KR100574563B1 - Plant high-efficiency expression promoter derived from Arabidopsis thaliana and plant high-efficiency expression vector comprising the same - Google Patents

Abstract

The present invention provides a plant high efficiency expression promoter and intron derived from the microsomal oleic acid desaturase gene of Arabidopsis thaliana and a plant high efficiency expression vector containing them and transformed with the expression vector. It is about a plant.

According to the present invention, the expression of the gene introduced in the transgenic plant is about 10 times or more in flowers and seed tissues, and about 3 times in roots and stems, compared to the CaMV 35S promoter derived from Cauliflower mosaic virus, which is widely used. Can be increased over. In addition, the present invention can be usefully used in the development of transformed plants to mass-produce useful materials.

Description

Plant high-efficiency expression promoter derived from Arabidopsis thaliana and plant high-efficiency expression vector comprising the same}

Figure 1 shows a promoter derived from the Arabidopsis microsomal oleic acid unsaturated agent gene of the present invention and the nucleotide sequence of the intron present in the gene.

Figure 2a is a schematic diagram showing a binary vector, pBI101 (Cat. # 6017-1, Clontech, USA) containing β-glucuronidase (hereinafter abbreviated as 'GUS') reporter gene.

Figure 2b is a schematic diagram showing a binary vector (hereinafter, abbreviated as 'pAtW6-P2.4') containing a promoter derived from the Arabidopsis microsomal oleic acid unsaturated agent gene of the present invention and the intron present in the gene.

Figure 3 shows that six kinds of binary vectors were digested with restriction enzymes Hin dIII and Bam HI, followed by electrophoresis on 0.7% agarose gel.

FIG. 4 shows Arabidopsis transformed by Agrobacteria containing six binary vectors of FIG. 3 and a pBI121 (Cat #: 6018-1, Clontech, USA) binary vector.

FIG. 5 shows histochemical method of GUS activity using whole tissues of roots, stems, leaves, cauliflowers, flowers, developmental seeds and pods of Arabidopsis plants transformed with pAtW6-P2.4 and pBI121. It is dyed with.

FIG. 6 shows the activity of GUS enzymes using the entire tissues of plants-roots, stems, leaves, cauline, flowers, developmental seeds, and pods in the seven types of transgenic Arabidopsis obtained.

The present invention provides a plant high efficiency expression promoter and intron derived from the microsomal oleic acid desaturase gene of Arabidopsis thaliana and a plant high efficiency expression vector containing them and transformed with the expression vector. It is about a plant.

Fatty acids in plants are important components of the cell membrane and the storage oil in the seeds. In particular, the microsomal oleic acid desaturase is present in the membrane of the endoplasmic reticulum, and the polyunsaturated monounsaturated fatty acid, oleic acid, present in the sn- 1 and sn- 2 positions of phosphatidylcholine It is an enzyme that catalyzes the conversion of fatty acids into linoleic acid.

Microsomal oleic acid desaturator genomic genes have been reported in Arabidopsis, petunia, and cotton, with one gene present in the Arabidopsis genome and two or more genes in other plants (Okuley et al., 1994; Verwoert et al., 2000; Pirtie et al., 2001). In particular, the microsomal oleic acid unsaturated gene gene of Arabidopsis is expressed in all tissues of plants and is involved in linoleic acid synthesis, which constitutes plant membranes and storage oils in seeds.

To date, microsomal oleic acid desaturator genomic genes have been reported from the three types of plants described above, but no studies have been made worldwide on their promoters and introns present in their genomic genes.

Introns present on several plant genomic genes, including the first intron of corn alcohol dehydrogenase gene in maize, have been reported to increase their gene expression in plants (Callis et al. , 1987; Norris et al., 1993; Currie et al., 1991 and 1993). However, no studies have been made on the regulation of their gene expression by introns present in the microsomal oleic acid desaturator genomic gene.

Recently, many studies have been conducted to improve plant traits using genetic engineering techniques. In this process, when a foreign useful gene is to be expressed in a transgenic plant, a promoter involved in gene expression is required. For this purpose, a promoter derived from cauliflower mosaic virus (CaMV35S), which is expressed in all tissues of plants, has been widely used. However, when the CaMV35S promoter is used, the expression of foreign genes is low in other tissues of the plant except the leaf and the active cell division of the plant. Therefore, there is a strong demand for a promoter capable of increasing the expression of foreign transgenes in transgenic plants.

Therefore, the present inventors focused on the expression of microsomal oleic acid desaturants derived from Arabidopsis edodes from all tissues of plants, and cloned the promoters and introns. As a result, the promoter of the Arabidopsis-derived microsomal oleic acid desaturator gene and the intron present in the gene were compared to the CaMV 35S promoter derived from Cauliflower mosaic virus, which is widely used. The present invention has been completed by revealing that expression can be increased about 10 times or more in flower and seed tissues, and about 3 times or more in roots and stems.

Accordingly, it is an object of the present invention to provide a plant high efficiency expression promoter and / or intron for enhancing expression derived from the microsomal oleic acid unsaturated agent gene of Arabidopsis.

Another object of the present invention is to provide a plant high efficiency expression vector and a plant transformed with the expression vector containing the above high efficiency expression promoter and / or intron for enhancing expression.

Still another object of the present invention is to provide a method for efficiently expressing a foreign gene in a transgenic plant by using the above-described high efficiency expression promoter and / or intron for enhancing expression when a large amount of useful substance is to be produced in a plant.

In order to achieve the object of the present invention, the present invention provides a high efficiency expression promoter of plants comprising the nucleotide sequence of the 1 to 959 (-959 to -1) region of SEQ ID NO: 1. Parentheses are numbered minus in the upstream direction from the transcription initiation site.

The high-efficiency expression promoter of the present invention can significantly increase the expression of the gene introduced in the transgenic plant as compared to CaMV 35S promoter derived from Cauliflower mosaic virus, which is widely used in the past.

In order to achieve the object of the present invention, the present invention provides an intron for enhancing expression of a plant comprising a nucleotide sequence of +144 to +1274 site from the transcription initiation site of SEQ ID NO.

The expression enhancing intron of the present invention can significantly increase the expression of a target gene by helping to initiate transcription of a gene introduced from a plant or by increasing the stability of the transcribed mRNA.

In order to achieve another object of the present invention, the present invention provides a high-efficiency expression vector of a plant containing a high-efficiency expression promoter and / or intron for enhancing expression of the plant.

The plant high efficiency expression vector of the present invention may contain only the promoter of the present invention or a combination of a general plant expression promoter such as a CaMV 35S promoter in the intron of the present invention, but preferably contains both the promoter and the intron of the present invention. It is good to express transgenes in plants with high efficiency.

The plant high efficiency expression vector of the present invention may be a transient expression vector capable of temporarily expressing a foreign gene in the introduced plant, but preferably, the plant can be permanently expressed in the introduced plant. Is a binary vector

The binary vector that can be used in the present invention may be any binary vector containing BR and BL of T-DNA capable of transforming plants when present with Ti plasmid of A. tumefaciens , but preferably in the art. PBI101 (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121, pBIN20, BIBAC vectors, and the like, which are frequently used in the present invention, are recommended.

In the plant seed specific expression vector of the present invention, the promoter and / or intron of the present invention is located in front of the foreign gene contained in the binary vector. In the present invention, pAtW6-P2.4 (Fig. 2b) in which the promoter and intron of the present invention were inserted into a binary vector (pBI101) containing a GUS reporter gene as an example of a foreign gene was produced, but the GUS reporter gene was used for other purposes It will be apparent to those skilled in the art that substitution with a foreign gene is possible.

In order to achieve another object of the present invention, the present invention provides a plant transformed with the plant high efficiency expression vector of the present invention.

When the plant high efficiency expression vector is a binary vector, for example, the plant is transformed according to a floral dip method (Clough and Bent, 1998, The Plant Journal), and in the case of a transient expression vector, for example, a particle bombardment method (Lacorte et al. , 1997, Plant Cell Reports Plants can be transformed. The plant seed specific expression vector of the present invention can transform any plant irrespective of dicotyledonous or monocotyledonous plants, but in the present invention, only Arabidopsis was transformed.

In order to achieve another object of the present invention, the present invention provides a method for efficiently expressing a foreign gene in a transgenic plant using the plant high efficiency expression vector of the present invention.

The foreign gene may be any gene for mass expression in a plant, and may be located after the promoter and / or intron in a plant high efficiency expression vector of the present invention and fused with a reporter gene as necessary.

The present invention relates to a promoter of the microsomal oleic acid desaturant gene derived from Arabidopsis thaliana and an intron present in the gene, which is compared to the CaMV35S promoter derived from Cauliflower mosaic virus, which is widely used. The expression of genes introduced in transgenic plants can be increased by at least about 10 times in flower and seed tissues and at least about 3 times in roots and stems. Therefore, the present invention can be used for the development of transgenic plants to mass produce useful substances.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Intron Acquisition and Sequence Analysis of a Promoter from Microsomal Oleic Acid Unsaturated Gene Derived from Arabidopsis

Arabidopsis genomic sequence (http://mips.gsf.de/proj/thal/index.html, At3g12120) to secure the promoter of the Arabidopsis microsome oleic acid desaturator gene (FAD2) and introns present in the gene Based on GenBank Accession No: NM_112047, forward primer (AraW6F1: 5'-CCG AAG CTT GGT GTG TCG TCG TTA TAC GCG GTA AA-3 ') and reverse primer (AraW6R4: 5'-CGA GGA TGA CTG CAG AAA ACC AAA AGC AAA AG-3 ') was produced, genomic DNA was extracted from the young leaves of Arabidopsis subsidiary, followed by polymerase chain reaction (hereinafter, abbreviated as'PCR') to microsomal oleic acid unsaturated gene. The intron present in the promoter of and its gene was isolated. The isolated 2.4 kb DNA fragment was cloned into a pGEM-T vector (Promega, USA) and then sequenced using an ABI Bigdye cycle sequencing kit (PE Applied Biosystems, USA) (FIG. 1). The analyzed sequences were compared with the existing Arabidopsis genome database (http://mips.gsf.de/proj/thal/index.html, At3g12120, GenBank Accession No: NM_112047) and found to be exactly the same clones. Italicized sequences represent intron sites present in the 5 'untranslated region of the gene. In addition, the protein synthesis start codon 'ATG' is shown in bold type, and the base (red) "A", which is a transcription initiation site, is expressed as '+1'.

SEQ ID NO: 1 shows the upstream of the transcription start site in FIG. 1, and SEQ ID NO: 2 shows the downstream from the transcription start site in FIG.

Example 2 Confirmation of transcription start site of Arabidopsis-derived microsomal oleic acid unsaturated agent gene

To investigate the activity of the promoter region of the Arabidopsis microsomal oleic acid desaturator gene ( At- FAD2), the exact transcriptional initiation site of this gene must be identified, so cRACE ( c ircular first-strand cDNA-mediated r apid a mplication of c According to the DNA e nds) method (Nucleic Acid Research 23: 3796-3797, 1995), a study was performed to identify the transcription initiation site of the At- FAD2 gene. After isolation of total RNA from Arabidopsis flower, cDNA pools were prepared using reverse transcriptase (Super Script ^TM II RT, G _IBCO BRL), and then the gene-specific primers (AraW6R2; 5'-) were used as templates. CAG CGT TTG AAA CAA TGC GGC GGG-3 'and AraW6R3; 5'-CGG CAT TCT TCC ACC TGC ACC CAT G-3') and adapter primers of the cDNA library (AP1; 5'-CCA TCC TAA TAC GAC TCA CTA TAG PCR was performed using GGC-3 'and AP2; 5'-ACT CAC TAT AGG GCT CGA GCG GC-3'), resulting in the nucleotide sequence preceding 147 base from the translation initiation codon (ATG). A "was identified as the transcription initiation site. This is indicated as '+1' in FIG.

Example 3 Preparation of binary vector using active fragment of Arabidopsis-derived microsomal oleic acid unsaturated gene gene promoter expressed in all tissues of plant and intron present in genomic gene

Gene specific primers based on the nucleotide sequence of FIG. 1 to analyze the activity of the At -FAD2 gene promoter, the analysis of promoter active fragments, and the effect of intron present in the gene on the transgenic plants (Table 1) After the preparation, the promoter site was partially removed from the original size and 5 'end by PCR. The obtained PCR product is a binary vector containing p-glucuronidase (hereinafter abbreviated as 'GUS') gene using Hin dIII and Bam HI restriction enzyme sites, pBI101 (Cat. # 6017-1, Clontech, USA) (Fig. Inserted into 2a) to prepare a binary vector called pAtW6-P2.4 (Fig. 2b). Wherein GUS is a reporter gene encoding β-glucuronidase, NPTII is a kanamycin resistance marker gene encoding Neomycin Phosphotransferase II, and Nos-pro and Nos-ter are plant expression promoters and terminators of NPTII. The GUS reporter gene is expressed in plants by the inserted promoter and the Nos (Nopaline synthase) terminator (Nos-ter).

Each of the prepared binary vectors was introduced into Escherichia coli ( DH5α ), and then plasmids were extracted using alkaline lysis method (Sambrook et al., 2001) and digested with restriction enzymes Hin dIII and Bam HI, followed by 0.7% agarose. Electrophoresis on the gel (Fig. 3). Here, pAtW6-P2.4 is a binary vector containing an existing promoter region amplified by gene specific primers, AraW6F1 and AraW6R4, and introns present in the gene, and pAtW6-PF1 are gene specific primers, AraW6F1 and AraW6R1. PAtW6-PF2 is a binary vector containing a promoter region amplified by, pAtW6-PF2 is a binary vector containing a promoter region amplified by AraW6F2 and AraW6R1, and pAtW6-PF3 is a gene specific primers, AraW6F3 and AraW6R1 PAtW6-PF4 is a binary vector containing a promoter region amplified by pAtW6-PF4 is a gene-specific primers, a binary vector containing promoter sites amplified by AraW6F4 and AraW6R1, and pAtW6-PF5 is a gene specific primers, AraW6F5 And a binary vector containing a promoter region amplified by AraW6R1.

Table 1 below shows the nucleotide sequences of the gene specific primers used in the PCR reaction.

(Example 4) Transformation of Arabidopsis plants using the prepared binary vector

The six types of binary vectors prepared as described above (pAtW6-P2.4, pAtW6-F1, pAtW6-F2, pAtW6-F3, pAtW6-F4, pAtW6-F5) and pBI121 binary vectors (Cat. # 6018-1, The introduction of Clontech, USA into Agrobacterium tumefaciens C58C1 (Suh et al., 2002, Planta) was performed based on the Freeze-thaw method (An, G. 1987, Methods in Enzymology). Agrobacteria tumefaciens a suspended C58C1 strain after suspension culture in YEP medium, 20 mM CaCl ₂ solution to a OD = 0.5, and then, after allowed to stand for 1 minute in liquid nitrogen by mixing seven types of binary vector with a respective, 37 ^o C Introduced into Agrobacterium tumefaciens C58C1 by incubation for 2 minutes. Each transformed Agrobacteria was incubated at 28 ^o C for 2 days, and then immediately before flowering at Arabidopsis thaliana cv. Columbia based on the floral dip method (Clough and Bent, 1998, The Plant Journal). By inoculating the Arabidopsis transformed.

Example 5 Histochemical staining and enzymatic analysis of transformed Arabidopsis vulgaris

Seeds were harvested from the seven Arabidopsis transformants prepared above, and then plated in MS medium containing kanamycin (30 mg / L) to select transgenic plants having resistance. GUS activity from each tissue of selected transgenic plants was examined using histochemical staining and enzymatic methods. To stain the entire tissue of each transgenic plant, each tissue of the plant was subjected to 1 mM X-glu (5-bromo-4-chloro-3-indolyl-β-glucuronide), 100 mM sodium phosphate (pH 7.0), 10 mM It was immersed in a solution containing EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% Triton X-100, reacted at 37 ° C for 12 hours, and then removed from the solution and added to the tissue by adding 100% ethanol. Chlorophyll was removed. As a result, as shown in FIG. 5, the entire tissues of the Arabidopsis transformed with pAtW6-P2.4 (AtW6P2.4)-GUS in roots, stems, leaves, cauline, flowers, developmental seeds and pods Activity was confirmed. In FIG. 5, Control is a non-transformed Arabidopsis, CaMV35S is a Arabidopsis transformed with pBI121 binary vector, and AtWP2.4 is Arabidopsis transformed by pAtW6-P2.4. Seed 1 is 5-7 days after flowering, seed 2 10-15 days after flowering, and seed 3 is 23-25 days after flowering.

In addition, to quantitatively investigate the activity of GUS, Jefferson et. al. According to the method of (EMBO J. 6: 3901-3907, 1987), each tissue of the transgenic plant was treated with 50 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 0.1% sodium lauroylsarcosine, and The supernatant was taken by grinding in a solution containing 10 mM β-mercaptoethanol and then centrifugation at 12,000 X g. The obtained supernatant was mixed with 1 mM MUG (4-methylumbelliferyl glucuronide) and reacted at 37 ° C., and 0.2 M Na ₂ CO ₃ was added to terminate the reaction. The reaction solution was terminated using a fluorometer at 365 nm and 455 nm. After measuring the value at the wavelength, the measured value was calculated by comparing the GUS activity by using a standard curve made using MUG standard solution, and is shown in Fig. 6. In Fig. 6, the GUS activity shown was 10 obtained for each construct. The result obtained by analyzing the transformants (T ₁ plants), as a result, the promoter of the microsomal oleic acid desaturator gene showed the strongest expression of GUS in the stems, flowers, and developmental seeds in the plant. The full-length promoter containing the gene's intron was found to have a higher expression of GUS than the CaMV35S promoter. Similarly, it can be increased by about 10 times in flower and seed tissues, and about 3 times in roots and stems, and when introns in the microsomal oleic acid unsaturated gene are used with their promoters. It has been shown that GUS activity can be increased at least twofold in all tissues of the plant, especially at least about 10-fold increase in GUS expression in developing seeds 5-7 days after flowering, and part of the microsomal oleic acid desaturator gene promoter. When gradually eliminated, it was confirmed that the GUS activity is reduced in all tissues of the plant.

As described above, in the present invention, as a result of expressing a foreign gene in a plant using a promoter derived from the microsomal oleic acid desaturator gene ( At- FAD2) of the Arabidopsis and its gene, it is widely used. It was confirmed that it is a novel promoter capable of increasing the expression of foreign genes introduced at least three to ten times in all tissues of plants except leaves and foliar leaves than the CaMV35S promoter. In addition, introns present in the microsomal oleic acid desaturator gene ( At- FAD2), when used in conjunction with its promoters, were found to increase the expression of foreign genes in the whole plant tissue by about three times or more.

<110> KOREA CHUNGANG EDUCATIONAL FOUNDATION <120> Plant high-efficiency expression promoter derived from          Arabidopsis thaliana and plant high-efficiency expression vector          configuring the same <130> PN051409 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 959 <212> DNA <213> Arabidopsis thaliana <220> <221> promoter (222) (1) .. (959) <400> 1 ggtgtgtcgt cgttatacgc ggtaaataga tagatagaaa aatagaagtc caatgcaaga 60 gacttaactt aatcatccca attaattgat tgcattaact tgtacttgta ttttccgtcc 120 gccacctaat ttgattaata atataataaa gattacaatt gaaaacataa acaagagaaa 180 atccgcacga atctaccaaa gtgcatcacg tttgggtatc catacacgtg accaccagtc 240 caccacaaca caatgtctgt agatatttta atgtttcaca tgatagaaga agccaaacgt 300 aagaactctc ttttccactt ttagcccttt ccccgcctac cactgcttac gacttgtgta 360 agtggcaaac tagtaataat agagacgaaa cttaaatata aaaaagttga atccaaccaa 420 gttggtgtta atcaaatggt taagttataa tggtgaaaga tttgccatgt gtattgtatt 480 aagagttaag accaaggttt ggttcccatc acttacgatt ctttcttttc atatgattct 540 aaagttagtt attataaaca tcttaattta ctacacaata ttcggtaatt tctacatatt 600 ttagagatta gtttgagttt caatccatac tttactagtg attataaatt aatatacgta 660 cttttcgact ataaagtgaa actaagtaaa ttagaacgtg atattaaaaa gttaatgttc 720 actgttatat ttttttcaca agtaaaaaat gggttatttg cggtaaataa aaataccaga 780 tattttgaat tgattaaaaa ggttgaaata agagaggagg ggaaagaaaa gaaggtgggg 840 gcccagtatg aaagggaaag gtgtcatcaa atcatctctc tctctctctc tctaccttcg 900 acccacgggc cgtgtccatt taaagccctg tctcttgcca ttccccatct gaccaccag 959 <210> 2 <211> 2577 <212> DNA <213> Arabidopsis thaliana <220> <221> intron (222) (144) .. (1274) <400> 2 aagaagagcc acacactcac aaattaaaaa gagagagaga gagagagaga cagagagaga 60 gagagattct gcggaggagc ttcttcttcg tagggtgttc atcgttatta acgttatcgc 120 ccctacgtca gctccatctc caggtccgtc gcttctcttc catttcttct cattttcgat 180 tttgattctt atttctttcc agtagctcct gctctgtgaa tttctccgct cacgatagat 240 ctgcttatac tccttacatt caaccttaga tctggtctcg attctctgtt tctctgtttt 300 tttcttttgg tcgagaatct gatgtttgtt tatgttctgt caccattaat aataatgaac 360 tctctcattc atacaatgat tagtttctct cgtctacaaa acgatatgtt gcattttcac 420 ttttcttctt tttttctaag atgatttgct ttgaccaatt tgtttagatc tttattttat 480 tttattttct ggtgggttgg tggaaattga aaaaaaaaaa aacagcataa attgttattt 540 gttaatgtat tcattttttg gctatttgtt ctgggtaaaa atctgcttct actattgaat 600 ctttcctgga ttttttactc ctattgggtt tttatagtaa aaatacataa taaaaggaaa 660 acaaaagttt tatagattct cttaaacccc ttacgataaa agttggaatc aaaataattc 720 aggatcagat gctctttgat tgattcagat gcgattacag ttgcatggca aattttctag 780 atccgtcgtc acattttatt ttctgtttaa atatctaaat ctgatatatg atgtcgacaa 840 attctggtgg cttatacatc acttcaactg ttttcttttg gctttgtttg tcaacttggt 900 tttcaatacg atttgtgatt tcgatcgctg aatttttaat acaagcaaac tgatgttaac 960 cacaagcaag agatgtgacc tgccttatta acatcgtatt acttactact agtcgtattc 1020 tcaacgcaat cgtttttgta tttctcacat tatgccgctt ctctactctt tattcctttt 1080 ggtccacgca ttttctattt gtggcaatcc ctttcacaac ctgatttccc actttggatc 1140 atttgtctga agactctctt gaatcgttac cacttgtttc ttgtgcatgc tctgtttttt 1200 agaattaatg ataaaactat tccatagtct tgagttttca gcttgttgat tcttttgctt 1260 ttggttttct gcagaaacat gggtgcaggt ggaagaatgc cggttcctac ttcttccaag 1320 aaatcggaaa ccgacaccac aaagcgtgtg ccgtgcgaga aaccgccttt ctcggtggga 1380 gatctgaaga aagcaatccc gccgcattgt ttcaaacgct caatccctcg ctctttctcc 1440 taccttatca gtgacatcat tatagcctca tgcttctact acgtcgccac caattacttc 1500 tctctcctcc ctcagcctct ctcttacttg gcttggccac tctattgggc ctgtcaaggc 1560 tgtgtcctaa ctggtatctg ggtcatagcc cacgaatgcg gtcaccacgc attcagcgac 1620 taccaatggc tggatgacac agttggtctt atcttccatt ccttcctcct cgtcccttac 1680 ttctcctgga agtatagtca tcgccgtcac cattccaaca ctggatccct cgaaagagat 1740 gaagtatttg tcccaaagca gaaatcagca atcaagtggt acgggaaata cctcaacaac 1800 cctcttggac gcatcatgat gttaaccgtc cagtttgtcc tcgggtggcc cttgtactta 1860 gcctttaacg tctctggcag accgtatgac gggttcgctt gccatttctt ccccaacgct 1920 cccatctaca atgaccgaga acgcctccag atatacctct ctgatgcggg tattctagcc 1980 gtctgttttg gtctttaccg ttacgctgct gcacaaggga tggcctcgat gatctgcctc 2040 tacggagtac cgcttctgat agtgaatgcg ttcctcgtct tgatcactta cttgcagcac 2100 actcatccct cgttgcctca ctacgattca tcagagtggg actggctcag gggagctttg 2160 gctaccgtag acagagacta cggaatcttg aacaaggtgt tccacaacat tacagacaca 2220 cacgtggctc atcacctgtt ctcgacaatg ccgcattata acgcaatgga agctacaaag 2280 gcgataaagc caattctggg agactattac cagttcgatg gaacaccgtg gtatgtagcg 2340 atgtataggg aggcaaagga gtgtatctat gtagaaccgg acagggaagg tgacaagaaa 2400 ggtgtgtact ggtacaacaa taagttatga ggatgatggt gaagaaattg tcgacctttc 2460 tcttgtctgt ttgtcttttg ttaaagaagc tatgcttcgt tttaataatc ttattgtcca 2520 ttttgttgtg ttatgacatt ttggctgctc attatgttat gtgggaagtt agtgttc 2577 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward primer AraW6F1 <400> 3 ccgaagcttg gtgtgtcgtc gttatacgcg gtaaa 35 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward primer AraW6F2 <400> 4 ccgaagcttc caccagtcca ccacaacac 29 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer AraW6F3 <400> 5 ccgaagcttg gtgaaagatt tgccatgtgt attg 34 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Forward primer AraW6F4 <400> 6 ccgaagcttg aacgtgatat taaaaagtta atgttcactg 40 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer AraW6F5 <400> 7 ccgaagcttc tctctacctt cgacccacgg 30 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer AraW6R1 <400> 8 caaggatccg gagatggagc tgacgtaggg gcg 33 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer AraW6R4 <400> 9 cgaggatccc tgcagaaaac caaaagcaaa ag 32

Claims (8)

A high efficiency expression promoter of a plant comprising the nucleotide sequence of the 1 to 959 (-959 to -1) region of SEQ ID NO: 1. An intron for enhancing expression of a plant comprising a nucleotide sequence of +144 to +1274 region from the transcription initiation site of SEQ ID NO. A high efficiency expression vector of a plant containing the high efficiency expression promoter of claim 1 and the intron for enhancing expression of claim 2. 4. The high efficiency expression vector of a plant according to claim 3, wherein the promoter is inserted into a binary vector. The high efficiency expression vector of the plant of Claim 4 which is pAtW6-P2.4 shown in FIG. 2B. E. coli or Agrobacterium tumefaciens C58C1 containing the high efficiency expression vector of any one of claims 3 to 5. A plant transformed with the high efficiency expression vector of any one of claims 3 to 5. A method of expressing a foreign gene in a transgenic plant using the seed specific expression vector of any one of claims 3 to 5.

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