JP3611083B2 - Promoter specific to plant meristem - Google Patents

Description

【図面の簡単な説明】
【図１】図１は、プラスミドｐＥｇＨ３−ＧＵＳおいて、植物ゲノムＤＮＡに組込まれる部分の模式図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a promoter that specifically promotes expression of a structural gene under its control in plant meristems.
[0002]
[Prior art]
With the development of plant molecular biology in recent years, it has become possible to breed plant varieties and the like having useful traits such as disease resistance and herbicide resistance using sense genes and antisense genes. That is, a structural gene involved in expression of a target trait is linked to a promoter that can be expressed in a plant in the sense direction or antisense direction to create a chimeric gene, and this is introduced into a plant as a vector. It promotes and suppresses the expression of traits. At present, such a technique has been applied to, for example, a disease-resistant plant in which an insecticidal BT toxin gene derived from Bacillus thuringensis has been introduced in the sense direction (DA Fischoff et al., Bio / Technology, vol. 232: 738, 1987). ) And polygalacturonase genes related to overripe tomato fruit have been produced in the long-lasting tomato (CJ Smith et al., Nature, vol. 334: 724, 1988).
[0003]
On the other hand, cauliflower mosaic virus (CaMV) 35S promoter is exclusively used as the promoter of the structural gene thus introduced. Although this CaMV35S promoter has a function of strongly promoting the transcription of structural genes, its promoting action is nonspecific, and in plant cells into which it is introduced, regardless of the growth stage of the plant or the type of tissue, It always promotes transcription of structural genes under its control. For this reason, depending on the type of structural gene, its overexpression may lead to metabolic abnormalities in the transgenic cells, which may cause malformation or growth inhibition of the plant tissue or the plant body itself.
[0004]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a promoter capable of controlling the expression of a structural gene in a tissue-specific and time-specific manner.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the histone H3 structural gene promoter isolated from Eucalyptus genus is specifically found in meristems such as plant stem apex, root tip, and callus. The present invention has been completed by finding that it promotes transcription of structural genes.
[0006]
That is, the above-mentioned problem of the present invention is that of this promoter, specifically, a DNA having the base sequence shown in SEQ ID NO: 1, or a structural gene that hybridizes with this DNA under stringent conditions and is under its control. Expression is solved by DNA that has the function of specifically promoting in the meristem of plants. Here, as stringent conditions, 6 × SSC (0.9 M NaCl, 0.09 M sodium citrate) and a temperature of 55 ° C. are used as a buffer solution.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
As the promoter of the present invention, any DNA having the base sequence shown in SEQ ID NO: 1 can be used without limitation. In addition, DNA that hybridizes with this DNA under stringent conditions also has a function of specifically promoting the expression of the structural gene under its control in the meristem of a plant, such as stem apex, root tip, callus, etc. Can be used.
[0008]
Such DNA may be of any origin. Plants such as those isolated from Eucalyptus genus such as Eucalyptus globulus (hereinafter abbreviated as E. globulas) and Eucalyptus grandis can be used, but the phosphite triester method (M. Hunkapiller et al., Nature, etc.) can also be used. , 310: 105, 1984), etc., can also be used.
[0009]
The structural gene to be controlled by the promoter of the present invention is linked to the downstream region of the promoter. Usually, the distance between the end of the promoter region and the ATG translation initiation codon of the structural gene is preferably within 10 bp. Further, immediately after the ATG translation initiation codon, the promoter activity of the present invention is further improved by linking a part containing the first intron of the Eucalyptus histone H3 structural gene between this codon and the structural gene to be controlled. Can be made.
[0010]
In the present invention, gene introduction into plant cells can be carried out by a conventional method using a vector prepared by linking a promoter and a structural gene to be controlled thereby as described above. That is, using this vector, viruses and bacteria that infect plants, such as cauliflower mosaic virus, gemini virus, tobacco mosaic virus, brom mosaic virus, Agrobacterium tumefaciens (hereinafter abbreviated as A. tumefaciens), Agrobacterium. It is also possible to introduce genes into plant cells indirectly through um lysogenes, etc., and physical methods such as microinjection, electroporation, polyethylene glycol, fusion, and high-speed ballistic penetration -It is also possible to introduce a gene directly into a plant cell by a chemical method (I. Potrykus, Annu. Rev. Plant Physiol. Plant Mol. Biol., 42: 205, 1991). Furthermore, from plant cells into which genes have been introduced by these methods, plant bodies can be redifferentiated by culturing them under conditions suitable for the type of the plant. The plant cells / plants thus obtained are plant cells into which a structural gene controlled by the promoter of the present invention has been introduced, and are plant bodies.
[0011]
The promoter of the present invention can control the expression of not only the histone H3 gene but also structural genes encoding various other proteins. Therefore, in the present invention, various structural genes, such as genes that give agriculturally superior traits, genes that are involved in the production of substances useful in the pharmaceutical industry, etc., genes that are required for the study of gene expression mechanisms, etc. Depending on the purpose, it can be used as a sense gene or an antisense gene.
[0012]
Moreover, the kind of plant which can use the promoter of this invention is not specifically limited. In addition to eucalyptus, the promoter of the present invention can be used for tobacco, herbaceous plants such as rice, Arabidopsis, and petunia, and woody plants such as porcupine, poplar, acacia, cedar, and pine.
[0013]
[Action]
Histones are basic proteins bound to DNA in the nucleus of eukaryotic cells, and five types of H1, H2A, H2B, H3, and H4 are known to exist in the S phase (DNA synthesis phase) during the cell cycle. Newly synthesized DNA plays an important role in constructing chromatin. Therefore, it is considered that genes encoding these have high homology in eukaryotic cells and are specifically expressed in the S phase during the cell cycle.
[0014]
The present inventors have found that among these histones, the promoter of a structural gene encoding H3 promotes its expression in the meristem of a plant regardless of the type of plant or the type of structural gene. The characteristics of such a histone H3 structural gene promoter are also considered to be related to such characteristics of histone structural genes, that is, high homology among organisms and cell cycle-specific expression.
[0015]
【Example】
Hereinafter, the present invention will be described based on examples.
[0016]
[Example 1]
1. Isolation and purification of Eucalyptus genomic DNA library E. coli grown for about 6 months after sowing. The leaves before fully developing from the globulae were collected and pulverized under liquid nitrogen. Using this crushed leaf as a material, according to the method of Doyle et al. (Focus, 12:13, 1989), genomic DNA was isolated and purified so as not to suffer physical damage as much as possible. About 1 mg of genomic DNA could be collected per 10 g (fresh weight).
[0017]
2. Eucalyptus genomic DNA library H buffer eucalyptus genome DNA20μg obtained in created 1 _{(50mM Tris-HCl / pH7.5,10mM MgCl} 2, 100mM NaCl, 1mMDTT) was dissolved in 200 [mu] l, this ^{1 -4} unit Restriction enzyme EcoRI (manufactured by Toyobo Co., Ltd.) was added and partial digestion was performed at 37 ° C. After 1 hour, 50 mM ethylenediaminetetraacetate (EDTA) and 0.5% sodium dodecyl sulfate (SDS) were added to the reaction solution. In addition, the reaction was stopped. Here, the unit is a unit having 1 unit of enzyme activity that completely decomposes 1 μg of lambda DNA dissolved in 50 μl of the buffer solution at 37 ° C. for 60 minutes. The partially digested DNA was precipitated by adding 2.5 times the amount of ethanol to the reaction solution after stopping the reaction and cooling at −80 ° C. for 1 hour, and this was recovered by centrifugation. As a result, 14 μg of a partially digested genomic DNA that can be ligated to a vector was obtained.
[0018]
This eucalyptus genomic DNA partial digest was then ligated to a lambda phage vector. Specifically, 2 μg of a substituted lambda phage vector ZAPII (manufactured by STRATAGENE) digested with EcoRI and dephosphorylated with alkaline phosphatase, and 5 μg of the genomic DNA partial digest obtained as described above were combined with ATP 1 mM and T4 DNA ligase. (Toyobo Co., Ltd.) In the presence of 2 units, the reaction was performed overnight at 16 ° C. in 20 μl of a buffer solution (10 mM Tris-HCl / pH 7.5, 10 mM MgCl ₂ , 1 mM DTT), and then 5 ° C. at 65 ° C. The reaction was stopped by heat treatment for 5 minutes to obtain recombinant phage DNA. Here, the unit is a unit having 1 unit of enzyme activity for ligating 6 μg of lambda DNA HindIII digested product dissolved in 20 μl of the above buffer solution at 16 ° C. for 30 minutes for 30% or more. The obtained recombinant phage DNA was mixed with lambda phage coat protein, left at 22 ° C. for 2 hours to perform in vitro packaging, and lambda phage having the recombinant DNA was regenerated (in vitro package manufactured by STRATAGENE). Ngkit Gigapack GOLD.).
[0019]
On the other hand, E. coli XL1-Blue MRF ′ strain was inoculated into 10 ml of LBMM medium (tryptone 1%, salt 1%, yeast extract 0.5%, magnesium sulfate 10 mM and maltose 0.4%) and shaken at 37 ° C. for 12 hours. And precultured. After preculture, the cells are collected by centrifugation, suspended in 10 ml of 10 mM magnesium sulfate previously cooled to 4 ° C., mixed with the above-mentioned recombinant lambda phage, making lambda phage easy to infect, 37 This was left at 15 ° C. for 15 minutes to infect it. The infected bacteria are suspended in LBMM medium containing 0.7% agarose, and this suspension is further spread thinly on the surface of LBMM medium solidified with 1.5% agar and cultured at 37 ° C. for 12 hours. went. After culturing, 10 ml of SM buffer (50 mM Tris-HCl / pH 7.5, 0.1 M NaCl, 7 mM MgSO ₄ , 0.01% gelatin) was added to the petri dish and kept at 4 ° C. for 8 hours. The liquid was recovered, and the subsequent operation was performed using the recovered liquid as a eucalyptus genomic DNA library.
[0020]
3. The DNA fragment containing the Eucalyptus histone H3 gene was amplified by the PCR method using the Eucalyptus genomic DNA library obtained in the determination 2 of the base sequence of the Eucalyptus histone H3 gene as a template.
[0021]
That is, as a primer, primer B (5′-CGCGAGCTGGATGTTCCTTG) corresponding to a common region existing 365 to 384 bp downstream of the ATG translation initiation codon in the Arabidopsis, rice, and kidney bean histone H3 genes, and beside the cloning site of the ZAPII vector Using the KS primer (5′-TCGGGTCGACGGGTTAG) corresponding to one of the existing regions, PCR was performed using the above library as a template, and the amplified DNA fragments were separated by agarose gel electrophoresis. Amplification of the DNA fragment was confirmed. Therefore, both ends of the DNA fragment were smoothed by T4 DNA polymerase treatment, phosphorylated by polynucleotide kinase, and subcloned by incorporating into the dephosphorylated SmaI restriction enzyme site of plasmid pUC19. For the DNA fragment, the base sequence was determined by the dideoxy method using a DNA sequencer (DNA sequencer Model 373S (manufactured by Perkin-Elmer)). In this DNA fragment, the structural gene region downstream of the ATG translation initiation codon present at a position of 660 bp from the 5 ′ end has very high homology with other known plant histone H3 structural genes. The hexamer sequence, which is a cis element of the histone H3 promoter region and is considered to be involved in the S phase-specific expression of the histone structural gene, is 34 bp, OLS-1 and OLS-2 are 45 and 56 bp, respectively, and the octamer sequence. And nonamer sequences are present at 229 and 256 bp, respectively (protein / nucleic acid / enzyme, vol. 37: 1146, 1992). From the above, it was confirmed that this DNA fragment contains the Eucalyptus histone H3 gene, that is, the Eucalyptus histone H3 structural gene and its promoter.
[0022]
4). For the DNA fragment whose nucleotide sequence was determined in Confirmation 3 of the expression control activity of the Eucalyptus histone H3 promoter, PCR was performed using a primer containing a HindIII restriction enzyme site and a primer containing a BamHI restriction enzyme site, and Eucalyptus histone H3 A promoter region of about 670 bp was amplified and inserted between the HindIII-BamHI restriction enzyme sites of the plant transformation vector pBI101 (manufactured by CLONTHEC), and this was designated as plasmid pEgH3-GUS. In this pEgH3-GUS, the eucalyptus histone H3 promoter is located adjacent to the upstream side of the β-glucuronidase (GUS) structural gene, and when it functions as a promoter, it controls the expression of this GUS structural gene. . The nucleotide sequence of a full-length 662 bp DNA extending upstream from the ATG translation initiation codon in the region of about 670 bp amplified by the PCR method is shown in SEQ ID NO: 1. In this base sequence, the ATG translation initiation codon is located at 660 to 662 bp. Moreover, in pEgH3-GUS, the schematic diagram of the part integrated in plant genomic DNA is shown in FIG. In the figure, NPTII is a kanamycin resistance gene, and Nos-ter is a polyadenylation signal of nopaline synthase.
[0023]
Subsequently, pEgH3-GUS was introduced into tobacco ( Nicotiana tabacum L cv. SR-1) by the Agrobacterium method. That is, pEgH3-GUS was electroporated by A.I. After introduction into Tumefaciens EHA105 (in 10% glycerol, 2500 V, 25 μF was applied as an electric pulse. Use GENE PUSER II manufactured by Bio-Rad). Tumefaciens was selected by culturing at 28 ° C. for 2 days in an LB agar medium (tryptone 1%, salt 0.5%, yeast extract 0.5%) containing kanamycin 50 mg / l, and this was aseptic for 4 weeks after seed germination. Cultured tobacco leaves were infected.
[0024]
For the infection, the tobacco leaf cut into about 5 mm square was transformed into pEgH3-GUS-introduced A. It was carried out by immersing in the culture medium of tumefaciens for 1 to 3 minutes with the epidermis of the leaves facing down. After removing the culture solution adhering to the leaf pieces after the infection treatment with a sterilized paper towel or the like, callus induction medium (Murashige-Skoog basic medium (hereinafter abbreviated as MS medium), 3% sucrose, 0. Placed on 8% agar, 1 mg / l naphthalene acetic acid, 0.1 mg / l benzyladenine, cultured for 3 days under continuous illumination at 25 ° C., and medium for shoot formation (MS basic medium, 3% sucrose, 0.8% Agar, 0.1 mg / l naphthalene acetic acid, 1 mg / l benzyladenine, 100 mg / l kanamycin, 500 mg / l carbenicillin) and continued to culture under the same temperature and light conditions to differentiate the foliage. The differentiated foliage was cut out about 4 weeks after culturing in the shoot-forming medium, and MS basic medium containing 100 mg / l kanamycin and 500 mg / l carbenicillin (3% sucrose, 0.8% agar or 0.25% gellan gum) After regenerating the plant by culturing for about 4 weeks under the same temperature and light conditions, vermiculite (manufactured by Japan Fireproof Industries) and peat moss (manufactured by Izumi Agricultural Materials) It transplanted to the culture soil mixed in the ratio of 1: 2, and was made to grow in a 25 degreeC greenhouse.
[0025]
The obtained pEgH3-GUS-introduced tobacco, that is, a tobacco having a eucalyptus histone H3 promoter-GUS fusion gene, was subjected to the method of Jefferson et al. (Plant Mol. Biol. Rep.) For the stem apex, stem, leaf, root and callus. , Vol.5: 387, 1987), the GUS gene expression test was performed. As a result, particularly strong GUS gene expression was confirmed in the root, stem apex, and callus meristem, where the eucalyptus histone H3 promoter inserted into pEgH3-GUS was a structural gene plant placed under its control. Promotes meristem-specific expression, and its function is as a promoter of the GUS structural gene, which is completely different from the structural gene from which it originally functions as a promoter, even in a plant different from the plant from which it is derived (eucalyptus). Even when used, it was shown to be demonstrated.
[0026]
[Example 2]
Introduction of plasmid pEgH3-GUS obtained in 4 of Example 1. In the same manner as in Example 1, tumefaciens was infected with the petiole of aseptically cultured hybrid porcupine ( Populus sieboldii x P. grandidentata ) Y63.
[0027]
The petiole after the infection treatment shows A. After removing the tumefaciens culture solution from the surface, a modified MS medium (3% sucrose, 0.8% agar) containing zeatin 0.5 mg / l and NH ₄ ⁺ and NO ₃ ⁻ concentrations of 10 mM and 30 mM, respectively. ) And then transplanted to a modified MS medium (3% sucrose, 0.8% agar) supplemented with 0.5 mg / l zeatin, 100 mg / l kanamycin, and 500 mg / l carbenicillin, The stems and leaves were differentiated by culturing for 4 months. Crossed porcupine into which pEgH3-GUS was introduced was prepared by using MS medium (sucrose 3%, agar 0.8% or gellan gum 0.25%, kanamycin 100 mg / l, carbenicillin) It was regenerated by transplanting to 500 mg / l) and rooting. Note that the temperature and light conditions at this time are the same as those employed in 4 of Example 1.
[0028]
The resulting pEgH3-GUS-introduced porcupine was tested for GUS gene expression in the same manner as in Example 1. As in the case of tobacco, the GUS gene was particularly strong in the root, stem apex, and callus meristem. Expression was confirmed.
[0029]
[Example 3]
Introduction of plasmid pEgH3-GUS obtained in 4 of Example 1. In the same manner as in Example 1, the tumefaciens was inoculated into the hypocotyl of Eucalyptus globulus seeded and germinated under aseptic conditions.
[0030]
The hypocotyl after infection treatment is the same as that of A. After removing the tumefaciens culture solution from the surface, it was placed on a modified MS medium (3% sucrose, 0.8% agar) containing 1.0 mg / l of zeatin and cultured for 3 days. l, callus was formed by culturing in a modified MS medium (3% sucrose, 0.25% gellan gum) supplemented with 50 mg / l kanamycin and 500 mg / l ticarcillin.
[0031]
When the obtained pEgH3-GUS-introduced eucalyptus was subjected to the GUS gene expression test in the same manner as in Example 1, strong GUS gene expression was confirmed.
[0032]
From the above results, the DNA of the present invention, ie, the DNA having the base sequence shown in SEQ ID NO: 1 isolated as the Eucalyptus histone H3 promoter is not limited to the type of plant into which it is introduced, and this is essentially the promoter. It was revealed that meristem-specific expression can be promoted even for structural genes that are completely different from the structural genes that act as.
[0033]
【The invention's effect】
The promoter of the present invention, that is, the DNA having the base sequence shown in SEQ ID NO: 1, has the ability to control the expression of structural genes in a tissue-specific and time-specific manner. That is, by using this, the structural gene placed under the control can be specifically expressed in the meristem of the plant. In addition, this promoter may be any kind of plant / structural gene that can be used.
[0034]
Therefore, if a structural gene is selected purposefully and placed under the control of the promoter of the present invention to create a vector, and transformation of a desired plant using this vector is carried out, the meristem of the plant is used. Since only the structural gene can be expressed, it is possible to avoid problems caused by the expression of the structural gene introduced for imparting the desired trait in an unplanned tissue / time.
[0035]
In addition, as long as the DNA hybridized under stringent conditions with the DNA having the base sequence shown in SEQ ID NO: 1 also has the function of specifically promoting the expression of the structural gene under its control in the meristem of the plant, The above problem can be avoided.
[0036]
Therefore, if a structural gene is selected purposefully and placed under the control of the promoter of the present invention to create a vector, and transformation of a desired plant using this vector is carried out, the meristem of the plant is used. Since only the structural gene can be expressed, it is possible to avoid problems caused by the expression of the structural gene introduced for imparting the desired trait in an unplanned tissue / time.
[0037]
In addition, as long as the DNA hybridized under stringent conditions with the DNA having the base sequence shown in SEQ ID NO: 1 also has the function of specifically promoting the expression of the structural gene under its control in the meristem of the plant, The above problem can be avoided.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic diagram of a portion of a plasmid pEgH3-GUS that is integrated into plant genomic DNA.

Claims (5)

DNA having the base sequence shown in SEQ ID NO: 1. A DNA that hybridizes with a DNA having the base sequence shown in SEQ ID NO: 1 under stringent conditions and has a function of specifically promoting expression of a structural gene under its control in a meristem of a plant. A vector comprising the DNA according to claim 1 or 2 and a structural gene whose expression is controlled by the DNA. A plant cell into which the DNA according to claim 1 or 2 and a structural gene whose expression is controlled by these DNAs are introduced. A plant regenerated from the plant cell according to claim 4.

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