WO2004016775A2 - Vegetaux a croissance modifiee et leur methode d'elaboration - Google Patents

Vegetaux a croissance modifiee et leur methode d'elaboration Download PDF

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WO2004016775A2
WO2004016775A2 PCT/EP2003/009142 EP0309142W WO2004016775A2 WO 2004016775 A2 WO2004016775 A2 WO 2004016775A2 EP 0309142 W EP0309142 W EP 0309142W WO 2004016775 A2 WO2004016775 A2 WO 2004016775A2
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seq
plant
nucleic acid
availability
nucleic acids
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PCT/EP2003/009142
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WO2004016775A3 (fr
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Dénes DUDITS
Valerie Frankard
Vladimir Mironov
Eva Csordas-Toth
Gábor HORVATH
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Cropdesign N.V.
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Priority to AU2003255457A priority Critical patent/AU2003255457A1/en
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Publication of WO2004016775A3 publication Critical patent/WO2004016775A3/fr

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    • 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

  • the present invention relates generally to the field of molecular biology. More specifically, the invention concerns a method for modifying the growth characteristics of a plant by reducing or substantially eliminating availability of retinoblastoma 1 (Rb1) in a plant. The invention also concerns plants having reduced or substantially eliminated availability of Rb1 , which plants have modified growth characteristics relative to corresponding wild-type plants.
  • Rb1 retinoblastoma 1
  • Crop yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality.
  • Crop yield is influenced to a high degree by the typical stresses to which plants or crops are subjected. Such stresses include environmental (abiotic) stresses (such as temperature stresses caused by atypical high or low temperatures; stresses caused by nutrient deficiency; stresses caused by lack of water (drought)) and biotic stresses (which can be imposed on plants by other plants (weeds), animal pests and pathogens). Crop yield may not only be increased by combating one or more of the 5 stresses to which the crop or plant is subjected, but may also be increased by modifying the inherent growth mechanisms of a plant.
  • the inherent growth mechanisms of a plant reside in a highly ordered sequence of events collectively known as the 'cell cycle'. Progression through the cell cycle is fundamental to the growth and development of all multicellular organisms and is crucial to cell proliferation.
  • Cell proliferation or cell division is fundamental for growth in humans, animals and plants.
  • eukaryotic cells including plant cells
  • the cell cycle is typically divided into 4 distinct phases:
  • - S DNA replication or synthesis
  • - G2 the gap between S and mitosis
  • Cell division is completed after cytokinesis, the last step of the M-phase. Cells that have exited the cell cycle and have become quiescent are said to be in the GO phase. Cells in the GO stage can be stimulated to re-enter the cell cycle at the G1 phase. Completion of the cell cycle process allows each daughter cell during cell division to receive a full copy of the parental genome.
  • the underlying mechanism controlling the cell cycle in all eukaryotic systems, including plants, is based on two key families of proteins which regulate the essential process of cell division, namely protein kinases (cyclin dependent kinases or CDKs) and their activating associated subunits, cyclins.
  • CDKs protein kinases
  • CDKs cyclin dependent kinases
  • the activity of these protein complexes is switched on and off at specific points of the cell cycle.
  • Particular CDK- cyclin complexes activated at the G 1 /S transition trigger the start of DNA replication.
  • the transition between the different phases of the cell cycle is typically driven by the sequential activation/inactivation of CDKs. Activation requires cyclins which are also important for targeting kinase activity to a given (subset of) substrate(s).
  • CDK inhibitors CKIs or ICKs, KIPs, CIPs, INKs
  • CAK CDK activating kinases
  • CDK phosphatases Cdc25
  • CDK subunit CKS
  • the Rb protein is central to regulation of G1 to S transition. In mammals, the activity of Rb depends on its phosphorylation state. During most of G1 , Rb is in a hypophosphorylated state, but in late G1 , phosphorylation of Rb is carried out by CDKs. The multiple phosphorylations of Rb disengage it from inhibitory complexes with proteins such as E2F/DP and MCM7 that are involved in the activation of S phase-specific transcription and initiation of DNA replication. The transcriptional repression of E2F-regulated genes occurs through both histone deacetylase (HDAC)-dependent and -independent mechanisms.
  • HDAC histone deacetylase
  • the HDAC enzymes modify the chromatin environment of the target genes by removing acetyl groups from the histones, thereby rendering them less accessible to transcription.
  • the MSI-related proteins thought to be transcription co- regulators, which bind to HDACs and target them to specific promoters via interaction with other proteins such as Rb.
  • Rb transcription co- regulators
  • cyclin A dependent kinase complexes The binding of E2F, CDK/cyclins and geminivirus proteins (such as replicases) occurs via the A/B pocket domain, which is conserved between animal and plant species.
  • Rb-like proteins in plants might be among nuclear targets of plant CDKs (Boniotti and Gutierrez, 2001). Sequential phosphorylation of Rb proteins by cyclin D in G1 and by cyclin E-dependent kinases in late G1 renders it inactive as a repressor of the S phase and thereby promotes DNA replication.
  • the Rb protein-binding motif LXCXE (where X denotes any amino acid) is found in most known plant D cyclins.
  • LXCXE-dependent interactors between D cyclins from Arabidopsis and maize Rb proteins have been demonstrated in vitro and in a yeast two-hybrid assay.
  • the maize Rb proteins contain multiple putative CDK phosphorylation sites, and ZMRB-1 is efficiently phosphorylated in vitro by mammalian G1- and S-specific CDKs. Furthermore, maize Rb proteins are known to undergo changes in phosphorylation during the transition to endoreduplication in the endosperm.
  • European patent application EP 0 914436 describes the isolation and characterisation of a DNA sequence encoding an Rb protein from a plant. The isolation and preliminary characterisation of an Rb homologue from maize has also been described (Xie ef al. (1996 EMBO J. 15: 4900-4908) and Grafi et al. (1996 Proc. Natl. Acad. Sci. USA 93: 8962-8967). The EST sequence was described in Shen ef al., 1994.
  • a method for modifying the growth characteristics of a plant by reducing or substantially eliminating availability of Rb1 in a plant Dicotyledonous plants typically have only one retinoblastoma gene, whereas monocotyledonous plants typically have two retinoblastoma genes, suggesting that the retinoblastoma gene function in monocotyledons and dicotyledons may differ. See for example Figure 2a which shows the percentage similarities and identities between OsRbl , OsRb2, and Rb from Arabidopsis. Percentage identities are shown in bold.
  • OsRbl and the Arabidopsis Rb have 56.9 percent sequence identity, whilst OsRbl and OsRb2 have only 51.6 percent sequence identity. It therefore is likely that the two retinoblastoma genes in monocotyledons may also have distinct functions.
  • Rb1 in corn more closely resembles an Rb2 rather than an Rb1
  • Rb3 in corn more closely resembles an Rb1.
  • a sequence falling within the definition of an Rb1 may be identified in the first instance by determining whether the sequence in question is an Rb. This may be carried out by determining the presence of a "pocket region" which consists of A and B domains separated by a spacer, which is conserved among all metazoan Rb protein family members.
  • the amino acid motif LXX(D/N)RH(I/L)DQ(I/L)XXXCXXXYXXK is a consensus sequence present in all Rb sequences identified to date.
  • the presence of an Rb1 may be identified by aligning (on a nucleotide level) the sequence of interest with an Rb1 from rice (SEQ ID NO: 1) and an Rb2 from rice (SEQ ID NO: 21) and determining whether the sequence has a greater percentage identity to the Rb1 sequence or to the Rb2 sequence. This should be easily identifiable since the percentage identities of the Rb1 and Rb2 sequences are not close in value. If the sequence in question has a greater percentage identity to rice Rb1 than rice Rb2, it is to be encompassed in the definition of Rb1 as used herein. Such alignments can easily be performed by a person skilled in the art using known techniques.
  • Reduction or substantial elimination of availability of Rb1 refers to reduced or substantially eliminated levels and/or activity of an Rb1 protein in a plant or to a decrease or substantial elimination of expression levels of an Rb1 gene in a plant, which gene may be an endogenous Rb1 gene and/or an Rb1 gene introduced into a plant and which reduction or substantial elimination of Rb1 is relative to levels and/or activity of Rb1 in wild type plants.
  • Rb1 may also result from elevated or ordinary (comparable to wild type) levels and/or activity of an Rb1 protein or enhanced or ordinary (comparable to wild type) levels of expression of an Rb1 gene relative to expression levels in corresponding wild type plants, but where the Rb1 protein or gene is in a substantially non-functional form.
  • the Rb1 protein or gene may be in a non-functional form due to, for example, a mutation such that the Rb1 is unable to perform its role in the cell cycle.
  • Various methods for reducing or substantially eliminating levels of Rb1 in plants are described hereinafter.
  • the growth characteristics of a plant may be modified by recombinant means, i.e. by introducing, into a plant, a nucleic acid capable of reducing or substantially eliminating availability of Rb1 , and/or by chemical means, i.e. the exogenous application of a substance capable of reducing or substantially eliminating Rb1 availability in a plant.
  • the availability of Rb1 in plants may be reduced or substantially eliminated by chemical means, such as by the exogenous application of a substance (which may be one or more elements and/or compounds) capable of reducing or substantially eliminating availability of Rb1 in a plant.
  • the exogenous application may comprise contacting, administering or exposing cells, tissues, organs or organisms to one or more plant hormones and/or contacting, administration of or exposure to antibodies to the gene product in cells, tissues, organs or organisms in which levels of the gene product and/or gene product activity are to be modulated.
  • Such antibodies comprise "plantibodies", single chain antibodies, IgG antibodies and heavy chain camel antibodies as well as fragments thereof. Additionally or alternatively, administering or exposing cells, tissues, organs or organisms to an inhibitor of the gene product or to the activity thereof is another exogenous or chemical means for reducing or substantially eliminating availability of Rb1 in a plant.
  • inhibitors include proteins (comprising, for example, proteinases and kinases) and chemical compounds.
  • a method for modifying the growth characteristics of a plant comprising exogenous application of a substance capable of reducing or substantially eliminating availability of Rb1 in a plant.
  • the availability of Rb1 in a plant may be reduced or substantially eliminated using a combination of recombinant DNA technology and exogenous application of one or more substances capable of reducing or substantially eliminating Rb1 availability in plants.
  • the availability of Rb1 in a plant may be reduced or substantially eliminated using recombinant DNA technology.
  • Rb1 availability may be carried out using one or more of several gene silencing techniques well known in the art.
  • Gene silencing or “downregulation” of expression, as used herein, refers to lowering levels of gene expression and/or levels of active gene product and/or levels of gene product activity.
  • the reduction or substantial elimination may be a reduction or substantial elimination of Rb1 protein levels and/or activity and/or Rb1 gene expression levels (which gene may be an endogenous Rb1 gene and/or may be an Rb1 gene previously introduced into a plant).
  • the reduction or substantial elimination is effected by introducing into a plant a double stranded RNA molecule (RNAi), which RNAi molecule is substantially homologous to a target gene encoding a protein capable of either directly or indirectly increasing Rb1 availability in a plant and wherein the RNAi molecule substantially prevents or diminishes functioning of the target gene.
  • RNAi double stranded RNA molecule
  • the target gene may be the Rb1 gene itself or any other gene which interacts directly or indirectly with Rb1.
  • the target gene in a preferred embodiment of the present invention is an Rb1 gene.
  • a reduction or substantial elimination of Rb1 expression may be accomplished by, for example, the introduction of coding sequences or parts thereof in a sense orientation into a plant cell (if it is desired to achieve co-suppression). Therefore, according to one aspect of the present invention, the growth characteristics of a plant may be modified by introducing into a plant an additional copy (in full or in part) of an Rb1 gene already present in a host plant. The additional gene will silence the endogenous Rb1 gene, giving rise to a phenomenon known as co-suppression.
  • Genetic constructs aimed at silencing gene expression may comprise at least a part of an Rb1 nucleotide sequence, for example as depicted in any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18) in a sense and/or antisense orientation relative to the promoter sequence.
  • Sense or antisense copies of at least part of the endogenous gene in the form of direct or inverted repeats may be utilised in the methods according to the invention.
  • Sense strand refers to a DNA strand that is homologous to an mRNA transcript thereof;
  • anti-sense strand refers to an inverted sequence which is complementary to the "sense strand”.
  • the growth characteristics of plants may also be modified by introducing into a plant at least part of an antisense version of the nucleotide sequence depicted in any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
  • Rb1 nucleic acids or any other Rb1 nucleic acid
  • Another method for the reduction or substantial elimination of Rb1 comprises the use of ribozymes, for example as described in Atkins et al. 1994 (WO 94/00012), Lenee et al. 1995 (WO 95/03404), Lutziger ef al. 2000 (WO 00/00619), Prinsen ef al. 1997 (WO 97/3865) and Scott ef al. 1997 (WO 97/38116).
  • Rb1 may also be achieved by insertion mutagenesis (for example, T-DNA insertion or transposon insertion) or by gene silencing strategies as described by, among others, Angell and Baulcombe 1998 (WO
  • Expression of an endogenous Rb1 gene may also be reduced or substantially eliminated if there is a mutation on the endogenous Rb1 gene and/or a mutation on an Rb1 gene subsequently introduced into a plant.
  • the reduction or substantial elimination of Rb1 availability may be caused by a non-functional Rb1 , for example because of a mutation or truncation that may still allow for binding to the E2F/DP complex, but not for inhibition of gene transcription.
  • the endogenous functional Rb1 would therefore be out-titred by overexpression of the non-functional Rb1.
  • the reduction or substantial elimination of Rb1 availability in a plant may be effected by introduction into a plant of a nucleic acid sequence encoding one or more proteins capable of directly or indirectly decreasing Rb1 availability in a plant.
  • introduction and subsequent expression of CDKA and cyclin D will result in hyperphosphorylation of Rb1 and transcription of S phase genes, thereby resulting in inhibition of expression of an Rb1 gene in a plant.
  • co-expression of E2F and DP genes may titre out the Rb1 protein in the cell, such that some E2F/DP complexes still bind to promoters of the S phase genes allowing cell cycle progression.
  • specific protein degradation mechanisms may exist, via the SCF (Skp/Cullin/F-box) complex or the APC (Anaphase Promoting Complex) complex. Up-regulation of these complexes could also lead to an increase in Rb1 breakdown rate.
  • the reduction or substantial elimination of Rb1 may be achieved by lowering, the level of active gene product or level of gene product activity as discussed in the first embodiment of the invention concerning chemical means for the reduction or substantial elimination of Rb1.
  • the essence of the present invention resides in the advantageous and surprising results found upon reduction or substantial elimination of the availability of Rb1 in plants and is not limited to any particular method for the reduction or substantial elimination of the availability of Rbl Other such methods will be well known to the skilled man.
  • the gene silencing techiniques used for the reduction or substantial elimination of the availability of Rb1 require the use of Rb1 sequences from monocotyledonous plants for transformation into monocotyledonous plants.
  • the reduction or substantial elimination of availability of Rb1 comprises introducing into a plant a nucleic acid capable of reducing or substantially eliminating the availability of Rb1 in a plant, wherein the nucleic acid is most preferably an Rb1 nucleic acid.
  • the methods according to the present invention are best performed by introducing into a monocotyledonous plant an Rb1 nucleic acid derived from a monocotyledonous plant.
  • an Rb1 nucleic acid from any given species is introduced into into that same species.
  • an Rb1 nucleic acid from rice (be it a full length Rb1 sequence or a fragment) is transformed into a rice plant.
  • the Rb1 nucleic acid need not be introduced into the same plant variety.
  • nucleic acid sequences or “nucleic acid molecules”, are used interchangeably herein and encompass a polymeric form of a deoxyribonucleotide or a ribonucleotide polymer of any length, either double- or single-stranded, or analogues thereof, that have the essential characteristic of a natural ribonucleotide in that they can hybridise to nucleic acids in a manner similar to naturally occurring polynucleotides.
  • the nucleic acid capable of reducing or substantially eliminating availability of Rb1 comprises at least a part of any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
  • the nucleic acid capable of reducing or substantially eliminating availability of Rb1 is the sequence represented by SEQ ID NO: 3.
  • the sequence depicted in SEQ ID NO: 3 is a fragment of the sequence of SEQ ID NO: 1 , thus clearly illustrating that the full length sequence is by no means necessary to achieve a gene silencing effect and to thereby practise the methods according to the invention.
  • the nucleic acid capable of reducing or substantially eliminating availability of Rb1 may also encode a homologue, derivative or active fragment of the sequence represented by SEQ ID NO: 2.
  • Encoding or “encodes”, with respect to a specified nucleotide sequence, refers to the information for translation into a specified protein.
  • a nucleic acid encoding a protein may contain non-translated sequences such as 5' and 3' untranslated regions (5' and 3' UTR) and introns or it may lack intron sequences, such as in cDNAs.
  • sequences suitable for performing the methods according to the invention include the sequence represented as SEQ ID NO: 4 (which is the 5' UTR of the sequence represented as SEQ ID NO: 1); SEQ ID NO: 5 (which is the 3' UTR of the sequence represented as SEQ ID NO: 1); SEQ ID NO: 6 (which is a partial sequence from sugar cane (Saccharum ssp.) located at the NH 2 end of the corresponding protein); SEQ ID NO: 7 (which is from the same clone as the sequence of SEQ ID NO: 6 but which depicts the 5' UTR); SEQ ID NO: 8 (which is from a second sugar cane clone and which is located at the COOH end of the corresponding protein); SEQ ID NO: 9 (which is from the same clone as that of SEQ ID NO: 8 but which represents the 3' UTR); SEQ ID NO: 10 (which is a full length cDNA of Rb3 from corn), SEQ ID NO: 11 (is the corresponding protein sequence for SEQ ID
  • the methods according to the present invention are not limited to the use of the sequence of SEQ ID NO: 3, nor to any of the sequences depicted by SEQ ID NO: 1 , SEQ ID NO: 2 or any of SEQ ID NO: 4 to SEQ ID NO: 18, which are for illustration purposes only.
  • alternative splice variants of an Rb1 nucleic acid or of any of the aforementioned sequences.
  • alternative splice variant encompasses variants of a nucleic acid in which selected introns and/or exons have been excised, replaced or added. Such variants will be ones in which the functional activity remains unaffected, i.e. such that plants having modified growth characteristics are obtained upon introduction of the splice variant into a plant.
  • Such splice variants may be found in nature or can be manmade. Methods for making such splice variants are well known in the art.
  • allelic variants may also be practised using allelic variants of an Rb1 nucleic acid or of any of the aforementioned sequences.
  • Allelic variants exist in nature and encompassed within the methods of the present invention is the use of these natural alleles.
  • Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp). SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
  • allelic variants in conventional breeding programmes, such as in marker-assisted breeding is also encompassed by the present invention; this may be in addition to their use in the methods according to the present invention.
  • breeding programmes sometimes require the introduction of allelic variations by mutagenic treatment of the plants.
  • One suitable mutagenic method is EMS mutagenesis.
  • Identification of allelic variants may then take place by PCR. This is followed by a selection step for selection of superior allelic variants of the Rb1 sequence and which give rise to altered growth characteristics in a plant.
  • Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the Rb1 sequence, for example, different allelic variants of SEQ ID NO: 1. Monitoring growth performance can be done in a greenhouse or in the field. Further optional steps include crossing plants, in which the superior allelic variant was identified, with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
  • the methods according to the present invention may also be practised by introducing into a plant at least a part of a (natural or artificial) chromosome (such as a Bacterial Artificial Chromosome (BAC)), which chromosome contains at least a gene/nucleic acid sequence encoding an Rb1 protein.
  • a chromosome such as a Bacterial Artificial Chromosome (BAC)
  • BAC Bacterial Artificial Chromosome
  • the method according to the present invention may also be practised using a fragment of an Rb1 DNA or nucleic acid sequence, which fragment is capable of reducing or substantially eliminating availability of Rb1 in a plant.
  • fragment refers to a piece of DNA derived or prepared from an original (larger) DNA molecule.
  • the fragment comprises at least a part of any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
  • the methods according to the present invention may also be practised using nucleic acids capable of hybridising with an Rb1 nucleic acid or with other nucleic acids capable of reducing or substantially eliminating the availability of Rb1 in a plant.
  • the hybridising sequence is capable of hybridising to at least a part of any one of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
  • hybridisation is a process wherein substantially homologous complementary nucleotide sequences anneal to each other.
  • the hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution.
  • Tools in molecular biology relying on such a process include the polymerase chain reaction (PCR; and all methods based thereon), subtractive hybridisation, random primer extension, nuclease S1 mapping, primer extension, reverse transcription, cDNA synthesis, differential display of RNAs, and DNA sequence determination.
  • the hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin.
  • Tools in molecular biology relying on such a process include the isolation of poly (A+) mRNA.
  • the hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitrocellulose or nylon membrane or immobilised by e.g. photolithography to e.g. a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips).
  • Tools in molecular biology relying on such a process include RNA and DNA gel blot analysis, colony hybridisation, plaque hybridisation, in situ hybridisation and microarray hybridisation.
  • the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
  • the stringency of hybridisation is influenced by conditions such as temperature, salt concentration and hybridisation buffer composition.
  • High stringency conditions for hybridisation include high temperature and/or low salt concentration (salts include NaCI and Na 3 -citrate) and/or the inclusion of formamide in the hybridisation buffer and/or lowering the concentration of compounds such as SDS (detergent) in the hybridisation buffer and/or exclusion of compounds such as dextran sulphate or polyethylene glycol (promoting molecular crowding) from the hybridisation buffer.
  • hybridisation conditions are described in, for example, Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York, but the skilled craftsman will appreciate that numerous different hybridisation conditions can be designed in function of the known or the expected homology and/or length of the nucleic acid sequence.
  • Sufficiently low stringency hybridisation conditions are particularly preferred to isolate nucleic acids heterologous to the DNA sequences of the invention defined supra.
  • An example of low stringency conditions is 4-6x SSC / 0.1-0.5% w/v SDS at 37-45°C for 2-3 hours.
  • alternative conditions of stringency may be employed, such as medium stringent conditions.
  • Examples of medium stringent conditions include 1-4x SSC / 0.25% w/v SDS at ⁇ 45°C for 2-3 hours.
  • An example of high stringency conditions includes 0.1-1x SSC / 0.1% w/v SDS at 60°C for 1-3 hours.
  • Elements contributing to heterology include allelism, degeneration of the genetic code and differences in preferred codon usage.
  • SEQ ID NO: 1 The sequences represented by SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 were hitherto unknown.
  • an isolated Rb1 nucleic acid molecule comprising: (a) a nucleic acid represented by any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:
  • SEQ ID NO: 18 or the complement of any of the aforementioned sequences; (b) a nucleic acid encoding an amino acid sequence represented by SEQ ID NO: 2;
  • a nucleic acid encoding a homologue, derivative or active fragment of the amino acid sequence represented by SEQ ID NO: 2, preferably wherein the homologue, derivative or active fragment has, in increasing order of preference, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the sequence of SEQ ID NO: 2;
  • nucleic acid capable of hybridising with a nucleic acid of any one of (a) to (c) above, preferably wherein the hybridising sequence has, in increasing order of preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one or more of the nucleic acids defined in (a) to (c) above;
  • nucleic acids of (a) to (f); nucleic acids having, in increasing order of preference, at least 65%, 70%, 75%,
  • nucleic acids encoding a protein which has, in increasing order of preference, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 2;
  • an isolated Rb1 polypeptide comprising at least part of:
  • GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximises the number of matches and minimises the number of gaps.
  • the BLAST algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information.
  • “Homologues” encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or additions relative to the protein in question and having similar biological and functional activity as an unmodified protein from which they are derived.
  • amino acids of the protein may be replaced by other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break ⁇ -helical structures or ⁇ -sheet structures).
  • Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company).
  • orthologous and paralogous Two special forms of homology, orthologous and paralogous, are evolutionary concepts used to describe ancestral relationships of genes.
  • the term "parologous” relates to gene- duplications within the genome of a species leading to paralogous genes.
  • the term “orthologous” relates to homologous genes in different organisms due to ancestral relationship.
  • the term “homologues” as used herein also encompasses paralogues and orthologues of the proteins according to the invention.
  • substitutional variants of a protein of the invention are those in which at least one residue in an amino acid sequence has been removed and a different residue inserted in its place.
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1-10 amino acid residues, and deletions will range from about 1-20 residues.
  • amino acid substitutions will comprise conservative amino acid substitutions.
  • Insertions of a protein of the invention are those in which one or more amino acid residues are introduced into a predetermined site in said protein. Insertions can comprise amino-terminal and/or carboxy-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino- or carboxy-terminal fusions, of the order of about 1 to 10 residues.
  • amino- or carboxy-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine) 6 -tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag «100 epitope, c-myc epitope, FLAG ® -epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
  • a transcriptional activator as used in the yeast two-hybrid system
  • phage coat proteins phage coat proteins
  • glutathione S-transferase-tag glutathione S-transferase-tag
  • protein A maltose-binding protein
  • dihydrofolate reductase Tag «100 epitope
  • “Deletion variants” of a protein of the invention are characterised by the removal of one or more amino acids from the protein.
  • Amino acid variants of a protein of the invention may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. The manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art.
  • substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
  • Derivatives of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise naturally occurring altered, glycosylated, acylated or non- naturally occurring amino acid residues compared to the amino acid sequence of a naturally- occurring form of the polypeptide.
  • a derivative may also comprise one or more non-amino acid substituents compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence such as, for example, a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring Rb1 protein.
  • Rb1 encompasses at least five contiguous amino acid residues of an Rb1 protein, which residues retain similar biological and/or functional activity to the naturally occurring Rb1.
  • the nucleic acid capable of reducing or substantially eliminating the availability of Rb1 in a plant is preferably comprised within a plant-expressible vector. Therefore, according to a fifth embodiment of the present invention, there is provided a plant-expressible vector comprising:
  • a nucleic acid sequence capable of reducing or substantially eliminating availability of Rb1 in a plant (i) a nucleic acid sequence capable of reducing or substantially eliminating availability of Rb1 in a plant; (ii) a seed-preferred promoter capable of driving expression of the nucleic acid sequence defined in (i); and optionally (iii) a transcription terminator sequence.
  • vector refers to a nucleic acid used for transfection or transformation of a host cell and into which a nucleic acid sequence can be inserted. Expression vectors allow transcription and/or translation of a nucleic acid inserted therein. Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors.
  • the term "expressible” or "plant-expressible” relates to the presence of control sequences which promote adequate expression of genes and/or proper translation of said sequences into a protein.
  • Rb1 nucleic acid or any of the aforementioned nucleic acids are suitable for reducing or substantially eliminating availability of Rb1 in a plant.
  • Most preferred is the sequence depicted in SEQ ID NO: 3.
  • the sequence depicted in any of SEQ ID NO: 1 and SEQ ID NO: 4 to SEQ ID NO: 18 may be suitable for reducing or substantially eliminating availability of Rb1 in a plant.
  • regulatory element refers to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated.
  • regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated.
  • transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • transcriptional regulatory sequence of a classical prokaryotic gene in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences.
  • regulatory element also encompasses a synthetic fusion molecule or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
  • the nucleic acid sequence capable of decreasing or substantially eliminating availability of Rb1 is preferably under the control of a seed-preferred promoter.
  • a seed- preferred promoter is a promoter that is expressed predominantly in seed tissue, but not necessarily exclusively in seed tissue. Seed tissue encompasses any part of the seed including the seed coat, the aleurone layer, the endosperm (for monocots and endospermic dicots), the embryo (scutellum, epiblast, plumule, radicle for monocots; cotyledons, hypocotyl, and radicle for dicots).
  • seed-preferred promoter is used herein because the promoters used to exemplify the methods of the invention also exhibited some expression in parts other than seed tissue, such as in young organs and in meristematic tissue. It is therefore likely that the methods of the invention may also be performed by using a promoter that is a non seed preferred pomoter, but is one that predominanatly drives expression in young organs and in meristematic tissue.
  • the invention is exemplified by use of the seed- preferred promoters: oleosin (SEQ ID NO: 19) and prolamin (SEQ ID NO: 20), however any seed-preferred promoter may be used in the methods according to the invention, as can a promoter predominanatly driving expression in young organs and in meristematic tissue.
  • the oleosin promoter drives expression preferentially in the embryo and in the aleurone layer, but is also very slightly expressed in young developing leaves.
  • promoters listed below are provided for exemplification only and the applicability of the present invention is not limited by the choice of any given promoter. Those skilled in the art will readily be in a position to provide additional promoters that are useful in performing the present invention. The promoters listed may also be modified to provide specificity of expression as required.
  • Inducible promoters are promoters that have induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus.
  • stress-inducible promoter are activated when a plant is exposed to various stress conditions.
  • Examples of stress-inducible promoters, which are also suitable to practise the method according to the invention, are given in Table B as shown below.
  • Such promoters may also be useful in practising the methods of the invention since modified growth (such as increased growth) induced in times of stress may have many advantages.
  • a terminator sequence may also be used in the construct introduced into a plant.
  • the term "terminator” encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription.
  • Additional regulatory elements may include transcriptional as well as translational enhancers.
  • the recombinant DNA constructs for use in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art.
  • the recombinant gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells.
  • the gene of interest may be associated with a selectable marker gene.
  • a selectable marker gene encodes a trait or a phenotype which allows the selection of a plant or plant cell containing the marker. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance. Cells containing the recombinant DNA will thus be able to survive in the presence of antibiotic or herbicide concentrations that kill untransformed cells.
  • selectable marker genes include the bar gene which provides resistance to the herbicide Basta; the npt gene which confers resistance to the antibiotic kanamycin; the hpt gene which confers hygromycin resistance.
  • Visual markers such as the Green Fluorescent Protein (GFP) may also be used as selectable markers.
  • the gene of interest is introduced into a plant by transformation. The term
  • transformation encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.
  • the polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid, or alternatively, may be integrated into the host genome.
  • the resulting transformed plant cell can then be used to regenerate a transformed plant in a manner known to persons skilled in the art. Transformation of plants is now a fairly routine technique.
  • any of several transformation methods may be used to introduce the gene of interest into a cell.
  • Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection.
  • Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1882, Nature 296, 72-74; Negrutiu I. ef al., June 1987, Plant Mol. Biol. 8, 363-373); electroporation of protoplasts (Shillito R.D. et al., 1985 BioTechnol 3, 1099-1102); microinjection into plant material (Crossway A. ef al., 1986, Mol.
  • a preferred method according to the present invention comprises the protocol according to Hiei ef al.1994.
  • plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co- transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
  • putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation.
  • expression levels of the newly introduced DNA may be undertaken using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
  • the present invention also provides plants obtainable by the methods according to the present invention.
  • the present invention also provides transgenic plants having modified growth characteristics, which transgenic plants have reduced or substantially eliminated availability of Rb1 than corresponding wild type plants.
  • a method for modifying the growth characteristics of a plant which method comprises:
  • plant as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and plant cells, tissues and organs.
  • plant cell also encompasses suspension cultures, embryos, meristematic regions, callus tissue, leaves, seeds, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • the methods according to the present invention are applicable to any monocotyledonous plant, preferably cereals.
  • Particularly preferred plants include rice, maize, wheat, barley and millet.
  • plants having reduced or substantially eliminated expression of Rb1 exhibit modified growth characteristics.
  • modified growth characteristics in a plant may be manifested by modified morphology, physiology or biochemistry.
  • plants having reduced or substantially eliminated availability of Rb1 exhibit modified growth characteristics selected from any one or more of:
  • altered cell cycle encompasses, but is not limited to, an altered (increased or decreased) rate of completion of the cell cycle (i.e., progression through the sequential phases G1 - S - G2 - M) relative to corresponding wild-type plants, or a faster rate of completion through one or more, or part of one or more, of the different phases of the cell cycle relative to corresponding wild-type plants.
  • these four phases occur sequentially, however, also encompassed is a modified cell cycle in which one or more of the phases are substantially absent possibly resulting in phenomena such as endomitosis, acytokinesis, polyploidy, polyteny and endoreduplication.
  • Rb is an essential regulator of cell cycle progression, modifying its availability will have an effect on how cell cycle proceeds, in both its duration and its total number of cycles before differentiating. This may have immediate effect on both cell number and ratio of differentiated versus undifferentiated cells in a delimited area (meristem size, for example).
  • the term "increased yield” as used herein encompasses, but is not limited to, an increase in biomass (weight) in one or more parts of a plant relative to the biomass of corresponding wild-type plants.
  • the term also encompasses an increase in seed yield, which includes an increase in biomass of the seed (seed weight) and/or an increase in the number of (filled) seeds and/or in the size of the seeds and/or an increase in seed volume, each relative to corresponding wild-type plants.
  • An increase in seed size and/or volume will also likely influence the composition of seeds.
  • An increase in seed yield could be due to an increase in the number and/or size of flowers.
  • An increase in yield might also increase the harvest index, which is expressed as a ratio of the total biomass over the yield of harvestable parts, such as seeds. Since the transgenic plants according to the present invention have increased yield, it is apparent that these plants exhibit a modified growth rate relative to the growth rate of corresponding wild type plants.
  • modified growth rate encompasses, but is not limited to, a faster rate of growth in one or more parts of a plant (including seeds), or throughout the whole plant, at one or more stages in the life cycle of a plant. Increased growth rate during the early stages in the life cycle of a plant may give rise to enhanced vigour.
  • enhanced vigour or “early vigour” as used herein encompasses, but is not limited to, faster plant growth and/or development in the early stages in the life cycle of a plant (for example after germination) relative to the growth and/or development of corresponding wild- type plants.
  • the increase in growth rate may also alter the harvest time of a plant allowing plants to be harvested sooner than would otherwise be possible.
  • sowing further seeds of the same plant species for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period
  • different plants species for example the sowing and harvesting of rice plants followed by, for example, the sowing and optional harvesting of soy bean, potatoes or any other suitable plant
  • the faster rate of growth may be determined by deriving various parameters from growth curves derived from growth experiments, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size), T-90 (time taken for plants to reach 90%> of their maximal size).
  • An increase in yield also encompasses a better performance of the plant under non- stress conditions and under stress conditions compared to wild-type plants. Plants typically respond to exposure to stress by growing more slowly. However, since the transgenic plants according to the present invention have increased yield, and therefore increased growth rate (see above), it is apparent that transgenic plants will also grow faster during stress conditions than corresponding wild type plants also exposed to the same stress conditions.
  • the stress conditions will typically be the everyday abiotic or environmental stresses to which a plant may be exposed. Typical environmental stresses include temperature stresses caused by atypical hot or cold/freezing temperatures; salt stress; water stress (drought or excess water).
  • modified shoot and root growth and development encompasses, but is not limited to, a morphological change in the shoots and/or roots relative to corresponding wild-type plants, for example, altered number of roots and/or root hairs, altered number and formation of lateral roots, altered root length and/or altered root mass.
  • modified embryo and/or seed development encompasses, but is not limited to, modified size and/or shape of the seed and/or embryo and/or any other part of the seed; altered timing of seed and/or embryo formation/development; altered number of seeds produced.
  • Figure 1 is a diagrammatic representation of the role of Rb in the cell cycle. Hypo- phosphorylated Rb binds to the E2F/DP complex, thereby preventing transcription of the genes controlled by this complex. At the G1/S phase, a CDC2a/cylinD complex phoshorylates Rb, thereby releasing it from the E2F complex. This allows transcription of the E2F responsive genes to proceed. E2F responsive genes may for instance be involved in the S phase of the cell cycle. From Meijer and Murray (2001) Curr Op Plant Biol 4:44-49.
  • Figure 2a shows the percentage similarities and identities at a protein level between OsRbl, OsRb2, and Rb from Arabidopsis. Percentage identities are shown in bold. OsRbl and the Arabidopsis Rb have 56.9 percent sequence identity, whilst OsRbl and OsRb2 have only 51.6 percent sequence identity. Alignments were made using the stretcher program from the EMBOSS package using the following parameters Matrix: EBLOSUM62, gap penalty: 12, extended penalty: 2.
  • Figure 2b shows the percentage identities at a nucleotide level between OsRbl ,
  • Figure 3 is a map of the chimeric gene construct used for downregulation of Rb1 expression.
  • the oleosin promoter (SEQ ID NO: 19) is located upstream of two inverted repeats of OsRbl separated by a fragment of a matrix attachment region (MAR).
  • MAR matrix attachment region
  • Figure 4 is a map of the chimeric gene construct used for downregulation of Rb1 expression.
  • the prolamin promoter (SEQ ID NO: 20) is located upstream of two inverted repeats of OsRbl separated by a fragment of a matrix attachment region (MAR).
  • MAR matrix attachment region
  • a termination signal from the nos gene is located at the other end of the inverted repeat.
  • Figure 5 shows pictures of T1 rice plants from a transgenic event, either homo- or heterozygous for the transgene.
  • FIG. 6 shows pictures of T1 Nullizygote rice plants segregating from the transgenic event. T1 rice plants nullizygous for the transgene were used as internal controls to measure the effect of the transgene on plant growth and yield.
  • Figure 7 shows growth curves during plant development. For each time point, the leaf area was measured for all T1 plants issued from the 5 events. The average data from all positives (Pos, black square; homo- or heterozygous for the transgene) was compared to the average of data obtained from all negatives (Neg, black circle; nullizygous for the transgene).
  • the 90% confidence interval of the averages is represented in both cases by the dotted line.
  • Figure 8 shows the Rb1 sequences useful in practising the methods according to the present invention.
  • Example 1 Cloning of the Rice Rbs A homology based screening was performed using a radioactively labelled probe representing the Medicago sativa Rb gene. The probe was 1521 bp long, including the C- terminus but not the N-terminus (not full length).
  • the cDNA bank screened was made from polyA+ extracted from rice cell suspension cultures harvested at different time points after refreshing the medium: 0, 3, 6, 9 and 12 hours. Total RNA was extracted from each time point individually and equimolar amounts were pooled. The reverse transcription step was performed on this mix. The inserts were ligated into the Stratagene HybridZap vector following the manufacturer's instructions. The initial titer consisted of 2 million clones with an average insert size of approximately 1 KB.
  • RRb1A4, RRb3A4 and RRb4A4 were isolated: RRb1A4, RRb3A4 and RRb4A4.
  • RRb4A4 also known as Rb1
  • Rb1 had an insert of about 3500 bp (SEQ ID NO: 1)
  • RRb1A4 and RRb3A4 were both approximately 900 bp and were thus partial.
  • Sequencing of the three clones revealed that the two smaller inserts originated from the same gene, and were different from Rb1.
  • Example 2 Design of the Chimeric Construct- Down Regulation (Rb1: oleosin) Reduction of expression of endogeneous Rb1 levels was performed using RNAi (RNA interference). An inverted repeat conformation was designed in which the two identical Rb1 fragments were separated by a partial MAR sequence from Nicotiana tabacum (accession number NTU67919).
  • Genomic DNA was isolated from N. tabacum plantlets, and used as a template for PCR amplification using the following primers:
  • p0240 (pUC18-Rb1-MARs) was constructed by cutting a part of Rb1 out of p0229 (gift plasmid pSK RRb1 (Os)ES, which contains the 3' end of Rb1) with Hincll- Ecl136ll. This 743 bp-fragment was. ligated into p156 (pUC18-MARs), opened with Ecl136ll- EcoRI (downstream from the MARs-sequence).
  • the same Rb1 -sequence was cloned into p0240, but in the opposite orientation, upstream from the MARs-sequence.
  • p0240 was opened with Hindi.
  • the Rb1 -fragment used to construct p0240 was used once more, but filled in to get a blunt-end fragment. This resulted in p0241 (pUC18-Rb1-MARs-Rb1 (as)).
  • the Rb1-MARs-Rb1(as)-cassette was then taken out of the p0241 -vector by using Eael and EcoRI. A fill in was performed to blunt the fragment. The fragment was put in p0106 (pCambia1301-Gos2-Gus), opened with the Gus-flanking enzymes Ncol-Pmll to result in p0252: pCambial 301 -Gos2- Rb1 -MARs-Rb1 (as).
  • the 18kDA oleosin promoter was utilised (Genbank accession number of oleosin gene is AF019212). This promotor was picked up from genomic DNA isolated from the rice cultivar IR36 (subsp. Indica) with the following primers:
  • Antisense primer (prm0204, one mismatch inserted for cloning purposes (Ncol site generated in bold))
  • the 1262-bp band was cloned into pUC18, Smal opened (resulting plasmid is called p0272).
  • the oleosin promotor was cut out again from p0272 using Ncol-EcoRl and this fragment was cloned into Ncol-EcoRl opened p0252.
  • the final construct was made by replacing the Gos2-promotor (in p0252) with the Oleosin promotor.
  • Agrobacterium strain LBA4404 harbouring binary T-DNA vectors were used for cocultivation.
  • Agrobacterium was inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28°C.
  • the bacteria were then collected and suspended in liquid co- cultivation medium to a density (OD600) of about 1.
  • the suspension was then transferred to a petri dish and the calli immersed in the suspension for 15 minutes.
  • the callus tissues were then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25°C.
  • Co-cultivated calli were grown on 2,4-D-containing medium for 4 weeks in the dark at 28°C in the presence of a selection agent. During this period, rapidly growing resistant callus islands developed. After transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and shoots developed in the next four to five weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks on an auxin- containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse. Seeds were then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50 % (Alde ita and Hodges1996, Chan ef al. 1993, Hiei et al. 1994).
  • T1 seedlings containing the transgene were selected by PCR.
  • the selected T1 plants were transferred to a greenhouse. Each plant received a unique barcode label to link unambiguously the phenotyping data to the corresponding plant.
  • Transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. From the stage of sowing untill the stage of maturity the plants were passed 10 times through a digital imaging cabinet (examples of pictures are shown in Figures 5 and 6). At each time point, digital images (2048x1536 pixels, 16 million colors) were taken of each plant from at least 6 different angles. The parameters described below were derived in an automated way from the digital images using image analysis software.
  • Plant aboveground area was determined by counting the total number of pixels from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments show that the aboveground plant area measured this way correlates with the biomass of plant parts above ground.
  • Plant height was determined by the distance between the horizontal lines going through the upper pot edge and the uppermost pixel corresponding to a plant part above ground. This value was averaged for the pictures taken on the same time point from the different angles and was converted, by calibration, to a physical distance expressed in mm. Experiments showed that plant height measured this way correlates with plant height measured manually with a ruler.
  • the mature primary panicles were harvested, bagged, barcode-labeled and then dried for three days in the oven at 37°C.
  • the panicles were then threshed and all the seeds collected.
  • the filled husks were separated from the empty ones using an air-blowing device. After separation, both seed lots were then counted using a commercially available counting machine. The empty husks were discarded.
  • the filled husks were weighed on an analytical balance and the cross-sectional area of the seeds was measured using digital imaging. This procedure resulted in the set of seed-related parameters described below.
  • the total seed number was measured by counting the number of filled husks harvested from a plant.
  • the total seed yield was measured by weighing all filled husks harvested from a plant.
  • the average seed weight was determined by dividing the values for total seed yield per plant by the total seed number per plant.
  • the average seed area was measured through analysis of digital images of seeds poured on a petridish. For each object recognised as a seed, the number of pixels contrasted from the background was counted. The average seed area was expressed as the average number of pixels per seed averaged over all the seeds on the petridish.
  • a two factor ANOVA corrected for the unbalanced design was used as statistical model for the overall evaluation.
  • An F-test was carried out to verify for an overall effect of the gene on all the parameters measured.
  • a t-test was performed within each event between data sets from the transgenic plants and the null plants.
  • Aboveground area was measured as a function of time.
  • a curve was fitted according to a three-parameter logistic model yielding parameters for curve slope, time to reach half- maximum value and the maximum value.
  • Results of the maximum aboveground area values are summarized in Table 1.
  • the t- test shows that for one event, transgenics plants are significantly larger (approximately 60% larger) than the nullizygotes, with a probability of the populations being equal of 0.0576.
  • absolute values show an 80% increase of leaf area in transgenic plants versus nullizygous plants, but the t-test results are less significant, with a t Prob of 0.0918.
  • results from the F-test give a probability of 0.0204, indicating that this difference is highly significant and that there is therefore an overall gene effect on maximal plant area.
  • Table 1 Maximum aboveground area.
  • Each row corresponds to one event, for which average maximum aboveground area (expreseed in mm 2 ) was determined for the 10 transgenics and the 5 null lines.
  • T Prob stands for the probability produced by the t-test.
  • the sub-table presents the average numbers for all 5 events.
  • F-Prob stands for the probability yielded by the F-test.
  • Figure 7 represents the growth curves based on aboveground area throughout plant development, with a 90% confidence interval. There is a significant increase in plant aboveground for the transgenics as compared to the nullizygotes.
  • Plant height was measured as a function of time.
  • a curve was fitted according to a three-parameter logistic model yielding parameters for curve slope, time to reach half- maximum value, and the maximum value.
  • Results of the maximum values for plant height measurements are summarized in Table 2.
  • the t-test shows that the best probability obtained for one event is 0.0957. Absolute values for this same event show that a difference in height of 10% is observed between the transgenics lines and the null lines of the event.
  • Table 2 Maximum plant height observed during life cycle.
  • Each row corresponds to one event, for which the average maximum plant height has been determined for the 10 transgenics and the 5 null lines, expressed in mm.
  • T Prob stands for the probability produced by the t-test.
  • the sub-table presents the average numbers for all 5 events.
  • F-Prob stands for the probability yielded by the F-test.
  • Results of the seed number measurements are summarized in Table 3.
  • the absolute average seed number values are double for the transgenics compared to the null plants.
  • Table 3 Number of seeds per plant Each row corresponds to one event for which the number of filled seeds has been determined for the 10 transgenics and the 5 null lines.
  • T Prob stands for the probability produced by the t-test.
  • the sub-table presents the average numbers for all 5 events.
  • F-Prob stands for the probability yielded by the F-test.
  • Results from the average seed area measurements are presented in Table 4.
  • the presence of the transgene has no overall effect, i.e., the seed area is not significantly modified between plant harbouring the transgenes and the null lines (F-test). No "line” effect can be observed either, as shown with the t-test results.
  • T Prob stands for the probability produced by the t-test.
  • the sub-table presents the average numbers for all 5 events.
  • F-Prob stands for the probability yielded by the F-test.
  • Table 5 Averaged seed weight measurements. Each row corresponds to one event, for which the average seed weight (in gram) has been determined for the 10 transgenics and the 5 null lines. T Prob stands for the probability produced by the t-test. The sub-table presents the average numbers for all 5 events. F-Prob stands for the probability yielded by the F-test.
  • Each row corresponds to one event, for which the average weight (g) has been determined for the 10 transgenics and the 5 null lines.
  • T Prob stands for the probability produced by the t-test.
  • the sub-table presents the average numbers for all 5 events.
  • F-Prob stands for the probability yielded by the F-test.
  • RNAi RNA interference
  • An inverted repeat conformation was designed in which the two identical Rb1 fragments were separated by a partial MAR sequence from Nicotiana tabacum (accession number NTU67919).
  • Genomic DNA was isolated from N. tabacum plantlets, and used as a template for PCR amplification using the following primers: Sense primer (prm0122)
  • the fragment of 316 bp (sequence shown below, with primer sequence in bold) was bluntend-cloned into a pUC18 vector and the resulting plasmid was called p0156.
  • p0240 (pUC18-Rb1-MARs) was constructed by cutting a part of Rb1 out of p0229 (gift plasmid pSK RRb1 (Os)ES, which contains the 3' end of Rb1) with Hincll- Ecl136ll. This 743 bp-fragment was ligated into p156 (pUC18-MARs), opened with Ecl136ll- EcoRI (downstream from the MARs-sequence).
  • the same Rb1 -sequence was cloned into p0240, but in the opposite orientation, upstream from the MARs-sequence.
  • p0240 was opened with Hindi.
  • the Rb1 -fragment used to construct p0240 was used once more, but filled in to get a blunt-end fragment. This resulted in p0241 (pUC18-Rb1-MARs-Rb1 (as)).
  • the Rb1-MARs-Rb1(as)-cassette was then taken out of the p0241 -vector by using Eael and EcoRI. A fill-in was performed to blunt the fragment. The fragment was put in p0106 (pCambia1301-Gos2-Gus), opened with the Gus-flanking enzymes Ncol-Pmll to result in p0252: pCambia1301-Gos2- Rb1-MARs-Rb1 (as).
  • the Prolamin RP6 13 kDa promoter was utilised (accession number D63901). This promoter was picked up from genomic DNA Oryza sativa (subsp. Japonica) cultivar Nipponbare with the following primers:
  • Sense primer (prm0173) 5' GAATTCCTTCTACATCGGCTTAGGTGTAGC 3' Antisense primer (prm0172, one mismatch inserted for cloning purposes (Ncol site generated in bold))
  • the 673-bp band was cloned into pUC18, Smal opened (gives p0230).
  • the Prolamin promoter was cut out again from p0230 using Ncol-EcoRl and this fragment was cloned into Ncol-EcoRl opened p0252.
  • the final construct (Fig. 4) was made by replacing the Gos2- promoter (in p0252) with the Prolamin promoter.
  • Example 8 Statistical analysis of the field trial results Single trail analysis was done by analysis of the variance of a randomised complete block design involving 5 replicates with, in the case of location 2, a fertility trend as a linear covariate. The combined analysis of the two trials was approached as a hierarchical model through a mixed model analysis based on a REML algorithm.
  • Table 7 Yield performance of RB1 :prolamin in the field trials performed in location 2, expressed in kg/ha.
  • the transgenic plants were compared to their null segregants. Absolute difference stands for the difference between the two populations in kg/ha. This difference is expressed as a percentage of the null yield in the following column.
  • the PT event is the probability obtained after statistical analysis, that the two populations are identical. The PT value obtained of 0.0087 reveals that the two populations are significantly different.
  • Event plants segregants difference difference PT.event
  • Table 8 Yield performance of Rb1 :prolamin in the field trials performed in location 1 , expressed in kg/ha.
  • the transgenic plants were compared to their null segregants. Absolute difference stands for the difference between the two populations in kg/ha. This difference is expressed as a percentage of the null yield in the following column.
  • the PT event is the probability obtained after statistical analysis, that the two populations are identical. The value obtained here, of 0.03, reveals that the two populations are significantly different.
  • Saccharum ssp clones SCCCCL6001 F07.g and SCBGHR1061 B04.g] are cloned under control of a seed-preferred promoter, such as the oleosin, prolamin or glutelin promoter, in a plant transformation vector suitable for y4grobacfer/ ⁇ v/77-mediated corn transformation.
  • a seed-preferred promoter such as the oleosin, prolamin or glutelin promoter
  • Vectors and methods for corn transformation are selected from those described in any of: EP0604662, EP0672752, EP0971578, EP0955371 , EP0558676, Ishida et al. 1996; and Frame ef al., 2002.
  • Transgenic plants made by these methods are grown in the greenhouse for T1 seed production. Inheritability and copy number of the transgene is checked by quantitative realtime PCR and Southern blot analysis. Expression levels of the transgene are determined by reverse PCR and Northern analysis. Transgenic lines with single copy insertions of the transgene and with varying levels of transgene expression are selected for T2 seed production. Progeny seeds are germinated and grown in a greenhouse in conditions adapted for maize (16:8 photoperiod, 26-28°C daytime temperature and 22-24°C night time temperature) as well under water-deficient, nitrogen-deficient, and excess NaCI conditions.
  • null segregants from the same parental line, as well as wild type plants of the same cultivar are used as controls.
  • the progeny plants resulting from the selfing or the crosses are evaluated for different biomass and growth parameters, including plant height, stem thickness, number of leaves, total above ground area, leaf greenness, time to maturity, flowering time, ear number, harvesting time.
  • the testing of maize for growth and yield-related parameters in the field is conducted using well-established protocols.
  • introgressing specific loci such as transgene containing loci

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Abstract

L'invention concerne des végétaux présentant des caractéristiques de croissance modifiée et leur méthode d'élaboration. Cette méthode implique la diminution ou l'élimination substantielle du rétinoblastome 1 (Rb1) chez un végétal.
PCT/EP2003/009142 2002-08-14 2003-08-13 Vegetaux a croissance modifiee et leur methode d'elaboration WO2004016775A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006131547A1 (fr) * 2005-06-08 2006-12-14 Cropdesign N.V. Plantes a caracteristiques de croissance ameliorees et procede pour produire de telles plantes
WO2011130815A2 (fr) * 2010-04-22 2011-10-27 Universidade Federal Do Rio De Janeiro - Ufrj Procédé pour favoriser une augmentation exacerbée de la biomasse végétale
CN102864167A (zh) * 2012-09-26 2013-01-09 浙江大学 一种植物表达载体及培育低植酸水稻的方法

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WO1998042851A1 (fr) * 1997-03-26 1998-10-01 Cambridge University Technical Services Ltd. Plantes presentant une croissance modifiee
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WO2002004649A2 (fr) * 2000-07-10 2002-01-17 Pioneer Hi-Bred International, Inc. Methodes destinees a augmenter la frequence de transformation des plantes
WO2002074909A2 (fr) * 2001-03-16 2002-09-26 Pioneer Hi-Bred International, Inc. Acides nucleiques et polypeptides intervenant dans le cycle cellulaire, et leurs utilisations

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WO1998042851A1 (fr) * 1997-03-26 1998-10-01 Cambridge University Technical Services Ltd. Plantes presentant une croissance modifiee
WO2001004285A2 (fr) * 1999-07-13 2001-01-18 Pioneer Hi-Bred International, Inc. Polynucleotides msi du maïs et modalites d'utilisation
WO2002004649A2 (fr) * 2000-07-10 2002-01-17 Pioneer Hi-Bred International, Inc. Methodes destinees a augmenter la frequence de transformation des plantes
WO2002074909A2 (fr) * 2001-03-16 2002-09-26 Pioneer Hi-Bred International, Inc. Acides nucleiques et polypeptides intervenant dans le cycle cellulaire, et leurs utilisations

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DATABASE EMBL [Online] 3 April 2000 (2000-04-03), BHALERAO, R.P., ET AL.: "Populus tremula x Populus tremuloides retinoblastoma-related protein 1 (RB1) mRNA, complete cds." XP002265442 Database accession no. AF133675 *
DATABASE EMBL [Online] 9 April 2003 (2003-04-09), JANTASURIYARAT, C., ET AL.: "OSIIEb02J16.f OSIIEb Oryza sativa (indica cultivar-group) cDNA clone OSIIEb02J16 5', mRNA sequence" XP002265444 Database accession no. CB627553 *
DATABASE EMBL [Online] 9 November 1999 (1999-11-09), WING, R.A. ET AL.: "nbeb0012K13f CUGI Rice BAC library (EcoRI) Oryza sativa genomic clone nbeb0012K13f, genomic survey sequence" XP002265443 Database accession no. AQ858184 *
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006131547A1 (fr) * 2005-06-08 2006-12-14 Cropdesign N.V. Plantes a caracteristiques de croissance ameliorees et procede pour produire de telles plantes
US7956240B2 (en) 2005-06-08 2011-06-07 Cropdesign N.V. Plants having improved growth characteristics and method for making the same
CN101189342B (zh) * 2005-06-08 2013-04-03 克罗普迪塞恩股份有限公司 具有改良生长特性的植物及其制备方法
WO2011130815A2 (fr) * 2010-04-22 2011-10-27 Universidade Federal Do Rio De Janeiro - Ufrj Procédé pour favoriser une augmentation exacerbée de la biomasse végétale
WO2011130815A3 (fr) * 2010-04-22 2012-05-10 Universidade Federal Do Rio De Janeiro - Ufrj Procédé pour favoriser une augmentation exacerbée de la biomasse végétale
CN102864167A (zh) * 2012-09-26 2013-01-09 浙江大学 一种植物表达载体及培育低植酸水稻的方法
CN102864167B (zh) * 2012-09-26 2014-04-09 浙江大学 一种植物表达载体及培育低植酸水稻的方法

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