WO2007105002A2 - Molecules d'acides nucleiques - Google Patents

Molecules d'acides nucleiques Download PDF

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Publication number
WO2007105002A2
WO2007105002A2 PCT/GB2007/000939 GB2007000939W WO2007105002A2 WO 2007105002 A2 WO2007105002 A2 WO 2007105002A2 GB 2007000939 W GB2007000939 W GB 2007000939W WO 2007105002 A2 WO2007105002 A2 WO 2007105002A2
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Prior art keywords
nucleic acid
acid molecule
seq
amino acid
sequence
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PCT/GB2007/000939
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English (en)
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WO2007105002A3 (fr
Inventor
Cristel Munster
Cecilie Anita Mathiesen
Anne Kathrine Hvoslef-Eide
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Norwegian University Of Life Sciences
Webber, Philip, Michael
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Publication of WO2007105002A2 publication Critical patent/WO2007105002A2/fr
Publication of WO2007105002A3 publication Critical patent/WO2007105002A3/fr

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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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8266—Abscission; Dehiscence; Senescence
    • 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 nucleic acid molecules that are involved in flower abscission and to the use of such molecules in the production of transgenic plants.
  • Abscission is an active process whereby a plant sheds one of its organs. After a process of senescence, this organ is superfluous to the plant.
  • abscission may be of value to the plant in several ways. It can be a process of self-pruning, removal of injured, diseased, or senescent parts or dispersal of seeds. Abscised plant parts are recycled and their mineral nutrients are returned to the soil. It also functions to maintain the homeostasis of the plant, keeping in balance roots and leaves, and vegetative and reproductive parts. It helps the plant to adapt to the environment in which it lives.
  • Abscission of flowers can be triggered by environmental stress stimuli like rapid temperature changes and unbalances in light or water supply. Ethylene is suggested to be the mediator of flower abscission (van Doom WG: Effect of ethylene on flower abscission: a survey. Annals of Botany 89: 689-693 (2002)). Although many studies on abscission has been done, little is still known about what exactly causes the onset of flower abscission, which genes that are involved and in what order they appear (Roberts et al., "Abscission, dehiscence, and other cell separation processes", Annual Review of Plant Biology 53 131-158 (2002)). Even in the fully sequenced Arabidopsis thaliana, only some genes linked to abscission have been identified.
  • the pot plant Euphorbia pulcherrima commonly known as poinsettia, is the largest ornamental crop in Norway. Six million plants are produced for Christmas every year (Norwegian Growers Association, NGF 3 yearly Statistics). In Northern Europe and North America, the consumption per inhabitant is not as high as in Norway, but poinsettia has a very good ranking among the most sold plants Qerardo A: "Floriculture and nursery crops situation and outlook yearbook”. In: USDA (ed), pp. 131pp. Electronic
  • Poinsettia has large sparkling leaves (bracts), but if the petalless yellow flowers (cyathia) in the middle are missing, growers define this as a serious reduction in quality.
  • the wholesalers would assume that the plants have been grown under poor conditions or exposed to stress during shipment. This would lower the price or make the plant unsaleable (Miller and Heins: "Factors influencing premature cyathia abscission In poinsettia", 'Annette Hegg Dark Red 1 Journal of the American Society for Horticultural Science 111: 114-121 (1986)).
  • auxin has been shown to increase ethylene production and abscission of the corolla (Brown, ibid), but there is also one published example of auxin inhibition of petal or corolla abscission (F. Addicott, American Midland Naturalist 97 (1977) 321-332).
  • the cells need to be at a developmental stage where they are responsive to an ethylene signal (L. Dolan, Journal of experimental botany 48 (1997) 201-210).
  • the ethylene-insensitive -4r ⁇ f ⁇ i ⁇ ps/s mutants etrl and ein2 show a considerable delay in floral abscission and comparative studies demonstrate that these mutants virtually undergo the same developmental progression as wild type (WT) plants.
  • ethylene may be moderated, at least in the case of Arabidopsis floral organ abscission, and it is conceivable that developmental pathways independent of ethylene may regulate floral organ abscission (A.B. Bleecker & S.E. Patterson, Plant Cell 9 (1997) 1169-79, S.E. Patterson, S.M. Huelster & A. Bleecker, Plant Physiol 105, (1994) 43).
  • A.B. Bleecker & S.E. Patterson Plant Cell 9 (1997) 1169-79, S.E. Patterson, S.M. Huelster & A. Bleecker, Plant Physiol 105, (1994) 43.
  • ACC oxidase ACC oxidase
  • up to six different genes encoding ACC synthase (ACS) have been identified in this species.
  • ASC2 is expressed in the abscission zone (AZ) 3 and also at high levels in roots, at moderate levels in flowers and at low levels in siliques, and the other ACC synthase genes are expressed at much lower levels in these organs Q.M. Alonso & J.R. Ecker, Sci STKE 2001 (2001) REl).
  • Anionic silver thiosulphate can inhibit corolla abscission in different plant families. Since silver ions prevent the action of ethylene (E.M.J. Beyer, Plant Physiol 58 (1976) 268-271), the effect of STS in delaying the onset of wilting or abscission is considered to be evidence for the involvement of ethylene in these processes. STS is widely used in the ornamental industry to block the action of ethylene and increase longevity of pot plants and cut flowers. However, STS is a heavy metal compound and hence is an environmental risk. As a result, there is considerable interest within the ornamentals industry in reducing the amounts of STS used Q. Q. Wilkinson et al., Nat. Biotechnol. 15 (1997) 444-7).
  • the invention therefore provides an isolated nucleic acid molecule coding for a molecule which is involved in or associated with abscission, wherein the isolated nucleic acid molecule comprises or consists of a nucleic acid molecule selected from the group consisting of: (i) a nucleic acid molecule whose nucleotide sequence is set forth in any one of SEQ ID NOs: I 5 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28;
  • nucleic acid molecule whose nucleotide sequence has at least 95% sequence identity, preferably at least 98% and more preferably at least 99% sequence identity, with any one of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28; (iii) a nucleic acid molecule that hybridizes with (i) under the following hybridisation conditions: 0.
  • Ix SSC, 0.1% SDS, 65 0 C, and wash conditions 2x SSC, 0.1% SDS, 65 0 C, followed by O.lx SSC, 0.1% SDS, 65 0 C; and (iv) a nucleic acid molecule whose nucleotide sequence is complementary to the sequence of a nucleic acid molecule of (i) or (ii).
  • nucleic acid molecule refers to a polymer of RNA or DNA that is single or double stranded, optionally including synthetic, non-natural or altered nucleotide bases. Examples of such polynucleotides include cDNA, genomic DNA and dsRNA, inter alia.
  • the nucleic acid molecule is dsRNA.
  • nucleic acid sequences referred to herein comprise thymidine ("t") nucleotides, it will be understood that the invention also relates to corresponding sequences wherein thymidine is replaced by uridine (V).
  • t thymidine
  • V uridine
  • the invention provides an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule whose nucleotide sequence is set forth in any one of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28.
  • the invention also relates to variants and derivatives of such nucleic acid molecules.
  • variant includes nucleic acid molecules which have single or multiple nucleotide changes compared to the nucleic acid molecules of the invention.
  • the variants might have 1, 2, 3, 4, or 5 additions, substitutions, insertions or deletions of one or more nucleotides.
  • the invention also relates to nucleic acid molecules whose sequences are degenerate to the sequences of the invention, i.e. molecules whose sequences contain nucleotide changes that do not result in a change in the encoded amino acid sequence.
  • Sequence identity may be assessed by any convenient method. However, for determining the degree of sequence identity between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson, J. D., D. G. Higgins, et al. (1994).
  • the invention also provides an isolated nucleic acid molecule coding for a molecule that is involved in or associated with abscission, wherein the isolated nucleic acid molecule hybridizes with a nucleic acid molecule whose nucleotide sequence is set forth in any one of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 under high stringency conditions, such as for example, O.lx SSC, 0.1% SDS, 65 0 C, and wash conditions 2x SSC, 0.1% SDS, 65 0 C followed by O. lx SSC, 0.1% SDS 3 65 0 C. Such conditions can be used to find similar fragments, such as homologous molecules from related organisms.
  • the nucleic acid molecules of the invention all code for molecules which are involved in or associated with abscission, preferably floral abscission.
  • the "molecule which is involved in or associated with abscission” might be an RNA molecule, such as a rRNA or a tRNA, or a polypeptide or peptide.
  • the molecules may be involved in degradation of the plant cell wall, plant cell-adhesion, in endo-reduplication (nuclear duplication without cell division, causing the cells to increase in size), or protein manufacturing in the ribosomes, inter alia. Some molecules may code for structural proteins; others may have regulatory functions.
  • SEQ ID NO: 1 The functions of some of the molecules of the invention which are involved in or associated with abscission are given in more specific terms below:
  • the polypeptide encoded by SEQ ID NO: 1 has 89% amino acid sequence identity to an A. thaliana immunophilin and hence SEQ ID NO: 1 also codes for an immunophilin or a fragment thereof.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule which codes for an immunophilin whose amino acid sequence has at least 90%, preferably at least 95%, at least 98% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 1.
  • the invention also relates to an isolated polypeptide coding for an immunophilin whose amino acid sequence has at least 90%, preferably at least 95%, at least 98% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 1.
  • SEQ ID NO: 2 has 99% nucleotide sequence identity to a Euphorbia pulcherrima chloroplast tRNA-Leu (trnl) gene and hence SEQ ID NO: 2 also codes for a tRNA-Leu or a fragment thereof.
  • the polypeptide encoded by SEQ ID NO: 3 has 94% amino acid sequence identity to an Atropa. belladona apocytochrome f precursor gene and hence SEQ ID NO: 3 also codes for an apocytochrome f precursor or a fragment thereof.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule which codes for an apocytochrome f precursor whose amino acid sequence has at least 95%, preferably at least 98% or at least 99% sequence identity, with the amino acid sequence encoded by SEQ ID NO: 3.
  • the invention also relates to an isolated polypeptide coding for an apocytochrome f precursor whose amino acid sequence has at least 95%, preferably at least 98% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 3.
  • SEQ ID NO: 4 has 92% nucleotide sequence identity to a Nicotiana tabacum NADH dehydrogenase subunit 2 DNA and hence SEQ ID NO: 4 also codes for a mitochondrial respiratory chain Complex I subunit or a fragment thereof.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule which codes for a mitochondrial respiratory chain Complex 1 subunit and whose nucleotide sequence has at least 95%, preferably at least 98% or at least 99%, sequence identity with SEQ ID NO: 4.
  • the invention also relates to an isolated polypeptide coding for a mitochondrial respiratory chain Complex 1 subunit wherein a nucleotide sequence coding for the polypeptide has at least 95%, preferably at least 98% or at least 99%, sequence identity with SEQ ID NO: 4.
  • polypeptide encoded by SEQ ID NO: 5 has 40% amino acid sequence identity to a Zea mays putative pol protein and hence SEQ ID NO:
  • 5 also codes for a pol protein or a fragment thereof.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule which codes for a pol protein whose amino acid sequence has at least 80%, preferably at least 90%, at least 95% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 5.
  • the invention also relates to an isolated polypeptide coding for a pol protein whose amino acid sequence has at least 80%, preferably at least 90%, at least 95% or at least 99% sequence identity, with the amino acid sequence encoded by SEQ ID NO: 5.
  • the polypeptide encoded by SEQ ID NO: 6 has 81% amino acid sequence identity to a Trifolium pratense ultraviolet-B-repressible protein mRNA and hence SEQ ID NO: 6 also codes for a ultraviolet-B-repressible protein or a fragment thereof or a fragment thereof.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule which codes for an ultraviolet-B-repressible protein whose amino acid sequence has at least 90%, preferably at least 95%, at least 98% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 6.
  • the invention also relates to an isolated polypeptide coding for an ultraviolet-B-repressible protein whose amino acid sequence has at least 90%, preferably at least 95%, at least 98% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 6.
  • the polypeptide encoded by SEQ ID NO: 7 has 72% amino acid sequence identity to an Oryza sativa adenylate kinase and hence SEQ ID NO: 7 also codes for an adenylate kinase or a fragment thereof.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule which codes for an adenylate kinase whose amino acid sequence has at least 80%, preferably at least 90%, at least 95% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 7.
  • the invention also relates to an isolated polypeptide coding for an adenylate kinase whose amino acid sequence has at least 80%, preferably at least 90%, at least 95% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 7.
  • the polypeptide encoded by SEQ ID NO: 8 has 82% amino acid sequence identity to A. thaliana translation initiation factor 3 and hence SEQ ID NO: 8 also codes for a translation initiation factor 3 or a fragment thereof.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule which codes for a translation initiation factor 3 whose amino acid sequence has at least 90%, preferably at least 95%, at least 98% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 8.
  • the invention also relates to an isolated polypeptide coding for a translation initiation factor 3 whose amino acid sequence has at least 90%, preferably at least 95%, at least 98% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 8.
  • the polypeptide encoded by SEQ ID NO: 9 has 71% amino acid sequence identity to an Oryza sativa integral membrane protein and hence SEQ ID NO: 9 also codes for an integral membrane protein or a fragment thereof.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule which codes for an integral membrane protein whose amino acid sequence has at least 80%, preferably at least 90%, at least 95% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 9.
  • the invention also relates to an isolated polypeptide coding for an integral membrane protein whose amino acid sequence has at least 80%, preferably at least 90%, at least 95% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 9.
  • the polypeptide encoded by SEQ ID NO: 10 has 83% amino acid sequence identity to an A. thaliana transcription factor and hence SEQ ID NO: 10 also codes for a transcription factor or a fragment thereof.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule which codes for a transcription factor whose amino acid sequence has at least 90%, preferably at least 95%, at least 98% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 10.
  • the invention also relates to an isolated polypeptide coding for a transcription factor whose amino acid sequence has at least 90%, preferably at least 95%, at least 98% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 10.
  • polypeptide encoded by SEQ ID NO: 11 has 72% amino acid sequence identity to an A. thaliana V-ATPase G subunit 1 and hence SEQ ID NO: 11
  • NO: 11 also codes for a V-ATPase G subunit 1 or a fragment thereof.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule which codes for a V- ATPase G subunit 1 whose amino acid sequence has at least 80%, preferably at least 90%, at least 95% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 11.
  • the invention also relates to an isolated polypeptide coding for a V- ATPase G subunit 1 whose amino acid sequence has at least 80%, preferably at least 90%, at least 95% or at least 99%, sequence identity with the amino acid sequence encoded by SEQ ID NO: 11.
  • the invention particularly relates to an isolated nucleic acid molecule comprising or consisting of a nucleic acid molecule whose nucleotide sequence has at least 80%, preferably at least 90%, at least 95% or at least 99%, sequence identity with the nucleotide sequence set forth in any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28, and which codes for a molecule involved in or associated with abscission.
  • the invention also relates to an isolated polypeptide whose amino acid sequence has at least 80%, preferably at least 90%, at least 95% or at least 99%, sequence identity with the amino acid sequence encoded by any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28, and which is involved in or associated with abscission. Further details of the sequence identities between the above-mentioned sequences and those of the prior art are given in Figure 5.
  • variants and derivatives of the invention of the above-specified sequences will have the same function as the above- specified sequences.
  • the invention also relates to a nucleic acid molecule comprising a fragment of a nucleic acid molecule whose nucleotide sequence is set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28.
  • the fragment consists of at least 20, more preferably at least 40 and most preferably at least 60 contiguous nucleotides obtained from a nucleic acid molecule whose nucleotide sequence is set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or
  • a further embodiment of the invention provides a chimeric gene comprising an isolated nucleic acid molecule coding for a molecule which is involved in or associated widi abscission, wherein the isolated nucleic acid molecule comprises or consists of a nucleic acid molecule selected from the group consisting of:
  • nucleic acid molecule whose nucleotide sequence is set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28;
  • nucleic acid molecule whose nucleotide sequence has at least 95% sequence identity, preferably at least 98% and more preferably at least 99% sequence identity, with any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28;
  • one or more of the regulatory sequences are heterologous regulatory sequences, for example, a heterologous promoter.
  • operably linked refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences may be operably linked to regulatory sequences in sense or antisense orientation.
  • coding sequence in this context applies not only to nucleotide sequences which encode polypeptides, but also nucleotide sequences which encode RNA, for example, rRNA and tRNA.
  • regulatory sequences refers to nucleotide sequences located upstream (5 1 non-coding sequences), within, or downstream (3 1 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 may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. Preferably, the regulatory elements are ones that are operational in plants, for example, a plant promoter and plant polyadenylation recognition sequence.
  • promoter refers to a nucleotide sequence capable of controlling the expression of a coding sequence or RNA.
  • a coding sequence is located 3' to a promoter sequence.
  • the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an “enhancer” is a nucleotide sequence that 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. 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 comprise synthetic nucleotide segments.
  • promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1- 82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.
  • a further embodiment of the invention provides a vector comprising a nucleic acid molecule coding for a molecule which is involved in or associated with abscission, wherein the isolated nucleic acid molecule comprises or consists of a nucleic acid molecule selected from the group consisting of:
  • nucleic acid molecule whose nucleotide sequence is set forth in any one of SEQ ID NOs: I 5 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28;
  • nucleic acid molecule whose nucleotide sequence has at least 95% sequence identity, preferably at least 98% and more preferably at least 99% sequence identity, with any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28;
  • a nucleic acid molecule whose nucleotide sequence is complementary to the
  • Vectors comprising the instant isolated nucleic acid molecule of the invention (or chimeric gene of the invention) may be constructed.
  • the choice of vector is dependent upon the method that will be used to transform host plants or on another intended use of the vector.
  • the skilled person is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells containing the nucleic acid molecule or chimeric gene of the invention.
  • the skilled person will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) MoI. Gen.
  • the invention particularly relates to a vector comprising an isolated nucleic acid molecule of the invention, wherein the vector is suitable for the production of a ssRNA or dsRNA form of the nucleic acid molecule of the invention.
  • the vector may, for example, comprise two opposing RNA promoters that flank the nucleic acid molecule of the invention, for the production of dsRNA.
  • the invention particularly relates to a vector comprising an isolated nucleic acid molecule of the invention for use in co- suppression.
  • the invention also provides the use of an isolated nucleic acid molecule of the invention in the production of an antisense form of the nucleic acid molecule or a dsRNA form of the nucleic acid molecule.
  • the invention also provides an isolated host cell which has been transformed or transfected with an isolated nucleic acid molecule of the invention or a chimeric gene or vector of the invention.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, plant or mammalian cells.
  • the host cells are monocotyledonous or dicotyledonous plant cells.
  • host cells are Arabidopsis cells and Euphorbia cells, cells of crop plants, and cells from Begonia x cheimantha, Begonia x hiemalis, Begionia x tuberhybrida, Begonia semperflorens, Campanula ssp., Brassica ssp., or Lycopersium esculentum.
  • the term "host cell” does not include human embryonic cells.
  • the host cell is a heterologous host cell, i.e. it is of a species which is different from the species of origin of the nucleic acid molecule of the invention.
  • the present invention also provides a process for producing an isolated host cell comprising an isolated nucleic molecule of the present invention or a chimeric gene or vector of the present invention, the process comprising transforming or transfecting a host cell with an isolated nucleic molecule of the present invention or a chimeric gene or vector of the present invention.
  • transformation or "transformed” or
  • transfected refers to the transfer of a nucleic acid molecule into the genome of a host organism, resulting in genetically stable inheritance.
  • methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al.
  • isolated nucleic acid molecules of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell.
  • a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence or RNA in a given host cell.
  • a number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, for example, Pouwels et al «1985; Supp.
  • plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5 1 and 3 1 regulatory sequences and a dominant selectable marker.
  • Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • a promoter regulatory region e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific expression
  • a further embodiment of the invention relates to a transgenic plant which comprises a nucleic acid molecule or chimeric gene or vector of the invention.
  • the nucleic acid molecule or chimeric gene or vector of the invention is integrated within the genome of the transgenic plant, most preferably stably integrated within the genome such that the nucleic acid molecule or chimeric gene or vector of the invention is passed on to successive generations of the plant.
  • the transgenic plant comprises a co-suppression vector, i.e. a vector comprising a nucleic acid molecule of the invention that is capable of producing a ssRNA or dsRNA form of one or more of the nucleic acid molecules of the invention.
  • a co-suppression vector i.e. a vector comprising a nucleic acid molecule of the invention that is capable of producing a ssRNA or dsRNA form of one or more of the nucleic acid molecules of the invention.
  • the plant is a monocot or dicot.
  • the plant is a crop plant, Arabidopsis ssp. ⁇ Euphorbia ssp. particularly Euphorbia pulcherrima, Begonia x cheimantha, Begonia x hiemalis, Begionia x tuberhybrida, Begonia semperflorens, Campanula ssp., Brassica $$p., or Lycopersium esculentum.
  • the invention also provides a method for making a transgenic plant, comprising transforming or transfecting a plant cell with a nucleic acid molecule or a chimeric gene or vector of the invention, and regenerating a plant from the transformed plant cell.
  • the term “genome” encompasses not only chromosomal DNA found within the nucleus, but also organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the plant cell.
  • plant includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of same.
  • transgenic plants are to be understood within the scope of the invention to comprise, for example, plant cells, protoplasts, tissues, callus 3 embryos, flowers, stems, fruits, leaves, roots originating in transgenic plants or their progeny previously transformed with a nucleic acid molecule of the invention and therefore consisting at least in part of transgenic cells which comprise the nucleic acid molecules of the invention, are also an object of the present invention.
  • plant cell includes seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores, inter alia.
  • the class of plants that can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
  • progeny comprises any subsequent generation of a plant and parts of that plant.
  • the invention particularly relates to seeds of plants of the invention which contain a nucleic acid molecule of the invention, or chimeric gene or vector of the invention.
  • the host cell or plant will express the molecule which is involved in or associated with abscission at a higher level or lower level compared to a host cell or plant that has not been transformed or transfected with the isolated nucleic acid molecule of the invention.
  • the plant will express the isolated nucleic acid molecule of the invention in cell types or at developmental stages in or at which the isolated nucleic acid molecule of the invention is not normally found.
  • the nucleic acid molecule is dsRNA.
  • the nucleic acid molecule of the invention will be transformed or transfected into a heterologous host cell or plant.
  • heterologous in reference to a nucleic acid molecule is a nucleic acid molecule that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • the invention provides a transgenic plant which contains a disruption in a gene whose sequence comprises a sequence as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28, or a sequence which has at least 80%, preferably at least 90% or at least 95% identity thereto, wherein the disruption results in the prevention of abscission or delay in abscission of one or more organs of that plant.
  • Overexpression of the nucleic acid molecules of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development.
  • the chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3' non-coding sequences encoding transcription termination signals may also be provided.
  • the instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.
  • Cells may be transformed by an appropriate method and then the cells that have been transformed may be grown into plants in accordance with conventional ways. (See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84). These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved.
  • a chimeric gene of the invention can be constructed by linking a nucleic acid molecule of the invention to an appropriate plant promoter sequence.
  • a chimeric gene designed to express antisense RNA for all or part of the current nucleic acid molecule can be constructed by linking the nucleic acid molecule of the invention in reverse orientation to an appropriate plant promoter sequence. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated. Elimination or reduction of expression may also be obtained by T-
  • the T-DNA transformation vector may, for example, be pROK 2 (Baulcombe et at, "Expression of biologically-active viral satellite RNA from the nuclear genome of transformed plants”. Nature 321 (6068): pp. 446-449, May 22 1986).
  • RNA in situ hybridisation can be used to localise with precision the area of expression of a nucleic acid molecule of the invention. It is a technique that allows specific nucleic acid molecules to be detected in morphologically preserved tissue sections, cells or chromosomes (Dahiya et al., Plant Phys 138 (2005) 1383-1395). Combined with immunocytochemistry, in situ hybridisation can relate gene activity at the
  • DNA, mRNA or protein level to microscopic topological information.
  • Positive hybridisation with an anti-sense probe in abscission zone of the plant of interest may be used to confirm the expression of a nucleic acid molecule.
  • digoxigenin (DIG) labelling of probes generated with specific (qRT-PCR) primers from cDNA template is used, followed by indirect detection by anti-DIG alkaline phosphatase conjugate.
  • the invention also provides antibodies which specifically bind to the polypeptides of the invention.
  • the antibodies may be of any suitable source, e.g. monoclonal, polyclonal, chimeric, bispecific, single-chain or fragments thereof.
  • Antibody fragments include, but are not limited to, Fab, Fab 1 and
  • Antigen- binding antibody fragments including single-chain antibodies, may comprise the variable region (s) alone or in combination with the entirety or a portion of the following: hinge region, CHl, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHl, CH2, and CH3 domains.
  • the antibodies of the invention may be from any animal origin including birds and mammals, or derived from phage or ribosome display libraries.
  • the antibodies are human, murine (e.g. mouse or rat), donkey, rabbit, goat, guinea pig, camel, horse, or chicken.
  • the antibodies of the invention may be monospecific, bispecific, trispecific or of greater multisperificity.
  • the antibodies may be labelled in any suitable manner, thus allowing for detection in a suitable assay.
  • Figure 1 illustrates Poinsettia abscission induction.
  • the line show the place for decapitation and the box the dissection area containing the AZ, indicating the pedicel area used for total RNA preparations.
  • Figure 2 shows the Real-Time RT-PCR primers for confirmation of DD expression of sequences isolated from poinsettia flower abscission zone.
  • the primers were synthesized by Invitrogen.
  • Figure 3 shows DD-PAGE sections of cDNA PCR products that were differentially expressed.
  • Poinsettia flower abscission zone total RNA samples were prepared from day 0 to day 7(0-7).
  • Column marked d include distal part of the pedicel as an internal control.
  • Left column indicate specific clone numbers. For clarity arrows indicating specific bands isolated.
  • Figure 4 shows the temporal expression patterns of DD clones monitored by Real-Time RT-PCR.
  • ⁇ Ct on the y axis refers to the fold difference of a particular DD clone mRNA level relative to its lowest expression. Expressions were normalized to the 18S ribosomal RNA endogenous control during an induced abscission process of poinsettia pedicel, whereas distal is an internal control sample dissected from above the
  • Figure 5 shows Real-Time RT-PCR results and similarity searches of DD clones from poinsettia flower abscission.
  • nr GenBank non-redundant
  • GenBank nr GenBank non-redundant
  • GenBank nr GenBank non-redundant
  • SwissProt with blastx.
  • Internal control Distal part of the abscising pedicel above the abscission zone.
  • d These genes were isolated as positive controls.
  • Figure 6 shows the Differential Display primers by GenHunter.
  • Figure 7 shows the sequences identified as differentially expressed during poinsettia flower abscission. All sequences were found using an upstream primer including an EcoRI site (first 6 bases in the submitted sequences) except for 125c and 208 and a downstream polyT-primer (sequences end therefore with polyA).
  • Abscission zones were obtained from flowering E. pulcherrima 'LiIo'. The poinsettias were induced to flower in short day conditions (1Oh) and 20°C night temperatures. The vents were set at 23°C during the day. Cyathia of the 3rd order (all male flowers) was chosen to standardise the plant material RNA extraction for differential display (DD). Abscission was induced by decapitation of the 3rd order buds with a razor blade, precisely under the stamens, leaving no traces of floral organs, but the floral bottom intact (Fig. 1). Abscission zones of 1-2 mm were dissected from the pedicel from Day 0 until Day 7.
  • RNAlater 0.1 g ml
  • RNAlater AZ tissue in RNAlater from the different stages of abscission development was thawed, the liquid removed and the tissue frozen again in liquid nitrogen.
  • a pistil for eppendorf tubes were cooled in liquid nitrogen and used to homogenize the tissue in the eppendorf tube.
  • the first solution in the RNeasy Plant Kit (Qiagen) was immediately added and samples stored on ice.
  • Total RNA was immediately prepared from the homogenized AZs following the manufacturers instructions, including DNase I treatment using RNase-Free DNaseI Set (Qiagen).
  • the quantity and quality of the RNA was measured by NanoDrop ND- 1000 (NanoDrop Technologies) spectrophotometer. Only RNA samples with a 260/280 nm ratio between 1.9 and 2.1 and a concentration higher than 30 ng ⁇ F were used.
  • Fluorescent DD was performed with the RNAspectra kit (GenHunter, Nashville, TN).
  • the mRNA in the total RNA samples were converted into DNA by reverse transcriptase with anchor primers (H-Tl IA 3 H-Tl IG or H- Tl 1 C) .
  • the resulting cDNA was amplified by DNA Polymerase
  • DyNAzymell (Finnzyme, Espoo, Finland) with the anchor primers and arbitrary primers from RNA spectral kits red and green #1, 4, 5 and 8 (for primer sequences see Figure 6). Besides those 32 arbitrary primers from GenHunters kits, four primers were designed on conserved areas of polygalactorunase and beta- 1 ,3-glucanase:
  • PG-A 5'-AAGCTTATTATGGAGG-S' (SEQ ID NO: 29), PG-T S'-AAGCTTATTTTGGAGG ⁇ 1 (SEQ ID NO: 30), GlucC 5'-AAGCTTTATGGAATG-S' (SEQ ID NO: 31) and GlucA 5'- AAGCTTTATGGCATG -3' (SEQ ID NO: 32).
  • the amplification products were separated on 6% denaturing polyacrylamide gels casted between low fluorescence glass plates (Amersham Bioscience). Parallel amplification products with fluoricin or rhodamin anchor primes were separated on different gels.
  • the gels were scanned on Typhoon 8600 (Amersham Bioscience) using the following laser settings: flouricin, excitation 495 nm, emission 520 nm, green laser (532 nm), emission filter 526 SP and rhodamine, excitation 570 nm, emission 590 nm, green laser (532 nm), emission filter 580 BP 30.
  • Kapton tape (Amersham Bioscience) was used for gel orientation.
  • the digital gel image was printed on paper 1 : 1 size and used for gel orientation and band identification. Bands appearing as differentially expressed were excised from the saran wrap covered gel with a surgical blade.
  • DNA from the fragments was eluted into distilled water, precipitated and reamplified by PCR as described by the fluorescent DD kit manufacturer.
  • the PCR product was purified on an agarose gel. Single bands larger than 200 bp were excised and subcloned into plasmid pCR 2.1-TOPO (Invitrogen) and chemically transferred into Top 10 Escherichia coli cells as described by the manufacturer. Twelve positive E. coli colonies were selected, restreaked and analysed by colony PCR. The insert was confirmed by separating the PCR products on an agarose gel. Plasmids from eight E.
  • coli clones were prepared using Montage plasmid miniprep 96 (Millipore) and Jetquick Plasmid Purification Spin Kit (Genomed). The insert was sequenced with BigDye Terminator Cycle Sequencing Kit v3.1 and ABIprism 3100 (Applied Biosystems). Sequences were visualized and processed in BioEdit sequence alignment editor (Hall TA: BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium 41 (1999)).
  • Real-Time RT-PCR primers were constructed using Primer Express (Applied Biosystems) ( Figure 2). The primers were tested on both cDNA and genomic DNA. Functioning primer pairs were used to confirm differential expression by using Superscript III Platinum Two-Step qRT-PCR Kit (Invitrogen) 3 a Real-Time quantitative RT-PCR kit containing ROX reference dye on an 7900HT Fast Real-Time PCR System (Applied Biosystems). An 18S primer pair was used to make correlative gene expression measurements. The relative expression of the selected sequences was calculated using Calibrator Normalized Relative Quantification (Anon:
  • RNA samples used in the DD screening were prepared from AZ tissue at eight different stages: every 24 h from Day 0 (control) and until abscission on Day 7.
  • the first visible signs of the forthcoming abscission event could be seen as a lighter green band at the place of the AZ and the onset of senescence related colour changes of the proximal end of the cyathia (flowers) (Fig. 1).
  • the proximal end normally falls off after a carefully prepared separation of the cell layers of the plant in the AZ.
  • the time lap from induction to separation (abscission) is normally seven days during the poinsettia season, which is the period before Christmas (advent) and the Christmas holidays.
  • RNA preparations of total RNA using RNeasy showed variable results due to difficulties homogenising poinsettia pedicel tissue and not with a specific stage of the abscission process.
  • the amount of total RNA was quantified by NanoDrop apparatus to be able to calibrated to equal amounts of template in the different cDNA reactions.
  • Each DD kit (GenHunter) contained eight arbitrary primers. Large difference could be observed in the ability of each primer to dissolve differentially displayed bands, only 10 out 32 primers did. In contrast to our own primers, those were all able to produce DD bands that were picked up for identification. Fluorescing primers were just as effective as radioactivity (data not shown), rhodamine signal was generally stronger than fluorescein. Examples of DD bands can be seen in Figure 3, selected due to their especially good correlation with the following confirmation analyses and clear PAGE results.
  • RNAi constructs are then made of the most conserved regions of the genes and transformed into poinsettia through electrophoresis DNA transfer and Arabidopsis with Agrobacterium floral dip to down-regulate the genes. 35S and abscission zone specific promoter constructs are used to up-regulate (over-express) the genes.

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Abstract

La présente invention concerne des molécules d'acides nucléiques qui sont impliquées dans l'abscission des fleurs, et l'utilisation de telles molécules pour la production de plantes transgéniques.
PCT/GB2007/000939 2006-03-16 2007-03-16 Molecules d'acides nucleiques WO2007105002A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994023043A2 (fr) * 1993-03-31 1994-10-13 Nickerson Biocem Limited Regulation de l'abscission et de la dehiscence des gousses dans les vegetaux
WO2004057004A2 (fr) * 2002-12-23 2004-07-08 Melinka Butenko Gene de plante

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994023043A2 (fr) * 1993-03-31 1994-10-13 Nickerson Biocem Limited Regulation de l'abscission et de la dehiscence des gousses dans les vegetaux
WO2004057004A2 (fr) * 2002-12-23 2004-07-08 Melinka Butenko Gene de plante

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Planter og miljo - summary" INTERNET ARTICLE, [Online] 2 February 2006 (2006-02-02), XP002444065 Retrieved from the Internet: URL:www.umb.no/print.php?viewID=14532> [retrieved on 2007-07-25] *
FERNANDEZ D E ET AL: "The embryo MADS domain factor AGL15 acts postembryonically: inhibition of perianth senescence and abscission via constitutive expression" PLANT CELL, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 12, February 2000 (2000-02), pages 183-197, XP002973250 ISSN: 1040-4651 *
GONZALES-BOSCH C. ET AL: "Inmmunodetection and characterization of tomato endo-beta-1,4-glucananse Cel1 protein in flower abscission zones" PLANT PHYSIOLOGY, vol. 114, 1997, pages 1541-1546, XP002444061 *
KANDASAMY M. ET AL: "Arabidopsis ARP7 is an essential actin-related protein required for normal embryogenesis, plant architecture, and floral organ abscission" PLANT PHYSIOLOGY, vol. 138, no. 4, August 2005 (2005-08), pages 2019-2032, XP002444060 *
MUNSTER C: "ON THE FLOWER ABSCISSION OF POINSETTIA (EUPHORBIA PULCHERRIMA WILLS. EX KLITZSCH), - A MOLECULAR AND PLANT HORMON STUDY" THESIS, XX, XX, 2006, page 120PP, XP001536511 *
Poster presentation at the X cell wall meeting Sorrento Italy 29/8-3/9 2004 "Abscission of poinsettia flowers studied by differential display" XP002444063 *
Poster presentation at the X cell wall meeting Sorrento Italy 29/8-3/9 2004 "The quest for genes involved in abscission using poisettia as the model plant" XP002444062 *

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