JP3882952B2 - Plant promoter - Google Patents

Description

【００４６】

【００４７】

【００４８】

【００４９】

【図面の簡単な説明】
【図１】形質転換体、Escherichia coli JM109/pBI18:GUSの構築を示す図である。
【図２】形質転換シロイヌナズナ（暗所生育、１週間目のシロイヌナズナ）のＧＵＳ染色を示す図に代わる写真である。
【図３】形質転換シロイヌナズナ（７週間目のシロイヌナズナ）のＧＵＳ染色を示す図に代わる写真である。
【図４】形質転換シロイヌナズナ（９週間目のシロイヌナズナ）のＧＵＳ染色を示す図に代わる写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a plant promoter having a promoter function in a plant. More specifically, the present invention relates to a plant promoter that regulates the expression of cotton fiber and the like. The base sequence having the plant promoter function can regulate the expression of foreign genes in eukaryotes or prokaryotes.
[0002]
[Prior art]
A promoter is a signal on DNA that initiates mRNA synthesis using DNA as a template, and has a common sequence of characteristic bases. In particular, in eukaryotes, there is a common sequence called a TATA box upstream of about 20 bases upstream of the transcription start point, and this site is considered to be a site necessary for transcription initiation. In order to produce a large amount of the target protein, it is considered advantageous to use a stronger promoter. Generally, since the activity is strong in plants, the cauliflower mosaic virus, CaMV 35S promoter is often used. In fact, it is used to produce herbicide-tolerant plants and virus-resistant plants. However, the 35S promoter does not have tissue specificity, and tissue specificity may be required depending on the purpose. Research has been conducted on cis and trans factors as promoters having plant tissue specificity. When a promoter having plant tissue specificity is used, a transformed plant body that can regulate the expression of the introduced gene in a desired organ can be produced.
[0003]
Currently, cotton fiber is produced by cultivating a cotton plant belonging to the genus Gossypium and collecting it from the obtained fruit (cotton ball). In cotton fibers, there are various characteristic values indicating fiber characteristics, and particularly important ones include fiber length, fineness, and strength. Conventionally, great efforts have been made to improve the properties of cotton fibers. Today, with the development of genetic engineering, it is possible to transform cotton plants and change their fiber properties. In that case, it is very important to express the target gene in a desired time and tissue. However, the formation and elongation mechanism of cotton fibers has not been fully elucidated, and the genes and promoters involved are not well understood. In order to express a target gene in a target tissue or time, it is necessary to use a more specific promoter instead of a constant expression like the CaMV35S promoter. Such promoters are indispensable for improving cotton fibers.
[0004]
Cotton fiber is an extension of each epidermal cell of the ovule, and one fiber is made of one cell. The fiber is formed through the stages of initiation, elongation, secondary wall deposition, and maturation. To date, several cotton-derived promoters have been discovered and reported to be useful in improving fiber specificity (WO 94/12014), and E6 or B8 promoters are disclosed. In particular, E6 has been studied in detail, and it has been shown that E6 mRNA is strongly expressed in the fiber after 15 days after flowering. In addition, long exposures of northern blotting give weak signals to flowers, ovules, and leaves (Proc. Natl. Acad. Sci. USA 89, 5769-5773, 1992). Furthermore, it has been shown that the FbL2A promoter is strongly expressed on the 25th to 30th days after flowering on cotton fibers (Plant Physiol. (1996) 112: 1331-1341).
In addition, it is known that promoter strength, working time, and the like vary depending on each promoter. However, after the 15th day of flowering, in the fiber formation of cotton, after the latter half of the elongation, these promoters are not sufficient for improving the fiber characteristics, but the promoters are expressed immediately after flowering until the end of fiber elongation. Is also necessary. However, such a promoter has not yet been discovered.
[0005]
[Problems to be solved by the invention]
One of the objects of the present invention is to provide a promoter useful for improving the fiber properties of cotton fibers. Furthermore, another object of the present invention is to provide a novel transformed plant that expresses a target gene in a desired tissue or organ in other plants.
[0006]
[Means for Solving the Problems]
The present inventors have already intensively studied to improve the fiber characteristics of cotton fibers or improve productivity, and isolated several cDNAs from cotton fibers (Japanese Patent Application No. 8-31987). The timing and tissue-specific expression of these isolated cDNAs were examined, and the upstream sequence of one of these genes, pKC18, was cloned and analyzed. As a result, the fiber properties of cotton fiber were improved or the productivity was improved. The present inventors have found a plant promoter to be achieved and have reached the present invention.
[0007]
That is, the present invention provides the following (a): Or It is a plant promoter containing the DNA of (b).
(A) DNA comprising the base sequence described in SEQ ID NO: 1
(B) In DNA comprising the base sequence of (a), one or more base DNA comprising a nucleotide sequence deleted, substituted or added, and having the ability to act as a plant promoter
[0008]
The present invention also relates to a plant expression vector in which the above plant promoter is introduced into a vector.
[0009]
Furthermore, the present invention is a transformed plant cell obtained by introducing the above plant expression vector into a host plant cell.
[0010]
The present invention also provides a transformed plant regenerated from the transformed plant cell.
[0011]
The present invention is a plant seed obtained from the transformed plant body.
[0012]
The present invention introduces a plant expression vector into which a plant promoter is inserted into a host plant cell to obtain a transformed plant cell, regenerates the transformed plant from the transformed plant cell, and a plant is obtained from the obtained transformed plant. A method for producing a plant body comprising obtaining seeds and producing a plant body from the seeds.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
The “plant promoter” of the present invention is DNA having the ability to act as a promoter (promoter function) in plants. “Promoter” means a specific base sequence on DNA that initiates mRNA synthesis (transcription) using DNA as a template. It has a common base sequence and recognizes this to synthesize mRNA (RNA polymerase). Synthesize mRNA. In the present invention, “promoter function” refers to the action of DNA polymerase binding to a specific region on DNA and initiating transcription. The plant promoter of the present invention specifically includes the following (a): Or (B) DNA.
(A) DNA consisting of the base sequence described in SEQ ID NO: 1.
(B) In DNA comprising the base sequence of (a), one or more base DNA comprising a nucleotide sequence deleted, substituted or added and having the ability to act as a plant promoter.
[0014]
In order to obtain the plant promoter of the present invention, the present inventors cloned the upstream region of pKC18 (Japanese Patent Application No. 8-31987), a gene that gave a strong signal in the ovule and the growing fiber by Northern blotting. As a result of Northern blotting, pKC18 showed a strong signal in the ovule on the 10th day of flowering, the strongest signal in the fibers on the 14th day after flowering, and no signal in the mature seeds, seedlings, and leaves. Thus, it can be predicted that a very specific promoter acting during fiber elongation exists in the upstream region of pKC22. Therefore, the plant promoter of the present invention has a nucleotide sequence (SEQ ID NO: 1) derived from cotton (Gossypium genus). Furthermore, the base sequence described in SEQ ID NO: 1 is a base sequence located upstream of pKC18 that is expressed in cotton fiber tissue in the early stage of cotton fiber elongation.
[0015]
The plant promoter of the present invention is a DNA comprising the base sequence set forth in SEQ ID NO: 1, wherein base Is a DNA consisting of a base sequence deleted, substituted or added and having the ability to act as a plant promoter. The plant promoter of the present invention includes those obtained by deleting the 5 ′ end of the base sequence described in SEQ ID NO: 1 as long as the promoter activity exists.
[0017]
The “plant expression vector” of the present invention is obtained by introducing the above plant promoter into a vector. Examples of the vector include a vector derived from E. coli, such as a pT7Blue vector, a plasmid containing a β-glucuronidase gene (GUS), pBI101- Hm2, shuttle vector, helper plasmid, pRK2013 and the like. Also, plant viruses such as calflower mosaic virus can be used. The vector is selected according to each host cell.
The method for introducing a plant promoter into a vector follows the method for introducing a normal gene into a vector.
[0018]
The “transformed plant cell” of the present invention is a transformed plant cell obtained by introducing the plant expression vector into a host plant cell. Examples of host plant cells include Arabidopsis thaliana Wassilewskija strain, tomato, tobacco, petunia, wheat, rice, corn, pumpkin, cucumber, cotton and the like.
Examples of transformation methods for introducing plant expression vectors into host plant cells include electroporation method, protoplast fusion method, microinjection method, polyethylene glycol method and particle gun method.
[0019]
The “transformed plant body” of the present invention is a transformed plant body regenerated from the transformed plant cell. As a regeneration method, there is a method in which callus-like transformed cells are transferred to a medium with different types and concentrations of hormones and cultured to form somatic embryos to obtain complete plants. As a medium to be used, cotton uses MSIC medium (MS salt, 0.75% MgCl ₂ , 1.9g / l KNO _Three , 30 g / l glucose, 2.0 g / l gellan gum, pH 5.8), and carrots include Kamada & Harada medium.
[0020]
In the “method for producing a plant” of the present invention, a plant expression vector having the plant promoter inserted therein is introduced into a host cell to obtain a plant cell, and a transformed plant body is regenerated from the plant cell. A step of creating a plant seed from the converted plant and producing the plant from the seed is included.
The step of creating plant seeds from the transformed plant is to take the transformed plant from the rooting medium, plant it in a pot containing water-containing soil, and grow it at a constant temperature. The process of growing, forming flowers, and finally forming seeds. In addition, the process of producing a plant from seeds is to isolate the seed formed on the transformed plant when it has matured, sow it in soil containing water, and grow it under a constant temperature and illuminance. The process of producing a plant body.
[0021]
The following processes are illustrated as a specific process example of the present invention.
(1) Cloning of upstream region of cotton fiber tissue specific gene, pKC18
(2) Fusion with β-glucuronidase gene (GUS gene)
(3) Introduction of plasmid into Agrobacterium
(4) Cultivation of sterile Arabidopsis
(5) Agrobacterium infection
(6) Disinfection
(7) Selection of transformed plants
(8) Regeneration of transformed plants
(9) Acquisition of antibiotic-resistant strains
(10) Measurement of GUS activity
[0022]
<1> Isolation of promoter region of genes involved in cotton fiber formation and elongation
A synthetic oligonucleotide is prepared from the base sequence of the tissue-specific gene pKC18 (Japanese Patent Application No. 8-31987) obtained from cotton fiber, and subjected to inverse PCR. Genomic DNA is extracted from cotton fiber, cut with restriction enzyme EcoRI, self-ligated, and used as a PCR template. After performing PCR using the first primer group, the reaction product is further subjected to PCR using the second primer group, and the upstream region is cloned.
After obtaining a clone of the desired length by agarose gel electrophoresis, it is subcloned into a TA cloning vector and sequenced using an autosequencer. Since a part of the sequence of the obtained PCR fragment completely coincided with the sequence of pKC18, it was determined to be upstream of pKC18 (plant promoter).
[0023]
<2> Utilization of promoter region of gene for cotton fiber formation and elongation
The plant promoter obtained by the above method can be used to control the expression of proteins involved in fiber formation and elongation by creating a chimeric gene contract in cotton plants or plants other than cotton. Furthermore, when combined with a DNA sequence encoding a signal peptide, cell wall components can be modified by the expression of various proteins on the cell wall, and can be applied to breeding new plants with added disease resistance.
[0024]
For example, when a gene involved in fiber formation and elongation is connected to the plant promoter of the present invention and introduced into cotton plants or other plants, the content of the target protein can be increased. On the other hand, when a negative strand (sequence complementary to the coding sequence) at least partly connected to the promoter in the reverse direction is introduced into a plant and so-called antisense RNA is expressed, the content of the target protein is reduced. Can do.
[0025]
<3> Introduction of plant promoter into vector and transformation of host plant cell
Plant cell transformation methods include electroporation, in which plasmids are introduced into plant cells by electropulsing protoplasts, fusion of protoplasts with small cells, cells, lysosomes, microinjection method, polyethylene glycol method, etc. Alternatively, a method such as a particle gun method can be used.
Moreover, the target gene can be introduced into a plant by using a plant virus as a vector. As a plant virus to be used, for example, cauliflower mosaic virus (CaMV) can be used. That is, first, a viral genome is once inserted into a vector derived from E. coli to prepare a recombinant, and then these target genes are inserted into the viral genome. These target genes can be inserted into the plant body by excising the viral genome thus modified from the recombinant with a restriction enzyme and inoculating the plant body [Hohn et al., Molecular Biology of Plant Tumors, Academic Press, New York, 549-560 (1982), US Pat. No. 4,407,956].
[0026]
Furthermore, there is a method using the Agrobacterium Ti plasmid, which utilizes the property that when a bacterium belonging to the genus Agrobacterium infects a plant, a part of the plasmid DNA possessed by the bacterium is transferred into the plant genome. These target genes can also be introduced into plants. Among the bacteria belonging to the genus Agrobacterium, Agrobacterium tumefaciens infects plants and develops a tumor called crown gall. However, during infection, a region called a T-DNA region (Transferred DNA) on a plasmid present in each bacterium called a Ti plasmid or Ri plasmid is transferred into the plant during infection, and is transferred into the plant genome. Due to being incorporated into Furthermore, on the Ti or Ri plasmid, there is a region called a vir region that is essential for the T-DNA region to be transferred into the plant and integrated into the plant genome. The vir region itself is not transferred into the plant, and this vir region can function even on a different plasmid from that in which the T-DNA region exists [Nature, 303, 179, (1983). ].
[0027]
If the DNA to be integrated into the plant genome is inserted into the T-DNA region on the Ti or Ri plasmid, the target DNA is transferred into the plant genome when a bacterium belonging to the genus Agrobacterium infects the plant body. Can be incorporated. Here, the crown gall in the T-DNA of the Ti or Ri plasmid or the part causing the hairy root can be removed without impairing the intended migration function, and the resulting product can be used as a vector. In the present invention, such various vectors can be used.
For example, a vector such as pBI101 (Clontech) called a binary vector, in which a gene involved in fiber formation and elongation is connected in the sense or antisense direction to the promoter of the present invention, is inserted into plant cells. can do. Note that these vectors do not have the vir region described above, and the Agrobacterium bacterium used by introducing the vector needs to contain another plasmid having the vir region. is there.
[0028]
In addition, these vectors are shuttle vectors that can be amplified not only in bacteria belonging to the genus Agrobacterium but also in E. coli. Therefore, the recombination operation of the Ti plasmid can be performed using E. coli. Furthermore, these vectors contain an antibiotic resistance gene, so that transformants can be easily selected when transforming E. coli, Agrobacterium bacteria, plant cells, or the like. In addition, these vectors contain the cauliflower mosaic virus, CaMV 35S promoter, and the genes inserted into these vectors can be expressed in a non-regulatory manner after being integrated into the plant genome.
[0029]
<4> Regeneration of plant bodies from transformed plant cells
Below, the introduction of the gene of interest by Agrobacterium in Arabidopsis thaliana and the method of regenerating transformed cells into plants will be described in detail.
Arabidopsis seeds are sown on a GM plate according to a conventional method and cultivated aseptically. Callus culture is performed on CIM plates using rooted hypocotyl sections. The target gene is connected to the plant promoter of the present invention, Agrobacterium transformed with a plasmid having a kanamycin and hygromycin resistance gene is cultured, and the diluted one is dispensed into a tube, and a callus-shaped hypocotyl section is obtained. Soak and co-cultivate on CIM plate for several days. When Agrobacterium is sufficiently grown until it can be observed with the naked eye, sterilization is performed and culture is performed on a SIM plate for several days. These sections are finally cultured on SIM plates and transplanted to new plates every week. Transformed sections continue to grow and callus appears. Non-transformed sections turn brown because of selection with antibiotics. Incubate until the transformants are about 5 mm in size and form shoots. When the complete shoot shape is exhibited, the root of the shoot is cut with a scalpel so as not to include the callus portion and transplanted to the RIM plate. When a large callus is attached, the roots come out through the callus even after rooting, and the vascular bundle is often not connected to the rosette. After rooting, inorganic salt medium [5 mM KNO _Three , 2.5 mM K-phosphate buffer (pH 5.5), 2 mM MgSO _Four , 2mM Ca (NO _Three ) ₂ , 50μM Fe-EDTA, 1000 × Trace element (70mM H _Three BO _Three , 14mM MnCl ₂ , 0.5mM CuSO _Four , 1mM ZnSO _Four , 0.2mM NaMoO _Four , 10mM NaCl, 0.01mM CoCl ₂ ) Planted on rock wool soaked in 1 ml / liter].
[0030]
The rooted plant can be transplanted to soil soaked in an inorganic salt medium to obtain seeds. The seed can be sterilized, seeded in a GM medium and germinated to obtain a transformant. From this transformant, DNA is extracted according to a conventional method, this DNA is cleaved with an appropriate restriction enzyme, and Southern hybridization is performed using a gene involved in fiber formation and elongation as a probe to confirm the presence or absence of transformation. can do.
[0031]
In addition, RNA is extracted from transformants and non-transformants according to a conventional method, and probes having sense or antisense sequences of genes involved in fiber formation and elongation are prepared. It is possible to examine the state of expression of the target gene by performing the initialization.
[0032]
【The invention's effect】
In the present invention, when a GUS gene widely used as a reporter gene in plants is connected to the 3 ′ end of the promoter of the present invention and used, the strength of the promoter can be easily evaluated by examining the GUS activity. The reporter gene is not limited to the GUS gene, and luciferase and green fluorescein protein can also be used.
Genes involved in fiber formation and elongation are expressed in cotton fiber cells in the process of cotton fiber formation and are involved in fiber elongation. By using the upstream region of this base sequence, etc., transcription factors involved in cotton fiber elongation May be identified. Therefore, the plant promoter of the present invention is a promoter involved in cotton fiber formation and elongation, establishment of technology for inducing fiber formation and elongation, isolation of cis and trans factors involved in fiber elongation, fiber formation and elongation. It can be used for elucidation of the mechanism of or the isolation of genes that regulate it, and is extremely useful in the technical fields of cell formation and elongation.
[0033]
Furthermore, the structure of the plant cell wall of a specific tissue can be changed by producing a chimeric gene in which a base sequence encoding a protein involved in fiber formation and elongation and the plant promoter of the present invention is combined. Useful for processing plant materials used in the field. In general, by using the 35S promoter of cauliflower mosaic virus, it is possible to cause morphological changes throughout the life cycle of the whole plant organ. If a regulatory promoter such as light, heat, or injury is used, a plant body whose form can be changed according to the growth environment can be produced. In addition, if a promoter specific to an organ or tissue is used, a morphological change can be caused only in a specific organ or tissue. That is, since the promoter of the present invention is organ-specific, by using the promoter of the present invention, for example, fiber formation can be controlled, and changes in fiber characteristics can be brought about.
[0034]
By using the plant promoter of the present invention, a target gene can be expressed specifically in a specific plant tissue. In particular, the target gene can be expressed during the Arabidopsis vascular system and cotton fiber elongation. Furthermore, by using the plant promoter of the present invention and expressing a specific gene in cotton fiber, it becomes possible to improve the properties (fiber length, fineness, strength, etc.) of cotton fiber and increase the yield. Furthermore, by using the plant promoter of the present invention, a new cotton variety having superior fiber characteristics and high productivity can be produced.
[0035]
【Example】
Hereinafter, the present invention will be specifically described by way of examples.
Example 1
(1) Cloning of cotton fiber tissue gene, upstream region of pKC18 (GKC18) After seeding cotton plant (genus Gossypium), genomic DNA was extracted by using Murray and Thompson's improved method using seedlings on the 18th Then, genomic DNA was cloned by the inverse PCR method.
Specifically, 1 μg of genomic DNA was cleaved with the restriction enzyme HindIII and then self-ligated with T4 ligase. The upstream region of pKC18 (hereinafter abbreviated as GKC18) was amplified using a PCR reagent kit (LA-PCR kit, Takara Shuzo) using 500 μg of the ligated DNA as a template. The reaction was carried out for 28 cycles at 94 ° C. for 15 seconds and 65 ° C. for 12 minutes using the first primer group having the nucleic acid sequences shown in SEQ ID NOs: 2 and 3. Next, using this PCR product, PCR was performed under the same conditions using a second primer group having the base sequences shown in SEQ ID NOs: 4 and 5. The amount and length of this PCR product were confirmed by electrophoresis, the obtained DN fragment was introduced into a vector, pT7BlueT, and Escherichia coli JM109 was transformed and cloned. Thereafter, the base sequence of GKC18 was determined using a Sequenase sequence kit (Amersham). The nucleotide sequence was completely consistent with the cDNA from the primer sequence, confirming that it was a genomic region corresponding to the cDNA. This base sequence is shown in SEQ ID NO: 1.
[0036]
(2) Fusion with β-glucuronidase gene (GUS gene)
A plasmid containing the β-glucuronidase gene (GUS), pBI101-Hm2 (obtained from Nara Institute of Science, Prof. Yasuhiko Shinna) was cleaved with restriction enzymes, HindIII and SacI, and subjected to agarose gel electrophoresis. The GUS gene was removed. This GUS gene was inserted into a plasmid, restriction enzyme of pUC19, HindIII and SacI sites to obtain a plasmid, pGUS.
Next, the plasmid, pGKC18, was cleaved with a restriction enzyme, NcoI, blunt-ended, cleaved with HindIII, and inserted into the plasmid, pGUS restriction enzyme, HindIII / SmaI site. It was confirmed whether or not it was correctly inserted from the base sequence of the inserted EcoRI-SacI fragment, and the correctly inserted plasmid was named pGKC18: GUS. Furthermore, this plasmid, pGKC18: GUS was cleaved with restriction enzymes, HindIII and SacI, and after electrophoresis, the HindIII-SacI fragment was inserted into the above plasmid, pBI101-Hm2 restriction enzyme, HindIII and SacI sites, and a novel plasmid PBI18: GUS was obtained. Subsequently, Escherichia coli JM109 / pBI18: GUS was obtained by transforming E. coli JM109 with the plasmid, pBI18: GUS. This process is shown in FIG.
[0037]
(3) Introduction of plasmid into Agrobacterium
The Escherichia coli obtained in (2), Escherichia coli JM109 / pBI101-18: GUS and a helper plasmid, pRK2013, were cultured overnight in an LB medium containing 59 mg / l kanamycin at 37 ° C. overnight. Separately, Agrobacterium EHA101 strain (obtained from Prof. Shinna, Nara Institute of Science) was cultured overnight at 37 ° C. in LB medium containing 50 mg / l kanamycin.
1.5 ml of each culture solution was taken in an Eppendorf tube, collected, and washed with LB medium. These cells were suspended in 1 ml of LB medium, 100 μl of 3 kinds of bacteria were mixed, spread on LB agar medium, and cultured at 28 ° C. to transfer the plasmid to Agrobacterium. One or two days later, a portion was scraped with a platinum loop and spread on an LB agar medium containing 50 mg / l kanamycin, 20 mg / l hygromycin and 25 mg / l chloramphenicol. After culturing at 28 ° C. for 2 days, a single colony was selected. The resulting transformant was named EHA101 / pBI18: GUS.
[0038]
(4) Cultivation of sterile Arabidopsis thaliana
Ten seeds of Arabidopsis Wassilewskija strain (hereinafter referred to as WS strain) (obtained from Nara Institute of Science, Professor Shinhiko Shinna) were placed in a 1.5 ml tube, 1 ml of 70% ethanol was added and left for 3 minutes. Subsequently, after immersing in a sterilizing solution (5% sodium hypochlorite, 0.02% TritonX-100) for 3 minutes and washing 5 times with sterilized water, GM plates (4.3 g Murashige-Skoog inorganic salt per liter, 10 g of sugar, 0.1 g of myo-inositol, 5 g of gellan gum, pH 5.7). This plate was allowed to stand at 4 ° C. for 2 days, followed by low-temperature treatment, and then cultured in a plant incubator (manufactured by Sanyo, MLR-350HT) at 22 ° C. under low light for 7 days.
[0039]
(5) Agrobacterium infection
Prepare several WS hypocotyls cultured for 7 days in (4) above, cut them to about 1.0 cm with a scalpel, CIM plate (3.9 g of Gambog B5 salt per liter, 20 g of glucose, 0.1 g of myo-inositol). Gellan gum 5 g, 2,4-dichlorophenoxyacetic acid 0.5 μg, and kinetin 0.05 μg were added and arranged at pH 5.7). Cultured for 2 days in light intensity 3000 lux, 24 hours light period, and the transformant obtained in (3) above, EHA101 / pBI18: GUS cultured for 1 day at 28 ° C, MS dilution (Murashige Dissolve 3 times diluted with sukuog inorganic salts 6.4g / l, pH6.3) into each tube 1ml and callus hypocotyl (cultured in CIM medium for 2 days) for 10 minutes. Soaked. They were arranged on two sterilized filter papers to remove excess water, and about 20 each were placed on a new CIM plate. Co-cultured for 2 days under the same conditions.
[0040]
(6) Disinfection
Sections that had grown sufficiently until each strain was observable with the naked eye were transferred to a sterilization solution (MS dilution solution added with kraforan to a final concentration of 200 μg / ml), and gently shaken and washed for 60 minutes. . After repeating this operation 5 times, water was removed on a sterile filter paper, and SIM plate (3.9 g of Gambog B5 salt per liter, glucose 20 g, myo-inositol 0.1 g, gellan gum 5 g, kanamycin 50 mg, hygromycin B 20 mg, claforan 0.2 g , Vancomycin 0.5 g, 2 iP 5 mg, IAA was added to a final concentration of 0.15 mg, placed at pH 5.7), and cultured at a light intensity of 4000 lux for 2 days.
[0041]
(7) Selection of transformed plants
The sections cultured for 2 days as described above were transplanted to a SIM plate and cultured at a light intensity of 4000 lux. Thereafter, it was transplanted to a new SIM plate every week. Transformed sections continued to grow and became dome-shaped calli, while non-transformants browned. The transformant had a green callus after about 2 weeks, and a shoot was formed after about 1 month.
[0042]
(8) Regeneration of transformed plants
Cut the root of the plant that became the shoot with a razor blade or scalpel so as not to include the callus part, and RIM plate (MS salt 4.3 g per liter, sucrose 10 g, myo-inositol 0.1 g, gellan gum 3.5 g, indole buty Rick Acid 0.02 mg, pH 5.7) was inserted lightly. About 1 month later, a few roots of about 1 to 2 cm were taken out with tweezers, pearlite and vermiculite (manufactured by TES) were mixed 1: 1 and replanted in soil soaked in an inorganic salt mixed medium. . After about one month, several hundred seeds were obtained per strain. This is hereinafter referred to as T1 seed.
[0043]
(9) Acquisition of antibiotic-resistant strains
About 100 T1 seeds were sterilized by the same method as in (4) above and sown on a GM plate (with kanamycin 50 mg and hygromycin B 20 mg). Kanamycin and hygromycin B resistant strains germinated at a ratio of approximately 3: 1.
[0044]
(10) Measurement of GUS activity
The resulting homozygous transformed Arabidopsis thaliana is cultivated, the tissue is cut with a scalpel, and the section is placed in a few ml of fixative (0.03% formalin, 10 mM MES (pH 5.6), 0.3 M mannitol) for 45 minutes at room temperature. Immerse. Next, it was washed several times with 50 mM phosphate buffer (pH 7.0). Immerse the sections in GUS staining solution (50 mM phosphate buffer, 0.5 M potassium ferricyanide, 0.5 M potassium ferrocyanide, 1 mM X-Gluc) and suck the solution well into the sample by vacuuming, and incubate at 37 ° C overnight. did. To remove chlorophyll, the sample was immersed in 5% formalin for 10 minutes, then immersed in 5% acetic acid for 10 minutes, 50% ethanol for 10 minutes, and 100% ethanol for 10 hours. When the blue-stained tissue was observed under a microscope, promoter activity was also observed in Arabidopsis thaliana. In particular, seedling expression was observed (FIG. 2). In addition, expression was observed in the leaves of flower stems (FIG. 3). Furthermore, expression was also observed at the base of the pod at 9 weeks (FIG. 4). From these results, it was found that the DNA of the present invention functions as a tissue-specific promoter even in Arabidopsis thaliana plants other than cotton.
[0045]

[0046]

[0047]

[0048]

[0049]

[Brief description of the drawings]
FIG. 1 shows the construction of a transformant, Escherichia coli JM109 / pBI18: GUS.
FIG. 2 is a photograph replacing a figure showing GUS staining of transformed Arabidopsis thaliana (growth in the dark, Arabidopsis thaliana at 1 week).
FIG. 3 is a photograph replacing a graph showing GUS staining of transformed Arabidopsis thaliana (Arabidopsis thaliana at 7 weeks).
FIG. 4 is a photograph replacing a figure showing GUS staining of transformed Arabidopsis thaliana (Arabidopsis thaliana at 9 weeks).

Claims (7)

The plant promoter containing DNA of the following (a) or (b).
(A) DNA comprising the base sequence described in SEQ ID NO: 1
(B) a DNA comprising the base sequence of (a), comprising a base sequence in which one or more bases have been deleted, substituted or added, and having the ability to act as a plant promoter A plant expression vector in which the plant promoter according to claim 1 is introduced into a vector. A transformed plant cell obtained by introducing the plant expression vector according to claim 2 into a host plant cell. A transformed plant body regenerated from the transformed plant cell of claim 3. Plant seeds obtained from the transformed plant body of claim 4. A plant expression vector into which a plant promoter according to claim 1 is inserted is introduced into a host cell to obtain a transformed plant cell, a transformed plant is regenerated from the transformed plant cell, and a plant is obtained from the obtained transformed plant. A method for producing a plant comprising obtaining seeds and producing a plant from the seeds. An expression vector into which a plant promoter containing DNA comprising the nucleotide sequence of SEQ ID NO: 1 is inserted is introduced into a host cell to obtain a transformed cell, and a transformed cotton plant is regenerated from the transformed cell. A method for producing a cotton plant comprising producing a cotton plant seed from the converted cotton plant and producing a cotton plant from the cotton plant seed.

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