JP4100494B2 - Late seed maturation and germination stage specific inducible promoters - Google Patents

Description

【００６２】
【配列表フリーテキスト】
配列番号２及び３は、合成プライマーである。
【図面の簡単な説明】
【図１】ノーザンブロット分析におけるハイブリダイゼーションの結果を示す電機泳動写真である。
【図２】形質転換植物体におけるGUS活性測定の結果を示す特性図である。
【図３】形質転換植物体から収穫した種子に対する吸水処理後0〜1日の写真である。
【図４】形質転換植物体から収穫した種子に対する吸水処理後2日の写真である。
【図５】形質転換植物体から収穫した種子に対する吸水処理後4日の写真である。
【図６】形質転換植物体から収穫した種子に対する吸水処理後21日の写真である。
【図７】形質転換植物体から収穫した種子に吸水処理を行わなかったときの写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel promoter exhibiting specific inducibility in late seed maturation and germination stages.
[0002]
[Prior art]
Arabidopsis thaliana has been extensively studied as a model plant in gene sequencing projects, etc., and the complete genome sequence of two chromosomes has been determined (Lin, X. et al., (1999 ) Nature 402, 761-768 .; Mayer, K. et al., (1999) Nature 402, 769-777.), Estimated to have about 26,000 genes.
[0003]
Based on such genomic information, it is desired to elucidate the functions of important genes throughout plants by using plant-specific genes and various mutants of Arabidopsis thaliana. It is possible to understand the role of useful genes shared by plants from the genome function research on Arabidopsis thaliana. In particular, among the genes of Arabidopsis thaliana, the functions of genes characteristic of plants involved in environmental response, morphogenesis, developmental differentiation, photosynthesis, substance metabolism, etc. have been clarified. Furthermore, through genome function analysis research, it is desired to search for useful genes related to the production of crops, trees, useful substances and energy.
[0004]
[Problems to be solved by the invention]
By the way, in plants such as Arabidopsis thaliana, the seed formation process is an initial stage in which cell division is actively repeated after fertilization to form larvae, and cell division is stopped to accumulate storage substances and acquire drought tolerance. It is divided into the middle and late stages of entering a dormant state. It is considered that immature seeds after the initial stage can be germinated if cultured under appropriate conditions, and the embryonic development is interrupted to enter the middle / late stage.
[0005]
However, even with the above-described studies on Arabidopsis, there is no known example in which a gene that is specifically expressed in a predetermined period in the seed formation process is identified.
An object of the present invention is to provide a promoter exhibiting specific inducibility in late seed maturation and germination stages by identifying genes that are specifically expressed in a predetermined period in the seed formation process.
[0006]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have succeeded in identifying a gene that is specifically expressed in the late stage of seed maturation and isolating its promoter region, thereby completing the present invention. It came.
(1) A promoter having specific inducibility in the late stage of seed maturation and germination, which comprises the following DNA of (a), (b) or (c).
(a) DNA consisting of the base sequence of SEQ ID NO: 1
(b) DNA consisting of a base sequence in which one or more bases have been deleted, substituted or added in the base sequence of SEQ ID NO: 1, and functioning as a late seed maturation and germination stage specific inducible promoter
(c) DNA that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and functions as an inducible promoter specific for late seed maturation and germination stages
[0007]
(2) The promoter according to (1), wherein the late stage of seed maturation is a period from the start of abscisic acid synthesis in the seed to the completion of a fully matured seed.
(3) The promoter according to (1), wherein the germination period is a period from the completion of mature seeds to the 12th day after germination.
(4) An expression vector comprising the promoter according to (1).
(5) An expression vector in which any gene is further incorporated into the expression vector according to (4).
(6) A transformant comprising the expression vector according to (5).
(7) (5) A transgenic plant comprising an expression vector.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The present inventor has found that the AtNCED2 gene present in the Arabidopsis thaliana genome is expressed according to the amount of abscisic acid in the seed. In plants such as Arabidopsis thaliana, abscisic acid is involved in important physiological functions such as desiccation, acquisition of low-temperature stress response and tolerance, seed maturation, dormancy, and germination. Abscisic acid is not synthesized in the early stage of the seed formation process, but begins to be synthesized in the middle and late stages.
[0009]
Therefore, the AtNCED2 gene promoter induces the expression of a downstream gene specifically at least in the late stage of seed maturation. The nucleotide sequence of 1.9 kbp upstream of the AtNCED2 gene is shown in SEQ ID NO: 1.
That is, the promoter according to the present invention includes the following DNA (a), (b) or (c), and can specifically express a gene arranged downstream in the late stage of seed maturation and germination.
[0010]
(a) DNA consisting of the base sequence of SEQ ID NO: 1
(b) DNA consisting of a base sequence in which one or more bases are deleted, substituted or added in the base sequence of SEQ ID NO: 1
(c) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence of SEQ ID NO: 1
[0011]
In the DNA shown in (b) above, “plurality” means lacking for the DNA of SEQ ID NO: 1 as long as the function of specifically expressing the downstream gene in the late stage of seed maturation and germination is lost. It means that the number of bases to be lost, substituted or added is not limited. Specifically, even if the base sequence has about several hundred bases deleted, substituted, or added to the DNA of SEQ ID NO: 1, the downstream gene is transferred to the late stage of seed maturation and germination. In the case where the function of specifically expressing is not lost, the promoter according to the present invention is included.
[0012]
In the DNA shown in (c) above, stringent conditions mean a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 42 to 68 ° C, preferably 42 to 65 ° C. More specifically, stringent conditions mean 5 × SSC (83 mM NaCl, 83 mM sodium citrate) and a temperature of 42 ° C. That is, even a base sequence that does not completely match the DNA of SEQ ID NO: 1 can be hybridized under the above stringent conditions, and the downstream gene can be placed in the late seed maturation and germination stages. In the case where the function of specifically expressing is not lost, the promoter according to the present invention is included.
[0013]
Here, “specific expression in the late stage of seed maturation and germination stage” refers to the function of RNA polymerase binding to the promoter and initiating transcription when the promoter is exposed in the late stage of seed maturation and germination stage. Further, the late seed maturation period means a period from the start of abscisic acid synthesis in the seed to the completion of the fully mature seed. The germination period means the period from the completion of fully matured seeds to the 12th day after germination.
[0014]
Once the base sequence of the promoter is determined, the present invention is followed by chemical synthesis, PCR using a cloned probe as a template, or hybridization with a DNA fragment having the base sequence as a probe. Such a promoter can be obtained. Furthermore, a mutant form of the promoter according to the present invention having a function equivalent to that of the promoter before mutation can be synthesized by site-directed mutagenesis.
[0015]
In order to introduce a mutation into the promoter sequence, a known method such as the Kunkel method or the Gapped duplex method, or a method based thereon can be employed. For example, using a mutagenesis kit (for example, Mutant-K (TAKARA) or Mutant-G (TAKARA)) using site-directed mutagenesis, or TAKARA LA PCR in vitro Mutagenesis Mutation is introduced using a series kit.
[0016]
1. Promoter isolation
The promoter of the present invention is a cis element existing upstream of the AtNCED2 gene that is specifically expressed in the late stage of seed maturation and germination in the seed formation process, and activates transcription of the downstream gene by binding to a transcription factor. It has a function to convert.
In isolating the promoter of the present invention, the AtNCED2 gene is first identified as follows in Arabidopsis thaliana as a gene that is specifically expressed in the late stage of seed maturation in the seed formation process.
[0017]
In maize, an ABA synthesis ability deficient strain is known in the late stage of seed maturation, and its gene has already been isolated as a Vp14 gene. Genes highly homologous to this Vp14 gene were collected from the Arabidopsis genome sequence by Blast search. Among the obtained genes, the same pattern as the increase in the ABA amount of the seed width, that is, a gene expressed at the seed maturation stage was identified by Northern hybridization.
[0018]
Thus, after identifying the AtNCED2 gene as a gene that is specifically expressed in the late stage of seed maturation and germination in the seed formation process, the base sequence of 1.9 kbp upstream of the AtNCED2 gene is isolated as a promoter. When isolating the base sequence of 1.9 kbp upstream of the AtNCED2 gene, any known technique may be used. Specifically, the AtNCED2 gene is obtained by extracting a genomic DNA of Arabidopsis thaliana and specifically amplifying and purifying a 1.9 kbp base sequence by PCR using the genomic DNA as a template and a pair of primers having a predetermined base sequence. A base sequence of 1.9 kbp upstream of can be obtained. A pair of primers used in the PCR can be appropriately designed from information on the genomic base sequence of Arabidopsis thaliana, and can be prepared by chemical synthesis based on this design.
[0019]
Whether or not the isolated 1.9 kbp base sequence upstream of the isolated AtNCED2 gene has a function of specifically expressing the gene arranged downstream thereof in the late stage of seed maturation and germination stage is described in “2. Can be examined using the expression vector constructed. That is, an expression vector incorporating a gene serving as a marker downstream of the 1.9 kbp upstream sequence of the AtNCED2 gene is transformed into a predetermined plant cell, and the resulting transformed plant is grown. At this time, the expression level of the marker gene is observed at each stage of growing the transformed plant, and it is confirmed whether the marker gene is specifically expressed in the late stage of seed maturation and germination. Then, by confirming that the marker gene is not expressed in the growth stage other than the late stage of seed maturation and germination, but only in the late stage of seed maturation and germination, 1.9 upstream of the isolated AtNCED2 gene is confirmed. It can be demonstrated that the kbp base sequence has a function of specifically expressing a gene arranged downstream thereof in the late seed maturation and germination stages.
[0020]
2. Construction of expression vector
The expression vector according to the present invention can be obtained by linking (inserting) the promoter isolated in the above “1” to an appropriate vector. The vector for inserting the promoter is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, shuttle vectors, and helper plasmids.
[0021]
As plasmid DNA, plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YCp50, etc.) And λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.
[0022]
In order to insert a promoter into a vector, first, a method in which purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and linked to the vector is employed. .
In the present invention, in order to express an arbitrary gene, the arbitrary gene can be further inserted into the expression vector. The method for inserting an arbitrary gene is the same as the method for inserting a promoter into a vector. Any gene is not particularly limited.
[0023]
When the promoter according to the present invention is used by linking a reporter gene, for example, a GUS gene widely used in plants, to its 3 ′ end, the strength of the promoter can be easily evaluated by examining GUS activity. it can. In addition to the GUS gene, luciferase, green fluorescein protein, and the like can be used as the reporter gene.
[0024]
Thus, various vectors can be used in the present invention. Furthermore, the promoter of the present invention can be prepared by connecting a desired gene of interest in the sense or antisense direction and inserted into a vector such as pBI101 (Clonetech) called a binary vector.
[0025]
3. Production of transformants
The transformant according to the present invention can be obtained by introducing the expression vector obtained in the above “2” into a host. Here, the host is not particularly limited as long as it can express a promoter or a target gene, but a plant is preferable. When the host is a plant, a transformed plant (transgenic plant) can be obtained as follows.
[0026]
Plants to be transformed include whole plants, plant organs (e.g. leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g. epidermis, phloem, soft tissue, xylem, vascular bundles, etc.) or plants Any of cultured cells is meant. Examples of plants used for transformation include plants belonging to the Brassicaceae, Gramineae, Solanum, Legumes, etc. (see below), but are not limited to these plants.
[0027]
Brassicaceae: Arabidopsis thaliana
Solanum: Tobacco (Nicotiana tabacum)
Gramineae: maize (Zea mays), rice (Oryza sativa)
Legumes: Soybean (Glycine max)
The recombinant vector can be introduced into a plant by a conventional transformation method such as electroporation (electroporation), Agrobacterium method, particle gun method, PEG method and the like.
[0028]
For example, when the electroporation method is used, the gene is introduced into the host by treatment with an electroporation apparatus equipped with a pulse controller under conditions of a voltage of 500 to 1600 V, 25 to 1000 μF, and 20 to 30 msec.
When the particle gun method is used, a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. The sample thus prepared can be processed using a gene introduction apparatus (for example, PDS-1000 / He manufactured by Bio-Rad). The treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 1000 to 1800 psi and a distance of about 5 to 6 cm.
[0029]
Moreover, a target gene can be introduced into a plant body by using a plant virus as a vector. Examples of plant viruses that can be used include cauliflower mosaic virus. That is, first, a virus genome is inserted into a vector derived from E. coli to prepare a recombinant, and then these target genes are inserted into the virus genome. The gene of interest can be introduced into a plant host by excising the viral genome thus modified from the recombinant with a restriction enzyme and inoculating the plant host.
[0030]
In the method using the Agrobacterium Ti plasmid, when a bacterium belonging to the genus Agrobacterium infects a plant, it takes advantage of the property of transferring a part of the plasmid DNA that it has into the plant genome. The target gene is introduced into the plant host. Among the bacteria belonging to the genus Agrobacterium, Agrobacterium tumefaciens infects plants to form a tumor called crown gall, and Agrobacterium rhizogenes infects plants. To generate hairy roots. These are because the region called T-DNA region (Transferred DNA) on the plasmid that is present in each bacterium called Ti plasmid or Ri plasmid at the time of infection is transferred into the plant and integrated into the genome of the plant. This is due to
[0031]
If the DNA to be incorporated 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 Agrobacterium bacteria infect the plant host. Can be incorporated.
Tumor tissue, shoots, hairy roots, and the like obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture, and can be used at an appropriate concentration using a conventionally known plant tissue culture method. Of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).
[0032]
The expression vector obtained in the above “2” is not only introduced into the plant host, but also Escherichia such as Escherichia coli, Bacillus such as Bacillus subtilis, or Pseudomonas ptida (Pseudomonas Introduced into bacteria belonging to the genus Pseudomonas such as putida), yeast such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, animal cells such as COS cells and CHO cells, or insect cells such as Sf9 Thus, a transformant can also be obtained. When a bacterium such as Escherichia coli or yeast is used as a host, the recombinant vector of the present invention can autonomously replicate in the bacterium, and at the same time, is composed of the promoter of the present invention, a ribosome binding sequence, a target gene, and a transcription termination sequence. It is preferable. Moreover, the gene which controls a promoter may be contained.
[0033]
The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.
When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe and the like are used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. Examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.
[0034]
When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells and the like are used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.
When insect cells are used as hosts, Sf9 cells and the like are used. Examples of methods for introducing a recombinant vector into insect cells include the calcium phosphate method, lipofection method, electroporation method and the like.
[0035]
Whether or not the gene has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization or the like. For example, DNA is prepared from the transformant, PCR is performed by designing a DNA-specific primer. PCR is performed under the same conditions as those used for preparing the plasmid. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, Confirm that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, a method of binding the amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence or enzyme reaction may be employed.
[0036]
4). Plant production
The transgenic plant according to the present invention can be obtained by regenerating a transformed plant from the transformed plant cell obtained in the above “3”. As a regeneration method, a method is employed in which callus-like transformed cells are transferred to a medium with different hormone types and concentrations and cultured to form somatic embryos to obtain complete plants. Examples of the medium to be used include LS medium and MS medium.
[0037]
The obtained transgenic plant can express a gene arranged downstream of the promoter according to the present invention specifically at the late stage of seed maturation in the seed formation process. Thereby, a desired trait can be imparted to a mature seed, for example, a seed having resistance to various environments, a seed with a controlled germination time, a seed accumulated with a desired substance, and the like can be obtained. . For example, seed germination can be suppressed by introducing a gene related to germination suppression downstream of the promoter according to the present invention. For example, by introducing a gene related to dormancy downstream of the promoter according to the present invention, a deep dormant state can be maintained as compared with the wild type. For example, a useful substance can be accumulated in mature seeds by introducing a gene involved in the production of a substance useful for the human body downstream of the promoter according to the present invention.
[0038]
In order to obtain plant seeds from transformed plants, for example, the transformed plants are collected from the rooting medium, transplanted to pots containing water-containing soil, and grown at a constant temperature to form flowers. And finally seeds are formed. In order to produce a plant from seeds, for example, when the seed formed on the transformed plant has matured, it is isolated, sown in water-containing soil, and grown under constant temperature and illuminance. As a result, a plant body is produced.
[0039]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[1] Isolation of promoter
First, it was confirmed by Northern blot analysis that the AtNCED2 gene was specifically expressed in late seed maturation in Arabidopsis (Colombia). After flowering, total RNA was extracted from Arabidopsis buds on the 5th, 8th, 11th, 13th and 15th days, respectively. RNA was extracted according to the method of S. Naito et al. (Plant. Physiol. 104: 497-503 (1994)). 15 μg of the extracted RNA was electrophoresed on a 1.2% agarose gel containing 0.66 M formaldehyde. Thereafter, the gel was subjected to capillary blot for two days and nights on a nylon membrane filter (Nylon Membranes, positively charged; manufactured by Roche) using 10 × SSC (containing 1.5M NaCl and 0.15M sodium citrate). Thereafter, the filter was baked at 80 ° C. for 2 hours.
[0040]
As the probe, antisense RNA of AtNCED2 gene was used. Antisense RNA of AtNCED2 gene was obtained from DIG-11-dUTP from a DNA fragment obtained by treating pBluescriptII SK (-)-AtNCED2 (Iuchi, S et al, Plant Journal 27, 325-333 (2001)) with NheI. Was synthesized by an in vitro transcription method.
[0041]
Hybridization was performed using the filter and the probe after baking. Hybridization was performed using a Roche DIG Northern Starter Kit according to the method described in the instructions attached to the kit. The result of hybridization is shown in FIG.
[0042]
From FIG. 1, it was revealed that the AtNCED2 gene began to express around the 13th day after flowering, and the expression level increased on the 15th day. From these results, it was revealed that the AtNCED2 gene was specifically expressed in late seed maturation of Arabidopsis thaliana. Therefore, we isolated a promoter that was thought to regulate the expression specific to the late stage of seed maturation of the AtNCED2 gene.
[0043]
First, the chromosomal DNA of Arabidopsis thaliana (Colombia) was extracted as follows. That is, put a part of Shiroizuna tissue into a 1.5 ml tube and add 200 μl of 3% CTAB buffer [3% CTAB, 100 mM Tris-HCl (pH 8.0), 1.4 M NaCl, 20 mM EDTA (pH 8.0)]. In addition, it was ground with a homogenizer. Next, 300 μl of 3% CTAB is added and the suspension is incubated at 60 ° C. for 30 minutes. Next, add 500 μl of chloroform, mix by inverting, centrifuge, and transfer the supernatant to a new tube. Add 0.7 volume of isopropanol to this, mix by inverting, centrifuge to remove the supernatant, add 1 ml of 70% ethanol to the pellet and immediately centrifuge to remove the supernatant. After drying the pellet, chromosomal DNA was extracted by adding 50 μl of TE [10 mM Tris-HCl (pH 8.0), lmM EDTA (pH 8.0)] and suspending.
[0044]
Next, using the obtained chromosomal DNA as a template, NCED2P HindIII5 ′ (AAGCTTGAAT TCGATTATGG ACTACGGTTT TAGAGTTGCA AGA: SEQ ID NO: 2) as a 5 ′ primer and NCED2P SpeI3 ′ (ACTAGTCTTC GGTATAGAGA GGCTTGAGAC AGTG: SEQ ID NO: 3) as a 3 ′ primer PCR using was performed. PCR was performed for 25 cycles using 50 μl of the reaction solution per reaction, with a denaturation reaction at 95 ° C. for 30 seconds, an annealing reaction at 56 ° C. for 30 seconds, and an extension reaction at 72 ° C. for 3 minutes. The reaction solution contains 200 nM dNTP mixed solution, 1 μM each of the above primers, and LA buffer containing 1.25 units of LA Taq DNA polymerase (Takara Shuzo).
[0045]
Primers NCED2P HindIII5 ′ and NCED2P SpeI3 ′ used for PCR were designed based on the already determined chromosomal DNA nucleotide sequence information of Arabidopsis (Colombia). The DNA fragments amplified by these primers NCED2P HindIII5 ′ and NCED2P SpeI3 ′ are 1977 bp DNA fragments existing upstream of the AtNCED2 gene, and were considered to control the time-specific expression of the AtNCED2 gene.
[0046]
[2] Construction of expression vector
In order to confirm that the DNA fragment isolated in [1] above is a promoter that controls time-specific expression, an expression vector into which the DNA fragment was introduced was constructed as follows. The obtained DNA fragment was treated with HindIII, and ligated with pBluescriptII SK (−) (Stratagene) previously treated with SpeI. The reaction product was SK (-)-NCED2. By determining the base sequence of SK (-)-NCED2, it was confirmed that the DNA fragment isolated in [1] contains the base sequence shown in SEQ ID NO: 1.
[0047]
Next, SK (−)-NCED2 was treated with HindIII and SpeI, and the resulting fragment was treated with pIG121 (Akama, K. et al., Plant Cell Rep., 12, 7-11 ( 1992)). The obtained vector was designated as pIG121-NCED2p. pIG121-NCED2p contains the GUS gene downstream of the DNA fragment isolated in [1] above.
[0048]
[3] Preparation of transformants
In order to confirm that the DNA fragment isolated in the above [1] is a promoter that controls time-specific expression, the expression vector pIG121-NCED2p obtained in the above [2] was used as follows. Arabidopsis thaliana was transformed.
[0049]
First, the expression vector pIG121-NCED2p obtained in [2] above was introduced into Agrobacterium GV3101. Next, Agrobacterium GV3101 was used to transform Arabidopsis (Colombia) by vacuum infiltration. Specifically, Agrobacterium GV3101 having pIG121-NCED2p is first suspended in 3 ml LB medium containing 100 μl / ml rifampicin, 50 μl / ml gentamicin and 50 μl / ml kanamycin, and cultured with shaking at 28 ° C. for 2 days. Next, the culture solution is transferred to 250 ml LB medium containing 100 μl / ml rifampicin, 50 μl / ml gentamicin and 50 μl / ml kanamycin, and cultured with shaking at 28 ° C. until the OD600 value becomes 1.2 to 1.5.
[0050]
Centrifugation of the resulting culture solution causes the cells to settle, discard the supernatant, and 250 ml suspension for infiltration [1/2 Murashige-Skoog salt, 1/2 Gamborg B5 vitamin, 5% sucrose, 0.5 mg / ml MES (pH 5.7), 44 nM benzylaminopurine, 0.02% Silwet L-77]. Plants are immersed in the resulting suspension by inverting the pot. In that state, the pressure is adjusted to 400 mm Hg (15 inches Hg, 50 kPa) and the pressure is reduced for about 10 minutes. After releasing the vacuum, the plant was taken out of the suspension and the seed was harvested in about 2 to 4 weeks to obtain transformed seed.
[0051]
The transformed Arabidopsis thaliana was grown as follows to obtain a transformed plant body. That is, first, the seeds obtained by transformation were soaked in 70% ethanol for 2 minutes, in 5% bleach and 1% SDS for 15 minutes, and then surface sterilized by washing with sterile water 3-5 times. . The seed after surface sterilization is suspended in 0.1% sterilized agar solution, then selected plate medium [Murashige-Skoog salt, Gamborg B5 vitamin, 1% sucrose, 0.5mg / ml MES (pH5.7), 200mg / ml kuraforan, 100mg / ml kanamycin], a transformant was obtained by selecting a plant that grew by stationary culture at 22 ° C. for 2 weeks.
[0052]
The leaves of the resulting transformed plant, immature cocoons, yellowed cocoons and fully ripe seeds were collected, and GUS activity was measured. In addition, GUS activity was also measured for wild-type Arabidopsis (colombia) seeds that were not transformed as targets.
[0053]
GUS activity was measured as follows. That is, first, a sample to be measured was frozen with liquid nitrogen and then pulverized until it became powdery. Next, the powdered sample was suspended in a GUS extraction buffer (100 mM sodium phosphate (pH 7.2), 5 mM DTT, 20 μg / ml leupeptin) in a tube placed in ice. Thereafter, the mixture was centrifuged at 4 ° C. and 15000 rpm for 15 minutes, and the supernatant was collected in a new tube placed in ice. The solution in this tube was used as an analysis sample for GUS activity measurement.
[0054]
The measurement of GUS activity using this analysis sample was performed by first measuring 4-methylumbelliferyl-β-D-glucronide (4MUG) buffer (20% methanol, 1 mM 4 MUG, 50 mM sodium phosphate (pH 7.0), 1 mM Na ₂ 75 μl of analysis sample was added to 112.5 μl (containing EDTA, 0.1% Triton X-100) and incubated at 37 ° C. At 15 minutes, 75 minutes and 195 minutes after the start of incubation, 50 μl of the reaction solution was collected and 4 ml of 2% Na ₂ CO _Three Was added and mixed well to stop the enzyme reaction, and samples for measuring GUS activity at 15 minutes, 75 minutes and 195 minutes after the start of incubation were prepared.
[0055]
Next, the prepared GUS activity measurement sample was measured using a spectrofluorometer (F-2000, manufactured by Hitachi, Ltd.) with an excitation wavelength of 365 nm and a fluorescence wavelength of 455 nm. As a standard sample, 50 nM 4-methylumbelliferone (4MU) sodium carbonate solution was used. The value of GUS activity was normalized by the amount of protein contained in the analysis sample, and was defined as GUS activity per 1 μg of protein. The results are shown in FIG.
[0056]
From FIG. 2, almost no GUS activity is observed in the seeds of wild-type Arabidopsis (colombia) and the leaves of the transformed plant. In addition, almost no GUS activity is observed even in immature cocoons. On the other hand, GUS activity was observed in cocoons and fully-ripened seeds that were yellowed and opened. Thus, the DNA fragment isolated in [1] above was a promoter that expressed a downstream gene in a time-specific manner from the late stage of seed maturation in the seed formation process.
[0057]
Next, seeds harvested from the resulting transformed plant were germinated, and the promoter activity at the germination stage of the DNA fragment isolated in [1] was examined. Examination of the promoter activity at the germination stage was carried out by absorbing seeds to promote germination and observing GUS activity by GUS staining over time. GUS staining was performed by immersing a sample to be stained in a GUS staining solution, removing the GUS staining solution with a vacuum apparatus, and allowing to stand overnight at 37 ° C. after degassing. For observation, the chlorophyll pigment was removed by immersing the dyed sample in an ethanol solution. Thereby, GUS staining could be observed more clearly.
[0058]
The results are shown in FIGS. 3 is a photograph of 0 to 1 day after the water absorption treatment, FIG. 4 is a photograph of 2 days after the water absorption treatment, and FIGS. 5 (A) and 5 (B) are photographs of the 4th day after the water absorption treatment. (A) and (B) are photographs 21 days after the water absorption treatment. 5 and 6, (B) is an enlarged photograph showing the root of (A). In addition, the photograph of the seed which performed GUS dyeing | staining without performing a water absorption process is shown in FIG.
[0059]
As shown in FIGS. 3 to 5, the promoter activity of the DNA fragment isolated in the above [1] could be confirmed in the germination stage. Further, as shown in FIG. 6, the promoter activity of the DNA fragment isolated in the above [1] could not be confirmed on the 21st day after the water absorption treatment after the germination period. Furthermore, as shown in FIG. 7, it was possible to confirm the promoter activity of the DNA fragment isolated in the above [1] in seeds that had not been subjected to water absorption treatment. Therefore, the DNA fragment isolated in the above [1] was a promoter that specifically expresses a downstream gene in a fully mature seed and also specifically expresses the downstream gene even at the germination stage.
[0060]
【The invention's effect】
As described above in detail, according to the present invention, it is possible to provide a promoter exhibiting specific inducibility in the late stage of seed maturation and the germination stage.
[0061]
[Sequence Listing]

[0062]
[Sequence Listing Free Text]
SEQ ID NOs: 2 and 3 are synthetic primers.
[Brief description of the drawings]
FIG. 1 is an electrophoretogram showing the results of hybridization in Northern blot analysis.
FIG. 2 is a characteristic diagram showing the results of GUS activity measurement in transformed plants.
FIG. 3 is a photograph of 0 to 1 day after water absorption treatment on seeds harvested from a transformed plant body.
FIG. 4 is a photograph two days after water absorption treatment for seeds harvested from transformed plants.
FIG. 5 is a photograph 4 days after water absorption treatment for seeds harvested from a transformed plant body.
FIG. 6 is a photograph 21 days after water absorption treatment for seeds harvested from a transformed plant body.
FIG. 7 is a photograph when water-absorbing treatment was not performed on seeds harvested from a transformed plant body.

Claims (7)

A promoter comprising the following DNA (a), (b) or (c) and exhibiting specific inducibility at the late stage of seed maturation and germination.
(a) DNA consisting of the base sequence of SEQ ID NO: 1
(b) a DNA consisting of a base sequence in which 1 base is deleted, substituted or added in the base sequence of SEQ ID NO: 1 and which functions as an inducible promoter specific to late seed maturation and germination stages
(c) DNA that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and functions as an inducible promoter specific for late seed maturation and germination stages   2. The promoter according to claim 1, wherein the late seed maturation period is a period from the start of abscisic acid synthesis in the seed to the completion of a fully matured seed.   2. The promoter according to claim 1, wherein the germination period is a period from the completion of a mature seed to the 12th day after germination.   An expression vector comprising the promoter according to claim 1.   An expression vector in which an arbitrary gene is further incorporated into the expression vector according to claim 4.   A transformant comprising the expression vector according to claim 5.   A transgenic plant comprising the expression vector according to claim 5.

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