stage on the 7th day of germination, GUS blue coloration was observed in the root of PAtRA166 transgenic Arabidopsis, whereas GUS blue coloration was not observed in non-transgenic Arabidopsis. Was observed in. In the PAtRA166 transgenic Arabidopsis, blue color was not observed in various tissues of plants, such as leaves, stems, flowers, and pods, but only in the roots, so PAtRA166 appears to be specifically expressed in the roots.

5. GUS Blue color  Quantitative Analysis

In order to investigate the expression of GUS blue color expression of PAtRA166, the total protein was isolated from the leaves, roots, stems and flowers of the transgenic Arabidopsis T2 generation 2 control and control group transformed with PAtRA166. To isolate the whole protein, transfer 50 mg of the tissue-specific sample ground in a mortar with liquid nitrogen to a 1.5 ml tube, and then 100 µl of protein extraction buffer (50 mM sodium phosphate, pH 7.0, 10 mM dithiothreitol (DTT), 1 mM disodium EDTA, 0.1% sodium dodecyl sulfate, 0.1% triton X-100) were added and mixed vigorously. The mixed solution was centrifuged at 4 ° C. and 15,000 rpm for 5 minutes, and the supernatant was transferred to a new tube. 50 μl of this was collected and 50 μl of assay solution (1.2 mM MUGluc (4-methylumbelliferyl β-glucuronide), 10.0 was adjusted to 37 ° C.). 1x Reaction buffer (1x Reaction buffer), FluorAce β-glucuronidase reporter assay kit (Bio-Rad) of mM β-mercaptoethanol (β-mercaptoethanol) were mixed and reacted for 30 minutes in a 37 ° C. water bath. In addition, 100 μl of the 1 × Stop buffer was mixed well with each sample, and GUS activity was quantified using a fluorophotometer (excitation filter 355 nm, emission filter 460 nm). Figure 3 extracts the whole protein from the root and other tissues of the Arabidopsis pole expressing the Arabidopsis promoter PAtRA166 and quantify the expression of the blue chromogenic protein compared with the expression of the blue chromogenic protein in the non-transforming Arabidopsis, a positive control .

6. RT - PCR  By reaction PAtRA166 Root Expression of

To investigate the expression patterns of PAtRA166, total RNA was isolated from leaves, roots, stems, and flowers of P35S-transformed Arabidopsis and non-transgenic Arabidopsis (T2) genotypes and controls. In detail, the whole RNA separation process is described. Approximately 1 g of each sample is crushed into a mortar with liquid nitrogen, transferred to a 2 ml tube, and 500 μl of RNA extraction buffer (50 mM sodium acetate pH 5.5) is added to each tube. Add 150mM LiCl, 5mM EDTA, 0.5% SDS (sodium dodecyl sulfate) and 500µl phenol and mix. The mixed solution was treated at 65 ° C. for 10 minutes and then mixed using a stirrer (rotary shaker) at room temperature for 15 minutes. After centrifugation at 4 ° C. (10000 rpm, 10 minutes), the supernatant layer is carefully transferred to a new tube, 500 μl of chloroform is added and mixed well, followed by centrifugation again to obtain the supernatant. 0.6M volume of 8M lithium chromide (LiCl) was added thereto, and then stored at -20 ° C for at least 2 hours. After centrifugation at 4 ° C. and 12,000 rpm for 20 minutes, the RNA precipitate was washed once with 4M LiCl and 80% ethanol, respectively, and the final RNA precipitate was dissolved in 50 μl of distilled water. RNA eluate was quantified by measuring A ₂₆₀ / A ₂₈₀ using a UV spectrophotometer. Reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out using 50 μM oligo dT primers for 50 minutes at 50 ° C and 5 minutes at 85 ° C. After cooling, 1 μl of RNase H was added thereto and treated for 20 minutes at 37 ° C. to synthesize cDNA (10X RT buffer, 25 mM MgCl ₂ , 0.1M DTT, RNase OUT, SuperScript ^TM RT), and synthesized cDNA template. Blue color gene GUS specific primer set, ie, GUS-F primer of SEQ ID NO: 6 (5'-ACCTGCGTCAATGTAATGTTCTGC-3 ') and GUS-R primer set of SEQ ID NO: 7 (5'-CTCCCTGCTGCGGTTTTTCA-3') EF1α-F primers and SEQ ID NO: 8 (5'-GTTTCACATTAACATTGTGGTCATT-3 '), a set of Arabidopsis elongation factor-1α (EF-1α) primers to demonstrate uniformity of the relative amount of RNA 94 using EF1α-R primer of 9 (5'-GAGGTACCAGTAATCATGTTCTTG-3 ') After denaturing at 5 ° C. for 5 minutes, reacting for 30 seconds at 94 ° C., annealing at 55 ° C. for 30 seconds, and performing a series of 25 reactions at 72 ° C. for 30 seconds, followed by expansion reaction at 72 ° C. for 7 minutes. PCR was performed. As a result of the reverse transcription PCR reaction, the transcript of the blue-colored protein gene GUS was clearly detected in the root of the PAtRA166 transgenic Arabidopsis, but not in other tissues such as leaves, stems, flowers and pods. Systemic expression of P35S detected GUS in all tissues, including roots. Arabidopsis elongation element EF1α demonstrated a relatively uniform concentration of RNA used.

1 is a schematic diagram of the plasmid pBGWFS7-PAtRA166 gene map.

≪ Description of reference numerals &

LB-Left border

Tnos-nos Terminator

Bar-herbicide resistance gene

Pnos-nos promoter

attB1-Adapter sequence B1 used for gateway cloning

PAtRA166-Arabidopsis Root-Specific Promoter of the Present Invention

attB2-Adapter sequence B2 used for gateway cloning

P35S-Cauliflower Virus 35S Promoter

EGfp-green fluorescent protein gene

Gus-β-glucuronidase gene

T35S-Cauliflower Virus 35S Terminator

RB-Right border

SpR-Spectinomycin Resistance Gene

      Figure 2 is a photograph observing the expression of the blue color gene in seedlings and adults of Arabidopsis transformed with pBGWFS7-PAtRA166.

≪ Description of reference numerals &

Seedlings (water): 7 days old seedling

Seedlings (soil): Seedlings germinated in soil (5 weeks)

Mature plant: Shows entire tissues such as leaves, flowers, stems, pods, roots, etc. about 3 weeks after seedling observation.

pBGWFS7-PAtRA166: Root-specific expression of Arabidopsis transformed with pBGWFS7-PAtRA166, both seedlings and adults

Col-0: non-transgenic Arabidopsis, non-expression control

pBGWFS7-P35S: Arabidopsis transformed with pBGWFS7-P35S, a systemic expression control

Figure 3 shows the expression of blue chromogenic protein in non-transgenic Arabidopsis as a positive control by extracting the whole protein from the root and other tissues of the Arabidopsis pole expressing the Arabidopsis promoter PAtRA166 of the present invention. It is a graph comparing.

≪ Description of reference numerals &

Col-0: Non Transgenic Arabidopsis

RA166: Arabidopsis transformed with PAtRA166

Figure 4 extracts the entire RNA from the root and other tissues of the Arabidopsis promoter expressing the Arabidopsis promoter PAtRA166 of the present invention, and after the reverse transcription reaction, the elongation factor 1α gene of the Arabidopsis endogenous gene as a blue control protein gene GUS and a positive control, respectively An electrophoretic photograph showing gene bands amplified by primers that amplify the targets.

≪ Description of reference numerals &

GUS: β-glucuronidase gene

EF1α: elongation factor 1α

RA166: Arabidopsis transformed with PAtRA166

Col-0: Nontransforming Babylon Pole

P35S: Arabidopsis transformed with P35S

M: 100bp ladder DNA marker

L: leaf

F: Flower

St: Stem

R: Root

Si: pod

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> A ROOT-SPECIFIC PROMOTER R <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 1926 <212> DNA <213> Arabidopsis thaliana <400> 1 aatgctggca aaactacatt acttcacatg ctcaaagacg aggtttgatt agtttcttcg 60 aattctcatt tttttggtag atctatcgat tcatgccttc tatgcattgc ttttgatgag 120 aaattatttg taaaaattga gatctgacat tttttgtctg tgtttgtgtg aatttggtgt 180 tggtggtgtt tgattagaga ttagttcagc atcagccgac gcaacatcca acgtctgaag 240 aacttagcat tggaaaaatc aagttcaagg cttttgattt gggtggtcat cagattgctc 300 gtagggtttg gaaagattac tatgccaagg ttaaatctct ctttctacat tctcctttta 360 gtaatccttt tggaagatga atatcgaaaa agcgcggtgt tttatcggat tcatcgagct 420 atatatttgt tccttgtttt aactccaagt attcttaaag tttagacttt taatgatttg 480 ttgttggcat tgctgataga agcttagatg ggagtttctt tattagatca taatcctatt 540 agaagatgca aatcgaaaac caatgcttga atctttgtat aaatgttcac ttacccttat 600 aggattcatc gaactctttt cttttacttg aaggtgtttg tttagtagct aaaactaaaa 660 ccataacatt tgttgtttgt ttctgttgca aatattctta aattttagac ctttaatgat 720 tgtagatggc acttgtcatg ctaatagaag ttttgatgga agtttctttt tttccatcac 780 aatctaatta cgcatgaatt tgcttcaaga tgatgtttca atggtacaaa ttaccaattt 840 tatgaagaaa gttttagtaa tttttacctt agttctagcc agttagtgga gttatgtttt 900 ggacctatga attatgttgg ggacgaactt cttggtaatt ttatttctct ggtgacatta 960 gaatgtatct gatatgattg taggcattga gccaatgtat atcgtaccag agttgtttat 1020 cgtattgacc tagagtttat ggtgttatca aactggaata ttttcatatc agtgatataa 1080 aatatggcta tgagtagtgt agagcgaact agaagtatgt ttgcgtggat ctaattgttt 1140 tgttagcatg ttaggatcta gtattgtctt taatcaaatg tattctcttt gctgatagaa 1200 actctttttt tttttgttgt ataaaggttg atgctgttgt ctaccttgtg gatgcttatg 1260 acaaagaaag atttgcagag tcaaaaagag agcttgatgc tcttctctcg gatgaagccc 1320 ttgcaactgt cccgtttctg attcttggga acaagattga tatcccttat gcagcttctg 1380 aagacgaact gaggtatcac ttgggtctca ctaacttcac caccggtaag ggcaaagtga 1440 cgctcggaga ctctggtgtt cgcccacttg aggtcttcat gtgcagcatt gtccgtaaaa 1500 tgggttatgg cgagggattc aaatggctct ctcagtacat caactaaaga cagacgtcaa 1560 gtcttttatc atattttcaa accaaaccca agaagatttt ccgtacaatt tgtctatttc 1620 gagctaaaag atttgtgatt gacgatgtca catccttccc ttagaagact tgagaacact 1680 tttttctcat atttgttctt tttgttgttt tctgtgtgga acataagttt ggtggcttgc 1740 atttccaaac ttattcattt ctcttgtcca catttttgtc aactgcttat gtttcattga 1800 acattattcc caagttagac caccaacttt tttgaacata ttctcacttt atattaacgt 1860 gtgccctaca taaacccaaa ttcaaatcga ttcaacaatc aatctctctc tgcttatttc 1920 tctccc 1926 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RA166 B1 <400> 2 aaaaagcagg ctaatgctgg caaaactaca tt 32 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RA166 B2 <400> 3 agaaagctgg gtgggagaga aataagcaga ga 32 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> adapter primer B1 <400> 4 ggggacaagt ttgtacaaaa aagcaggct 29 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> adapter primer B2 <400> 5 ggggaccact ttgtacaaga aagctgggt 29 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GUS-F primer <400> 6 acctgcgtca atgtaatgtt ctgc 24 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GUS-R primer <400> 7 ctccctgctg cggtttttca 20 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> EF1a-F primer <400> 8 gtttcacatt aacattgtgg tcatt 25 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EF1a-R primer <400> 9 gaggtaccag taatcatgtt cttg 24  

Claims (4)

Root specific promoter, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. A primer for amplifying a root-specific promoter according to claim 1, comprising an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 3. Expression vector pBGWFS7-PAtRA166 (KACC 95080P) comprising a promoter sequence of SEQ ID NO: 1. Plant cell, characterized in that transformed with the expression vector pBGWFS7-PAtRA166 (KACC 95080P) of claim 3.

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