KR20050022491A - Ethanol-inducible expression vector - Google Patents

Abstract

PURPOSE: An ethanol-inducible expression vector is provided, thereby easily obtaining an interest gene from plants at a certain culturing period or stage by using ethanol as an inducing chemical which can be sprayed in a large scale due to its strong volatility. CONSTITUTION: The ethanol-inducible expression vector comprises the nucleotide sequence(SEQ ID NO:1) encoding the ADR1 site which is a transcription factor of Saccharomyces cerevisiae; the nucleotide sequence(SEQ ID NO:2) encoding a promoter of ADH2(alcohol dehydratase 2); and an interest gene operably linked to the promoter, wherein the ADR1 site essentially comprises the nucleotide sequence(SEQ ID NO:3) encoding the TADI site which is the zinc finger site; the ADH2 promoter essentially comprises the nucleotide sequence(SEQ ID NO:4) encoding the UAS site(200bp DNAs); the ADH2 promoter is ADH2-35S fusion promoter having the nucleotide sequence of SEQ ID NO:15; and the alcohol is ethanol.

Description

Ethanol-derived plant expression vector {ETHANOL-INDUCIBLE EXPRESSION VECTOR}

Technical Field

The present invention relates to a vector system for regulating the expression of genes by foreign chemicals. More specifically, the present invention relates to ethanol-derived plant gene expression vectors.

Prior art

Vector systems that regulate the expression of genes by chemicals have advantages not only in the function of genes but also in the application of biotechnology research products, which are already common in many multicellular organisms, from yeast to animals. Hartley et al, 2002, Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system.Pro. Natl. Acad. Sci. USA 99: 1377-1382).

In plants, expression of the target genes has been reported by the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, which is consistently highly expressed throughout the entire plant development and in all organs (Ref .: Odell et al., 1985, Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter.Nature 313: 810-812. Sustained expression of foreign genes by these promoters in plants results in a loss of normal growth of the plant and, in severe cases, fatal damage to the plant in genes that convert primary products into storage compounds (Ref. Stitt, M. et al., 1995, Regulation of metabolism in transgenic plants.Annu. Rev. Plant Physiol.Plant Molec. For example, yeast invertase genes may be modified under the control of the CaMV 35S promoter (Sonnewald U. et al., 1991, Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast). : a powerful tool for studying sucrose metabolism and sink / sucrose interactions.Plant J. 1: 95-106) and tobacco (Bussis, D. et al, 1997, Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast Such is the case with transformed invertase either in the apoplast, vacuole or cytosol.Planta 202: 126-136).

To overcome this drawback, a patatin promoter that induces potato tuber specific expression (Grerson, C. et al, 1994. Separate cis sequences and transfactors direct metabolic and developmental regulation of a potato tuber storage A number of promoters have been developed that induce organ-specific and developmental-specific expression, such as the protein gene.Plant J. 5: 815-826 (Ref. Kuhlemeier, C., et al., 1987. gene expression in higher plants.Ann. Rev. Plant Physiol.Plant Molec.Biol. 38: 221-257). However, even in this case, it was impossible to artificially control the timing of expression, and thus developmental stage specific expression could not be performed.

In addition, vector systems in which expression is induced by chemicals have been studied and developed. Major chemical inducers developed until recently include copper (Mett, V. et al., 1993. Copper-controllable gene expression system for whole plants.Proc. Natl. Acad. Sci. USA. 90: 4567-4571) And rat gluco-corticoids (Aoyama, T. et al., 1997. A glucocorticoid-mediated transcriptional induction system in transgenic plants.Plant J. 11: 605-612). However, it is not easy to apply these derivatives to plants in large quantities, and copper is particularly toxic and its use is limited. In addition, steroids (Mark, S. et al., 1991. A steroid-inducible gene expression system for plant cells.Proc. Natl. Acad. Sci. USA. 88: 10421-10425) and ecdyson (Reference: Martinez, A. et al., 1999. Ecdysone agonist inducible transcription in transgenic tobacco plants.Plant J. 19: 97-106) was used as an expression inducer, but it was significantly more expressed than a promoter inside the plant. Did not show. In addition, a system using tetracycline as an inducer has been reported, but it has not provided specificity and fine control of expression for studying the function of the inducer system gene, and in certain plants, for example, Arabidopsis, etc. Its general purpose is poor because it cannot be induced, and the inducer is relatively expensive, and the physiological inactivation inside the plant has not been verified (Ref. Weimann, P. et al., 1994. A chimeric transactivator allows tetracycline-responsive) gene expression in whole plants.Plant J. 5: 559-569). Thus, there has been a continuing need for the development of new expression inducers and their associated vector systems that are degradable, inexpensive, and capable of large scale spreading in plants (Ref. Hairul, AR et al., 2001. Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana.Plant J. 28: 225-235).

As a result of this research, recent studies have been carried out in Arabidopsis, tobacco and potatoes using Alc regulon, an alcohol-degrading gene family of Aspergillus nidulans . Mark, XC et al., 1997, An ethanol inducible gene switch for plants used to manipulate carbon metabolism, Nature Biotechnology 16: 177-180). Alc regulators consist of alcA, which encodes an ALCR transcriptional regulator, and promoter palcR derived from alcA (FIG. 1), where alcR in A. nidulans encodes alcohol dehydrogenase I (ADHI) and aldehyde dehydrogenase (AldDH), respectively. It is known to modulate a large number of structural gene activities, such as alcA and aldA (Gwynne, FP et al., 2001. Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana. Plant J. 28: 225-235). In FIG. 1 the construct comprises a complete CaMV35S promoter (p35S) which induces alcR cDNA located upstream of tnos (nos terminator). The three genes used, GUS, GFP and LUC, consist of the CaMV35S minimum promoter (-31 to +5) fused to the upstream promoter gene of alcA at the TATA box position. The CaMV35S terminator (t35S) is located downstream from the reporter gene. This construct is introduced into the plant by the transformation vector pGPTVhyg.

In the present invention, an alcohol-induced expression vector using an alcohol degradation gene derived from yeast ( S. cerevisiae ) is provided. Yeast basically uses glucose as a carbon source, but in the absence of glucose, in order to use alcohol as a substitute (FIG. 2), ethanol is finally expressed by expressing the transcriptional activator Adr1 to convert alcohol to acetaldehyde. Is used as the carbon source (Gleeson, MA and PE Sudbery. 1988. The Methylotrophic Yeasts. Yeast 4: 1-15). Figure 2 depicts glycolysis and gluconeogenesis pathways in S. cerevisiae , wherein the use of alcohol as a carbon source by yeast Saccharomyces cerevises is associated with the inhibition of alcohol dehydrogenase encoded by the ADH2 gene ( Ciriacy, M., 1975, Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae.II.Two loci controlling synthesis of the glucose-repressible ADH II.Mol Gen Genet 138: 157-64). In contrast, ADH2 is completely inhibited in the presence of fermentable substrates (Lin et al., 2001, Enhanced intsfotion gluconeogenesis and increased energy storage as hallmarks of aging in Saccharomyces cerevisiae).

In the present invention, the promoter of ADH2 (Alcohol Dehydrogenase 2) of the alcohol fermentation gene of yeast, which is well known for establishing a plant gene expression system by ethanol (SEQ. ID No .: 2) (Reference: Scheer, N. and JA Camnos-Ortega. 1999.Use of the Gal4-UAS technique for targeted gene expression in the zebrafish.Mech. Dev. 80: 153-158) and the transcriptional activation site of the transcriptional activator ADR1 (SEQ. ID No .: 1). (Manuel, MS et al., 1995, A C-terminal region of the Saccharomyces cerevisiae transcription factor ADR1 plays an important role in the regulation of peroxisome proliferation by fatty acids.Mol. Gen. Genet. 249: 289 -296). ADR1, a transcriptional activator, is a zinc finger region that is involved in DNA binding in its entire sequence (Thukral, SK, A. Eisen, ET Young. 1991. Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression Cell Bio. 11: 1566-1577), as well as TAD (Trans-activation Domain) I, II, III sites, which are ethanol-induced transcriptional activation sites (Cook, WJ et al., 1994a, Dissection of the ADR1 protein reveals multiple, functionally redundant activation domains interspersed with inhibitory regions: evidence for a repressor binding to the ADR1 region.Mol. Cell Biol. 14: 629-640) (FIGS. 3A and 3B). In FIGS. 3A and 3B, both cis-acting factors (UAS1 and UAS2) required for maximal activity of the gene are identified in the ADH2 regulatory region (Shuster, J. et al., 1986, ADR1 -mediated regulation of ADH2 requires an inverted repeat sequence.Mol Cell Biol 6: 1894-902). UAS1 has been found to be a binding site of Adr1 (Ref .; Thukral et al., 1991, Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression.Mol . Cell Bio. 11: 1566-1577). It has been genetically identified as a positive regulator of ADH2 (Ref. Ciriacy et al., 1975. Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae II.Two loci controlling synthesis of the glucose-repressible ADH II, Mol Gen Genet 138: 157- 64). Adr1 contains two zinc finger motifs, which interact with each half of the palindromic UAS1 factor as monomers (Ref. Blumberg, H. et al., 1987, Two zinc fingers of a yeast regulatory protein shown by genetic evide- nce to be essential for its function Nature 328:. 443-45). Among TAD sites of ADR1, TAD I (SEQ. ID No .: 3) alone is sufficient to show the best expression of ADH2 (Ref .: Cook, WJ et al., 1994b, Mutations in the zinc-finger region of the yeast regulatory protein ADR1 affect both DNA binding and transcriptional activation J. Biol. Chem. 269: 9374-9379). TAD II and III alone do not perform their function but do additional functions of TAD I (Denis, CL et al., 1992, ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230.Mol.Cell Biol. 12: 1507-1514). In addition, 200 bp of DNAs (SEQ. ID No .: 4) corresponding to the UAS (Upstream Activation Sequence) in the ADH2 promoter to which the transcriptional activator ADR1 binds are very well conserved sequences in various yeast species (Ref. ET et al., 2000, Evolution of a glucose-regulated ADH gene in the genus Saccharomyces.Gen 245: 299-309), which acts as members of expression induction vectors in a variety of organisms as well as yeast (Ref. Scheer, N, and JA Camnos-Ortega. 1999.Use of the Gal4-UAS technique for targeted gene expression in the zebrafish.Mech. Dev. 80: 153-158. Scheer and Camnos Ortega).

In this study, two genes corresponding to transcriptional activators and their binding sites were isolated from yeast, inserted into plant expression vectors, transformed into the protoplasts of the plant of interest, and the ethanol of the reporter gene GFP gene. By inducing expression, the vector system induced by ethanol, which can be sprayed at large scale by strong volatility, was developed in plants.

The expression vector of the present invention is easy to use as a vector because the overall size is smaller than the conventional alcohol expression vector, the maximum expression induction time is 10 times faster and longer duration compared to the prior art.

An object of the present invention is to provide an ethanol-induced expression vector (system) using ethanol as an expression inducing substance which can be degraded in plants and can be sprayed in large units.

It is also an object of the present invention to provide a transformed plant in which the expression of the target gene is induced by ethanol.

In order to achieve the above object, the present invention isolated the alcohol degradation gene group from S. cerevisiae and prepared an alcohol induced expression vector. More specifically, an expression vector including TAD I, II and III sites of the transcriptional activator ADR1 and 200 bp of DNA corresponding to the UAS in the ADH2 promoter to which the transcriptional activator ADR1 binds was prepared.

Example

I. Biological Materials and Reagents

(1) plant material

In the present invention, Arabidopsis protoplasts were used as the plant material. Arabidopsis protoplasts soak Arabidopsis seeds in 0.1% (w / v) Triton X-100 solution for 1 hour, rinse once with ethanol and 10% in 1% (w / v) sodium hypochlorite. Soak for 15 minutes to disinfect the surface and washed 7-10 times with sterile distilled water. After low temperature treatment at 4 ° C. for about 2 to 3 days, the cells were cultured by germinating in MS (Murashige and Skoog, 1962) medium or sterile soil. Plant material was used Arabidopsis leaf grown for 2 to 3 weeks under the light intensity of 150 μmol m-2s-1 at 24 ° C./22° C. and a light cycle of 16/8 hours in a light incubator.

(2) reagent

Enzymes such as restriction enzymes SacI, BamH I, HindIII, BglII and EcoRI used in this study were produced by Boeheringer Mannheim (BM, Germany) or TaKaRa. ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit used for sequencing was used by Perkin-Elmer, and the remaining reagents were manufactured by Sigma (USA). Plasmid vectors for DNA cloning and genetic engineering include pGEM-T easy (Promega, USA), pEZ-T (RNA, Korea), pBI121 (Clonetech, USA), and pCAMBIA1303 (MRC Laboratory of Molecular Biology, UK) Was used.

(3) Strains and Characteristics

The strain used in the present invention and its properties are shown in Table 1 below. Agrobacterium strain tyumeo of Pacific Alliance (Agrobacterium tumefaciens) was used C58C1 (Molecular Research Center, USA) . E. coli was inoculated in LB medium containing ampicillin or kanamycin (10 g / L bacto-tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and incubated at 37 ° C. C58C1 was inoculated in YEP medium (10 g / L bacto-peptone, 10 g / L yeast extract, 5 g / L NaCl, pH 7.5) containing gentamicin (25 mg / L) and incubated at 28 ° C. . Solid medium was used by adding agar (15 g / L) to each liquid medium. Yeast strain is Y190 (clonetech, USA), these were used incubated for two days at 30 ℃ conditions in YEPD medium (10 g / L yeast extract, 20 g / L Bacto -peptone, 20 g / L glucose).

II. Isolation and Amplification of Nucleic Acids

(1) Isolation of Genomic DNA and RNA from Yeast

Yeast grown at 30 ° C and OD ₅₉₅ = 1.0-1.5 in YEPD (1% yeast extract, 2% Bacto-peptone, 2% glucose) was used. Yeast genomic DNA was isolated using the WizardR genomic DNA purification kit (Promega, USA), and yeast RNA was identified by Leeds et al. (Reeds, P., SW Peltz, A. Jacobson and MR Culbertson. 1991. The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon, Genes Dev. 5: 2303-2314 to modify AE buffer (50 mM sodium acetate, pH5.3, 10 mM EDTA), phenol and chloroform Was separated using. Quantitative and qualitative analysis after yeast RNA isolation was calculated by measuring absorbance (A260 / 280).

Table 1. E. coli strains and S. cerevisiae strains used in the present invention

Table 2. S. cerevisiae strains used in the present invention

(2) Isolation of Plasmid DNA

Isolation of small amounts of plasmid DNA was performed by alkaline digestion method (Brush, D. et al, 1985, Replacement variant histone genes cotain intervening sequences. Mol. Cell. Biol. 5: 1307-1317). In order to perform cloning, electrophoresis of plasmid DNA treated with restriction enzyme at the multicloning site at 3-4 V / cm on an agarose gel was carried out, and the gel region containing the desired DNA fragment was cut out, and the Jet Sorb Extraction Kit ( Genomed). The separated DNA was quantitatively and qualitatively analyzed by absorbance at 260 nm and 280 nm or by electrophoresis on an agarose gel containing EtBr.

(3) E. coli transformation

In order to transform plasmid DNA into Escherichia coli, XL1-Blue was replaced with a CaCl ₂ treatment method (Mandel, M. and A. Higa, 1970, Calcium-dependent bacteriophage DNA infection. Biotechnology 24: 198-201). competent) was used as E. coli. On the 37 ℃ not more than 1x10 ⁸ dogs per 1 mL shake culture and harvest of E. coli, 0.1 M CaCl ₂ and a turn washed with and suspended into 2 mL of 0.1 M CaCl ₂ was prepared kompi consultant coli. About 50 ng of DNA per 200 µL of this E. coli solution was added, and after 15 minutes on ice, heat shock was applied at 42 ° C. for 1 minute and 30 seconds. Next, the heat shocked E. coli was immediately transferred to ice and left for 1 to 2 minutes, followed by SOC (Tryptone 20 g / L, yeast extract 5 g / L, NaCl 0.5 g / L, 2.5 mM KCl, 10 mM MgCl ₂ , 20 mM glucose) medium was added to 800 μL shaking culture for 1 hour at 37 ℃. After shaking culture, the cells were plated in a selection medium containing antibiotics and cultured in a 37 ° C. incubator to select transformants.

(3) gene cloning

Ethane of the present invention was prepared by using a conventional gene cloning method, and an expression vector was prepared and shown in FIGS. 4A to 4D. In FIGS. 4A-4D 35S-Adr1-tnos obtained by the pBI121 vector is inserted into the Hind III site of pCAMBIA1303. Promoters associated with ADH2 are inserted into pCAMBIA1303 by Hind III and Bg1II. Inserted into the plasmid of Figure 4d DNA fragment of Figure 4a is the ADR1 I-III: pADH2 modified pCAMBIA1303 vector (3-7p). Inserting the DNA fragment of FIG. 4B into the plasmid of FIG. 4D is an ADR1 I: pADH2 modified pCAMBIA1303 vector (1-2p), and inserting the DNA fragment of FIG. 4C into the plasmid of FIG. 4D is an ADR1 I: pADH2-p35S modification. PCAMBIA1303 vector (1-2f). Primers used for gene cloning are shown in Table 3.

Table 3. Oligonucleotides Used in the Invention

III. ADH 2 promoters ADH Plant expression vector introduction of 2-35S fusion promoter

(1) Acquisition of ADH2 promoter in yeast by PCR

The pADH2 primer (Table 3) was used to synthesize a 312 bp DNA fragment corresponding to the UAS site of ADH2 to which ADR1 binds. Based on the Tm values calculated using the prepared primers, S. cerevisiae genomic DNA was used as a template, first denatured at 96 ° C for 5 minutes, then 1 minute at 94 ° C, 1 minute at 51 ° C, and 2 minutes at 72 ° C. After repeating the cycle of 30 times, the PCR was performed at 72 ° C. for 5 minutes to finally extend the reaction. PCR amplification fragments were inserted into the pEZ-T vector and transformed with E. coli to obtain clones corresponding to each amplification fragments, and subjected to sequencing.

(2) Fusion of 35S promoter to isolated ADH2 promoter

Since the isolated ADH2 promoter has the same TATA site as the 35S CaMV promoter, a part of the 35S promoter including a TATA box was prepared as a primer and fused using a fused pADH2 primer (Table 3). Fusion of these promoters is essential for robust expression of reporter genes (Ref. Hairul, AR et al, 2001, Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana. Plant J. 28: 225-235) . The reaction conditions were denatured at 95 ° C. for 3 minutes, followed by 40 minutes of stretching for 1 minute at 94 ° C., 50 seconds at 49 ° C., and 50 seconds at 72 ° C., followed by 5 minutes at 72 ° C. for the last extension. PCR was performed. The eluted DNA was inserted into the pGEM-T easy vector and transformed with E. coli to obtain colonies and sequencing. ADH2-35S fusion promoter was SEQ. It had a sequence of ID No .: 15.

(3) Introduction of plant expression vector of ADH2 promoter and ADH2-35S fusion promoter

The HindIII restriction enzyme recognition site was inserted at the 5 'primer end of the ADH2 promoter and the ADH2-35S fusion promoter, and the BglII restriction enzyme recognition site was inserted at the 3' end, respectively, to the Lac Z alpha and CaMV 35S promoter sites of the plant expression vector pCAMBIA1303.

(4) ADR1 acquisition from yeast mRNA via RT-PCR

2 μg of total RNA isolated from yeast was used as a template, and a reaction solution containing an oligo dT primer (Table 3) was added at 42 ° C using a Promega Access RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) system. Reverse transcription was performed for 1 hour. After inactivating the reverse transcriptase at 95 ° C. for 2 minutes using each ADRI primer and ADR1 III primers, 40 μL of the total reactants in the same tube were used as templates for the cDNA portion of 516 bp and 1,713 bp of the transcription activator ADR1. Sections were synthesized. Reaction conditions were denatured at 94 ° C. for 50 seconds, annealed at 42 ° C. for 1 minute 30 seconds, and PCR was performed to repeat the reaction of elongating at 72 ° C. for 50 seconds for 40 times. The PCR product was electrophoresed on a 1% agarose gel, and the eluted DNA was inserted into the pGEM-T easy vector, transformed into E. coli to obtain colonies, and sequencing was performed.

(5) Insertion of ADR1 fragment into plant expression vector

516 bp and 1713 bp of the transcription activator ADR1 were introduced into the Gam region of the plant expression vector pBI121 by introducing a BamHI restriction enzyme recognition site at the 5 'primer end and a SacI restriction enzyme recognition site at the 3' end, respectively. It was allowed to overexpress under the control of the promoter.

(6) Amplification of 35S-ADR1-tnos in pBI121 with ADR1 partial fragment inserted

Since there is an EcoRI restriction enzyme recognition site in the ADR1 fragment, pBI121 was inserted at the 5 'end and 3' end of 35'-ADR1-tnos without the EcoRI / HindIII from pBI121 inserted ADRI fragment. Primers were prepared and amplified 35S-ADR1-tnos.

(7) Ethanol Induction Vector Construction

In order to combine the two plant expression vectors thus prepared into one ethanol derived plant expression vector, 35S-ADR1-tnos containing ADR1 was digested with a HindIII restriction enzyme to the multi-cloning site (MCS) of pCAMBIA1303. Inserted.

IV. Introduction of Ethanol Expression Vectors into Arabidopsis Protoplasts by PEG Transformation

A transient assay was performed to confirm the action of the recombinant ethanol derived plant expression vector. The media and reagents used for the transient assay are shown in Table 4 below.

Table 4. Transient Assay Medium Compositions Used in the Present Invention

(1) Separation of Arabidopsis Protoplasts

The greenish area except the flowers of Arabidopsis was cut with sterile scissors and soaked in 20-30 mL of enzyme solution and shaken at 20-30 rpm for about 12 hours. To separate the protoplasts, the mixture was placed in 21% sucrose solution and centrifuged at 730 rpm for 10 minutes. Take the supernatant of sucrose solution, add 20-25 mL of W5 wash solution, centrifuge at 600 rpm for 8 minutes, discard the supernatant, and add W5 wash solution. This procedure was repeated once to obtain protoplasts.

(2) PEG transformation

The protoplasts separated from the Arabidopsis were centrifuged at 550 rpm for 5 minutes to collect the protoplasts. 0.3 mL of mannitol / Mg solution was added to the collected protoplasts. The ethanol-derived plant expression vector 10-30 ㎍ DNA was placed in 0.4 mL protoplast solution, and the protoplasts and DNA were mixed well. Next, 0.4 mL PEG solution was carefully added and returned to mix well. 3 to 5 mL of W5 solution was added to the PEG solution, mixed very slowly, centrifuged at 550 rpm for 5 minutes, the supernatant was removed, and 2 mL of W5 solution was added and placed at 22 ° C. for at least 12 hours. .

V. Ethanol Treatment and GFP Expression Observation

(1) Ethanol Treatment and GFP Expression in Transgenic Arabidopsis Protoplasts

After 24 hours of treatment with 2% ethanol to the transformed Arabidopsis protoplasts, the GFP of the Arabidopsis protoplasts transformed using a Confocal Laser Scanning Microscope of a common instrument was observed. GFP absorbs light from 395 nm to 470 nm and emits green fluorescence at 509 nm.

(2) GFP expression analysis

Three plant expression vectors induced by ethanol: modified pCAMBIA1303 vector (3-7p) inserted with ADR1 I-III: pADH2, modified pCAMBIA1303 vector (1-2p) inserted with ADR1 I: pADH2, and The modified pCAMBIA1303 vector (1-2f) into which ADR1 I: pADH2-p35S was inserted was analyzed for GFP expression after ethanol treatment. The program used for analysis was an image j program. This is an image analysis program provided by NIH and quantified GFP expression patterns by accessing the homepage (http://rsbweb.nih.gov/ij/).

VI. result

(1) Production of ethanol derived plant expression vector

i) Construction of the ADH2 promoter and pADH2-p35S subsection fusion promoter and insertion into the pCAMBIA1303 vector

The 200 bp DNA corresponding to the UAS (Upstream Activation Sequence) in the ADH2 promoter to which the transcriptional activator ADR1 binds is a very well conserved sequence in various yeast species (Ref. Young, ET et al, 2000. Evolution of a glucose -regulated ADH gene in the genus Saccharomyces.Gen 245: 299-309), since it functions as a member of expression induction vectors in various organisms as well as yeast (Scheer, N. et al., 1999. Use of the Ga14-UAS technique for targeted gene expression in the zebrafish.Mech. Dev. 80: 153-158). ADH2 promoter was prepared by PCR in the entire sequence of S. cerevisiae (FIG. 5A). FIG. 5B shows the results of electrophoresis on pADH2 fragments that did not contain restriction enzyme recognition sites (HindIII / Bg1II) obtained by PCR with specific primers. The PCR results were separated on 0.8% agarose gel: M is 100 bp ladder; Rows 1-4 show the pADH2 312 bp PCR product. FIG. 5C shows the results of electrophoresis on pADH2 fragments containing restriction enzyme recognition sites (HindIII / Bg1II) obtained by PCR with specific primers. The PCR product was isolated on 0.8% agarose gel: M is 100 bp Ladder; Columns 1-3 show the pADH2 267 bp PCR product. On the other hand, the ADH2 promoter alone is insufficient to express the reporter gene of the plant expression vector with a small amount of ethanol (Ref: Mark, XC et al., 1997, An ethanol inducible gene switch for plants used to manipulate carbon metabolism. : 177-180) A 60-mer primer including a TATA box of p35S was prepared using a TATA box portion matched with each other in the ADH2 promoter and the p35S promoter. Thus, pADH2-p35S partial fragment fusion promoter was constructed by PCR (FIG. 6A). FIG. 6B shows the results of electrophoresis on pADH2: 35S fusion promoter fragments containing restriction enzyme recognition sites (HindIII / Bg1II) obtained by PCR with specific primers, where the PCR results were isolated on 0.8% agarose gel. : M is the / HindIII DNA size marker and rows 1-3 show the pADH2 279 bp PCR product. Two final promoters of this (ADH2 promoter, ADH2-p35S partial fragment fusion promoter) were inserted into the pCAMBIA1303 plant expression vector, respectively.

ii) Construction of partial fragments of ADR1 and insertion into pBI121 vector

Of the TAD (Transactivation Domain) I, II, and III sites of ADR1, the transcriptional activator of S. cerevisiae , TAD II and III do not function alone, whereas TAD I is sufficient to induce the expression of ADH2. Cook, WJ et al., 1994b, Mutations in the zinc-finger region of the yeast regulatory protein ADR1 affect both DNA binding and transcriptional activation.J. Biol. Chem. 269: 9374-9379). Therefore, 516 bp of the ADR1-I portion including only TAD I, a zinc finger region involved in DNA binding, was prepared from the entire sequence of ADR1, a transcriptional activator of S. cerevisiae , and TAD I, II, a activating transcriptional region by ethanol. DNA fragments of 1,713 bp corresponding to the site III were prepared. FIG. 7A shows the results of electrophoresis on ADR1 fragments containing TAD I obtained by RT-PCR using specific primers, where PCR results were separated on 0.8% agarose gel: M is 100 bp ladder; Columns 1 to 2 show the ADR1-I 516 bp PCR product. FIG. 7B shows the results of electrophoresis on ADR1 fragments containing TAD III obtained by RT-PCR using specific primers, where PCR results were separated on a 0.8% agarose gel: M is a 1 kbp ladder; Column 1 shows the ADR1-III 1,713 bp PCR product. ADR1 synthesized from the RNA of Y190, a S. cerevisiae , was introduced into the GUS gene region located behind the 35S promoter of the plant expression vector pBI121 to produce an overexpression of the ADR1 gene.

iii) 35S-ADR1-tnos partial amplification into pBI121 vector and insertion into recombinant pCAMBIA1303

Only the 35S-ADR1-tnos portion containing ADR1 inserted into the pBI121 vector was amplified by PCR (FIGS. 8A and 9A) and inserted into pCAMBIA1303 with pADH2 promoter and pADH2-35S fusion promoter, respectively. The CAMV 35S promoter was included in the pBI121 vector for overexpression of ADR1. The size of the amplified 35S-ADR1 (TADI) -tnos and 35S-ADR1 (TADI ~ TADIII) -tnos were 1,605 bp and 2,815 bp, respectively. FIG. 8B shows the results of electrophoresis on 35S-ADR1-tnos fragments (1,605 bp, 2,815 bp) obtained by PCR with specific primers, wherein the PCR results were separated on 0.8% agarose gel: M is / HindIII DNA Size markers and column 1 are negative controls and columns 2 to 3 represent the 35S-ADR1 (TAD I) -tnos PCR product. FIG. 8B shows the results of electrophoresis on 35S-ADR1 (TADI-TADIII) -tnos fragments obtained by PCR with specific primers, where PCR results were separated on 0.8% agarose gel: M is 1 kbp ladder Row 1 is a negative control and rows 2 to 3 represent 35S-ADR1 (TADI-TADIII) -tnos PCR products (2,815 bp).

(2) Analysis of plant expression vector induced by ethanol

i) Observing GFP Expression in Transgenic Arabidopsis Protoplasts

After treating the Arabidopsis protoplasts transformed with the ethanol-derived plant expression vector for 2% ethanol for one day, GFP expression was observed through a confocal laser scanning microscope (FIG. 10a to 10d). Comparing Arabidopsis protoplasts transformed with pBI121 vector without GFP gene as a control and three plant expression vectors induced by ethanol, GFP expression was observed after ethanol treatment. In the (negative) control protoplasts that were not transformed with the ethanol derived plant expression vector, there was no significant change before and after ethanol treatment (FIG. 10A). However, Arabidopsis protoplasts transformed with ethanol-induced expression vectors showed increased GFP expression after ethanol treatment. 10B, 10C and 10D are transformants incorporating ethanol derived plant expression vectors treated with 2% ethanol. 10B is a transformant introducing p35S-ADR1 (TADI) -tnos-pADH2: p35S-pCAMBIA1303 [1-2f] and FIG. 10C shows p35S-ADR1 (TADI) -tnos-pADH2-pCAMBIA1303 [1-2p] It is a transformant introduced. 10D is a transformant into which p35S-ADR1 (TADI to TADIII) -tnos-pADH2-pCAMBIA1303 [3-7p] was introduced. Autofluorescence was present in the plant, but compared with the control group, Arabidopsis protoplasts transformed with ethanol-derived plant expression vectors showed strong green color after ethanol treatment, indicating that GFP was expressed.

Data quantifying the amount of GFP expression (FIG. 11) shows the action of ethanol derived plant expression vectors. Negative controls were transformed using the pBI121 vector (without the GFP gene). The transformed protoplasts used in the experiment were maintained for 24 hours before and after each use of 2% ethanol. Protoplasts transformed with 1-2p, 3-7p and 2-1f increased GFP expression after 2% ethanol treatment. However, among the three ethanol-induced plant expression vectors, GFP expression was the most increased after ethanol treatment in the vector fused with p35S fragment and the protoplast transformed with 1-2f . After the ethanol treatment, the 3-7p vector had less GFP expression than the 1-2p transformer because the 3-7p vector had a TADII region that inhibits ADH2 activity. Can be estimated.

As such, the 1-2p vector eliminated the possibility of inhibiting protein activity by removing the self-regulatory site located between TADI and TADII, compared to the previously developed alcohol expression induction vector. Therefore, the maximum expression induction time is shortened by 1 hour, which is 10 times faster than 16 hours of the existing vector, and the duration of inducing expression of 50% or more of the existing vector is about 24 hours, whereas the 1-2p vector is used. Lasted 48 hours or more, indicating a two-fold or longer expression duration (FIG. 12). In addition, it is easy to use because it is about 2 kb smaller than the existing vector.

As such, the plant expression vector provided according to the present invention can use ethanol, which can be sprayed at a large scale due to its high volatility, as an induction chemical, and thus, a desired gene from the plant can be obtained only at a specific time or stage of plant culture. . Thus, the present invention is useful for gene function analysis and for the production of gene expressing plants.

1 shows the structure of an alc induced gene expression system.

2 shows glycolytic and glucogenesis pathways in S. cerevisiae .

3A shows the functional region of ADR1 protein (SEQ. ID No .: 1).

3B shows the configuration of the ADH2 promoter region (SEQ. ID No. 2).

4A-4D show schematic diagrams of ethanol-derived expression vectors.

5A shows the configuration of pADH2.

Figure 5b shows an electrophoresis result of pADH2 fragments do not have a restriction enzyme recognition site (Hind III / Bg1II).

Figure 5c shows an electrophoresis result of the inserted fragment pADH2 restriction enzyme recognition sites (Hind III / Bg1II).

6A shows the configuration of pADH2-p35S fusion promoter fragments.

6B shows the results of electrophoresis of pADH2-p35S fusion promoter fragments.

7A shows the results of electrophoresis of ADR1 fragment (516 bp) (SEQ. ID No .: 3) containing the TAD I domain.

7B shows the results of electrophoresis of ADR1 fragments (1,713 bp) (SEQ. ID No .: 4) containing the TAD I-III domains.

8A shows the configuration of the 35S-ADR1 (TAD I) -tnos PCR result.

8B shows the results of electrophoresis of 35S-ADR1 (TAD I) -tnos PCR results.

Figure 9a shows the configuration of 35S-ADR1 (TAD I to TAD III) -tnos PCR results.

Figure 9b shows the results of electrophoresis of 35S-ADR1 (TAD I to TAD III) -tnos PCR results.

10A is a confocal micrograph of GFP expression before and after 2% ethanol treatment for the negative control (pBI121 protoplasts).

10B is a confocal micrograph of GFP expression before and after 2% ethanol treatment for the expression vector (1-2f) of the invention.

10C is a confocal micrograph of GFP expression before and after 2% ethanol treatment for expression vectors (1-2p) of the present invention.

10D is a confocal micrograph of GFP expression before and after 2% ethanol treatment for the expression vector (3-7p) of the present invention.

FIG. 11 shows the relative fluorescence intensity of GFP after ethanol treatment in transgenic Arabidox protoplasts.

12 is a comparison of the relative expression induction intensity by ethanol of the vector according to the prior art and the 1-2p vector of the present invention.

<110> Seoul National University Industry Foundation <120> ETHANOL-INDUCIBLE EXPRESSION VECTOR <130> IA23915 <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 7303 <212> DNA <213> Saccharomyces cerevisiae <300> Hartshorne, T. A. Blumberg, H. Young, E. T. <302> Sequence homology of the yeast regulator protein ADR1 with Xenopus transcription factor TFIIIA <303> Nature <304> 320 <305> 6059 <306> 283-287 <307> 1986-01-01 <313> 1166-7303 <400> 1 atccgagtta ttgcccaagt tgagacaaat tttccaacaa tttggcaagg atttgctggc 60 cacccatggt aatgacattc aggtgcccga atctcaggtg aaatctaact acacaagagg 120 taaccaaaaa agtagcttta ccgaaatcaa ggactccgct tcaaagccaa aaaagaatgc 180 acttccctct tctacttcaa cttcagcacc agtctccagc accaacaaag tcccccaaaa 240 cggaagcggc aatagtacat ctatttattt agaaccaact tttaatgtgc cttcctccga 300 gctctacgaa acgttccttg ataagcaacg catattggcc tggaccagat cagcacaatt 360 tttcaacagc ggaccgaagc tggagaccaa agaaaaattc gagctctttg gcggcaacgt 420 gatcagcgag ttggtttcat gcgaaaagga caagaaactt gttttccact ggaaactcaa 480 ggattggtct gcccctttca actctactat cgaaatgact ttccatgagt cgcaagaatt 540 ccatgaaacc aaattacagg tgaagtggac tggcataccc gttggcgagg aagatcgcgt 600 gcgcgctaat tttgaagaat actacgtgcg ttctattaaa ttgacattcg gctttggtgc 660 cgtattataa gatatattgt tctcatgttt caataaaagt atgcgtaaat atttacatac 720 cactattggc atagatagaa tgcaatacat catgtagttc tgtacggcat ccgcgcggct 780 ccgatcgccc gtcctacttt tgcggcccgg cgccgacggc ctccccggaa tttaccaacg 840 aagaggaaaa taaactggag ttataacgcc attggcggga aactccgaaa ccatagcgcc 900 gtggtctttc cccggttttt tggcgccggg tgtgcttgcg ttgtgtgaac ggcgccctgg 960 cagctgaggg ggaagggaaa cgccggaggc gacggcaagg atgaccatgg cgtcaaggaa 1020 catgcggaac atgctagaat gattgccgaa aggcgggccc tagtcaatgg catcatgcgg 1080 gaacgtgcag taaagctcaa tgtaacttga tctatcggcg ttgttcgagc taaggacgat 1140 gtgatagact gaggaagggt gaacgtcgat agttagccgt tgtgaactaa ataaaagtga 1200 tattttttcg ttgttctttt cttggtttgg cggtgttttc atttgttttc atgttttact 1260 tcgccacagg ttttcaaaga acaacgcctt aaaaatagga aaacgttttc gctacaggtg 1320 ttgttattat tgttgttgtg ctgttgttta ttgtgctata cttgtggtat ttattctgga 1380 cttccgatcg gaaattttct tcccttgaag accttttgaa gacaacagtt atatatcatt 1440 gatctgaatt tctcaggcta ttttcaaaat tccatacctc cttattccaa catttgctcg 1500 actactatag aaaagcctta ttcttttatc tttgaaagaa agaaaaggtg tcatagcaaa 1560 agtttattgt tactctgttt tgatatactc cctcttattc gttggaagta taagattgat 1620 ttgcataaat taaccaatca ttttgctact ttcccggttc tccctttatt ataaacactt 1680 cagaaaaata ttctgctact attccttact ttactataag aattttgttt tccaaaaaaa 1740 aaaaatataa aaaaaataat catactctat tactatggct aacgtagaaa aaccaaacga 1800 ttgttcaggc tttcccgttg ttgacttgaa ttcgtgcttt tctaacggct tcaataatga 1860 gaaacaagaa atagaaatgg aaacggatga ttcaccgatt ttattaatgt catcatcagc 1920 ttccagagaa aactcaaaca ctttctctgt gatacagagg acgccagatg gaaagatcat 1980 taccacaaat aataatatga actccaagat taacaagcaa ctggacaagt tgcccgaaaa 2040 tttaaggctt aatggtagaa cccccagtgg gaaactaagg tcatttgttt gcgaggtttg 2100 tacgagagcg ttcgcaagac aagagcactt gaaaagacat tacagatcgc atacaaatga 2160 aaaaccttat ccctgtggcc tctgcaacag atgctttact aggagggact tactgatcag 2220 gcatgctcaa aaaatccata gtggtaattt aggggaaacg atttcccata ccaagaaagt 2280 gtcgagaact ataactaaag ctcggaaaaa ttctgcatcc tcagtcaagt ttcaaactcc 2340 aacctatggt actccagata atggtaattt tttgaatcgc actactgcca atacaagaag 2400 aaaagcaagc cctgaagcta atgttaaacg taagtacttg aaaaaactga cgcgcagggc 2460 ttcatttagc gcacaatcag catccagcta tgctttgccc gaccaatctt cgctagaaca 2520 acatccaaag gatcgtgtta aattttctac gcctgaatta gttccacttg acttgaagaa 2580 tcctgaactt gactcttcgt ttgacctgaa tatgaatcta gatttaaacc taaatctaga 2640 ttccaatttc aatatagcat taaaccgttc tgattcttct ggatcaacaa tgaatttgga 2700 ttataaattg cccgaatcag caaataacta cacatattct tccggctcac caacccgcgc 2760 atatgtcggc gctaacacga attctaagaa cgcttcattt aatgacgcag acttattgtc 2820 gtcgtcgtac tggataaaag cctataatga tcatttgttt tcagtatctg aaagtgatga 2880 aacttctcca atgaactctg aattaaacga cactaaatta atcgtcccag attttaaatc 2940 gactatacat catttgaagg attcaaggtc ctcctcttgg actgttgcta tagataataa 3000 tagcaataac aataaggtat cagacaacca acctgatttc gtcgattttc aagaactgct 3060 ggataatgat actttaggta atgatttgtt agagaccact gccgttttaa aagaatttga 3120 acttttacat gatgatagcg taagtgctac cgccacgtca aatgagattg acctttccca 3180 tttgaaccta tcaaactctc caatttctcc tcataagtta atttataaga ataaagaggg 3240 gaccaatgac gatatgttga tttctttcgg actcgatcat ccttccaatc gcgaagatga 3300 tctggataag ctatgtaata tgaccagaga tgttcaagcc atattcagtc aatatttgaa 3360 aggagaagag tctaaacgat ccctggaaga ctttttatca acgtcaaaca ggaaagaaaa 3420 gccagatagc ggcaactata ctttttatgg gttagattgt ttaacgttat cgaaaatatc 3480 aagagctctg ccggcctcca ctgtgaacaa caatcagcca tcgcattcca tagaatcaaa 3540 gctatttaat gaaccaatga gaaatatgtg cattaaagtg cttagatact atgaaaagtt 3600 cagtcatgat agtagtgaga gtgtcatgga ctctaatcca aacttgctgt ccaaagaatt 3660 gttaatgcca gctgtgagtg aattgaacga atatttagat cttttcaaga ataatttcct 3720 tccccatttc cctattattc acccaagctt gcttgatttg gatttggata gcttgcaacg 3780 atatactaat gaggatgggt atgatgacgc tgaaaacgcg cagttgtttg atcgattaag 3840 tcaagggaca gataaagaat atgattacga gcactatcaa atcttgtcca tttcgaaaat 3900 cgtttgttta cccttattta tggccacatt tggttctttg cataagttcg gttacaaatc 3960 tcaaacaata gaattgtatg agatgagtag aagaattcta cattcttttt tggagactaa 4020 aagaaggtgt cgcagtacaa cagtaaatga cagttatcag aacatttggt tgatgcaatc 4080 cctaatattg agcttcatgt tcgctctagt tgctgattat ttggagaaaa ttgactcctc 4140 tttgatgaaa aggcaattgt ccgcattatg ttcaacgatc agatcaaact gtttaccgac 4200 aatttctgca aattctgaga agagtatcaa taataacaat gaacctttaa catttggttc 4260 tcctcttcaa tacatcattt ttgagtcaaa aattagatgc accttaatgg cttatgattt 4320 ttgtcagttc ttgaaatgtt tcttccatat taaattcgat ttgtctataa aggaaaaaga 4380 tgttgaaacc atttatattc ccgacaatga gtcaaaatgg gccagtgaat cgataatatg 4440 taatgggcat gttgtgcaaa agcaaaattt ttatgatttt agaaactttt attacagttt 4500 cacgtatgga cacttacact caataccaga atttttaggg tcatctatga tttattatga 4560 atacgattta agaaaaggaa ccaaatcaca tgtgtttttg gatcgaatcg atacgaaaag 4620 gctagagagg agtcttgaca cttcttccta tggcaatgat aatatggcag caaccaataa 4680 aaatattgcg atcttaattg atgacaccat aattttgaaa aataatttaa tgtcaatgag 4740 attcatcaaa cagattgatc gctcgtttac tgagaaggtt agaaaaggac aaatagcaaa 4800 gatatatgat tcctttttga actctgtgag gttgaatttt ttgaagaatt attcagttga 4860 agtattgtgt gaatttttag tagcgttgaa cttttcaatc cgtaatattt cgtctttata 4920 cgtagaagaa gaaagtgatt gctcccaaag aatgaattct ccagagctgc caaggatcca 4980 cctgaataat caagcgcttt ctgtcttcaa tttacaaggc tattactatt gcttcatcct 5040 aattatcaaa tttttattgg attttgaagc aactccaaat tttaagttac tgagaatttt 5100 tattgagttg agaagccttg cgaattctat tttacttccc acactttcaa gattgtatcc 5160 gcaagagttt tctggatttc ctgatgttgt atttacgcaa caatttataa ataaagataa 5220 tggtatgctt gtccctggtt tatccgcaaa tgaacaccat aatggtgcaa gtgcagctgt 5280 taagactaag ttagccaaaa agatcaatgt tgaagggctt gcaatgttta ttaatgaaat 5340 cctagttaac tcttttaacg atacctcttt tttgaatatg gaggatccta ttcgaaatga 5400 attttccttt gataatgggc acagggcagt gacagacttg cctcgttcag cacatttcct 5460 atcggatacc ggcctagaag gtattaactt cagcggctta aatgattcgc atcaaactgt 5520 ttctactttg aatcttttac gttacgggga aaatcattca tcaaaacata aaaatggtgg 5580 aaaggggcaa ggatttgccg aaaagtacca attatctctg aaatatgtta ctattgccaa 5640 gttatttttc accaatgtta aagaaaacta cattcattgt cacatgttag ataagatggc 5700 aagtgatttc cacactttgg aaaatcatct aaagggaaac agttgacgat tatgtctcgt 5760 tgctgatatg tgtcgtccca gtactcaact ctttctgatg agattttgaa tatctcattt 5820 tatttctcat caaacagcca tgcatgcgac tgaagtggac tactgcggga gaacaccgat 5880 cttatctttg aaattgttgc acaaaaatat ttcactacct tggcgtttct catctcaccc 5940 attggggtga atctcgttat tgctccttat tttggtccat tttatattaa atcctgtatc 6000 atgttcaaca tttttcatct tatacctcat acaaaaaaga aaagctattt atgaaagtta 6060 gttcattaca agatgcaagc ctaaaatata tgcttgatct taaaattcat gtatagaaga 6120 aaaagacctt accaacgttg ttggttaccg tgtataacta ataccaatct tgaacattaa 6180 ccaatcctgg cgtgtgggag gatgttctta gacttaatta agaatctcta aatttttttt 6240 tatttaatcg tccctttcta tcaattatga gtttatatat ttttataatt tcatctaacc 6300 tcagaaatag tgttgtatat atcattgtcc gtaatatcat cgtgaaaacc agtgtcctcg 6360 ttaattattg tctgaattag ccattcttta gattcagtgt gaaatatgta attaaatttc 6420 ttaaatttca gtgatatttg acttctcaat ctttcgagaa gcttcatctg agatttacca 6480 ttattttcgt tagcatatat gagaaacttt aactttcgat tagataattc tgatcctatt 6540 ctgggtacta aagaatctaa ggcatttaac aatggctctg tatcatctac atcaatattg 6600 ggtaaatcaa ttgattgcat tctacctgcg ctcaaacatg caaaagcaaa tttgacaaaa 6660 ctgtccaact cagatctacc ccaaacaata acaatgtagt catttaacat tttcttcact 6720 ccacatatct gatctcttaa attagatccg cgtaagaatt gtgtataaaa tgaaaaaata 6780 ttgttggatt taatgattcc tccctttgat ggagaatcta acgataaccg aaaactttca 6840 cccgaaggta ataggtattg gtatattaaa tgtggtacta ttcttttctt ttcaatgcat 6900 gcacttatga atttccagtg tagtgtaggc caccccaacg ccaaagtttc taagtacttt 6960 aagcttctta aatgccgttt tgtgattagg caagcaaatc tgcagtctgc aaatagggaa 7020 tgaggttcca ggctagcttc aaggccaatt ctcgaatatt atctgtatta cctttattga 7080 ctaaagattt agctagcgga tgagtgaagt taaataaagt ggaaaatcct gactcaatta 7140 cagtgccgcc ttgcgattca atggtctgtc gaagttcctc tctattttcg aataggcttg 7200 tcaaaacaaa aatacattta tcaaatacat tccctgttct gatttcacca gaagaaaaaa 7260 gcaggcttgg cgctcctgca tgttcagcta atgcgtctct aga 7303 <210> 2 <211> 312 <212> DNA <213> Saccharomyces cerevisiae <220> <221> promoter (222) (1) .. (165) <223> S. cerevisiae ADH2 gene promoter <300> <301> Leplatois, P. Loison, G. Pessegue, B. Shire, D. <302> Artificial promoter for protein expression in yeast <308> <310> EP <311> 1991-07-03 <313> 1-165 <400> 2 gtcttcatta acggctttcg ctcataaaaa tgttatgacg ttttgcccgc aggcgggaaa 60 ccatccactt cacgagactg atctcctctg ccggaacacc gggcatctcc aacttataag 120 ttggagaaat aagagaattt cagattgaga gaatgaaaaa aaaaaaaaaa aaaaaggcag 180 aggagagcat agaaatgggg ttcacttttt ggtaaagcta tagcatgcct atcacatata 240 aatagagtgc cagtagcgac ttttttcaca ctcgaaatac tcttactact gctctcttgt 300 tgtttttatc ac 312 <210> 3 <211> 516 <212> DNA <213> Saccharomyces cerevisiae <220> <221> gene (222) (1) .. (516) <223> Saccharomyces cerevisiae Adr1 TADI <400> 3 atggctaacg tagaaaaacc aaacgattgt tcaggctttc ccgttgttga cttgaattcg 60 tgcttttcta acggcttcaa taatgagaaa caagaaatag aaatggaaac ggatgattca 120 ccgattttat taatgtcatc atcagcttcc agagaaaact caaacacttt ctctgtgata 180 cagaggacgc cagatggaaa gatcattacc acaaataata atatgaactc caagattaac 240 aagcaactgg acaagttgcc cgaaaattta aggcttaatg gtagaacccc cagtgggaaa 300 ctaaggtcat ttgtttgcga ggtttgtacg agagcgttcg caagacaaga gcacttgaaa 360 agacattaca gatcgcatac aaatgaaaaa ccttatccct gtggcctctg caacagatgc 420 tttactagga gggacttact gatcaggcat gctcaaaaaa tccatagtgg taatttaggg 480 gaaacgattt cccataccaa gaaagtgtcg agaact 516 <210> 4 <211> 1713 <212> DNA <213> Saccharomyces cerevisiae <220> <221> gene (222) (1) .. (1713) <223> Saccharomyces cerevisiae Adr1 TADI-TADIII <400> 4 atggctaacg tagaaaaacc aaacgattgt tcaggctttc ccgttgttga cttgaattcg 60 tgcttttcta acggcttcaa taatgagaaa caagaaatag aaatggaaac ggatgattca 120 ccgattttat taatgtcatc atcagcttcc agagaaaact caaacacttt ctctgtgata 180 cagaggacgc cagatggaaa gatcattacc acaaataata atatgaactc caagattaac 240 aagcaactgg acaagttgcc cgaaaattta aggcttaatg gtagaacccc cagtgggaaa 300 ctaaggtcat ttgtttgcga ggtttgtacg agagcgttcg caagacaaga gcacttgaaa 360 agacattaca gatcgcatac aaatgaaaaa ccttatccct gtggcctctg caacagatgc 420 tttactagga gggacttact gatcaggcat gctcaaaaaa tccatagtgg taatttaggg 480 gaaacgattt cccataccaa gaaagtgtcg agaactataa ctaaagctcg gaaaaattct 540 gcatcctcag tcaagtttca aactccaacc tatggtactc cagataatgg taattttttg 600 aatcgcacta ctgccaatac aagaagaaaa gcaagccctg aagctaatgt taaacgtaag 660 tacttgaaaa aactgacgcg cagggcttca tttagcgcac aatcagcatc cagctatgct 720 ttgcccgacc aatcttcgct agaacaacat ccaaaggatc gtgttaaatt ttctacgcct 780 gaattagttc cacttgactt gaagaatcct gaacttgact cttcgtttga cctgaatatg 840 aatctagatt taaacctaaa tctagattcc aatttcaata tagcattaaa ccgttctgat 900 tcttctggat caacaatgaa tttggattat aaattgcccg aatcagcaaa taactacaca 960 tattcttccg gctcaccaac ccgcgcatat gtcggcgcta acacgaattc taagaacgct 1020 tcatttaatg acgcagactt attgtcgtcg tcgtactgga taaaagccta taatgatcat 1080 ttgttttcag tatctgaaag tgatgaaact tctccaatga actctgaatt aaacgacact 1140 aaattaatcg tcccagattt taaatcgact atacatcatt tgaaggattc aaggtcctcc 1200 tcttggactg ttgctataga taataatagc aataacaata aggtatcaga caaccaacct 1260 gatttcgtcg attttcaaga actgctggat aatgatactt taggtaatga tttgttagag 1320 accactgccg ttttaaaaga atttgaactt ttacatgatg atagcgtaag tgctaccgcc 1380 acgtcaaatg agattgacct ttcccatttg aacctatcaa actctccaat ttctcctcat 1440 aagttaattt ataagaataa agaggggacc aatgacgata tgttgatttc tttcggactc 1500 gatcatcctt ccaatcgcga agatgatctg gataagctat gtaatatgac cagagatgtt 1560 caagccatat tcagtcaata tttgaaagga gaagagtcta aacgatccct ggaagacttt 1620 ttatcaacgt caaacaggaa agaaaagcca gatagcggca actatacttt ttatgggtta 1680 gattgtttaa cgttatcgaa aatatcaaga gct 1713 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pADH2 primer F-primer <400> 5 acgcttttca ttaacggc 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pADH2 primer-R primer <400> 6 agatctcgct actggcactc 20 <210> 7 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Fused pADH2 R primer <400> 7 agatctcgtg ttctctccaa atgaaatgaa cttccttata tgtgataggc atgctatagc 60 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Oligo dT primer <400> 8 tttttttttt ttttt 15 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ADR1 F primer <400> 9 ggatccatgg ctaacgta 18 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Adr1 R primer <400> 10 gagctcagct ttagttatag ttc 23 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Adr1III F primer <400> 11 ggatccatgg ctaacgta 18 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Adr1III R primer <400> 12 gagctcagct cttgatattt tc 22 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PBI121 F primer <400> 13 gattacgcca agcttgcatg cctg 24 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PBI121 R primer <400> 14 atgcaagctt gtcacgacgt tgtaaaacg 29 <210> 15 <211> 306 <212> DNA <213> Artificial Sequence <220> <223> ADH2-35S fusion promoter <400> 15 gtcttcatta acggctttcg ctcataaaaa tgttatgacg ttttgcccgc aggcgggaaa 60 ccatccactt cacgagactg atctcctctg ccggaacacc gggcatctcc aacttataag 120 ttggagaaat aagagaattt cagattgaga gaatgaaaaa aaaaaaaaaa aaaaaggcag 180 aggagagcat agaaatgggg ttcacttttt ggtaaagcta tagcatgcct atcacatata 240 aatagaagat ctcgtgttct ctccaaatga aatgaacttc cttatatgtg ataggcatgc 300 tatagc 306

Claims (9)

Chemical inducible plant according to an expression vector, saccharose as MY unrestricted access celebrity DNA sequence encoding the part design ADR1 transcription activity of (Saccharomyces cerevisiae) (SEQ. ID No .: 1) and coding for the alcohol dehydrogenase ADH2 promoter An alcohol-derived plant expression vector in which a DNA sequence (SEQ. ID No .: 2) and a target gene are operably linked. The expression vector according to claim 1, wherein ADR1 essentially comprises a DNA sequence (SEQ. ID No .: 3) encoding a TADI region which is a zinc finger region. The expression vector of claim 1 or 2, wherein the ADH2 promoter essentially comprises a DNA sequence (SEQ. ID No .: 4) encoding a UAS site (200 bp DNAs). The expression vector of claim 1 or 2, wherein the ADH2 promoter is an ADH2-35S fusion promoter (SEQ. ID No. 15). The expression vector according to claim 1 or 2, wherein the alcohol is ethanol. A plant transformed with the vector of claim 1. i) providing protoplasts of plant cells, ii) a DNA sequence encoding the ADR1 site, a transcriptional activator of Saccharomyces c erevisiae (SEQ. ID No .: 1) and a DNA sequence encoding the ADH2 promoter, an alcohol dehydrogenase (SEQ. ID No .: 2) and providing an alcohol-derived plant expression vector to which the target gene is operably linked, iii) introducing the plant expression vector prepared in ii) into the protoplast of i), iv) culturing the plant into which the expression vector of iii) has been introduced, and v) a method of expressing a gene of interest from the plant by alcohol induction, comprising contacting the alcohol with the plant. 8. The expression vector of claim 7, wherein the alcohol is ethanol. A plant transformed by the method of claim 7 or 8.