JP2005295958A - Plant absorbing co2 and nox, and method for growing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plant absorbing CO<SB>2</SB>and NO<SB>X</SB>, having excellent absorbing capacity of not only the NO<SB>X</SB>but also the CO<SB>2</SB>; and to provide a method for growing the plant. <P>SOLUTION: The method for growing the plant absorbing the CO<SB>2</SB>and the NO<SB>X</SB>is characterized by regeneration of a plant body by incorporating a cDNA of an NO<SB>2</SB><SP>-</SP>transporter of a cucumber to a vector pBI121 to obtain a recombinant vector, introducing the recombinant vector to an Agrobacterium, infecting a cell of Arabidopsis thaliana with the Agrobacterium to obtain a transformant, and culturing the transformant. The NO<SB>2</SB><SP>-</SP>transplanter is expressed on a plasma membrane of the cell of the plant body of the CO<SB>2</SB>and NO<SB>X</SB>-absorbing plant, and the plant has ≥1.2 times CO<SB>2</SB>-absorbing capacity and ≥1.1 times NO<SB>2</SB>-absorbing capacity compared to those of an untransformed plant body in which the NO<SB>2</SB><SP>-</SP>transplanter is not expressed. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a CO ₂ and NO _X absorption plant excellent in not only NO _X but also CO ₂ absorption capacity and a method for growing the same.

Nitrogen gas in the atmosphere is chemically inert and not harmful to living organisms. However, when fossil fuel, which is a reducing agent, is burned in a compressed atmosphere, nitrogen oxides (NO _x ) are discharged from the exhaust gas into the atmosphere. In addition, it is known that such nitrogen oxides, for example, NO ₂ are contained in a large amount in photochemical smog. It has been pointed out that such NO ₂ causes acid rain and has a strong oxidizing power, which may cause an adverse effect of destroying the ozone layer. Moreover, such NO ₂ dissolves in water to produce NO ₂ ⁻ , which is combined with hemoglobin to promote methemoglobinization. In addition, the photodynamic effect of this hemoglobin / NO ₂ complex causes in vivo It is known that the generation of radical species may have a harmful effect on the living body.

To remove air pollutants such as such NO _X, various chemical to date, although physical methods have been developed, chemical is conventional, rather than physical methods, air pollutants Biological methods have been developed to absorb and remove air pollutants with plants that have the ability to absorb (Patent Document 1 and Non-Patent Document 1 below). That is, NO ₂ ^- is also produced as an intermediate of nitric acid assimilation in which plants absorb nitric acid and reduce it to ammonia, but the produced NO ₂ ^- is immediately transported to chloroplasts and reduced to ammonia for consumption. Is done. If NO ₂ ⁻ in the atmosphere can be removed by utilizing such a metabolic function of the plant, air pollution can be solved. In relation to the air purification method by such a biological method, Patent Document 1 listed below shows that the size of guard cells is increased or tetraploidy compared to ordinary homologous plants. A plant with improved ability to absorb pollutants and a method for growing the same are described.

JP-A-8-317739 Plant Cell Engineering Supplement 1 "Environmental Problems and Plant Biotechnology", p. 76-86 (1994)

The present invention has been made in view of the above circumstances, not NO _X only, and to provide an excellent CO ₂ and NO _X absorption plants and their growing method absorptivity of CO _2.

The present inventors have a result of intensive studies, exogenous NO ₂ ^- was introduced transporter gene NO ₂ ^- transformed plants that expressed transporters, not only excellent in the NO _X absorption ability, Quite surprisingly, CO _{2 The} present invention was completed by finding out that it has excellent absorbency.

The present invention is as follows.
[1] NO ₂ ^- to obtain a transformant by DNA encoding the transporter is introduced into the plant cell, then CO ₂ and NO _X absorption plant characterized by regenerating a plant from said transformant How to train.
[2] the NO ₂ ^- DNA encoding transporter, the NO ₂ into a vector ^- a recombinant vector DNA encoding the transporter is connected, [1] as claimed introduced into the plant cell A method for growing CO ₂ and NO _X absorbing plants.
[3] the NO ₂ ^- DNA encoding transporter, the NO ₂ to the vector ^- by Agrobacterium into which a recombinant vector has been introduced that DNA encoding the transporter is connected to infect the plant cells The method for growing a CO ₂ and NO _X- absorbing plant according to [1], which is introduced into the plant cell.
[4] the NO ₂ ^- transporter, CO ₂ and NO _X method for growing absorption plant according to any one of [1] to [3] to be expressed on the cell plasma membrane of the plant.
[5] above [1] to [4] of either the CO ₂ and NO _X absorbent plants characterized in that it is obtained by the method for growing a CO ₂ and NO _X absorption plant according.
[6] the NO ₂ ^- transporters, CO ₂ and NO _X absorption plant of [5], wherein expressed on the plasma membrane of a cell of the plant.
[7] the NO ₂ ^- above having CO ₂ absorption capacity and 1.1 times the NO ₂ absorption capacity of more than 1.2 times compared to non-transformed plant that is not to express transporters [5] or [6] The CO ₂ and NO _X absorbing plant according to [6].

According to the growing method of the present invention, it is possible to easily grow a plant excellent not only in NO _X absorption capacity but also in CO ₂ absorption capacity.
Since the CO ₂ and NO _X absorption plant of the present invention is excellent in NO _X absorption ability, it can efficiently absorb and remove NO _X that is a pollutant in the atmosphere, and the plant body is nitrogen. Can be used as part of fertilizer. Moreover, since the CO ₂ and NO _X absorption plant of the present invention is also excellent in CO ₂ absorption ability at the same time, it can absorb CO ₂ that causes global warming, and accordingly, the growth of the plant body is increased. Can be promoted.

(1) In the CO ₂ and NO _X method for growing absorption plant CO ₂ and NO _X method for growing absorption plant of the present invention, NO ₂ ^- by a gene encoding a transporter is introduced into a plant cell, a transformant obtain.
The NO ₂ ^- transporter is typically present envelope of chloroplasts, the chloroplast from the cytoplasmic side NO ₂ ^- is a known to have a function of transporting against a concentration gradient. In the present invention, the NO ₂ ^- are not particularly limited to the type of the DNA encoding the transporter, a variety of NO ₂ if necessary ^- can be used DNA encoding the transporter. Typically, NO ₂ from high nitrate assimilation ability cucumber ^- DNA encoding a transporter is used. Also, the NO ₂ ^- transporters, NO ₂ contained in the plant regenerated from the plant cell is a host ^- may be a transporter akin, it may be heterologous. For example, using the rice or Arabidopsis cell as a plant cell is a host, the NO ₂ ^- as DNA encoding the transporter, cucumber NO ₂ ^- can be used DNA encoding the transporter. Note that the NO ₂ ^- information of the base sequence or the like of the DNA encoding the transporter can be obtained by utilizing various databases such as Genebank managed by European Molecular Biology Laboratory (EMBL). For example, NO ₂ cucumber ^- transporter cDNA has been registered in the slip number Z693702 the gene bank.

The NO ₂ ^- DNA encoding transporter, the NO ₂ ^- obtained from plants transporter is expressed, the NO ₂ ^- is synthesized by reverse transcriptase from the total RNA encoding a transporter, needs Accordingly, it may be a cDNA obtained by screening or amplification by PCR or the like. The method for obtaining the total RNA from the plant body is not particularly limited, and can be performed by a conventionally known method. Also, the NO ₂ from the cDNA obtained by the above method ^- a method of isolating cDNA encoding the transporter is not particularly limited, for example, may be performed by a conventionally known cloning methods, also, the NO ₂ ^- on the basis of the information of the DNA encoding the transporter, to prepare appropriate primers may be prepared by methods such as PCR.

Also, the NO ₂ ^- DNA encoding a transporter may be used as it is synthesized from commercially available or RNA cDNA, or may be used as necessary was recombinant gene recombinant DNA it can. Furthermore, the NO ₂ ^- in the range that does not impair the properties of the transporter, base deletion, it may be carried out substitution or addition. For example, NO ₂ from cucumber ^- transporter gene (CsNitr1) utilizes the hydrogen ion concentration gradient, NO ₂ generated in the cytoplasm ^- together with encoding transport protein for feeding the chloroplast, the chloroplast to the N-terminus It has a body transit signal sequence. Then, NO ₂ CsNitr1 is expressed ^- the transporter is synthesized, NO ₂ ^- integrated into the chloroplast envelope is where the transporter to function, functions the N-terminal signal sequence is removed when the considered to be a structure, at the stage of a differentiation of cucumber, cell membrane where the signal sequence is removed NO ₂ ^- transporter (CsNitr1-S) is present. This cell membrane transfer type recombinant DNA may be used.

Also, the NO ₂ ^- for DNA encoding the transporter, to connect the various promoter and terminator for expression in plants may be used a recombinant DNA capable of modulating the expression frequency and location. Such recombinant DNA, for example, NO ₂ ^- transporter cDNA, particularly NO ₂ from cucumber ^- the transporter cDNA, CaMV35S (35sRNA of cauliflower mosaic virus) promoter from cauliflower mosaic virus, such as, rbcS ( Examples thereof include a recombinant cDNA in which a promoter such as riborose 1,5-bisphosphate carboxylate) or cab (chlorophyll a / b binding protein) is linked. Using such recombinant cDNA, NO ₂ ^- constitutively transporter cDNA, also can be expressed tissue-specifically for the latter two, as a result, to improve the CO ₂ and NO _X absorbent It is preferable because it is possible.

There is no particular limitation on the type of the plant cell, the NO ₂ ^- can be appropriately selected depending on the method for introducing such a DNA encoding the transporter. As the plant cell, for example, the use of cells of plants reproduction system from protoplasts has been established, the foreign NO ₂ ^- can be easily introduced transporter gene, resulting in the present invention CO ₂ And it is preferable because NO _X- absorbing plants can be easily grown. Furthermore, the Agrobacterium method described below the foreign NO ₂ ^- When introducing transporter gene, as the plant cell is preferably a cell of plants showing infectious Agrobacterium, for example, various types of twins Examples thereof include monocotyledonous plants when leaf plants and acetosyringone coexist. Specific examples of the plant cell include cells such as Arabidopsis thaliana, tobacco, and rice. The plant cells may be plant tissues or plant cells as they are, or include callus and protoplasts.

In method for growing CO ₂ and NO _X absorption plant of the present invention, the NO ₂ ^- is not particularly limited, the DNA encoding the transporter to a method of introducing into the plant cell, according to the type, etc. of the plant cells The method can be selected. For example, methods for directly introducing foreign genes into plant cells include electroporation, particle gun, and polyethylene glycol methods.

The electroporation method is a method of introducing DNA by mixing the target DNA and protoplasts and making holes in the cell membrane by electrical stimulation. Since this method has high reproducibility, culturing protoplasts is stable and easy, and can be suitably used for plant cells in which plant bodies can be easily regenerated from protoplasts.
The particle gun method is a method of introducing DNA by attaching the target DNA to fine metal particles and driving it into cells at high speed. Since this method is not limited to host cells, for example, it is suitably used for plant cells that are difficult to infect Agrobacterium, plant cells that are difficult to prepare protoplasts, or for which regeneration systems from protoplasts have not been established. be able to.
The polyethylene glycol method is a method of introducing DNA by mixing the target DNA and protoplast and treating with polyethylene glycol.

Also, the NO ₂ ^- DNA encoding a transporter as a method for introducing into the plant cell, can be used a method of introducing using a vector. That is, the NO ₂ ^- to obtain a recombinant vector incorporating a DNA encoding the transporter into the vector by introducing it into the plant cells, the NO ₂ ^- the plant cell a DNA encoding the transporter Can be introduced.

In this case, the type of vector to be used is not particularly limited, and usually a plasmid vector, more preferably a binary vector is used. Specific examples of the vector include pUC18, pUC19, pBR322, pBR325, pBluescript, pBI121, and pBI221. Among these, pBI121 is particularly preferably used. In order to facilitate selection of a recombinant vector, the vector preferably has a selection marker for antibiotic resistance such as kanamycin resistance, streptomycin resistance, chloramphenicol resistance, and the like. Furthermore, the vector, the NO ₂ in the plant cells ^- may contain a nucleotide sequence of the promoter and enhancer that can control the expression of the DNA encoding the transporter. For example, the binary vector pBI121 described above incorporates the CaMV35S promoter described above in the vector itself.

The recombinant vector of the vector DNA and the NO ₂ ^- limit the DNA encoding the transporter enzymes, for example XbaI and BamHI, SmaI, and then digested by the action of SacI like, the resulting DNA fragment with T4 ligase It can obtain by connecting by well-known methods, such as.

The NO ₂ ^- as a method of introducing a DNA encoding a transporter to the plant cells, when introduced using vectors, electroporation method, the particle gun method or the polyethylene glycol method or the like, as the host of the recombinant vector However, the Agrobacterium method using Agrobacterium is usually used. The Agrobacterium method is the NO ₂ ^- Agro a recombinant vector comprising a DNA encoding the transporter tumefaciens (. Agrobacterium tumefaciens, hereinafter referred to as "Agrobacterium tumefaciens") was introduced into obtained transformants by infecting the plant cells, the NO ₂ ^- DNA encoding the transporter is a method of introducing into the plant cell.

  In the Agrobacterium method, there are no particular limitations on the types of Agrobacterium and the vector constituting the recombinant vector, and various types having a T-DNA region can be used as necessary. For example, Agrobacterium tumefaciens LBA4404 or the like can be used as the Agrobacterium. Moreover, as a vector which comprises the said recombinant vector, vectors, such as said pBI121, can be used, for example.

In the above Agrobacterium method, the NO ₂ ^- is not particularly limited to the method of introducing a recombinant vector comprising a DNA encoding the transporter into Agrobacterium tumefaciens. For example, E. coli having a recombinant plasmid, E. coli having a conjugation ability (helper plasmid) and Agrobacterium are mixed and cultured, and introduced using the conjugation ability of E. coli. Alternatively, the recombinant vector can be directly introduced into Agrobacterium by electroporation.
The Agrobacterium transformant obtained by introducing the recombinant vector is obtained, for example, by culturing in a medium containing an antibiotic such as kanamycin or streptomycin and selecting colonies that have grown due to antibiotic resistance. be able to.

In the Agrobacterium method, there is no particular limitation on the method of infecting the plant cell with the obtained Agrobacterium transformant. For example, the suspension of the Agrobacterium transformant and the plant cell are brought into contact with each other by a method such as immersing the plant cell as a host in the suspension of the Agrobacterium transformant. It can be infected by incubating as necessary. Alternatively, a vacuum infiltration method may be used in which a grown plant is infected by reducing the pressure in a suspension of the Agrobacterium transformant and then returning to normal pressure. The vacuum infiltration method is easier to use than conventional tissue culture methods, and can be used to treat a large number of plants at the same time with less effort, and thus is particularly suitable for treating a large number of plants. be able to.
In the Agrobacterium method, after infection, Agrobacterium can be removed by transferring to a medium containing an antibiotic such as claforan.

In the method for growing a CO ₂ and NO _X absorbing plant of the present invention, a transformant is obtained by the above method. Usually, the transformant having antibiotic resistance is selected by transferring the plant cells after the introduction treatment to a medium containing an antibiotic such as kanamycin and culturing.

The method for growing CO ₂ and NO _X absorption plant of the present invention, by regenerating plants from transformants obtained by the method described above, to foster CO ₂ and NO _X absorption plant.
There is no particular limitation on the method for regenerating the plant from the transformant. For example, a plant can be regenerated by culturing the transformant under appropriate conditions. More specifically, for example, the above transformant is transformed into a known plant differentiation-inducing agent (for example, auxins such as indoleacetic acid, indolepropionic acid, indolebutyric acid, naphthaleneacetic acid, and kinetin such as zeatin, kinetin, and benzyladenine). Selected from callus dedifferentiated in a medium containing an antibiotic corresponding to the selectable marker gene of the vector in which the gene is incorporated, and then other as necessary A young plant body is obtained by culturing in the above medium, and the plant body is regenerated by transplanting and growing the young plant body in soil, vermiculite or the like, and the CO ₂ and NO _X absorbing plant of the present invention Can be obtained.

In addition, in the case of obtaining a transformant by the vacuum infiltration method, after infection, the plant is grown and collected as it is, and the resulting seed is transferred to a medium containing an appropriate antibiotic and grown, The CO ₂ and NO _X absorbing plant of the present invention can be selected and grown. In this case, if necessary, the plant can be transplanted to a medium not containing antibiotics and continued to grow, and when the plant begins to be drawn, it can be transplanted to rock wool or soil for each individual and grown and seeded.

It can be confirmed, for example, by the method described below that the target CO ₂ and NO _X absorbing plant can be grown by the method for growing the CO ₂ and NO _X absorbing plant of the present invention. Namely, oligonucleotides designed and produced based on the known base sequence of the gene (for example, nit-S [SEQ ID NO: 1] and nit-As [SEQ ID NO: 2]) are used as primers and are known from the prepared plant. The synthesized DNA is separated by agarose gel electrophoresis by performing a polymerase chain reaction (hereinafter referred to as PCR) using the DNA prepared by the method as a template, and the presence of the expected length of the DNA You can confirm the introduction.

(2) CO ₂ and NO _X absorption plant CO ₂ and NO _X absorbent plants present invention is obtained by method of growing CO ₂ and NO _X absorption plant of the present invention. In CO ₂ and NO _X absorption plant of the present invention, the NO ₂ ^- transporter usually is expressed on the plasma membrane of cells, there is no particular limitation on the portions expressing.

CO ₂ and NO _X absorption plant of the present invention, NO ₂ foreign ^- expressing transporters, especially by expressing the plasma membrane of a cell, excellent in the NO _X absorbent, is excellent in CO ₂ absorbent. For example, CO ₂ and NO _X absorption plant of the present invention, the exogenous NO ₂ ^- as compared to non-transformed plant that is not to express transporters least 1.2 times, preferably 1.4 times or more, more preferably It has 1.6 times or more CO ₂ absorption capacity. Further, for example, CO ₂ and NO _X absorption plant of the present invention, the exogenous NO ₂ ^- as compared to non-transformed plant that is not to express transporters 1.1 times or more, preferably 1.15 times or more, more The NO ₂ absorption capacity is preferably 1.2 times or more, particularly preferably 1.3 times or more. This NO ₂ absorption capacity and CO ₂ absorption capacity can be measured by the methods described in the examples.

Hereinafter, the present invention will be specifically described with reference to examples.
(1) cell membrane translocation type NO ₂ ^- transporter cDNA (CsNitr1-S) Preparation cucumber seeds 4 days (Cucumis sativus, cv "Haiti"), allowed to absorb water germinated in the dark, then light irradiation the seeds The cotyledons were collected 6-12 hours later. The cotyledon part was ground in liquid nitrogen, and total RNA was obtained using “RNeasy Plant Mini kit” (manufactured by Qiagen). Then, CsNitr1-S was synthesized using 2 μg of the above total RNA and reverse transcriptase (manufactured by Toyobo, hereinafter abbreviated as “RT”) using oligo (dT) ₁₆ as a primer. PCR using rTaq DNA polymerase (manufactured by Toyobo) with 2 μL of the RT reaction as a template and oligonucleotide primers (nit-S / Xa [SEQ ID NO: 3] and nit-As / Sc [SEQ ID NO: 4]) shown below Was used to amplify CsNitr1-S. The PCR was performed under the conditions of 94 ° C. for 4 minutes, (94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 1 minute) 40 cycles, 72 ° C. for 7 minutes.

(2) Restriction enzyme treatment of CsNitr1-S The PCR reaction solution was added with 3 volumes of ethanol at room temperature and centrifuged to precipitate the PCR amplification product. The obtained precipitate was washed with 70% ethanol, and then the pressure was reduced to remove the remaining ethanol. The obtained precipitate was dissolved in a restriction enzyme XbaI buffer (M buffer), and 4 units of restriction enzyme SacI (manufactured by Toyobo) and 4 units of XbaI (manufactured by Toyobo) were added thereto, followed by treatment at 37 ° C. for 6 hours. After adding 1/10 volume of 10 × gel loading solution (manufactured by Toyobo) to the reaction solution, the entire volume was subjected to 1% agarose gel electrophoresis. Then, a piece of agarose gel containing a band of about 1500 base pairs (bp) is cut out on an ultraviolet illuminator, and DNA is recovered from the gel using a “Quiaquick Gel Extraction Kit” (manufactured by Qiagen) according to the manual of this product. And dissolved in 10 μL of sterilized water. 1 μL of this DNA solution was subjected to agarose electrophoresis with a standard marker DNA, and the concentration of ethidium bromide was compared to determine the DNA concentration.

(3) Preparation of vector pBI121 (Clontech) was used as a vector for transformation. The pBI121 was treated with SacI and XbaI under the same reaction conditions as the restriction enzyme treatment of the PCR product. After the treatment, the same volume of a phenol / chloroform (1: 1) solution was added, and the mixture was stirred vigorously for 1 minute, and then centrifuged at 10,000 × g for 5 minutes to recover the aqueous layer. To the obtained aqueous layer, 3 volumes of ethanol was added to obtain a DNA precipitate, and the DNA was washed with 70% ethanol. The DNA was then dissolved in alkaline phosphatase buffer and 10 units of alkaline phosphatase (CIAP manufactured by Toyobo) was added to dephosphorylate. Then, 1/10 volume of 10 × gel loading buffer was added to the reaction solution, and DNA was separated with 0.8% agarose gel. Recovery of pBI121 from which the β-glucuronidase (GUS) gene has been removed from the gel and determination of the concentration are the same procedures as in the above CsNitr1-S purification.

  The restriction enzyme-treated pBI121 and CsNitr1-S were mixed at an equimolar number to prepare a 1 μL solution. To this solution, 4 μL of A solution and 1 μL of B solution of “DNA ligation kit” (Takara Ver.1) were added, and reacted at 16 ° C. for 3 hours, so that pBI121 :: CsNitr1-S (caMV35S promoter of pBI121 and nopaline) A recombinant vector in which CsNitr1-S was incorporated in the sense direction between synthase (NOS) gene terminators was prepared.

(4) Transformation and regeneration of plant body 2 μL of the above recombinant vector solution was taken and E. coli was obtained using the calcium chloride method. The recombinant vector was introduced into E. coli DH5α competent cells. The transformation cell is selected as a colony that can grow on an LB agar plate containing 50 μg / mL kanamycin, and the plasmid prepared by alkaline lysis is treated with SacI and XbaI, and contains a DNA fragment corresponding to CsNitr1-S. Was stored at -85 ° C as a 16% glycerol solution.

  E. The above recombinant vector prepared by alkaline lysis from E. coli DH5α as needed is electroporated together with 25 μL of Agrobacterium tumefaciens LBA4404 (manufactured by Clontech) prepared in advance and stored at −85 ° C. It was placed between the electrodes of the deposition chamber and introduced into Agrobacterium tumefaciens LBA4404 by applying a pulse of 2.7 kV using a cell porator (manufactured by GibcoBRL). Then, the transformed Agrobacterium was selected by culturing on an LB agar medium containing 50 μg / mL kanamycin and 25 μg / mL streptomycin at 30 ° C. for 3 to 4 days, and selecting growing colonies.

Using the transformed Agrobacterium, vacuum infiltration (vacuum infiltration) method (reference: Bechtold, N. and Pelletier, G. (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Plant cells were transformed based on Arabidopsis Protocols (Martinez-Zapater, JM and Salinas, J. eds.) Pp. 259-266, Humana Press, New Jersey. In addition, Arabidopsis thaliana was used as a plant to be transformed. The transformed Arabidopsis seeds obtained by the above vacuum infiltration method were sterilized, and then the seeds were placed in a cooled agarose solution and overlaid on a plate medium with good mixing. After the agarose had completely solidified, the plate was covered and sealed with sadical tape. And after overnight at 4 ° C., it was transferred to 22 ° C. and grown for about 1 week. As a result, transformed Arabidopsis plants were regenerated. Transplanted Arabidopsis thaliana (S1-3 and S4-1 strains) of this example, which is a CO ₂ and NO _X- absorbing plant, was obtained by transplanting them individually to soil.

For the S1-3 and S4-1 strains, RNA was extracted from the leaves, RT reaction was performed according to the above-described method, and nit-S (SEQ ID NO: 1) and nit-As ( the RT-PCR method to confirm the amplification of CsNitr1-SDNA by SEQ ID NO: 2) was used as a primer PCR, cucumber origin of the NO ₂ ^- was confirmed that transporter is expressed.

(5) CO using S1-3 strain and S4-1 strain obtained by the measurement method described above ₂ and NO _X absorption ability, by the method described below was measured to measure the CO ₂ and NO _X absorption ability. The measurement results are shown in Table 1 below. As a comparative example, untransformed wild type Arabidopsis thaliana was used.

Using an infrared gas analyzer, measure the CO ₂ concentration of air exhausted from the closed chamber where the irradiated Arabidopsis thaliana of this embodiment is placed, subtract from the value before introduction into the chamber, and reduce the concentration by 5 minutes And the amount of CO ₂ absorbed was determined. This measurement was repeated three times, and the CO ₂ absorption amount was determined based on the average value. Further, using the same chamber as the chamber used in the CO ₂ absorption measurement, chemiluminescent NO _X analyzer (Horiba Apna-350E type) similar to the measurement of the CO ₂ absorption with, before and after injecting the chamber NO , And NO _x concentration were measured for 5 minutes, respectively, and the NO release amount and the NO _x absorption amount were integrated from the change in concentration and the flow rate. The same measurement was subsequently performed for the comparative Arabidopsis thaliana and the medium alone, and the absorption or release of this example and the comparative example was made except for absorption by the medium alone.
The leaf area of the plant was measured using NIH image software for photographic images, and based on this, the amount of CO ₂ and NO _X absorbed and the amount of NO released per certain leaf area were calculated.

(6) Results of Examples From Table 1, the S1-3 strain and S4-1 strain into which CsNitr1-S was introduced had a NO release amount of about 40 to 50% lower than that of the wild type that was not transformed. It can be seen that the NO ₂ absorption is about 15 to 35% higher. Also, compared to wild type untransformed, S1-3 strain and S4-1 strain was introduced CsNitr1-S is, CO ₂ absorption is higher than 60%, it is particularly excellent in CO ₂ absorption capacity I understand.
From the above, it can be seen that the S1-3 strain and the S4-1 strain into which the CsNitr1-S of the present invention has been introduced are not only excellent in NO _X absorption capacity but also particularly excellent in CO ₂ absorption capacity. .

  In addition, in this invention, it can be set as the Example variously changed according to the objective and a use, without being restricted to what is shown to the said specific Example.

The CO ₂ and NO _X absorption plant of the present invention can promote air purification by plants by being used for road greening, landscaping, afforestation and the like. In addition, the CO ₂ and NO _X absorption plant of the present invention absorbs nitrogen and can be used as a part of nitrogen fertilizer. Furthermore, the CO ₂ and NO _X absorption plant of the present invention can absorb CO ₂ that causes global warming, and also absorbs CO ₂ , so that the growth of the plant body is fast and the amount of the plant body is increased. And in particular, increased amounts of components that produce absorbed CO ₂ such as starch can also be expected.

SEQ ID NO ^: 1 is an oligonucleotide designed as a primer used for confirmation of expression of NO ₂ -transporter.

SEQ ID NO ^: 2 is an oligonucleotide designed as a primer used for confirmation of expression of NO ₂ -transporter.

SEQ ID NO: 3, the cell membrane translocation type NO ₂ ^- an oligonucleotide designed as a primer used for amplifying the transporter cDNA.

SEQ ID NO: 4, the cell membrane translocation type NO ₂ ^- an oligonucleotide designed as a primer used for amplifying the transporter cDNA.

Claims (7)

NO ₂ ^- to obtain a transformant by DNA encoding the transporter is introduced into plant cells and then the CO ₂ and NO _X absorption plant characterized by regenerating a plant from said transformant growing method . The NO ₂ ^- DNA encoding transporter, the NO ₂ into a vector ^- a recombinant vector DNA encoding the transporter is connected, according to claim 1, wherein is introduced into the plant cell CO ₂ and NO _A method for growing _X- absorbing plants. The NO ₂ ^- DNA encoding transporter, the NO ₂ to the vector ^- by Agrobacterium into which a recombinant vector has been introduced that DNA encoding the transporter is connected to infect the plant cells, said plant The method for growing a CO ₂ and NO _X- absorbing plant according to claim 1, which is introduced into a cell. The NO ₂ ^- transporter, CO ₂ and NO _X method for growing absorption plant according to any one of claims 1 to 3 is expressed on the plasma membrane of a cell of the plant. CO ₂ and NO _X absorbent plants characterized in that it is obtained by the method for growing a CO ₂ and NO _X absorption plant according to any one of claims 1 to 4. The NO ₂ ^- transporters, CO ₂ and NO _X absorption plant of claim 5, wherein expressed on the plasma membrane of a cell of the plant. The NO ₂ ^- CO according to claim 5 or 6, wherein with a CO ₂ absorption capacity and 1.1 times the NO ₂ absorption capacity of more than 1.2 times compared to non-transformed plant that is not to express transporters ₂ and NO _X absorption plants.