JPH11341928A - Incorporation of c4 photosynthesis circuit in c3 plant using malic enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for genetically transforming a C3 plant by incorporating a C4 photosynthesis circuit therein, to give the excellent characters of the C4 plant, e.g. those for improving photosynthesis capacity, dry product productivity, resistance to dryness, resistance to strong light and photosynthesis capacity at a low CO2 condition, to the C3 plant by driving the circuit. SOLUTION: This method provides a C3 plant with a C4 photosynthesis circuit by incorporating a gene coding for phosphoenolpyruvate carboxylase (PEPC), another gene coding for NADP-malic enzyme (NADP-ME) and still another gene coding for pyruvate phosphate dikinase (PPDK). The NADP-ME is preferably of a chloroplast type, which is manifested to a high extent in the chloroplast of the C3 plant under the influence of a promoter capable of manifesting it peculiarly to the photosynthesis organ, in order to incorporate a part of the C4 photosynthesis circuit, similar to that of the NADP-ME type C4 plant, into the C3 plant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for introducing C4 plants by introducing a plurality of enzymes that contribute to the C4 photosynthetic cycle into C3 plants.
The present invention relates to a method for transforming C3 plants to which four circuits are provided.

[0002]

BACKGROUND ART Photosynthetic pathways of higher plants include C3 type and C4 type.
There are three types: type and CAM type. The leaf tissue of a plant that performs C4 photosynthesis (C4 plant) is composed of mesophyll cells and vascular sheath cells around the vascular bundle, and has a special leaf tissue structure called a Kranz-type leaf structure. C4 plants fix atmospheric carbon dioxide in the form of a C4 compound by phosphoenolpyruvate carboxylase (hereinafter sometimes referred to as PEPC) localized in the cytoplasm of mesophyll cells, and decarboxylate in vascular sheath cells. Ribulose-1,5, a true carbonic anhydrase, releasing carbon dioxide by the action of enzymes
-Diphosphate carboxylase / oxygenase (hereinafter referred to as
Rubisco. ) The concentration of carbon dioxide in the vicinity is increased. The metabolite decarboxylated in the vascular sheath cells is transported to mesophyll cells, and ATP is consumed by the action of pyruvate phosphate dikinase (hereinafter sometimes referred to as PPDK) located there. It is converted to phosphoenolpyruvate (hereinafter sometimes referred to as PEP) which is a substrate of PEPC. Therefore, the two types of cells of the green leaf of the C4 plant are functionally differentiated, and the mesophyll cells generate the C4 compound and regenerate the substrate of PEPC by the initial carbon fixation, and the vascular sheath cells perform the decarboxylation from the C4 compound. True carbon fixation is performed by the Calvin-Benson circuit.

[0003] Carbon fixation by these PEPCs, release of carbon dioxide near Rubisco by decarboxylase,
The three processes of regeneration of the substrate of PEPC that consumed ATP form a circuit reaction called C4 photosynthesis circuit. This circuit reaction is responsible for the carbonic acid concentration function of C4 plants, and avoids a decrease in the efficiency of the photochemical system under strong light conditions (avoids light damage).
It provides a function, water stress tolerance function, which is not found in plants that perform normal photosynthesis (C3-type photosynthesis) (C3 plants), so that C4 plants do not have the apparent light respiration observed in C3 plants. And photosynthetic capacity under dry, intense and high temperature conditions are smaller than those of C3 plants. It is generally said that C4 plants have a higher maximum photosynthetic rate. Therefore, the C4 plant is C
It can be said that the photosynthetic ability is superior to the three plants.

[0004] As an attempt to impart a C4 photosynthetic circuit to C3 plants, introduction by cross breeding is conceivable. However, a species having a C4 photosynthetic circuit and a normal C3 photosynthetic circuit is a genus that is difficult to cross with current crossing techniques. Most are classified by level. Attempts to introduce C4 photosynthetic traits by crossing C3 and C4 plants within the same genus have been made using the plants of the genus Hamaoka, but failed to introduce the traits of the C4 photosynthetic circuit (Osugi Tatsunogyo Technology (1995) 50 pp.
30-36).

On the other hand, Hudspeth et al. (Hudspeth et a
l., Plant Physiol., (1992) 98: 458-464) includes a tobacco chlorophyll a / b binding protein promoter (cab
It has been reported that the PEPC gene was inserted downstream of the promoter (promoter) into tobacco, the PEPC activity of green leaves increased twice, and the malic acid content of the leaves increased.
mi et al. (Kogami et al., Transgenic Research (199
4) 3: 287-296) contains cauliflower mosaic virus 35
It has been reported that when a PEPC gene was connected downstream of the S promoter and introduced into tobacco, the leaf PEPC activity was increased two-fold. However, none of the documents
Was independently introduced into tobacco, a C3 plant, and accumulation of malic acid, a C4 compound, was confirmed, but no change in photosynthetic ability was observed. In addition, C3 plants lack the ability to decarboxylate C4 compounds in the plant and supply them to the Calvin cycle. Therefore, the introduction of the PEPC gene alone into the C3 plant cannot reproduce the carbonic acid concentration function, the photoinjury avoidance function, and the like found in the C4 photosynthetic circuit, and does not improve the photosynthetic ability of the C3 plant.

Japanese Patent Application Laid-Open No. 8-80197 discloses a phosphoenolpyruvate carboxykinase (hereinafter referred to as PC) to which a transit peptide necessary for translocation to chloroplasts is added.
The gene has been introduced into rice as a C3 plant, and the detection of enzyme activity in the crude green leaf extract and the transfer of PCK protein to chloroplasts have been confirmed. From this,
It is suggested that PCK activity can be localized in chloroplasts. However, it does not mention the rotation of the C4 photosynthetic cycle and changes in photosynthetic activity in the transformed plant.

[0007] Further, Ichikawa et al.
63, Appendix 2 (1994), p.247)
Into the C3 plants Arabidopsis and tomato,
Although it has been confirmed that the present enzyme protein accumulates, it does not mention the rotation of the C4 photosynthetic cycle and changes in photosynthetic activity in the transformed plant. In addition, Japanese Patent Publication No. 6-1299
In 0 JP, carbonic anhydrase (hereinafter sometimes to CA.) Proteins were incorporated the Rikoperishikon esculentum (Lycopersico n escu
lentum ) has been reported to change the photosynthetic efficiency of cotyledon protoplasts, see Majeau et al. (Plant Mol. Biol.
94) 25: 337-385) also reports that there was no change in photosynthetic activity even when CA was overexpressed in actual plants by gene transfer.

[0008] Ku et al. (Maurice SB Ku, et al., Plant
Physiol. (1997) 114: S-300) introduced the maize PEPC gene into rice and compared it with non-transformed plants by 0.5-90.
A rice expressing twice the PEPC activity was produced. Rice plants overexpressing PEPC showed lower photorespiration and oxygen damage than non-transformed plants, and improved photosynthetic rate. But,
In this study, only the carbonic anhydrase PEPC was highly expressed in the cytoplasm, and there was no mention of the formation of the C4 photosynthetic circuit.

Ku et al. (Maurice SB Ku, et al., Sixth
Western Regional PhotosynthesisConference (1997)
p.52) transformed rice with NADP-ME to produce rice having 2 to 20 times the activity of the non-transformant. However, there is no mention of the formation of the C4 photosynthesis circuit.

The international patent application WO 94/28180 “FRUIT WITH
MODIFIED NADP-LINKED MALIC ENZYME ACTIVITY "
By introducing a cDNA encoding NADP-ME of tomato (C3 plant) in a sense or antisense orientation,
It discloses the production of a transformed plant with increased or decreased NADP-ME activity. However, the purpose of this invention is to modify the malic acid content of the fruits,
It is not intended to provide a four-photosynthesis circuit.

Japanese Patent Laid-Open Publication No. Hei 8-89250 discloses a cDNA encoding NADP-ME of aloe (CAM plant) and a transgenic plant into which the cDNA has been introduced. However, Aloe NADP-M introduced into transformed plants
The activity of E has not been confirmed.

As described above, attempts to introduce genes of enzymes involved in the C4 photosynthetic cycle into C3 plants by genetic engineering techniques include PEPC, PCK, PPDK, and NAD.
There is only an example in which P-ME and CA were introduced alone and their expression or enzyme activity was examined, and a case in which the C4 photosynthetic cycle was rotated in a transformed plant to achieve a remarkable change in photosynthetic activity was reported. It has not been.

On the other hand, NADP-ME (EC 1.1.1.40) is widely distributed in animals, plants, and microorganisms, and catalyzes the reaction of decarboxylating malic acid to produce pyruvic acid using NADP as a coenzyme.
In plants, two types of isozymes, cytoplasmic and chloroplast localized, are known. The cytoplasmic type has functions such as regulation of intracellular pH, ripening of fruits, supply of NADPH and pyruvate to biological reactions in C3 plants, and decarboxylation of malic acid accumulated at night in CAM plants. It is believed that there is. The chloroplast type is localized in the vascular sheath cells of certain C4 plants (NADP-ME type) such as corn,
It contributes to the carbonic acid enrichment mechanism unique to C4 plants, namely the supply of CO ₂ to isco. Recently, a C3 type Flavelaria plant (Barb
el Lipka, Klaus Steinmuller, Elke Roshe, Dagmer Bo
rsch andPeter Wethoff (1994), Plant Mol. Biol. 26
: 1775-1783) and wheat (Veronica G. Maurino, Maria)
F. Drincovich, Paula Casati, Carlos S. Andreo, Ger
ald E. Edwards, Maurice SB Ku, Sanjay K. Gupta a
nd Vincent R. Franceschi (1997), J. Exp. Bot., 48:
799-811) reported the presence of chloroplast NADP-ME, but its function has not been clarified.

[0014]

As described above, there have been no reports on the provision of a C4 photosynthetic circuit to C3 plants by genetic engineering techniques. No successful case has been reported for the introduction of the C4 photosynthetic trait by crossing. Therefore, providing a C3 photosynthesis circuit to a C3 plant is an unsolved problem in the related art.

An object of the present invention is to improve the photosynthetic ability of C3 plants by using NADP-ME involved in the C4 photosynthesis circuit.
By introducing a plurality of enzymes including
It is an object of the present invention to provide a method for transforming a C3 plant for providing a photosynthetic circuit.

More specifically, the present invention relates to the use of NA in C3 plants.
Drive the C4 photosynthetic circuit of DP-ME type C4 plants to improve photosynthetic ability, dry matter productivity, drought tolerance ability, strong light tolerance ability, photosynthesis ability under low CO ₂ conditions, etc. Provided is a method for transforming a C3 plant in order to confer the excellent traits of the C4 plant.

The present invention also provides a method for transforming C4
It is another object to provide a transformed plant to which a circuit has been added and a vector for this transformation.

[0018]

SUMMARY OF THE INVENTION The method of the present invention comprises the steps of:
E (preferably chloroplast NADP-ME, more preferably chloroplast NADP-ME of a C4 plant) is highly expressed in the chloroplast of a C3 plant under the control of a promoter that specifically expresses photosynthesis; C4 similar to NADP-ME type C4 plant
Part of the photosynthetic cycle (Decarboxylate malic acid to convert CO ₂ into Rubisco
This is a method for imparting the excellent photosynthetic properties of C4 plants to C3 plants. In the method of the present invention, in addition to NADP-ME, pyruvate phosphate dikinase (PPDK), which is one of the C4 photosynthesis genes, is simultaneously highly expressed in the cytoplasm or chloroplast under the control of a similar promoter. By simultaneously expressing phosphoenolpyruvate carboxylase (PEPC), one of photosynthetic genes, in the cytoplasm under the control of a similar promoter, NADP-ME type C4
Create C3 plants driven by a more efficient C4 photosynthesis circuit, similar to C4 photosynthesis of plants.

As described above, in the conventional attempts, the gene involved in C4 photosynthesis is expressed alone, but it has not been successful to introduce a C4 photosynthesis circuit into C3 plants.
The present inventors hypothesized that the C4 photosynthetic circuit was not activated in C3 plants because the decarboxylase NADP-ME did not express sufficient activity in places where it could contribute to photosynthesis (chloroplasts). . So, if you introduce NADP-ME into C3 plants,
It was thought that this enzyme functions to drive the C4 photosynthesis circuit. At that time, the three genes of NADP-ME, PPDK and PEPC can be simultaneously expressed at the target place at a high level.
Predicted to be important for the provision of the C4 photosynthetic circuit to plants (rice and the like). That is, in the C4 plant, the initial carbon fixation reaction, the decarboxylation reaction, and the true carbon fixation reaction are performed by separately differentiated cells, but in the C3 plant, the chloroplasts in the same cell are regarded as different cells, and NADP -ME (preferably C4
It was expected that the C4 photosynthetic circuit would be formed if the plant chloroplast type was highly expressed.

Therefore, as a result of intensive studies, a C4 photosynthesis circuit shown in FIG. 3 was constructed in C3 plants. As shown in this figure, NADP, a decarboxylase in the C4 photosynthetic cycle
-ME is highly expressed in chloroplasts of C3 plants (rice, etc.),
The four-photosynthesis circuit is driven. In addition to NADP-ME, C
In the three plants, the expression of the low activity PPDK or PEPC to make up for them makes the driving of the C4 photosynthetic circuit more reliable. If PPDK is expressed in the cytoplasm or chloroplast of green mesophyll cells, pyruvate generated by the decarboxylation reaction can be converted to PEPC.
It is expected to drive the C4 photosynthetic circuit closer to C4 plants and more efficiently because it is converted to PEP, which is a substrate of P4. Plus PP
It is also expected that ATP is consumed by the reaction of DK, and a function to avoid photodamage is imparted. In addition, by expressing PEPC in the cytoplasm of green leaf mesophyll cells, the initial carbon fixation reaction of the C4 photosynthetic circuit can be strengthened, and an efficient driving of the C4 photosynthetic circuit closer to C4 plants is expected.

As described above, according to the method of the present invention, NADP-ME is expressed in chloroplasts, and PEPC is expressed in the cytoplasm of green leaf mesophyll cells.
By expressing DK in the cytoplasm or chloroplast of green leaf mesophyll cells, in the green leaf mesophyll cells of C3 plants,
Using the cytoplasm as mesophyll cells of C4 plants and the chloroplast as vascular sheath cells of C4 plants, a carbon fixation pathway similar to the tissue differentiation of C4 plants is created. This allows C
(4) To enhance the activity of three enzymes required for the photosynthetic cycle, maximize the driving force of C4 photosynthesis, and confer to C3 plants the excellent traits of C4 plants, including carbonic acid concentration function and light damage avoidance function. It is possible.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
In the method of the present invention, the NADP-ME gene is introduced into a C3 plant, preferably so as to be expressed in chloroplasts.
Thus, the NADP-ME gene is preferably a chloroplast NAD that functions naturally in the chloroplast of C4 plants.
P-ME gene. Such genes include, for example, Beverly A. Rothermel and Timothy Nelson, "Prima
ry Structure of the Maize NADP-dependent Malic Enz
yme ", J. Biol. Chem. (1989) 264: 19587-19595, discloses the nucleotide sequence of the full-length NADP-ME cDNA of corn. Discloses a cDNA encoding rice (C3 plant) NADP-ME.

Genes encoding PEPC are known to be derived from bacteria, protozoa and plants. For example, bacteria-derived bacteria include those derived from coryneform / glutamic acid-producing bacteria (Japanese Patent Publication No. 7-83714). However, preferred PEPCs are derived from plants, for example, those derived from corn (Japanese Patent Publication No. 6-30587,
And Hudspeth, RL et al., Plant Mol. Biol. (198
9) 12: 579-589), derived from amaranth (Rydzik,
E. and Berry, JO, Plant Physiol., (1995) 110: 71
3) from Flaveria Trinerbia (Poetsch,
W., et al., FEBSLett., (1991) 292: 133-136), from tobacco (Koizumi, N. et al., Plant Mol. Biol.,
(1991) 17: 535-539), derived from soybean (Japanese Unexamined Patent Publication No.
319567), those derived from oilseed rape (JP-A-6-90766)
Publication), those derived from potatoes (Merkelbach, S. et.
al., Plant Mol. Biol., (1993) 23: 881-888), derived from alfalfa (Pathariana, SM et al., Plant
Mol. Biol., (1992) 20: 437-450), from Mesembriantemum crystallinum (Cushman, JCand B
ohnart, HJ, Nuc. Acid Res., (1989) 6743-6744)
And the like, and among them, those derived from corn are particularly preferable.

As the gene encoding PPDK, a maize C4 type PPDK gene (Matsuoka, M. et al.
et al., J. Biol. Chem., (1988) 263: 11080-1108.
3), from rice (Japanese Patent Laid-Open No. 7-184657), from Flaveria pringley (Rosche, E. et al., Plant
Mol. Biol., (1994) 26: 763-769), derived from Mesembriantemum crystallinum (Fisslthaler, B. et al.
et al., Planta, (1995) 196: 492-500), and in the present invention, the maize C4-type PPDK gene is preferred. The PPDK gene may be expressed in chloroplasts, but may be expressed in the cytoplasm if necessary. When expressing in the cytoplasm, the transit peptide-encoding sequence may be removed before use.

In the present invention, a gene encoding CA may be introduced into C3 plants to supply bicarbonate ions, which are direct substrates of PEPC, to the cytoplasm. Although many genes encoding CA are known to be derived from animals and plants, genes encoding CA derived from higher plants and those derived from other organisms have low similarities in nucleotide sequences. Enzyme activity of higher plant CA is controlled by inorganic phosphate (Sultemeyer, D. et al., Physio
l. Plant., (1993) 88: 179-190). Therefore, the genes used are preferably those derived from higher plants, and those derived from higher plants include those derived from spinach (Bur
nell et al., Plant Physiol. (1990) 92: 37-40), from pea (Roeske, CA and Ogren, WL, Nuc.
Acid Res., (1990) 18: 3413), derived from Arabidopsis thaliana (Raines, CA et al., Plant Mol. Biol., (199
2) 20: 1143-1148), derived from rice (WO 95/1197)
9) and those derived from corn (WO 95/11979), and preferably those derived from spinach.
Since spinach CA is an enzyme localized in chloroplasts,
A transit peptide sequence is added to the gene encoding this enzyme. Therefore, in the construction of the gene, as described in Example 1 of Japanese Patent Application No. 9-26658 of the applicant of the present invention, the region coding for the transit peptide was removed by partial mutagenesis and described in SEQ ID NO: 3. It is good to use a gene.

The promoter sequence used in the gene encoding each of the above-mentioned enzymes is not particularly limited, but a promoter sequence that specifically expresses the gene in a photosynthetic organ is preferable. Such promoter sequences include, for example, corn C4 type PPDK.
Promoter sequence (Glackin et al., (1990) Proc. Nat
l. Acad. Sci. USA 87: 3004-3008, and Matsuoka,
M. et al., Proc. Natl. Acad. Sci. USA (1993) 90: 95.
86-9590), a maize C4 type PEPC promoter sequence (Hudspeth, RL and Grula, JW, Plant Mol. Bi.
ol., (1989) 12: 579-589), a rice Rubisco small subunit (rbcS) promoter sequence (Kyozuka, J. et al., Plant
Physiol., (1993) 102: 991-1000), a rice chlorophyll a / b binding protein promoter sequence (Sakamoto,
M. et al., Plant Cell Physiol., (1991) 32: 385-39
3) and the like. In the present invention, corn C
A type 4 PPDK promoter or rice rbcS promoter sequence is preferred.

In the present invention, the genes encoding the enzymes involved in the C4 cycle may be separately introduced and transformed into C3 plants as construction genes for gene introduction.
Preferably, each gene is ligated on the same gene-introduced construction gene, and this is introduced into a C3 plant and transformed. There is no particular restriction on the order of the genes.

In order to transform a C3 plant cell with a transgenic construct containing one or more required genes linked to each other, the above-described construct is inserted into the selected C3 plant cell by a conventional method. What is necessary is just to introduce a gene. Examples of the method of introduction include conventional methods such as electroporation, electroinjection, a method using a chemical treatment such as PEG, and a method using a gene gun.
In particular, it is preferable to introduce and transform each gene into C3 plants using the Agrobacterium method. The Agrobacterium method is well known in the art, and can transform both dicotyledonous plants (for example, JP-A-4-330234) and monocotyledonous plants (WO 94/00977). Plants that have been successfully transformed can be selected by the method described below.

The genetic trait of the transformed plant can be fixed by a general breeding method, and the introduced gene can be transmitted to progeny plants.

As a C3 plant to be transformed, this technique can be applied to any C3 plant, especially rice, wheat, barley, soybean, potato, tobacco,
It is useful for crops such as rape that are expected to improve dry matter productivity by improving photosynthetic ability. In the present invention, it is preferably applied to monocotyledonous plants, and particularly preferably applied to rice.

The C4 photosynthesis circuit referred to in the present invention is:
As described above, carbonic acid fixation by PEPC, NADP-M
Emission of carbon dioxide near Rubisco by E, A
It is formed by three processes of regeneration of the substrate of PEPC that consumed TP.

This C4 photosynthesis circuit is transformed with C3
The method of determining whether or not it is functioning in a plant will be described in detail in Examples described later. In summary, it is performed by at least one of the following methods. The produced transformants and control cut leaves were made to incorporate radioactively labeled carbon dioxide ( ¹⁴ CO ₂ ) for a short period of time, and the ratio of labeled carbon compounds was compared. PEPC functioned in the transformed plants. To determine whether C4 compounds, which are the initial carbon dioxide fixation products of the C4 photosynthesis circuit, are produced. In addition, the changes over time of the labeled carbon compound are compared to investigate whether the introduced C4 photosynthetic pathway functions in the transformed plant. Malic acid ([ ¹⁴ C] malic acid) labeled with a radioisotope was incorporated into the cut leaves of the produced transformant and control, and after a certain period of time, the ratio of photosynthetic metabolites including sucrose labeled was compared. NADP-M in transformed plants
It is investigated whether E functions to decarbonate the C4 compound. The photosynthetic activity of the resulting transformant is measured to determine whether the photosynthetic ability has changed.

EXAMPLES Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

[0034]

EXAMPLES (1) A DNA fragment containing a maize PPDK promoter and a rice Rubisco small subunit (rbcS) promoter that is specifically expressed in rice in a photosynthetic organ was isolated. (2) NADP-ME, P from maize green leaf cDNA library
EPC and PPDK cDNAs were isolated. (3) The terminator region was isolated, a gene cassette was constructed in which a promoter was connected upstream of the cDNA and a terminator was connected downstream, and these were connected as described below to construct a vector to be introduced into rice. 1) Maize NADP-ME 2) Maize NADP-ME + Maize PPDK (Chloroplast type) 3) Maize NADP-ME + Maize PPDK (Cytoplasmic type) 4) Maize NADP-ME + Maize PEPC + Maize PEDP-Maize NADPME (Chloroplast type) 6) Maize NADP-ME + Maize PEPC + Maize PPDK (Cytoplasmic type) (4) Using 6 types of construct vectors, each gene was introduced into rice by Agrobacterium method,
A transformant was obtained. The expression of the transgene in each transformant was investigated by Western blotting using a specific antiserum, and high-expressing individuals were selected. For some of the high expression individuals, it was confirmed by enzyme activity measurement that the transgene protein was expressed in an active form. (5) Carbon dioxide ( ¹⁴ CO ₂ ) labeled with radioisotope under light irradiation on the cut leaves of the created transformants and controls
Was incorporated for a short time, and the ratio of the labeled carbon compound was compared to confirm the fixation to the C4 compound, which is the initial carbonic acid fixation product of C4 photosynthesis. (6) Incorporate malic acid ([ ¹⁴ C] malic acid) labeled with a radioisotope into the cut leaves of the transformants and controls under light irradiation, and after a certain period of time, labeled sucrose and photorespiration Intermediate metabolites (serine, glycine) and calvin cycle intermediate metabolites (3-phosphoglycerate, sugar phosphate)
Of malic acid and CO
It was confirmed that ₂ was fixed by the photosynthetic carbonic acid fixing reaction. Next, the method of these experiments is shown in detail.

Example 1 In order to express various genes
Vector Construction Known sequences as PPDK promoters (Matsuoka,
M., Tada, Y., Fujimura, T., and Kano-Murakami, Y.,
"Tissue-specific light-regulated expression direc
ted by the promoter of a C4 gene, maize pyruvate,
orthophosphatedikinase, in a C3 plant, rice. ", Pro
Natl. Acad. Sci. USA (1993) 90: 9586-9590) was digested with EcoRI and NdeI, and a DNA fragment of about 1.6 kbp was used for gene construction.
However, when connecting to PEPC cDNA, an NcoI linker was inserted into the SacI site upstream of the NdeI site,
A DNA fragment digested with coRI and NcoI was used.

The promoter region of rice rbcS was obtained as follows. Known base sequences (Kyozuka, J., McEloy,
D., Hayakawa, T., Xie, Y., Wu, R. and Shimamoto,
K., "Light-regulated and cell-specific expression
of tomato rbcS-gusA and ricerbcS-gusA fusion genes
in transgenic rice. ", Plant Physiol. (1993) 102: 9
91-1000) 5'-GCAAGCTTTTACTTGTACCAACTAATATAATGAGTG-3 '(5' side)
5'-TGGATCCTCTAGAGTACTTCTTGAGATGCACTGCTC-3 '(3' side)
PCR (Mcpher) using rice genomic DNA as a template
son, MJ, Quirke, P. and Taylor, GR ed .: PCR.A
practical approach.Oxford express press, Oxford N
Y (1991)) to obtain a DNA fragment of about 1.3 kbp. This was digested with HindIII and XbaI to obtain about 1.3 k
The bp DNA fragment was used for gene construction.

The isolation of corn NADP-ME cDNA was performed as follows. MRNA was prepared from the green leaves of corn A188 according to the usual method (edited by Tadashi Watanabe, edited by Masahiro Sugiura, Cloning and Sequence of Plant Biotechnology Manual, Rural Culture Co., Ltd. (1989)). This m
From RNA to TimeSaver cDNA syntheses
isKit (product name, Pharmacia, Sweden)
Was used to prepare cDNA and cDNA library. The nucleotide sequence of a known maize NADP-ME cDNA (Beverly A. Rothermel and Timothy Nelson, "
Primary Structure of the Maize NADP-dependent Mali
c Enzyme ", J. Biol. Chem. (1989) 264: 19587-19595)
5'-TACTTACGTGGCCTTCTTCCTCCG-3 '(5' side), 5'-GGTTTGGCT
Using GCTGGATTCAAAGGG-3 '(3' side), the corn green leaf cDNA was used as a template to perform PCR (Mcpherson, MJ
Et al., Supra), and a DNA fragment of about 900 bp, ME1
A fragment was obtained. Using this ME1 fragment as a probe, a conventional method (Sambrook, J., Fritsch, EF and Maniatis, T .: Mol
ecular cloning: A laboratory manual, 2nd ed., Cold
spring harbor laboratory press, Cold Spring Harbo
r NY (1989)).
The library was screened and plasmid pSK95B4
And pSK95B7 were obtained. In addition, a known base sequence (Beverly
A. et al., Supra) 5'-GTCGACCATATGCTGTCCACGCGCACCGCC-3 '(5' side), 5'-CAA
PCR (Mcpherson, MJ) using AAGGGTGTAACCGCTTGC-3 '(3' side) and the corn green leaf cDNA as a template
Supra), and a DNA fragment of about 300 bp, ME3
A fragment was obtained. In addition, known base sequences (Beverly A. et al.
5'-AGCCTACGAGCTCGGTCTG-3 '(5' side), 5'-CCCGGGCATGCGCA
Using AAATTGATCCCCGCAG-3 '(3' side) and the corn green leaf cDNA as a template, PCR (Mcpherson, MJ
Supra), a DNA fragment of about 120 bp, MEc
A fragment was obtained. The insert fragment of ME3 fragment and pSK95B7 was Not
At the I site, the inserted fragment of pSK95B7 and the inserted fragment of pSK95B4 were connected at the EcoRI site, and the inserted fragment of pSK95B4 and the MEc fragment were connected at the SacI site to prepare cDNA containing the entire coding region of the malic enzyme (FIG. 1). This cDNA
Was digested with NdeI and SphI to obtain about 1.9 kbp.
Fragment was used for gene construction.

The maize C4 type PPDK cDNA is
Known base sequences (Matsuoka, M., Ozeki, Y., Yamamoto,
N., Hirano, H., Kano-Murakami, Y. and Tanaka,
Y., "Primary structure of maize pyruvate, orthophos
phate dikinase as deduced from cDNA sequence. ", J.
Biol. Chem. (1988) 263: 11080-11083), using a synthetic oligonucleotide 5′-TAGCTCGATGGGTTGCACGATCATATGGAGCAAGG-3 ′ as a probe, according to a conventional method (Sambrook, J. et al., Supra).
It was isolated from a maize green leaf cDNA library prepared using a ZAP vector (Stratagene, USA). A DNA fragment of about 3 kbp obtained by digestion with NdeI and ClaI was used for gene construction as chloroplast-type PPDK cDNA.

To prepare a cytoplasmic type PPDK cDNA, a known sequence (Sheen, J .: Molecular mechanisms un
derlying the differential expression of Maize Pyru
vate, Orthophosphate dikinase genes.Plant Cell 3: 2
25-245 (1991)) 5'-TTTCATATGGCGCCCGTTCAATGTGCGCGTTCGCAGAGGGTGTTCCA
CTTCGGCAA-3 '(5' side), 5'-GTACTCCTCCACCCACTGCA-3 '(3'
Side) and PCR (Mcpherson, MJ et al., Supra) using the above-mentioned corn PPDK cDNA as a template,
A DNA fragment of about 250 bp, DK1 fragment, was obtained. This fragment was digested with NdeI and SacII and replaced with the portion between the NdeI and SacII sites of the chloroplast-type PPDK cDNA. A DNA fragment of about 2.9 kbp obtained by digestion with NdeI and ClaI was converted to cytoplasmic PPDKcD.
NA was used for gene construction.

The maize PEPC cDNA has a known nucleotide sequence (Hudspeth, RL and Grula, JW, "Struc
ture and expression of the maize gene encoding the
phosphoenolpyruvate carboxylase isozyme involved
in C4 photosynthesis. ", Plant Mol. Biol. (1989) 12:
579-589) using a synthetic oligonucleotide 5'-GCCATGGCGCGGCGGGAAGCTAAGCACGGAAGCGA-3 'as a probe and isolated from a maize green leaf cDNA library by a conventional method (Sambrook, J. et al., Supra). After digesting this clone with XhoI, a DNA fragment of about 3 kbp obtained by partial digestion with NcoI was used for gene construction.

The terminator region is pPGA643A (Gynheu
ng AN, Paul R. Ebert, Amilava Mirta and Sam B. HA:
binary vector, Plant Molecular Biology Manual A3:
1-19 (1988)) was digested with ClaI and KpnI, and a DNA fragment of the gene 7 terminator region, pG
L2 (Bilang R, Iida S, Peterhans A, Potrykus I, Pas
zkowski J: The 3'-terminal reagion of the hygromyc
in-B-resistance geneis important for its activity
in Escherichia coli and Nicotiana tabacum.Gene 10
0: 247-250 (1991)) and the DN of the CaMV35S terminator region obtained by digestion with SphI and EcoRI.
A fragment and pBI121 (Jefferson, RA, Kavanaugh, T.
A. & Bevan, MW GUS fusions: β-glucuronidase as
a sensitive and versatile gene fusion marker in h
igher plant. EMBO J. 6: 3901 (1987)) was digested with SacI and EcoRI, and three types of DNA fragments in the nos terminator region were used.

The obtained promoter, cDNA and terminator DNA fragments were ligated as follows (FIG. 2).・ PPDK promoter: NADP-ME cDNA: CaMV35S terminator (ME gene) ・ PPDK promoter: PPDK cDNA: gene7 terminator (P
(PDK gene)-rbcS promoter: cytoplasmic PPDK cDNA: gene7 terminator (cytoplasmic PPDK)-PPDK promoter: PEPC cDNA: nos terminator (PE
PC gene)

Each of the ligated genes was converted into pSB22 (Komari, T., H
iei, Y., Saito, H., Murai, N., Kumashiro, T .: Vecto
rs carrying two separate T-DNAs for co-transformat
ionof higher plants mediated by Agrobacterium tume
faciens and segregationoftransformants free from s
election markers. Plant J. 10: 165-174 (1996)) inserted upstream or downstream of the HPT gene, and used as super binary intermediate plasmids pSBmMH, pSBmHDM, pSBmHDcM, pSB.
mHMP, pSBmHMPD, and pSBmHMPDc were constructed. Each gene fragment was arranged as follows in T-DNA present in each plasmid (FIG. 2). (ME gene)-(HPT): (pSBmMH) (HPT)-(PPDK gene)-(ME gene): (pSBmHDM) (HPT)-(cytoplasmic PPDK)-(ME gene): (pSBmHDcM) (HPT) -(ME gene)-(PEPC gene) :( pSBmHMP) (HPT)-(ME gene)-(PEPC gene)-(PPDK gene) :( pSBmHM
(PD) (HPT)-(ME gene)-(PEPC gene)-(cytoplasmic PPDK gene): (pSBmHMPDc)

Escherichia coli D carrying each plasmid
E. coli HB101 and pSB1 carrying H5α and pRK2013 (Ko
Agrobacterium LBA4 carrying mari, T. et al., supra).
Introgression into Agrobacterium and homologous recombination (Komari, T. et al., Supra) were carried out by crossing the three lines of 404, and pSB1MH, pSB1
Agrobacterium having HDM, pSB1HDcM, pSB1HMP, pSB1HMPD, and pSB1HMPDc was prepared.

Example 2 Production of Transformants (1) Transformation All rice was transformed using the Japanese rice variety "Tsuki no Hikari". Transformation of rice is performed by the Agrobacterium method (Hiei,
Y., Ohta, S., Komari, T. and Kumashiro, T .: Efficie
nt transformation of rice (Oryza sativa L.) mediat
ed by Agrobacterium and sequence analysis of the b
oundaries of the T-DNA. Plant J. 6: 271-282 (199
4)). The resulting transformant was placed in an air-conditioned greenhouse (1
6 hours daylength, day: 28 ° C, night: 23 ° C).

(2) Detection of Enzyme Protein The expression of the transgene protein in each transformed rice plant was determined by
Detection was performed by Western blotting in the manner described below. Green leaf 10 mg 400 μl extract (20 mM Tris-HCl
pH6.8, 1% SDS, 140 mM 2-mercaptoethanol, 20%
(Glycerol), and a part of the obtained extract is subjected to SDS-P
Subjected to AGE. After electrophoresis, the proteins in the gel are electrically immobilized to Immobilon-P membrane (trade name, Millipore, USA)
Transcribed into corn PEPC protein, corn
Rabbit antiserum against PPDK protein or corn NADP-ME protein, alkaline phaosphatase-labeled goat anti-rabbit IgG antibody (Organon Technica, Belgium), enzyme using AP color kit (trade name, Biorad, USA) Protein was detected.

(3) Measurement of NADP-ME activity 0.1 g of green leaf was extracted with 1 ml of an extract (50 mM Hepes-KOH pH 7.5, 5 mM M
gCl ₂ , 5 mM dithiothreitol, 2% polychloral AT)
The mixture was ground using an ice-cooled mortar and pestle. 15,0
Centrifugation was performed at 00 × g at 4 ° C. for 15 minutes to obtain a supernatant. The supernatant was passed through a NAP5 column (trade name, Pharmacia, Sweden) equilibrated with a column buffer (50 mM Hepes / KOH pH 7.5, 5 mM dithiothreitol) to obtain a crude extract. 1 ml reaction solution containing 50 μl of crude extract (50 mM Tricine-KOH, 0.1
mM EDTA, 0.3 mM β-NADP, 5 mM L-malic acid, 2 mM MgCl ₂ )
The enzyme activity was calculated by increasing the absorbance of NADPH at 340 nm at 30 ° C. Chlorophyll can be determined by extraction with 96% ethanol using a trituration solution (Winermans and deMot
s (1965) Biochim. Biophys. Acta 109: 448-453).

(4) Measurement of PPDK activity 0.1 g of green leaf was extracted in 1 ml (50 mM Hepes-KOH pH 7.5, 5 mM
The mixture was ground with dithiothreitol, 1 mM EDTA, 10 mM MgCl ₂ , 1 mM pyruvic acid, 2 mM phosphoric acid, 20% glycerol, 5 mg / ml isoascorbic acid, 2% polychloral AT) using a mortar and pestle cooled with ice. The triturated liquid was centrifuged at 15,000 × g at 4 ° C. for 15 minutes to obtain a supernatant. This supernatant is added to the column buffer (50m
M Hepes-KOH pH7.5, 5mM dithiothreitol, 1mMEDT
A, a crude extract was obtained by passing through a NAP5 column (trade name, Pharmacia, Sweden) equilibrated with 10 mM MgCl ₂ , 1 mM pyruvic acid, 2 mM phosphoric acid, 20% glycerol). 1 ml of the reaction solution containing 200 μl of the crude extract (25 mM Hepes-KOH pH 8.
0, 10 mM dithiothreitol, 10 mM KHCO ₃ , 8 mM MgSO ₄ ,
340 nm at 25 ° C. using 1 mM ATP, 1 mM glucose 6-phosphate, 5 mM NH ₄ Cl, 5 mM pyruvate, 2.5 mM KH ₂ PO ₄ , 0.2 mM NADH, 2 units malate dehydrogenase, 2 units PEP carboxylase). The enzyme activity was calculated from the decrease in the absorbance of NADH. Chlorophyll was determined using a trituration solution.
% Ethanol extraction (Winermans et al., Supra).

(5) Tracer experiment using ¹⁴ CO ₂ Transformed rice grown in an air-conditioning greenhouse and control rice (moonlight) were cut at about 7 cm from the tip of the leaf in water so that air did not enter through the cut. The cut was wrapped with absorbent cotton soaked in water and set in a self-made assimilation box (capacity: about 50 ml). The experimental device is a system in which a tube (volume of about 40 ml) is connected to an assimilation box, the flow in the system is opened and closed by a solenoid valve, and the gas in the system is circulated in one direction by a pump (flow rate 5 liters per minute).

After bubbling outside air at a flow rate of 5 liters per minute for 30 minutes under irradiation of light at about 27,000 lux, about 100 μl of 60% perchloric acid and about 50 μCi of a NaH ¹⁴ CO ₃ solution (Amersham, UK) ) Was generated by mixing in a gas tight syringe
¹⁴ CO ₂ was injected into the closed system. After a 5 second pulse, the leaves were frozen in liquid nitrogen to stop bioactivity and immersed in 80% boiling ethanol for about 30 minutes to extract soluble material. The obtained extract was concentrated by an evaporator, and 80% ethanol was added to finally adjust the liquid volume to 50 to 100 µl. This is a thin plate of Funacell SF cellulose (trade name, 20cmX
(20 cm, Funakoshi Co., Ltd.). Phenol-water-acetic acid-0.5M EDTA (474: 84: 5.5: 1.14; V / V) as a one-dimensional developing solvent for development
Is used as a two-dimensional developing solvent in solution A (1-butanol: water = 7
4: 5; V / V) and solution B (propionic acid: water = 9:11; V / V)
Were mixed in equal volumes. The development was performed at room temperature. After the completion of the development, the plate was air-dried, and autoradiography, radioactivity quantification of each spot, and comparison were performed using a bioimage analyzer Bas1000 system (Fuji Photo Industry).

(6) Tracer using [ ¹⁴ C] malic acid
Leaves of transformed rice and control rice (moonlight) grown in an experimental air-conditioned greenhouse were cut in water so that no air could enter through the cut, and then put in 10 mM phosphate buffer (pH 6.4) to about 27,000 lux. Irradiated with light. Depending on the experiment, the respiration inhibitor sodium azide (NaN ₃ ) was added to the buffer to a final concentration of 1 mM prior to light irradiation. One hour later, [ ¹⁴ C] malic acid (Amersham, UK) was added at 1 μCi / 100 μl, and light irradiation was continued. After 15 minutes, the leaf pieces from which the part immersed in the buffer solution was excised were immersed in 80% boiling ethanol to stop the biological activity, and allowed to stand for about 30 minutes to extract soluble substances. In the same way as in the tracer experiment using ¹⁴ CO ₂ , the substance labeled with a radioisotope was quantified,
Compared.

Example 3 Analysis of Transformants The following construction genes: NADP-ME alone; NADP-ME + PPDK (chloroplast type); NADP-ME + PPDK (cytoplasmic type); NADP-ME + corn PEPC, NADP- For ME + PEPC + PPDK (chloroplast type); and NADP-ME + PEPC + PPDK (cytoplasmic type), a large number of transgenic individuals were produced, and the protein expression level of each gene was determined by Western blotting using specific antiserum. Investigation was performed to select individuals in which the target protein was relatively highly expressed. 238 rice plants expressing NADP-ME alone
From the callus, 30 regenerated plants were obtained, and 17 individuals were selected as high-expressing individuals. NADP-ME + PPDK (chloroplast type)
As the expression rice, 27 regenerated plants were obtained from 334 calli, and 7 individuals were selected as high expression individuals. NADP-ME + PPD
As for the rice expressing K (cytoplasmic type), 65 regenerated plants were obtained from 517 calli, and 4 individuals were selected as highly expressing individuals. As for the rice expressing NADP-ME + corn PEPC, 126 regenerated plants were obtained from 2199 calli, and 32 individuals were selected as high expression individuals. NADP-ME + PEPC + PPDK (chloroplast type)
As the expression rice, 159 regenerated plants were obtained from 2562 calli, and 31 individuals were selected as highly expressing individuals. NADP-ME +
PEPC + PPDK (cytoplasmic) -expressing rice is 2 from 2867 calli
49 regenerated plants were obtained, and 27 individuals were selected as high expression plants.

(1) ME, ME + PPDK, ME + cytoplasmic PPDK
As for the rice plants expressing the enzyme activity NADP-ME alone in the green leaves of rice, the progeny (R1 generation) of the individual that showed high expression in the current generation were replaced with NADP-ME + PPDK (chloroplast type), NADP-ME + PPDK With regard to the transformed rice into which (cytoplasmic type) was introduced, the enzymatic activity of the introduced gene was investigated using the transformed generation. Activity measurements of the expressed enzymes in three independent individuals show that the transgenes of both NADP-ME and both chloroplast and cytoplasmic PPDK express the active enzymes. The results were obtained (Table 1).

[0054]

[Table 1] Table 1. Enzyme activity in transformed rice green leaves ------------------------------------------- ------------------------- Enzyme activity (μmol / hour / mg Chl.) ME PPDK ------------ -------------------------------------------------- ------ ME introduced individual 1 14.9 nd ME introduced individual 2 17.4 nd ME introduced individual 3 64.8 nd ME + PPDK introduced individual 1 37.4 29.9 ME + PPDK introduced individual 2 20.0 23.3 ME + PPDK introduced individual 3 11.9 9.73 ME + cytoplasmic PPDK introduced individual 25.5 11.8 ME + cytoplasmic type PPDK introduced individual 2 16.0 4.02 ME + cytoplasmic type PPDK introduced individual 3 59.9 6.05 Non-transformant 7.39 1.20 ------------------------- ------------------------------------------- nd: No measurement

(2) ME + PEPC, ME + PEPC + PPDK, ME + PEPC +
Enzyme activity in rice green leaves into which cytoplasmic PPDK has been introduced 1 ml of extract (50 mM Hepes-KOH (pH 7.
5), 5 mM dithiothreitol, 1 mM EDTA, 10 mM MgCl ₂ ,
1 mM pyruvate, 2 mM phosphate, 20% glycerol, 5 mg /
The mixture was ground with an ice-cooled mortar and pestle using ml ascorbic acid (2% polychloral AT). 15,000 × g of grinding solution, 15 at 4 ° C
After centrifugation for minutes, the supernatant was obtained. Column buffer (50 mM Hep
es-KOH (pH7.5), 5 mM dithiothreitol, 1 mM EDTA,
A crude extract was obtained by passing the supernatant obtained through a NAP5 column (trade name, Pharmacia, Sweden) equilibrated with 10 mM MgCl ₂ , 1 mM pyruvic acid, 2 mM phosphoric acid, 20% glycerol).

The ME activity was determined using a 1 ml reaction solution containing 50 μl of the crude extract (50 mM Tricine-KOH (pH 8.3), 2 mM MgCl ₂ , 0.3 mM
340 nm NADP at 25 ° C using β-NADP, 5 mM malic acid)
It was calculated from the rate of increase of H absorption.

The PEPC activity was measured using 1 ml of a reaction solution containing 25 μl of the crude extract (25 mM Hepes-KOH (pH 8.0), 4 mM dithiothreitol, 5 mM KHCO ₃ , 5 mM MgSO ₄ , 5 mM phosphoenolpyruvate, 1 mM glucose 6 Phosphate, 0.25 mM NADH, 4 units malate dehydrogenase) and 340 nm NADH at 25 ° C
Calculated from the rate of decrease of the light absorption.

The PPDK activity was measured using 1 ml of a reaction solution containing 100 μl of the crude extract (25 mM Hepes-KOH (pH 8.0), 10 mM dithiothreitol, 10 mM KHCO ₃ , 8 mM MgSO ₄ , 1 mM ATP, 1 mM glucose 6-phosphate, 5 mM NH ₄ Cl, 5 mM pyruvate, 2.5 mM KH ₂ P
O ₄ , 0.2 mM NADH, 2 units malate dehydrogenase, 2 units PEP carboxylase) before and after ATP addition.
It was calculated from the rate of decrease in the absorbance of 340 nm NADH at 5 ° C.

The protein in the crude extract was quantified by a protein assay kit (trade name, Bio-Rad, USA)
This was performed using

[0060]

[Table 2] Table 2. Enzyme activity in transformed rice green leaves ------------------------------------------- --------------------------- Enzyme activity (μmol / hour / mg protein) ME PEPC PPDK ---------- -------------------------------------------------- ---------- ME + PEPC introduced individual 1 27.5 19.6 ND ME + PEPC introduced individual 2 5.81 7.15 ND ME + PEPC + PPDK introduced individual 1 14.7 30.5 1.67 ME + PEPC + PPDK introduced individual 2 10.6 22.5 1.55 ME + PEPC + cytoplasmic PPDK introduced individual 1 25.5 31.5 0.209 ME + PEPC + cytoplasm Type PPDK-introduced individual 2 4.03 22.6 0.241 Non-transformed 0.90 2.10 0 ------------------------------------ ---------------------------------- ND: Not measured

Example 4 C4 light in each transformed rice
Confirmation of the driving of synthesis. R1 generation of transgenic rice into which only NADP-ME was introduced, R0 generation of other transgenic rice, and non-transformed rice (moonlight) were given ¹⁴ CO ₂ to the cut leaves. Was stopped and the proportion of labeled C4 compounds was investigated. As a result, in the transformant, the C4 compound (malic acid + aspartic acid)
Occupied 11 to 22%, which was 5 to 13 times higher than that of non-transformed rice (Table 3-1). This indicates that the transformed rice is efficient in fixing CO ₂ in the atmosphere as a C4 compound.

[ ^14C ] Malic acid was given to the cut leaves of the transformed rice R1 generation into which only NADP-ME was introduced, the R0 generation of another transformed rice, and the untransformed rice (moonlight) for 15 minutes. Later, the biological reaction of the tissue was stopped and the proportion of labeled substance was investigated. As a result, the ratio of photosynthetic metabolites was 2 to 6 times higher in the transformed rice than in the non-transformed rice (Table 3-2). Even when respiratory inhibitors are added,
The above ratio was 2 to 13 times higher than that of the untransformed rice (Table 3-3). This indicates that transformed rice can efficiently metabolize malic acid to photosynthetic products without depending on respiration.

When the above experimental results are combined, in the transformed rice, both the initial carbon fixation reaction and the decarboxylation fixation reaction of the C4 photosynthesis circuit are functioning, and it is determined that the C4 photosynthesis circuit is provided. Was.

[0064]

[Table 3]

[0065]

Industrial Applicability According to the method of the present invention, in a C3 plant, malic acid can be decarboxylated in chloroplasts by expression of NADP-ME to increase the CO ₂ concentration in chloroplasts and improve photosynthetic ability. Become. In addition, NADP-M
Pyruvic acid generated by E can be converted to phosphoenolpyruvate (PEP), a substrate of PEPC, by consuming ATP. By promoting the supply of PEP to PEPC, in addition to driving C4 photosynthesis more efficiently, it is expected that C3 plants having a function to avoid photodamage due to ATP consumption can be produced. In addition, the expression of PEPC can enhance the initial carbon fixation reaction of C4 photosynthesis, and can increase the driving force of the C4 photosynthesis circuit. Therefore, by driving the C4 photosynthesis circuit in the C3 plant, the C4 photosynthesis ability, the dry matter productivity, the drought tolerance ability, the strong light tolerance ability, and the photosynthesis ability under low CO ₂ conditions can be improved. The excellent traits of the plant can be imparted to the C3 plant.

[0066]

[Sequence list]

[0067] SEQ ID NO: 1 sequence Length: 930 SEQ types: the number of nucleic acid strands: double-stranded Topology: linear sequence type: Genomic DNA Origin: Organism Name: thia Mace (Zea mays) cultivars Name: features of inbred B73 sequence: nature: C4 type PPDK partial sequence of the gene promoter region CTAAAGACAT GGAGGTGGAA GGCCTGACGT AGATAGAGAA GATGCTCTTA GCTTTCATTG 60 TCTTTCTTTT GTAGTCATCT GATTTACCTC TCTCGTTTAT ACAACTGGTT TTTTAAACAC 120 TCCTTAACTT TTCAAATTGT CTCTTTCTTT ACCCTAGACT AGATAATTTT AATGGTGATT 180 TTGCTAATGT GGCGCCATGT TAGATAGAGG TAAAATGAAC TAGTTAAAAG CTCAGAGTGA 240 TAAATCAGGC TCTCAAAAAT TCATAAACTG TTTTTTAAAT ATCCAAATAT TTTTACATGG 300 AAAATAATAA AATTTAGTTT AGTATTAAAA AATTCAGTTG AATATAGTTT TGTCTTCAAA 360 AATTATGAAA CTGATCTTAA TTATTTTTCC TTAAAACCGT GCTCTATCTT TGATGTCTAG 420 TTTGAGACGA TTATATAATT TTTTTTGTGC TTACTACGAC GAGCTGAAGT ACGTAGAAAT 480 ACTAGTGGAG TCGTGCCGCG TGTGCCTGTA GCCACTCGTA CGCTACAGCC CAAGCGCTAG 540 AGCCCAAGA G GCCGGAGTGG AAGGCGTCGC GGCACTATAG CCACTCGCCG CAAGAGCCCA 600 AGAGACCGGA GCTGGAAGGA TGAGGGTCTG GGTGTTCACG AATTGCCTGG AGGCAGGAGG 660 CTCGTCGTCC GGAGCACAGG CGTGGAGAAC GTCCGGGATA AGGTGAGCAG CCGCTGCGAT 720 AGGCGCGTGT GAACCCCGTC GCGCCCCACG GATGGTATAA GAATAAAGGC ATTCCGCGTG 780 CAGGATTCAC CCGTTCGCCT CTCACCTTTT CGCTGTACTC ACTCGCCACA CACACCCCCT 840 CTCCAGCTCC GTTGGAGCTC CGGACAGCAG CAGGCGCGGG GCGGTCACGT AGTAAGCAGC 900 TCTCGGCTCC CTCTCCCCTT GCTCCATATG 930

SEQ ID NO: 2 Sequence length: 697 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin: Biological name: Spinatia oleracea (Spinacia oleracea) Characteristics: Property: mRNA sequence of carbonic anhydrase from which the region encoding the transit peptide has been removed CC ATG GAG TTA GCC GAC GGT GGC ACA CCA TCC GCC AGT TAC CCG GTT 47 Met Glu Leu Ala Asp Gly Gly Thr Pro Ser Ala Ser Tyr Pro Val 5 10 15 CAG AGA ATT AAG GAA GGG TTT ATC AAA TTC AAG AAG GAG AAA TAC GAG 95 Gln Arg Ile Lys Glu Gly Phe Ile Lys Phe Lys Lys Glu Lys Tyr Glu 20 25 30 AAA AAT CCA GCA TTG TAT GGT GAG CTT TCT AAG GGC CAA GCT CCC AAG 143 Lys Asn Pro Ala Leu Tyr Gly Glu Leu Ser Lys Gly Gln Ala Pro Lys 35 40 45 TTT ATG GTG TTT GCG TGC TCA GAC TCC CGT GTG TGT CCC TCG CAC GTA 191 Phe Met Val Phe Ala Cys Ser Asp Ser Arg Val Cys Pro Ser His Val 50 55 60 CTA GAT TTC CAG CCC GGT GAG GCT TTC ATG G TT CGC AAC ATC GCC AAC 239 Leu Asp Phe Gln Pro Gly Glu Ala Phe Met Val Arg Asn Ile Ala Asn 65 70 75 ATG GTG CCA GTG TTT GAC AAG GAC AAA TAC GCT GGA GTC GGA GCA GCC 287 Met Val Pro Val Phe Asp Lys Asp Lys Tyr Ala Gly Val Gly Ala Ala 80 85 90 95 ATT GAA TAC GCA GTG TTG CAC CTT AAG GTG GAG AAC ATT GTC GTG ATT 335 Ile Glu Tyr Ala Val Leu His Leu Lys Val Glu Asn Ile Val Val Ile 100 105 110 GGA CAC AGT GCT TGT GGT GGA ATC AAG GGG CTT ATG TCT TCT CCA GAT 383 Gly His Ser Ala Cys Gly Gly Ile Lys Gly Leu Met Ser Ser Pro Asp 115 120 125 GCA GGA CCA ACC ACA ACT GAT TTT ATT GAG GAT TGG GTC AAA ATC TGC 431 Ala Gly Pro Thr Thr Thr Asp Phe Ile Glu Asp Trp Val Lys Ile Cys 130 135 140 TTG CCT GCC AAG CAC AAG GTG TTA GCC GAG CAT GGT AAT GCA ACT TTC 479 Leu Pro Ala Lys His Lys Val Leu Ala Glu His Gly Asn Ala Thr Phe 145 150 155 GCT GAA CAA TGC ACC CAT TGT GAA AAG GAA GCT GTG AAT GTA TCT CTT 527 Ala Glu Gln Cys Thr His Cys Glu Lys Glu Ala Val Asn Val Ser Leu 160 165 170 170 175 GGA AAC TTG TTG ACT TAC TAC CCA TTT GTA AGA GAT GGT TTG GTG AAG AAG 575 Gly Asn Leu Leu Thr Tyr Pro Phe Val Arg Asp Gly Leu Val Lys Lys 180 185 190 ACT CTA GCT TTG CAG GGT GGT TAC TAC GAT TTT GTC AAT GGA TCA TTC 623 Thr Leu Ala Leu Gln Gly Gly Tyr Tyr Asp Phe Val Asn Gly Ser Phe 195 200 205 GAG CTA TGG GGA CTC GAA TTC GGC CTC TCT CCT TCC CAA TCT GTA 668 Glu Leu Trp Gly Leu Glu Phe Gly Leu Ser Pro Ser Gln Ser Val 210 215 220 TGAACCAACA CAACCATTTG ACTGCATGC 697

SEQ ID NO: 3 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GCAAGCTTTT ACTTGTACCA ACTAATATAA TGAGTG 36

SEQ ID NO: 4 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence TGGATCCTCT AGAGTACTTC TTGAGATGCA CTGCTC 36

SEQ ID NO: 5 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence TACTTACGTG GCCTTCTTCC TCCG 24

SEQ ID NO: 6 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GGTTTGGCTG CTGGATTCAA AGGG 24

SEQ ID NO: 7 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GTCGACCATA TGCTGTCCAC GCGCACCGCC 30

SEQ ID NO: 8 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence CAAAAGGGTG TAACCGCTTG C 21

SEQ ID NO: 9 Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence AGCCTACGAG CTCGGTCTG 19

SEQ ID NO: 10 Sequence length: 30 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence CCCGGGCATG CGCAAAATTG ATCCCCGCAG 30

SEQ ID NO: 11 Sequence length: 35 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: synthetic DNA sequence TAGCTCGATG GGTTGCACGA TCATATGGAG CAAGG 35

SEQ ID NO: 12 Sequence length: 56 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence TTTCATATGG CGCCCGTTCA ATGTGCGCGT TCGCAGAGGG TGTTCCACTT CGGCAA 56

SEQ ID NO: 13 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GTACTCCTCC ACCCACTGCA 20

SEQ ID NO: 14 Sequence length: 35 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GCCATGGCGC GGCGGGAAGC TAAGCACGGA AGCGA 35

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the isolation and construction of corn NADP-ME cDNA.

FIG. 2 is a schematic diagram showing a constructed gene used for transformation.

FIG. 3 is a schematic diagram showing a photosynthetic circuit in transformed rice.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 9/88 C12N 15/00 ZNAA (C12N 5/10 C12R 1:91) (72) Inventor Keisuke Kasaoka Toyotamachi Higashi, Iwata-gun, Shizuoka Prefecture 700 Hara Japan Tobacco Inc. Genetics Breeding Research Institute

Claims (7)

[Claims] 1. a gene encoding phosphoenolpyruvate carboxylase (PEPC); a gene encoding NADP-malic enzyme (NADP-ME);
By introducing a gene encoding pyruvate phosphate dikinase (PPDK) into a C3 plant,
4. A method for transforming a C3 plant that imparts a photosynthetic circuit. 2. The method according to claim 1, wherein the NADP-ME gene is introduced into a C3 plant and expressed in chloroplasts. 3. The NADP-ME gene is a chloroplast NA
The transformation method according to claim 1 or 2, which is a DP-ME gene. 4. The method according to claim 1, wherein the promoter sequence used for the transformation is a promoter sequence for specifically expressing a gene in a photosynthetic organ. 5. The method according to any one of claims 1 to 4, wherein two or more or all of the genes are ligated on the same construction gene for gene introduction, and the gene is introduced into a C3 plant. Transformation method. 6. A gene encoding phosphoenolpyruvate carboxylase (PEPC), a gene encoding NADP-malic enzyme (NADP-ME),
A chimeric gene linked to a gene encoding pyruvate phosphate dikinase (PPDK). 7. A transformed plant transformed by the transformation method according to any one of claims 1 to 5 and provided with a C4 photosynthesis circuit.