DE4306627C1 - Method for increasing the sugar and starch content in plants - by a vector including transcription regulatory sequences and a cDNA fragment from Poty virus for inclusion proteins - Google Patents

Abstract

Claimed is a method for increasing the sugar and starch content of plants by (a) producing a construct combining constitutive or tissue specific transcription regulatory signals with a cDNA fragment of a Poty virus encoding the cytoplasmic inclusion protein CI or nucleic inclusion proteins NIa or NIb and (b) plant transformation with (a). Pref. the construct is stably integrated into the plant genome by (i) transformation using Agrobacterium, (ii) particle gun bombardment, (iii) naked DNA transfer. By depositing specific proteins as inclusion bodies in plants, the content of free amino acids is lowered and the influx of saccharose is increased. This is partic. important for sugar beet where free amino acids inhibit the quantitative crystallisation of the sugar. USE/ADVANTAGE - An increase in the starch and sugar content of plants increases their value as food. It may be used in sugar-storing plants, starch-storing plants and in grains. The method is independent of the plant species.

Description

The invention relates to a method for increasing the sugar and starch content of plants through the expression of genes, those for proteins with the property of being deposited in the Plant cell in quasi-crystalline structures (inclusion bodies) encode, as well as transgenic plants, transgenic plant parts and Seeds of transgenic plants by the through the Erfin increased sugar and / or starch content are drawn.

It is generally known that genetic engineering Techniques to express foreign genes in plants to let. This can be done once by known methods Genes can be integrated into the plant genome. This as sta bile transformation known technique is routinely based on ver carried out different ways, e.g. B. by "particle gun bom bardment "(see M.E. Fromm and others" Inheritance and expression of chimeric genes in the progeny of transgenic maize plants ". Bio / Technology 8: 833-839, 1990), by "naked DNA transfer" (see P. Meyer et al. "A new petunia flower color generated by transformation of a mutant with a maize gene ". Nature 330: 677-678, 1987) or Agrobacterium -mediated stable integra tion of genes or gene segments in the genome of the recipient  plant (see R. Walden "Genetic Transformation in Plants", Open University Press, Milton Keynes, 1988). As alternative for chromosomal integration of foreign genes z. B. extra chromosomally replicating vectors derived from plant viruses (see J.W. Davies and J. Stanley: "Geminivirus genes and vectors ". Trends Genet. 5: 77-81, 1989) to express foreign genes without integration into plants Ren. The foreign genes to be expressed in plants must to the for this purpose under the control of regulatory signals (promo tor, terminator), which are either for a consti tutive, tissue and / or development specific transcription to care. It is also desirable to use a strong promoter to an increased mRNA synthesis of the foreign gene cause.

Carbohydrates such as starch and sugar are supplying plants As is well known, it is of great importance for food rich, but also invaluable for industrial applications fertilize.

It is also known that the phloem as the conduction tissue Assimilate, d. H. mainly the disaccharide sucrose Photosynthesis in plants from their place of synthesis, the leaf a storage organ, where it from the phloem in Par enchym cells and either in the form of sugar, e.g. B. in the beet of the sugar beet, or after enzymatic poly merization in strength, e.g. B. in the tuber of the potato, abge is stored. The molecular mechanisms of this transport of Sucrose and nitrogen compounds, from leaf to Storage organ, have not yet been clarified in detail (cf. M.B. Peoples and R.M. Gifford: "Long-distance transport of ni trough and carbon from sources to sinks in higher plants ", in: D.T. Dennis and D.H. Turpin: "Plant Physiology, Biochemistry and Molecular Biology ". pp. 434-447, Longman Group, Singapore, 1990).

Finally, it is also known that the potyviruses which the largest and most economically important group of plant viruses  and infect both mono- and dicotyledons, three of their viral gene products, namely the cytoplasmic one Inclusion protein CI and the nuclear inclusion proteins NIa (one of the viral proteases) and NIb (the putative viral Replicase) as quasicrystalline compounds (inclusion bodies) in the cytoplasm (CI) or in the nucleus (NIa, NIb) of the infected Deposit cell (see R.I.B. Francki et al .: "Atlas of Plant Viruses ". Vol. II, CRC Press, Boca Raton, 1985).

The object of the invention is to plant a generally applicable species-independent process to increase the sugar and / or To provide starch in transgenic plants.

Surprisingly, it was found that the task can be solved with a method that is labeled by

• a) the production of a construct by combining kon statutory or commercial transcription regulations signals with a cDNA fragment of a Potyvirus, which for the cytoplasmic inclusion protein CI or the nuclear inclu ion proteins encoded NIa or NIb, and
• b) the transformation of plants with the construct obtained in a manner known per se.

Without a specific theory of how it works of the process according to the invention can be bound suspect that with an assumed passive sugar transport other intracellular metabolites such as amino acids nen positive influence on the sugar diffusion gradient ha ben when the concentration of free amino acids by an ef fective conversion into protein (s) is decreased intracellularly, which is particularly the case when there is a small pro content such as B. in the sugar beet (1.2%) or potato (2%) noticeable. Hence the cellular synthesis of Proteins that are in the form of quasicrystalline structures in the Deposit the plant, a dramatic effect on the sucrose- Inflow as these proteins do not depend on cellular proteases are built and therefore they raise the free amino acid level lower.  

According to the invention, expression of a protein is also used certain physicochemical properties, namely the ability speed, as a quasi-crystalline structure (inclusion body) in Plants deposit as a result of the free amino acid content decreased and the influx of sucrose increased. You can the same for carrying out the method according to the invention Constructs used in the older German Pa tent registration P 42 39 026.5 described and to increase the Protein content of plants can be used.

According to a first embodiment of the invention, the Kon structures into Agrobacterium-mediated transformation Plant genome stably integrated.

According to a second embodiment of the invention, the Kon structures through particle gun bombardment into the plant genome stably integrated.

According to a third embodiment of the invention, the Kon is stable through bare DNA transfer into the plant genome integrated.

According to a fourth embodiment of the invention, the Construct with the help of extrachromosomally replicating, viral Vectors expressed in the plant.

The method according to the invention is of particular importance for the increase in the starch content of tuber plants whose the most important representatives at the moment are: batate, potatoes, yam, Cassava, taro and tania.

The process according to the invention is of very special importance However, for highly sugar-storing crops such as the sugar beet. This procedure results in one in addition to the increased sugar content, a desirable side effect. The industrial processing of sugar beet for production of sugar takes place in such a way that the sucrose by Kri  stallization is obtained from an aqueous solution (molasses). Free amino acids in the molasses prevent the quantitative Crystallization of the sugar. The procedure described increases but not only the sucrose inflow into storage organs, son at the same time it lowers the proportion of free amino acids.

Although the method according to the invention is of particular importance for sugar-storing crops, it can be also use in the case of other plants, such as the plants mentioned and in the case of cereals by deposition of the protein in the storage organs (grains).

A compilation of currently known potyviruses, which are known as Suppliers of cDNA fragments are eligible for the method according to the invention is suitable can be found in the R.I.B. Francki, R.G. Milne, T. Hatta: "Atlas of Plant Viruses", Vol. II, CRC Press, Boca Raton 1985.

Agrobacterium strains into which this is found in stage a) posed construct can be introduced, z. B. Agrobacterium tumefaciens and Agrobacterium rhizogenes.

The total sequence is for four of the potyviruses known to date of the viral RNA genome and the location of each Sequences encoding viral proteins from an overview articles by Riechmann u. a. J. Gen. Virol. 73: 1-16 (1992) be knows. So the sequence for the CI gene was accessible, namely for the potyviruses plum pox virus (PPV), tobacco vein mottling virus (TVMV), tobacco etch virus (TEV) and potato vi rus Y (PVY).

According to the z. B. as in the following example Potato will be shown the starch content of the potato Increase by up to 50% while maintaining concentration of free amino acids is reduced by a factor of 6.5 to 7.

The invention will be based on the example of the expression of Potyviral CI protein in the potato explained in more detail the. The method for example shown below The ex shows an increase in the starch content in the potato tuber Pression of the CI gene of a tobacco potyvirus (TVMV) in the hete rologist host potato.

The figures show:

Fig. 1 above: the schematic structure of the RNA genome TVMV
bottom: the TVMV cDNA fragment used for the expression of the cytoplasmic inclusion protein, obtained by DraIII (D) / EcoRV (EV) restriction digest from the TVMV cDNA clone pTVBam7. E: EcoRI restriction sites. The numbers indicate the number of nucleotides in kb.

Fig. 2 The binary plant transformation vector pGDW32. Pnos nopaline synthase gene promoter: pad g4 poly adenylation signals of the T _L -DNA gene 4 : ori _v - and ori _T replication and conjugation transfer. Origins of the plasmids ColE1 and RK2; RB and LB right and left T-DNA border sequences; HPT hy gromycin phosphotransferase gene; Amp ^r Ampicil lin resistance marker. Restriction enzymes: C ClaI; E EcoRI; P PstI; K KpnI; S SacI; Sa SalI; Sm SmaI and XB XbaI.

From a work by
LL Domier; SM Franklin; M. Shahabuddin; GM Hellmann; JH Overmeyer; ST Hiramath; MFE Siaw; GP Lomonosoff; JG Shaw and RE Rhoads: "The nucleotide sequence of tobacco vein mottling virus". Nucleic Acids Res. 14: 5417-5430 (1986) the overall sequence of the TVMV is known. For this purpose, DNA copies (cDNA) of the genomic TVMV-RNA were made and their sequence determined. A 4.7 kb TVMV cDNA in plasmid pTVBam7 (corresponding to the cDNA sequence between the BamHI cleavage sites, nucleotides 1934 to 6648; FIG. 1 above) was developed by Dr. Rhoads, and a 3.3 kb DraIII / EcoRV fragment was isolated from this plasmid ( FIG. 1 below). According to the information in the cited work, this 3.3 kb cDNA from the clone pTVBam7 is defined in more detail by a nucleic acid and a resulting amino acid sequence and contains a continuous open reading frame (ORF) which is suitable for the P3 protein ( 42 kDa), a 6K1 protein (6 kDa) and the cytoplasmic inclusion protein CI (67 kDa).

This 3.3 kb fragment was brought under the control of transcriptional regulatory signals which (1) cause strong constitutive expression in all plant tissues (CaMV 35S promoter and terminator), or (2) predominantly tissue-specific expression in the tuber (promoter of the waxy gene from the potato, (see W. Rohde, D. Becker, B. Kull, F. Salamini: "Structural and functional analysis of two waxy gene promoters from potato". J. Genet. 44: 311-315, 1990) These cDNA fragments provided with regulatory signals have now been cloned into the binary Agrobacterium tumefaciens vector pGDW32 ( FIG. 2), introduced into Agrobacteria cells and stably incorporated using the work technique referred to as transformation transferred the genome of the potato (Solanum tuberosum, cv. Desiree) The calli obtained after selection with the antibiotic hygromycin were regenerated to plants in tissue culture and in vitro for a first protein test high sucrose concentrations stimulated the formation of tubers. Although the results of the protein determinations on the tubers of 10 independent transformants with the waxy / CI construct and of 6 transformants with the CaMV 35S / CI construct showed a dramatic increase in the protein concentration, all transformants showed a comparison with the non-transformed one Control Desiree an absolutely abnormal phenotype. To rule out an artifact, the in vitro tubers for the wx / CI lines 1 , 4 , 8 , 9 , 10 were germinated in the greenhouse in normal soil and the ripened tubers were subjected to a further analysis. In the phenotype, these tubers were indistinguishable from those of the non-transgenic control plant, and the protein values showed good agreement with the values obtained for in vitro tubers.

Embodiment 1) Media used 1a) Media for the cultivation of bacteria

YEB medium: 0.5% sucrose, 0.5% peptone, 0.5% beef extract and 0.1% yeast extract, pH 7.4;
1.5% agar agar was added for solid medium.

1b) Media for plant tissue culture

Murashige and Skoog developed the plant tissue culture (1962) described base salt mixture used.

MSI medium (after agrobacterial transformation):
MS medium with 3% glucose.

MSII medium (callus and shoot medium):
MS medium with 3% glucose, 2 mg / l zeatin riboside, 0.02 mg / l naphthylacetic acid, 0.02 mg / l Gibberelic acid A ₃ , 500 mg / l cefotaxime and 10 mg / l hy gromycin.

MSIII medium (root medium):
analogous to MSII medium but without hormones.

MS8 medium (tuber medium):
MS medium with 8% sucrose and antibiotics.

0.8% agar agar was added for solid media.

2) Subcloning the sequence of the cytoplasmic inclusion sionsprotein (CI) of the Tobacco Vein Mottling Virus (TVMV) 2a) Isolation of the 3.3 kb CI fragment

The starting material was that of Dr. RE. Rhoads (Universi Kentucky) provided plasmid clone pTVBam7. The It contains ≈4.7 kb (corresponding to nucleotides 1934 to 6648) of the TVMV sequence (Domier et al., Nucleic Acids Res. (1986), 14: pp. 5417-5430, 1986) in the BamHI site of the pUc119. DNA of this clone was extracted with the restriction enzymes DraIII and EcoRV cut. After separation of this restriction approach a DNA band of 3.3 kb was found on a 1% agarose gel cut out. The DNA was electroeluted from the gel and cleaned over DEAE cellulose. The 3.3 kb DraIII / EcoRV Fragment comprises the sequence for the helper component (120 3′- terminal nucleotides), the complete P3 sequence and the ge encode all of the cytoplasmic inclusion protein (CI) de sequence except for the 3'-terminal 23 nucleotides. To the subclo The 3.3 kb DraIII / EcoRV fragment with the Enzyme mung bean nuclease treated. For easier understanding the DraIII / EcoRV fragment is referred to as a CI fragment.

2b) Connection of the binary vector system pGDW32 with Trans description signals

From a pBR plasmid which contains the promoter and the terminator of the Carries 35S transcripts of the Cauliflower Mosaic Virus (CaMV), was the CaMV 35S promoter / Ter with the restriction enzyme EcoRI minator fragment (0.79 kb) cut out and as before ter 2a), cleaned.

Furthermore, from another pBR plasmid which carries the promoter sequence of the waxy locus (Wx) (from Solanum tuberosum, cv. Granola) (Rohde et al., J. Genet. 44: 311-315, 1990) became a 0.9 kb large Wx promoter fragment isolated. For this purpose, the Wx plasmid was treated with the restriction enzymes EcoRI and BamHI, ie an EcoRI / BamHI-Wx promoter fragment was obtained. The necessary terminator sequence was taken from the previously mentioned pBR plasmid ( 1 ). For this purpose, the plasmid ( 1 ) with the restriction enzyme BamHI was opened between the CaNV 35S promoter and terminator sequence and cut with the restriction enzyme EcoRI. The 0.25 kb BamHI / EcoRI terminator fragment was purified as described under 2a). A new promoter / terminator unit consisting of Wx promoter and CaMV 35S terminator was constructed using the overlapping BamHI interface.

The binary vector system was used for plant transformation by Wing and co-workers (Mol. Gen. Genet. 219: 9-16, 1989) used. This vector pGDW32 was with the restriction enzyme EcoRI opened in the T-DNA and with the calfintestine phospha tase (CIP) dephosphorylated. Each one of the above described promoter / terminator units (CaMV 35S promoter / terminator or Wx promoter / CaMV 35S terminator) was inserted into this EcoRI Interface subcloned. Thus, the two plant vectors ren pPTGDW with CaNV 35S promoter / terminator) and pGWX (pGDW32 with Wx promoter / CaMV 35S terminator).

2c) Subcloning of the CI fragment into the plant vectors pPTGDW and pGWX

Each of the plant vectors described in 2b) was compared with the Restriction enzyme BamHI between the corresponding promoter and terminator sequence opened and with the enzyme mung bean nu treated and dephosphorylated. In the so pretreated Plant vectors became the 3.3 kb described under 2a) CI fragment subcloned. These subclonings were carried out on 3'-end of the CI coding sequence generated a stop codon.

2d) Introduction of the CI constructs into Agrobacterium tume faciens

Both promoter / CI constructs were made using the direct method ten Agrobacterium tumefaciens transformation in the Koncz and Schell in Mol. Gen. Genet. 204: 383-396 (1986) an agrobacterial strain GV3101 (pMP90RK) was transformed. Therefor a bacterial colony of the strain was transferred to 5 ml of YEB medium Dressed at 28 ° C overnight. The next day, this was Bak in suspension of 100 ml YEB medium and inoculated for 4 h at 28 ° C shaken, in a Heraeus Minifuge RF at 6000 rpm centri fugiert and the pellet resuspended in 1 ml of YEB medium. To each because 200 ul of this suspension were 200 ng of 2c) be added written constructs and in liquid nitrogen frozen. After a heat shock of 5 min at 37 ° C 0.8 ml of YEB medium was added and incubated at 28 ° C. for 1 h. By Growth at 28 ° C under selective conditions (100 mg / l rifam picin, selection for the bacterial chromosome; 25 mg / l Kanamy cin, selection for the helper plasmid pMP90RK; 100 mg / l carbeni cillin, selection for plasmid pGDW32) were then plasmid containing bacterial cultures isolated.

By means of the "Southern blot" analysis it was checked that the bacterial clones isolated the recombinant plasmids of pGDW32 with the corresponding intact transcription units sat.

3) Transformation of Solanum tuberosum with the selected agrobacteria 3a) Cultivation of bacteria

Individual bacterial cultures were grown in 5 ml of YEB medium with the antibiotics mentioned in 2d) at 28 ° C. for 2 days. With this culture, 100 ml of antibiotic-containing YEB medium was inoculated and shaken at 28 ° C. overnight. The bacterial culture was centrifuged for 10 min at 4 ° C and 6000 rpm in a Heraeus Minifuge RF. The pellet was resuspended in 20 ml of a 10 mM MgSO ₄ solution, centrifuged again and resuspended in 20 ml of MSI medium.

3b) Leaf disc transformation

Solanum tuberosum plants of the variety D´sir´e were on MSII Medium (without antibiotics) has been attracted at a day / night Alternation of 16 h light / 24 ° C and 8 h dark / 18 ° C. leaves were cut off at the base under sterile conditions and with each of the above bacterial suspensions for 1 hour incubated in the dark. The leaves were then placed on MSI Plates were placed and incubated for two days in the dark at 24 ° C. Then the leaves were washed in MSI medium with cefotaxime and put on MSII plates (with antibiotics) and at the above described day / night change held. Since the T-DNA of the Plasmid pGDW32 carrying a hygromycin resistance gene was unable to Hygromycin can be selected on those plant cells that had taken up this T-DNA. The leaves were removed every 14 days transferred to fresh plates. After the shoots were formed these are cut off from the callous tissue and on MSIII plates rooted. For induction of tubers in vitro internodes of the rooted shoots on MS8 Plates set. Similarly, internodes of were not transformed wild-type potato plants (cv. D´sir´e) Brought MS8 medium (without antibiotics).

4) Evidence of the expression of the TVMV CI fragment under Kon troll the CaMV 35S promoter or the Wx promoter Solanum tuberosum (cv. Granola)

Transformed plants were checked by DNA analysis ("Southern blotting") for the presence of the corresponding promoter / 3.3 kb TVMV CI fragment / terminator constructs. The expression of the CI fragment (P3 and CI protein) was detected by analyzing a total protein extract from transgenic lines in the polyacrylamide gel. For this purpose, 400 mg of plant material was crushed with quartz sand (cf. C. Lawson et al.: "Engineering resistance to mixed virus infection in a commer cial potato cultivar: resistance to potato virus X and potato virus Y in transgenic Russet Burbank." Bio / Technology 8 : 127-134, 1990). After adding 3 ml of Laemmli buffer (62.5 mM TRIS-HCl pH 8, 2% SDS, 10% glycerin, 1% DTT, 5% mercapto ethanol), the samples were heated at 100 ° C for 15 minutes and then after quartz sand and solid plant residues removed by centrifugation (15 minutes, 4000 rpm). Proteins were precipitated from each 2.5 ml of the supernatant with 12.5 ml acetone and sedimented by subsequent centrifugation (15 minutes, 4000 rpm). The protein precipitate was washed twice with 70% acetone and then dissolved in 300 μl MTPBS (150 mM NaCl, 16 mM Na ₂ HPO ₄ , 4 mM NaH ₂ HPO ₄ , 8 urea, pH 7.3). The protein determination was carried out according to Bradford (cf. MM Bradford: "A rapid and sensitive method for quantitation of microgram quantities of proteins using the principle of protein-dye bin ding." Anal. Biochem. 72: 248-254, 1976). For the gel electrophoretic detection of the expressed TVMV proteins (P3 + CI, CI, P3; note: the P3 protein is obviously a protease and cleaves from the P3 + CI precursor), part of the extract was precipitated again, in Laemmli Buffer denatured by heating and separated on a 10% polyacrylamide gel (cf. UK Laemmli: "Cleavage of structural proteins during the assembly of the head of bacteriophage T4." Nature 227: 681-685, 1970). The separated proteins were made visible by staining with Coomassie blue.

5) Studies on the total nitrogen and starch content of Tubers of CI-transgenic potato lines

Parts of three tubers of CI transgenic plants and the Desiree's non-transgenic starting line were ge under vacuum dries. The determination of total nitrogen and starch was held in the analytical laboratory of the company Roquette Freres (Lestrem, France) performed. There were those in the get the following table:  

table

Determination of total nitrogen, protein and starch

Claims (14)

1. Process for increasing the sugar and starch content of plants, characterized by

• a) the production of a construct by combining constitutive or tissue-specific transcription regulation signals with a cDNA fragment of a potyvirus, which codes for the cytoplasmic inclusion protein CI or the nuclear inclusion proteins NIa or NIb, and
• b) the transformation of plants with the construct obtained in a manner known per se.

2. The method according to claim 1, characterized shows that the construct can be identified by Agrobacterium mediated transformation into the plant genome stable inte freezes. 3. The method according to claim 1, characterized draws that the construct by particle gun bombardment is stably integrated into the plant genome. 4. The method according to claim 1, characterized shows that the construct can be identified by naked DNA Transfer integrated into the plant genome stably.   5. The method according to claim 1, characterized net that the construct with the help of viral vectors increased by extrachromosomal replication and for expression brings. 6. The method according to claim 1, characterized by

• a) the production of a construct by combining constitutive or tissue-specific transcription regulation signals with a cDNA fragment of a potyvirus which codes for the cytoplasmic inclusion protein CI or the nuclear inclusion proteins NIa or NIb,
• b) introducing the construct obtained into an Agrobacterium, and
• c) Transformation of plants with the selected agrobacteria.

7. The method according to any one of claims 1, 2, or 6, characterized characterized in that one of a cDNA fragment the potyviruses TEV, TVMV, PPV or PVY runs out. 8. The method according to any one of claims 1 to 7, characterized characterized in that one of a cDNA fragment which is obtained by cutting the cDNA clone pTVBam7 of the potyvirus TVMV with the restriction enzymes DraIII and EcoRV. 9. The method according to any one of claims 1 to 8, characterized characterized in that one of a cDNA fragment assumes that the information for the TVMV protein P3 contains.   10. The method according to claim 6, characterized records that in stage b) the construct in agro bacterium tumefaciens or Agrobacterium rhizogenes. 11. The method according to any one of claims 1 to 5, characterized characterized in that the construct in the Genome of sugar and starch-storing crops stable integrated. 12. The method according to any one of claims 1 to 9, characterized characterized in that in step c) with the selected Agrobacteria storing sugar and starch Cultivated plants transformed. 13. The method according to claim 11, characterized draws that the construct into the genome of Potato plants stably integrated. 14. Transgenic plants, transgenic parts of plants and seeds of transgenic plants produced by the method according to one of the Claims 1 to 14 were obtained.