JP2001512685A - Methods for increasing yield in plants - Google Patents

Abstract

(57) SUMMARY Disclosed is a method for increasing yield in a plant, wherein the nucleotide sequence is expressed under the control of a companion cell-specific promoter. The nucleotide sequence encodes a protein whose expression triggers the stimulation of photoabsorptive loading on the phloem.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for increasing the yield in plants, the recombinant nucleic acid molecules used in these methods, their use and to plants with increased yield.

In the field of agriculture and forestry, in particular, plants with increased yields, in order to ensure the supply of food to the ever-increasing world population and to ensure the supply of renewable raw materials Efforts are constantly being made to produce Usually, breeding attempts to obtain plants with increased yield, but this requires a lot of time and effort. In addition, appropriate breeding programs need to be implemented for each relevant plant species. Partial progress has been made by genetic engineering of plants, ie, by introducing and expressing recombinant nucleic acid molecules in plants.
Such an approach has the advantage that it is usually not limited to one plant species but can be transferred to another plant species. For example, EP-A0511979 discloses that the expression of prokaryotic asparagine synthetase in plant cells causes, inter alia, increased biomass production. For example, WO 96/21737 discloses the production of plants with increased yields by increasing the rate of photosynthesis due to the expression of deregulated or unregulated fructose-1,6-bisphosphatase. . Nevertheless, there is still a need for generally applicable methods for improving yield in plants that are of interest to agriculture or forestry.

[0003] The problem underlying the present invention is therefore to provide further methods for increasing the yield in plants. This problem is solved by the present invention by providing the embodiments characterized in the claims.

[0004] Accordingly, the present invention provides (a) a region specifically enabling transcription in a companion cell, and (b) (i) a protein having an enzymatic activity for cleaving sucrose. (Ii) a sucrose transporter, (iii) a protein having an activity to stimulate a proton gradient located on the plasma membrane of a plant cell, and (iv) citrate synthase (EC4.1.3.7.). Comprising a nucleotide sequence encoding the selected polypeptide,
The present invention relates to a method for increasing the yield in a plant, characterized by expressing a recombinant DNA molecule stably integrated into the genome of the plant.

[0005] Surprisingly, the expression of said protein specifically in the phloem of the plant,
A dramatic increase in yield was found to be caused.

The term “increased yield” preferably relates to increased biomass production, especially as determined as freshness of the plant. Such an increase in yield is preferably
It is related to the so-called "sink" organs of plants, which are the organs that take up the photo-assimilates produced during photosynthesis. Particularly preferred are seeds, fruits, storage roots, roots, tubers, flowers,
Harvestable plant parts, such as buds, shoots, stems or xylem. The increase in yield according to the invention, when grown under the same conditions, is at least 3%, preferably 10%, particularly preferably at least 20% relative to the biomass compared to a non-transformed plant of the same genotype. It is.

[0007] The proteins have in common that when expressed in the phloem, their biological activity causes increased loading of the photoabsorptive on the phloem. In the context of the present invention, photoassimilates are to be understood as sugars and / or amino acids.

According to the invention, the nucleotide sequence referred to in (b) can usually encode a plant protein or a bacterial protein or a fungal or animal derived protein.

In a preferred embodiment, the nucleotide sequence is a sucrose synthase (EC
2.4.1.13), preferably encodes a plant sucrose synthase, especially a sucrose synthase from Solanum tuberosum, particularly preferably a form expressed in the tubers of S. tuberosum. Such arrays are, for example, Salanoubat and Belliard (Ge
ne 60 (1987), 47-56) and has accession number X67125 at EMBL Gene Bank.
Available at

[0010] In a further preferred embodiment, the nucleotide sequence encodes sucrose phosphorylase (EC 2.4.1.7). The sequence coding for sucrose phosphorylase is known, for example, from WO 96/24679.

[0011] In another preferred embodiment, the nucleotide sequence is an invertase (E.
C.3.2.1.26), preferably encoding an invertase from a microorganism, especially from a fungus of the genus Saccharomyces, preferably from S. cerevisiae. Cytosolic invertase (Sonnewa
ld et al., Plant J. 1 (1991), 95-106) are particularly preferred.

According to the invention, a sucrose transporter is understood as a transporter that transports sucrose across membranes in plant systems. Such transporters are
Preferably, it is derived from a plant (for example, EMBL gene bank registration number G21319). Particularly preferably, the sequence described in (b) is particularly useful, for example, in Riesmeier et al (EMBO
J. 11 (1992), 4705-4713) encodes a sucrose transporter from spinach (Spinacia oleracea) having the sequence of clone SoSUT1.

[0013] In a particularly preferred embodiment, the protein that stimulates a proton gradient located at the plasma membrane is a proton ATPase. In this case, the sequence described in (b) preferably encodes a protein from a microorganism, in particular from a fungus of the genus Saccharomyces, preferably from S. cerevisiae. In a particularly preferred embodiment, the sequence is a proton ATPase PMA1 from S. cerevisiae (Serrano et al., Nature 319 (1986), 689-693; EMBL
gene bank) or the 3′-terminal truncated form of the proton ATPase derived from S. cerevisiae, particularly the ATPase ΔPAM1 described in Example 3 of the present invention.

[0014] Alternatively, the nucleotide sequence may encode a proton ATPase from a plant, preferably a proton ATPase from Solanum tuberosa.

Particularly preferred is the proton ATPase PHA2 from potato (Harms et al., Pla
Biol. 26 (1994), 979-988; EMBL Gene Bank X76535) or the 3′-terminal truncated form of this potato-derived proton ATPase, particularly the ATPase ΔPHA described in Example 4 of the present invention.
An array that codes 2.

According to the present invention, the citrate synthase can be any citrate synthase, for example, a citrate synthase from a bacterium, fungus, animal or plant. For example, the following organisms: Bacillus subtilis (U05256 and U05257), Escherichia coli (V01501), R. prowazekii (M17149), P. aeruginosa (M29728)
), A. anitratum (M33037) (Schendel et al., Appl. Enviro
n. Microbiol. 58 (1992), 335-345 and the references cited therein),
Haloferax volcanii (James et al., Biochem. S
oc.Trans, 20 (1992), 12), Arabidopsis thaliana
(Z17455) (Unger et al., Plant Mol. Biol. 13 (1989), 411-418), B. coagulans (M74818), C. burnetti (M36338) (H
einzen et al., Gene 109 (1990), 63-69), M. smegmatis (X60
513), T. acidophilum (X55282), T. thermophila (T. th
ermophila) (D90117), pig (M21197) (Bloxham et al., Proc. Natl. Acad. Sci.
USA 78 (1981), 5381-5385), N. crassa (M84187) (Ferea et al
., Mol. Gen. Genet. 242 (1994), 105-110), S. cerevisiae (Z11113, Z23259, M1468)
6, M54982, X00782) (Suissa et al., EMBO J. 3 (1984), 1773-1781) and DNA sequences encoding citrate synthase from potato (EP95913066.7) are known.

The numbers in parentheses are the corresponding registration numbers in the GenEMBL database.

[0018] The nucleotide sequence according to the present invention generally refers to any organism, especially plants, fungi,
It may encode any suitable protein from bacteria or animals. The sequence preferably encodes a protein from a plant or a fungus. Preferably, the plants are higher plants, especially useful plants that store starch or oils and fats, such as potatoes or rice, corn, wheat, barley, rye, triticale, oats, millet and the like, spinach, tobacco, sugar beet, Soybeans, cotton, etc. The fungi are preferably fungi of the genera Saccharomyces, Schizosaccharomyces, Aspergillus or Neurospora, in particular Saccharomyces cerevisiae, Saccharomyces pombe spellae, Saccharomyces pombe sp. ), Aspergillus nigar or Neurospora crassa.

In a preferred embodiment of the method according to the invention, the region referred to in (a) which ensures companion cell-specific transcription is Agrobacterium rhizogenes.
This is the promoter of the rolC gene derived from Rizogenes. This promoter is described, for example, in Schmulling et al. (Plant Cell (1989), 665-671) and Kuhn (Charac
terization and localization of the sucrose carrier SUT1 in Solanaceae, Do
ctoral Thesis (1991), Freie Universitat Berlin, biology department). Preferably, a region of the promoter having the nucleotide sequence set forth in SEQ ID NO: 1 is used.

In addition to the rolC promoter described above, one skilled in the art can use other promoters for companion cell-specific expression without undue experimentation. Arabidopsis thaliana sucrose transporter promoter (Truernit and Sauer, Plant
Further companion cell-specific promoters have been disclosed in the literature, such as a 196 (1995), 564-570).

In addition, their specific presence in companion cells for different RNAs and proteins has been disclosed in the literature (eg Foley et al., Plant Mol. Biol. 30 (1
996), 687-695; DeWitt, Plant J. 1 (1991), 121-128; Stadler et al., Plant Cell 7 (
1995), 1545-1554). One of skill in the art can isolate a cDNA, starting from a known protein, without undue experimentation, by antibodies or by using oligonucleotides derived from amino acid sequences (eg, Sambrook et al.). , Molec
ular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (198
9), Cold Spring Harbor, NY.). Starting from the cDNA thus obtained, it is possible to further screen established genomic libraries from the corresponding organism and identify genomic fragments. By comparing the nucleotide sequence of the cDNA with the nucleotide sequence of the genomic clone, the location of the promoter can be roughly determined. Promoter specificity can be confirmed in a transgenic environment by using a chimeric gene consisting of the promoter and an indicator gene such as β-glucuronidase (eg, Kertbund).
USA et al., Proc. Natl. Acad. Sci. USA 88 (1991), 5212-5216).

The method according to the invention can in principle be applied to any plant. Accordingly, monocot and dicot species are particularly preferred. The method is preferably used with plants that are of interest for agriculture, horticulture and / or forestry. Examples are cucumber, melon, pumpkin, eggplant, zucchini, tomato, spinach, cabbage,
Vegetable plants such as peas, kidney beans and the like and fruits such as pears and apples. Furthermore, plants storing oils and fats, such as rapeseed, sunflower, and soybean, are suitable. In particularly preferred embodiments, plants that store starch, such as cereals (rice, corn, wheat, rye, oats, triticale, millet, barley), potatoes, tapiocanoki, sweet potatoes, and the like, are preferred. The method can be applied to plants that store sucrose, such as, for example, sugar beet and sugarcane, or to other useful plants, such as, for example, cotton, tobacco, wood molds, wine, hops, and the like.

The present invention further provides (a) a region that specifically enables transcription in a companion cell of a plant, and (b) (i) sucrose synthase, (ii) sucrose which is operably linked thereto. A phosphorylase, (iii) a sucrose transporter, (iv) a protein having an activity of causing stimulation of a proton gradient located in the plasma membrane of a plant cell, and (v) a polypeptide selected from the group consisting of: citrate synthase And a recombinant nucleic acid molecule comprising the nucleotide sequence of

With respect to preferred embodiments of such molecules, the same as described above in connection with the method of the invention applies to the region referred to in (a) and to the nucleotide sequence referred to in (b).

The present invention also relates to vectors containing the recombinant nucleic acid molecules of the invention, in particular vectors suitable for the transformation of plant cells and vectors suitable for the integration of foreign DNA into the plant genome.

The present invention further relates to plant cells which have been transformed with the nucleic acid molecules of the invention and which have stably integrated into the genome. These cells differ from naturally occurring plant cells in that the nucleic acid molecules of the invention are integrated into the genome of the cell at a non-naturally occurring location.

The present invention further provides for the expression of a recombinant nucleic acid molecule integrated into the genome in a companion cell of a plant comprising a plant cell of the present invention and comparing it to a corresponding untransformed plant grown under the same conditions. And transgenic plants that exhibit increased yields.

The present invention further relates to a propagation material of the plant of the present invention comprising the above-mentioned plant cell of the present invention. The term "breeding material" refers, in particular, to seeds, nuts, tubers, rhizomes, cuttings, potash (ca).
lli), cell cultures and the like.

Finally, the present invention relates to a region which specifically enables transcription in a companion cell of a plant, and a protein having an enzymatic activity for cleaving sucrose, which is operatively linked thereto, (ii) A) a sucrose transporter; (iii) a protein having an activity to cause stimulation of a proton gradient located at the plasma membrane; and (iv) a nucleotide sequence encoding a polypeptide selected from the group consisting of: citrate synthase. The present invention relates to the use of nucleic acid molecules for expression in transgenic plants to increase the yield.

[0030] Methods for the transformation of monocots and dicots are known to those skilled in the art.

Various techniques can be used for introducing DNA into a plant host cell. These techniques include Agrobacterium tumefaciens (Ag
r-bacterium tumefaciens) or T-DNA using Agrobacterium rhizogenes
Transformation of plant cells, fusion of protoplasts, injection, electroporation of DNA, introduction of DNA by biolistic methods and further possibilities.

For DNA injection and electroporation in plant cells, the plasmid need not meet certain requirements. Simple plasmids such as pUC derivatives can be used. The use of Agrobacteria for the transformation of plant cells
It has been extensively reviewed and described in EP-A120516, Hoekema (In: The Bi
nary Plant Vector System Offsetdrukkerij Kanters BV, Alblasserdam (1985)
, Chapter V), Fraley et al. (Crit. Rev. Plant. Sci. 4, 1-46) and An et al. (EMBO J. 4 (1985),
277-287).

With respect to the transfer of DNA into plant cells, plant explants
Can be co-cultured with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From infected plant material (eg, leaf explants, stem segments, roots, as well as protoplasts or suspension-cultured plant cells), containing antibiotics or biozides for selection of transformed cells Whole plants can be regenerated on any suitable medium. The plants thus obtained can then be tested for the presence of the introduced DNA. Other possibilities for the introduction of foreign DNA using the biolistic method or protoplast transformation are known (eg, W
illmitzer, L., 1993 Transgenic plants.In:Biotechnology,A Multi-Volume Comp
rehensive Treatise (HJ Rehm, G. Reed, A. Puhler, P. Stadler) Vol. 2, 627-659, VC
H Weinheim-New York-Basel-Cambridge).

[0034] Transformation of dicotyledonous plants with the Ti plasmid-vector system assisted by Agrobacterium tumefaciens is well established. Recent studies have suggested that monocotyledonous plants can also be transformed with Agrobacterium-based vectors (Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et
al., Plant J. 6 (1994), 271-282; Deng et al., Science in China 33 (1990), 28-34;
Wilmink et al., Plant Cell Reports 11 (1992), 76-80; May et al., Bio / Technolo
gy 13 (1995), 486-492; Conner and Domisse; Int.J. Plant Sci. 153 (1992), 550-555
Ritchie et al., Transgenic Res. 2 (1993), 252-265).

Another system for the transformation of monocotyledonous plants is the transformation by the biolistic method (Wan and Lemaux, Plant Physiol. 104 (1994), 37-48; Vasil et al., Bio / Technolo).
gy 11 (1993), 1553-1558; Ritala et al., Plant Mol. Biol. 24 (1994), 317-325; Spen
cer et al., Theor. Appl. Genet. 79 (1990), 625-631), protoplast transformation,
Electroporation of partially permeabilized cells and introduction of DNA by glass fiber.

In particular, transformation of corn has been described several times in the literature (eg WO 95/0
6128, EP 0 513 849; EP 0 465 875; Fromm et al., Biotechnology 8 (1990), 833-8
44; Gordon-Kamm et al., Plant Cell 2 (1990), 603-618; Koziel et al., Biotechno
logy 11 (1993), 194-200). EP292435 and Shillito et al. (Bio / Technology 7 (1989),
No. 581) discloses a process by which a torsionous plant can be obtained starting from a soft (friable) corn callus free of mucus. Prioli and Sondahl (B
io / Technology 7 (1989), 589) discloses the regeneration and acquisition of torsion plants from corn protoplasts of the Catate maize inbred line Cat100-1.

The success of transformation of other cereal species has also been described, for example, in barley (Wan and Lemaux, see above; Rit.
ala et al., supra) and wheat (Nehra et al., Plant J. 5 (1994), 285-297).

After the introduced DNA is integrated into the genome of the plant cell, it is usually stable there, and is also included in the progeny of the originally transformed cell. It usually contains a selectable marker that renders the transformed plant cells resistant to biocides or antibiotics such as kanamycin, G418, bleomycin, hygromycin or phosphinotricin. Thus, individually selected markers are:
Allows selection of transformed cells from cells that do not contain the introduced DNA.

The transformed cells grow in plants in the usual way (McCormick et al., P.
lant Cell Reports 5 (1986), 81-84). The obtained plant can be cultured by a usual method. Seeds can be obtained from plants. More than one generation should be cultivated to ensure that phenotypic characteristics are maintained and transmitted stably. Seeds should be harvested to ensure that the corresponding phenotypic qualities or properties therefor are maintained.

Hereinafter, the present invention will be described with reference to examples.

Example 1 Preparation of Plasmid pBinRolC-SS and Preparation of Transgenic Potato Plant The plasmid pBinRolC-SS was prepared using the binary vector pBin19 (Bevan, Nucl.
s.12 (1984), 8711) contains three fragments A, B and C (see FIG. 1).

Fragment A contains the rolC promoter from Agrobacterium rhizogenes. r
The olC promoter is a 1138 bp EcoRI / Asp718 DNA fragment (Lerchl et al., Plant Cell
7 (1995), 259-270) TL-DN of Ri agropine-type plasmid derived from A. rhizogenes
A DNA region (positions 11306 to 12432) (Slightom et al., J. Biol. Chem.
261 (1986), 108-121). Fragment A was constructed with the EcoRI cleavage site of the polylinker of pBin19 and Asp
Insert at 718 cleavage site. Fragment B contains the coding region of the cDNA for sucrose synthase (SS) from Solanum tuberosum (positions 76 to 2493) (Salanoub
at and Belliard, Gene 60 (1987), 47-56).

Fragment B is the vector pBluescrip inserted into the BamHI cleavage site of the polylinker.
tSK ^- was obtained as a BamHI fragment of 2427bp from. Fragment B was ligated in the sense orientation to the BamHI cleavage site of vector pBin19, i.
It was inserted downstream of the olC promoter.

Fragment C was used for the gene 3 polyadenylation signal of T-DNA of Ti plasmid pTiACH5 (Gi
elen et al., EMBO J. 3 (1984), 835-846), especially plasmid pAGV40 (Herrera-Estr
ella et al., Nature 303 (1983), 209-213) and isolated by addition of a Sphl linker to the SphI cleavage site of the polylinker of pBin19 and Hi
Nucleotides 11749-1 cloned into the PvuII cleavage site between the ndIII cleavage site
Including 1939.

The resulting plasmid pBinRolC-SS was introduced into potato plant cells by Agrobacterium tumefaciens-mediated gene transfer. For this purpose, ten leaflets of a potato sterile culture wounded with a scalpel were combined with 50 μl of Agrobacterium tumefaciens culture containing 2% sucrose that had been selected overnight. 10 ml of MS medium (Murashige and Skoog, Physiol.
(1962)). After gentle shaking for 3 to 5 minutes, further incubation was performed in the dark for 2 days. Next, for callus induction, 1.6% glucose, 5m
g / l naphthyl acetate, 0.2 mg / l benzylaminopurine, 250 mg / l claforan
ran), leaves were placed on MS medium containing 50 mg / l kanamycin and 0.8% Bactoagar. After one week incubation at 25 ° C. and 3000 lux, 1.6% glucose, 1.4 mg / l zeatin ribose, 20 μg / l naphthyl acetate, 20 μg
Leaves were placed on MS medium containing / l gibberellic acid, 250 mg / l claforan, 50 mg / l kanamycin and 0.8% Bacto agar.

Analysis of the leaves of a number of plants transformed with this vector system clearly showed the presence of increased sucrose synthase activity. This is a result of the expression of the potato-derived sucrose synthase gene contained in pBinRolC-SS (FIG. 2a).
See).

Analysis of the tuber yield (fresh weight of tubers in grams) of plants transformed with this vector system and exhibiting increased sucrose synthase activity clearly showed increased tuber yield. This is also a result of the expression of the potato-derived sucrose synthase gene contained in pBinRolC-SS (see FIG. 2b).

The starch content of potato tubers is linearly dependent on tuber density (von Sche
ele et al., Landw. Vers. Sta. 127 (1937) 67-96). Analysis of the density of transgenic tubers of plants transformed with the vector system pBinRolC-SS, which has increased sucrose synthase activity, surprisingly showed an increased starch content. This is the result of the expression of the potato-derived sucrose synthase gene contained in pBinRolC-SS (see FIG. 2c).

Example 2 Preparation of Plasmid pBinRolC-Suc2 and Preparation of Transgenic Potato Plant The plasmid pBinRolC-Suc2 was placed in the binary vector pBin19 (Bevan, supra).
It contains three fragments A, B and C and is illustrated in FIG.

Fragments A and C correspond to fragments A and C described in Example 1.

Fragment B contains the coding region (positions 845 to 2384) of the cytosolic invertase gene from yeast (Saccharomyces cerevisiae). Fragment B, the vector is inserted into the polylinker BamHI cleavage site pBluescriptSK ^- it was obtained from a BamHI fragment of the length of 1548 bp. Fragment B is inserted in the sense orientation into the BamHI cleavage site of vector pBin19.

The plasmid pBinRolC-Suc2 was introduced into potato plant cells by Agrobacterium-mediated gene transfer. Whole plants were regenerated from the transformed cells. Such plants have increased yield (compared to untransformed plants).
Increased biomass).

Example 3 Construction of Plasmid pBinRolC-ΔPMA1 and Construction of Transgenic Potato Plant Plasmid pBinRolC-ΔPMA1 contains three fragments A, B and C in the binary vector pBin19 (Bevan, supra), and Is schematically illustrated in FIG.

Fragments A and C correspond to fragments A and C described in Example 1.

Fragment B contains the coding region of the gene for the proton ATPase PMA1 from yeast Saccharomyces cerevisiae (positions 937 to 3666) (Serrano et al., Nature 319).
(1986), 689-693). Fragment B was obtained by the polymerase chain reaction (PCR). For this purpose, the 3 'end of the coding region of the gene PMA1 was deliberately shortened by 27 bp, while at the same time introducing a required new stop codon. The DNA fragment modified in this manner was named ΔPMA1. Fragment B was ligated into the vector pBin19 at the BamHI cleavage site (a compatible insertion site for the BclI restriction site) and the XbaI cleavage site (a compatible insertion site for the SpeI restriction site) in the sense orientation, a length of 2739 bp. It was inserted as a BclI-SpeI fragment.

Fragment B was obtained as a BclI / SpeI fragment from the vector pBluescriptSK ⁻ inserted via the polylinker cleavage sites NtoI and PstI (see FIG. 5). Plasmid pBinRolC-ΔPMA1 was introduced into potato plant cells via Agrobacterium-mediated gene transfer. Whole plants were regenerated from the transformed cells.

The stable integration of ΔPMA1 in the genome of the transgenic plant obtained by using the vector system pBinRolC-ΔPMA1 allows the polymerase chain reaction (PC
R) (see FIG. 6).

The transformed plants show an increased yield (increased biomass) compared to the untransformed plants (see FIGS. 13 and 14).

Example 4 Preparation of Plasmid pBinRolC-ΔPHA2 and Preparation of Transgenic Potato Plant The plasmid pBinRolC-ΔPHA2 contains the three fragments A, B and C in the binary vector pBin19 (Bevan, supra), and Is schematically illustrated in FIG.

Fragments A and C correspond to fragments A and C described in Example 1.

Fragment B contains the coding region of the cDNA for the proton ATPase PHA2 (positions 64 to 2672) (Harms et al., Plant Mol. Biol. 26 (1994), 979-988). Fragment B was obtained by polymerase chain reaction (PCR). For this purpose, the 3 'end of the coding region of the gene PHA2 was deliberately shortened by 249 bp while simultaneously introducing two new stop codons. The DNA fragment modified in this way was named ΔPHA2. Fragment B was ligated with the BamHI cleavage site of the vector pBin19 (a compatible insertion site for the BglII restriction site) and the XbaI
It was inserted as a 2631 bp BglII-SpeI fragment in the sense direction into the cleavage site (a compatible insertion site for the SpeI restriction site).

[0062] Fragment B, the vector is inserted into the EcoRI cleavage site and PstI cleavage site of the polylinker sequence pBluescriptSK ^- was obtained from a BglII / SpeI fragment (Figure 8: reference to cloning methods ΔPHA2).

The plasmid pBinRolC-ΔPHA2 was introduced into potato plant cells via Agrobacterium-mediated gene transfer. Whole plants were regenerated from the transformed cells.

The stable integration of ΔPHA2 into the genome of transgenic plants obtained by using the vector system pBinRolC-ΔPHA2
R) (see FIG. 9). Transformed plants show increased yield (increased biomass) compared to untransformed plants (see FIG. 15).

Example 5 Construction of Plasmid pBinRolC-SoSUT1 and Construction of Transgenic Potato Plant Plasmid pBinRolC-SoSUT1 contains three fragments A, B and C in the binary vector pBin19 (Bevan, supra), and Is schematically illustrated in FIG. Fragments A and C are
Corresponds to fragments A and C described in Example 1.

Fragment B contains a cDNA (positions 1 to 1969) encoding a sucrose transporter from spinach (Spinasia oleracea) (Riesmeier et al., EMBO J. 11 (1992)
), 4705-4713; accession numbers X67125 and S51273). Fragment B, the vector pBluescriptSK being inserted through a NotI linker sequence ^- was obtained as a NotI fragment. For cloning into the SmaI cleavage site of the vector pBin19, the cohesive ends of the fragment obtained from NtoI digestion were converted to blunt ends and inserted into pBin19 in the sense orientation. The resulting plasmid was named pBinRolC-SoSUT1.

It was introduced into potato plant cells via Agrobacterium-mediated gene transfer. Whole plants were regenerated from the transformed cells. Plants so transformed have an increased yield (as compared to untransformed plants).
Increased biomass).

Example 6 Production of Plasmid pBinRolC-CiSy and Production of Transgenic Potato Plant The plasmid pBinRolC-CiSy was prepared using the binary vector pBin19 (Bevan19) modified according to Becker (Nucl. Acids Res. 18 (1990), 203). , Nucl. Acids Res. 12 (1984), 8711)
It contains three fragments A, B and C (see FIG. 11).

Fragment A contains the rolC promoter from Agrobacterium rhizogenes. r
The olC promoter is a 1143 bp EcoRI / Asp718 DNA fragment (Lerchl et al., Plant
Cell 7 (1995), 259-270) contains the TL-DNA DNA region (positions 11306 to 12432) of the Ri agropine-type plasmid derived from A. rhizogenes (Slightom et al., J. Bio).
l. Chem. 261 (1986), 108-121). Fragment A is inserted into the EcoRI and Asp718 cleavage sites of the polylinker of pBin19.

Fragment B is a citrate synthase from fission yeast Saccharomyces cerevisiae (C
iSy) cDNA coding region. Fragment B was obtained as a 1400 bp long BamHI fragment from vector pBluescriptSK- inserted into the BamHI cleavage site of the polylinker (Landschutze, Studies on the influence of the acetyl-CoA synth
esis and use in transgenic plants, Doctoral Thesis, Freie Universitat Berl
in, (1985) D83 / FB15 No.028).

Fragment C is the gene 3 polyadenylation signal of T-DNA of Ti plasmid pTiACH5 (Gi
elen et al., EMBO J. 3 (1984), 835-846), PvuII / HindII from plasmid pAGV40.
I fragment (Herrera-Estrella et al., Nature 303 (1983), 209-213) and, after addition of the Sphl linker to the PvuII cleavage site, the SphI and HindIII cleavage sites of the polylinker of pBin19. Nucleotides 11749-1193 cloned between
Including 9

The plasmid pBinRolC-CiSy has a length of about 13 kb.

The plasmid pBinRolC-CiSy was introduced into potato plants via Agrobacterium tumefaciens-mediated gene transfer. Whole plants were regenerated from the transformed cells.

Analysis of a large number of plants transformed with this vector system clearly showed increased biomass. This is the result of the expression of yeast-derived CiSy cDNA contained in pBinRolC-CiSy.

Example 7 Grafting Experiment For grafting, the shoots of the receiver plant are replaced with the shoots of the donor plant. In this experiment, shoots of a transgenic plant (RolC-Suc2 # 25) are grafted to rootstocks of a wild-type plant (Solanum tsuberosam desily variety). In a control experiment, wild-type shoots are grafted to a wild-type rootstock to eliminate differences in culture in the experiment (self-grafting). The purpose of the experiment was to examine the exclusive effects of photosynthetic activity and the photoabsorptive distribution of transgenic shoots in the organs of wild-type rootstocks (in this case tubers). Potato plants were transferred from tissue culture to soil and placed in a greenhouse. After about 5 weeks (the plant has not yet induced tuber formation at this stage), the plants were grafted. For this purpose, unnecessary shoots of the receiver plant are cut off and wedge-shaped cuts are made in the stems of the receiver plant. The grafted donor shoot is cut at the end of the stem in a suitable manner and inserted into the wedge of the receiver plant. Secure the graft with adhesive tape.

Next, the grafted potato plants are maintained for about one week in the shade under increased air humidity. Within 7 to 10 days, gradually adapt to normal greenhouse conditions. At this stage, the plants are 7 weeks old.

Here, to remove all leaves of the receiver plant and to ensure that only the photosynthetic activity and the photo-assimilate distribution of the donor shoots nourish the rootstock of the grafted plant,
Cover the stems with light from a sheet of aluminum.

The plants are maintained in a greenhouse and potato tubers are harvested about 2 months after grafting and about 3 months after planting in soil. The results of such a grafting experiment are illustrated in FIG.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the outline of construction of plasmid pBinRolC-SS.

FIG. 2a shows an analysis of sucrose synthase (SS) activity in leaves of transgenic potato plants transformed with a RolC-SS construct. Enzyme activity is Zr
(Plant J. 7 (1995), 97-107). Activity is given as μmol hexose equivalent / (min × g fresh weight). Columns represent the average of three samples per genotype. The standard deviation is also shown.

FIG. 2b shows an analysis of tuber yield of transgenic potato plants transformed with the RolC-SS construct. Columns represent the average of 10 to 15 plants per genotype. The standard deviation is also shown. Tuber yield is given in g by fresh weight.

FIG. 2c shows the analysis of tuber starch of transgenic potato plants transformed with the RolC-SS construct. For this purpose, tubers collected from 10 to 15 plants per genotype are collected and the tuber starch content is determined according to Von Scheele et al. (Landw. Vers. Sta. 127 (1937), 67-96). It has been determined.

FIG. 3 shows an outline of the construction of plasmid pBinRolC-Suc2.

FIG. 4 shows the outline of construction of plasmid pBinRolC-ΔPMA1.

FIG. 5 shows an outline of a cloning method of ΔPMA1. Steps from A to B: H ⁺ -ATPase ΔPMA1 having a shortened 3 ′ end is used as a substrate for ΔP
Amplification was performed by PCR using MA1 cDNA and complementary internal primer (A). The adjacent synthetic site of the PCR product (B) was introduced with the corresponding synthetic primer. Steps B to C: PstI / NotI degradation and cloning of the PCR fragment into the E. coli vector SK- via the PstI / NotI cleavage site (C). Steps C to D: BclI / SpeI digestion of plasmid SK-ΔPMA1 and cloning of the fragment into the compatible BamHI / XbaI cleavage site of pBinRolC (D).

FIG. 6 shows the results of the polymerase chain reaction with specific oligonucleotides, showing the stable uptake of ΔPMA1 in the genome of transgenic plants obtained by transformation with the rolC-ΔPMA2 construct. PCR product size = 730 bp; WT = wild type; M = marker.

FIG. 7 shows an outline of construction of plasmid pBinRolC-ΔPHA2.

FIG. 8 shows an outline of a method for cloning ΔPHA2. Steps from A to B: H ⁺ -ATPase ΔPHA2 having a shortened 3 ′ end
Amplification was performed by PCR using HA2 cDNA and complementary internal primer (A). The adjacent synthetic site of the PCR product (B) was introduced with the corresponding synthetic primer. Steps B to C: PstI / EcoRI digestion and cloning of PCR fragment into E. coli vector SK- via PstI / EcoRI cleavage site (C). Steps C to D: BglII / SpeI digestion of plasmid SK-ΔPHA2 and cloning of the fragment into the appropriate BamHI / XbaI cleavage site of pBinRolC (D).

FIG. 9 shows the results of the polymerase chain reaction with specific oligonucleotides, showing stable uptake of ΔPHA2 in the genome of transgenic plants obtained by transformation with the rolC-ΔPHA2 construct. PCR product size = 758 bp; WT = wild type; M = marker.

FIG. 10 shows an outline of construction of plasmid pBinRolC-SoSUT1.

FIG. 11 shows an outline of the construction of plasmid pBinRolC-CiSy.

FIG. 12 shows the results of determining the sucrose content in tuber parenchyma samples of grafted potato plants enriched in vascular tissue. The genotype used for grafting was line Ro
1C-Suc2- # 25 (cytosol invertase) and wild-type Solanum tuberosum var. Desiree (Solanum tuberosum var. Desiree). Sucrose content was determined according to Stitt et al. (Methods Enzymol. 174 (1989), 518-522). Columns represent the average of 12 samples per grafted mold. The sucrose content is given in μmol hexose equivalent / g fresh weight.

FIG. 13 shows an analysis of phloem exudates of ΔPMA1 leaves left in the light for 6 hours under a 14 CO ₂ atmosphere. Sucrose content was determined according to Stitt et al. (Supra). Columns represent the average of 4 to 5 samples per genotype. Standard deviations are shown.

FIG. 14 shows tuber yield (gram fresh weight) of ΔPMA1 plants. Columns represent the average of 6 plants per genotype. Standard deviations are shown. Tuber yield is given in g fresh weight.

FIG. 15 shows tuber yield (gram fresh weight) of ΔPHA2 plants. Columns represent the average of 4 to 5 plants per genotype. Standard deviations are shown. Tuber yield
g Shown in fresh weight.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] February 7, 2000 (2000.2.7)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/88 C12N 5/00 C 15/09 15/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) , MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, F I, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG , MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Wilmitzer Rosser Germany Arnold-Knob Lauch Ring 1F Term (Reference) 2B030 AD07 AD08 CA15 CA16 CA17 CD17 4B024 AA08 BA07 BA11 BA12 CA04 DA01 EA04 FA02 GA11 GA17 4B050 CC03 DD04 DD13 LL10 4B065 AA11Y AA80Y AA88Y AB01 AC14 BA02 CA27 CA29 CA31 CA53

Claims (15)

[Claims] 1. (a) a region specifically enabling transcription in a companion cell, and (b) (i) a protein having an enzymatic activity for cleaving sucrose, (ii) A sucrose transporter, (iii) a protein having an activity to cause stimulation of a proton gradient located at the plasma membrane of a plant cell, and (iv) a nucleotide sequence encoding a polypeptide selected from the group consisting of: citrate synthase. ,
A method for increasing the yield of a plant, characterized by expressing a recombinant DNA molecule stably integrated into the genome of the plant. 2. The method according to claim 1, wherein the nucleotide sequence encodes a plant protein.
The described method. 3. The method of claim 1, wherein the nucleotide sequence encodes a protein from bacteria or fungi. 4. The method according to claim 1, wherein the nucleotide sequence is sucrose synthase or sucrose synthase.
The method according to claim 1, which encodes a protein having an enzymatic activity for cleaving sucrose, selected from the group consisting of phosphorylase and invertase. 5. The method according to claim 1, wherein the nucleotide sequence is Spinacia oleracea.
2. The method of claim 1 which encodes a sucrose transporter from racea). 6. The method of claim 1, wherein the nucleotide sequence encodes a proton ATPase.
The described method. 7. The method according to claim 1, wherein the nucleotide sequence is Solanum tuberosum.
7. A method according to claim 6, which encodes a proton ATPase from Saccharomyces cerevisiae or Saccharomyces cerevisiae. 8. The method according to claim 1, wherein the region referred to in (a) is the rolC promoter from Agrobacterium rhizogenes. 9. (a) a region specifically enabling transcription in a companion cell of a plant, and operably linked thereto (b) (i) sucrose synthase, (ii) sucrose phosphorylase, (Iii) a sucrose transporter, (iv) a protein having an activity of causing stimulation of a proton gradient located on the plasma membrane of a plant cell, and (v) a nucleotide encoding a polypeptide selected from the group consisting of: citrate synthase A recombinant nucleic acid molecule comprising a sequence. 10. A vector comprising the recombinant nucleic acid molecule according to claim 9. 11. The vector according to claim 10, which is suitable for transformation of a plant cell and integration of a foreign DNA into a plant genome. 12. A plant cell transformed with and containing the recombinant nucleic acid molecule of claim 9. 13. A plant comprising a plant cell according to claim 12, wherein the expression of the recombinant nucleic acid molecule in a companion cell of the plant shows an increased yield compared to the corresponding non-transformed plant. 14. A plant propagation material according to claim 13, comprising the plant cell according to claim 12. 15. A region specifically enabling transcription in a companion cell of a plant, and operably linked thereto (i) a protein having an enzymatic activity of cleaving sucrose, (ii) a sucrose transporter, (Iii) the yield of a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide selected from the group consisting of: (i) a protein located at the plasma membrane and having the ability to stimulate a proton gradient; and (iv) a citrate synthase. Use for expression in a transgenic plant to increase expression.