KR20010021566A - Processes for increasing the yield in plants - Google Patents

Abstract

The present invention relates to a method for increasing plant yield, a recombinant nucleic acid molecule used in the method, a plant having increased yield as well as their use,

A method of increasing the yield of a plant in which a nucleotide sequence is expressed under the control of a half cell specific promoter is described.

The nucleotide sequence is characterized by encoding a protein that accumulates photosynthetic products in the phloem.

Description

PROCESSES FOR INCREASING THE YIELD IN PLANTS

The present invention relates to a method for increasing plant yield, a recombinant nucleic acid molecule used in the method, a plant having increased yield as well as their use.

In agriculture and forestry, there has been a steady effort to produce plants with increased yields, in particular to continuously feed the growing world population, and to provide renewable raw materials. Conventionally, efforts have been made to obtain plants that have increased yields by breeding, but this is a waste of time and labor. Moreover, suitable breeding plans should be implemented for each relevant plant species.

Partial advances have been made in plant engineering, ie by introducing recombinant nucleic acid molecules into plants and expressing them in plants. This approach has the advantage that it is not limited to one plant species but can be transferred to another plant species. European patent EP-A 0 511 979 describes the expression of prokaryotic asparagine synthase in plant cells, in particular to increase biomass.

WO 96/21737 describes the production of plants with increased production by expression of regulated or disordered fructose-1,6-bisphosphatase, which increases the rate of photosynthesis.

Nevertheless, there is still a need for commonly applied methods for increasing plant production in agriculture or forestry.

Thus, the basic problem of the present invention is to further provide a method for increasing plant yield.

The problem is solved according to the present invention by providing an embodiment characterized within the claims of the present invention.

Therefore, the present invention relates to a method for increasing plant yield,

(a) a region that specifically transduces in half cells; And operably coupled thereto

(b) a protein having enzymatic activity to remove sucrose;

(Ii) sucrose carriers;

(Iii) a protein having an activity of stimulating proton gradient present in the plasma membrane of plant cells; And

(Iii) Citrate synthase (E.C.4.1.3.7) consisting of a nucleotide sequence encoding a polypeptide selected from the group consisting of, characterized in that the recombinant DNA molecules are stably integrated into the genome of the plant is expressed.

It was found that the expression of the protein specifically in the phloem of the plant dramatically increased the yield.

The term "increase in yield" means increasing biomass production, as measured by the plant's live weight. Said increase in production preferably means the so-called "sink" organ of the plant, which is the organ that absorbs the photosynthetic product produced during photosynthesis. Particularly preferred are parts of plants that can be harvested such as seeds, fruits, storage roots, roots, tubers, flowers, snow, bird branches, stems or timber. The yield growth rate according to the invention, when cultivated under the same conditions, is at least 3%, preferably at least 10% and particularly preferably at least 20% relative to the biomass compared to untransformed plants of the same genotype.

When the proteins are usually expressed in the phloem, their biological activity increases the rate of accumulation of photosynthetic products into the phloem.

In the context of the present invention, photosynthetic products are understood as sugars and / or amino acids.

According to the present invention, the nucleotide sequence referred to in (b) can encode proteins or bacterial proteins or plant proteins that are usually of fungal or animal organisms.

In a preferred embodiment, the nucleotide sequence in particular comprises a sucrose synthase (EC 2.4.1.13), preferably a plant sucrose synthase, expressed in the tubers of solanium tuberosum, preferably in solanium tuberosum Coding Such sequences are described, for example, in Salanoub and Belliard (Gene 60 (1987), 47-56) and can be used in the EMBL Genbank under accession number X67125.

In a further preferred embodiment, the nucleotide sequence encodes sucrose phosphorylase (E.C.2.4.1.7).

Sequences encoding sucrose phosphorylase are known, for example, from WO 96/24679.

In another preferred embodiment, the nucleotide sequence is invertase (EC3.2.1.26), preferably the invertase of the microorganism, in particular the genus Saccharomyces fungi, preferably Saccharomyces cerevisiae (S. cerevisiae). Particularly preferred are sequences encoding cytoplasmic invertases (Sonnewald et al., Plant J. 1 (1991), 95-106).

According to the present invention, sucrose carriers are understood as carriers that carry sucrose in plant systems across membranes. The carrier is preferably derived from a plant (eg EMBL GenBank Accession No. G21319). Particularly preferred sequences of (b) are spinach (Spinacia oleracea) having the sequence of clone SoSUT1, as described in particular by Reismeier et al. (EMBO J. 11 (1992), 4705-4713). oleracea)) encodes a sucrose carrier.

In another preferred embodiment, the protein that stimulates the proton gradient present in the plasma membrane is proton ATPase.

In this case, the sequence described in (b) encodes a microorganism, in particular a fungus of the genus Saccharomyces, preferably a protein of Saccharomyces cerevisiae.

In a particularly preferred embodiment, the sequence is cleaved from Saccharomyces cerevisiae at proton ATPase PMA1 (Serrano et al., Nature 319 (1986), 689-693; EMBL gene bank), or 3 ′ end. From the Saccharomyces cerevisiae a variant of said proton ATPase, in particular the ATPase ΔPAM1 as described in Example 3 of the present invention.

Optionally, the nucleotide sequence may also encode proton ATPases from plants, preferably proton ATPases from solanium tubulosum.

Particularly preferred are proton ATPases from potatoes PHA2 (Harms et al., Plant Mol. Biol. 26 (1994), 979-988; EMBL Genbank X76535), or proton ATPases from potatoes cut at the 3 ′ end Is a sequence encoding an ATPase ΔPHA2 as described in Example 4 of the present invention.

According to the invention, the citrate synthase may be a specific citrate synthase, for example citrate synthase from bacteria, fungi, animals or plants. DNA sequences encoding citrate synthase are known, examples include Bacillus subtilis (U05256 and U05257), E. coli (V01501), egg. R. prowazekii (M17149), p. P. aeruginosa (M29728), A. A. anitratum (M33037) (Schendel et al., Appl. Environ. Microbiol. 58 (1992), 335-345 and references thereof), Haloferax volcanii (James) (James) et al., Biochem. Soc. Trans. 20 (1992), 12), Arabidopsis thaliana (Z17455) (Unger et al., Plant Mol. Biol. 13 (1989), 411- 418), b. B. coagulans (M74818), p. C. burnetti (M36338) (Heinzen et al., Gene 109 (1990), 63-69), M. M. Smegmatis (X60513), T. T. acidophilum (X55282), t. T. thermophila (D90117), pig (M21197) (Bloxham et al., Proc. Natl. Acad. Sci. USA 78 (1981), 5381-5385), N. N. crassa (M84187) (Ferea et al., Mol. Gen. Genet. 242 (1994), 105-110), S. et al. S. cerevisiae (Z11113, Z23259, M14686, M54982, X00782) (Suissa et al., EMBO J. 3 (1984), 1773-1781) and potatoes (EP 95 91 3066.7). .

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

Nucleotide sequences according to the invention can encode suitable proteins from microorganisms, in particular from plants, fungi, bacteria or animals. The sequence preferably encodes a protein from a plant or fungus. Plants are high-grade plants, especially plants useful for storing starch or oils, such as potatoes or cereals, such as rice, corn, wheat, barley, rye, rye wheat, oats, sorghum, etc., as well as spinach, tobacco, sugar beets, soybeans, cotton Etc. are preferable.

Fungi include the genus Saccharomyces, Schizosaccharomyces, Aspergillus or Neurospora, in particular Saccharomyces cerevisiae, Schizocaromyces pombe (Schizosaccharomyces pombe), Aspergillus flavus, Aspergillus niger or Neurospora crassa are preferred.

In a preferred embodiment of the method according to the invention, the region mentioned in (a) which ensures anti-cell specific transcription is the promoter of the rolC gene from Agrobacterium rhizogenes.

Such promoters include, for example, the characterization and alignment of the sucrose carrier SUT1 in Solanaceae (Schmuelling et al. (Plant Cell (1989), 665-671) and Quinn), Doctoral Thesis (1991), Freie Universitaet Berlin, biology department). It is preferred to use a promoter region having the nucleotide sequence set forth in SEQ ID NO: 1.

Unlike the rolC promoter mentioned above, one skilled in the art can readily use other promoters for anti-cell specific expression. Other half cell specific promoters, such as the promoter of sucrose carriers from Arabidopsis thaliana, are described in Truernit and Sauer, Planta 196 (1995), 564-570.

And their specific incidence in semi-cells for other RNAs and proteins is described in, for example, Foley et al., Plant Mol. Biol. 30 (1996), 687-695; DeWitt, Plant J. 1 ( 1991), 121-128; see Stadler et al., Plant Cell 7 (1995), 1545-1554). Starting from known proteins, those skilled in the art can readily isolate cDNAs by antibodies or using oligonucleotides derived from amino acid sequences (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual). , Cold Spring Harbor Laboratory Press (1989), Cold Spring Harbor, NY). Starting from the cDNA obtained in the method, genome libraries formed from the organism can be fractionated and genomic fragments identified. The position of the promoter can be approximately determined by comparing the nucleotide sequence of the cDNA and the nucleotide sequence of the genomic clone. The specificity of a promoter may vary within the transduction site using a chimeric gene consisting of a promoter and an indicator gene, such as β-glucuronidase (eg, Kertbundit et al., Proc. Natl Acad.Sci. USA 88 (1991), 5212-5216).

The method according to the invention can be applied to any plant inherently. Therefore, dicotyledonous species as well as monocotyledonous species are particularly suitable. The method is preferably used with plants of interest for agriculture, horticulture and / or forestry.

Examples include vegetable plants such as cucumbers, melons, pumpkins, egg plants, zucchini, tomatoes, spinach, cabbage varieties, peas, beans and the like, as well as fruits, such as pears and apples.

And oil storage plants are suitable, for example, rape, sunflower, soybeans. In a particularly preferred embodiment, the starch stock is particularly suitable for cereals (rice, corn, wheat, rye, oats, rye wheat, sorghum, barley), potatoes, cassava, sweet potatoes and the like.

The method can also be applied to sucrose stocks such as sugar beets and sugar cane as well as other useful plants such as cotton, tobacco, timber, wine, hops and the like.

The present invention also provides

(a) a region that specifically transduces in plant half cells; And operably coupled thereto

(b) (iii) sucrose synthase;

(Ii) sucrose phosphorylase;

(Iii) sucrose carriers;

(Iii) a protein having an activity of stimulating a proton gradient present in the plasma membrane of the plant cell; And

(Iii) Citrate synthase relates to a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide selected from the group consisting of.

With respect to the preferred embodiments of such molecules, the same applies to the nucleotide sequences mentioned in (b) and to the regions mentioned in (a) already mentioned above in connection with the methods of the invention.

The present invention also relates to a vector containing the nucleic acid molecule of the present invention, which is suitable for transforming plant cells as well as integrating foreign DNA into the plant genome.

The present invention also relates to plant cells containing the molecule transformed with the nucleic acid molecule of the present invention and stably integrated into the genome. The cells differ from naturally occurring plant cells in that the nucleic acid molecules of the present invention are integrated into the cell's genome in a non-naturally occurring position.

The invention also results in the expression of recombinant nucleic acid molecules integrated into the genome within the plant half-cells, which show increased production compared to the corresponding non-transgenic plants grown under the same conditions, and contain the plant cells of the invention. It relates to a transgenic plant.

The present invention also relates to a propagation material of the plant of the present invention containing the plant cell of the present invention. The term "propagation material" includes especially seeds, fruits, tubers, underground diameters, crops, kali, cell cultures and the like.

Finally, the present invention relates to a region specifically transcribed in plant half cells; And (iii) a protein having an enzymatic activity to remove sucrose, which is operably bound thereto; (Ii) sucrose carriers; (Iii) a protein having an activity of stimulating proton gradient present in the plasma membrane; And (iii) a nucleotide sequence encoding a polypeptide selected from the group consisting of citrate synthase, and relates to the use of recombinant nucleic acid molecules for expression in transgenic plants to increase production.

The encoded protein is preferably the protein described above.

Methods of transforming monocotyledonous and dicotyledonous plants are known to those of ordinary skill in the art.

Various techniques can be used to introduce DNA into plant host cells. The techniques include the use of Agrobacterium lithium faciens or Agrobacterium lysine as a means of transformation to transform plant cells into T-DNA, to fuse protoplasts, to inject, to electroporate DNA, Biodegradation as well as techniques for introducing DNA by other possible methods.

In order to inject and electroporate DNA into plant cells, the plasmid does not have to meet certain requirements. Simple plasmids such as pUC can be used. The use of Agrobacterium to transform plant cells is described in European Patent EP-A 120 516, Hoekema (In: The Binary Plant Vector System Offsetdrukkerij Kanters BV, Alblasserdam (1985), Chapter V), Praray (Fraley et al., Crit. Rev. Plant. Sci. 4, 1-46) and An et al. (EMBO J. 4 (1985), 277-287) and are fully disclosed.

To deliver DNA to plant cells, the plant graft may be co-cultured with Agrobacterium lithium faciens or Agrobacterium lysine. From infectious plants (e.g., leaf grafts, stem fragments, roots as well as suspensions of protoplasts or plant cells cultured), the whole plant is in a suitable medium containing antibiotics or biozides for selecting transformed cells. Can be recycled. Plants obtained in this method can be tested in the presence of introduced DNA. Other possible methods of introducing foreign DNA using biodegradation or protoplast transformation are known (e.g. Willmitzer, L., 1993 Transgenic plants.In: Biotechnology, A Multi-Volume Comprehensive Treatise (HJ Rehm, G. Reed, A. Puehler, P. Stadler, eds), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).

Techniques for transforming dicotyledonous plants through the Ti-plasmid-vector system by Agrobacterium zumfaciens are well established. Recent studies have also found that monocots can be transformed with Agrobacterium-based vectors (Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al. Many, 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 / Technology 13 (1995), 486-492; Conner and Domisse; Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al., Transgenic Res. 2 (1993), 252-265).

Alternative systems for transforming monocots are described by biodegradation (Wan and Lemaux, Plant Physiol. 104 (1994), 37-48; Vasil et al., Bio / Technology 11 (1993), 1553). -1558; Ritala et al., Plant Mol. Biol. 24 (1994), 317-325; Spencer et al., Theor.Appl. Genet. 79 (1990), 625-631), protoplast traits Conversion, electroporation of some fluidized cells, as well as introduction of DNA by glass fibers.

In particular, the transformation of maize has been described several times in the literature (eg, WO95 / 06128, EP 0 513 849; EP 0 465 875; Fromm et al., Biotechnology 8 (1990), 833-844; Gordon-Kamm et al., Plant Cell 2 (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200). From the mucus-free soft (friable) corn callus by the methods described in EP 292 435 and Shillito et al. (Bio / Technology 7 (1989), 581), the forest plants Can be obtained. In Bio / Technology 7 (1989), 589 of Prioli and Soendahl, regeneration and obtaining of forest plants from the corn protoplasts of Catto maize incest cat 100-1 Described.

Successful transformation of other cereal species is also described, for example, for barley (Wan and Lemaux, supra; Ritala et al., Supra) and wheat (Nehra et al., Plant J. 5 (1994), 285-297). Is disclosed.

Once the introduced DNA is integrated into the plant cell genome, it is usually stable and also included in the descendants of the originally transformed cell. Usually, they contain a selection marker that forms transgenic plant cells that are resistant to antibiotics or biozides such as kanamycin, G 418, bleomycin, hygromycin or phosphinothricin. Therefore, each selected marker must select for transformed cells from cells that have not been introduced with DNA.

Transformed cells are grown in plants by conventional methods (see also McCormick et al., Plant Cell Reports 5 (1986), 81-84). The plant obtained can be grown normally. Seeds can be obtained from plants.

Two or more generations must be cultivated to ensure that the phenotype remains stable and is transmitted. Seeds should be harvested to ensure that the corresponding phenotype or other properties are maintained.

1 shows the structure of plasmid pBinRolC-SS.

Figure 2a shows the analysis of the sucrose synthase (SS) activity in the leaves of the transgenic potato plants transformed with the RolC-SS construct, the enzyme activity is Zrenner et al. (Plant J. 7 (1995) , 97-107), and the activity is expressed in hexose equivalent (μmol) / (min × live weight (g)). The column shows the average of three samples per genotype. Standard deviation is also shown.

Figure 2b shows the tuber production of transgenic potato plants transformed with the RolC-SS construct, the column shows the mean value of 10 to 15 plants per genotype, also shows the standard deviation, and the tuber production is in vivo (g )

Figure 2c shows the analysis of tuber starch of the transgenic potato plants transformed with the RolC-SS construct, for this purpose to collect the tubers harvested from 10 to 15 plants per genotype, the starch content of tubers von Schiller It was measured according to (Von Scheele) et al. (Landw. Vers. Sta. 127 (1937), 67-96).

3 shows the structure of plasmid pBinRolC-Suc2.

4 shows the structure of plasmid pBinRolC-ΔPMA1.

5 shows a cloning scheme of ΔPMA1,

Step A-B:

H ⁺ -ATPase ΔPMA1 cleaved at the 3 ′ end was amplified by PCR with PMA1 cDNA as a substrate and complementary internal primers (A). The flanking removal site of the PCR product (B) was introduced via correspondingly synthesized primers.

Step B-C:

Pstl / Notl digested and cloned PCR fragment into E. coli vector SK- via Pstl / Notl removal site (C).

Step C-D:

Plasmid SK-ΔPMA1 was digested with Bcll / Spel and the fragment cloned into the appropriate BamHl / Xbal removal site of pBinRolC (D).

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

7 depicts the structure of plasmid pBinRolC-ΔPHA2.

8 shows a cloning scheme of ΔPHA2.

Step A-B:

The H ⁺ -ATPase ΔPHA2 cleaved at the 3 'end was amplified by PCR with PHA2 cDNA as a substrate and complementary internal primers (A). The flanking removal site of PCR product (B) was introduced through the correspondingly synthesized primers.

Step B-C:

Pstl / EcoRl digested and cloned PCR fragment into E. coli vector SK- via Pstl / EcoRl removal site (C).

Step C-D:

Plasmid SK-ΔPHA2 was digested with Bglll / Spel and the fragment cloned into the appropriate BamHl / Xbal removal site of pBinRolC (D).

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

10 depicts the structure of plasmid pBinRolC-SoSUT1.

Figure 11 shows the structure of plasmid pBinRolC-CiSy.

Figure 12 shows the results of measuring the sucrose content in the milk tissue sample of tubers of grafted potato plants rich in ductal tissue. Genotypes used for grafting were lineage RolC-Suc2- # 25 (cytoplasmic invertase) and wild type solanium tuberosum var. It is Desiree. The content as souk was determined according to Stitt et al. (Methods Enzymol. 174 (1989), 518-522). The column shows the average of 12 samples per graft. Standard deviations are also shown. Sucrose content is expressed in hexose equivalent (μmol) / live weight (g).

FIG. 13 shows the analysis of phloem exudates of ΔPMA1 leaves placed in the presence of light for 6 hours in a 14CO ₂ atmosphere. Sucrose content was determined according to Stuttg et al. The columns represent the mean values of 4-5 samples per genotype. Standard deviations are also shown.

FIG. 14 shows tuber production (bioweight in g) of ΔPMA1 plants. The column shows the average of six plants per genotype. Standard deviations are also shown. Tuber production is expressed as live weight (g).

Fig. 15 shows tuber production (bioweight in g) of the ΔPHA2 plant. Columns represent the mean values of 4 to 5 plants per genotype. Standard deviations are also shown. Tuber production is expressed as live weight (g).

The following examples detail the invention.

Example 1

Preparation of Plasmid pBinRolC-SS and Preparation of Transgenic Potato Plants

Plasmid pBinRolC-SS contains three fragments A, B and C in binary vector pBin19 (Bevan, Nucl. Acids Res. 12 (1984), 8711) (see FIG. 1).

Fragment A consists of the rolC promoter of Agrobacterium lysine. The rolC promoter is a 1138 bp EcoRI / Asp718 DNA fragment (Lerchl et al., Plant Cell 7 (1995), 259-270) as a TL-DNA (Slightom et al. Of Ri-Agropin-type plasmid from Agrobacterium rizogen , J. Biol. Chem. 261 (1986), 108-121). The fragment A is inserted at the EcoRI and Asp718 removal sites of the polylinker of pBin19.

Fragment B contains the coding region (position: 76-position 2493) in the cDNA of sucrose synthase (SS) from solanium tuberosum (Salanoubat and Belliard, Gene 60 (1987), 47-56). Fragment B was obtained from the vector pBluescript SK ⁻ as a BamHI fragment of 2427 bp, where it is inserted at the BamHI clearance site of the polylinker. Fragment B was inserted at the BamHI elimination site in the sense orientation in the vector pBin19, ie downstream of the rolC promoter in an orientation that transcribed the translatable RNA.

Fragment C comprises the polyadenylation signal of Gene 3 (Gielen et al., EMBO J. 3 (1984), 835-846), in particular nucleotides 11749-11939, in the T-DNA of Ti plasmid pTiACH 5, It was isolated from plasmid pAGV 40 into PvuII / HindIII fragments (Herrera-Estrella et al., Nature 303 (1983), 209-213) and removed SphI and HindIII in the polylinker of pBin19 when SphI was added. The linker was cloned to the PvuII removal site between the sites.

Results The plasmid pBinRolC-SS was introduced into potato plant cells via gene transfer mediated by Agrobacterium lithium faciens. For this purpose, scalar in 10 ml MS medium (Murashige and Skoog, Physiol. Plant. 15 (1962), 473) with 2% sucrose containing 50 μl of cultured Agrobacterium lithium faciens daily culture. Ten small leaves (solarium tuberosum L. cv. Desiree) of potato sterile cultures damaged with Pel were added. After shaking carefully for 3-5 minutes, the cells were further incubated for 2 days in the dark. Then on MS medium containing 1.6% glucose, 5 mg / l naphthyl acetic acid, 0.2 mg / l benzylaminopurine, 250 mg / l claporan, 50 mg / l kanamycin and 0.8% bacto-agar for callus induction Put the leaves on. After incubation for 1 week at 25 ° C. and 3000 lux, 1.6% glucose, 1.4 mg / l of zetin ribose, 20 μg / l of naphthyl acetate, 20 μg / l of gibberelic acid, 250 mg / l of claporan, 50 mg of kanamycin The leaves were placed on MS medium containing / l and 0.8% bacto-agar.

Analysis of the leaves of several plants transformed with the vector system clearly demonstrated an increase in sucrose synthase activity. This is the result of expressing the sucrose synthase gene from potatoes contained in pBinRolC-SS (see FIG. 2A).

Analysis of tuber yield (g tuber biomass) of plants transformed with the vector system shows increased sucrose synthase activity clearly showing that tuber yield was increased. It is also the result of expressing the sucrose synthase gene from potatoes contained in pBinRolC-SS (see FIG. 2B).

The starch content of potato tubers is linearly proportional to the density of tubers (Von Scheele et al., Landw. Vers. Sta. 127 (1937), 67-96). Density analysis of transgenic tubers of plants transformed with the vector system pBinRolC-SS with increased sucrose synthase activity showed increased starch content. This is the result of expressing the sucrose synthase gene from potatoes contained in pBinRolC-SS (see FIG. 2C).

Example 2

Preparation of Plasmid pBinRolC-Suc2 and Preparation of Transgenic Potato Plants

Plasmid pBinRolC-Suc2 contains three fragments A, B and C in binary vector pBin19 (Bevan, cited) and is shown in FIG. 3.

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

Fragment B comprises a coding region (position: 845 to position: 2384) in the gene of cytoplasmic invertase from yeast (Saccharomyces cerevisiae). Fragment B is obtained as a BamHI fragment having a length of 1548 bp from the vector pBluescript SK ⁻ inserted into the BamHI elimination site in the polylinker. Fragment B is inserted in the sense orientation into pBin19 at the BamHI clearance site.

Plasmid pBinRolC-Suc2 was introduced into potato plant cells via gene transfer mediated by Agrobacterium. The whole plant was regenerated from the transformed cells. The plants show increased production (increased biomass) compared to non-transformed plants.

Example 3

Preparation of Plasmid pBinRolC-ΔPMA1 and Preparation of Transgenic Potato Plants

Plasmid pBinRolC-ΔPMA1 contains three fragments A, B and C in binary vector pBin19 (Bevan, cited) and is shown in FIG. 4.

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

Fragment B comprises a coding region (position: 937 to position 3666) in the gene of the proton ATPase PMA1 from yeast Saccharomyces cerevisiae (Serrano et al., Nature 319 (1986), 689-693 ). Fragment B was obtained by polymerase chain reaction (PCR). For this purpose, the 3 'end of the coding region in gene PMA1 was cleaved to 27 bp and at the same time the necessary stop codon was introduced. DNA fragments modified by this method are called ΔPMA1. Fragment B, like the BclI / SpeI fragment with a length of 2739 bp, is inserted in the sense orientation at the BamHI (compatible position for BclI restriction) and XbaI (compatible position for SpeI restriction) deletions of the vector pBin19. It became.

Fragment B was obtained as a BclI / SpeI fragment from the vector pBluescript SK ⁻ which was inserted through the removal sites NotI and PstI in the polylinker (see FIG. 5).

Plasmid pBinRolC-ΔPMA1 was introduced into potato plant cells via gene transfer mediated by Agrobacterium. The whole plant was regenerated from the transformed cells.

The stable integration of ΔPMA1 into the genome of transgenic plants obtained using the vector system pBinRolC-ΔPMA1 was detected by polymerase chain reaction (PCR) (see FIG. 6).

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

Example 4

Preparation of Plasmid pBinRolC-ΔPHA2 and Preparation of Transgenic Potato Plants

Plasmid pBinRolC-ΔPHA2 contains three fragments A, B and C in binary vector pBin19 (Bevan, cited) and is shown in FIG. 7.

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

Fragment B comprises the coding region (position: 64 to position: 2672) in the cDNA of the proton-ATPase PHA2 (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 in the gene PHA2 was cut to 249 bp and two new stop codons were introduced at the same time. DNA fragments modified by this method are called ΔPHA2. Fragment B is a BglII / SpeI fragment with a length of 2631 bp, inserted in the sense orientation at the BamHI (compatible position for BglII restriction) and XbaI (compatible position for SpeI restriction) deletions of the vector pBin19. .

Fragment B was obtained as a BglII / SpeI fragment from the vector pBluescript SK ⁻ which is inserted into the EcoRI and PstI removal sites in the polylinker sequence (see FIG. 8: ΔPHA2 cloning scheme).

Plasmid pBinRolC-ΔPHA2 was introduced into potato plant cells via gene transfer mediated by Agrobacterium. The whole plant was regenerated from the transformed cells.

The stable integration of ΔPHA2 into the genome of transgenic plants obtained using the vector system pBinRolC-ΔPHA2 was detected by polymerase chain reaction (PCR) (see FIG. 9).

The transformed plants show increased production (increased biomass) compared to untransformed plants (see FIG. 15).

Example 5

Preparation of Plasmid pBinRolC-SoSUT1 and Preparation of Transgenic Potato Plants

Plasmid pBinRolC-SoSUT1 contains three fragments A, B and C in binary vector pBin19 (Bevan, cited) and is shown in FIG. 10.

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

Fragment B comprises a cDNA (position: 1 to position: 1969) encoding a sucrose carrier from spinach (Spinacia oleracea) (Riesmeier et al., EMBO J. 11 (1992), 4705-4713; accession numbers X67125 and S51273). Fragment B was obtained as a Notl fragment from the vector pBluescript SK ⁻ , which is inserted through the NotI linker sequence. To clone to the SmaI elimination site in the vector pBin19, the sticky ends of the fragments obtained from NotI digestion are converted to blunt ends and inserted into the sense orientation of pBin19. The resulting plasmid is called pBinRolC-SoSUT1.

It was introduced into potato plant cells through gene transfer mediated by Agrobacterium. The whole plant was regenerated from the transformed cells.

Plants transformed by this method show increased production (increased biomass) compared to untransformed plants.

Example 6

Preparation of Plasmid pBinRolC-CiSy and Preparation of Transgenic Potato Plants

The plasmid pBinRolC-CiSy is expressed in three fragments A in binary vector pBin19 (Bevan, Nucl. Acids Res. 12 (1984), 8711) modified according to Becker's (Nucl. Acids Res. 18 (1990), 203). , B and C (see FIG. 11).

Fragment A consists of the rolC promoter from Agrobacterium lysine. The rolC promoter is an EcoRI / Asp718 DNA fragment (Lerchl et al., which has a length of 1143 bp in the DNA region (position: 11306 to position: 12432) in the TL-DNA of Ri-Agropin type plasmid from Agrobacterium rizogen. The Plant Cell 7 (1995), 259-270) (Slightom et al., J. Biol. Chem. 261 (1986), 108-121). Fragment A is inserted within the EcoRI and Asp718 removal sites in the polylinker of pBin19.

Fragment B comprises a coding region in the cDNA of citrate synthase (CiSy) from cleavage yeast Saccharomyces cerevisiae. Fragment B was obtained as a BamHI fragment with a length of 1400 bp from the vector pBluescript SK ⁻ , which was inserted at the BamHI removal site of the polylinker (Landschutze, Studies on the influence of the acetyl-CoA synthesis and use in transgenic plants, Doctoral Thesis). , Freie Universitat Berlin, (1985) D83 / FB15 No.028).

Fragment C is isolated from the polyadenylation signal of gene 3 (Gielen et al., EMBO J. 3 (1984), 835-846), plasmid pAGV40 as PvuII / HindIII fragment in T-DNA of Ti plasmid pTiACH 5 Nucleotides 11749-11939 (Herrera-Estrella et al., Nature 303 (1983), 209-213), and SphI and HindIII in the polylinker of pBin19 after addition of the SphI linker at the PvuII removal site. It was cloned between removal sites.

Plasmid pBinRolC-CiSy is about 13 kb in length.

The plasmid pBinRolC-CiSy was inserted into potato plants via gene transfer mediated by Agrobacterium lithium faciens. The whole plant was regenerated from the transformed cells.

Analysis of the various plants transformed with the vector system clearly shows that the biomass was increased, which is the result of expressing CiSy cDNA from yeast contained in pBinRolC-CiSy.

Example 7

Grafting experiment

For grafting, the branches of the acceptor plant are replaced with the branches of the donor plant.

In this experiment, the branch of the transgenic plant (RolC-Suc2 # 25) is grafted onto the large tree of the wild type plant (solarium tuberosum, var. Desiree). In the control experiment, the wild type branches are grafted onto the wild type tree (autograft) to know the culture difference in the experiment. The purpose of the experiment was to investigate the effect of photosynthetic product distribution and photosynthetic activity of transgenic branches on organs (in this case, tubers) of wild type trees. Potato plants are transferred from the tissue culture solution to the soil and placed in a greenhouse. After about 5 weeks (plants do not induce tuber formation at this stage), the plants are grafted. To do this, the branches of the unwanted receptor plant are cut off, and the wedge is cut into the stems of the receptor plant. The grafted donor branch is cut at the end of the stem in a suitable manner and inserted into the wedge of the receptor plant. Fix the grafting seat with adhesive tape.

The grafted potato plants are then shaded for about a week under increased atmospheric humidity. Within 7-10 days, they gradually adapt to normal greenhouse conditions. In this step, the plant is left for 7 weeks.

To demonstrate that the photosynthetic activity of the donor branch and the distribution of photosynthetic products nourished the plants' grafted plants, all leaves of the recipient plant were removed and the stems covered with aluminum sheets to block from light.

About 2 months after grafting and about 3 months after planting in soil, the plants were placed in a greenhouse until potato tubers were harvested. The result of the grafting experiment is shown in FIG.

Claims (15)

In a method for increasing plant yield, Recombinant DNA molecules; (a) a region that specifically transduces in half cells; And operably coupled thereto (b) a protein having enzymatic activity to remove sucrose; (Ii) sucrose carriers; (Iii) a protein having an activity of stimulating proton gradient present in the plasma membrane of plant cells; And (Iii) A recombinant DNA molecule comprising a nucleotide sequence encoding a polypeptide selected from the group consisting of citrate synthase, which is stably integrated into the genome of a plant. The method of claim 1, The nucleotide sequence encodes a plant protein. The method of claim 1, Nucleotide sequences encode proteins from bacteria or fungi. The method of claim 1, The nucleotide sequence encodes a protein having an enzymatic activity to remove sucrose, selected from the group consisting of sucrose synthase, sucrose phosphorylase and invertase. The method of claim 1, The nucleotide sequence encodes a sucrose carrier from Spinacia oleracea. The method of claim 1, The nucleotide sequence encodes a proton ATPase. The method of claim 6, The nucleotide sequence encodes a proton ATPase from either solanium tuberosum or Saccharomyces cerevisiae. The method according to any one of claims 1 to 7, The region mentioned in (a) is a rolC promoter of Agrobacterium rizogen. (a) a region that specifically transduces in plant half cells; And operably coupled thereto (b) (iii) sucrose synthase; (Ii) sucrose phosphorylase; (Iii) sucrose carriers; (Iii) a protein having an activity of stimulating a proton gradient present in the plasma membrane of the plant cell; And (Iii) A recombinant nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide selected from the group consisting of citrate synthase. A vector comprising the recombinant nucleic acid molecule of claim 9. The method of claim 10, The vector is characterized in that it is suitable for transformation of plant cells and integration of foreign DNA into the plant genome. A plant cell transformed with the recombinant nucleic acid molecule of claim 9 and containing the same. The method of claim 12, Said plant is characterized in that it exhibits an increased production compared to the corresponding non-transforming plant due to the expression of the recombinant nucleic acid molecule in the plant half cell. A propagation material of a plant of claim 13 containing a plant cell of claim 12. Regions that specifically transduce in plant half cells; And (iii) a protein having an enzymatic activity to remove sucrose, which is operably bound thereto; (Ii) sucrose carriers; (Iii) a protein having an activity of stimulating proton gradient present in the plasma membrane; And (iii) a recombinant nucleic acid molecule wherein the recombinant nucleic acid molecule containing a nucleotide sequence encoding a polypeptide selected from the group consisting of citrate synthase is used for expression in a transgenic plant for increasing production.