WO2005048693A2 - Plante a tolerance accrue a la secheresse - Google Patents

Plante a tolerance accrue a la secheresse Download PDF

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WO2005048693A2
WO2005048693A2 PCT/IB2004/052440 IB2004052440W WO2005048693A2 WO 2005048693 A2 WO2005048693 A2 WO 2005048693A2 IB 2004052440 W IB2004052440 W IB 2004052440W WO 2005048693 A2 WO2005048693 A2 WO 2005048693A2
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plant
gene
cbp20
homologue
drought tolerance
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PCT/IB2004/052440
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WO2005048693A3 (fr
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István Papp
Csaba Koncz
Ferenc Nagy
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Agricultural Biotechnology Center
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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
    • C12N15/8273—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 for drought, cold, salt 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

Definitions

  • the present invention relates to a plant with improved drought tolerance that is transgenic and/or mutagenized chemically and/or physically, wherein the function of the Cap Binding Protein (CBP20) gene or a homologue thereof is decreased or blocked due to genetic manipulation and/or mutagenization.
  • the invention further relates to constructs used for the manipulation and/or mutagenezis, as well as methods for the production of the plant with improved drought tolerance.
  • Agronomicallyimportant genes can be isolated in several different ways.
  • a generally used strategy is direct mapping of genes responsible for favorable traits in crop species, followed by map based cloning. This approach is, however, hampered by the relatively scarce information available in these species.
  • Activation or inactivation of an important signaling pathway in a plant may cause multiple phenotypic changes beside the primary biochemical or physiological defect (pleiotropy). These changes may affect the plant's stature, growth, developmental phases or other parameters that can be scored relatively easily. Based on these subtle changes, mutants often can be spotted easily in a mutant plant population. In a functional genomic screen these mutants may be tested further to reveal the primary nature of the mutation, with special emphasis on the scientifically or agronomically useful traits they may carry. Practically, this means a second round of rigorous screening, where the selected mutants are tested under a number of different stress conditions.
  • a method that has been employed to this end is conventional breeding, where crossings and selection is used to improve the characteristics of the particular variety (see e.g. Hungarian Patent No. 218 309 and US Patent No. P9885).
  • transgenic techniques are used more and more often to tailor the physiology of a plant to resist environmental stresses, such as drought.
  • a few examples for this method overproduction of a molybdenum-cofactor sulphurase, which yields the cofactor of the last enzyme of abscisic acid (ABA) biosynthesis (US patent application No. 2003/0084485); increasing the raff ⁇ nose content of a plant by transgenic means (US patent application No. 2003/0074696A1); overproduction of the ABI5 protein - which is a stress-induced transcription factor - (US patent application No. 2002/0174454A1).
  • ABA abscisic acid
  • nCBC nuclear cap binding complex
  • the core of the nCBC complex consists of the Cap Binding Proteins CBP80, CBP20 (with molecular weights of 80 and 20 kDa, respectively) bound by an additional handful of proteins, including eIF4G [McKendrick, L., Thompson, E., Ferreira, J., Morley, S.J., Lewis, J.D., Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m 7 guanosine cap., Mol. Cell. Biol. 21, pp. 3632-3641 (2001)].
  • eIF4G McKendrick, L., Thompson, E., Ferreira, J., Morley, S.J., Lewis, J.D.
  • CBP20 has also been used in the literature to designate the chitin binding protein, unrelated to the subject of the present invention; see Ponstein A.S. et al., Plant Physiology 104(1), pp. 109-118 (1994)].
  • CBP80 and CBP20 are able to bind the 5' cap structure together.
  • the full biological function of nCBC however is probably achieved in a complex interaction with all the proteins participating in the complex.
  • CBP20 is the subunit that binds the 5' cap directly, and the details of this interaction are well documented [Calero G. et al., Nat. Struct. Biol. 9(12), pp. 912-917 (2002)].
  • nCBC is implicated in mRNA splicing, 3' end maturation and in the export of U snRNA from the nucleus [Izaurralde, E., Lewis, J., McGuigan, C, Jankowska, M., Darzynkiewicz, E., Mattaj, I.W., A nuclear cap binding protein complex involved in pre-mRNA splicing., Cell 78, pp. 657-668 (1994); Flaherty, S., Fortes, P., Izaurralde, E., Mattaj, I.W., Gilmartin, G:M., Participation of the nuclear cap binding complex in pre-mRNA 3' processing., Proc. Natl. Acad. Sci. USA 94, pp. 11893-11898 (1997)].
  • nCBC itself is known to be regulated posttranscriptionally by protein phosphorylation in mammals.
  • the rice homolog for the CBP80 protein is detailed in WO02/081696. It is therefore likely that, at least structurally, nCBC is evolutionally conserved in dicotyledonous and monocotyledonous plants and animals (see references above), as well as in yeast [e.g. see Colot et al., The yeast splicing factor Mudl3p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex., Genes Dev. 10, pp. 1699-1708 (1996)].
  • Clark et al. have investigated the effects of different mutations on alternative splicing in yeast by splicing specific microarrays [Clark, T.A., Sugnet, C.W. and Ares, M., Genom wide analysis of mRNA processing in yeast using splicing-specif ⁇ c microarrays., Science 296, pp. 907-910 (2002)].
  • the gcr3 and mudl3 yeast mutants (corresponding to cbp80 and cbp20, respectively) do not have the same effect on the accumulation of the spliced RNA set of the yeast. Consequently the two proteins may have at least partially different functions in the cell.
  • the Arabidopsis mutant line with improved drought tolerance made by the present inventors carried a lesion in the CBP20 gene, which is likely a member of the nCBC complex.
  • the nucleotide and amino acid sequence of CBP20 gene is presented on SEQ. ID. NOs. 1 and 2, respectively.
  • the Arabidopsis mutant line with improved drought tolerance made by the present inventors carried a lesion in the CBP20 gene, which is likely a member of the nCBC complex.
  • the nucleotide and amino acid sequence of CBP20 gene is presented on SEQ. ID. NOs. 1 and 2, respectively.
  • the term 'gene' refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including its regulatory sequences.
  • the term 'native gene' refers to gene as found in nature.
  • a 'transgene' refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes.
  • the term 'endogenous gene' refers to a native gene in its natural location in the genome of an organism.
  • the term 'coding sequence' refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences.
  • the terms 'open reading frame' and 'ORF' refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
  • the terms 'initiation codon' and 'termination codon' refer to units of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).
  • the terms 'regulatory sequences' refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences, as well as sequences which may be a combination of synthetic and natural sequences. Some regulatory sequences useful in the present invention will include, but are not limited to constitutive plant promoters, plant tissue-specific promoters, plant developmental stage-specific promoters, inducible plant promoters and viral promoters.
  • the '3' region' means the 3' non-coding regulatory sequences located downstream of a coding sequence. This DNA can influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
  • the term 'promoter' refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
  • the term 'promoter' includes a minimal promoter that is a short DNA sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression.
  • 'Promoter' also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA.
  • promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an 'enhancer' is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter.
  • enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments.
  • a promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.
  • the term 'homologue' or 'variant' of a nucleic acid sequence refers to a sequence having at least 50%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, and still more preferably at least 95% sequence identity with the said sequence.
  • the FastDB program of EMBL or SWISSPROT data bases can be used.
  • Other algorithms and computerized embodiments thereof well known in the art may also be used for the determination of this homology.
  • the term 'homologue' or 'variant' of an amino acid sequence is defined similarly to that of nucleic acid sequences. Therefore, the term 'homologue' or 'variant' of an amino acid sequence refers to a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and still more preferably at least 98% sequence identity with the said sequence. To determine whether the sequences are homologous, the above-mentioned programs and algorithms may be used.
  • 'Isolated' means altered 'by the hand of man' from natural state. If an 'isolated' composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not 'isolated', but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is 'isolated', as the term employed herein.
  • a nucleic acid molecule is regarded 'hybridizable' with another nucleic acid molecule if it can specifically be bound to the other molecule (i.e., the binding can give rise to a signal that is distinguishable from the background noise and from the signal caused by the non-specific binding of any random sequenced nucleic acid molecule), preferably a nucleic acid molecule is regarded as hybridizable if it specifically binds to another nucleic acid molecule under stringent conditions.
  • a regulatory sequence is 'operably linked' to a structural gene within a DNA construct if the regulatory sequence is able to influence the expression rate or manner of said structural gene under conditions suitable for the expression of said structural gene and for the functioning of said regulatory sequence.
  • the terms 'transformed', 'transformant' and 'transgenic' refer to plants or calli that have been through the transformation process and contain a foreign gene integrated into their chromosome.
  • the term 'untransformed' refers to normal plants that have not been through the transformation process.
  • 'transgenic plant' includes reference to a plant, which comprises within its genome a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • 'Transgenic' is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
  • transgenic' does not encompass the alteration of the genome (chromosomal or extra- chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non- recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
  • the function of a gene is 'decreased', 'inhibited' or 'blocked' if the expression or transcription of the gene, the splicing of the RNA transcribed from the gene, or the transport, membrane integration, activation or activity of the protein encoded by the gene, or any other molecular, cellular or plant level process having a role in the expression or function of the gene or the product encoded, is lowered, such as about 75%), preferably about 50%, more preferably about 25%, even more preferably about 10%, more preferably even about 5%, or most preferably completely blocked, that is about 0%, compared to the same function of the wild type plant.
  • the present invention relates to a plant with improved drought tolerance that is transgenic and/or mutagenized chemically and/or physically, wherein the function of the Cap Binding Protein (CBP20) gene or a homologue thereof is decreased or blocked due to genetic manipulation and/or mutagenization.
  • CBP20 Cap Binding Protein
  • the present invention relates to a plant wherein the function of the CBP20 gene or a homologue thereof is decreased or blocked by addition, deletion and/or substitution of one or more nucleotide within the regulatory and/or coding region of the gene.
  • the invention relates to a plant wherein the function of the CBP20 gene or a homologue thereof is decreased or blocked by introduction of a re- combinant construct that leaves the regulatory and/or coding region of the gene intact.
  • the said plant is dicotyledonous, preferably the plant belongs to the order of Brassicales, and more preferably to the family of Brassicaceae. In a highly preferred embodiment, the plant is Arabidopsis.
  • the plant is monocotyledonous.
  • the invention also relates to a cell, tissue, organ, part, seed, fruit, product, progeny or propagation material of the plant according to the invention.
  • the invention relates to a recombinant nucleic acid construct comprising a nucleic acid inhibiting or blocking the CBP20 gene upon its expression, operably linked to a regulatory sequence capable of directing transcription in a plant.
  • the construct according to the invention comprises a nucleic acid sequence encoding the CBP20 gene or a homologue thereof in its entirety or a part thereof in antisense direction with respect to the regulatory sequence.
  • the construct according to the invention comprises a nucleic acid sequence encoding the CBP20 gene or a homologue thereof in its entirety or a part thereof in sense direction with respect to the regulatory sequence.
  • the construct according to the invention comprises a nucleic acid sequence encoding the CBP20 gene or a homologue thereof in its entirety or a part thereof in an inverted repeat arrangement.
  • the invention further relates to a method for the production of a plant with improved drought tolerance comprising the preparation a mutagenized plant population by random mutagenezis; selection of the mutagenized plants by molecular biological techniques capable of recognizing mutations within the CBP20 gene or homologues thereof; and optional sexual or asexual propagation of the resulting plants with improved drought tolerance.
  • the invention relates to a method for the production of a plant with improved drought tolerance comprising the preparation a mutagenized plant population by introducing a nucleic acid construct suitable for the mutagenesis or functional blocking of the CBP20 gene or homologue thereof; selection of the mutagenized plants with improved drought tolerance; and optional sexual or asexual propagation of the resulting plant with improved drought tolerance.
  • the invention relates to a method wherein a construct according to the invention is used for the mutagenesis or functional blocking of the gene.
  • the invention relates to a method wherein a wherein the mutation within the CBP20 gene or homologue thereof is preferably detected hybridization, sequencing, PCR, SSCP, and/or TILLING.
  • the invention also relates to a method wherein during the selection step the plant population is screened in advance for visible pleiotropic features being characteristic of plants comprising a mutant CBP20 gene or a homologue thereof.
  • Figure 1 shows wild type (on the left) and cbp20 mutant (on the right) plants and leaves.
  • Figure 2 shows leaves of cbp20 and serrate (se) plants, and their FI progeny.
  • Figure 3 shows the result of T-DNA specific PCR on F2 plants with wild type (panel A) and mutant (panel B) phenotype.
  • Figure 4 shows Northern hybridization on total RNA samples prepared from wild type (on the left) and cbp20 mutant (on the right) plants.
  • Figure 5 shows wild type and cbp20 mutant plants following 8 days of water deprivation.
  • drought resistant plant according to the invention can be obtained by damaging (i.e. inhibiting or completely blocking) the CBP20 gene or gene expression. Damaging of the CBP20 gene or gene expression can be achieved by introducing DNA constructs into the plant.
  • Ti derived plasmids transform a wide range of higher plants, including monocot and dicot plants such as soybean, cotton, rape, tobacco and rice [Pacciotti et al., Bio/Technology 3, p. 241 (1985); Byrne et al., Plant Cell, Tissue and Organ Culture 8, p. 3 (1987); Sukhapinda et al., Plant Mol. Biol. 8, pp. 209-216 (1987); Lorz et al., Mol. Gen. Genet. 199, p. 178 (1985); Potrykus et al., Mol. Gen. Genet. 199, p. 183 (1985); Park et al., J. Plant Biol. 38, pp.
  • RNA construct is designed covering the full or partial sequence of the target gene (may include exons, introns, 5' or 3' untranslated regions of mRNA) in antisense orientation, operably linked to a promoter suitable in the plant manipulated.
  • a minimum of 40-50 base pair homology is generally thought to be required to achieve silencing, in general practice a ⁇ 300-400 base pair long DNA sequence is used.
  • antisense RNA is synthesized from this construct that may disturb the expression of the target gene at different levels.
  • the nucleic acid delivered to the cell may be antisense DNA or RNA oligonucleotide as well.
  • the antisense nucleic acid may hybridize with the target mRNA, yielding double stranded RNA that is degraded by the host cell, preventing the expression of the gene [Matzke and Matzke, Plant Physiology 107, pp. 679-685 (1995), Vaucheret et al., The Plant Journal 16, pp. 651-659 (1998), Mlotshwa et al., Plant Cell 14 Suppl, pp. 289-301 (2002)].
  • the silencing construct need not be fully homologous with the target sequence, moreover one construct may silence more than one target genes.
  • Sense suppression The target gene or a part of its sequence is overexpressed in the plant, driven by a promoter that is functional in the plant. In some cases this leads to silencing of the introduced and endogenous genes, or any homolog sequences in the genome. Presumably there is a control mechanism in the cell degrading the mRNA species in excess [Taylor C.B., Plant Cell 9, pp. 1245-1249 (1997), Jorgensen et al., Trends in Genetics 15, pp. 11-12 (1999), Fagard and Vaucheret, Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, pp. 167-194 (2000)].
  • the silencing construct may contain the promoter of the gene, exons, introns, 5' or 3' non-translatable regions or coding sequences of the target gene or parts of these elements [Brummell et al., Inverted repeat of a heterologous 3'-nontranslated region for high-efficiency, high-throughput gene silencing., Plant J. 33, pp. 793-800 (2003); Aufsatz et al., RNA-directed DNA methylation in Arabidopsis., Proc. Natl. Acad. Sci. USA 99 Suppl 4, pp. 16499-16506 (2002)].
  • the size of the silencing construct required is similar to that of used in the antisense method, there is no need for full homology, and multiple genes can be targeted at once, as well.
  • the regulatory (promoter) elements controlling the expression of the above constructs may work constitutively, but can also be regulated. Stress induced transgene expression is described in response to wounding by Rizhsky L. and Mittler R. [Plant Mol. Biol. 46, pp. 313-323 (2001)], pathogen attack [Strittmatter G. et al., Bio/ Technology 13, pp. 1085-1089 (1995)], drought or ABA induction [Su J. et al., Plant Physiol. 117, pp. 913-922 (1998)]. Regulated expression has the obvious advantage that any secondary effect that would reduce the fitness of the whole plant is less pronounced.
  • a DNA construct can be designed that is capable of specific integration into the target region bringing about targeted mutation in the gene.
  • several favorable kind of mutations may be created, such as loss of function, dominant negative, altered substrate specificity, etc.
  • 5' and 3' noncoding regions, as well as other regulatory regions may be used. In plants so far the efficiency of this method is low, however it is expected that it can be improved in the future [Offriga et al., Transgenic Res. 1, pp. 114-123 (1992), Vergunst and Hooykaas, Crit. Rev. In Plant Sci. 18, pp. 1-31 (1999)].
  • Oligonucleotide chimeras.Targeted mutation can be obtained by introducing short DNA or RNA nucleotides into the cell that are homologous to the target sequence. Efficiency of this method is also low at present [Hohn and Puchta, Proc. Natl. Acad. Sci. USA 96, pp. 8321-8323 (1999)].
  • a dominant negative variant of a protein may disturb the function of the target polypeptide by binding to it [Ferrario S. et al., Plant Cell 16, pp. 1490-1505 (2004)], or alternatively, similar effect can be achieved by binding or changing the substrate of the protein [Niki T. et al., Plant Physiol. 126, pp. 965-972 (2001)].
  • Recombinant antibodies may also be expressed in plant cells [Conrad and Manteuffel, Trends in Plant Sci. 6, pp. 399-402 (2001)], where the antibody can recognize and inactivate the antigene that may be a protein or other.
  • the DNA construct intended to abolish the expression of a target gene may be needed to propagate and select those cells.
  • the transgenic cells are selected on appropriate media, then grown into calli by tissue culture methods. Shoot development is induced from the calli on appropriate media, followed by regeneration of the whole plant. Certain parts of the plant (e.g. buds) can be transformed directly by Agrobacterium at a competent developmental stage. In this case, seeds are selected to obtain transgenic progeny.
  • Transgene constructs may be linked to selectable markers in order to differentiate between transformed and wild type genotypes. Useful markers are different antibiotics (e.g.
  • kanamycin, G418, bleomycin, hygromycin, chloramfenicol, etc.) or some herbicides e.g. BASTA.
  • Components of DNA constructs introduced may derive from the same host plant (endogen), or from foreign organism (exogen origin). 'Foreign' means that the DNA sequence is not found in the genome of the host plant. Heterologous constructs contain at least one region of foreign origin.
  • transgenic plants can be propagated and harvested, and their seeds may be collected.
  • Heritable alterations have been made in their genome that all offspring will carry. In appropriate genetic context, the tissues and organs affected will show the desired phenotype.
  • the introduced transgenes and genetic alterations may be transferred into appropriate recipients by conventional crossings.
  • random mutagenesis In addition to the targeted transgenic constructs, destruction of a gene can be achieved by random mutagenesis as well. With some of these methods it is easier to introduce mutations, but this advantage is balanced by the more difficult selection procedures to obtain the desired, in our case drought tolerant, mutants. Methods for random mutagenesis are well known to the person skilled in the art. Non-limiting examples include irradiation, chemical mutagenesis, treatment with modified nucleotides, etc. [Negrutiul., In vitro mutagenesis., pp. 19-38., in: Plant Cell Line Selection, Dix, P.J. (ed.), VCH Verlag GmbH. (1990)]. Random mutagenesis can be done by using mutator DNA constructs with known sequence.
  • T-DNA mutagenesis where large scale plant transformation is used to generate a mutant population [e.g. Chen et al., Distribution and characterization of over 1000 T-DNA tags in rice genome., Plant J. 36, pp. 105-113 (2003)].
  • this population may contain mutants with loss of function, gain of function, or mutants with altered functions [Alonso et al., Genome-wide insertional mutagenesis of Arabidopsis thaliana., Science 301, pp. 653-657 (2003), Sessions et al., Ahigh- throughput Arabidopsis reverse genetics system., Plant Cell 14, pp.
  • transposons may be used to obtain mutant populations.
  • the transposons may be endogenous or foreign (heterolog) to the species, and can be introduced either by one of the transformation methods outlined above or traditional sexual crossing [May et al., Maize-targeted mutagenesis: A knockout resource for maize., Proc. Natl. Acad. Sci. USA 100, pp.
  • Selection of the mutant with improved drought tolerance from the random mutagenized population can be achieved by direct screening for the trait, or in one of the following ways. If we already possess an induced transgenic mutant obtained by any of the ways described above, it may help in the selection procedure. As we mentioned, in addition to the improved drought tolerance, the morphology of the cbp20 mutant is also slightly changed compared to wild type. These visible pleiotropic traits may aid in selecting the mutant from a large mutant population, with more effective screening of the visible phenotype. Pleiotropic effects may vary among species, which can be tested by a transgenic mutant created by silencing.
  • the desired mutant may be selected from the mutant population created by random mutagenizer DNA constructs by several way. PCR reactions may be performed based on the sequences of the target gene (including promoter and other regulatory regions) and that of the mutator sequence [for example see: Krysan et al., Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport., Proc. Natl. Acad. Sci. USA 93, pp. 8145-8150 (1996)]. Screening may be accomplished with degenerate primers if the exact sequence of the target gene is unknown, but a characteristic motif is chosen (e.g.
  • RNA binding, kinase domain, etc. [Young et al., Efficient screening of Arabidopsis T-DNA insertion lines using degenerate primers., Plant Physiol. 125, pp. 513-518 (2001)].
  • sequences flanking the mutator DNA may be amplified by PCR. From among these sequences the gene of interest can be selected. For an overview of the method see Maes et al., Plant tagnology Trends in Plant Sci. 4, pp. 90-96 (1999).
  • a further possibility to select a particular mutant from a mutant population is the use of the TILLING method [(McCallum et al., Targeting Induced Local Lesions IN Genomes (TILLING) for plant functional genomics., Plant Physiol. 123, pp. 439-442 (2000)].
  • TILLING Targeting Induced Local Lesions IN Genomes
  • the targeted gene is specifically amplified from individuals of a random mutagenized population, then a wild type target sequence hybridized. With the help of an enzyme specifically cutting mismatched heteroduplex DNA, individuals carrying point mutations or small sequence changes in the gene of interest can be selected.
  • Wild type Arabidopsis (ecotype Columbia) have been randomly mutagenized by Agrobacterium infiltration with a T-DNA construct [pPCV6NFHyg; Koncz, C, Mayerhofer, R., Koncz-Kalman, Z., Nawrath, C, Reiss, B., Redei, G.P. ⁇ s Schell, J., Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana., EMBO J . 9, pp. 1337-1346 (1990)], creating a mutant plant population. Hygromycin resistant transformants were grown, their seeds were harvested. Using phenotypic screening of approx. 10,000 T2 progeny, a plant line has been selected showing more compact stature than wild type, slightly delayed development and serrated leaf margins (Figure 1). The mutant was fertile and in most characteristics very similar to the wild type.
  • the plant genomic sequences flanking the T-DNA have been cloned by cutting the genomic DNA with an appropriate restriction enzyme (Hindlll), religating and transforming it into competent Escherichia coli.
  • the plasmid construct rescued has been sequenced with T-DNA specific primers. This way in vitro was found out that the T-DNA was inserted into the second exon of gene At5g44200 on chromosome 5, which codes Cap Binding Protein 20.
  • the T-DNA substituted a 36 base sequence between base pairs 62147 and 62183 of the MLNl PI clone.
  • the integrated large piece of T-DNA disrupts the transcription and translation of the gene at this point. In the mutant only the first exon and part of the second exon can be transcribed. The produced truncated mRNA is degraded, we could not detect it in our Northern experiment ( Figure 4).
  • mutant plants were tested under different biotic and abiotic stresses. They showed considerable differences from wild type under water deprivation stress. Mutant and wild type plants were sawn and grown under normal conditions for 8 weeks then they were left without watering. The phenotype of the mutants were markedly different from wild type after approx. 6-8 days of drought (Figure 5); they tolerated water deprivation better. As mutants develop somewhat slower than wild type, in the above experiment we used plants of approx. the same developmental stage, but at slightly different age. By using plants of exactly the same age however, similar results could be achieved.
  • Example 2 Example 2
  • Random mutagenesis is performed by conventionally used mutagens (e.g. irradiation, chemical mutagenesis). The mutant population screened phenotypically for the visible traits of the cbp20 mutant, whereby a small number of mutants will be selected. This small population is screened extensively for water stress resistance, yielding the transgene-free drought tolerant mutant.
  • mutagens e.g. irradiation, chemical mutagenesis

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  • General Health & Medical Sciences (AREA)
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Abstract

Plante à tolérance accrue à la sécheresse, ladite plante étant transgénique et/ou mutagénisée sur le plan chimique et/ou physique, et la fonction du gène de la protéine de liaison à la coiffe (CBP20) ou d'un homologue de ce gène étant réduite ou bloquée par manipulation génétique et/ou mutagenèse. L'invention concerne en outre des structures de synthèse utilisées pour la manipulation et/ou la mutagenèse, ainsi que des procédés de production de ladite plante à tolérance accrue à la sécheresse.
PCT/IB2004/052440 2003-11-19 2004-11-16 Plante a tolerance accrue a la secheresse WO2005048693A2 (fr)

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HUP0303778 2003-11-19
HU0303778A HUP0303778A2 (en) 2003-11-19 2003-11-19 Plant with increased drought resistance

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WO2005048693A2 true WO2005048693A2 (fr) 2005-06-02
WO2005048693A3 WO2005048693A3 (fr) 2005-10-27

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008053059A1 (fr) * 2006-10-31 2008-05-08 Calantia Biotech, S.L. Utilisation de la mutation d'ocp3 comme régulateur de la résistance à la sécheresse des plantes
WO2010046422A2 (fr) 2008-10-22 2010-04-29 Basf Se Utilisation d'herbicides de type auxine sur des plantes cultivées
WO2010046423A2 (fr) 2008-10-22 2010-04-29 Basf Se Utilisation d'herbicides sulfonylurées sur des plantes cultivées
WO2014053395A1 (fr) 2012-10-01 2014-04-10 Basf Se Utilisation de composés de n-thio-anthranilamide sur des plantes cultivées
WO2014079820A1 (fr) 2012-11-22 2014-05-30 Basf Se Utilisation de composés d'anthranilamides pour réduire les infections virales véhiculées par les insectes
EP3028573A1 (fr) 2014-12-05 2016-06-08 Basf Se Utilisation d'un triazole fongicide sur des plantes transgéniques
WO2016091674A1 (fr) 2014-12-12 2016-06-16 Basf Se Utilisation de cyclaniliprole sur des plantes cultivées
WO2016162371A1 (fr) 2015-04-07 2016-10-13 Basf Agrochemical Products B.V. Utilisation d'un composé de carboxamide insecticide contre les nuisibles sur des plantes cultivées
EP3338552A1 (fr) 2016-12-21 2018-06-27 Basf Se Utilisation d'un fongicide tetrazolinone sur des plantes transgéniques

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EP1033405A2 (fr) * 1999-02-25 2000-09-06 Ceres Incorporated Fragments d'ADN avec des séquences déterminées et polypeptides encodées par lesdits fragments
WO2001096585A2 (fr) * 2000-06-14 2001-12-20 The Regents Of The University Of California Modulation de la transduction du signal de l'acide abscisique dans des plantes
WO2002081696A2 (fr) * 2001-04-06 2002-10-17 Syngenta Participations Ag Proteine 80 se liant a une coiffe nucleaire oryza sativa

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EP1033405A2 (fr) * 1999-02-25 2000-09-06 Ceres Incorporated Fragments d'ADN avec des séquences déterminées et polypeptides encodées par lesdits fragments
WO2001096585A2 (fr) * 2000-06-14 2001-12-20 The Regents Of The University Of California Modulation de la transduction du signal de l'acide abscisique dans des plantes
WO2002081696A2 (fr) * 2001-04-06 2002-10-17 Syngenta Participations Ag Proteine 80 se liant a une coiffe nucleaire oryza sativa

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HUGOUVIEUX VERONIQUE ET AL: "Localization, ion channel regulation, and genetic interactions during abscisic acid signaling of the nuclear mRNA cap-binding protein, ABH1." PLANT PHYSIOLOGY (ROCKVILLE), vol. 130, no. 3, November 2002 (2002-11), pages 1276-1287, XP002328697 ISSN: 0032-0889 *
KMIECIAK M ET AL: "Cloning and characterization of two subunits of Arabidopsis thaliana nuclear cap-binding complex" GENE: AN INTERNATIONAL JOURNAL ON GENES AND GENOMES, ELSEVIER SCIENCE PUBLISHERS, BARKING, GB, vol. 283, no. 1-2, 23 January 2002 (2002-01-23), pages 171-183, XP004339904 ISSN: 0378-1119 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008053059A1 (fr) * 2006-10-31 2008-05-08 Calantia Biotech, S.L. Utilisation de la mutation d'ocp3 comme régulateur de la résistance à la sécheresse des plantes
ES2304304A1 (es) * 2006-10-31 2008-10-01 Universidad Politecnica De Valencia Uso dea mutacionen ocp3 como regulador de la resistencia a sequia en plantas.
WO2010046422A2 (fr) 2008-10-22 2010-04-29 Basf Se Utilisation d'herbicides de type auxine sur des plantes cultivées
WO2010046423A2 (fr) 2008-10-22 2010-04-29 Basf Se Utilisation d'herbicides sulfonylurées sur des plantes cultivées
WO2014053395A1 (fr) 2012-10-01 2014-04-10 Basf Se Utilisation de composés de n-thio-anthranilamide sur des plantes cultivées
WO2014079820A1 (fr) 2012-11-22 2014-05-30 Basf Se Utilisation de composés d'anthranilamides pour réduire les infections virales véhiculées par les insectes
EP3028573A1 (fr) 2014-12-05 2016-06-08 Basf Se Utilisation d'un triazole fongicide sur des plantes transgéniques
WO2016091674A1 (fr) 2014-12-12 2016-06-16 Basf Se Utilisation de cyclaniliprole sur des plantes cultivées
WO2016162371A1 (fr) 2015-04-07 2016-10-13 Basf Agrochemical Products B.V. Utilisation d'un composé de carboxamide insecticide contre les nuisibles sur des plantes cultivées
EP3338552A1 (fr) 2016-12-21 2018-06-27 Basf Se Utilisation d'un fongicide tetrazolinone sur des plantes transgéniques

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HU0303778D0 (en) 2004-03-01
HUP0303778A2 (en) 2007-06-28

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