JPH10191976A - Aluminum-resistant plant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new plant, comprising a plant cell into which a gene capable of promoting the synthesis of an organic acid forming a chelate with Al is transduced and capable of manifesting a high secretion ability of the organic acid, absorbing the phosphoric acid in the presence of the Al and improving the productivity of field crops in oligotrophic soil. SOLUTION: This new aluminum resistant plant cell comprises a gene, capable of promoting the synthesis of an organic acid forming a chelate with aluminum (e.g. a phosphoenolpyruvate carboxylase gene) and transferred thereinto and is capable of manifesting a high ability to secrete the organic acid and providing a transgenic plant capable of absorbing phosphoric acid even in the presence of the aluminum and further improving the productivity of field crops in oligotrophic soil. The aluminum-resistant plant is obtained by treating the phosphoenolpyruvate carboxylase gene capable of promoting the synthesis of the organic acid forming the chelate with the aluminum with a restriction enzyme, binding the resultant fragment to a plasmid and transducing the resultant plamid into a tobacco cultured cell, etc., according to a particle gun method, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to the field of genetic engineering. In particular, it relates to the production of useful transformed plants using genetic engineering.

[0002]

BACKGROUND OF THE INVENTION Plant growth is inhibited by oligotrophic soils in a wide range of arable lands around the world, and has a great influence on crop production. The major causes of such plant growth inhibition are, in particular, aluminum damage due to soil acidification,
That is, there is cited a phosphate deficiency disorder in plants by binding of aluminum in soil to phosphoric acid, which is a nutrient of plants, and inhibiting absorption of phosphate by plants.

There are varieties of plants that exhibit high tolerance to oligotrophic soils. And it is reported that secretions of organic acids such as citric acid and malic acid are observed in such varieties with high tolerance to oligotrophic soil (Miyasaka C.
S. et al. Plant Physiol. (1991) 96 , 737-743), Delhaiz
e E.et al.Plant Physiol. (1993) 103, 695-702, Kochi
an LVAnnu Rev.Plant Pysiol.Plant Mol.Biol. (199
5) 46 , 237-260). It has also been observed that varieties that actually have high levels of malic acid have increased aluminum tolerance (Ryan PR et al. Aust. J. Plant Physio).
l. (1995) 22 , 531-536). From this, it is thought that there is some relationship between the secretion of organic acids by plants and the resistance to oligotrophic soil from the viewpoint of phenomena. For example, it is envisioned that organic acids secreted by plants combine with aluminum in the soil to convert phosphate into a form that can be absorbed by the plant, thereby causing the plant to acquire aluminum tolerance. Also, a mechanism has been envisioned in which an organic acid forms a chelate with aluminum to prevent aluminum ions from being absorbed into the plant body and prevent harm to the plant body due to the aluminum ion. However, to date, there have been no reports that demonstrated that the secretion of organic acids is directly related to oligotrophic soil tolerance, and conferring aluminum tolerance to plants by increasing the ability of plants to secrete organic acids. There have been no successful cases. It has been confirmed at the laboratory level that malic acid and the like form chelates with aluminum (Delh
aize E.et al.Plant Physiol. (1993) 103, 695-702).

On the other hand, phosphoenolpyruvate carboxylase (PEPC) is an enzyme involved in the replenishment reaction of the TCA cycle in which citric acid and malic acid are synthesized.
It has been reported that overexpression of this gene causes an increase in malic acid concentration in tobacco plants (RL
Hudspeth et al, Plant Physiol. (1992) 98 , 458-46
Four). However, the research described in the literature is not aimed at producing aluminum-tolerant plants by secreting organic acids, but is aimed at converting C3-type photosynthetic tobacco into C4-type photosynthetic plants. Nothing is shown about the extracellular secretion of acids. That is, exogenous PEPC
There are no reports that the gene was expressed in plant cells to successfully secrete organic acids out of plant cells.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a plant having an enhanced ability to secrete organic acids and having tolerance to oligotrophic soil, and a method for producing the plant.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, introduced a gene that promotes the synthesis of an organic acid that forms a chelate with aluminum into a plant cell, Was found to be able to enhance the ability of plants to secrete organic acids by highly expressing in plant cells, thereby conferring aluminum tolerance on plants. That is, the present inventors have succeeded for the first time to artificially secrete an organic acid out of a plant cell by introducing a gene that promotes organic acid synthesis into a plant cell. For the first time, and thus completed the present invention.

[0007] That is, the present invention relates to a transgenic plant having a high ability to secrete an organic acid and exhibiting aluminum tolerance, and a method for producing the plant. More specifically, the present invention relates to the synthesis of an organic acid which forms a chelate with aluminum. Aluminum-tolerant plant cells into which a gene that promotes
The present invention relates to the plant cell, wherein the gene that promotes the synthesis of an organic acid that forms a chelate with aluminum is a phosphoenolpyruvate carboxylase gene. The present invention also relates to a plant containing the plant cell.

Further, the present invention provides a method for producing an aluminum-tolerant transgenic plant, which comprises introducing into a plant cell a gene that promotes the synthesis of an organic acid that forms a chelate with aluminum. The present invention relates to the production method, wherein the gene that promotes the synthesis of the organic acid that forms is a phosphoenolpyruvate carboxylase gene.

In the present invention, “aluminum resistance” refers to the ability of a plant (or plant cell) to grow even in the presence of aluminum. The “aluminum” of the present invention also includes those in an ionized form and those in a salt form.

[0010]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a transgenic plant having high ability to secrete organic acids and having aluminum tolerance. The transgenic plant of the present invention can be produced by introducing a gene that promotes the synthesis of organic acid into a plant cell and redifferentiating the plant cell, or by directly introducing the gene into a plant.

The organic acid which forms a chelate with aluminum in the present invention includes, for example, citric acid and malic acid. The gene that promotes the synthesis of these organic acids is not particularly limited as long as it is a gene of an enzyme involved in the metabolism of organic acids. For example, a phosphoenol pyruvate carboxylase gene is preferred.

The gene for promoting the synthesis of an organic acid of the present invention is preferably incorporated into an appropriate expression vector and introduced into a plant cell or plant.

The expression vector is not particularly limited as long as the transgene can be highly expressed in plant cells. For example, pBI121 (CLONTECH), pBI221 (CLONTEC)
H) and the like.

The plant into which the gene that promotes the synthesis of organic acids is introduced is not particularly limited. Examples thereof include herbaceous dicotyledonous plants such as Arabidopsis thaliana and tobacco, monocots such as rice and corn, eucalyptus and poplar. And other woody plants.

Gene transfer into a plant cell or plant can be carried out by
For example, it can be performed using a particle gun method, an electroporation method (electroporation method), an Agrobacterium infection method, or the like.

The transfected plant cells can be redifferentiated, for example, by the method described in the literature (Pillon et al. Plant J. (1996) 9 , 573-577), whereby the transgene of the present invention can be obtained. It is possible to create genic plants.

The transformed plant or transformed plant cell can be prepared by incorporating a drug resistance gene for kanamycin or hygromycin into the recombinant DNA used for the transformation, or an agar medium containing these drugs, or Cultivation or cultivation in a liquid medium enables stable maintenance.

The transgenic plant of the present invention thus obtained has a higher ability to secrete organic acids as compared with a wild-type plant. In acidic soils, aluminum in soil binds to phosphate and inhibits the use of phosphate by plants, but the transgenic plants of the present invention can absorb phosphate by releasing organic acids that can chelate aluminum. It can be changed to a different form. Therefore, the transgenic plant of the present invention can grow by absorbing phosphate even in an oligotrophic soil where aluminum is present in the soil.

The transgenic plant of the present invention is capable of preventing aluminum from entering the plant body regardless of the presence or absence of surrounding phosphate, and is generally resistant to aluminum ions under the presence of aluminum ions. Needless to say.

[0020]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to the examples. The methods required for general gene recombination such as DNA cutting, ligation, and transformation of Escherichia coli in the Examples are commercially available reagents used for each operation, manuals attached to mechanical devices, etc., and experimental manuals. (For example, “Molecular cloning (Maniatis T.
et al. Cold Spring Harbor Laboratory Press) ").

Example 1 Construction of Transformation Construct and Overexpressing Transformed Plant The overexpression construct (p35S-PEPC) is pBI121 (C
The GUS gene of LONTECH) was cut with BamHI and SacI and removed, and then blunted, and the phosphoenolpyruvate carboxylase gene (PEPC # 16: T. Sugimoto et al, Plant Mo
An EcoRI fragment (including soybean PEPC # 16 full length) of a full-length cDNA clone of lecular Biology (1992), 20 , 743-747) was ligated.

Example 2 Introduction of PEPC Gene into Cultured Tobacco Cells A plasmid (p35S-PEPC) constructed for overexpressing the PEPC gene was introduced into cultured tobacco cells (BY-2) by a particle gun method. . Next, cultured cells were selected on MS agar medium containing 50 μg / ml kanamycin, and calli into which the plasmid was stably introduced were selected. The selected transgenic strain was used in the following citrate secretion measurement experiment.
As a control for this experiment, tobacco BY-2 cells into which pBI121 had been introduced were used.

In order to measure the amount of citrate secreted from the transformed tobacco cultured cells, kanamycin-resistant transformed cultured cells were cultured in an R2 liquid medium (pH 5.10) containing 50 μg / ml kanamycin.
7: Ohira K et al. Plant Cell Phsiol. (1973), 14,1113-
1121) for about 2 weeks, followed by transplantation into an R2 liquid medium of 0.5 mM sodium dihydrogen phosphate containing 50 μg / ml kanamycin, followed by shaking culture for about 1 week. Thus, suspension culture was performed.

Example 3 Analysis of Citric Acid and Malic Acid Secretion in PEPC-Overexpressing Tobacco Cultured Cells The cells cultured in suspension in Example 2 were mixed with 4 mM aluminum chloride and 30 mM MES (the final concentration above) in an R2 basic medium. ) And add p
The cells were transplanted to a medium adjusted to H5.6. At this time, 1-2g / 8-9ml
At a cell density of 120 rpm at 120 rpm. The culture solution was sampled after 3 hours and 24 hours, and the cultured cells were sedimented by centrifugation, and the citrate concentration and malate concentration of the supernatant were measured. A cycling reaction was used to measure the concentration.
Specifically, the pH was adjusted to 12 with 1N NaOH, and the mixture was treated at 60 ° C. for 15 minutes. Next, 1N HCl was added to the medium to adjust the pH to 2, and the temperature was adjusted to 3 at room temperature.
After standing for minutes, NAD and NADH in the medium were degraded. This medium was passed through an ion exchange column (Dowex 50w-x8) to remove aluminum ions, neutralized with 1N NaOH, and adjusted to pH 6-8. The above sample was used as a reaction solution (for citric acid, imidazole hydrochloride (pH 7.0), NADH, ZnCl ₂ , MDH, Citrate Lya
se: 2-amino-2-methylpropanol for malic acid
(pH9.9), NAD, glutamic acid, MDH, Glutamate-OAA-Tra
nsaminase) and stirred, and reacted at 25 ° C. for 30 minutes. Next, hydrochloric acid (when measuring citric acid) or sodium hydroxide (when measuring malic acid) was added, and the reaction was further stopped by boiling at 100 ° C. for 5 minutes. In the above reaction, the relevant amount is NAD for citric acid, and the relevant amount is N for malic acid.
Each solution was converted to ADH, and this solution was added to a cycling reaction solution containing alcohol dehydrogenase to give NAD.
Alternatively, NADH was quantified to measure the amounts of citric acid and malic acid, respectively. As a result, compared to pBI121 transfected cells, p3
The citrate secretion of the 5S-PEPC-introduced cells increased about twice and the malate secretion about three times.

Example 4 Analysis of Aluminum Resistance of Cultured Tobacco Cells Overexpressing PEPC Cells used in the measurement of aluminum resistance used were cells in a stationary phase (stationary phase). The cultured cells are collected by spontaneous sedimentation, and suspended in a liquid medium of aluminum phosphate (aluminum phosphate, MES) to a concentration of about 0.2 g / ml. And cultured at 170 rpm. After 3 days, the culture solution was sampled, and the cultured cells were collected by filtration, and the wet weight was measured. As a result, the wet weight of the cells overexpressing PEPC was heavier than that of the control cells, which proved that the PEPC overexpressing tobacco cells had aluminum resistance.

[0026]

According to the present invention, there has been provided a transgenic plant having a high ability to secrete organic acids and capable of absorbing phosphate even in the presence of aluminum. Therefore, if the transgenic plant of the present invention is applied to useful agricultural crops, it is possible to improve the productivity of these agricultural crops under poor nutrition soil.

──────────────────────────────────────────────────続 き Continuing on the front page (51) Int.Cl. ⁶ Identification symbol FIC12R 1:91) (72) Inventor Hiroyuki Koyama 1-1, Yanagido, Gifu City, Gifu Prefecture Gifu University Faculty of Agriculture Department of Biofunctional Engineering, Department of Bioresource Utilization

Claims (5)

[Claims] 1. An aluminum-tolerant plant cell into which a gene that promotes the synthesis of an organic acid that forms a chelate with aluminum has been introduced. 2. The aluminum-tolerant plant cell according to claim 1, wherein the gene that promotes the synthesis of an organic acid that forms a chelate with aluminum is a phosphoenolpyruvate carboxylase gene. 3. A plant comprising the plant cell according to claim 1 or 2. 4. A method for producing an aluminum-resistant transgenic plant, comprising introducing a gene that promotes the synthesis of an organic acid that forms a chelate with aluminum into a plant cell. 5. The method according to claim 4, wherein the gene that promotes the synthesis of an organic acid that forms a chelate with aluminum is a phosphoenolpyruvate carboxylase gene.