PCK-type C4 cycle

Abstract

The present invention relates a method for transforming a C3 plant to provide it with a C4 photoshynthetic pathway by way of the introduction of some genes participating in a C4 photosynthetic pathway. To this end, the method of the invention comprises introducing a phosphoenolpyruvate carboxylase (PEPC) and a gene coding for a phosphoenolpyruvate carboxykinase (PCK) which has been connected with a DNA fragment coding for a transit peptide into a C3 plant.

Description

[0001] The present invention relates to a process for transforming a C3 plant to provide it with a C4 cycle by introducing two or more enzymes which participate in the C4 photosynthetic pathway.PRIOR ART

[0002] Three types of photosynthetic pathway are known in higher plants, namely, C3, C4 and CAM types. Leaf tissues of plants having a C4 type photosynthetic pathway (hereafter sometimes referred to as C4 plants) comprise mesophyll cells and bundle sheath cells existing around fibro vascular bundles, forming the specific leaf tissue structure called Kranz-type anatomy. C4 plants fix carbon dioxide into a C4 compound by the action of a phosphoenolpyruvate carboxylase (hereafter sometimes referred to as PEPC) located in the cytoplasm of mesophyll cells. The fixed carbon dioxide is released by decarboxylase in bundle sheath cells, which increases the level of carbon dioxide in the vicinity of ribulose-1,5-biphosphate carboxylase / oxygenase (hereafter sometimes referred to as Rubisco) whi...

Examples

example 1

Construction of Transgenes

[0047] (1) Promoter sequence

[0048] The DNA fragment of the corn C4 type PPDK promoter region was obtained by the PCR method (Mcpherson, M. J., Quirke, P. and Taylor, GR. ed.: PCR. A practical approach, Oxford Express Press, Oxford NY (1991)) with the use of the following two synthetic primers prepared on the basis of a known nucleotide sequence (Glackin, C. A. and Grula, J. W. (1990) Proc. Natl. Acad. Sci. USA 87:3004-3008):

1 5'-CTAAAGACATGGAGGTGGAAG-3' (5' side) (SEQ ID NO:6) 5'-GTAGCTCGATGGGTGCACG-3'. (3' side) (SEQ ID NO:7)

[0049] The amplification was carried out by using maize genomic DNA as the template, which had been obtained by extracting total nucleic acids from a maize inbred B73 green leaf by the SDS-phenol method followed by purification by cesium chloride-ethidium bromide ultracentrifugation. The DNA fragment thus obtained was inserted into the cloning site of a plasmid vector pCR1000 (manufactured by Invitrogen, USA). The plasmid thus obtained...

example 2

Construction of Transformants

[0082] A Japonica rice (cultivar "Tsukinohikari") was used throughout the transformation study of rice.

[0083] Rice transformants having pDPN, pDKS and pDCS introduced thereinto were constructed by the electroporation method previously described (Japanese Patent Public Disclosure Hei 8-80197).

[0084] Rice transformants having pSB4PK, pSB4CPK and pSB4CiPiDiKi introduced thereinto were constructed by the Agrobacterium method reported described (Hiei, Y. et al. (1994) Plant J. 6:271-282).

[0085] These transformants were grown in an air-conditioned green house (daylight period: 16 hours, daytime: 28.degree. C., nighttime: 23.degree. C.).

example 3

Detection of Enzyme Proteins and Measurement of Enzymatic Activities

[0086] About 0.1 g of the green leaves of the transformants or control rice ("Tsukinohikari") was homogenized in 1 ml of an ice-cold extraction buffer (50 mM HEPES-KOH pH 7.0, 10 mM magnesium chloride, 2 mM manganese chloride, 1 mM sodium pyruvate, 1 mM phosphoric acid, 1 mM EDTA, 0.1% 2-mercaptoethanol, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM 6-amino-n-caproic acid, 0.2% (w / w) isoascorbic acid, and 2% (w / v) polyclar AT). The homogenate was centrifuged at 15,000.times.g for 20 minutes at 4.degree. C. Then the obtained supernatant was desalted by passing through an NAP5.TM. column (manufactured by Pharmacia, Sweden) which had been equilibrated at room temperature with a column buffer (50 mM HEPES-KOH pH 7.0, 10 mM magnesium chloride, 2 mM manganese chloride, 1 mM EDTA, 0.1% 2-mercaptoethanol, and 20% glycerol) to thereby give a crude extract. The chlorophyll content in the homogenate ...

[0001] The present invention relates to a process for transforming a C3 plant to provide it with a C4 cycle by introducing two or more enzymes which participate in the C4 photosynthetic pathway.PRIOR ART

[0002] Three types of photosynthetic pathway are known in higher plants, namely, C3, C4 and CAM types. Leaf tissues of plants having a C4 type photosynthetic pathway (hereafter sometimes referred to as C4 plants) comprise mesophyll cells and bundle sheath cells existing around fibro vascular bundles, forming the specific leaf tissue structure called Kranz-type anatomy. C4 plants fix carbon dioxide into a C4 compound by the action of a phosphoenolpyruvate carboxylase (hereafter sometimes referred to as PEPC) located in the cytoplasm of mesophyll cells. The fixed carbon dioxide is released by decarboxylase in bundle sheath cells, which increases the level of carbon dioxide in the vicinity of ribulose-1,5-biphosphate carboxylase / oxygenase (hereafter sometimes referred to as Rubisco) whi...

Examples

example 1

Construction of Transgenes

[0047] (1) Promoter sequence

[0048] The DNA fragment of the corn C4 type PPDK promoter region was obtained by the PCR method (Mcpherson, M. J., Quirke, P. and Taylor, GR. ed.: PCR. A practical approach, Oxford Express Press, Oxford NY (1991)) with the use of the following two synthetic primers prepared on the basis of a known nucleotide sequence (Glackin, C. A. and Grula, J. W. (1990) Proc. Natl. Acad. Sci. USA 87:3004-3008):

1 5'-CTAAAGACATGGAGGTGGAAG-3' (5' side) (SEQ ID NO:6) 5'-GTAGCTCGATGGGTGCACG-3'. (3' side) (SEQ ID NO:7)

[0049] The amplification was carried out by using maize genomic DNA as the template, which had been obtained by extracting total nucleic acids from a maize inbred B73 green leaf by the SDS-phenol method followed by purification by cesium chloride-ethidium bromide ultracentrifugation. The DNA fragment thus obtained was inserted into the cloning site of a plasmid vector pCR1000 (manufactured by Invitrogen, USA). The plasmid thus obtained...

example 2

Construction of Transformants

[0082] A Japonica rice (cultivar "Tsukinohikari") was used throughout the transformation study of rice.

[0083] Rice transformants having pDPN, pDKS and pDCS introduced thereinto were constructed by the electroporation method previously described (Japanese Patent Public Disclosure Hei 8-80197).

[0084] Rice transformants having pSB4PK, pSB4CPK and pSB4CiPiDiKi introduced thereinto were constructed by the Agrobacterium method reported described (Hiei, Y. et al. (1994) Plant J. 6:271-282).

[0085] These transformants were grown in an air-conditioned green house (daylight period: 16 hours, daytime: 28.degree. C., nighttime: 23.degree. C.).

example 3

Detection of Enzyme Proteins and Measurement of Enzymatic Activities

[0086] About 0.1 g of the green leaves of the transformants or control rice ("Tsukinohikari") was homogenized in 1 ml of an ice-cold extraction buffer (50 mM HEPES-KOH pH 7.0, 10 mM magnesium chloride, 2 mM manganese chloride, 1 mM sodium pyruvate, 1 mM phosphoric acid, 1 mM EDTA, 0.1% 2-mercaptoethanol, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM 6-amino-n-caproic acid, 0.2% (w / w) isoascorbic acid, and 2% (w / v) polyclar AT). The homogenate was centrifuged at 15,000.times.g for 20 minutes at 4.degree. C. Then the obtained supernatant was desalted by passing through an NAP5.TM. column (manufactured by Pharmacia, Sweden) which had been equilibrated at room temperature with a column buffer (50 mM HEPES-KOH pH 7.0, 10 mM magnesium chloride, 2 mM manganese chloride, 1 mM EDTA, 0.1% 2-mercaptoethanol, and 20% glycerol) to thereby give a crude extract. The chlorophyll content in the homogenate ...

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