EP1220936A2 - Amelioration de la tolerance au stress dans le mais par manipulation des genes de regulation du cycle cellulaire - Google Patents

Amelioration de la tolerance au stress dans le mais par manipulation des genes de regulation du cycle cellulaire

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EP1220936A2
EP1220936A2 EP00965435A EP00965435A EP1220936A2 EP 1220936 A2 EP1220936 A2 EP 1220936A2 EP 00965435 A EP00965435 A EP 00965435A EP 00965435 A EP00965435 A EP 00965435A EP 1220936 A2 EP1220936 A2 EP 1220936A2
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Prior art keywords
expression
plant
promoter
construct
expression construct
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Yuejin Sun
Jeffrey E. Habben
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Pioneer Hi Bred International Inc
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Pioneer Hi Bred International Inc
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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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • This invention relates generally to the field of plant molecular biology. More specifically, this invention relates to methods and reagents for the temporal and spatial expression of genes that enhance cell division in plants, especially transgenic plants, to increase yield and health of crop plants in general as well as in periods of stress.
  • Cell division plays a crucial role during all phases of plant development.
  • the continuation of organogenesis and growth responses to a changing environment require precise spatial, temporal and developmental regulation of cell division activity in meristems (and in cells with the capability to form new meristems, such as in lateral root formation).
  • Control of cell division is also important in organs themselves (i.e., separate from meristems per se), for example, in leaf expansion, secondary growth, and endoreduplication.
  • a complex network controls cell division in eukaryotes.
  • Various regulatory pathways communicate environmental constraints such as nutrient availability, mitogenic signals such as growth factors or hormones, or developmental cues such as the transition from vegetative to reproductive growth. Ultimately, these regulatory pathways control the timing, rate, plane, and position of cell division.
  • Cell division in higher eukaryotes is controlled by two main checkpoints in the cell cycle which prevent the cell from entering either M- or S-phase prematurely.
  • Evidence from yeast and mammalian systems has repeatedly shown that over-expression of key cell cycle genes can either trigger cell division in non- dividing cells, or stimulate division in previously dividing cells (i.e., the duration of the cell cycle is decreased and cell size is reduced). Examples of genes whose over-expression has been shown to stimulate cell division include cyclins (see, e.g.,
  • the basic mechanism of cell cycle control is conserved among eukaryotes.
  • a catalytic protein kinase and an activating cyclin subunit control progress through the cell cycle.
  • the protein kinase is generally referred to as a cyclin - dependent-kinase (CDK), whose activity is modulated by phosphorylation and dephosphorylation events and by association with regulatory subunits called cyclins.
  • CDKs require association with cyclins for activation, and the timing of activation is largely dependent upon cyclin expression.
  • Eukaryote genomes typically encode multiple cyclin and CDK genes. In higher eukaryotes, different members of the CDK family act in different stages of the cell cycle.
  • Cyclin genes are classified according to the timing of their appearance during the cell cycle.
  • CDKs are often physically associated with other proteins which alter localization, substrate specificity, or activity.
  • CDK interacting proteins are the CDK inhibitors, members of the Retinoblastoma-associated protein (Rb) family, and the Constitutive Kinase Subunit (CKS).
  • the protein kinase activity of the complex is regulated by feedback control at certain checkpoints. At such checkpoints the CDK activity becomes limiting for further progress. When the feedback control network senses the completion of a checkpoint, CDK is activated and the cell passes through to the next checkpoint. Changes in CDK activity are regulated at multiple levels, including reversible phosphorylation of the cell cycle factors, changes in subcellular localization of the complex, and the rates of synthesis and destruction of limiting components. Regulation of the cell cycle by the cyclin/CDK complex is noted particularly at the
  • Plants have unique developmental features that distinguish them from other eukaryotes. Plant cells do not migrate, and thus only cell division, expansion, and programmed cell death determine morphogenesis. Organs are formed throughout the entire life span of the plant from specialized regions called meristems. In addition, many differentiated cells have the potential both to dedifferentiate and to reenter the cell cycle. There are also numerous examples of plant cell types that undergo endoreduplication, a process involving nuclear multiplication without cytokinesis. The study of plant cell cycle control genes is expected to contribute to the understanding of these unique phenomena. O. Shaul et al., Regulation of Cell Division in Arabidopsis, Critical Reviews in Plant Sciences, 15(2):97-112 (1996).
  • Anthesis is generally recognized as the critical period of ear and kernel development in maize. Varied experimental approaches demonstrate that treatments, which decrease the cell division around anthesis, decrease grain yield. For example, large yield losses occur when maize plants are subjected to abscisic acid (ABA) (Myers, P.N. et al, 1990; Mambelli and Setter, 1998), thermal stress (Jones, R.J. et al, 1985; Cheikh and Jones, 1994), water-deficits (Artlip, T.S. et al, 1995) or exposed to high plant density around anthesis. (See Zinselmeier, C. and J.E.
  • ABA abscisic acid
  • kernel number and size may be limited by cell division, particularly during drought or high density stress at anthesis.
  • enhancing cell division of the immature ear and grain would maintain ear and seed growth, and as a consequence, buffer this important vulnerable period of yield formation.
  • the tissues targeted for transgenes are in the maize female inflorescence, since relative to other organs, it is frequently the most sensitive to abiotic stress.
  • transient water stress prior to pollination has been shown to arrest the growth of ears, embryo sacs, and silks.
  • drought stress can inhibit endosperm cell division, which peaks at 8 to 10 days after pollination.
  • both kernel set and endosperm development are inhibited. This effect is most pronounced in the apical region of the ear.
  • Retarded endosperm development can result in aborted apical kernels, because of reduced cell division and decreased endoreduplication.
  • both of these events have been shown to be controlled by cyclin dependent protein kinases.
  • Barrennesss (the lack of ear development) is one of the most common manifestations of maize plants grown at high densities. Another prevalent trait in density stressed plants is an increase in the anthesis/silking interval, which has been shown to be the result of retarded ear growth. Based on this and other information, one key to producing a viable ear under plant-population stress is to maintain its growth rate. Since cell division is a key component of organ growth, the cell cycle regulatory mechanism in the female inflorescence is the target for expression of transgenes.
  • the present invention comprises the spatial and temporal expression of a nucleotide sequence which will enhance stress tolerance (buffer female inflorescence), particularly high density and drought stresses, in plants at critical times in plant development such as the vulnerable time of anthesis.
  • this invention relates to polynucleotides which encode proteins involved in the regulation of the cell cycle. More particularly, the polynucleotides encode proteins which enhance cell division in maize ears and kernels by directly increasing the activities of cyclin dependent protein kinases or indirectly by augmenting the activity of enzymes which control CDK activity.
  • Cell division in higher eukaryotes is controlled by a well-conserved mechanism.
  • the principal control factor of this mechanism is a protein threonine/serine kinase complex that is composed of cyclin (the regulatory subunit) and CDK (the catalytic subunit).
  • This complex controls cell division by phosphorylating target proteins.
  • Eukaryotes have evolved an elaborate regulatory network to safeguard the fluctuation of CDK activities in the cell cycle. Cyclins oscillate in abundance as a result of both transcriptional and post- transcriptional regulation. This provides an on/off control for CDK, since the association of cyclin is absolutely required for kinase activity. Phosphorylation and dephosphorylation of CDK occurs.
  • CDK activating kinase activates CDK
  • phosphorylation of Thrl4 and Tyrl ⁇ by Mytl and Weel respectively, inactivates CDK
  • CDC25 a protein tyrosine phosphatase dephosphorylates Tyrl ⁇ and activates CDK (Kumagai, A. and Dunphy, W.G., Cell 70, 139 (1992).
  • Both Weel and CDC25 are in turn regulated by phosphorylation.
  • Niml a protein kinase identified in S. pombe is able to phosphorylate Weel (this inhibits Weel activity), while Plxl is able to use CDC25 as a substrate and enhance CDC25 activity, a positive feedback loop for CDK regulation.
  • the CDK complex interacts with CDK inhibitors (CKIs).
  • CKIs CDK inhibitors
  • a number of proteins can physically bind to CDK and inhibit CDK activity.
  • Well- characterized inhibitors in human systems include p21, p27, p57, pl6, and pl9. Identification of rate-limiting pathways influenced by abiotic stresses are important in determining which ones to target.
  • Apical kernel abortion is a common characteristic of maize subjected to drought stress.
  • Research has shown that the plant hormone cytokinin, is able to reduce apical kernel abortion. Concurrently, it was shown that cytokinin can enhance CDK activities by reducing the extent of CDK phosphorylation at Tyrl ⁇ .
  • Other research has shown that the cell cycle regulatory mechanism is highly conserved among all eukaryotes. Cell cycle genes from maize, Arabidopsis, and alfalfa are able to rescue yeast mutants that are defective in cell cycle genes. Likewise, yeast cell cycle genes, such as CDC25, are able to promote cell division in higher plants. Thus, heterologous genes will work in transgenic maize events.
  • the invention comprises a genetic construct which upon expression in plant cells provides a DNA sequence encoding a gene product useful for directing the phosphorylation or activation state of CDK of a plant or plant tissue. Particularly, B-type and D-type cyclins, CDC25, Niml, and Plxl will be over expressed in order to promote cell division under stress.
  • the invention comprises a genetic construct which provides a DNA sequence encoding a gene product useful for co-suppressing Weel in order to promote cell division of a plant or plant tissue. Kernel abortion increases when unfavorable environments occur around flowering, thereby decreasing genetic yield potential in plants.
  • CDKs are critical enzymes that determine maize floral cell division. Modification of female cell division by altering the activation of CDKs in a tissue and temporal specific manner should increase the likelihood of vigorous female floral development and also improve the consistency of seed set under unfavorable conditions.
  • the invention contemplates expression of cell division enhancing nucleotide sequences during vulnerable periods, primarily those involved with anthesis development, where yield is most significantly affected by stress.
  • anthesis development shall include any period in plant development where yield may be more significantly impacted by stress. This can include the exponential growth phase of the ear during which biomass is accumulated and the lag phase of kernel development as more fully described herein and in the following references.
  • ears shall not be limited to maize and shall include any developing female inflorescence from a plant.
  • kernel shall also not be limited to maize but shall include grain, or seed within a fruit.
  • cell division enhancing nucleotide sequence shall mean any nucleotide sequence, (DNA, RNA, coding and/or antisense) the expression of which increases the rate of a particular plant tissue's cell division as compared to the rate without the expression of said sequence.
  • a genetic construct which causes expression of the cell division enhancing nucleotide sequence at a time and location to maximize cell division typically during very vulnerable periods primarily, around anthesis.
  • the spatial and temporal expression of genes affecting cell division of tissues can be achieved using different types of promoters.
  • Promoters useful for the invention are promoters which would cause the temporal and spatial expression of a gene product during anthesis as defined herein and can be constitutive, inducible, or tissue specific.
  • seed specific promoters can be used to enhance cell division during seed development, pre-pollination promoters can also be used or stress inducible promoters can be used to enhance cell division during periods of stress.
  • the optimization of promoters to achieve the objectives of the invention is considered routine and easily ascertainable by those of skill in the art and is intended to be within the scope of the invention.
  • Figure 1 is a schematic diagram (reproduced from Shaw, Robert "Climate Requirement", Corn Improvement, 3 rd ed., Chapter 10, pp. 609-638). As shown in Figure 1, reprinted from Corn and Corn Improvement from p. 614) of the relationship between age of crop and percentage yield decrement due to 1 day of moisture stress. The top and bottom lines represent the highest and lowest yield reductions obtained in stress experiments, the middle line the average reduction.
  • Figure 2 is a chart depicting expression timing of various promoters useful for the present invention.
  • the present invention is based on isolation and characterization of genes affecting CDKs or enzymes which control CDKs which control cell division in plants.
  • Any nucleotide sequence encoding an enzyme in the CDK activation/modulation (phosphorylation/dephosphorylation) pathways may be used in accordance with the present invention. Nucleotide sequences encoding these enzymes are easily ascertainable to those of skill in the art through Genbank or the references disclosed herein. Other reactions and pathways may be utilized by different organs in a plant or by different plant species. By changing the levels or activity of a component in the activation/deactivation/modulation pathway, it is possible to affect the levels of cell division in the plant, plant organ, or plant tissue.
  • CDKs have been identified in plants.
  • cDNAs encoding functional homologs of cdc2 kinase have been isolated by reduced stringency hybridization or reverse transcription coupled polymerase chain reaction from a number of plant species, including pea (Feiler and Jacobs, 1990), alfalfa (Hirt et al., 1991, 1993), Arabidopsis (Ferreira et al., 1991; Hirayama et al., 1991), soybean (Miao et al., 1993), Antirrhinum (Fobert et al., 1994), and maize (Colasanti et al., 1991). ). Soni, R. et al., "A Family of Cyclin D Homologs from Plants Differentially Controlled by Growth Regulators and Containing the
  • At least three different types of cyclins have been identified in plants: A- type homologs, B-type homologs, and D-type homologs (Renaudin, J-P et al.,
  • A-type cyclins are broken down into three structural groups (Al, A2, and A3). Cyclin Al has been isolated from maize. (Renaudin et al., Table 1). B-type cyclins are broken down into two structural groups (Bl, and B2). Cyclins Bl and B2 have been isolated from maize. (Renaudin et al., Table 1). D-type cyclins contain three structural groups (Dl, D2, and D3).
  • a number of cDNA sequences encoding plant mitotic cyclins with A- or B-type characteristics of having mixed A- and B-type features have been isolated from various species, including carrot (Hata et al., 1991), soybean (Hata et al., 1991), Arabidopsis (Hermerly et al., 1992; Day and Reddy, 1994), alfalfa (Hirt et al, 1992), Antirrhinum (Fobert et al, 1994) and maize (Redaudin et al, 1994; Sun, Y. et al, 1997, CycZmln from maize endosperm (GenBank #U66607), CycZmel, GenBank #U66608). Soni, R. et al, "A Family of Cyclin D Homologs from Plants Differentially Controlled by Growth Regulators and Containing the conserveed Retinoblastoma Protein Interaction Motif', The
  • the invention comprises a nucleotide construct comprising a cell division enhancing nucleotide sequence, a regulatory promoter to regulate temporal tissue and spatial expression during anthesis development and termination sequences operably linked to said cell division enhancing sequence.
  • a non-exclusive list of enzymes that might be candidates for such intervention include Mytl, Weel, Niml, CDC25, Plxl, CKIs, CAK, and cyclins.
  • Identification of other polynucleotides which may be useful in the invention will typically be based on screening for procaryotic or eucaryotic organisms with altered levels of cell division using assays standard in the art and described herein.
  • plant hormones such as cyktokinins
  • polynucleotides useful in the invention can be formed from a variety of different polynucleotides (e.g., genomic or cDNA, RNA, synthetic oligonucleotides, and polynucleotides), as well as by a variety of different techniques.
  • a polynucleotide is a sequence of either eukaryotic or prokaryotic synthetic invention.
  • the invention comprises use of one or more nucleotide sequences which, when expressed together enhance reproductive cell division.
  • the invention comprises a DNA sequence encoding B- or D-type cyclins, CDC2 ⁇ , Niml, and or Plxl capable of promoting cell division by activating or modulating the activity of CDKs in critical, stress sensitive periods of plant development.
  • DNA sequence encoding for suppression of Weel capable of promoting cell division by modulating the activity of CDKS is provided for increasing yield, seed development, flowering or resistance to stress.
  • the invention is not limited to any plant type and can be used for any crop or ornamental plant species for which it is desirable to increase yield.
  • the methods of the invention may be applicable to any species of seed-bearing plant to enhance yield potential by affecting the cell division in seed tissue.
  • the nucleotide constructs of the present invention will share similar elements, which are well known in the art of plant molecular biology.
  • the DNA sequences of interest will preferably be operably linked (i.e., positioned to ensure the functioning of) to a promoter which allows the DNA to be transcribed (into an RNA transcript) and will comprise a vector which includes a replication system.
  • the DNA sequence of interest will be of exogenous origin in an effort to prevent co- suppression of the endogenous genes.
  • Promoters may be heterologous (i.e., not naturally operably linked to a DNA sequence from the same organism).
  • Promoters useful for expression in plants are known in the art and can be inducible, constitutive, tissue-specific, derived from eukaryotes, prokaryotes, or viruses, or have various combinations of these characteristics.
  • tissue-specific or developmentally regulated promoter is a DNA sequence which regulates the expression of a DNA sequence selectively in the cells/tissues of a plant critical to seed set and/or function and/or limits the expression of such a DNA sequence to the period of seed maturation in the plant.
  • Any identifiable promoter may be used in the methods of the present invention which causes expression during anthesis development as defined herein. It may also be advantageous to use a stress inducible promoter to provide expression of the construct during periods of stress.
  • Differential screening techniques can be used to isolate promoters expressed in developing female reproductive organs (kernels and/or immature ears) from around 14 days before pollination to approximately 12 days after pollination. Promoters predicted to operate in this manner include LTP2, gamma- zein, and ZAG2.
  • Promoters preferred for the invention would be acceptably timed to 14 days before and 12 days after anthesis when both immature ear and mitotically active kernel are most susceptible to the stress. Promoters predicted to operate during these developmental stages include LTP2, MZE40, nucl and ZAG2.
  • LTP2 promoter from Barley confers the specificity of aleurone expression. Pioneer researchers have shown that this promoter is also functional in maize. When fused with a GUS reporter gene, LTP2 promoter directed aleurone specific expression of GUS activity in maize kernels (Niu and Tome, unpublished).
  • Aleurone is a single celled, out-most layer of endosperm that retains mitotic activity when the central region of endosperm ceased division and committed to endoreduplication. Therefore, LTP2 promoter will allow us to manipulate endosperm cell division when fused with cell division regulatory genes.
  • Zag2 transcripts can be detected 5 days prior to pollination to 7 to 8 DAP, and directs expression in the carpel of developing female inflorescences and Ciml which is specific to the nucellus of developing maize kernels. Ciml transcript is detected 4 to 5 days before pollination to 6 to 8 DAP.
  • Other useful promoters include any promoter which can be derived from a gene whose expression is maternally associated with developing female florets.
  • Figure 2 also depicts the timing of various preferred promoters and kernel development.
  • a construct useful for the present invention might include the maize B-cyclin gene operably linked to the ZAG2 promoter for expression of B- cyclin ⁇ 0 to 22 days after pollination.
  • promoters which are seed or embryo specific and may be useful in the invention include patatin (potato tubers) (Rocha-Sosa, M. et al (1989) EMBO ⁇ L 8:23-29), convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W.G., et al. (1991) Mol. Gen. Genet. 2 ⁇ 9:149-l ⁇ 7; Newbigin, E.J., et al (1990) Planta 180:461- 470; Higgins, T.J.V., et al. (1988) Plant. Mol. Biol.
  • Promoters of seed-specific genes operably linked to heterologous coding regions in chimeric gene constructions maintain their temporal and spatial expression pattern in transgenic plants.
  • Such examples include Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and Brassica napus seeds (Vanderkerckhove et al, Bio/Technology 7:L929-932 (1989)), been lectin and bean ⁇ -phaseolin promoters to express luciferase (Riggs et al, Plant Sci. 63:47- ⁇ 7 (1989)), and wheat glutenin promoters to express chloramphenicol acetyl transferase (Colot et al, EMBO J 6:3559-3564 (1987)).
  • any inducible promoter can be used in the instant invention to temporarily express a particular construct during reproductive development. See Ward et al. Plant Mol. Biol.22: 361-366 (1993).
  • Exemplary inducible promoters include, but are not limited to, that from the ACE1 system which responds to copper (Mett et al PNAS 90: 4567-4 ⁇ 71 (1993)); In2 gene from maize which responds to benzenesulfonamide herbicide safeners (Hershey et al, Mol. Gen. Genetics 227: 229-237 (1991) and Gatz et al, Mol Gen. Genetics 243: 32-38 (1994)) or Tet repressor from TnlO (Gatz et al, Mol Gen.
  • a particularly preferred inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond.
  • An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone. Schena et al, Proc. Natl. Acad. Sci. U.S.A. 88: 0421 (1991).
  • constitutive promoters include, but are not limited to, the promoters from plant viruses such as the 35S promoter from CaMV (Odell et al, Nature 313: 810-812 (1985) and the promoters from such genes as rice actin (McElroy et al, Plant Cell 2: 163-171 (1990)); ubiquitin (Christensen et al, Plant Mol. Biol 12: 619-632 (1989) and Christensen et al, Plant Mol Biol. 18: 67 ⁇ -689 (1992)): pEMU (Last et al, Theor. Appl. Genet. 81: ⁇ 81- ⁇ 88 (1991)); MAS
  • the ALS promoter a Xbal/Ncol fragment ⁇ ' to the Brassica napus ALS3 structural gene (or a nucleotide sequence that has substantial sequence similarity to said Xbal/Ncol fragment), represents a particularly useful constitutive promoter. See PCT application WO96/30 ⁇ 30. Transport of protein produced by transgenes to a subcellular compartment such as the nucleus, chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondrion, or for secretion into the apoplast, is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the 5' and or 3' region of a gene encoding the protein of interest.
  • Targeting sequences at the 5' and/or 3' end of the structural gene may determine, during protein synthesis and processing, where the encoded protein is ultimately compartmentalized.
  • the presence of a signal sequence directs a polypeptide to either an intracellular organelle or subcellular compartment or for secretion to the apoplast.
  • Many signal sequences are known in the art.
  • an expression vector contains (1) prokaryotic DNA elements encoding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) DNA elements that control initiation of transcription, such as a promoter; (3) DNA elements that control the processing of transcripts such as transcription termination/polyadenylation sequences; and (4) a reporter gene.
  • Useful reporter genes include ⁇ -glucuronidase, ⁇ -galactosidase, chloramphynical acetyltransferase, luciferase, kanamycin or the herbicide resistance genes PAT and BAR.
  • the selectable marker gene is kanamyacin or the herbicide resistance genes PAT and BAR.
  • the BAR or PAT gene is used with the selecting agent Bialaphos, and is used as a preferred selection marker gene for plant transformation (Spencer, et al. (1990) J. Thero. Appl'd Genetics 79:62 ⁇ -631). ( ⁇ ) The target or structural gene of interest.
  • nptll neomycin phosphotransferase II
  • nptll neomycin phosphotransferase II
  • Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al, Plant Mol. Biol, 5: 299 (1985).
  • Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside- 3' -adenyl transferase, the bleomycin resistance determinant. Hayford et al, Plant Physiol. 86: 1216 (1988), Jones et al, Mol. Gen. Genet., 2K_- 86 (1987), Svab et al, Plant Mol. Biol. 14: 197 (1990), Hille et al, Plant Mol. Biol. 7: 111 (1986).
  • Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or broxynil.
  • marker genes for plant transformation require screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include ⁇ -glucuronidase (GUS), ⁇ -galactosidase, luciferase and chloramphenicol acetyltransferase. Jefferson, R.A., Plant Mol Biol Rep. 5: 387 (1987)., Teeri et al, EMBO J.
  • GUS ⁇ -glucuronidase
  • luciferase luciferase
  • chloramphenicol acetyltransferase chloramphenicol acetyltransferase.
  • GFP Green Fluorescent Protein
  • GFP and mutants of GFP may be used as screenable markers.
  • Genes included in expression vectors must be driven by a nucleotide sequence comprising a regulatory element, for example, a promoter.
  • a regulatory element for example, a promoter.
  • Several types of promoters are now well-known in the transformation arts, as are other regulatory elements that can be used alone or in combination with promoters.
  • Expression vectors containing genomic or synthetic fragments can be introduced into protoplast or into intact tissues or isolated cells. Preferably expression vectors are introduced into intact tissue.
  • General methods of culturing plant tissues are provided, for example, by Maki, et al (Maki, et al. (1993) Procedures for Introducing Foreign DNA into Plants: In: Methods in Plant Molecular Biology & Biotechnology; Glich et al. eds. (CRC Press), pp. 67-88; Philips, et al (1988) Cell-Tissue Culture and In Vitro Manipulation. In Corn & Corn Improvement, 3rd ed. Sprague, et al. eds. (American Society of Agronomy Inc.), pp. 34 ⁇ -387).
  • Methods of introducing expression vectors into plant tissue include the direct transfection or co-cultivation of plant cell with Agrobacterium tumefaciens (Horsch et al. (198 ⁇ ) Science, 227:1229). Descriptions oi Agrobacterium vector systems and methods for Agrob ⁇ cte um-mediated gene transfer are provided by Gruber et al (supra).
  • A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells.
  • the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C.I., Crit. Rev. Plant. Sci.10: 1 (1991).
  • the expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes.
  • a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes.
  • Transformed plants can be screened by biochemical, molecular biological, and other assays.
  • Various assays may be used to determine whether a particular plant, plant part, or a transformed cell shows an increase in enzyme activity.
  • the change in expression or activity of a transformed plant will be compared to levels found in wild type (e.g., untransformed) plants of the same type.
  • the effect of the introduced construct on the level of expression or activity of the endogenous gene will be established from a comparison of sibling plants with and without the construct.
  • Cyclin, CDC25, Niml, and Plxl transcript levels can be measured, for example, by Northern blotting, primer extension, quantitative or semi-quantitative PCR (polymerase chain reaction), and other methods well known in the art (See, e.g.,
  • Protein can be measured in a number of ways including immunological methods (e.g., by Elisa or Western blotting).
  • CDK activity can be measured in various assays as described in Sun et al, Proc. Nat'l. Acad. Sci. U S A. 96(7):4180-85 (1999).
  • Cell division of a plant cell or tissue can be measured in a variety of ways including those described in Myers et al, Plant Physiol. 94:1330-36 (1990) and Artlip, et al, Plant Cell and Environ 18:1034-40 (1995).
  • regeneration means growing a whole plant from a plant cell, a group of plant cells, a plant part, or a plant piece (e.g., from a protoplast, callus, or a tissue part).
  • the foregoing methods for transformation would typically be used for producing transgenic inbred lines.
  • Transgenic inbred lines could then be crossed, with another (non-transformed or transformed) inbred line, in order to produce a transgenic hybrid maize plant.
  • a genetic trait which has been engineered into a particular maize line using the foregoing transformation techniques could be moved into another line using traditional backcrossing techniques that are well known in the plant breeding arts.
  • a backcrossing approach could be used to move an engineered trait from a public, non-elite line into an elite line, or from a hybrid maize plant containing a foreign gene in its genome into a line or lines which do not contain that gene.
  • crossing can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.
  • Various plants will be suitable targets for enhancing cell division in female reproductive organs with the identified genes.
  • the methods of the invention described herein may be applicable to any crop species including but not limited to barley, sorghum, wheat, maize, soybean, and rice.
  • transformation is carried out in maize plants according to the method of Agrobacterium.
  • Parts obtained from the regenerated plant such as flowers, pods, seeds, leaves, branches, fruit, and the like, are covered by the invention, provided that these parts comprise cells which have been so transformed. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention, provided that these parts comprise the introduced DNA sequences. Cyclin, CDC25, Niml,and Plxl levels and the activity of CDK are preferably determined as set forth in the examples.
  • transgenic plant Once a transgenic plant is produced having a desired characteristic, it will be useful to propagate the plant and, in some cases, to cross to inbred lines to produce useful hybrids.
  • mature transgenic plants may be self crossed to produce a homozygous inbred plant.
  • the inbred plant produces seed containing the genes for the newly introduced trait. These seeds can be grown to produce plants that will produce the selected phenotype. All articles cited herein and in the following list are hereby expressly incorporated in their entirety by reference.

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Abstract

Cette invention se rapporte à un procédé transgénique servant à accroître la division cellulaire dans des organes reproducteurs femelles de plantes. Les gènes sont exprimés temporairement et spatialement pour modifier l'activation et/ou la modulation des kinases dépendantes de la cycline dans un organe ou un tissu végétal. Cette invention concerne également des produits d'expression et des procédés servant dans la production de plantes de culture ayant des phénotypes héritables qui sont utiles pour des programmes d'amélioration génétique conçus pour accroître le potentiel de rendement dans un large éventail de conditions ambiantes.
EP00965435A 1999-09-27 2000-09-26 Amelioration de la tolerance au stress dans le mais par manipulation des genes de regulation du cycle cellulaire Withdrawn EP1220936A2 (fr)

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US156222P 1999-09-27
PCT/US2000/026405 WO2001023594A2 (fr) 1999-09-27 2000-09-26 Amelioration de la tolerance au stress dans le mais par manipulation des genes de regulation du cycle cellulaire

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Publication number Priority date Publication date Assignee Title
WO2015181823A1 (fr) * 2014-05-28 2015-12-03 Evogene Ltd. Polynucléotides isolés, polypeptides et procédés de leur utilisation pour augmenter la tolérance au stress abiotique, de la biomasse et le rendement de végétaux
US10975383B2 (en) 2014-05-28 2021-04-13 Evogene Ltd. Isolated polynucleotides, polypeptides and methods of using same for increasing abiotic stress tolerance, biomass and yield of plants

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HUP0202626A3 (en) 2004-09-28
WO2001023594A2 (fr) 2001-04-05
AU780301B2 (en) 2005-03-17
WO2001023594A3 (fr) 2001-12-06
CA2374431A1 (fr) 2001-04-05
AU7615500A (en) 2001-04-30
MXPA02003254A (es) 2002-09-30

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