WO2001011062A2 - Accumulation de polyamine dans les plantes - Google Patents

Accumulation de polyamine dans les plantes Download PDF

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WO2001011062A2
WO2001011062A2 PCT/IB2000/001185 IB0001185W WO0111062A2 WO 2001011062 A2 WO2001011062 A2 WO 2001011062A2 IB 0001185 W IB0001185 W IB 0001185W WO 0111062 A2 WO0111062 A2 WO 0111062A2
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plant
transgenic
levels
promoter
polyamine
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PCT/IB2000/001185
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WO2001011062A3 (fr
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Paul Christou
Teresa Capell
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John Innes Centre
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Definitions

  • PAs Polyamines
  • PA putrescine
  • ADC arginine decarboxylase
  • ODC ornithine decarboxylase
  • spermidine and spermine synthases which add propylamino groups generated from decarboxylated S-adenosyl-methionine (dcSAM) by SAM decarboxylase (SamDC; Mahnberg et al., Critical Rew. in Plant Sci.. 17(2): 199-224 (1998)).
  • Polyamines are oxidatively deaminated by the action of amine oxidases.
  • Amine oxidases include, e.g., copper diamine oxidase (EC 1.4.3.6); these enzymes are characterized by their substrate specificity towards diamines.
  • the flavoprotein polyamine oxidases (EC 1.5.3.3), oxidize Spd and Spm at their secondary amino groups (Tiburcio, et al, Physiol. Plant, 100:664- 674 (1997)).
  • Diamine oxidase oxidizes the primary amino group of Put and Spd with the formation of pyrroline (from Put) and aminopropylpyrroline (from Spd) along with ammonia and hydrogen peroxide (Smith, T.A., Biochem. Soc. Trans., 13 (1988) 319-322).
  • Polyamine oxidase yields pyrroline and aminopropylpyrroline, from Spd and Spm, respectively, along with 1,3-diaminopropane and hydrogen protein.
  • Diaminopropane can be converted into ⁇ -alanine, whereas pyrroline can be further catabolized to ⁇ -aminobutyric acid in a reaction catalyzed by pyrroline dehydrogenase
  • PAs have been implicated in a wide range of biochemical processes in plants and animals including DNA replication, transcription, protein synthesis, membrane stabilization and RNA and protein turnover and PAs may mediate or regulate processes such as, e.g., cell growth and division, morphogenesis and development and stress responses (Evans and Malmberg, Annu Rev Plant Phvsiol. Plant Mol. Biol. 40: 235-269 (1989): Bardoc etal.. Nutr. Biochem., 4:66-70 (1993)).
  • Burtin and Michael (Biochem. J.. 325:331-337 (1997)) generated transgenic tobacco plants constitutively expressing oat adc cDNA. These accumulated agmatine, a precursor of Put in the ADC pathway, to levels 20 to 65-fold higher than control plants, but there was no significant change in PA levels.
  • Masgrau et al. (The Plant J.. l l(3):465-473 (1997)) generated transgenic tobacco plants carrying the tetracycline- inducible oat adc cDNA. Put levels in these plants increased only after tetracycline administration to excised leaf discs from primary transformants.
  • transgenic plants, plant parts, and propagules that have an enhanced amount of total polyamines as compared to non-transgenic plants of the same species. It also is an object of the present invention to provide transgenic plants, plant parts, and propagules that have an enhanced level of one or more particular polyamines as compared to the average level of the particular polyamines in like non-transgenic plants or plant parts of the same species.
  • transgenic plant comprising an integrated nucleic acid molecule, which encodes an enzyme of the polyamine metabolic pathway.
  • the activity of the enzyme in the transgenic plants is increased as compared to the activity in a non-transfo ⁇ ned plant and the level of total polyamines or the level of particular polyamines, e.g., putrescine, spermine or spermidine, in the transgenic plants is increased over the level of the polyamines in a non-transformed plant of the same species.
  • transgenic plants or plant parts of this invention include, e.g., single cells, callus tissue, leaf discs, and irnmature or mature embryos, hypocotyls and cotyledons, shoots, roots, leaves, and seeds, and propagules thereof having significantly elevated levels of one or more polyamines, e.g., putrescine, spermine, or spermidine, as compared to the levels in control plants.
  • polyamines e.g., putrescine, spermine, or spermidine
  • the transgenic plants or plant parts of this invention may be dicotyledonous or monocotyledonous plants, preferably monocotyledonous and more preferably the plants, plant parts or propagules are selected from the group consisting of barley, corn, millet, oat, rye, rice, wheat or amaranth.
  • the total polyamine levels are increased in the transformed plants or plant parts.
  • the plants or plant parts contain at least 1.5 times the level of polyamines generally found in control plants. More preferably the polyamine levels in the transgenic plants or plant parts are at least 2.5 times the level found in the control plants or plant parts, most preferably the levels are at least four to six times the levels found in the control plants or plant parts.
  • the levels of putrescine, spermidine and spe ⁇ riine are all elevated in the transgenic plant as compared to the control plants or plant parts.
  • the transgenic plants and plant parts of this invention have levels of spermine that are at least 1.5 times the level found in the control plants, preferably the level of spermine in the transgenic plants are at least three times the level and most preferably at least four to six times the level of spermine in the control plants.
  • the transgenic plants and plant parts of this invention may also have elevated levels of spermidine as compared to a control plant.
  • the level of spermidine is at least 1.5 times that of a control plant or plant part, more preferably the level is at least three times the level in the control plant or plant part and most preferably the level of spermidine is at least four times the level in control plants or plant parts.
  • the plants or plant parts of this invention may also contain elevated levels of both spermidine and spermine.
  • the transgenic plants and plant parts of this invention have more than 1.5 times the level of putrescine as compared to a control, e.g., a like plant or plant part that is not transgenic.
  • the transgenic plant has a level of putrescine that is greater than three times the level in a control plant or plant part that is not transgenic. More preferred is a transgenic plant having a level of putrescine that is more than five times the level in a control plant or plant part that is not transgenic.
  • These plants may also have an elevated level of more than one polyamine, e.g., putrescine and spermidine, or putrescine and spermine, or all three.
  • the level of spermidine is at least about 1.5 times the level found in a control plant or plant part that is not transgenic and more preferred the level of spermidine is more than three times the level in a control plant or plant part.
  • the spermine level is more than at least about 1.5 times and more preferably more than at least about three times the level of spermine in a control plant or plant part.
  • transgenic plant comprising an integrated nucleic acid molecule that produces an antisense RNA, e.g., a transcribed strand that is complementary to an endogenous mRNA in the plant, wherein the endogenous mRNA encodes an enzyme of the polyamine catabolic pathway.
  • an antisense RNA e.g., a transcribed strand that is complementary to an endogenous mRNA in the plant, wherein the endogenous mRNA encodes an enzyme of the polyamine catabolic pathway.
  • the transgenic plants of this invention containing an antisense RNA may have total polyamine levels that are at least about 1.5 times the level of the total polyamine in non-transformed plant or the polyamine levels of particular polyamines, e.g., putrescine, spermine or spermidine, are at least about 1.5 fold the levels of the particular polyamines in a non-transformed plant, or both the levels of the total polyamines and particular polyamines are at least about 1.5 times the levels found in non-transformed plants.
  • the levels of particular polyamines are at least about 3.5 times the levels and more preferably at least about 6 times the levels found in non-transformed plants or plant parts or the same species.
  • an aspect of this invention are seeds of the transgenic plants that have total polyamine levels that are at least about 1.5 times the level of the total polyamine in non- transformed seed or wherein the polyamine levels of particular polyamines are at least about twice the levels of the particular polyamines in a non-transformed plant, or both.
  • This invention also relates to methods for producing transgenic plants, plant parts, and propagules thereof having elevated levels of polyamines by altering the activity levels of enzymes in the catabolic and metabolic polyamine pathways.
  • One embodiment of this invention relates to methods comprising the introduction of a nucleic acid molecule, which encodes an enzyme of the polyamine biosynthetic pathway, into a plant cell wherein the nucleic acid molecule is in operable linkage with a promoter.
  • a suitable enzyme of the polyamine biosynthetic pathway may be, e.g., arginine decarboxylase or ornithine decarboxylase.
  • the promoter has a strength that is sufficient to increase the level of the enzyme to a level such that spermidine or spermine, or both spermidine and spermine accumulate within the transgenic plant or plant part to at least about 1.5 times the level of spermidine or spermine in control plants or plant parts.
  • the promoter is a strong promoter and may be constitutive or inducible.
  • the promoter may also be a tissue specific promoter.
  • the promoter has a promoter strength that is not significantly weaker than the maize ubiquitin- 1 promoter.
  • the promoter is a strong constitutive promoter. More preferably the promoter is the maize ubiquitin- 1 promoter or the pea nodulin promoter.
  • the tissue specific promoter is a seed specific promoter such as, e.g., a glutelin promoter. More, preferably, the promoter is the rice glutelin promoter or the wheat low molecular weight glutenin promoter.
  • This invention also relates to vectors, which are suitable for transforming plant cells, comprising a nucleic acid molecule, which encodes an enzyme of the polyamine metabolic pathway, wherein the nucleic acid molecule is in operable linkage with a promoter.
  • the promoter has a strength sufficient to increase the production of the enzyme to a level such that spermine accumulates within the transgenic plant or plant part to at least 1.5 times the level of spermine in control plants or plant parts.
  • the level of polyamines in the transgenic plant is 3.5 times the level and more preferred at least 6 times the level as compared to the levels in control plants or plant parts.
  • Another aspect of this invention is an antisense expression vector which provides the antisense molecules, e.g., transcripts that are complementary to an endogenous mRNA in the plant, wherein the endogenous mRNA encodes an enzyme of the catabolic pathway.
  • the antisense molecule inhibits the production of the enzyme.
  • the expression vector comprises a nucleic acid molecule in operable linkage with a promoter and may also comprise additional sequences that control the processing of transcripts, e.g., a polyadenylation/termination sequence or an enhancer sequence.
  • the level of antisense expression is sufficient to reduce the catabolic enzyme activity to a level sufficient to increase the level of total polyamines or particular polyamines.
  • the level of antisense expression is sufficient to reduce the catabolic enzyme activity by at least about 30% of the level in a control plant or plant part. More preferably the antisense expression is sufficient to reduce the level of the catabolic enzyme by at least about 60% of the level in a control plant or plant part.
  • the methods of this invention also include fransforming plants cells with a nucleic acid molecule, which produces an antisense RNA molecule.
  • the antisense RNA is complementary to an endogenous nucleic acid molecule that encodes an enzyme of the polyamine catabolic pathway, e.g., diamine oxidase (DAO) or polyamine oxidase (PAO).
  • DAO diamine oxidase
  • PAO polyamine oxidase
  • the "antisense" nucleic acid molecule, which produces the antisense RNA comprises a cDNA that encodes an enzyme of the polyamine catabolic pathway and is in operable linkage with a promoter.
  • the cDNA is in antisense orientation relative to its endogenous orientation.
  • the cDNA encodes a diamine oxidase.
  • Antisense RNA useful in this invention is of sufficient length that it inhibits synthesis of the enzyme encoded by the endogenous RNA.
  • the antisense RNA is at least about 100 nucleotides in length. More preferably the antisense RNA is at least about 150 nucleotides in length and most preferably the antisense RNA is at least about 200 nucleotides in length.
  • the antisense RNA should share significant homology with the sequence of the endogenous nucleic acid molecule that encodes the enzyme of the polyamine catabolic pathway.
  • the cDNA may be prepared from RNA isolated from a mammalian or plant source. The plant source may be either dicotyledonous or monocotyledonous, preferably monocotyledonous.
  • the nucleic acid molecule is a cDNA that encodes an enzyme, or fragment thereof, of a polyamine catabolic pathway, preferably, a plant polyamine catabolic pathway.
  • the cDNA may also be from a 5' untranslated region (UTR) sequence of the mRNA encoding the enzyme.
  • the enzyme of the polyamine catabolic pathway is a diamine or polyamine oxidase, more preferably a plant diamine or polyamine oxidase.
  • transgenic plant or plant part having elevated levels of total polyamines or one or more particular polyamines, produced by fransforming a plant cell with a first nucleic acid molecule, which codes for an enzyme of the polyamine metabolic pathway, and a second nucleic acid molecule, which produces an antisense RNA that has sufficient length that it interferes with the production of a polyamine catabolic enzyme; selecting transformed plant cells, and; regenerating transgenic plant having elevated levels of at least one polyamine selected from the group consisting of putrescine, spermidine and spermine.
  • the plant cell is a monocotyledonous or dicotyledonous plant cell and more preferably the plant cells is selected from the group consisting of barley, corn, milo, oat, rice, wheat and amaranth.
  • the plant part is a seed.
  • the plant cell may be co-transformed simultaneously with the first and second nucleic acid molecules or sequentially so that the plant cells are transformed with either the first or second nucleic acid molecule and subsequently transformed with the other molecule.
  • Plant cells may be transformed by using any suitable DNA transfer technology, such as, e.g., Agrobacterium mediated transfection, particle or microprojectile bombardment (US 5100792, EP-A-444882, EP- A-434616, Altpeter et al., Plant Cell Rep.. 16: 12-17 (1996) or Klein et al., Nature.
  • Agrobacterium mediated transfection, particle or microprojectile bombardment US 5100792, EP-A-444882, EP- A-434616, Altpeter et al., Plant Cell Rep.. 16: 12-17 (1996) or Klein et al., Nature.
  • the plant cells are transformed by particle or microprojectile bombardment.
  • the DNA may be in the form of a minimal transgene expression cassette as described in co-pending application Serial No. 60/144,513.
  • transgenic plants from transformed cells in culture.
  • a plant may be regenerated, e.g., from single cells, callus tissue, leaf discs, and immature or mature embryos, hypocotyls and cotyledons, as is standard in the art.
  • any plant e.g., rice, wheat, corn, oat, barley, sorghum, legumes, and woody species can be entirely regenerated from cells, tissues and organs of the plant.
  • the generation of fertile transgenic plants has been achieved in the cereals, e.g., rice, maize, wheat, oat, and barley
  • Vasil et al. Bio/Technology, 10:667-674, (1992); Vain et al., Biotechnology Advances, 13:4:653-671 , (1995); Vasil, Nature Biotechnology, 14, page 702 (1996)).
  • Available techniques are also reviewed in Vasil et al., "Cell Culture and Somatic Cell Genetics of Plants", Vol I, II, and III, Laboratory Procedures and Their Applications,
  • Plants according to this invention include any plant part or propagule thereof, seed, selfed or hybrid progeny and descendants.
  • a plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders' Rights. It is noted that a plant need not be considered a "plant variety” simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.
  • a plant according to this invention may also be one that is sterile. For example, a genetically engineered sterile seed and plant that developed from said seed are also contemplated by this invention
  • Fig. 1 Schematic representation of the construct pUbiADCs containing the 2.1 Kb oat adc cDNA in the sense orientation under the control of the maize ubiquitin 1 promoter (Ubi-1) and nos ternrinator (nos tcr ). Restriction sites flanking the cDNA are shown.
  • the probe used for Southern and northern blot analysis was a 1.5 Kb EcoRI fragment DIG- labeled PCR product derived from within the adc coding sequence.
  • Fig. 2 RT-PCR analysis of cDNA ( 1.5 Kb) from total RNA extracted from callus transformed with pUbiADCs in PM. 1.6 Kb maker size is shown in the figure. Lane 1 : molecular size marker Lane 2: positive control pUbiADCs Lane 3 : negative control water
  • Lane 4 wild type transformed with hpt gene
  • Lane 5 to 9 clones 6, 18, 20, 30 and 31 callus
  • Fig. 3 (A) Southern blot analysis of transgenic callus lines. 10 ⁇ g of genomic DNA was digested with Hindlll and hybridized with a 1.5Kb DIG-labeled adc PCR product. Lane 1 : molecular size marker Lane 2: control plants transformed with hpt gene Lane 3 to 10: transgenic clones 6, 15, 18, 20, 30, 31, 34 and 73
  • Fig. 3(B) Northern blot analyzes of total RNA extracted from callus transformed with pUbiADCs. Each lane was loaded with 30 ⁇ g RNA. Lane 1 : control plants transformed with hpt gene
  • Lane 2 to 9 transgenic callus lines fransformed pUbiADCs: 6, 15, 18, 20, 30, 31, 34 and 73
  • Fig. 4 Enzyme ADC (Fig. 4A) and ODC (Fig. 4B)activity in different transgenic plant lines compared to control plants transformed with hpt gene. Values are mean ⁇ SE for three replicates. ADC activity was significantly different from control at p ⁇ 0.05. ODC activities were not significantly different from control.
  • Fig. 5 Cellular PA levels in control plants (C) transformed with hpt gene and different transgenic callus lines. Values are mean ⁇ SE for three replicates. Total PA levels (5D), Put (5 A), Spd (5B) and Spm (5C) levels when were significantly different from control were at O.05.
  • Fig. 6 Polyamine levels in leaves from z t-control plants (1) and transgenic plants (R Q ) containing adc driven by the ubi promoter (2 and 3).
  • Fig. 7 Polyamine levels in seeds from /z/?t-control plants (1) and transgenic plants containing adc driven by the ubi promoter (R )(2).
  • Fig. 8 Polyamine levels in seed from /z/?t-control plants (1) and transgenic plants containing the oat ADC cDNA driven by the 35S promoter (2-7).
  • Fig. 9 Polyamine content in rice seeds from transgenic plants boiled for up to 10 minutes.
  • Fig. 10 Southern blot analysis of transgenic callus lines. Ten micrograms of genomic DNA were digested with Sphl and probed with the 1.8 Kb DIG-labeled PCR product. Lane 1 : molecular size marker ( 1 Kb DNA Ladder, GIBCO BRL, UK) Lanes 2-5 and 7-8: representative transgenic clones Lane 6: wild-type control
  • Fig. 11 RT-PCR analysis of cDNA (1.8 Kb) from total RNA extracted from callus transformed with p35S ⁇ a.
  • Lane 1 molecular size marker (1 Kb DNA Ladder, GIBCO BRL, UK)
  • Lane 2 positive control, plasmid p35S ⁇ oa
  • Lane 3 negative control (water)
  • Lane 4 wild-type control callus
  • Lanes 5 to 1 1 transgenic callus lines transformed with p35s ⁇ a: clones 6, 1 1, 12, 14, 20,
  • Fig. 12 Northern blot analyses of total RNA extracted from callus transformed with pEdaoa. Each lane was loaded with 40 ⁇ g of total RNA and probed with the 1.8 Kb DIG- labeled PCR product.
  • Lane 1 molecular size marker (0.24-9.5 Kb Ladder GIBCOBRL, UK)
  • Lanes 3 to 9 transgenic callus lines: clones 11, 12, 13, 17, 19, 22, and 23, respectively
  • Fig. 13(B) Displays DAO enzyme activity in different transgenic callus lines transformed with pE ⁇ zoa. DAO activity in clones 7, 13, 17, 19, 22 and 13 was significantly different from controls at P ⁇ 0.01. Remaining values were not significantly different from control levels at P > 0.05.
  • 14(B) Demonstrates that the Spd levels in callus lines 14 and 37 are significantly different from controls at P ⁇ 0.01 ; that the Spd level in clone 6 is significantly different from levels in controls at P ⁇ 0.05. Remaining values were not significantly different from control levels at P > 0.05.
  • 14(C) Demonstrates that the Spm levels in clone 6 significantly different from controls at P ⁇ 0.001 ; the Spm levels in clones 22 and 34 are significantly different from the levels in controls at P ⁇ 0.01 , and; the Spm levels in clones 19 and 26 are significantly different from levels in controls at P ⁇ 0.05. Remaining values were not significantly different from control levels at P > 0.05.
  • CaMV 35S promoter suggested a correlation between the level of PA accumulation and the inability of dedifferentiated tissue to undergo morphogenesis (Capell et al., Theoret. Appl. Genet, 97:246-254 (1998)).
  • transgenic rice callus lines and regenerating shoots expressing the oat adc cDNA under the control of the maize ubiquitin 1 promoter we describe transgenic rice callus lines and regenerating shoots expressing the oat adc cDNA under the control of the maize ubiquitin 1 promoter.
  • the maize ubiquitin 1 promoter is a strong constitutive promoter and expression of the ubiquitin- 1 /adc construct resulted in significantly higher levels of expression of ADC, as compared to the weaker CaMV 35S promoter, the transformed plant cells containing the ubiquitin- 1 promoter did not arrest in the dedifferentiation process. As discussed below, the PA levels in these ubiquitin- 1 transformed plants differed significantly from those where ADC expression was controlled by the CaMV 35S promoter.
  • DAO enzyme activity in transgenic rice cell lines suggests that there is an adequate degree of homology between the endogenous rice mRNA encoding the DAO enzyme and the heterologous transcript encoding the pea DAO enzyme for the antisense transcript to be effective. It was demonstrated previously that region as short as 150 contiguous base pairs of identity between sequences can result in an observable antisense effect (Fray and Grierson, TIG, 9:438-443 (1993)). All transgenic lines which did not show a reduction in DAO enzyme activity contained basal levels of PAs. Lines which exhibited a reduction on DAO activity displayed differences in the accumulation pattern of the various polyamines.
  • Genomic PCR amplifications were carried out in a total reaction volume of 50 ⁇ l, comprising 100 ng genomic DNA (Creissen and Mullineaux, Planta, 197:422-425
  • DNA and RNA were isolated from callus and plant material according to the procedure of Creissen and Mullineaux, Planta, 197:422-425 (1995). Following digestion with appropriate restriction enzymes (EcoRI or Hindlll) and electrophoresis on a 1 % TA ⁇ agarose gel (Sambrook et al., Molecular cloning; a laboratory manual, 2nd edition. New York: Cold Spring Harbor Press 1989) DNA was transferred to a positively charged membrane (Roche, UK). Nucleic acids were fixed by baking at 80 °C for 2 h. Filters were washed in 2xSSC for 30 min and then pre-hybridized at 38 °C for 30 min using the DIG easy hybridization solution (Roche, UK).
  • the 2.1 Kb EcoRI adc fragment from pUbiADCs was labeled with the DIG-system using the PCR DIG probe synthesis kit (Roche, UK). Alkali-labile DIG- 1 1 -dUTP (Roche, UK) was incorporated into the probe as follows: PCR was set up in a final volume of 50 ⁇ l containing 200 ⁇ M dATP, dCTP and dGTP; 160 ⁇ M dTTP, 40 ⁇ M DIG-1 1-dUTP, lOx PCR buffer (50 mM KC1, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100); 2.5 U of Taq DNA polymerase (Roche, UK), 150 ng of both forward and reverse sequence primers (Genosys, Cambridge, UK) and 100 pg of 2.1 Kb EcoRI Adc fragment from pUbiADCs as a template.
  • the 1.5 Kb probe was purified using the QIAquick Gel Extraction Kit (Quiagen, UK) and denatured at 68 ° C for ten minutes prior to use. Hybridization was performed at 38 ° C overnight in hybridization buffer (Roche, UK). The membranes were washed twice for 5 min in 2xSSC, 0.1% wt/vol SDS at room temperature, and then two times ( 15 min) in 0.5xSSC, 0.1 %> wt/vol SDS at 68 °C. Chemilurninescent detection was performed according to the manufacturer ' s instructions using DIG Luminescent Detection Kit (Roche, UK).
  • RNA manipulations were carried out following the same procedures described for DNA. Membranes were exposed to X-ray film (Fuji Photofilm Co., Ltd, Kanawa, Japan) for 30 min at 37 °C.
  • the reaction mixture to dete ⁇ riine ODC activity contained 20 ⁇ l of extraction buffer, 160 ⁇ l of crude enzyme and 20 ⁇ l of the substrate (20 ⁇ l of DL-(I- 14 C) orn (20Ci/mmol, Amersham International pic, UK)) diluted with 20 ⁇ l cold om (2.5 M) and 60 ⁇ l of distilled water to give a final concentration of 50 mM orn.
  • bands were scraped into 6 ml of ethyl acetate and quantified using a Kontron SFM 25 spectrofluorophotometer at an excitation wavelength of 350 nm and an emission wavelength of 495 nm.
  • Immature rice embryos were co-bombarded with plasmid pUbiADCs, containing the oat adc cDNA driven by the maize ubiquitin 1 promoter and first intron (Fig. 1), and a plasmid containing the hygromycin phosphotransferase (hpt) gene as a selectable marker (Capell et al., Theoret. Appl. Genet.. 97:246-254 (1998)). Thirty independently- derived transgenic callus lines were recovered. H. Molecular characterization of transgenic lines
  • Hindlll digested genomic DNA (Hindlll cuts once in pUbiADCs) indicated a different integration pattern of the gene of interest in all transgenic lines, confirming their independent origin (Fig. 3A).
  • Fig. 3B shows that the maize ubiquitin 1 promoter efficiently transcribed the oat adc cDNA in all lines analyzed. Transgene copy number and integration patterns had no effect on apparent mRNA levels.
  • ADC and ODC activities were measured in lines that had been co-transformed with pUbiADCs and hpt (Figs. 4A and 4B).
  • Background ADC activity in wild type control callus was of the order of 1-3 ⁇ mol of 14 C0 2 per min per g fresh weight.
  • Put levels were measured in callus lines after subculturing for 12 days on PM, which maintains the callus in a dedifferentiated state. Compared to hpt- trans formed controls, Put levels decreased from 2000 ⁇ mol per g fresh weight (basal level in hpt- control tissue) to 600- 1200 ⁇ mol per g fresh weight in half the lines analyzed. In the rest of the lines, Put was a 1800-2800 nmol per g fresh weight; however, these values were not significantly different to those in /jt/?-controls. We detected a significant increase of 4500-5500 ⁇ mol per g fresh weight in one particular line.
  • Put levels were transferred to MM and Put levels were assayed. Put levels were in the range of 2300 to 13000 ⁇ mol per g fresh weight in the transgenic lines, compared to Put 1200 ⁇ mol per g fresh weight for the equivalent ⁇ j Dt-control. These values were in most cases higher than the levels observed in corresponding fully dedifferentiated callus grown on PM. In fully dedifferentiated callus, the corresponding values for Put were 600 to 5500 ⁇ mol per g fresh weight.
  • Spd levels in callus lines on PM showed a significant decrease in all the lines analyzed.
  • the Apt-control Spd levels were about 550-650 ⁇ mol per g fresh weight, while the Spd levels in transgenic lines were between 200 and 450 ⁇ mol per g fresh weight.
  • Callus lines were transferred to MM and the levels of Spd were assayed.
  • Spd levels were about 550 to 1600 ⁇ mol per g fresh weight in transgenic lines, compared to 300 ⁇ mol per g fresh weight in Apt-control lines. These values were in most cases higher than levels observed on PM.
  • Spm levels measured in dedifferentiated callus tissue on PM increased significantly in 85% of the transformed lines, about 550-2200 ⁇ mol per g fresh weight.
  • Spm levels in Apt-control tissue were about 400-500 ⁇ mol per g fresh weight.
  • the reverse trend was observed for lines growing on MM.
  • the Spm levels in the transformed lines on MM (700 to 1000 ⁇ mol per g fresh weight) were significantly lower than the Spm levels in Apt-control lines (1300 ⁇ mol per g fresh weight) and almost all the lines had lower Spm compared to dedifferentiated tissue on PM.
  • Spm levels in regenerating shoots on SM were significantly increased compared to the levels in hpt controls (shoots; 1584 ⁇ mol per g fresh weight).
  • RNA accumulation in transgenic rice callus containing the adc gene driven by CaMV 35S promoter indicated that only a very small number of transgenic lines accumulated messenger.
  • the steady-state transcription of RNA in these lines (35S-adc) was only detectable in lines that were terminally dedifferentiated, i.e. not able to regenerate plants (Capell et al., Theoret. Appl. Genet., 97:246-254 ( 1998)).
  • messenger accumulation was detected in all rice callus lines transformed with pUbiADCs. This translated to functional protein with activity increases in all lines ⁇ Fig. 3(B)).
  • the PA levels of regenerated transgenic plants was determined in young leaves from two month old plants. As shown in Figure 6, compared to Apt-control plants, Put levels increase significantly up to 4-fold (1350 ⁇ mols per gram fresh weight) in one of the four lines analyzed. Put levels in hpt-plants and in the rest of the lines ranged between 300 and 400 ⁇ mols per gram fresh weight. No significant variation was found in Spd (30 to 60 ⁇ mols per gram fresh weight) and Spm levels (40 to 60 ⁇ mols per gram fresh weight) in transgenic plants compared with Apt-control. L. Polyamine levels of R Q seeds
  • the PA content of rice seeds generated from transgenic plants was deteimined after collection of mature seeds and dessication. Dehusked dry seeds were analyzed from all fertile lines. Seeds recovered from clone 20 containing pUbiADCs displayed a significant increase in Put (10-fold increase, 560 ⁇ mols per gram fresh weight) and Spm (1.5-fold increase, 75 ⁇ mols per gram fresh weight) compared to the Apt-confrols (50 ⁇ mols Put per gram fresh weight and 50 ⁇ mols Spm per gram fresh weight; Figure 7).
  • Spm levels in the transgenic plants were about 50 to 100 ⁇ mols per gram fresh weight (up to 2-fold mcrease) as compared to the Apt-controls (35 to 45 ⁇ mols per gram fresh weight). 20% of the seedlings also showed a significant increase in Put, Spd and Spm levels, about 1.5 to 3 fold increase, within the same plant.
  • the 2.249 Kb pea dao cDNA (Tipping and McPherson, Journal of Biological Chemistry, 270: 16939-16946 (1995)), was excised as an Nhel/Ndel fragment frompBS SK(-) (Promega). The fragment was blunt ended and subcloned in opposite orientation into the Smal site of pJIT60 (Gurineau and Mullineaux, Plant Mol. Biol.. 18:815-818 (1992)), which contains a CaMV 35S promoter with duplicated enhancer sequences and a CaMV transcriptional termination region. This plasmid is referred to as p35S ⁇ oa.
  • the 2.249 Kb pea dao cDNA was excised as EcoRI fragment from pBS SK (-) and subcloned into pENOD12 in the EcoRI site in antisense orientation to the pea ENOD12 nodulrn promoter (Wisniewskietal. unpublished).
  • the resulting plasmid was designated pE ⁇ oa.
  • Rice transformation and plant regeneration were carried out essentially as described in Example 1.
  • Rice tissues were co-transformed with a plasmid containing the Apt gene as the selectable marker and plasmid p35S ⁇ a or pE ⁇ oa using previously published procedures (Sudhakar et al., Trans . Res., 7:371-378 (1998); Capell et al, Theor. Appl. Genet.. 97:246-254 (1998)).
  • Fifteen transgenic callus clones were analyzed with PCR and Southern blots for the presence of the constructs.
  • Apt-transformed lines which contained the selectable marker gene, were used as controls.
  • PCR reactions to detect the pea dao cDNA were carried out in 50 ⁇ l total reaction volume containing 100 ng of genomic DNA, lx PCR buffer (50 mM KC1, 1.5 mM MgC , 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100); 400 ⁇ M deoxynucleoside triphosphates, 100 nM of both forward and reverse sequence primers (Genosys, Cambridge, UK) and 2.5 U of Taq polymerase (Roche, UK).
  • the thermocycler was programmed for denaturation at 96°C for 40 sec, annealing at 55°C for 30 sec and extension at 72°C for 2 min. The reaction was carried out for 35 cycles.
  • the forward sequence primer started from position 137 in the pea dao cDNA open reading frame and consisted of 5'-GCAGTTGTCTCAGTTACACCG-3' (SEQ ID NO: 3); the reverse sequence consisted of 5'-CAGCTCAAATGAGGTGCTCAATAG-3' (SEQ ID NO: 4) resulting in a fragment of 1.8 Kb.
  • the size of the final product was confirmed on a 1% agarose gel.
  • RNA samples were used in each RT-PCR reaction. Reverse transcription was performed according to standard procedures (Sambrook et al., Molecular cloning: a laboratory manual, 2nd edition. New York: Cold Spring Harbor Press (1989)). cDNAs were amplified for 35 cycles in 50- ⁇ l volumes. Conditions and primer sequences used were the same as those in the PCR reaction.
  • DNA and RNA were isolated from callus tissues transformed with both p35s ⁇ oa and pEdaoa constructs according to the procedure of Creissen and Mullineaux, Planta, 197:422-425 (1995).
  • PCR DIG probe synthesis kit (Roche, UK). Alkali-labile DIG-11-dUTP was incorporated into the probe as follows: PCR was set up in a final volume of 50 ⁇ l containing 400 ⁇ M dATP, dCTP and dGTP; 320 ⁇ M dTTP, 80 ⁇ M DIG-11-dUTP, lxPCR buffer (50 mM KC1, 1.5 mM MgC , 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100); 2.5 U of Taq DNA polymerase, 100 nM of both forward and reverse sequence primers (the primers used in the PCR reactions, supra, SEQ ID NO: 3 and 4) and 100 pg of 2.2 Kb NAel/Ndel dao fragment from p35S ⁇ oa as a template.
  • the membranes were washed twice for 5 min in 2xSSC, 0.1%o wt/vol SDS at room temperature, and then two more times (15 min) in 0.5xSSC, 0.1% wt/vol SDS at 68°C.
  • Chemilurninescent detection was performed according to the manufacturer' s instructions using the DIG Luminescent Detection Kit. After washing, the membranes were incubated with CSPDTM Chemilurninescent Substrate (Roche, UK) and subsequently exposed to X- ray film for 1 h at 37°C.
  • Northern blots of RNA isolated from the plant callus lines were hybridized with similar probes under similar washing conditions as described above. Membranes were exposed to X-ray film for 2 h at 37°C and analyzed. E. RT-PCR analyses and Northern blot hybridization
  • 5'-CAGCTCAAATGAGGTGCTCAATAG-3' (SEQ ID NO: 4) were used to amplify a 1.8 Kb fragment inside the coding region of the pea dao cDNA.
  • a Northern blot of total mRNA from transgenic callus lines was hybridized with the 1.8 Kb DIG-labeled fragment from p35S ⁇ oa. No hybridization signals were detected in Northern blots in lines that contained p35Sctooa. From 12 lines that contained pE ⁇ a, six lines accumulated levels of steady-state mRNA that were high enough to be detected on Northern blots (lines 7, 13, 17, 19, 22, and 23; representative lines shown in Fig. 12). Steady-state mRNA was detected in the same pEdaoa lines by both RT-PCR and Northern blot analyses.
  • Transformed callus lines expressing p35S ⁇ oa compared to wild-type callus or callus lines expressing the selectable marker gene alone displayed a two-fold decrease in DAO activity (P ⁇ 0.01).
  • Callus tissue was used for DAO enzyme activity measurements. Tissue was extracted in buffer (0.1M K-phosphate pH 7.5 and 2 mM dithiothreitol) at a ratio of lg per 3 ml buffer. Polyvinylpyrrolidone (100 mg) was added during grinding. Following centrifugation at 27,000 x g for 10 min, the supernatant was used directly in enzyme activity assays. The reaction mixture contained 250 ⁇ l of crude enzyme, 12 ⁇ l of Put 0.1M and up to 1 ml of 0.1 M K-phosphate pH 7.5.
  • One unit (U) represents the amount of enzyme catalyzing the formation of 1 ⁇ mol of ⁇ -pyrroline min "1 .
  • Seventy-five % of the lines transformed with p35S ⁇ roa (7 of 9)(P ⁇ 0.05) and 65% of the lines transformed with pEdaoa. (6 of 9)(P ⁇ 0.01) showed a significant reduction in endogenous DAO activity.
  • Reduction in enzyme activity for the p34Scf ⁇ a transformants ranged between 30%> (9 nmol ⁇ pyrroline min "1 g "1 fw in clone 34, P ⁇ 0.001) to 50%) (6 nmol ⁇ pyrroline min " 'g 'fvv in clone 37, P ⁇ 0.01).
  • the increased levels ranged from a 1.8-fold increase in clone 37 (2290 nmol/g fw; P ⁇ 0.05) to a 6-fold increase in clone 11 (7848 nmol/g fw, P ⁇ 0.001) compared to wild-type and Apt-transformed controls (1000 to 15000 nmol/g fw, Fig. 14A).
  • Put levels were increased significantly (P ⁇ 0.05) in 60% of the lines (7 of 11).
  • the Spd levels in p35S ⁇ oa- transformed callus line increased significantly (P ⁇ 0.05) in 25%o of the lines (3 out of 12). Increases varied from approximately 2-fold in clone 37 (970 nmol/g fw, P ⁇ 0.05) to 8-fold in clone 6 (3300 nmol g fw, P ⁇ 0.05) as compared to wild-type or Apt-transformed controls (340 to 480 nmol/g fw; Fig. 14(B) In pE ⁇ oa-transformed callus lines, Spd levels increased significantly (P ⁇ 0.05) in 60% of the lines (7 from 11).
  • Wheat tissue was prepared, transformed and regenerated using standard conditions essentially as described by Stoger et al., Plant Mol. Bio., 42:583-590 (2000), incorporated herein by reference. Transformed wheat cells were selected for resistance to phosphinothricin. The conditions for RT-PCR, Southern and Northern blots to assay for integration and expression of the genes of interest were essentially as described supra.
  • polyamine content dete ⁇ r ⁇ ned essentially as described in Example 1, was assayed in desiccated mature seeds harvested from primary transformants.
  • Three of twenty independent transformants provided seeds with a significant increase in Put levels as compared to the average levels in seeds from control non-transformed and bflr-transformed plants (6 lines each).
  • the increase in Put levels in the Rj seeds ranged from 2-fold in T6 (1500 nmol/g fresh weight (fw); P ⁇ 0.05) to 3-fold in T 11 (2300 nmol/g fw; PO.001) as compared to the levels in control seeds (800nmol/g fw).
  • the spermidine level in seeds of Tl 1 was 1.5 times the level in control seeds (850 nmol/g fw).
  • the speimine levels in seeds from 10% of the transgenic lines (2 of 20) were 1.5 fold the levels in control seeds.
  • the increased spermine levels ranged from 270 nmol/g fw in Tl 1 to 300nmol/g fw in T6 as compared to a basal level of 200 nmol/g fw.
  • a maximum of 2-fold increase in the concentration of total PAs was detected in the seeds of Tl 1.

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Abstract

La présente invention concerne des procédés permettant de produire des plantes possédant des niveaux sensiblement élevés de polyamines en modifiant l'expression d'enzymes dans les voies métaboliques et cataboliques de la polyamine. L'invention se rapporte également à des plantes, des parties de plantes et des propagules de ces dernières présentant des niveaux sensiblement élevés de polyamines totales ou particulières.
PCT/IB2000/001185 1999-08-10 2000-08-07 Accumulation de polyamine dans les plantes WO2001011062A2 (fr)

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WO2002023974A1 (fr) * 2000-09-20 2002-03-28 Toyobo Research Center Co., Ltd. Plante ayant une meilleure tolerance a diverses contraintes environnementales, procede de production de cette plante et gene d'enzyme relatif au metabolisme de la polyamine
WO2003084314A1 (fr) * 2002-04-08 2003-10-16 Toyobo Research Center Co., Ltd. Plante presentant une organogenese amelioree et procede de creation de cette plante
WO2006005603A1 (fr) * 2004-07-15 2006-01-19 Dsm Ip Assets B.V. Synthese biochimique de 1,4-butanediamine
WO2006005604A1 (fr) * 2004-07-15 2006-01-19 Dsm Ip Assets B.V. Synthese biochimique de 1,4-butanediamine
EP2100962A1 (fr) 2008-03-12 2009-09-16 Biogemma Plantes dotées d'une résistance améliorée aux pathogènes
WO2010004070A1 (fr) * 2008-07-11 2010-01-14 Universidad De Barcelona Plante présentant une résistance au stress à basses températures et procédé de production de celle-ci

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Title
CAPELL T ET AL: "OVER-EXPRESSION OF THE OAT ARGININE DECARBOXYLASE CDNA IN TRANSGENIC RICE (ORYZA SATIVA L.) AFFECTS NORMAL DEVELOPMENT PATTERNS IN VITRO AND RESULTS IN PUTRESCINE ACCUMULATION IN TRANSGENIC PLANTS" THEORETICAL AND APPLIED GENETICS,SPRINGER, BERLIN,DE, vol. 97, no. 1/02, July 1998 (1998-07), pages 246-256, XP000942574 ISSN: 0040-5752 cited in the application *
KUMAR A ET AL: "POTATO PLANTS EXPRESSING ANTISENSE AND SENSE S-ADENOSYLMETHIONINE DECARBOXYLASE (SAMDC) TRANSGENES SHOW ALTERED LEVELS OF POLYAMINES AND ETHYLENE: ANTISENSE PLANTS DISPLAY ABNORMAL PHENOTYPES" PLANT JOURNAL,GB,BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, vol. 9, no. 2, 1 February 1996 (1996-02-01), pages 147-158, XP002043133 ISSN: 0960-7412 cited in the application *
LI Z ET AL: "Comparison of promoters and selectable marker genes for use in Indica rice transformation." MOLECULAR BREEDING, vol. 3, no. 1, 1997, pages 1-14, XP002162549 ISSN: 1380-3743 *
MASGRAU C ET AL: "Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants." PLANT JOURNAL, vol. 11, no. 3, 1997, pages 465-473, XP002162548 ISSN: 0960-7412 cited in the application *
NOURY M ET AL: "A TRANSGENIC RICE CELL LINEAGE EXPRESSING THE OAT ARGININE DECARBOXYLASE (ADC) CDNA CONSTITUTIVELY ACCUMULATES PUTRESCINE IN CALLUS AND SEEDS BUT NOT IN VEGETATIVE TISSUES" PLANT MOLECULAR BIOLOGY,NIJHOFF/JUNK, THE HAGUE,NL, vol. 43, 2000, pages 537-544, XP000942473 ISSN: 0167-4412 *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7888554B2 (en) 2000-09-20 2011-02-15 Toyo Boseki Kabushiki Kaisha Plants having improved tolerance to various types of environmental stress, their production, and polyamine metabolism-related enzyme genes
US7238861B2 (en) 2000-09-20 2007-07-03 Toyo Boseki Kabushiki Kaisha Plant having improved tolerance to various environmental stresses, method of constructing the same and polyamine metabolism-relating enzyme gene
WO2002023974A1 (fr) * 2000-09-20 2002-03-28 Toyobo Research Center Co., Ltd. Plante ayant une meilleure tolerance a diverses contraintes environnementales, procede de production de cette plante et gene d'enzyme relatif au metabolisme de la polyamine
WO2003084314A1 (fr) * 2002-04-08 2003-10-16 Toyobo Research Center Co., Ltd. Plante presentant une organogenese amelioree et procede de creation de cette plante
US8455713B2 (en) 2002-04-08 2013-06-04 Toyo Boseki Kabushiki Kaisha Plants with improved morphogenesis and method of constructing the same
US8053629B2 (en) 2002-04-08 2011-11-08 Toyo Boseki Kabushiki Kaisha Plants with improved morphogenesis and method of constructing the same
US7446242B2 (en) 2002-04-08 2008-11-04 Toyo Boseki Kabushiki Kaisha Plants with improved morphogenesis and method of constructing the same
EA010179B1 (ru) * 2004-07-15 2008-06-30 ДСМ АйПи АССЕТС Б.В. Биохимический синтез 1,4-бутандиамина
AU2005261861B2 (en) * 2004-07-15 2010-08-26 Dsm Ip Assets B.V. Biochemical synthesis of 1,4-butanediamine
EP2236613A1 (fr) * 2004-07-15 2010-10-06 DSM IP Assets B.V. Synthèse biochimique de 1,4-butanediamine
EA011232B1 (ru) * 2004-07-15 2009-02-27 ДСМ АйПи АССЕТС Б.В. Биохимический синтез 1,4-бутандиамина
WO2006005604A1 (fr) * 2004-07-15 2006-01-19 Dsm Ip Assets B.V. Synthese biochimique de 1,4-butanediamine
WO2006005603A1 (fr) * 2004-07-15 2006-01-19 Dsm Ip Assets B.V. Synthese biochimique de 1,4-butanediamine
US8497098B2 (en) 2004-07-15 2013-07-30 Dsm Ip Assets B.V. Biochemical synthesis of 1,4-butanediamine
EP2949755A1 (fr) * 2004-07-15 2015-12-02 DSM IP Assets B.V. Synthèse biochimique de 1,4-butanediamine
US9523083B2 (en) 2004-07-15 2016-12-20 Dsm Ip Assets B.V. Biochemical synthesis of 1,4-butanediamine
EP2100962A1 (fr) 2008-03-12 2009-09-16 Biogemma Plantes dotées d'une résistance améliorée aux pathogènes
WO2010004070A1 (fr) * 2008-07-11 2010-01-14 Universidad De Barcelona Plante présentant une résistance au stress à basses températures et procédé de production de celle-ci
ES2344877A1 (es) * 2008-07-11 2010-09-08 Universidad De Barcelona Planta con resistencia a estres por bajas temperaturas y metodo de produccion de la misma.
US9139841B2 (en) 2008-07-11 2015-09-22 Universidad De Barcelona Plant having resistance to low-temperature stress and method of production thereof

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