JP4606140B2 - Seed coat-specific inducible promoter - Google Patents

Description

  The present invention relates to a seed coat-specific inducible promoter.

  Many of the vacuolar proteins are synthesized as inactive precursors in the endoplasmic reticulum (ER) and are subject to strict control that they are processed into the active mature form after being transported to the vacuole. It is known that the maturation mechanism involves vacuolar processing enzyme (VPE).

  In Arabidopsis thaliana, three types of VPE (αVPE, βVPE, γVPE) are known to exist (Non-patent Documents 1 and 2). From the analysis of these genes, it has been clarified that βVPE is expressed in a seed-specific manner, and αVPE and γVPE are expressed in a nutritional organ-specific manner (Non-patent Document 3).

Recently, a fourth VPE was newly discovered from the Arabidopsis gene database and named δVPE (Non-patent Document 4). Non-Patent Document 4 clarifies that δVPE is a new VPE that is different from any of the VPEs known so far, and is expressed in the process of generating seed coats from nacre. However, its specific properties are still unclear.
Kinoshita, T. et al., Plant Cell Physiol., 36, 1555-1562 (1995) Kinoshita, T. et al., Plant Mol. Biol., 29, 81-89 (1995) Kinoshita, T. et al., Plant J., 19, 43-53 (1999) Gruis, DF et al., Plant Cell, 14, 2863-2882

  An object of the present invention is to provide a seed coat-specific inducible promoter.

  In order to elucidate the physiological function of the δVPE, the present inventors consider that it is necessary to examine in detail the place where the δVPE functions as a first step. Therefore, a specific antibody against the δVPE is generated, and immunoblotting and fluorescence are performed. Its localization was examined by antibody staining. As a result, it was found that δVPE was localized in specific cell layers (ii2 and ii3 layers) of the inner nacre at the early stage of seed development. Therefore, when the localization mechanism of δVPE in the inner nacre was further examined, mRNA that is a transcription product of δVPE gene or δVPE protein that is the translation product of mRNA is controlled by degradation in tissues other than inner nacre. As a result, it was revealed that the localization of δVPE in the inner nacre is unexpectedly due to the seed coat-specific inducibility of the δVPE promoter. Under the circumstances described above, the present inventors have discovered the presence of a seed coat-specific inducible promoter and completed the present invention.

That is, the present invention
[1] (a) DNA consisting of the base sequence of SEQ ID NO: 1, (b) consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence of SEQ ID NO: 1, and seed coat DNA that functions as a specific inducible promoter, or (c) DNA that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and functions as a seed coat-specific inducible promoter Seed coat-specific inducible promoter,
[2] An expression vector comprising the promoter according to [1],
[3] A transformant comprising the expression vector according to [2],
[4] A transformant comprising the promoter according to [1], and [5] a transgenic plant comprising the promoter according to [1],
About.

  According to the present invention, a seed coat-specific inducible promoter can be provided. By using such a promoter, for example, a seed coat-specific expression of a gene encoding a protein effective in improving the properties of plant seeds such as resistance to pathogenic bacteria and pests is improved, and the digestibility of seed coat of food seeds is improved. To express seedling-specific enzymes, seed-specific expression of metal binding proteins effective for capturing heavy metals, etc., seed-specific expression and accumulation of nutritionally effective protein components, etc. Therefore, it is useful for improving the properties of plant seeds.

  The seed coat-specific inducible promoter of the present invention is capable of specifically inducing expression of a downstream gene in the seed coat, preferably the inner nacre at the early stage of seed development. Specifically, the promoter comprises (a) DNA comprising a base sequence at positions 1 to 1294 upstream of the δVPE gene as a main component. The base sequence is shown in SEQ ID NO: 1.

  As long as the promoter of the present invention can function as a seed coat-specific inducible promoter, (b) from a base sequence in which one or several bases are deleted, substituted or added in the base sequence of SEQ ID NO: 1. Or (c) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 1.

  In the DNA of (b), “one or several” means that the function of inducing the expression of an arbitrary gene located downstream of the promoter is not lost so long as the function of losing the seed coat is not lost. It means that bases may be deleted, substituted or added without limitation to the number of base sequences.

The “stringent conditions” in the DNA of (c) are the temperature and salt concentration at the time of hybridization, preferably at the time of washing, depending on the DNA hybridized to the DNA consisting of the nucleotide sequence of SEQ ID NO: 1. Although it is set by appropriately determining, stringent conditions, e.g., Zanburuku (Sambrook) et al., eds., "Molecular cloning: A Laboratory manual 2nd Edition (Molecular cloning: A Laboratory manual 2 nd Ed.) " [(Cold Spring Harbor Laboratory Press (1989)] and the like. Specifically, for example,
(i) 6 × SSC (composition of 1 × SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhardt, 100 μg / mL denatured fragmented salmon sperm DNA Incubate at 42 ° C. overnight with the probe in a solution containing 50% formamide,
(ii) a step of removing the non-specifically hybridized probe by washing, from the standpoint of further improving the accuracy, where a lower ionic strength, for example, 2 × SSC, or more stringent, 0.1 × SSC And / or higher temperature, for example, at 40 ° C. of the Tm value of the nucleic acid used, more stringent at 30 ° C., more stringent at 25 ° C., more stringent at 10 ° C. At 25 ° C., specifically depending on the Tm value of the nucleic acid used, it is 25 ° C. or higher, more stringent 37 ° C. or higher, more stringent 42 ° C. or higher, and even more stringent 50 More than stringent, more stringent, wash under conditions such as 60 ° C or more,
The condition is mentioned.

Tm is, for example, the following formula:
Tm = 81.5 + 16.6 (log [Na ⁺ ]) + 0.41 (% G + C)-(600 / N)
(Where N is the length of the oligonucleotide and% G + C is the content of guanine and cytosine residues in the oligonucleotide)
It is calculated by.

  The hybridization operation may be performed with reference to the above-mentioned book, for example. Note that the operations described in this specification can be performed as appropriate by referring to the book.

  The promoter of the present invention can be obtained, for example, by isolating a DNA portion having a base sequence at positions 1 to 1294 upstream of the δVPE gene of Arabidopsis thaliana. Isolation of the DNA portion can be performed according to a known method. Information on the base sequence of the δVPE gene of Arabidopsis thaliana is available, for example, as Genebank Accession No. AV555212.

  For example, the genomic DNA of Arabidopsis thaliana is extracted, and the DNA portion is obtained by specifically amplifying and purifying the desired base sequence by PCR using a pair of primers having a predetermined base sequence as a template. Can do. A pair of primers used for PCR can be appropriately designed from information on the genome base sequence of Arabidopsis thaliana, and can be prepared according to a known method.

  Further, the promoter of the present invention may be obtained by appropriately introducing mutations into the base sequence of the isolated DNA portion according to a known method. Examples of the mutagenesis method include a known method such as the Kunkel method or a method equivalent thereto. Furthermore, the promoter of the present invention can be obtained by using an isolated DNA portion as a probe and screening an arbitrary genomic DNA library, for example.

  Whether or not the promoter of the present invention has a desired function can be examined using, for example, the expression vector of the present invention described later. That is, an expression vector incorporating a gene serving as a marker downstream of the promoter of the present invention is introduced into Arabidopsis cells, and the resulting transformed plant is grown. Then, the expression level of the marker gene in the seed coat of the transformed plant is observed. If the expression of the marker gene is confirmed, it can be determined that the used promoter has a desired function.

  The expression vector of the present invention can be obtained by linking (inserting) the promoter of the present invention to an appropriate vector. The vector for inserting the promoter is not particularly limited as long as it can replicate in the host. For example, the following vectors are used.

  Examples of plasmid vectors include plasmids derived from E. coli (eg, pBR322, pUC118, pUC18, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YCp50, etc.), and the like. Can be mentioned. Examples of the phage vector include λ phage (for example, Charon4A, EMBL3, λgt10, λZAP, etc.). Animal virus vectors such as retrovirus or vaccinia virus and insect virus vectors such as baculovirus can also be used. As the vector, binary vectors such as pBI101 and pBI121 may be used.

  As a method for inserting a promoter into a vector, for example, purified DNA constituting the promoter is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a desired vector DNA, and ligated to the vector. Methods and the like. In this case, any gene that is desired to be expressed in the host can be inserted in the same manner. The gene for which expression is desired is not particularly limited. Such a gene may be inserted in either the sense direction or the antisense direction as desired. Also, depending on the host to which the expression vector is to be introduced, a replication origin, a ribosome binding sequence, a transcription termination sequence, a promoter control sequence, etc. are selected as appropriate, and any of these components (base sequences) are further inserted. May be.

  In the expression vector of the present invention, if a reporter gene, for example, a GUS gene widely used in plants, is linked as an arbitrary gene downstream of the promoter of the present invention, for example, GUS activity in the host plant can be increased. By examining it, it is possible to easily evaluate the function and strength of the promoter.

  The transformant of the present invention can be obtained by introducing an arbitrary gene desired to be expressed in the host into the host together with the promoter of the present invention. Here, the host is not particularly limited as long as the promoter of the present invention can be operated and expression of a desired gene can occur.

  Examples of the host include Escherichia coli, bacteria, yeast, animal cells, insect cells, and the like in addition to plants, among which plants are preferred.

  When the host is a plant, the transformation can be performed, for example, as follows. For example, S.J.Clough and A.F.Bent, Plant Journal, 16, 735-743 (1998) may be referred to for plant transformation operations.

  Plants to be transformed include whole plants, plant organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg epidermis, phloem, soft tissue, xylem, vascular bundles, etc.) And plant cultured cells. Examples of plant species used for transformation include plants belonging to the Brassicaceae, Gramineae, Solanum, Legumes, etc., but are not limited to these plants.

  Examples of cruciferous plants include, for example, Arabidopsis thaliana, grasses such as corn (Zea mays) and rice (Oryza sativa), and solanaceous plants, for example, tobacco (Nicotiana tabacum), legumes. Examples thereof include soybean (Glycine max).

  The expression vector of the present invention can be suitably used for producing the transformant of the present invention. The method for introducing the expression vector of the present invention into the host is not particularly limited, and examples thereof include direct introduction methods such as a particle gun method, an electroporation method (electroporation method), and a PEG method.

  On the other hand, when the host is a plant, the transformant of the present invention can also be produced by a biological introduction method such as a method using Ri plasmid, cauliflower mosaic virus or the like in addition to Ti plasmid contained in Agrobacterium. . In the transformed plant produced by these methods, the promoter of the present invention and any gene desired to be expressed in the host are incorporated into the genome.

  Therefore, according to the present invention, a transformant comprising the expression vector of the present invention and a transformant comprising the promoter of the present invention in its genomic DNA can be obtained.

  The transformed plant of the present invention is obtained as a plant tissue, cell, somatic embryo, embryo, scutellum tissue, and a redifferentiated plant or germinated individual depending on the plant used as a host. Then, they can be used for cell culture, tissue culture, and organ culture as appropriate.

  Whether or not a desired gene has been introduced into the host can be confirmed by a known method such as PCR, Southern hybridization, Northern hybridization and the like.

  The transgenic plant of the present invention can be produced by regenerating a plant body from the cells of the transformed plant of the present invention. Regeneration may be carried out in accordance with a known method. Usually, a method 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 a complete plant is preferable. is there.

  The resulting transgenic plant can express a desired gene located downstream of the promoter of the present invention in a seed coat-specific manner. Thereby, a desired character can be imparted to the seed. For example, seeds that are resistant to pests and pathogens, and food seeds that are easy to digest can be obtained by softening the seed coat by expressing the cell wall component degrading enzyme only in the seed coat. Moreover, metallothionein, which is a metal binding protein effective for capturing heavy metals and the like, can be expressed specifically in the seed coat. For example, the accumulation of cadmium in rice is regarded as a problem, but the problem of cadmium accumulation in rice could be solved by specifically expressing cadmiumthionein in the rice seed coat and capturing cadmium in the seed coat. . Furthermore, seeds and the like that accumulate nutritionally effective protein components and other desired substances in a seed coat-specific manner can be obtained. In addition, as a transgenic plant of this invention, the seed obtained by doing in that way and also the progeny of this plant are included.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1 Isolation of a promoter from Arabidopsis thaliana
One leaf is sampled from seedling grown on agar for 10 days, ground in 500μL extraction buffer (0.2M Tris-HCl, 0.4M LiCl, 25mM EDTA, 1% (w / v) SDS), crude extraction A liquid was obtained. To the crude extract, 500 μL of a mixture of phenol and chloroform at a volume ratio of 1: 1 was added to remove proteins, and 350 μL of genomic DNA dissolved from the aqueous layer was obtained. 350 μL of isopropanol was added thereto to precipitate genomic DNA, washed with 70% EtOH, and concentrated by dissolving in 100 μL of TE. Of this, 20 μL was used as a template for PCR. PCR was performed using the primer shown in SEQ ID NO: 2, the primer shown in SEQ ID NO: 3 with the restriction enzyme site SmaI added to its 5 ′ end, and Ex-Taq DNA polymerase. The DNA fragment amplified by PCR was ligated to pT7 blue T-vector (Novagen) (hereinafter referred to as T-vector). It was confirmed by sequencing that the sequence was free from PCR errors, and a δVPE promoter (SEQ ID NO: 1) was obtained.

Example 2 Construction of Ti Vector The T-vector obtained by cloning the δVPE promoter was cleaved with restriction enzymes HindIII and SmaI, and then subjected to agarose electrophoresis to extract the δVPE promoter sequence in the plasmid. This δVPE promoter sequence was introduced into the restriction enzyme HindIII-SmaI site on Ti-vector pBI121 (Clontech). pBI121 is a binary vector having a GUS gene.

  Ti-vectors incorporating a fusion gene (GUS gene linked downstream of the δVPE promoter) can be directly transformed into Agrobacterium tumefaciens by electroporation (Plant Cell Engineering Vol.14 No.3 1992, p48-58). , EHA105). 1 ng of vector was added to 80 μL of Agrobacterium suspended in water, and pulsed at a maximum voltage of 2400 V using a CELJECT BACIC electroporation system (EquiBio) and a cuvette with an electrode width of 2 nm (electric field 12 KV / cm, pulse time 5.28 msec) ). Incubate in 1mL LB medium (10g / L NaCl, 10g / L bactotryptone, 5g / L yeast-extract) at 28 ° C for 1 hour, and then apply to LB agar medium containing 50mg / L kanamycin at 28 ° C. Cultured for 2 days, transformed Agrobacterium was selected.

Example 3 Preparation of transformed plant body and measurement of GUS activity The transformation method was according to the method of Clough et al. (Plant Journal, 16, 735-743 (1998)). Arabidopsis transgenic plants were obtained using the flower buds.

That is, the transformed Agrobacterium obtained in Example 2 was pre-cultured in 10 mL LB medium for 1 day, all of which was transferred to 200 mL LB medium and main-cultured for 1 day. The cultured Agrobacterium was recovered by centrifugation at 5 kG, 20 ° C. for 15 minutes, infiltration medium [1 bag of mixed salt for Murashige-Skoog medium (Nippon Pharmaceutical Co., Ltd.), 100 g Sucrose, 200 mL 0.05% MES・ Agrobacterium was diluted and suspended so that the absorbance at 600 nm was 0.8 with KOH pH5.7 / 2L]. The flower buds were immersed in this medium for about 10 minutes to infect Agrobacterium. Grown about the next three weeks, to recover the T ₁ generation of seeds. T _1st generation seed is MS solid medium (Murashige & skoog salt base, 1 mL / L B5 Vitamin stock (100 mg / mL myo-) containing 50 mg / L kanamycin, 200 mg / L Cefotaxime Sodium Salt (WAKO). inositol, 10 mg / L thiamin- HCl, 1 mg / L nicotinic acid, 1 mg / mL pyridoxin-HCl), to obtain a 30 g / L sucrose, 0.08% agar] selected transformants were T ₂ generation seeds This was used for analysis. Genomic DNA was extracted from the resulting transformed plant body in the same manner as in Example 1, and it was confirmed that the target DNA fragment was contained by sequencing.

From the transformed plant body, seeds (globular to heart type embryonic stage), seeds (torpedo type embryonic stage), pollen, flower stem, shoot apex, root, leaf stem, and leaf were collected, respectively, and GUS activity was measured. The tissue of the transformed plant was collected by the following method. T ₂ generation seeds are sown in vermiculite / perlite (1: 1), grown under continuous light at 25 ° C for 6-7 weeks, seeds (globular to heart type embryo stage), seeds (torpedo type embryo) Period), pollen was collected. Further, pollen, flower stem, shoot, root, leaf and stem, and tissues, such as leaves, a T ₂ generation seeds were sown in MS solid medium, placed dark 4 ° C. for 4 days, after vernalization treatment, continuous It was collected from plants that had been cultivated at 25 ° C for 1 week under light. As a control, GUS activity was also measured in the same manner for a wild-type Arabidopsis derived from untransformed.

Measurement of GUS activity was performed by observing GUS staining of each part of the transformed plant body. In GUS staining, a sample to be stained is prepared by using a GUS staining solution [50 nM X-Gluc (5-bromo-4-chloro-3-indlyl-β-D-glucuronide-cyclohexylammonium salt), 500 μM K ₃ [CN] ₆ / K ₄ [Fe (CN) ₆ ], 0.3% Triton-X, 50 μM NaH ₂ PO ₄ ] was immersed in the solution and incubated at 37 ° C. After 12 hours from the start of incubation, the enzymatic reaction was stopped by immersing the plant in 100% EtOH. After 100% EtOH was changed several times to sufficiently remove the chlorophyll dye, the blue staining was observed with a differential interference microscope, and the state was photographed.

  The results are shown in FIG. (A) is seed (globular to heart type embryonic stage), (b) and (b) ′ are seeds (torpedo type embryonic stage), (c) is pollen, (d) is flower stem, (e) is shoot tip, (F) is a root, (g) is a leaf stem, and (h) is a state of GUS staining in a leaf. As can be seen from the figure, GUS staining was observed in the seed coat only in (a), (b) and (b) '. In addition, GUS staining was not observed in wild-type Arabidopsis derived. From the above results, it can be seen that the expression of GUS was induced in a seed coat-specific manner by the δVPE promoter obtained in Example 1. This indicates that according to the promoter, expression of the gene linked downstream thereof can be specifically induced in the seed coat.

  The present invention provides a seed coat-specific inducible promoter.

The state of GUS staining at each site of an Arabidopsis transformed plant introduced with a GUS gene linked downstream of the δVPE promoter is shown.

Claims (5)

  (A) DNA consisting of the base sequence of SEQ ID NO: 1, (b) consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence of SEQ ID NO: 1, and seed coat-specific induction Seed coat-specific, comprising DNA that functions as a sex promoter, or (c) DNA that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and functions as a seed coat-specific inducible promoter Inducible promoter.   An expression vector comprising the promoter according to claim 1.   A transformant comprising the expression vector according to claim 2.   A transformant comprising the promoter according to claim 1.   A transgenic plant comprising the promoter according to claim 1.

Images