JP3905607B2 - Promoter sequences and uses thereof - Google Patents

Description

【００３７】
〔実施例４〕カーネーションでの一過的発現とＧＵＳ活性の測定
実施例２で得られたキメラＧＵＳ遺伝子を有するバイナリーベクターをエレクトロポ−レ−ション法によりAgrobacterium tumefaciens AGL0 へ導入し、これを3mlのYEB-km培地に接種し、28℃で16時間培養した後、遠心により集菌し10mlの感染培地に懸濁して、これを感染液とした。YEB-km培地及び感染培地の培地組成を以下に記載する。
【００３８】
YEB-km培地；5g/lビ−フエキス、1g/l酵母エキス、5g/lペプトン、5g/lスクロ−ス、2mM硫酸マグネシウム、50mg/lカナマイシン
感染培地； 1/2濃度のMS（Murashige & Skoog , Physiol. Plant. 15:473-497(1962))培地の無機塩及びビタミン類、15g/lスクロ−ス、10g/lグルコ−ス、10mM MES(pH5.4)
【００３９】
カ−ネ−ション（Dianthus caryophyllus）の品種スケニア(scania)の無菌個体の葉を5-7mm角に切断し、各種ベクタ−を導入したアグロバクテリア感染液に10分間浸し、過剰な感染液を濾紙上でふき取った後、共存培養培地に移植し25℃暗所で培養した。3日間培養した後、組織化学的なGUS活性測定に用いた。
培養に用いた共存培養培地の成分を以下に記載する。
MS培地（Physiol. Plant. 15 (1962) 473-497)の無機塩及びビタミン類、30g/lスクロ−ス、0.1mg/lナフタレン酢酸、1mg/lベンジルアデニン、8g/l寒天、5mM MES(pH5.8)、200μＭアセトシリンゴン
【００４０】
組織化学的なＧＵＳ活性の測定はCastle & Morris の方法(Plant Molecular Biology Manual, B5 (1994) 1-16 (Ed.)S.B.Gelvin&R.A.Schilperoort, Kluwer Academic Publishers) に従い行った。37℃で1晩インキュベ−トした後、70％メタノ−ルで脱色し、組織の青い染色を観察することによりGUS活性を測定した。その結果、 ipt遺伝子、6b遺伝子のプロモーターを用いた場合と同様に新規プロモーターでもGUS活性による青い染色を確認することができた。
【００４１】
【発明の効果】
本発明により、植物における転写を活性化するＤＮＡ（プロモーター配列）が提供された。本発明のＤＮＡを利用することにより、植物において、構造遺伝子の発現を誘導したり、構造遺伝子を高発現させることができる。
【００４２】
【配列表】
配列番号：１
配列の長さ：３７３
配列の型：核酸
鎖の数：２本鎖
トポロジー：直鎖状
配列の種類；Ｇｅｎｏｍｉｃ ＤＮＡ
起源：
生物名：Agrobacterium tumefaciens
株名： A281
配列

【００４３】
配列番号：２
配列の長さ：２４
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類；他の核酸 合成ＤＮＡ
配列
ATAAAGCTTA GGACTGTGAC TGGT 24
【００４４】
配列番号：３
配列の長さ：２６
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類；他の核酸 合成ＤＮＡ
配列
GCACTAGTCG CAATTTTCGT ATTTAC 26
【００４５】
配列番号：４
配列の長さ：３９
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類；他の核酸 合成ＤＮＡ
配列
CTAGACTTCT CTCAATCCAA CTTTTCTATG GCCATGGCA 39
【００４６】
配列番号：５
配列の長さ：３９
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類；他の核酸 合成ＤＮＡ
配列
GATCTGCCAT GGCCATAGAA AAGTTGGATT GAGAGAAGT 39
【００４７】
配列番号：６
配列の長さ：２７
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類；他の核酸 合成ＤＮＡ
配列
ATAAAGCTTC GCAATTTTCG TATTTAC 27
【００４８】
配列番号：７
配列の長さ：２６
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類；他の核酸 合成ＤＮＡ
配列
GCACTAGTTT CTTATTATGA GCCCTC 26
【００４９】
配列番号：８
配列の長さ：２５
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類；他の核酸 合成ＤＮＡ
配列
ATAAAGCTTA CAAGTATTGC ACGTT 25
【００５０】
配列番号：９
配列の長さ：２６
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類；他の核酸 合成ＤＮＡ
配列
GCACTAGTCG GTCTTGCGAT ATGTTG 26
【００５１】
配列番号：１０
配列の長さ：２６
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類；他の核酸 合成ＤＮＡ
配列
ATAAAGCTTG ACGATCTCAC GGCCAA 26
【００５２】
配列番号：１１
配列の長さ：２６
配列の型：核酸
鎖の数：２本鎖
トポロジー：直鎖状
配列の種類；Ｇｅｎｏｍｉｃ ＤＮＡ
起源：
生物名：Agrobacterium tumefaciens
株名： A281
配列
TGGCAGGATA TATTGGGGTG TCAATT
【図面の簡単な説明】
【図１】Agrobacterium tumefaciensA281株のTL-DNA領域と新規ORFおよびそのプロモーター領域を示す。
【図２】本発明のプロモーター配列と他のプロモーターで知られているOCS配列との相同性を示す。
【図３】レポーター遺伝子であるベータグルクロニダーゼ（ＧＵＳ）と新規プロモーターのキメラ遺伝子の構築法を概説した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a nucleotide sequence that activates transcription existing in T-DNA of Agrobacterium tumefaciens , that is, a DNA sequence that functions as a promoter and use thereof.
[0002]
[Prior art]
In order to express a foreign gene, it is necessary to use an appropriate promoter. Promoters such as promoter of cauliflower mosaic virus 35S, nopaline synthase, which is an opine synthase gene on Agrobacterium T-DNA, and mannopine synthase are known as those commonly used in plants. However, there are only a limited number of cases where effective (high expression level) can be obtained by simply linking them. For this reason, efforts have been made to improve the expression level by improving the 5 ′ untranslated region, using introns, or re-synthesizing genes (Plant Mol. Biol. 32 (1996) 393-405). In addition, since the activity of the cauliflower mosaic virus 35S promoter itself is low in monocotyledonous plants, new promoters of rice actin and corn ubiquitin have been obtained and used (Plant Cell Rep. 11 (1992) 586-591 , Plant. Phsiol. 100 (1992) 1503-1507, Plant. Phsiol. 102 (1992) 1077-1084).
[0003]
On the other hand, in chrysanthemum, there are reports that some transformed plants have been obtained, but there are reports that the expression is low compared to the expression in other plants, or the expression cannot be confirmed (Plant Cell Rep. 14 ( 1995) 649-698). The reason for this is that in chrysanthemum, the activity of the cauliflower mosaic virus 35S promoter most frequently used in plants is not high.
[0004]
Therefore, we tried to obtain a promoter highly active in chrysanthemum plants from Agribacterium T-DNA. Agrobacterium tumefaciens A281 is a supervirlence strain with a very wide host range (J. Bacteriol. 169 (1987) 4417-4425). Create a goal (Plant Cell Rep. 9 (1991) 505-508). Therefore, it is considered that the promoter in this T-DNA has a possibility of becoming a promoter that can be expressed over a wide host range. However, it is known as a plant hormone-related gene, and the isopentenyl transferase gene promoter (Mol. Gen. Genet. 216 (1989) 388-394) on the T-DNA of the already reported A281 strain and the 6b gene When a promoter (Plant. Mol. Biol. 29 (1995) 1299-1304) was tested, high activity was not obtained.
[0005]
[Problem to be Solved by the Invention]
Accordingly, an object of the present invention is to provide a promoter sequence that induces the expression of a structural gene when inserted on the 5 ′ side of the structural gene.
Another object of the present invention is to provide a DNA strand comprising a structural gene and the promoter sequence.
Another object of the present invention is to provide a host transformed with the above DNA strand, and a method for inducing the expression of the structural gene or high expression of the structural gene using the host.
[0006]
[Means for Solving the Problems]
The present inventors have found that a novel reading frame (ORF) exists in the T-DNA sequence of Agrobacterium tumefaciens A281 strain, and that the 5 ′ upstream sequence functions as a promoter that works effectively in the plant body. And by analyzing the expression pattern of the reporter gene in the transient expression experiment of carnation, the present invention was completed.
[0007]
Therefore, the present invention provides DNA consisting of the base sequence of SEQ ID NO: 1 or an equivalent thereof having promoter activity. The present invention also provides a DNA strand comprising the structural gene DNA and the above DNA or its equivalent incorporated at the 5 ′ site of the structural gene DNA in such a manner that the structural gene is expressed. To do. The DNA strand may further comprise a component selected from the group consisting of a translation enhancer, a translation stop codon, a terminator, and combinations thereof. Furthermore, the present invention provides a host transformed with the above DNA strand. The host may be a plant cell. Furthermore, the present invention provides a method for inducing the expression of a structural gene in a plant, characterized by culturing or cultivating the plant cell transformed with the above DNA strand so that the structural gene can be expressed. To do. In this method, the structural gene may be a foreign gene.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Promoter sequence The DNA (promoter sequence) of the present invention consists of the base sequence of SEQ ID NO: 1. This sequence is inserted 5 ′ to the translation start point of the structural gene, thereby inducing the expression of the structural gene or expressing the structural gene at a high level. Therefore, it can be used to induce the expression of structural genes, particularly foreign genes, or to increase the efficiency of expression.
When the nucleotide sequence of SEQ ID NO: 1 is used, the promoter sequence is not translated, but is translated from the start codon immediately thereafter.
[0009]
Moreover, the DNA which is the promoter sequence of the present invention includes an equivalent sequence of the base sequence of SEQ ID NO: 1. Here, “the DNA equivalent consisting of the base sequence of SEQ ID NO: 1” or “the equivalent sequence of the base sequence of SEQ ID NO: 1” refers to the insertion, substitution, or substitution of several bases in the base sequence of SEQ ID NO: 1. It shall have been deleted or added to both ends and still retain its translational activation activity (promoter activity). Maintaining the activity of translation activation in the equivalent or equivalent sequence means that in the actual usage mode using the activity, the base sequence having the entire sequence of SEQ ID NO: 1 can be used in substantially the same conditions. It means that a certain level of activity is maintained. Such an “equivalent” or “equivalent sequence” refers to the nucleotide sequence of SEQ ID NO: 1, according to the description in the literature such as Molecular Cloning (edited by Sambrook et al. (1989) Cold Spring Harbor Lab. Press, New York). Obviously, those skilled in the art can select and manufacture without any particular difficulty.
[0010]
The host cell in which the promoter sequence of the present invention is preferably used is a plant cell. Preferable specific examples thereof are plants that are infected with the A281 strain from which the promoter sequence of the present invention is isolated to form crown gall, and belong to the solanaceous plants to which tobacco belongs, such as tomatoes, potatoes, eggplants, peppers, Examples of useful plants of other families that have been reported to be transformed include plant cells such as chrysanthemum, cotton, soybean, rapeseed, rice, wheat, and corn.
[0011]
According to the present invention, there is further provided a structural gene DNA and a DNA consisting of the base sequence of SEQ ID NO: 1 incorporated in the 5 ′ site of the structural gene DNA in such a manner that the structural gene is expressed, or an equivalent thereof. A DNA strand comprising is provided.
Examples of the structural gene DNA include, but are not limited to, DNA encoding β-glucan elicitor receptor (Proc. Natl. Acad. Sci. USA 94 (1997) 1029-1034).
[0012]
The specific form of the DNA strand of the present invention may be, for example, a form in which the sequence of SEQ ID NO: 1 is inserted as part of a member in plasmid or phage DNA. Furthermore, it may be in a form that is present in a microorganism (particularly a bacterium), a phage particle, or a plant in a form inserted into a plasmid or phage or genomic DNA. In addition, it cannot be overemphasized that bacteria here contain colon_bacillus | E._coli and Agrobacterium.
[0013]
The preferred form of the DNA strand of the present invention is the sequence of SEQ ID NO: 1 or an equivalent sequence thereof, a translation enhancer so that the structural gene to be expressed in a gene product such as a protein can be stably expressed in a plant. , A structural gene DNA chain, a translation termination codon, and a terminator are integrally bound to each other and are present in the plant in a form inserted into the genome. As the translation enhancer, translation termination codon, and terminator, known ones can be used in appropriate combination. Specific examples of those considered to be available are known as translation enhancers, for example, tobacco mosaic virus, alfalfa mosaic virus RNA4, bromomosaic virus RNA3, potato virus X, and tobacco etch virus (Annu. Rev. Plant Physiol. Plant Mol. Biol. 44 (1993) 985-994) leader sequences and the like, and terminators include nopaline synthase terminator, octopine synthase terminator and the like.
[0014]
Acquisition of DNA strand The DNA consisting of the nucleotide sequence of SEQ ID NO: 1 of the present invention can be obtained by chemical synthesis according to the method of nucleic acid synthesis. Alternatively, only a part of the nucleic acid is synthesized as a primer, and it can be easily obtained by polymerase chain reaction (PCR) using the total DNA of Agrobacterium tumefaciens A281 strain as a template. Furthermore, the above-mentioned DNA strand comprising these sequences can be produced using a technique commonly used in the field of genetic engineering.
[0015]
Use / transformation of base sequence The base sequence of SEQ ID NO: 1 has use as a promoter sequence. These sequences are preferably used in the form of the above-described DNA strand of the present invention.
By transforming a host cell with such a DNA strand and culturing or cultivating the host cell, the expression of a structural gene including a foreign gene can be induced or the gene can be expressed with high efficiency.
[0016]
Preferred examples of the host include cells of the above-mentioned types of plants, and more specific plant materials include leaf pieces, stem pieces, tuber pieces, protoplasts, callus, pollen, pollen tubes, and the like.
As a method for introducing a gene into a host, various methods already reported and established can be used. Preferred examples include, for example, a method using an Agrobacterium Ti plasmid as a vector, a method of introducing a gene into a plant protoplast by electroporation (“Plant genetic transformation and gene expression; a laboratory manual”, Draper , J. et. Al. Eds., Blackwell Scientific Publication, 1988).
[0017]
When it is desired to isolate and use an expression product of a gene that has been induced or highly expressed, it may be isolated and purified from the culture by a method suitable for the expression product. Furthermore, when the growth or growth of the host cell is further modified by the presence of the expression product, and the properties of the cell are modified, such a host is cultured or the host is a dedifferentiated plant. By cultivating the plant body, the expression product of the target gene is induced or highly expressed.
[0018]
【Example】
The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.
[Example 1] Discovery of a novel ORF and determination of its promoter region
Total DNA was extracted from Agrobacterium tumefaciens A281 strain (J. Bacteriol. 129 (1977) 101-107) using ISOPLANT (manufactured by Nippon Gene). A DNA synthesizer (Perkin Elmer) was used to convert a part of the previously reported 6b gene sequence (Mol. Plant Microbe Interact. 8 (1995) 534-548) and IS1312 sequence (J. Bacteriol. 177 (1995) 2554-2559). Synthesized). The sequence is shown below.
5'-ATAAAGCTTAGGACTGTGACTGGT-3 '(SEQ ID NO: 2)
5'-GCACTAGTCGCAATTTTCGTATTTAC-3 '(SEQ ID NO: 3)
[0019]
PCR was carried out using the previously extracted total DNA as a template, and a fragment (approximately 2.0%) expected to contain the right border expected in the schematic diagram of the previous report (J. Bacteriol. 177 (1995) 2554-2559). Only amplification of DNA fragments (approximately 3.0 kbps) larger than (kbps) could be confirmed. The amplified fragment was subcloned into pBluescript SK + (Stratagene) and isolated using a combination of HindIII, BglII, SpeI, and EcoRV restriction enzymes. The structure of this DNA fragment was analyzed with a fluorescent sequencer (Perkin Elmer type) to determine the primary structure.
[0020]
Dang et al. (Mol. Plant Microbe Interact. 8 (1995) 534-548) report that a clear right border cannot be found in this region, but the DNA fragment we obtained is reported as a right border. There was a region showing extremely high homology with the sequence (Crit. Rev. Plant Sci. 10 (1991) 1-32), and this region was presumed to be a right border. The sequence was as shown in FIG. 1 and SEQ ID NO: 11.
[0021]
In FIG. 1, 5, 7, iaH, iaAM, ipt and 6p represent 5 gene, 7 gene, iaaH gene, iaaM gene, ipt gene and 6p gene, respectively. LB, RB, and IS represent left border, right border, and insertion sequence, respectively.
It came to discover newly that the reading frame (1001 bps) of the opposite direction to 6b gene exists between Right Border and 6b gene of the obtained arrangement | sequence.
[0022]
The obtained sequence contained a portion very similar to the promoter sequence of the 6b gene described previously (Plant. Mol. Biol. 29 (1995) 1299-1304). There was a difference of 10 base pairs between the previously reported sequence (826 base pairs) and the newly determined sequence (5 base pairs of 373 base pairs in the promoter region). This suggests that the previous authors could not find the reading frame.
[0023]
Further, it was found that a sequence similar to the OCS sequence (EMBO J. 8 (1989) 4197-4204) effective for the promoter activity shown in FIG. 2 exists in this promoter region.
In FIG. 2, CONSENSUS is a CONSENSUS (extracted common part) sequence described in EMBO J. 8 (1989) 4197-4204. In addition, ocs, mas1 ', mas2' and nos are OCS sequences on T-DNA of Agrobacterium extracted from the table of the above literature, and NEW is newly found by us that are similar to these OCS sequences. Is an array.
[0024]
[Example 2] Isolation of promoter region and construction of chimeric GUS gene Chimera gene of beta glucuronidase (GUS) which is a reporter gene and a novel promoter, specifically, chimeric gene expression plasmid pBINEW-EIG shown in FIG. Built.
[0025]
First, pBI35S-EIG was transferred from Professor Kenzo Nakamura of Nagoya University for the purpose of adding a translation enhancer (J. Biol. Chem. 270 (1995) 12466-12470) of the ferredoxin-binding subunit of tobacco: Plant Cell Physiol. 31 (1990) 805-813) GUS gene (including the intron of castor catalase gene) 5 'and XbaI and BamHI sites are cleaved with restriction enzymes XbaI and BamHI The following synthetic linker DNAs synthesized using Applied Biosystems) were annealed and ligated. As a result, a plasmid pBI35S-EIG having a GUS gene containing an intron added with a translation enhancer induced by the cauliflower mosaic virus 35S promoter was obtained.
5'-CTAGACTTCTCTCAATCCAACTTTTCTATGGCCATGGCA-3 '(SEQ ID NO: 4)
5'-GATCTGCCATGGCCATAGAAAAGTTGGATTGAGAGAAGT-3 '(SEQ ID NO: 5)
[0026]
pBINEW-EIG was created as follows. First, the following two kinds of oligonucleotide primers were synthesized, and PCR was carried out using the total DNA extracted from Agrobacterium tumefaciens A281 strain as a template.
5'-ATAAAGCTTCGCAATTTTCGTATTTAC-3 '(SEQ ID NO: 6)
5'-GCACTAGTTTCTTATTATGAGCCCTC-3 '(SEQ ID NO: 7)
[0027]
The thus amplified DNA fragment (new promoter) is purified from an agarose gel, excised with restriction enzymes HindIII and SpeI, plasmid pBI35S-EIG is treated with restriction enzymes HindIII and XbaI to replace the 35S promoter, and plasmid pBINEW-EIG It was created.
The control pBIipt-EIG was prepared as follows. Based on the previous report (Mol. Gen. Genet. 216 (1989) 388-394), the following two oligonucleotide primers were synthesized, and PCR was performed using the total DNA extracted from Agrobacterium tumefaciens A281 strain as a template.
[0028]
5'-ATAAAGCTTACAAGTATTGCACGTT-3 '(SEQ ID NO: 8)
5'-GCACTAGTCGGTCTTGCGATATGTTG-3 '(SEQ ID NO: 9)
The DNA fragment thus amplified (ipt promoter) is purified from an agarose gel, excised with restriction enzymes HindIII and SpeI, and plasmid pBI35S-EIG is treated with restriction enzymes HindIII and XbaI to replace the 35S promoter, and plasmid pBIipt-EIG It was created.
[0029]
The control pBI6b-EIG was prepared as follows. Based on the previous report (Plant. Mol. Biol. 29 (1995) 1299-1304), SEQ ID NO: 3 and the following oligonucleotide primers were synthesized, and PCR was performed using the total DNA extracted from Agrobacterium tumefaciens A281 strain as a template.
5'-ATAAAGCTTGACGATCTCACGGCCAA-3 '(SEQ ID NO: 10)
[0030]
The thus amplified DNA fragment (6b promoter) is purified from an agarose gel, excised with restriction enzymes HindIII and SpeI, and plasmid pBI35S-EIG is treated with restriction enzymes HindIII and XbaI to replace the 35S promoter and plasmid pBI6b-EIG It was created.
[0031]
[Example 3] Transformation of chrysanthemum and measurement of GUS activity The binary plasmid having the chimeric GUS gene obtained in Example 2 was transferred from Agrobacterium tumefaciens AGL0 (Dr. Ludwig, University of California, Santa Cruz) by electroporation. : Bio / Technology 9 (1991) 963-967), inoculated into 3 ml of YEB-km medium, cultured at 28 ° C for 16 hours, collected by centrifugation, suspended in 10 ml of infection medium This was used as an infectious solution. The medium composition of YEB-km medium and infection medium is described below.
[0032]
YEB-km medium: 5 g / l beef extract, 1 g / l yeast extract, 5 g / l peptone, 5 g / l sucrose, 2 mM magnesium sulfate, 50 mg / l kanamycin infection medium; 1/2 concentration of MS (Murashige & Skoog, Physiol. Plant. 15 (1962) 473-497) Medium inorganic salts and vitamins, 15 g / l sucrose, 10 g / l glucose, 10 mM MES (pH 5.4)
[0033]
Sterilized leaves of cultivar legan cultivar (Dendranthema grandiflora) were cut into 5-7mm squares, soaked in Agrobacterium infection solution with various vectors introduced for 10 minutes, and excess infection solution was wiped off on filter paper Thereafter, the cells were transplanted to a coculture medium and cultured in the dark at 25 ° C. After culturing for 3 days, it was transferred to a selective medium. Two weeks later, the cells were transplanted to a fresh selective medium, and further cultured for 2 weeks. The culture was performed under conditions of 25 ° C., 16 hours illumination and 8 hours no illumination. The obtained callus was cut out from the leaf and used for GUS activity measurement.
The components of the medium used for the culture are described below.
Co-culture medium: MS (Murashige & Skoog, Physiol. Plant. 15 (1962) 473-497) Medium salts and vitamins, 30 g / l sucrose, 1 mg / l naphthalene acetic acid, 2 mg / l benzyladenine, 8 g / l agar, 5 mM MES (pH 5.8), 200 μM acetosyringone selective medium; inorganic salts and vitamins of MS medium, 30 g / l sucrose, 1 mg / l naphthalene acetic acid, 2 mg / l benzyladenine, 8 g / l Agar, 5 mM MES (pH 5.8), 100 mg / l kanamycin, 300 mg / l cefotaxime [0034]
GUS activity measurement using a fluorescent substrate was performed according to the method of Castle & Morris (Plant Molecular Biology Manual, B5 (1994) 1-16 (Ed.) SBGelvin & R.A. Schilperoort, Kluwer Academic Publishers) using 50-100 mg of callus. It was. The amount of protein was measured according to the method of Bradford, and GUS activity was converted to the amount of protein (Anal. Biochem. 72 (1976) 248-254). The results were as shown in Table 1.
[0035]
Surprisingly, as is apparent from the table below, GUS activity was measured for each callus of chrysanthemum introduced with the respective chimeric genes, compared to controls (CaMV35S :: GUS, ipt :: GUS and 6b :: GUS). In addition, a high GUS activity was detected in those having a novel promoter (NEW :: GUS).
[0036]
[Table 1]

[0037]
[Example 4] Transient expression in carnation and measurement of GUS activity The binary vector having the chimeric GUS gene obtained in Example 2 was introduced into Agrobacterium tumefaciens AGL0 by electroporation, The YEB-km medium was inoculated and cultured at 28 ° C. for 16 hours, and then collected by centrifugation and suspended in 10 ml of an infection medium to obtain an infection solution. The medium composition of YEB-km medium and infection medium is described below.
[0038]
YEB-km medium: 5 g / l beef extract, 1 g / l yeast extract, 5 g / l peptone, 5 g / l sucrose, 2 mM magnesium sulfate, 50 mg / l kanamycin infection medium; 1/2 concentration of MS (Murashige & Skoog, Physiol. Plant. 15: 473-497 (1962)) Medium salts and vitamins of medium, 15 g / l sucrose, 10 g / l glucose, 10 mM MES (pH 5.4)
[0039]
Cut the leaves of a cultivar (Dianthus caryophyllus) cultivar, scania, into 5-7mm squares, immerse them in an Agrobacterium infection solution containing various vectors for 10 minutes, and filter the excess infection solution through filter paper. After wiping off, it was transplanted to a co-culture medium and cultured in the dark at 25 ° C. After culturing for 3 days, it was used for histochemical GUS activity measurement.
The components of the coculture medium used for the culture are described below.
Inorganic salts and vitamins of MS medium (Physiol. Plant. 15 (1962) 473-497), 30 g / l sucrose, 0.1 mg / l naphthalene acetic acid, 1 mg / l benzyladenine, 8 g / l agar, 5 mM MES ( pH 5.8), 200 μM acetosyringone [0040]
Histochemical GUS activity was measured according to the method of Castle & Morris (Plant Molecular Biology Manual, B5 (1994) 1-16 (Ed.) SBGelvin & R.A. Schilperoort, Kluwer Academic Publishers). After incubation overnight at 37 ° C., GUS activity was measured by decolorizing with 70% methanol and observing the blue staining of the tissue. As a result, blue staining due to GUS activity could be confirmed with the new promoter as well as with the promoters of the ipt gene and 6b gene.
[0041]
【The invention's effect】
The present invention provides DNA (promoter sequence) that activates transcription in plants. By using the DNA of the present invention, the expression of a structural gene can be induced or the structural gene can be highly expressed in a plant.
[0042]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 373
Sequence type: Number of nucleic acid strands: Double strand topology: Type of linear sequence; Genomic DNA
origin:
Name of organism: Agrobacterium tumefaciens
Stock Name: A281
Array

[0043]
SEQ ID NO: 2
Sequence length: 24
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence; Other nucleic acid Synthetic DNA
Array
ATAAAGCTTA GGACTGTGAC TGGT 24
[0044]
SEQ ID NO: 3
Sequence length: 26
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence; Other nucleic acid Synthetic DNA
Array
GCACTAGTCG CAATTTTCGT ATTTAC 26
[0045]
SEQ ID NO: 4
Sequence length: 39
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence; Other nucleic acid Synthetic DNA
Array
CTAGACTTCT CTCAATCCAA CTTTTCTATG GCCATGGCA 39
[0046]
SEQ ID NO: 5
Sequence length: 39
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence; Other nucleic acid Synthetic DNA
Array
GATCTGCCAT GGCCATAGAA AAGTTGGATT GAGAGAAGT 39
[0047]
SEQ ID NO: 6
Sequence length: 27
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence; Other nucleic acid Synthetic DNA
Array
ATAAAGCTTC GCAATTTTCG TATTTAC 27
[0048]
SEQ ID NO: 7
Sequence length: 26
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence; Other nucleic acid Synthetic DNA
Array
GCACTAGTTT CTTATTATGA GCCCTC 26
[0049]
SEQ ID NO: 8
Sequence length: 25
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence; Other nucleic acid Synthetic DNA
Array
ATAAAGCTTA CAAGTATTGC ACGTT 25
[0050]
SEQ ID NO: 9
Sequence length: 26
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence; Other nucleic acid Synthetic DNA
Array
GCACTAGTCG GTCTTGCGAT ATGTTG 26
[0051]
SEQ ID NO: 10
Sequence length: 26
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence; Other nucleic acid Synthetic DNA
Array
ATAAAGCTTG ACGATCTCAC GGCCAA 26
[0052]
SEQ ID NO: 11
Sequence length: 26
Sequence type: Number of nucleic acid strands: Double strand topology: Type of linear sequence; Genomic DNA
origin:
Name of organism: Agrobacterium tumefaciens
Stock Name: A281
Array
TGGCAGGATA TATTGGGGTG TCAATT
[Brief description of the drawings]
FIG. 1 shows a TL-DNA region of Agrobacterium tumefaciens A281 strain, a novel ORF and its promoter region.
FIG. 2 shows the homology between the promoter sequence of the present invention and OCS sequences known for other promoters.
FIG. 3 is a diagram outlining a method for constructing a chimeric gene of a reporter gene, beta glucuronidase (GUS), and a novel promoter.

Claims (5)

DNA comprising the nucleotide sequence of SEQ ID NO: 1, or DNA comprising one or several bases inserted, substituted, deleted, or added to both ends in the nucleotide sequence of SEQ ID NO: 1 , and having promoter activity .   A DNA strand comprising the structural gene DNA and the DNA of claim 1 incorporated in the 5 'site of the structural gene DNA in such a manner that the structural gene is expressed.   The DNA strand according to claim 2, further comprising a component selected from the group consisting of a translation enhancer, a translation termination codon, a terminator, and combinations thereof.   A host transformed with the DNA strand according to claim 2 or 3.   The host according to claim 4, wherein the host is a plant cell.

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