KR20080103716A - Anther specific promoter derived from acapulo lily, recombinant vector, transgenic plant and preparation method - Google Patents

Abstract

A specific promoter derived from Lilium hybrid cv. Acapulco, a recombinant vector and a transgenic plant is provided to improve quality of transformant by expressing many proteins of a target gene, to increase an efficiency, to obtain an exact expression and to be used for cross breeding after introducing a luxury gene. A plant specific promoter includes a nucleotide having a homoeology over 70 % with a base sequence of a sequence number 1 or a complement thereof. A base sequence of a sequence number 2 is included in the promoter. A vector includes the plant specific promoter. The plant is asexuality-propagated by a method of culturing plant tissue transformed to the vector, cuttage or meristele.

Description

TECHNICAL SPECIFIC PROMOTER DERIVED FROM ACAPULO LILY, RECOMBINANT VECTOR, TRANSGENIC PLANT AND PREPARATION METHOD

1 is a map of a recombinant vector for transforming petunias.

2 is a result of sudden analysis after the transformation of petunia. In FIG. 2, C (E) is control DNA digested with EcoRI and # 16, 20, 21, 53 and 62 are digested with EcoRI DNA of petunia transformants.

Figure 3 shows the results of analyzing GUS expression after the transformation of petunia.

Figure 4 shows the results of microscopic confirmation of GUS expression after the transformation of petunia.

Field of technology

The present invention relates to a plant drug specific promoter, and more particularly, a plant capable of inducing the expression of a gene in a monocotyledonous and dicotyledonous plant and a large amount of the target gene and / or protein using the promoter in the plant medicine. It relates to a method that can be expressed.

Prior art

Promoters used in the transformation of plants are usually promoters that are mainly expressed in the entire tissues or cells of the plant. These promoters lack tissue-selectivity or organ-selectivity, which causes a problem that the expressed gene is expressed in whole plants. In addition, due to the nature of the promoter, the expression of the introduced gene is limited to the target organ and cannot be sufficiently expressed, thereby reducing the economical efficiency of the transformant. However, currently, studies on promoters isolated from plants having organ-specific characteristics are insufficient.

Currently, the most commonly used promoters in transformation include 35S CaMV, and Ubi (Ubiquitin: maize, 1993), Act1 (actin; rice, 1994), OsCc1 (cytichrome C, 2002). However, these promoters work to ensure that genes are expressed in all organs of the plant.

Genes that must be expressed only at specific organs (e.g., medicines, petals, roots, drugs, etc.) or at specific times (e.g., pathogen attack) When converting, it is necessary to have a promoter that operates only at a specific institution or time. Such promoters have been reported in rice (medicine, seeds), Arabidopsis (seeds, petals, roots, medicine, etc.), tobacco (medicine), potatoes (wounds), and the like. However, these promoters are also difficult to apply between different crops.

In order to solve the problems of the prior art, an object of the present invention is to provide a promoter for inducing the expression of genes specifically in plant medicine.

Another object of the present invention is to provide a method of improving the quality of a transformant or storing or producing a target protein in a drug by expressing a large amount of a protein of a gene of interest using a promoter of a gene specifically expressed in a plant drug. It is.

Another object of the present invention is to provide a vector having plant drug specific expression.

Another object of the present invention is to provide a method for specifically expressing a gene of interest in a plant medicine.

It is still another object of the present invention to provide a male infertility and / or mating breeding method using the medicinal promoter of the plant.

Another object of the present invention is to provide a transformant in which the protein of interest is specifically expressed in plant medicines.

In order to achieve the above object, the present invention provides a plant drug tissue specific promoter comprising a nucleotide having at least 70% or more homology with the nucleotide sequence of SEQ ID NO: 1.

In another aspect, the present invention comprises preparing a vector linked to express the gene for the target protein in the 3 'direction of the plant drug specific promoter, and transforming the vector into a plant to induce the expression of the target gene in the plant medicine Provided are methods for producing target proteins in plant medicines.

The present invention also provides a vector comprising the plant drug specific promoter.

The present invention also provides a plant that is asexually propagated by the plant tissue culture method transformed with the vector.

Hereinafter, the present invention will be described in detail.

The present inventors have attempted to establish a system capable of specifically expressing or inhibiting a gene of interest by confining promoters capable of regulating plant tissue or organ specific gene expression. Therefore, the present inventors cloned the drug-specific promoter through the genome library of Lilium hybrid cv. Acapulco, a monocotyledonous plant, while studying the drug-specific promoter, and stably tested the drug-specific promoter in petunia. It has been found to function as a specific promoter and the present invention has been completed based on this.

"Plant drug specific promoter" referred to in the present invention means a promoter that is linked to the 3 'position of the promoter to specifically express a gene whose expression is regulated in plant medicine, and in plant tissues other than plant medicine, It means little expression.

Although the drug-specific promoter of the present invention has been isolated from Lilium, which is a monocotyledonous plant, it has been found to function successfully in the petunia, which is a dicotyledonous plant.

The plant may be a common monocotyledonous plant, dicotyledonous plant, herbaceous plant or woody plant, preferably a crop, a flower crop, a vegetable crop or an orchard crop, and more preferably a plant in which asexual reproduction is known. Examples of plants include rice, wheat, barley, oats, millet, crude, sorghum, corn, barley, asparagus, onions, bamboo, rye, sugar cane, pineapple, bananas, tomatoes, tobacco, cotton, grapes, peppers, lettuce , Legumes, cabbage, broccoli, sunflowers, carrots, eggplants, spinach, cucumbers, pumpkins, potatoes, sweet potatoes, apples, peaches, pears, poplars, pines and arabidopsis, but are not limited thereto. Asexual reproduction of plants includes cutting, dispensing, abandonment, tissue culture, and pollen culture. Tissue culture method is a method of taking a part of the leaves of the plant and cultivating in the medium, followed by culturing in the plant root formation medium to form the root and drinking it in the soil.

The plant drug specific promoter of the present invention is a nucleotide having at least 70% or more homology with the nucleotide sequence set forth in SEQ ID NO: 1 and having a drug specific promoter function. Preferably, the promoter is a nucleotide having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology and about specific promoter function. Examples of the sequence of the plant drug tissue specific promoter include, but are not limited to, the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

SEQ ID NO 1:

aa ctcggtggtt

tacactcagg ggacttgcta cgaaccaaat tttaaaactg ccagtatggt ggaaataaac

ttctctccgc ctcttctgtc cttggggaat aagattcaaa ccccttcaat ccgaggttat

cgatgccgcc acgttgctta gttaactgct tgttcccaac tatccatctc cctataaaac

ctttcgcccc atacaacctt cacttcatct ctctccatcc actacacctg ctctgcattc

tagtccgact ccgatagtat cttcggtt

SEQ ID NO 2:

1 gtcgaccaag ggcaagccga ggcctccaca tttcttatca ttagtgtgtg gtctgagtac

6l aattaggcca tcatgctaat ctaccaagtt cacccagata tgtccctgaa taggtcagca

121 gacaaagccc tgaccttcaa gttggacatc ataagccctg gctaactcct aggtgtgtcc

181 ctgaataggt tggcagtcag agatccgacc ttcaggccaa acttcctaag gccctactaa

241 ctcccaggca tttaagtccc taaattggtt atcgggcaaa gcctcgacct tcaggccaga

301 cttcctaaga tctggctaac tcccaggcat tcaagttcct aaatgagtgg gcggacagag

361 ctctgacctt caggcaagac ttcttaagtc ctgactaact cccaggcatg tccctgaata

421 ggtcggtggg caaaactcct aagccctatc taactcccaa gtgtgtcccc gaacgagtca

481 gcgggtagag ctctgatctt cagaccagac ttcctaagca ttggctaact cctaagggca

541 tccttgaatg ggtcaacggg cagagctctg accttcaggc tggactttct aagccctagc

601 taactctcaa gtattcatgt ccctgaacgg gcggcgggc aaagccctga ccttcaggct

661 attcatgtcc ctgaacgggt cggcggcaa agccctgacc ttcaggctag atatcctaag

721 ccctggctaa cccccaagtg tgtccctgaa tgggttgatg agcagatctc caaccttcat

781 gccagacttc ctaagccttg gctaactccc aggCattcaa gtcccttaat gggtcagtgg

841 gcagagatcc gaccttcaga ccggactttt taaaccttgg caaactccta ggggtgttcc

901 tgaacttgtt ggcgggcaga gctccgacct tcaggccaga ctttccaagc gctagctaac

961 tcccagggcc attcaaagcc cttgaattag tctatgggca tagccccgga cctttagttc

1021 aggcattcca agccctgact atactcccag gcacaataaa cccctgagtc gatctgcgag

1081 catagcaccg atcttcaggc tgggcatccc aaaccctggc tatactccca ggcatataag

1141 tcccagatgc caaaactttt cttcttgcat cgatcccaaa attctcaggc caagcgagca

1201 ttctccctca atccaccttg ccttctcctt aagaagcctg ggcaagaagg agggacaata

1261 gaccaaELgat caaaggatca actccgtgct gacttatccc cgacttctta gcacatgtgc

1321 agggtcaaca aggcgtctcc gacaaactca tacacgggca tacaggctgg cccatgccct

1381 ggtccaaacg acatcgaagg acagtctcct ccggctccgg cgggaagacc cgtgtgccca

1441 atcggtgggt atccctagga tatccatgcc cgacacccac aggatgaccc gtgtgccagg

1501 acaaactaca caataaccag taccttaact gacttctcga cataacatca catatcgcat

1561 ttaatgtgct gacctaggag ttgacgtgtt gtcccttctg ttggatcgct aggctacgta

1621 tgctcaacaa atagtcacac cgacctaagt gatttgacga ctcataggca tatgcgtgga

1681 tccttggaac tcgactccac agtcacccct agtgcatata aaaagcctcc tccactcccc

1741 tttgaggggg taagctttac agactctttg cacgtatata atatgtccgg atcggagcac

1801 aggtactctg ctcacctaag agcggtcagc tgcccagtat cgtagaaact cgacactcac

1861 tctcacatcg gaggagtgtc gcggggatac gtccatgcga ccatgtttag gtgtttcttg

1921 tgctcgcagg tctcgacgac gacctcatcc gtaccagcct gagctctccc agttctccaa

1981 ccactacgat ggaacgtttg tctccagttc gaccctgcga gcgccctgct cacccaggtc

2041 ggagctccac tagctcgtag cccggatctg actgtctcta cgcacacgcc cggctagagc

2101 tctaccagct cgcagcctgg atccgaccat ctccgcgtcc atgacaggcc agagctccac

2161 tagctcgccg gacaaatcct cgacgcccat gcccgtggtt ggccatttcc cgcatcctcg

2221 cctccgaaca aaactcaaca agccaggaat ctgaagcgta gtgtcacgtc agaccccgct

2281 gaagaaggac gtcatcacaa ccaatttatt ttgacgtcat caataagtaa tcttaaatct

2341 gcattctcca aaacatatat ttttgtcatt gaacttatta agtattagac atttctttta

2401 cttggtcatt gatgtcgccg aaatcattcc actgaactct aacaccaaat tgttcaacta

2461 tatctaattc taaaaataag attatatgca acacttcggt accaatttgt tcggctatgt

2521 caaactctac aaataaaatt aataaaatat atgtgatcga ccaattattc aaagagatca

2581 taatcggtcc caatgttaga aggtacacaa aatcgttgtc ccaaaaacaa ctcggtggtt

264l tacactcagg ggacttgcta cgaaccaaat tttaaaactg ccagtatggt ggaaataaac

2701 ttctctccgc ctcttctgtc cttggggaat aagattcaaa ccccttcaat ccgaggttat

2761 cgatgccgcc acgttgctta gttaactgct tgttcccaac tatccatctc cctataaaac

2821 ctttcgcccc atacaacctt cacttcatct ctctccatcc actacacctg ctctgcattc

2881 tagtccgact ccgatagtat cttcggttct aaca

In one embodiment, the drug specific promoter comprising the nucleic acid sequence of SEQ ID NO: 1 performs transcription of the gene predominantly in the drug tissue. In another embodiment, the drug specific promoter comprising the nucleic acid sequence of SEQ ID NO: 2 predominantly expresses in the drug tissue, but in some cases also expresses the gene in the room tissue.

The drug specific promoter according to the present invention may control specific traits such as removing pollen or exchanging pigments in the drug by transformation. Accordingly, it is possible to cultivate the parent plants that can be used for breeding and breeding of vegetables or flowers, or to improve the disadvantages of flower crops that are less commercially available due to potted plants. To this end, a plant having a desired trait can be prepared by linking the gene and / or protein of interest to the drug specific promoter according to the present invention so that it can be expressed and transformed.

To this end, the present invention provides a vector comprising the plant drug specific promoter. The vector may include a nucleic acid sequence encoding a protein of interest in the 3 'direction of the promoter in a form that is expressively linked, and also includes an origin of replication, a promoter regulatory region such as an enhancer, a transcription termination region, a selection marker, or a transcription. It may further comprise regulatory factors. Preferably, the vector includes a plant drug specific promoter, a target protein coding site, and a transcription termination site in a 5 'to 3' direction, and the promoter may be substituted with the plant drug specific promoter in a conventional vector. The conventional vector may be a pUC family vector such as pBR322, pBI121, pCAMBIA, Gateway vector, Ti-plasmid, and a vector derived therefrom, but is not limited thereto.

Said "expressively linked nucleic acid sequence" means that the transcription and translation of the nucleic acid sequence can be regulated by a promoter.

The "promoter 3 'direction" means that the nucleic acid sequence encoding the protein of interest is directly linked or adjacent to the 3' end of the promoter.

The selection marker is a nucleic acid sequence having properties that can be selected by conventional chemical methods, and all genes that can distinguish the transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, bleomycin, hygromycein, and chloramphenicol. There are, but are not limited to, resistance genes.

The transcription termination site may be a conventional transcription termination site, and examples thereof include a nopaline synthesis termination site, a phaseoline terminator, and octopine of agrobacterium tumefaciens. ) Terminators of genes, but are not limited thereto.

The transcriptional regulator may be known in the art, and may be used in appropriate combination depending on the target plant.

The target protein may be a protein derived from all kinds of plants or animals, and preferably, a protein derived from a plant to be used. For example, it is a protein associated with a phenotype of pollen, such as a pigment-related protein, or a protein used for cross breeding, a pigment-related protein such as anthocyanin pigments, a protein related to pollen production, such as a lipid transfer-related protein, and the like. Or subject related proteins, the subject protein is selected from, but is not limited to, kanamycin, β-glucrinidase (Gus), nopaline synthase, and Cauliflower Mosaic Virus promoter.

The vector can be easily prepared according to known methods by those skilled in the art.

A vector comprising the plant drug specific promoter of the present invention can be introduced into a plant according to a conventional method to produce a transformed plant. Transformation methods include, but are not limited to, transformation of protoplasts using calcium / polyethylene glycol method, electroporation, microinjection, (coated) particle bombardment, gene gun, and physical introduction, but are not limited thereto. It can be adjusted appropriately according to the type of. In addition to such direct gene transformation methods, transformation systems including vectors such as viral vectors (eg from Cauliflower Mosaic Virus (CaMV)) and bacterial vectors (eg from the genus Agrobacterium) are widely used. It is available. After selection and / or screening of the resulting transgenic plants, the transformed protoplasts, cells or plant parts can be regenerated into complete plants according to known methods (Horsch, R. b. Et al., 1985). The choice of transformation and / or regeneration techniques is apparent to those skilled in the art.

In plants, medicine is an important organ that has both coronary and reproductive, and the anther of the drug in the coronal side, the ornamental value is often lowered and the commercial value is often impaired. At this time, by producing a transgenic strain so that the expression-related gene is expressed under the control of the drug specific promoter of the present invention, it is possible to cultivate a flower crop having a normal shape and excellent commercial property without anther. In addition, when the crops of the vegetable crops, the crops of the maternal side of the crops to be mated are generally cross-breeding through the cross-breeding operation, at this time, by allowing the drug-related genes to be expressed under the control of the drug-specific promoter of the present invention There is an advantage that can perform a simple cross without the need of de-emulsion.

The present invention is described in detail with reference to the following examples. The following examples are merely to illustrate the invention, but not to limit the protection scope of the invention.

Example 1 Preparation of Genomic Library and Genomic Gene Isolation

Isolate genomic DNA from the leaves of Oriental Lilium hybrid cv.Acapulco according to the method of Rapid isolation of high-molecular-weight plant DNA (1980), and using the Lambda Fix II / XhoI kit ( Startagene) and ZAP-cDNA Gigapack cloning kit (Stratagene) were run according to the manufacturer's instructions to prepare a genomic library.

Probes were prepared by labeling ALCHS2 cDNA expressed in petals and drugs with [α- ³² P] dCTP according to the manufacturer's instructions using the Rediprime ^™ II DNA labeling system (Amersham). The genome library prepared above was screened with this probe to isolate gALCHS7 (Genome Acapulco Lily Chalcone Synthase 7: about 15.0 kbp), which was digested with EcoR I to obtain a 6.2 kbp clone containing ALCHS2.

Example 2 Sequencing and Transcription Regulation Sites of CHS Genomic Genes

In order to analyze the nucleotide sequence of a gALCHS7 clone of about 6.0 kbp, restriction enzyme maps were generated by cleavage with several restriction enzymes. According to this map, digested with salI and EcoRI, subcloned with pBluescript II SK (+) vector, and analyzed by ABI Prime 31O Genetic Anaiyzer (PE Apphed Biosystems) to obtain full length sequences. A total of 6203 bp sequences were identified by exon and intron sites using genescan (http: // genes. Mit. Edu), and PLACE database (PLAnt Cis-acting regulatory DNA Elememt: http // www.dna.affrc. go.jp) to analyze the transcriptional regulatory sites. The results are shown in Table 1 below (the following positions are described based on ATG).

Factor or site name location order Characteristic GT1CONSENSUS -362 GRWAAW GTGA motif found in the promoter of Tobacco late pollen gene g10 that harbors homology to pectate lyase GTGANTG10 -1264, -224 GTGA Conservative GT-1 Binding in Multiple Photo-regulatory Genes INRNTPSADB -1716, -1057 YTCANTYY "Inr (initiator)" element found in tobacco psaDb gene promoter without TATA box INTRONLOWER -1605 TGCAGG 3 'intron-exon splice junction: new intron subsequence MYB2AT -1397, -132 TAACTG Binding to ATMYB2, Arabidopsis MYB Homolog (TAACTG) MYBCORE -1315 CNGTTR Petunia MYB Binding Protein Involved in the Regulation of Flavonoid Biosynthesis MYBPZM -936 CCWACC The core of the conserved maize P (myb homolog) junction, the maize P gene, forms the red pigment of Kernel peric POLLEN1LELAT52 -1069 AGAAA Regulatory Factors Response to Pollen-specific Activation of the Tomato lat52 Gene QELEMENTZMZM13 -2811 AGGTCA Motif found in maize (Z.m.) ZM13 gene promoter involved in enhanced expression ROOTMOTIFTAPOX1 -556 ATATT Motif found in rolD and root specific wheat peroxidase promoter CAAT BOX -168 CAAT General sequence expressed at the 5'- flanking region of eukaryotic genes

Example 3: Vector Fabrication

In a 6.2 kbp genomic clone (gALCHS7), 270 bp (SEQ ID NO: 1) of the 5'- flanking region was amplified by PCR and used for vector construction. The genome gХ 隙 ◎ template was amplified with gSalI (forward primer: 5'-AACTCGGTGGTCGACACTCAGGGG-3 ') and gXbaI (reverse primer: 5'-ATTTCTAGAACCGAAGATACTA-3') primers, cleaved and isolated with SalI and XbaI, The gALCHS7-7 / GUS vector was constructed by connecting to the vector pBI101 digested with the same restriction enzyme (FIG. 1).

Example 4 Transformation and Convertor Analysis

A leaf disk method modified from the method of Horsh et al. (A Simple and general method for transferring genes into plants, 1985), and transformation into Petunia (Dreams Red, PanAmerican Seed) using Agrobacterium strain C58C1 Was carried out.

Specifically, petunia plants sown in the cabin are grown to a total length of about 7 cm, and only 1 to 3 leaves are harvested, cut to about 5 mm in length and length, cut into hormone-containing regeneration media for 1 day. Incubated. Agrobacterium strains cultured at 28 ° C. for 2 days were centrifuged at 5,000 rpm for 10 minutes to dilute the precipitate in a liquid medium. The bacterial dilutions and the cut leaves were mixed and inoculated for 10 minutes, and then cultivated in the regeneration medium and co-cultured in the dark for 2 days. Two days later, the co-cultured leaves were washed in a liquid medium containing 200 mg / L of Cytoxim and washed in transformant selection medium. In the transformant selection method, a shoot grown for 4 weeks in a medium containing kanamycin (50 mg / L) was transferred to a medium containing kanamycin (100 mg / L) and cultured for 3 weeks. Selected transformants were transferred to the rooting medium containing no kanamycin and induced rooting for about 4-6 weeks before being transferred to the greenhouse for purification. Genomic DNA was isolated from the leaves of the selected transformants using DNeasy Plant Mini Kit (Qiagen, Germany), followed by NPTII primers (primer 1: 5'- GAG GCT ATT CGG CTA TGA CTG-3 ', primer 2: 5'-ATC GGG AGC GGC GAT ACC GTA-3 ') to confirm the transformation. Five individuals confirmed to be transformed with NPTII primers were subjected to Southern hybridization by Southern method (1975). 10 μg of the isolated genomic DNA was digested with EcoR I and subjected to electrophoresis with 0.5 × TBE buffer for 18 hours at 1 V / cm in a 0.7% agarose gel. Beta-glucuronidase (GUS) using a Rediprime ^™ II DNA Labeling system (Amersham), a probe labeled with [α- ³² P] dCTP was used for a sudden hybridization reaction according to the manufacturer's instructions. As a result (FIG. 2), the insertion of beta-glucuronidase (GUS) was confirmed in three individuals, which were then purified in the greenhouse after proliferation.

Example  5: GUS  Expression analysis

GUS activity was examined using each organ of petunia transformants. Cut the leaves, petals, pills, pistils, and stalks of the petunia flower at the pre-opening stage with a razor, and then use Gallagher (GUS Protocols: Using the GUS gene as a reporter of gene expression, 1992) at 37 ° C for 12 hours It was immersed in the modified GUS solution. At this time, 20% methanol was added to suppress the similar expression of endogenous GUS in untransformed tissue (An improved assay for β-glucoronudase transfomed cells: methanol almost completely suppresses a putative endogenous β-glucoronudase activity, 1990).

As a result of GUS expression analysis (FIG. 3), strong expression was confirmed in two drugs among all the transformants used for the test. Other organs, such as leaf and petal operating tables, were unable to identify any visible GUS expression, as did plants without transformation. The remaining one of the three confirmed Southern populations was unable to confirm GUS expression in any organ other than the pre-drug drug.

Example  6: Dark - field  Using techniques GUS  Expression analysis

For dark field observation, samples were fixed by combining two methods (Initiation patterns of flower and floral Organ development in Arabidopsis thaliana, Development, 1996: Method in short protocols in molecular biology, Unit 14.2-3 ). Two drugs showing GUS activity were stained with GUS, and then fixed by immersion with the following solution at room temperature for 12 hours (50% ethanol, 5% acetic acid and 3.7% formaldehyde). Dehydrate with a series of ethanol dilutions (70%, 85%, 95%) and then 1 hour each with xylene and ethanol dilutions (1: 3, 1: 1, 3: 1) After soaking, the mixture was further changed twice with 1% of 100% xylene. About 5-10 ml of xylene was added and the same amount of paraffin (paraplast, Fisher) was added and placed in an oven at 60 ° C. for 12 hours. The molten paraffin was discarded and the new paraffin was poured 2-3 times, then poured into a mold, fixed and trimmed to a shape suitable for cutting. Using a paraffin cutter (Leica) was cut to 10-15 ㎛, fixed on a poly-prep slide (sigma) and dried for about 12 hours at 37 ℃. For microscopic observation, the slide on which the sample was fixed was dissolved in 100% xylene for 12 hours, then dehydrated using xylene and ethanol dilution (1: 3, 1: 1, 3: 1), and then applied Permount (Fisher). The cover slip was covered and fixed. After drying, it was observed at a ratio of 100 times on a microscope equipped with a dark field. Observations showed that in drugs with sufficiently developed pollen, strong GUS expression was observed not only in pollen but also in the supportive tissues of the drug (endothethium, epidermis) (FIG. 4). No significant expression was observed. Thus, it was confirmed that the gALCHS7-7 promoter is a promoter that operates only in the drug and its supportive tissue.

As described above, the drug-specific promoter of the present invention can obtain an accurate expression with increased efficiency when introducing a specific trait by transformation, and can be used for breeding breeding after introducing a specific gene into the drug. have. In addition, it can be applied to ornamental plants to cultivate flower crops that have a normal shape, but not anther, and have excellent commercial properties.In addition, vegetable reproductive genes can be expressed under the control of drug-specific promoters for vegetable crops. It is possible to cultivate crops that can be simply crossbreed without.

<110> Rural Development Administration <120> ANTHER SPECIFIC PROMOTER DERIVED FROM ACAPULO LILY, RECOMBINANT          VECTOR, TRANSGENIC PLANT AND PREPARATION METHOD <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 279 <212> DNA 213 Lilium hybrid cv. Acapulco <400> 1 aactcggtgg tttacactca ggggacttgc tacgaaccaa attttaaaac tgccagtatg 60 gtggaaataa acttctctcc gcctcttctg tccttgggga ataagattca aaccccttca 120 atcgaggtta tcgatgccgc cacgttgctt agttaactgc ttgttcccaa ctatccatct 180 ccctataaaa cctttcgccc catacaacct tcacttcatc tctctccatc cactacacct 240 gctctgcatt ctagtccgac tccgatagta tcttcggtt 279 <210> 2 <211> 1604 <212> DNA 213 Lilium hybrid cv. Acapulco <400> 2 gcacatgtgc agggtcaaca aggcgtctcc gacaaactca tacacgggca tacaggctgg 60 cccatgccct ggtccaaacg acatcgaagg acagtctcct ccggctccgg cgggaagacc 120 cgtgtgccca atcggtgggt atccctagga tatccatgcc cgacacccac aggatgaccc 180 gtgtgccagg acaaactaca caataaccag taccttaact gacttctcga cataacatca 240 catatcgcat ttaatgtgct gacctaggag ttgacgtgtt gtcccttctg ttggatcgct 300 aggctacgta tgctcaacaa atagtcacac cgacctaagt gatttgacga ctcataggca 360 tatgcgtgga tccttggaac tcgactccac agtcacccct agtgcatata aaaagcctcc 420 tccactcccc tttgaggggg taagctttac agactctttg cacgtatata atatgtccgg 480 atcggagcac aggtactctg ctcacctaag agcggtcagc tgcccagtat cgtagaaact 540 cgacactcac tctcacatcg gaggagtgtc gcggggatac gtccatgcga ccatgtttag 600 gtgtttcttg tgctcgcagg tctcgacgac gacctcatcc gtaccagcct gagctctccc 660 agttctccaa ccactacgat ggaacgtttg tctccagttc gaccctgcga gcgccctgct 720 cacccaggtc ggagctccac tagctcgtag cccggatctg actgtctcta cgcacacgcc 780 cggctagagc tctaccagct cgcagcctgg atccgaccat ctccgcgtcc atgacaggcc 840 agagctccac tagctcgccg gacaaatcct cgacgcccat gcccgtggtt ggccatttcc 900 cgcatcctcg cctccgaaca aaactcaaca agccaggaat ctgaagcgta gtgtcacgtc 960 agaccccgct gaagaaggac gtcatcacaa ccaatttatt ttgacgtcat caataagtaa 1020 tcttaaatct gcattctcca aaacatatat ttttgtcatt gaacttatta agtattagac 1080 atttctttta cttggtcatt gatgtcgccg aaatcattcc actgaactct aacaccaaat 1140 tgttcaacta tatctaattc taaaaataag attatatgca acacttcggt accaatttgt 1200 tcggctatgt caaactctac aaataaaatt aataaaatat atgtgatcga ccaattattc 1260 aaagagatca taatcggtcc caatgttaga aggtacacaa aatcgttgtc ccaaaaacaa 1320 ctcggtggtt tacactcagg ggacttgcta cgaaccaaat tttaaaactg ccagtatggt 1380 ggaaataaac ttctctccgc ctcttctgtc cttggggaat aagattcaaa ccccttcaat 1440 ccgaggttat cgatgccgcc acgttgctta gttaactgct tgttcccaac tatccatctc 1500 cctataaaac ctttcgcccc atacaacctt cacttcatct ctctccatcc actacacctg 1560 ctctgcattc tagtccgact ccgatagtat cttcggttct aaca 1604 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer, gSall <400> 3 aactcggtgg tcgacactca gggg 24 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer, gXbaI <400> 4 atttctagaa ccgaagatac ta 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gaggctattc ggctatgact g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atcgggagcg gcgataccgt a 21  

Claims (8)

A plant drug specific promoter comprising a nucleotide or a complement thereof having a homology of 70% or more with the nucleotide sequence of SEQ ID NO: 1. The promoter according to claim 1, wherein the promoter comprises a nucleotide sequence of SEQ ID NO: 2. A vector is prepared in which the gene for the target protein is expressed in the 3 'direction of the plant drug specific promoter according to claim 1 or 2, and the vector is transformed into a plant to convert the plant gene and / or protein in the plant medicine. A method for producing a plant, wherein the protein of interest comprising the induction of the expression of the specific expression in the drug. The method of claim 3, wherein the target protein is selected from the group consisting of kanamycin, β-glucrinidase (Gus), nopaline synthase, and Cauliflower Mosaic Virus promoter. A vector comprising a plant drug specific promoter according to claim 1. Plants that are asexually propagated by plant tissue culture, cutting, or aliquot transformed with the vector according to claim 5. A plant comprising a fragment of the plant obtained from the plant of claim 5, a plant cell and a descendant of the plant. 7. The plant according to claim 6, wherein the plant is selected from the group consisting of crops, flower crops, vegetable crops and fruit trees.

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