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WO2000037488A2 - Nouveaux genes a boite mads et utilisation de ces genes - Google Patents

Nouveaux genes a boite mads et utilisation de ces genes

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Publication number
WO2000037488A2
WO2000037488A2 PCT/EP1999/010116 EP9910116W WO0037488A2 WO 2000037488 A2 WO2000037488 A2 WO 2000037488A2 EP 9910116 W EP9910116 W EP 9910116W WO 0037488 A2 WO0037488 A2 WO 0037488A2
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WIPO (PCT)
Prior art keywords
nucleic acid
plant
acid molecule
expression
protein
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PCT/EP1999/010116
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English (en)
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WO2000037488A3 (fr
Inventor
Jorge Cacharron
Günther THEISSEN
Wim Deleu
Heinz Saedler
Original Assignee
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to AU30402/00A priority Critical patent/AU3040200A/en
Publication of WO2000037488A2 publication Critical patent/WO2000037488A2/fr
Publication of WO2000037488A3 publication Critical patent/WO2000037488A3/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
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • 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 encoding MADS-box proteins that are specifically expressed in the upper florets of spikelets of grass inflorescence as well as to regulatory sequences which naturally regulate the expression of such nucleic acid molecules.
  • the present invention also provides vectors comprising said nucleic acid molecules, wherein the nucleic acid molecules are operatively linked to regulatory elements allowing expression in prokaryotic and/or eukaryotic host cells as well as proteins encoded by said nucleic acid molecules, antibodies to said proteins and methods for their production.
  • the present invention also relates to recombinant DNA molecules and vectors comprising said regulatory sequences as well as to host cells transformed therewith.
  • the present invention further relates to kits and diagnostic compositions comprising the aforementioned nucleic acid molecules, proteins, antibodies, regulatory sequences, recombinant DNA molecules and vectors as well as antibodies.
  • the present invention also relates to methods for the identification of compounds being capable of activating or inhibiting the expression of genes specifically expressed in the upper florets of spikelets of grass inflorescence and/or their gene products.
  • the present invention relates to transgenic plant cells, plant tissue and plants containing the above-described nucleic acid molecules, regulatory sequences, recombinant DNA molecules and vectors as well as to the use of the aforementioned nucleic acid molecules, regulatory sequences, recombinant DNA molecules, vectors, proteins, antibodies and/or compounds identified by the method of the invention in plant cell and tissue culture, plant breeding and/or agriculture.
  • Inflorescence and flower development are determined by a network of regulatory genes which is organized in a hierarchical fashion (for recent reviews, see Okada and Shimura 1994; Thei ⁇ en and Saedler 1995). Near the top of that hierarchy there are 'late and early flowering genes' that start reproductive development, perhaps by activating meristem identity genes. Mehstem identity genes 'control' the transition from vegetative to inflorescence and from inflorescence to floral meristems.
  • cadastral genes set the boundaries of floral organ identity gene functions, thus defining the different floral whorls.
  • floral organ identity genes homeotic selector genes; "ABC genes”
  • Floral organ identity genes could be cloned meanwhile from several plant species (Coen and Meyerowitz 1991 ; Weigel and Meyerowitz 1994; Thei ⁇ en et al. 1996). Most of them, but also some of the meristem identity and cadastral genes belong to the family of MADS-box genes.
  • MADS-box which encodes the DNA-binding domain of the respective MADS-domain transcription factors
  • MADS-domain proteins have a very similar modular structure that includes a MADS (M-), intervening (I-), keratin-like (K-) and C-terminal (C-) domain (Thei ⁇ en et al. 1996).
  • MIKC-type MADS-box genes M ⁇ nster et al.
  • MADS-box genes Comparably less is known about MADS-box genes in monocotyledonous plants, though monocots include the cereals, being the globally most important crop plants. Since cereal inflorescence and flower development is of central agronomic importance, knowledge about MADS-box genes in these species might aid a future design of crops by transgenic technology (Meyerowitz 1994). Moreover, since the lineages that led to monocots and eudicots have already separated about 200 million years ago (Savard et al. 1994), a comparison of MADS- box gene structure and function between both taxa might tell a great deal about the conservation as well as the variability of the molecular control of flower development.
  • grass inflorescences and flowers are considered, which are very distinct from those of eudicots.
  • grasses such as maize (Zea mays ssp. mays) produce flowers in which the perianth is highly reduced or absent.
  • the provision of genes involved in the setting of floral organ identity and in particular their regulatory sequences may have applications in several aspects of agriculture.
  • the technical problem underlying the present invention was to comply with the need for genes and their regulatory sequences which are specific for a certain tissue and/or cells hitherto not available.
  • the invention relates to a nucleic acid molecule encoding a protein having the immunological and/or biological activity of a protein that is expressed in the upper florets of spikelets of grass inflorescences, said nucleic acid molecule being selected from the group consisting of:
  • nucleic acid molecules comprising a nucleotide sequence encoding a protein comprising the amino acid sequence as given in SEQ ID NO: 2 or 4;
  • nucleic acid molecules comprising the nucleotide sequence as given in SEQ ID NO: 1 or 3;
  • nucleic acid molecules hybridizing with the complementary strand of a nucleic acid molecule as defined in (a) or (b) under stringent hybridization conditions;
  • nucleic acid molecules encoding a protein having an amino acid sequence which has an identity of at least 90 % to the amino acid sequence encoded by the nucleic acid molecule of (a) or (b);
  • nucleic acid molecules encoding a protein comprising an amino acid sequence which is at least 75 % identical to the amino acid sequence from amino acid residue 76 to 86 or at least 60% identical to the amino acid sequence from amino acid residues 175 to 235 encoded by a nucleic acid molecule of (a) or (b);
  • nucleic acid molecules comprising a nucleotide sequence encoding at least the l-domain and/or C-domain of a protein encoded by a nucleic acid molecule of any one of (a) to (e); and (g) nucleic acid molecules, the nucleotide sequence of which is degenerate as a result of the genetic code to a nucleotide sequence of a nucleic acid molecule as defined in any one of (a) to (f).
  • expressed or expression in the upper florets of spikelets of grass inflorescences means that said protein or its encoding gene is to be predominantly, preferably exclusively expressed in upper florets of spikelets of inflorescences such as from the monocotyledonous plant maize and that no or substantially no expression is present in other organs such as the lower florets of the spikelets.
  • MADS-box genes Most floral meristem and organ identity genes of dicotyledonous plants belong to the MADS-box gene family. Since they are generally transcribed in those tissues and organs whose identity they determine, they are excellent markers for developmental processes. In accordance with the present invention, a novel pair of MADS-box genes, ZMM8 and ZMM14, from the monocotyledonous plant maize have been cloned.
  • ZMM8 is a novel MADS-box gene from maize, of which previously only a partial cDNA had been obtained among several other MADS-box genes (Fischer et al. 1995a). According to the amino acid sequence of 47 amino acids in length phylogeny reconstructions suggested that the corresponding gene is a member of the clade of GL2-like MADS-box genes (Thei ⁇ en et al. 1996; M ⁇ nster et al. 1997). While the function was not known, initial attempts to isolate genomic clones or full length cDNA clones were hampered by the fact that the conserved MADS-domain is shared by several gene family members.
  • cDNAs containing the complete ZMM8 coding region SEQ ID NO: 1
  • ZMM14 SEQ ID NO: 3
  • ZMM8 and ZMM14 mRNAs are expressed in the upper floret meristem and all its derivatives, but apparently not in the lower floret; see Figures 4 to 7.
  • Maize is a monoecious species, i.e. it forms male and female inflorescences separately on the same plant.
  • the male inflorescence (tassel) develops in a terminal position, whereas the female inflorescences (ears) grow in the axils of vegetative leaves.
  • the unisexual flower types of the tassel and ear are both derived from an initially bisexual state through the abortion of pistil phmordia in the tassel and stamen primordia in the ear (Cheng et al. 1983).
  • the three stamens or carpels (pistil) of each maize flower are surrounded by a pair of bract-like scales called palea (inner) and lemma (outer), thus constituting structures called florets.
  • a pair of bract-like scales called palea (inner) and lemma (outer)
  • florets In the flowers of the tassel also lodicules are formed, two knob-like perianth organs which are probably homologous to dicot petals.
  • the florets of maize are assembled into complex higher order structures. Two florets each are enclosed by another pair of bract-like scales called glumes, thus forming spikelets.
  • spikelets of female inflorescences In the spikelets of female inflorescences, only the upper floret develops due to abortion of the lower floret tissues at early developmental stages. In the spikelets of male inflorescences, both florets develop until maturity. Spikelets are formed in pairs along the ear and tassel inflorescence, with one spikelet being pedicellate, the other sessile.
  • ZMM8 and ZMM14 two cDNAs the underlying mRNA of which is specifically expressed in the upper florets of spikelets of grass inflorescences cell layer have been isolated, designated ZMM8 and ZMM14, respectively.
  • Their nucleotide and amino acid sequences are depicted in SEQ ID NOS: 1 and 3 and SEQ ID NOS: 2 and 4, respectively, and show sequence and structural homology (82.0% identity) to OSMADS1 from rice (Chung et al 1994); see Figure 1.
  • Phylogeny reconstructions based on all available MADS-domain protein sequences suggest that ZMM8 and ZMM14 might be paralogous genes, and that both genes are in an orthologous relationship to OSMADS1 from rice. Paralogy of ZMM8 and ZMM14 could be confirmed by determining the chromosomal map locations of the respective genes (Fig. 2).
  • nucleotide sequence as well as those encoding the amino acid sequences depicted in SEQ ID NOS: 2 and 4, it is possible to isolate identical or similar nucleic acid molecules which encode proteins from other species or organisms, in particular orthologous MADS-box genes from plant species other than maize.
  • orthologous as used herein means homologous sequences in different species that arose from a common ancestor gene during speciation. Orthologous genes may or may not be responsible for a similar function (see, e.g., the glossary of the "Trends Guide to Bioinformatics", Trends Supplement 1998, Elsevier Science).
  • the present invention also relates to nucleic acid molecules hybridizing with the above-described nucleic acid molecules and differ in one or more positions in comparison with these as long as they encode a MADS-box protein as defined above.
  • hybridizing it is meant that such nucleic acid molecules hybridize under conventional hybridization conditions, preferably under stringent conditions such as described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).
  • stringent conditions such as described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).
  • the hybridization conditions described in the appended examples are used.
  • Such molecules comprise those which encode fragments, analogues or derivatives and in particular orthologues of the above-described MADS-box proteins and differ, for example, by way of amino acid and/or nucleotide deletion(s), insertion(s), substitution(s), addition(s) and/or recombination(s) or any other modification(s) known in the art either alone or in combination from the above-described amino acid sequences or their underlying nucleotide sequence(s). Methods for introducing such modifications in the nucleic acid molecules according to the invention are well- known to the person skilled in the art.
  • novel nucleic acid molecules of the invention include all nucleotide sequences encoding proteins or peptides which have at least a part of the primary structural conformation for one or more epitopes capable of reacting specifically with antibodies to MADS-box proteins which are encodable by a nucleic acid molecule as set forth above.
  • the peptides and proteins encoded by a nucleic acid molecule of the invention are recognized by an antibody that specifically recognizes an epitope of the ZMM8 protein comprising the amino acid residues 76 to 86, 175 to 182 or 193 to 217 of SEQ ID NO: 2 or an epitope of the ZMM14 protein comprising the amino acid residues 81 to 88 or 190 to 213 of SEQ ID NO: 4.
  • the invention also relates to nucleic acid molecules the sequence of which differs from the nucleotide sequence of any of the above-described nucleic acid molecules due to the degeneracy of the genetic code.
  • the nucleic acid molecule of the invention is derived from a plant, preferably from a monocotyledonous plant, most preferably from maize.
  • the proteins encoded by the nucleic acid molecules identified according to the present invention in maize are expected to define a novel class of MADS-box proteins that play a role in regulating the floret number in grasses or upper floret determinacy or identity. Corresponding proteins displaying similar properties should therefore be present in other plants as well.
  • Nucleic acid molecules of the invention can be obtained, e.g., by hybridization of the above-described nucleic acid molecules with a (sample of) nucleic acid molecule(s) of any source.
  • Nucleic acid molecules hybridizing with the above-described nucleic acid molecules can in general be derived from any plant possessing such molecules, preferably form monocotyledonous plants, in particular from any plant of interest in agriculture, horticulture or wood culture, such as crop plants, namely those of the family Poaceae, any starch producing plants, such as potato, manioc, leguminous plants, oil producing plants, such as oilseed rape, linenseed, etc., plants using polypeptide as storage substances, such as soybean, plants using sucrose as storage substance, such as sugar beet or sugar cane, trees, ornamental plants as well as plants that can be used for the production of biomass, regenerative energy, or building materials such as cambric grass etc.
  • the nucleic acid molecules according to the invention are derived from plants belonging to the family Gramineae.
  • Nucleic acid molecules hybridizing to the above- described nucleic acid molecules can be isolated, e.g., from libraries, such as cDNA or genomic libraries by techniques well known in the art.
  • hybridizing nucleic acid molecules can be identified and isolated by using the above-described nucleic acid molecules or fragments thereof or complements thereof as probes to screen libraries by hybridizing with said molecules according to standard techniques.
  • Possible is also the isolation of such nucleic acid molecules by applying amplification techniques such as the polymerase chain reaction (PCR) using as primers oligonucleotides derived form the above-described nucleic acid molecules.
  • PCR polymerase chain reaction
  • Nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include fragments, derivatives and allelic variants of the above- described nucleic acid molecules that encode a MADS-box protein of the invention or an immunologically or biologically active fragment thereof. Fragments are understood to be parts of nucleic acid molecules long enough to encode the described protein or a biologically or immunologically active fragment thereof as defined above.
  • the nucleic acid molecule encodes at least the l-domain and/or C-domain of the above-described MADS-box protein of the invention.
  • the I- domain (meaning “intervening domain”) is the domain between the MADS- and the K-domain
  • the C-domain (“C-terminal domain”) is everything downstream of the K- domain (i.e., due to limited sequence similarity these domains are only defined relatively to the better defined MADS- and K-domains).
  • the MADS-domain is by far the most highly conserved region of the proteins (Purugganan, Genetics 140 (1995), 345-356). In most cases, it is found at the N-terminus of the putative proteins, although some plant proteins contain additional residues N-terminal to the MADS-domain (NMIKC-type proteins). Most authors consider amino acids 1 to 57 - 60 as belonging to the MADS-domain, if this domain is N-terminal.
  • the K-domain which is not present in any of the animal and fungal MADS-domain proteins known so far (Thei ⁇ en, J. Mol. Evol. 43 (1996), 484-516; Thei ⁇ en, Curr. Opin. Genet. Dev. 5 (1995), 628-639) is characterized by a conserved, regular spacing of hydrophobic residues, which is proposed to allow for the formation of an amphipathic helix.
  • Fig. 1 the I- and C-domain of the MADS-box proteins of the invention are quite different from those of the OSMADS1 protein on the amino acid sequence level.
  • the l-domain of ZMM8 and ZMM14 are 73% and 67% identical to that of OSMADS1 and the C-domain of ZMM8 and ZMM14 share 55% and 54% sequence identity with the corresponding domain of OSMADS1.
  • nucleotide sequence of these nucleic acid molecules differs from the sequences of the above-described nucleic acid molecules in one or more nucleotide positions and are highly homologous to said nucleic acid molecules.
  • Homology is understood to refer to a sequence identity of at least 80 %, particularly an identity of at least 85 %, preferably more than 90 % and still more preferably more than 95 %.
  • the deviations from the sequences of the nucleic acid molecules described above can, for example, be the result of nucleotide substitution(s), deletion(s), addition(s), insertion(s) and/or recombination(s); see supra.
  • nucleic acid molecules or encoded proteins are functionally and/or structurally equivalent.
  • the nucleic acid molecules that are homologous to the nucleic acid molecules described above and that are derivatives of said nucleic acid molecules are, for example, variations of said nucleic acid molecules which represent modifications having the same biological function, in particular encoding proteins with the same or substantially the same biological function. They may be naturally occurring variations, such as sequences from other plant varieties or species, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques.
  • allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants; see supra.
  • nucleic acid molecules are RNA or DNA molecules, preferably cDNA, genomic DNA or synthetically synthesized DNA or RNA molecules.
  • the present invention relates to a nucleic acid molecule which hybridizes with the complementary strand of the nucleic acid molecule of the invention and which encodes a mutated version of the protein as defined above which has lost its immunological and/or biological activity.
  • This embodiment may prove useful for, e.g., generating dominant mutant alleles of the above-described MADS-box proteins.
  • Said mutated version is preferably generated by substitution, deletion and/or addition of 1 to 5 or 5 to 10 amino acid residues in the amino acid sequence of the above-described MADS-box proteins.
  • the invention relates to nucleic acid molecules of at least 15 nucleotides in length hybridizing specifically with a nucleic acid molecule as described above or with a complementary strand thereof. Specific hybridization occurs preferably under stringent conditions and implies no or very little cross- hybridization with nucleotide sequences encoding no or substantially different proteins.
  • nucleic acid molecules may be used as probes and/or for the control of gene expression. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary in length.
  • said nucleic acid molecules comprise at least 15, more preferably at least 30, still more preferably at least 50 consecutive nucleotides of a nucleotide sequence as defined above.
  • nucleic acid probes of 16 to 35 nucleotides in length. Of course, it may also be appropriate to use nucleic acids of up to 100 and more nucleotides in length.
  • the nucleic acid probes of the invention are useful for various applications. On the one hand, they may be used as primers for amplification of nucleic acid sequences according to the invention. Another application is the use as a hybridization probe to identify nucleic acid molecules hybridizing with a nucleic acid molecule of the invention by homology screening of genomic DNA or cDNA libraries.
  • Nucleic acid molecules according to this preferred embodiment of the invention which are complementary to a nucleic acid molecule as described above may also be used for repression of expression of a MADS-box gene, for example due to an antisense or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201 , EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene comprising a nucleic acid molecule of the invention or part thereof.
  • nucleic acid molecules may either be DNA or RNA or a hybrid thereof.
  • said nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell.
  • Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell.
  • PNA peptide nucleic acid
  • the binding of PNAs to complementary as well as various single stranded RNA and DNA nucleic acid molecules can be systematically investigated using thermal denaturation and BIAcore surface-interaction techniques (Jensen, Biochemistry 36 (1997), 5072-5077).
  • the nucleic acid molecules described above as well as PNAs derived therefrom can be used for detecting point mutations by hybridization with nucleic acids obtained from a sample with an affinity sensor, such as BIAcore; see Gotoh, Rinsho Byori 45 (1997), 224- 228.
  • PNA peptide nucleic acids
  • PNAs for example as restriction enzymes or as templates for the synthesis of nucleic acid oligonucleotides are known to the person skilled in the art and are, for example, described in Veselkov, Nature 379 (1996), 214 and Bohler, Nature 376 (1995), 578-581.
  • the present invention also relates to vectors, particularly plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering that contain a nucleic acid molecule according to the invention.
  • vectors particularly plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering that contain a nucleic acid molecule according to the invention.
  • Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989).
  • the nucleic acid molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • the nucleic acid molecule present in the vector is linked to regulatory elements which allow the expression of the nucleic acid molecule in prokaryotic and/or eukaryotic cells.
  • Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA.
  • Regulatory elements ensuring expression in prokaryotic and/or eukaryotic cells are well known to those skilled in the art.
  • eukaryotic cells they comprise normally promoters ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript, for example, those of the 35S RNA from Cauliflower Mosaic Virus (CaMV).
  • Other promoters commonly used are the polyubiquitin promoter, and the actin promoter for ubiquitous expression.
  • the termination signals usually employed are from the Nopaline Synthase promoter or from the CAMV 35S promoter. Additional regulatory elements may include transcriptional as well as translational enhancers; see, e.g., Christensen, Plant Molecular Biology 18 (1992), 675-689; Taylor, Plant Cell Reports 12 (1993), 491- 495; Callis, Genes Dev. 1 (1987), 1183-1200; Toepfer, Meth. in Enzymology 217 (1993), 66-78. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the P L , lac, trp or tac promoter in E.
  • coli and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM ⁇ , pRc/CMV, pcDNAI , pcDNA3 (In-vitrogene), pSPORTI (GIBCO BRL).
  • the above-described vectors of the invention comprises a selectable and/or scorable marker.
  • Selectable marker genes useful for the selection of transformed plant cells, callus, plant tissue and plants are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J.
  • hygro which confers resistance to hygromycin
  • Additional selectable genes namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci.
  • mannose- 6-phosphate isomerase which allows cells to utilize mannose
  • ODC ornithine decarboxylase
  • DFMO ornithine decarboxylase
  • DFMO deaminase from Aspergillus terreus which confers resistance to Blasticidin S
  • Useful scorable marker are also known to those skilled in the art and are commercially available.
  • said marker is a gene encoding luciferase (Giacomin, PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or ⁇ -glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907).
  • luciferase PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121
  • green fluorescent protein Gerdes, FEBS Lett. 389 (1996), 44-47
  • ⁇ -glucuronidase Jefferson, EMBO J. 6 (1987), 3901-3907
  • the present invention furthermore relates to host cells comprising a vector as described above or a nucleic acid molecule according to the invention wherein the nucleic acid molecule is foreign to the host cell.
  • nucleic acid molecule is either heterologous with respect to the host cell, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the host cell but located in a different genomic environment than the naturally occurring counterpart of said nucleic acid molecule. This means that, if the nucleic acid molecule is homologous with respect to the host cell, it is not located in its natural location in the genome of said host cell, in particular it is surrounded by different genes. In this case the nucleic acid molecule may be either under the control of its own promoter or under the control of a heterologous promoter.
  • the vector or nucleic acid molecule according to the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained in some form extrachromosomally.
  • the nucleic acid molecule of the invention can be used to restore or create a mutant gene via homologous recombination (Paszkowski (ed.), Homologous Recombination and Gene Silencing in Plants. Kluwer Academic Publishers (1994)).
  • the host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant or animal cells.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae.
  • Another subject of the invention is a method for the production of a protein having the immunological and/or biological activity of a protein that is expressed in the upper florets of spikelets of grass inflorescences which comprises the cultivation of host cells according to the invention which, due to the presence of a vector or a nucleic acid molecule according to the invention, are able to express such a protein, under conditions which allow expression of the protein and recovering of the so- produced protein from the culture.
  • the protein may be recovered from the cells, from the culture medium or from both.
  • the present invention furthermore relates to a protein having the immunological and/or biological activity of a protein that is expressed in the upper florets of spikelets of grass inflorescences encoded by the nucleic acid molecules according to the invention or produced by the above-described method.
  • the proteins according to the invention may be further modified by conventional methods known in the art.
  • By providing the proteins according to the present invention it is also possible to determine fragments which retain biological activity, such as DNA binding, protein-protein interaction or transcriptional activation. Suitable fragments may comprise the different domains of MADS-domain proteins.
  • the MADS-domain is the major determinant of DNA-binding, but it also performs dimerization and accessory factor binding functions (Shore, Eur. J. Biochem.
  • CArG-box CC-A rich-GG
  • MADS-box genes bind to similar DNA sites based on the consensus sequence CC(AfT)eGG, which is called CArG-box (CC-A rich-GG).
  • CArG-boxes are present in the promoter regions of many genes that are probably regulated by MADS-box genes (Shore, loc. cit.; Tilly, Devlopment 125 (1998), 1647-1657).
  • the I- domain directly downstream of the MADS-domain, comprises approximately 30 amino acids, but is somewhat variable in length (Ma, Genes Dev. 5 (1991 ), 484- 495; M ⁇ nster, Proc. Natl. Acad. Sci. USA 94 (1997), 2415-2420). It is only relatively weakly conserved within plant MADS-domain proteins (Purugganan, loc. cit.). In case of some Arabidopsis MADS-domain proteins it was shown that the l-domain constitutes a key molecular determinant for the selective formation of DNA-binding dimers (Riechmann, Biol. Chem. 378 (1997), 1079-1101 ).
  • the K-domain which is not present in any of the animal and fungal MADS-domain proteins known so far (Thei ⁇ en, loc. cit.; Thei ⁇ en, loc. cit.), is characterized by a conserved, regular spacing of hydrophobic residues, which is proposed to allow for the formation of an amphipathic helix. It is assumed that such an amphipathic helix interacts with that of another K-domain containing protein to promote dimerization (Riechmann, loc. cit.; Shore, loc.cit.). The most variable region, both in sequence and length, is the C- domain at the C-terminus of the MADS-domain proteins.
  • this domain is unknown, and it turned out to be dispensable for DNA binding and protein dimerization in case of some floral homeotic MADS-domain proteins (see, e.g., ref. (Zachgo, Development 121 (1995), 2861-2875)). It could be involved in transcriptional activation or the formation of multimeric transcription factor complexes.
  • This allows the construction of chimeric proteins and peptides comprising an amino sequence derived from the protein of the invention, which is crucial for DNA binding, protein-protein interaction or transcription regulation activity and other functional amino acid sequences, e.g. GUS marker gene (Jefferson, EMBO J. 6 (1987), 3901-3907).
  • the other functional amino acid sequences may be either physically linked by, e.g., chemical means to the proteins of the invention or may be fused by recombinant DNA techniques well known in the art.
  • folding simulations and computer redesign of structural motifs of the protein of the invention can be performed using appropriate computer programs (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679).
  • Computer modeling of protein folding can be used for the conformational and energetic analysis of detailed peptide and protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45).
  • the appropriate programs can be used for the identification of interactive sites of the protein and, if present, its receptor, its ligand or other interacting proteins by computer assistant searches for complementary peptide sequences (Fassina, Immunomethods 5 (1994), 114-120. Further appropriate computer systems for the design of protein and peptides are described in the prior art, for example in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from the above-described computer analysis can be used for, e.g., the preparation of peptidomimetics of the protein of the invention or fragments thereof.
  • pseudopeptide analogues of the natural amino acid sequence of the protein may very efficiently mimic the parent protein (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224).
  • incorporation of easily available achiral ⁇ -amino acid residues into a protein of the invention or a fragment thereof results in the substitution of amide bonds by polymethylene units of an aliphatic chain, thereby providing a convenient strategy for constructing a peptidomimetic (Banerjee, Biopolymers 39 (1996), 769-777).
  • Superactive peptidomimetic analogues of small peptide hormones in other systems are described in the prior art (Zhang, Biochem. Biophys. Res. Commun.
  • peptidomimetic combinatorial libraries can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive amide alkylation and testing the resulting compounds, e.g., for their immunological properties. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709- 715.
  • a three-dimensional and/or crystallographic structure of the protein of the invention can be used for the design of peptidomimetic inhibitors of the biological activity of the protein of the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).
  • the present invention relates to antibodies specifically recognizing a MADS-box protein according to the invention or parts, i.e. specific fragments or epitopes, of such a protein.
  • These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc.
  • Monoclonal antibodies can be prepared, for example, by the techniques as originally described in K ⁇ hler and Milstein, Nature 256 (1975), 495 and Galfre, Meth. Enzymol. 73 (1981 ), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals.
  • antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. These antibodies can be used, for example, for the immunoprecipitation and immunolocalization of proteins according to the invention as well as for the monitoring of the synthesis of such proteins, for example, in recombinant organisms, and for the identification of compounds interacting with the protein according to the invention.
  • surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies selections, yielding a high increment of affinity from a single library of phage antibodies which bind to an epitope of the protein of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97- 105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • the nucleic acid molecules according to the invention are in particular useful for the genetic manipulation of plant cells in order to modify inflorescence and flower development and to obtain plants with modified, preferably with improved or useful phenotypes.
  • the present invention also relates to a method for the production of transgenic plants, plant cells or plant tissue comprising the introduction of a nucleic acid molecule or vector of the invention into the genome of said plant, plant cell or plant tissue.
  • the molecules are placed under the control of regulatory elements which ensure the expression in plant cells.
  • regulatory elements may be heterologous or homologous with respect to the nucleic acid molecule to be expressed as well with respect to the plant species to be transformed, in general, such regulatory elements comprise a promoter active in plant cells.
  • constitutive promoters are used, such as the 35 S promoter of CaMV (Odell, Nature 313 (1985), 810-812) or promoters of the polyubiquitin genes of maize (Christensen, Plant Mol. Biol. 18 (1982), 675-689).
  • tissue specific promoters see, e.g., Stockhaus, EMBO J. 8 (1989), 2245-2251 ).
  • tissue specific promoters which are specifically active in tubers of potatoes or in seeds of different plants species, such as maize, Vicia, wheat, barley etc.
  • Inducible promoters may be used in order to be able to exactly control expression.
  • An example for inducible promoters are the promoters of genes encoding heat shock proteins.
  • microspore-specific regulatory elements and their uses have been described (WO96/16182).
  • the chemically inducible Tet- system may be employed (Gatz, Mol. Gen. Genet. 227 (1991 ); 229-237).
  • the regulatory elements may further comprise transcriptional and/or translational enhancers functional in plants cells.
  • the regulatory elements may include transcription termination signals, such as a poly-A signal, which lead to the addition of a poly A tail to the transcript which may improve its stability; for literature see also supra.
  • nucleic acid molecule according to the invention is expressed in sense orientation it is in principle possible to modify the coding sequence in such a way that the protein is located in any desired compartment of the plant cell.
  • these include the endoplasmatic reticulum, the vacuole, the mitochondria, the plastids, the apoplast, the cytoplasm etc.
  • Methods how to carry out this modifications and signal sequences ensuring localization in a desired compartment are well known to the person skilled in the art.
  • An example is the localization in the nucleus (this is the normal mode of MADS-domain protein localization) or in mitochondria (Winning, Methods in Enzymology 260 (1995), 293-302).
  • Methods for the introduction of foreign DNA into plants are also well known in the art. These include, for example, the transformation of plant cells or tissues with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection, electroporation, biolistic methods like particle bombardment and other methods known in the art.
  • the vectors used in the method of the invention may contain further functional elements, for example "left border"- and "right border”-sequences of the T-DNA of Agrobacterium which allow for stably integration into the plant genome.
  • Suitable strains of Agrobacterium tumefaciens and vectors as well as transformation of Agrobacteria and appropriate growth and selection media are well known to those skilled in the art and are described in the prior art (GV3101 (pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic. Acid Res. 12(1984), 8711 ; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989), 8467-8471 ; Koncz, Plant Mol. Biol.
  • Agrobacterium tumefaciens is preferred in the method of the invention
  • other Agrobacterium strains such as Agrobacterium rhizogenes
  • Methods for the transformation using biolistic methods are well known to the person skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil, Bio/Technology 11 (1993), 1553-1558 and Christou (1996) Trends in Plant Science 1 , 423-431. Microinjection can be performed as described in Potrykus and Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, NY (1995).
  • the plants which can be modified according to the invention and which either show overexpression of a protein according to the invention or a reduction of the synthesis of such a protein can be derived from any desired plant species.
  • They can be monocotyledonous plants or dicotyledonous plants, preferably they belong to plant species of interest in agriculture, wood culture or horticulture interest, such as crop plants (e.g. maize, rice, barley, wheat, rye, oats etc.), potatoes, oil producing plants (e.g. oilseed rape, sunflower, pea nut, soy bean, etc.), cotton, sugar beet, sugar cane, leguminous plants (e.g. beans, peas etc.), wood producing plants, preferably trees, etc.
  • crop plants e.g. maize, rice, barley, wheat, rye, oats etc.
  • potatoes oil producing plants
  • oil producing plants e.g. oilseed rape, sunflower, pea nut, soy bean, etc.
  • the present invention relates also to transgenic plant cells which contain, preferably stably integrated into the genome, a nucleic acid molecule according to the invention linked to regulatory elements which allow expression of the nucleic acid molecule in plant cells and wherein the nucleic acid molecule is foreign to the transgenic plant cell.
  • a nucleic acid molecule according to the invention linked to regulatory elements which allow expression of the nucleic acid molecule in plant cells and wherein the nucleic acid molecule is foreign to the transgenic plant cell.
  • the presence and expression of the nucleic acid molecule in the transgenic plant cells leads to the synthesis of a MADS-box protein which has an influence on the regulatory gene network controlling development of the plant cells and leads to physiological and phenotypic changes in plants containing such cells.
  • the present invention also relates to transgenic plants and plant tissue comprising transgenic plant cells according to the invention. Due to the (over)expression of a MADS-box protein of the invention these transgenic plants may show various physiological, developmental and/or morphological modifications in comparison to wild-type plants. For example, in these transgenic plants the level or composition of protein(s) in the upper florets in the spikelets of grass inflorescence can be altered. Since the agronomically most relevant parts of maize plants are the kernels developing in the female inflorescence (the maize cob), and only the upper florets (not the lower ones) of female inflorescences develop kernels in normal maize plants, any agronomically favorable change of florets improving kernel development (e.g.
  • transgene expression could be restricted to the essential minimum, i.e. to the upper floret that eventually develops the maize kernel, with the only exception that the upper floret of the male inflorescence will probably undergo the same change, but not develop kernels.
  • promoter derivatives may be designed that drive gene expression only in the upper floret of the female inflorescence.
  • the invention relates to a transgenic plant cell which contains, preferably stably integrated into the genome, a nucleic acid molecule according to the invention or part thereof, wherein the transcription and/or expression of the nucleic acid molecule or part thereof leads to reduction of the synthesis of a MADS-box protein.
  • the reduction is achieved by an anti-sense, sense, ribozyme, co-suppression and/or dominant mutant effect.
  • nucleic acid molecules according to the invention opens up the possibility to produce transgenic plant cells with a reduced level of the protein as described above and, thus, with a defect in the development of the inflorescence, in particular of the upper floret.
  • Techniques how to achieve this are well known to the person skilled in the art. These include, for example, the expression of antisense- RNA, ribozymes, of molecules which combine antisense and ribozyme functions and/or of molecules which provide for a co-suppression effect; see also supra.
  • the nucleic acid molecule encoding the antisense-RNA is preferably of homologous origin with respect to the plant species used for transformation.
  • nucleic acid molecules which display a high degree of homology to endogenously occurring nucleic acid molecules encoding a MADS-box protein.
  • the homology is preferably higher than 80%, particularly higher than 90% and still more preferably higher than 95%.
  • the reduction of the synthesis of a protein according to the invention in a plant comprising the transgenic plant cells can result various physiological, developmental and/or morphological changes.
  • the present invention also relates to transgenic plants comprising the above- described transgenic plant cells. These may show, for example, an increase or a reduction of the number of florets or kernels in the spikelets.
  • An increase in the number of kernels leads to an increase in kernel yield and possibly to an increase in total yield per plant or standard area under cultivation. Plants with a decreased number of kernels may produce bigger kernels, or kernels of a higher quality or which are better suited for technical processing.
  • the present invention relates to transgenic plants wherein the reduction of the synthesis of the protein leads to an increase of the number of florets and/or kernels in the spikelets.
  • transformation of maize plants with a ZMM8 antisense construct resulted in a clear increase of florets per spikelets in male and female inflorescences.
  • the present invention also relates to cultured plant tissues comprising transgenic plant cells as described above which either show overexpression of a protein according to the invention or a reduction in synthesis of such a protein.
  • the invention also relates to harvestable parts and to propagation material of the transgenic plants according to the invention which either contain transgenic plant cells expressing a nucleic acid molecule according to the invention or which contain cells which show a reduced level of the described protein.
  • Harvestable parts can be in principle any useful parts of a plant, for example, leaves, stems, fruit, seeds, roots, kernels etc.
  • Propagation material includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks etc.
  • nucleic acid molecule and vectors of the invention are particularly useful for the production of plants which display an altered level of MADS-box protein(s).
  • the present invention relates to the use of a nucleic acid molecule or a vector of the invention for the production of plants which display an altered level or composition of protein(s) in the florets in the spikelets, for modulating the number of kernels, for improving disease resistance or for generating late or early flowering.
  • a further object of the present invention is the provision of means and methods for specifically expressing heterologous proteins and modulating gene expression in certain cells and tissues, in particular in the upper florets of spikelets of inflorescences.
  • the invention also relates to a regulatory sequence of a promoter naturally regulating the expression of a nucleic acid molecule of the invention described above or of a nucleic acid molecule homologous to a nucleic acid molecule of the invention, said regulatory sequence being capable of conferring expression of a heterologous DNA sequence in the upper florets of the spikelets of grass inflorescence.
  • regulatory sequence refers to sequences which influence the specificity and/or level of expression, for example in the sense that they confer cell and/or tissue specificity. Such regions can be located upstream of the transcription initiation site, but can also be located downstream of it, e.g., in transcribed but not translated leader sequences, or in introns.
  • promoter within the meaning of the present invention refers to nucleotide sequences necessary for transcription initiation, i.e. RNA polymerase binding and successful start of processive transcript formation, and may also include, for example, the TATA box.
  • nucleic acid molecule homologous to a nucleic acid molecule of the invention includes promoter regions and regulatory sequences of other MADS-box genes, such as genes from other species, for example, sorghum, millet, coix, barley, wheat and rice which are homologous to the maize MADS-box genes and which display substantially the same expression pattern.
  • promoters are characterized by their capability of conferring preferably exclusively expression of a heterologous DNA sequence in the upper florest of spikelets of grass inflorescences; for the meaning of "expressed or expression in the upper florets of spikelets of grass inflorescences" see supra.
  • regulatory sequences from other species can be used that are functionally homologous to the regulatory sequences of the promoter of the above defined MADS-box specific nucleic acid molecules, or promoters of genes that display an identical or similar pattern of expression, in the sense of being expressed in the upper florets of spikelets.
  • the expression conferred by the regulatory sequences of the invention may not be limited to the upper florets of spikelets but can include or be restricted to, for example, the spikelet meristem.
  • the particular expression pattern may also depend on the plant/vector system employed.
  • heterologous DNA sequences driven by the regulatory sequences of the invention predominantly occurs in the upper florets of spikelets unless certain elements of the regulatory sequences of the invention, were taken and designed by the person skilled in the art to control the expression of a heterologous DNA sequence in one of the above described or other cell types.
  • MADS-box genes Only very little about promoter functions of MADS-box genes.
  • detailed studies on the promoters of the AG and AP3 genes from Arabidopsis have started (Hill, Development 125 (1998), 1711-1721 ; Sieburth, Plant Cell 9 (1997), 355-365; Tilly, loc. cit.). It seems already clear that CArG-boxes (see above), where MADS-domain proteins bind to, play an important regulatory role. In case of the AG gene it became clear that important regulatory sites are located intragenically.
  • novel regulatory sequences of MADS-box genes designated ZMM8 and ZMM14, respectively, (see supra) can be isolated and have been exemplified for the regulatory sequence of the ZMM14 gene (SEQ ID NO: 5); see Example 3.
  • inverse PCR can be used.
  • genomic DNA can be digested with appropriate restriction enzymes, denatured and allowed to anneal to a reverse primer derived from the cDNA sequence of the invention.
  • a blunt-ended adaptor can be ligated and PCR can be performed using a nested reverse primer derived from the mentioned cDNA, and a forward primer derived from the adaptor sequence.
  • a physical map of the genomic sequences upstream the coding region can be constructed by mean of genomic southern analysis. With this information, genomic DNA can be digested with selected restriction enzymes, genomic fragments containing a piece of the upstream sequences and the coding sequence can be gel purified and self-ligated in a large volume to favour the formation of circular molecules, that can subsequently be amplified by PCR with forward and reverse primers, derived from the coding sequence of the MADS-box gene of the invention.
  • 100 micrograms genomic DNA from maize can be digested with EcoRI .
  • the digested DNA can be electrophoresed and the restriction fragments preferably comprising between 1.5 and 4 Kbp can be purified from the gel.
  • One tenth of the recovered DNA can be self-ligated in a volume of 50 microlitres, EtOH precipitated and resuspended in 10 microlitres of water.
  • One microlitre of the self- ligated DNA can be used for PCR with primers forward (5'-3'; SEQ ID NO:6) and reverse (5'-3'; SEQ ID NO: 7). PCR reaction can be performed according to the manufacturer's description.
  • An appropriate program for the PCR can be: 94°C, 2' followed by 30 to 40x: 94°C, 40"; 55 to 70°C 1 ', 72°C, 3', and finally a step at 72°C for 5'.
  • the amplified fragment can be cloned in a suitable plasmid, and can be sequenced then using standard procedures well known to everyone skilled in the art.
  • Primers for inverse PCR could be targeted e.g. against the 5'UTR (5' untranslated region) of the ZMM8 cDNA.
  • the transcription start site could be determined by standard procedures well known to everyone skilled in the art, such as 5'-RACE, primer extension or S1 mapping.
  • the respective region is fused to marker genes such as genes encoding GUS or GFP, and 5' deletion derivatives of these construct are generated. They are transformed into suitable plant material, and the expression of the marker gene depending on the remaining upstream sequence (putative promoter) is determined.
  • the regulatory sequences of the invention can be used to drive the expression of heterologous DNA sequences specifically in the upper florets of the spikelets of grass florescences.
  • the regulatory sequences of the invention may be capable of conferring expression with higher specificity for certain cells than it is observed for the natural MADS-box genes.
  • the present invention also relates to regulatory sequences which are substantially identical to that of SEQ ID NO: 5 or which are homologous thereto by way of their structure or to parts thereof and which are able to confer specific expression in upper florets of spikelets.
  • Such regulatory sequences differ at one or more positions from the above- mentioned regulatory sequence but still have the same specificity, namely they comprise the same or similar sequence motifs, preferably 6 to 10 nucleotides in length, responsible for the above described expression pattern.
  • Preferably such regulatory sequences hybridize to one of the above-mentioned regulatory sequences, most preferably under stringent conditions.
  • Particularly preferred are regulatory sequences which share at least 85%, more preferably 90-95%, and most preferably 96-99% sequence identity with one of the above-mentioned regulatory sequences and have the same or substantially the same specificity.
  • regulatory sequences also comprise those which are altered, for example by nucleotide deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination in comparison to the above-described nucleotide sequence.
  • Methods for introducing such modifications in the nucleotide sequence of the regulatory sequences of the invention are well known to the person skilled in the art. It is also immediately evident to the person skilled in the art that further regulatory elements may be added to the regulatory sequences of the invention.
  • transcriptional enhancers and/or sequences which allow for induced expression of the regulatory sequences of the invention may be employed.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gatz, supra.
  • the regulatory sequence of the invention comprises a DNA sequence selected from the group consisting of
  • the regulatory sequence of the invention may be derived from the MADS- box genes of maize (see Examples) although other plants may be suitable sources for such regulatory sequences as well.
  • the nucleotide sequences of the invention can be compared as appropriate computer programs known in the art such as BLAST, which stands for Basic Local Alignment Search Tool (Altschul, 1997; Altschul, J. Mol. Evol. 36 (1993), 290-390; Altschul, J. Mol. Biol.
  • BLAST produces alignments of nucleotide sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologues. With such means it is possible to identify conserved nucleotide sequences that may play a role in upper floret specific expression.
  • said regulatory sequence is part of a recombinant DNA molecule.
  • the regulatory sequence in the recombinant DNA molecule is operatively linked to a heterologous DNA sequence.
  • heterologous with respect to the DNA sequence being operatively linked to the regulatory sequence of the invention means that said DNA sequence is not naturally linked to the regulatory sequence of the invention.
  • Expression of said heterologous DNA sequence comprises transcription of the DNA sequence, preferably into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells, preferably plant cells are well known to those skilled in the art. They usually comprise poly-A signals ensuring termination of transcription and stabilization of the transcript, see also supra. Additional regulatory elements may include transcriptional as well as translational enhancers; see supra.
  • the heterologous DNA sequence of the above- described recombinant DNA molecules encodes a peptide, protein, antisense RNA, sense RNA and/or ribozyme.
  • the recombinant DNA molecule of the invention can be used alone or as part of a vector to express heterologous DNA sequences, which, e.g., encode proteins for, e.g., seed storage proteins, toxins, antibodies ("plantibodies") or diagnostics of MADS-box related gene expression.
  • the recombinant DNA molecule or vector containing the DNA sequence encoding a protein of interest is introduced into the cells which in turn produce the protein of interest.
  • the regulatory sequences of the invention can be operatively linked to sequences encoding Barstar and Barnase, respectively, for use in the production of male and female sterility in plants.
  • Applications of the chimeric promoter are evident to the person skilled in the art and can be derived from the literature, e.g., Strittmatter and Wegener, Zeitschrift f ⁇ r Naturutz 48c (1993), 673-688; Kahl, J. Microbiol. Biotechnol. 11 (1995), 449-460 and references cited therein.
  • said protein can be a scorable marker, e.g., luciferase, green fluorescent protein or ⁇ -galactosidase.
  • a scorable marker e.g., luciferase, green fluorescent protein or ⁇ -galactosidase.
  • This embodiment is particularly useful for simple and rapid screening methods for compounds and substances described herein below capable of modulating MADS-box gene expression.
  • a transgenic plant can be cultured in the presence and absence of a candidate compound in order to determine whether the compound affects the expression of genes which are under the control of regulatory sequences of the invention, which can be measured, e.g., by monitoring the expression of the above-mentioned marker.
  • other marker genes may be employed as well, encoding, for example, a selectable marker which provides for the direct selection of compounds which induce or inhibit the expression of said marker.
  • the regulatory sequences of the invention may also be used in methods of antisense approaches.
  • the antisense RNA may be a short (generally at least 10, preferably at least 14 nucleotides, and optionally up to 100 or more nucleotides) nucleotide sequence formulated to be complementary to a portion of a specific mRNA sequence and/or DNA sequence of the gene of interest. Standard methods relating to antisense technology have been described; see, e.g., Klann, Plant Physiol. 112 (1996), 1321-1330 and supra.
  • the antisense RNA binds to its target sequence within a cell, thereby inhibiting translation of the mRNA and down-regulating expression of the protein encoded by the mRNA.
  • the invention relates to nucleic acid molecules of at least 15 nucleotides in length hybridizing specifically with a regulatory sequence as described above or with a complementary strand thereof. Specific hybridization occurs preferably under stringent conditions and implies no or very little cross- hybridization with nucleotide sequences having no or substantially different regulatory properties. Such nucleic acid molecules may be used as probes and/or for the control of gene expression. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary in length.
  • nucleic acid probes of 17 to 35 nucleotides in length are preferred. Of course, it may also be appropriate to use nucleic acids of up to 100 and more nucleotides in length.
  • the nucleic acid probes of the invention are useful for various applications. On the one hand, they may be used as PCR primers for amplification of regulatory sequences according to the invention. Another application is the use as a hybridization probe to identify regulatory sequences hybridizing to the regulatory sequences of the invention by homology screening of genomic DNA libraries.
  • Nucleic acid molecules according to this preferred embodiment of the invention which are complementary to a regulatory sequence as described above may also be used for repression of expression of a gene comprising such regulatory sequences, for example due to an antisense, cosupression or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201 , EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene comprising a regulatory sequence of the invention. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith et al.
  • nucleic acid probe may either be DNA or RNA or a hybrid thereof.
  • nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches; see supra.
  • the present invention also relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a regulatory sequence or corresponding recombinant DNA molecule of the invention.
  • said vector is an expression vector and/or a vector further comprising a selection marker for plants.
  • selector markers see supra.
  • Methods which are well known to those skilled in the art can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989).
  • the recombinant DNA molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • the present invention furthermore relates to host cells transformed with a regulatory sequence, a DNA molecule or vector of the invention.
  • Said host cell may be a prokaryotic or eukaryotic cell.
  • the regulatory sequence, vector or recombinant DNA molecule of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. Preferred cells are plant cells.
  • the present invention provides for a method for the production of transgenic plants, plant cells or plant tissue comprising the introduction of a nucleic acid molecule, recombinant DNA molecule or vector of the invention into the genome of said plant, plant cell or plant tissue.
  • a nucleic acid molecule, recombinant DNA molecule or vector of the invention into the genome of said plant, plant cell or plant tissue.
  • further regulatory sequences such as poly A tail may be fused, preferably 3' to the heterologous DNA sequence, see also supra.
  • Further possibilities might be to add transcriptional or translational enhancers that increase gene expression, or sequences that increase mRNA stability.
  • the present invention relates also to transgenic plant cells which contain, preferably stably integrated into the genome, a regulatory sequence, a recombinant DNA molecule or vector according to the invention.
  • the present invention also relates to transgenic plants and plant tissue comprising the above-described transgenic plant cells.
  • These plants may show, for example, in comparison to a non-transformed or wild type plant an altered number and/or morphology of florets due to the stable or transient presence of a foreign DNA resulting in at least one of the following features:
  • said foreign DNA or part thereof is stably integrated into the genome of the transgenic plant.
  • the above described transgenic plant is a graminaecious monocotyledonous plant, preferably it belongs to the genus selected from the group consisting of Lolium, Zea, Triticum, Sorghum, Saccarum, Bromus, Oryza, Hordeum, Secale and Setaria, and more preferably said plant is maize.
  • the invention relates to harvestable parts and to propagation material of the transgenic plants according to the invention which contain transgenic plant cells described above.
  • Harvestable parts can be in principle any useful part of a plant, for example, leaves, stems, fruit, seeds, kernels, roots etc.
  • Propagation material includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks etc.
  • the present invention further relates to a method for the identification of an activator or inhibitor of genes specifically expressed in the upper florets in the spikelets of grass inflorescence comprising the steps of: (a) providing a plant, plant cell, or plant tissue comprising a recombinant DNA molecule comprising a readout system operatively linked to a regulatory sequence of the invention;
  • read out system in context with the present invention means a DNA sequence which upon transcription and/or expression in a cell, tissue or organism provides for a scorable and/or selectable phenotype.
  • read out systems are well known to those skilled in the art and comprise, for example, recombinant DNA molecules and marker genes as described above.
  • plurality of compounds in a method of the invention is to be understood as a plurality of substances which may or may not be identical.
  • Said compound or plurality of compounds may be inorganic or organic, naturally occurring or man made compounds and may be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms.
  • said compound(s) may be known in the art but hitherto not known to be capable of suppressing or activating and/or enhancing the transcription of a MADS-box gene.
  • the plurality of compounds may be, e.g., added to the culture medium or injected into the plant, plant cells or tissue or sprayed onto the plant or supplied in the soil.
  • a sample containing a compound or a plurality of compounds is identified in the method of the invention, then it is either possible to isolate the compound from the original sample identified as containing the compound capable of suppressing or activating and/or enhancing the transcription of a MADS-box gene, or one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample.
  • the steps described above can be performed several times, preferably until the sample identified according to the method of the invention only comprises a limited number of or only one substance(s).
  • said sample comprises substances of similar chemical and/or physical properties, and most preferably said substances are identical.
  • the compound identified according to the above described method is further formulated in a form suitable for the application in plant breeding or plant cell and tissue culture.
  • the compounds which can be tested and identified according to a method of the invention may be expression libraries, e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and references cited supra).
  • expression libraries e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs or the like
  • genes encoding a putative regulator of a MADS-box gene and/or which are located up- or downstream the inflorescence developmental pathway may be identified using, for example, insertion mutagenesis using, for example, gene targeting vectors known in the art (see, e.g., Hayashi, Science 258 (1992), 1350-1353; Fritze and Walden, Gene activation by T-DNA tagging. In Methods in Molecular biology 44 (Gartland, K.M.A. and Davey, M.R., eds). Totowa: Human Press (1995), 281-294) or transposon tagging (Chandlee, Physiologia Plantarum 78 (1990), 105-115).
  • Said compounds can also be functional derivatives or analogues of known inhibitors or activators.
  • Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA.
  • said derivatives and analogues can be tested for their effects according to methods known in the art.
  • peptidomimetics and/or computer aided design of appropriate derivatives and analogues can be used, for example, according to the methods described above.
  • said recombinant DNA molecule comprising said read out system is a recombinant DNA molecule of the invention as described in the embodiments hereinbefore.
  • said plant or plant cell or tissue is a plant, plant cell or tissue of the invention described in the embodiments hereinbefore.
  • Determining whether a compound is capable of suppressing or activating and/or enhancing the transcription of a MADS-box gene can be done, for example, in plants by monitoring the reporter gene. It can further be done by monitoring the phenotypic characteristics of the transgenic plant of the invention contacted with the compounds and compare it to that of wild-type plants. In an additional embodiment, said characteristics may be compared to that of a transgenic plant contacted with a compound which is either known to be capable or incapable of suppressing or activating and/or enhancing MADS-box gene expression or the activity of the protein.
  • the compounds identified according to the method of the invention are expected to be very beneficial since promoters that have been known so far are only of limited use due to the non or not tightly regulated tissue specificity of their regulatory sequences.
  • the inhibitor or activator identified by the above-described method may prove useful as a herbicide, pesticide and/or as a plant growth regulator.
  • the invention relates to a compound obtained or identified according to the method of the invention said compound being an activator or inhibitor of gene expression and/or function in the upper florets in the spikelets of grass inflorescence.
  • Such useful compounds can be for example transacting factors which bind to the regulatory sequence of the invention. Identification of transacting factors can be carried out using standard methods in the art (see, e.g., Sambrook, supra, and Ausubel, supra). To determine whether a protein binds to the regulatory sequences of the invention, standard DNA footprinting and/or native gel-shift analyses can be carried out. In order to identify a transacting factor which binds to the regulatory sequence of the invention, the regulatory sequence can be used as an affinity reagent in standard protein purification methods, or as a probe for screening an expression library.
  • transacting factor modulation of its binding to the regulatory sequences of the invention can be pursued, beginning with, for example, screening for inhibitors against the binding of the transacting factor to the regulatory sequences of the present invention.
  • Activation or repression of MADS- box genes could then be achieved in plants by applying of the transacting factor (or its inhibitor) or the gene encoding it, e.g. in a vector for transgenic plants.
  • the active form of the transacting factor is a dimer
  • dominant-negative mutants of the transacting factor could be made in order to inhibit its activity.
  • further components in the pathway leading to activation e.g.
  • the compound identified according to the above described method or its analog or derivative is further formulated in a form suitable for the application in plant breeding or plant cell and tissue culture.
  • a plant protection composition can be prepared by employing the above-described method of the invention and synthesizing the compound identified as inhibitor or activator in an amount sufficient for use in agriculture.
  • the present invention also relates to a method for the preparation of an agricultural plant protection composition comprising the above- described steps of the method of the invention and synthesizing the compound so identified or an analog or derivative thereof.
  • the compound identified by the above-described method may be preferentially formulated by conventional means commonly used for the application of, for example, herbicides and pesticides or agents capable of inducing systemic acquired resistance (SAR).
  • SAR systemic acquired resistance
  • certain additives known to those skilled in the art comprising stabilizers or substances which facilitate the uptake by the plant cell, plant tissue or plant may be used, for example, carborundum, or 0.01 % SDS (sodium dodecylsulfate) solution.
  • the present invention also relates to an antibody specifically recognizing a regulatory sequence of the invention or the compound identified according to the method of the present invention.
  • Antibodies can be prepared as described hereinbefore
  • the invention also relates to a diagnostic composition
  • a diagnostic composition comprising at least one of the aforementioned nucleic acid molecules and/or comprising a nucleic acid molecule which is complementary for such a nucleic acid molecule, a vector of the invention, a MADS-box protein of the invention or an immunologically or biologically active fragment thereof or an antibody specifically recognizing such a protein or fragment; a regulatory sequence or recombinant DNA, or a corresponding vector of the invention, a compound designed orientated according to the protein of the invention and/or identified according to the method described above and/or an antibody specifically recognizing such a compound or a regulatory sequence of the invention, and optionally suitable means for detection.
  • Said diagnostic compositions may be used for methods for detecting expression of MADS-box proteins by detecting the presence of corresponding mRNA which comprises isolation of mRNA from a cell and contacting the mRNA so obtained with a probe comprising a nucleic acid probe as described above under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the protein by the cell.
  • Further methods of detecting the presence of a protein according to the present invention comprises immunotechniques well known in the art, for example enzyme linked immunosorbent assay.
  • the present invention relates to a kit comprising at least one of the aforementioned nucleic acid molecules, vectors, proteins, compounds or antibodies of the invention.
  • the kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transgenic plant cells, plant tissue or plants. Furthermore, the kit may include buffers and substrates for reporter genes that may be present in the recombinant gene or vector of the invention.
  • the kit of the invention may advantageously be used for carrying out the method of the invention and could be, inter alia, employed in a variety of applications referred to herein, e.g., in the diagnostic field or as research tool.
  • the parts of the kit of the invention can be packaged individually in vials or in combination in containers or multicontainer units. Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art.
  • kit or its ingredients according to the invention can be used in plant cell and plant tissue cultures, for example, for any of the above described methods for detecting inhibitors and activators of MADS-box genes.
  • the kit of the invention and its ingredients are expected to be very useful in breeding new varieties of, for example, plants which display improved properties such as nutritial value or disease resistance.
  • nucleic acid molecules according to the invention can be employed to produce transgenic plants with a desired trait; see for review TIPTEC Plant Product & Crop Biotechnology 13 (1995), 312-397.
  • nucleic acid molecules according to the invention it is possible to use the nucleic acid molecules according to the invention as molecular markers in plant breeding.
  • the overexpression of nucleic acid molecules according to the invention may be useful for the alteration or modification of plant/pathogene interaction.
  • pathogene includes, for example, bacteria, viruses and fungi as well as protozoa.
  • the present invention relates to the use of a regulatory sequence, a recombinant DNA molecule, a vector, a compound and/or the antibody of the invention for the expression of heterologous gene products such as nucleic acids and proteins in the upper florets of the spikelets of grass inflorescence, for modification of solute partitionary in florets, for conferring or improving disease resistance, for the improvement of kernel derived products or for the expression of enzymes affecting the quality of any agronomic aspects of the kernel or the whole inflorescence, such as its suitability for industrial processing or storage, for generating late or early flowering, or for modifying fertility.
  • heterologous gene products such as nucleic acids and proteins in the upper florets of the spikelets of grass inflorescence, for modification of solute partitionary in florets, for conferring or improving disease resistance
  • enzymes affecting the quality of any agronomic aspects of the kernel or the whole inflorescence such as its suitability for industrial processing or storage, for generating late
  • the described nucleic acid molecules may also be used for several other applications, for example, for the identification of nucleic acid molecules which encode proteins which interact with the MADS-box proteins described above. This can be achieved by assays well known in the art, for example, as described in Scofield (Science 274 (1996), 2063-2065) by use of the so-called yeast "two-hybrid system". In this system the protein encoded by the nucleic acid molecules according to the invention or a smaller part thereof is linked to the DNA-binding domain of the GAL4 transcription factor.
  • a yeast strain expressing this fusion protein and comprising a lacZ reporter gene driven by an appropriate promoter, which is recognized by the GAL4 transcription factor, is transformed with a library of cDNAs which will express plant proteins or peptides thereof fused to an activation domain.
  • the complex is able to direct expression of the reporter gene.
  • the nucleic acid molecules according to the invention and the encoded peptide can be used to identify peptides and proteins interacting with MADS-box proteins. This method can also be employed for identifying inhibitors and activators as described above.
  • Fig. 1 Comparison of predicted amino acid sequences of ZMM8 and ZMM14 from maize and OSMADS1 from rice. Identical and similar amino acids are indicated by a dark or light grey background, respectively. The MADS- and K-domains are boxed. Nucleotide sequence data of the cDNAs corresponding to ZMM8 and ZMM14 have been deposited in the EMBL, GenBank and DDBJ databases under accession numbers Y09303 and AJ005338.
  • Fig. 2 Map locations of ZMM8 and ZMM14 in syntenic regions of maize chromosomes 1 and 9. The figure is based on the Brookhaven National Laboratory (BNL) map (URL: http://burr.bio.bnl.gov/acemaz.html). Only fragments of the chromosomes are shown. The orientation of the chromosome 9 fragment has been inverted relative to the standard orientation of chromosome 1. Duplicate molecular marker loci shared by both chromosomes are indicated and connected by thin lines. The map positions of the genes can be found on the BNL map under the marker designations MPIK28 (ZMM8) and MPIK43 (ZMM14). Also shown is the chromosomal region in which the ifa 1 locus was located (Laudencia-Chingcuanco and Hake 1998).
  • RNA gel blot analysis of ZMM8 expression contains total RNA from endosperm (1 ), embryo (2), female inflorescence (3), male inflorescence (4), leaf (5) and root (6), as indicated by the numbers above the lanes.
  • the blot was hybridized with a 3'-specific probe of ZMM8. It was subsequently hybridized with a GAPDH probe as a control for RNA quality and loading. The apparent lengths of the transcripts are indicated at the right margin.
  • Fig. 4 Expression of ZMM8 mRNA in inflorescences, as revealed by in situ hybridization with digoxigenin-labeled antisense riboprobes.
  • A Median longitudinal section through female inflorescence with spikelets at early developmental stages (stages B-E).
  • B Close up of median longitudinal section through two spikelets of a female inflorescence at developmental stage D, when expression of ZMM8 in the upper floret primordium starts.
  • C Transverse cross-section through a male spikelet at developmental stage F.
  • D Median longitudinal section through male inflorescence with spikelets at developmental stage H.
  • alf abortive lower floret
  • c carpel
  • gl glume
  • gy gynoecium
  • hu husk leaf
  • I lemma
  • Ifp lower floret primordium
  • p palea
  • si silk
  • st stamen
  • ufp upper floret primordium. Bars, 100 ⁇ m.
  • Fig. 5 Comparison of ZMM8 and ZMM14 expression patterns.
  • A Section through male spikelet at developmental stage H, probed with antisense riboprobe obtained from ZMM14.
  • B-E Sections through spikelets of a female inflorescence, probed with antisense riboprobes obtained from (B, D) ZMM8 or (C, E) ZMM14.
  • B, C Spikelets at developmental stage F;
  • D, E spikelets at developmental stage H-l. Key to label: See legend to Fig. 4.
  • Fig. 6. ZMM8 expression compared to expression patterns of ZMM2 and ZMM6.
  • A, B Median longitudinal sections through female spikelets displaying late stages of development (stages l-K), probed with antisense riboprobes obtained from ZMM8 (A) or ZMM2 (B) cDNA.
  • C, D Median longitudinal sections through developing male spikelets (stage F-G) probed with ZMM8 (C) or ZMM2 (D) antisense riboprobes.
  • E, F, G Consecutive transverse cross-sections through female spikelets at an intermediate developmental stage (stage F) probed with (E) ZMM6, (F) ZMM8 and (G) ZMM2 antisense riboprobes.
  • H Median longitudinal section through male spikelet at developmental stage H, probed with ZMM6 antisense riboprobe. Key to label: See legend to Fig. 4.
  • Fig. 7 Expression of ZMM8 in the development of maize spikelets.
  • the development of four florets originating from a single spikelet pair meristem is depicted schematically.
  • four different types of reproductive meristems (numbered on the left side; corresponding developmental stages according to Cheng et al. (1983) are indicated on the right side) are involved in maize inflorescence development.
  • the inflorescence meristem (IM) is the first reproductive meristem to arise. It generates many ranks of spikelet pair meristems (SPM) in an acropetal sequence on its flanks, of which only one is shown here for clarity.
  • SPM spikelet pair meristems
  • the spikelet pair meristems produce a single derivative, a spikelet meristem (SM), then converting to spikelet meristem activity itself.
  • SM spikelet meristem
  • two spikelet meristems result from each spikelet pair meristem.
  • pedicellate the pedicellate
  • sessile spikelet the spikelet meristem initiates a pair of lateral organs, glumes (gl), then initiates a single lower floret meristem (LFM), and finally converts to floret meristem activity itself, thus forming the upper floret meristem (UFM).
  • the floret meristems initiate only lateral organs: a palea (p), a lemma (I), and the floral organs, lodicules (not shown), stamens (st) and carpels (c). Meristems and organs expressing ZMM8 are marked in red.
  • Fig. 8 Male spikelet phenotype of a ZMM8 antisense maize plant.
  • Example 1 Isolation and structural evaluation of ZMM8 and ZMM14 cDNAs
  • a cDNA library was constructed from poly(A) + mRNA isolated from maize ears ranging in size from 2 to 30 mm, employing the ZAP-cDNA Gigapack II Cloning kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. 400.000 clones of the cDNA library were screened by plaque hybridization with a radioactive probe derived from plasmid clone BRACE9-22, containing a cDNA fragment of ZMM8 (Fischer et al. 1995a). Hybridization was performed under conditions of moderate stringency (58°C, 5xSSC) following standard procedures (Sambrook et al. 1989).
  • the hybridization conditions for the isolation of the ZMM8 and the ZMM14 cDNAs were 58 °C, 5 x SSC, 0.1 % SDS and 5 x Denhardt's reagent (50 x Denhardt's reagent containing 5 g ficoll (type 400, Pharmacia), 5 g polyvinylpyrrolidone, 5 g bovine serum albumin (fraction V, company Sigma) and water to a total volume of 500 ml).
  • the washing conditions were 60 °C, 0.2 x SSC, 0.1 % SDS. For the production of 5 x SSC and 0.2 x SSC 20 x SSC are used.
  • pSW72 and pSW24 Two ZMM8 cDNA clones were isolated, termed pSW72 and pSW24.
  • Conceptual translation of the cDNAs yields a protein of 240 amino acids (SEQ ID NO: 2) that shows the typical domain structure of a MIKC-type MADS-domain protein (Fig. 1 ).
  • SEQ ID NO: 2 shows the typical domain structure of a MIKC-type MADS-domain protein.
  • ZMM8 and ZMM14 are paralogous genes, and are very similar to OSMADS1 from rice.
  • the sequence comparisons among all available AGL2-like sequences revealed that the sequences most similar to that of ZMM8 are ZMM14 (overall identity 88.7%) and OSMADS1 from rice (Oryza sativa) (Chung et al. 1994) (82.0% identity). All other AGL2-like sequences known are already significantly less similar.
  • ZMM8 and ZMM14 could be confirmed by determining the chromosomal map locations of the respective genes (Fig. 2). As revealed by the colinear arrangement (synteny) of flanking molecular markers, ZMM8 and ZMM14 are located in duplicated (i.e. paralogous) regions of the maize genome, thus supporting paralogy of the genes (see Thei ⁇ en et al. (1995) for other pairs of paralogous MADS-box genes in maize).
  • Total RNA was prepared from a collection of developing male and female inflorescences at 0.2 to 3 cm in size, embryos and endosperm 18 days postpollination, roots and leaves, using the BIOMOL-kit (BIOMOL, Hamburg, Germany) according to the manufacturer's instructions. Total RNA samples (20 ⁇ g per lane) were separated by electrophoresis on 1 % agarose gels containing formaldehyde (Sambrook et al. 1989) and transfered to Hybond-N nylon membranes (Amersham Life Sciences, Cleveland, OH, USA).
  • Hybridization was performed at 42°C in a solution containing 5 x SSC, 5 x Denhardt's, 0.1 % SDS, 100 ⁇ g/ml herring sperm DNA and 50% formamide for 12 - 15 hours (Sambrook et al. 1989).
  • the radioactive probes used had been prepared by 'linear PCR' as described (Fischer et al. 1995a), employing BRACE9-22/ ⁇ g/ll (ZMM8) or pJC18/Sc/l (ZMM14) as templates. Filters were finally washed for 15 min in 0.1 x SSC, 0.1 % SDS at 68°C.
  • RNA gel blot hybridizations revealed that ZMM8 is strongly expressed in male and female inflorescences of maize (Fig. 3). In some experiments, a faint signal was also seen in the endosperm. No expression was detected in roots, leaves and embryos (Fig. 3). At that level of resolution, a very similar expression pattern was found for ZMM14. To more precisely determine the localization of ZMM8 mRNA expression in developing maize inflorescences, digoxigenin-labeled antisense RNA probes were hybridized to sections of immature inflorescences containing spikelets in a wide range of developmental stages (Figs 4-6).
  • Preparation of maize tissues and in situ hybridization were carried out essentially as described by De Block and Debrouwer (1993), with the following exceptions: fixation of the tissue in FAA was done for 24 h.
  • the probes were prepared by in vitro transcription with digoxigenin-labeled UTP (Boehringer Mannheim, FRG) according to the manufacturer's instructions.
  • the templates used for run-off transcription were BRACE9-1/Sfyl (ZMM2), BRACE9-12/ ⁇ g/ll (ZMM6), BRACE9-22/ ⁇ g/ll (ZMM8) (Fischer et al. 1995a) and pJC18/ ⁇ c/l.
  • the restriction enzymes used cut between the MADS-box and the phage promoters (T3 in case of BRACE9-22, T7 in all other cases) used for generating antisense transcripts.
  • T3 in case of BRACE9-22, T7 in all other cases
  • Sense transcripts were generated employing the alternative promoters. Hybridization under high stringency conditions was performed overnight at 50°C in a humidified box.
  • the hybridization mix consisted of 500 ⁇ g/ml tRNA from yeast, 100 ⁇ g/ml poly(A), 300 mM NaCI, 1x Denhardt's solution, 10% dextran sulfate and 50% deionized formamide, all in DEPC treated water. Partially hydrolized probes of about 150 bases length were added to the mix at a concentration of 1 ⁇ g/ml. High stringency washes were carried out twice for one hour at 60°C in O.lxSSC. As controls for specificity, consecutive sections were hybridized with sense and antisense probes of one and the same gene, or with antisense probes of different genes displaying distinguishable expression patterns. Photographs were made on a Zeiss-Axiophot using Nomarski-optics or brightfield. Brightness, contrast, and color balance were adjusted using Adobe Photoshop 3.0 (Adobe Systems Inc., Mountain View, CA, USA).
  • Figs 4C,D for male spikelets
  • Figs 5B and 6F for female spikelets
  • Lack of ZMM8 expression in the lower floret was observed at all developmental stages. It was also verified in different types of tissue sections (e.g., compare Figs 4D and C for a median longitudinal section and a cross-section through male spikelets; compare Figs 5B and 6F for female spikelets), excluding that absence of a ZMM8 signal in the lower floret is due to a special plane of a chosen tissue section.
  • ZMM8 expression does not appear in lower floret primordia, even when they have reached developmental stages that are far beyond the stage of the upper floret primordia at the onset of ZMM8 expression, indicating that the absence of ZMM8 expression in the lower floret primordia is not simply due to the fact that the lower floret lags in development behind the upper one.
  • ZMM8 expression becomes visible in all floret organs, i.e. lemma, palea, lodicules (only in male florets, but not visible in the sections shown here), stamens and carpels (Figs 4C, 4D, 5B, 6F).
  • ZMM8 expression is present everywhere in the developing upper floret, but apparently nowhere in the lower one.
  • the early expression pattern of ZMM14 (around developmental stage D) appears to be identical to that of ZMM8.
  • ZMM14 transcripts show the same spatial distribution as those of ZMM8, but the expression level seems to be significantly lower (compare Fig. 5A, showing ZMM14 expression, with Fig. 4D, showing ZMM8 expression; the spikelets in both figures are from male inflorescences and are at about the same developmental stage).
  • Figs 5B and C shows expression patterns of ZMM8 and ZMM14 start to differ at about developmental stage F (compare Figs 5B and C).
  • ZMM14 shows not a uniform expression in all organ primordia. Rather, expression is strong in carpels but weak (yet visible) in all other developing organs. That difference in expression is maintained also at later developmental stages (Figs 5D,E).
  • ZMM8 expression was directly compared to that of two other MADS-box genes, ZMM2 and ZMM6 (Fig. 6).
  • ZMM2 is a putative orthoiog of the Arabidopsis C-type floral homeotic gene AGAMOUS (Thei ⁇ en et al. 1995; Fischer et al. 1995b; Mena et al. 1996). Therefore, expression in stamens and carpels, but not in other plant organs was expected for ZMM2.
  • ZMM6 is another AGL2- ⁇ ke MADS-box gene from maize that is expressed in all organ primordia of the floret (Cacharr ⁇ n et al. 1995).
  • ZMM8 expression was found to be restricted to the upper floret (Figs 6A,C,F)
  • ZMM2 and ZMM6 showed their typical expression patterns in both, the upper and the lower floret of each spikelet (Fig. 6B,D,E,G,H).
  • Fig. 6B,D,E,G,H Within upper as well as lower florets, ZMM2 expression was indeed found to be restricted to stamen and carpel primordia of female (Figs 6B,G) as well as male (Fig. 6D) florets.
  • ZMM6 expression could be detected in all organs of both floret types of male (Fig. 6H) as well as female inflorescences (Fig. 6E) which is in strong contrast to the lack of ZMM8 expression in the lower floret primordium (compare Fig. 6H (ZMM6) with 4D (ZMM8), showing male spikelets at developmental stage H, and Fig. 6E (ZMM6) with 6F (ZMM8), showing female spikelets at developmental stage F).
  • ZMM14 could have a similar function, but might be more specialized to specify the identity of the carpel of the upper floret rather than that of the whole floret.
  • ZMM8 and ZMM14 may exert their function by encoding transcription factors that regulate sets of target genes important for special features of the upper floret or its carpel, respectively.
  • the upper floret is genetically different from the lower one, since some mutations affect only the lower, but not the upper floret.
  • the lower florets become aborted during ontogeny, they develop until maturity in the traditional sweet corn cultivar "Country Gentleman" (Huelsen and Gillis 1929).
  • floret meristems are generated from the inflorescence meristem in several steps (for details, see Fig. 7; Cheng et al. 1983).
  • the inflorescence meristem generates spikelet pair meristems (or primordia).
  • the spikelet pair meristems then generate two spikelet meristems (or primordia).
  • Each spikelet meristem initiates a pair of glumes, and then generates the upper and lower floret meristem (or primordium).
  • the inflorescence meristem, spikelet pair meristems, spikelet meristems and floret meristems represent a series of four different types of reproductive meristems in which increasing determinacy is confered by the conversion of meristems into derivative types.
  • Spikelet meristems, the third type of meristem in the series each initiate first a pair of lateral organs, glumes, and then produce only a single derivative, a (lower) floret meristem (the fourth meristem type in the series), before itself becoming converted to an (upper) floret meristem.
  • the lower and upper floret meristems are thus of different origin: whereas the lower floret meristem is a (lateral) derivative of the spikelet meristem, the upper floret meristem is a direct transformation product of the spikelet meristem, and thus is in a terminal position. If this model is correct, it could be the function of ZMM8 and ZMM14 to confer determinacy to the spikelet meristem and thus to transform it into an upper floret meristem. It is conceivable that a gene functionally similar to TERMINAL FLOWER1ICENTRORADIALIS from Arabidopsisl Antirrhinum (Bradley et al.
  • ZMM8 and ZMM14 function are needed throughout spikelet development to prevent the upper floret meristem to switch back to a less determined state.
  • Ts6 Tassel seed 6
  • remnants of the spikelet meristem may remain after the upper and lower floret meristems have been formed laterally. If so, IDS1 function may be needed to suppress indeterminate growth within the spikelet meristem and thus would probably be independent from ZMM8IZMM14 function. However, even a direct functional interaction between ZMM8/ZMM14 and IDS1 in some cells of the spikelet cannot be excluded at the moment. It will be interesting to determine whether the expression domains of ZMM8IZMM14 and IDS1 overlap, and how ZMM8 and ZMM14 are expressed in an idsl mutant plant.
  • the number of florets per spikelet varies considerably between different grass species, ranging from one in rice and Stipa to at least 40 in Eragrostis oxylepis (Clifford 1987). This variability could be explained by changes in the behavior of the spikelet meristem during evolution. Assuming that the "non-equivalent origin model" of floret ontogeny is true, species in which the spikelet meristems are transformed into terminal floret meristems before they have formed a derivative floret meristem on their flanks will then develop just one floret per spikelet.
  • Species in which the spikelet meristems form n derivative floret meristems before they are transformed into floret meristems will have n+1 florets per spikelet. Analogous calculations can be made when the "equivalent origin model" is applied, and similar considerations also apply to the intraspecific variability of the number of florets per spikelet in case of mutants. If expression of ZMM8/ZMM14 and its orthologs indicates that a switch from a spikelet to a terminal floret meristem has occurred, these genes always should be expressed only in the uppermost floret primordium of each spikelet.
  • ZMM8 and its close relatives are even causally involved in such a switch in meristem identity, temporal changes in the expression of these genes could have determined changes in the number of florets per spikelet during evolution.
  • genes like ZMM8 and ZMM14 may have played a role in regulating floret number in grasses during evolution. It would be interesting, therefore, to determine how ZMM8 and ZMM14 are expressed in mutants such as Ts6, and how orthologs of these genes are expressed in grass species with more than two florets per spikelet. Since the spikelets of grasses are phylogenetically highly derived structures it is clear that the upper floret specific expression of ZMM8 and ZMM14 represents a derived state as well.
  • the promoter from ZMM8 and ZMM14 can be isolated by screening a gene library.
  • a gene library of maize required for this purpose is, for example, commercially available (Company Stratagene, catalogue no. #946102). Approx. 100.000 to 500.000 clones of the genomic DNA library were screened by plaque hybridization with radioactive probes that are specific for ZMM8 or ZMM14. The probes are generated as templates by means of linear PCR on the clones pSW24 (ZMM8) and pWF1552 (ZMM14); see Examples 1 and 2. Therefor 20 pmol primers pSW24 (ZMM8): primer WD53: 5' CGCAGCTCCAACTACAGCACACAGG 3' (SEQ ID NO: 8)
  • pWF1552 (ZMM14): primer w258_ZMM 14: 5' GGACAAGGGTTGAATTCCTCCAAACTACAC 3' (SEQ ID NO: 9), 100 ng template, each 2 ⁇ M dATP, dGTP, dTTP, 2.5 U Taq polymerase (Promega) and 50 ⁇ Ci (1.7 ⁇ M) [ ⁇ - 32 P]dCTP (Amersham) are added to a total volume of 10 ⁇ l, which is adjusted to 1 x fmol sequence buffers (Promega).
  • any oligonucleotide can serve as primer that binds to the 3'-region of the cDNAs binds and that is oriented stream upwards.
  • the hybridization of the filters on which the gene library had been transferred with the radioactively labeled probes is carried out for 15 hours at 65 °C in a hybridization solution as follows: 3 x SSPE, 0.02 % ficoll, 0.02 % polyvinylpyrrolidone, 0.1 % SDS, 100 ⁇ g/ml salmon sperm-DNA. Then the filters are washed in 0.2 x SSPE, 0.1 % SDS for 3 x 20 minutes, the positive phage clones are identified by autoradiography and then separated by repeating this process. For the production of 3 x SSPE and 0.2 x SSPE 20 x SSPE is used.
  • the amplified fragment is sequenced via primer walking until several kb upstream from the transcription start point have been reached, if present on the clone, preferably more than 3 kb.
  • the transcription start site is determined by standard procedures well known to everyone skilled in the art, such as 5'-RACE, primer extension or S1 mapping. To define cis-regulatory elements upstream of the transcription start site (i.e.
  • the respective region is fused to marker genes such as genes encoding GUS or GFP, and 5' deletion derivatives of these construct are generated. They are transformed into suitable plant material, and the expression of the marker gene depending on the remaining upstream sequence (putative promoter) is determined.
  • Maize plants were generated which are assumed to express a specific part of the ZMM8 cDNA (IKC-domains, i.e. all domains excluding the highly conserved MADS- domain, nucleotides 330 to 773 of SEQ ID NO: 1 ) in reverse orientation under the control of the 35S promoter of the Cauliflower Mosaic Virus (CaMV) using a self- made vector that contains the 35S promoter of pRT100 (T ⁇ pfer, Nucl. Acids Res. 15 (1987), 5890) and the selectable marker gene BAR from pAHC25 (Taylor, Plant Cell Rep. 12 (1993), 491-495).
  • IKC-domains i.e. all domains excluding the highly conserved MADS- domain, nucleotides 330 to 773 of SEQ ID NO: 1
  • CaMV Cauliflower Mosaic Virus
  • ZMM8 antisense plants are expected to have a reduced expression of the ZMM8 gene. Transformation was done using a standard PEG method applied to maize protoplasts (M ⁇ rocz, Theor. Appl. Genet. 80 (1990), 721-726). Up to now 71 transformants have been obtained from 4 independent transformation events. DNA gel blot hybridization and PCR studies on plants derived from all 4 events confirmed that the putative transformants indeed contain the transgene. Phenotypic analyses showed that some (3%-6%) of the spikelets of the male inflorescences of transformants contained an increase in the number of florets per spikelet (Fig. 8B) compared to wild-type (Fig. 8A).
  • the number of florets is increased from 2 to 3-4, or only the number of a subset of the floral organs is increased.
  • the number of glumes is increased from 2 to 4.
  • increase in floret number was also clearly observed in plants from all 4 independent transformation events, but less frequently than in male inflorescences.
  • transformants have been crossed out to non-transgenic standard lines to test the heritability of the phenotype and for further analyses (e.g. ZMM8 expression studies by RNA gel blot hybridization and RT-PCR).

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Abstract

L'invention concerne des molécules d'acide nucléique codant pour de nouvelles protéines à boîte MADS qui sont exprimées dans les florules supérieures des épillets des inflorescences herbeuses ainsi que des séquences régulatrices qui régulent naturellement l'expression de ce type de molécules d'acide nucléique. L'invention concerne également des vecteurs comprenant ces molécules d'acide nucléique, dans lesquels les molécules d'acide nucléique sont associées de manière fonctionnelle à des éléments régulateurs permettant leur expression dans des cellules hôtes procaryotes ou eucaryotes ainsi que des protéines codées par ces molécules d'acide nucléique, des anticorps dirigées contre ces protéines, et des procédés permettant de produire ces vecteurs, protéines et anticorps. L'invention concerne en outre des molécules et des vecteurs d'ADN recombinant comprenant ces séquences régulatrices ainsi que des cellules hôtes transformées au moyen de ces séquences. L'invention concerne en outre des kits et des compositions de diagnostic comprenant les molécules d'acide nucléique, protéines, anticorps, séquences régulatrices, molécules et vecteurs d'ADN recombinant et anticorps cités ci-dessus. L'invention concerne encore des procédés permettant l'identification de composés capables d'activer ou d'inhiber les gènes ou produits géniques exprimés dans les florules supérieures des épillets des inflorescences des herbes ou leurs produits géniques. L'invention concerne enfin des cellules végétales, des tissus végétaux et des plantes transgéniques contenant les molécules d'acide nucléique, les séquences régulatrices, les molécules et vecteurs d'ADN recombinant décrits ci-dessus, ainsi que l'utilisation des molécules d'acide nucléique, séquences régulatrices, molécules et vecteurs d'ADN recombinant, protéines, anticorps, peptides et/ou des composés identifiés par un procédé relevant de l'invention dans des cultures de cellules et de tissus végétaux, la culture de plantes et/ou l'agriculture.
PCT/EP1999/010116 1998-12-21 1999-12-20 Nouveaux genes a boite mads et utilisation de ces genes WO2000037488A2 (fr)

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Cited By (12)

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WO2002033091A1 (fr) * 2000-10-19 2002-04-25 Agriculture Victoria Services Pty Ltd Manipulation de la floraison et de l'architecture vegetale
WO2002014486A3 (fr) * 2000-08-18 2002-11-28 Advanta Seeds Bv Inhibition de propagation procreatrice dans des graminees genetiquement modifiees
WO2003000904A3 (fr) * 2001-06-22 2003-12-04 Syngenta Participations Ag Identification et caracterisation de genes vegetaux
WO2004035797A3 (fr) * 2002-09-27 2005-11-10 Dlf Trifolium As Promoteurs specifiques aux tissus issus de plantes
GB2418917A (en) * 2004-09-30 2006-04-12 Malaysian Palm Oil Board B-type MADS box genes from oil palm
US7550579B2 (en) 2005-04-29 2009-06-23 Pioneer Hi-Bred International, Inc. Pericarp-preferred regulatory element
US8022274B2 (en) 1998-09-22 2011-09-20 Mendel Biotechnology, Inc. Plant tolerance to low water, low nitrogen and cold
WO2019204373A1 (fr) * 2018-04-18 2019-10-24 Pioneer Hi-Bred International, Inc. Protéines de mads box et amélioration de caractéristiques agronomiques dans des plantes
CN113429465A (zh) * 2021-05-24 2021-09-24 哈尔滨学院 一种桑黄MADS-box类转录因子PbMADS1及其编码基因与应用
CN114058630A (zh) * 2021-11-25 2022-02-18 仲恺农业工程学院 荔枝MADS-box转录因子LcMADS1及其在抑制植物器官脱落中的应用
US12077766B2 (en) 2018-04-18 2024-09-03 Pioneer Hi-Bred International, Inc. MADS box proteins and improving agronomic characteristics in plants
US12371702B2 (en) 2018-04-18 2025-07-29 Pioneer Hi-Bred International, Inc. Improving agronomic characteristics in maize by modification of endogenous mads box transcription factors

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WO1994000582A2 (fr) * 1992-06-30 1994-01-06 Bruinsma Seeds B.V. Procede d'obtention d'une plante a morphologie florale modifiee, et procede de protection des plantes contre les insectes nuisibles
US5859326A (en) * 1994-10-14 1999-01-12 Washington State University Gene controlling floral development and apical dominance in plants
US5990386A (en) * 1994-10-14 1999-11-23 Washington State University Research Foundation Genes controlling floral development and apical dominance in plants
US5795753A (en) * 1994-12-08 1998-08-18 Pioneer Hi-Bred International Inc. Reversible nuclear genetic system for male sterility in transgenic plants

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8022274B2 (en) 1998-09-22 2011-09-20 Mendel Biotechnology, Inc. Plant tolerance to low water, low nitrogen and cold
WO2002014486A3 (fr) * 2000-08-18 2002-11-28 Advanta Seeds Bv Inhibition de propagation procreatrice dans des graminees genetiquement modifiees
WO2002014524A3 (fr) * 2000-08-18 2002-12-05 Advanta Seeds Bv Inhibition de la propagation generative de gazons genetiquement modifies resistants aux herbicides
US7297846B2 (en) 2000-08-18 2007-11-20 Advanta Seeds B.V. Grasses expressing AtH1 exhibit delayed heading and reduced inflorescenses
WO2002033091A1 (fr) * 2000-10-19 2002-04-25 Agriculture Victoria Services Pty Ltd Manipulation de la floraison et de l'architecture vegetale
WO2003000904A3 (fr) * 2001-06-22 2003-12-04 Syngenta Participations Ag Identification et caracterisation de genes vegetaux
WO2004035797A3 (fr) * 2002-09-27 2005-11-10 Dlf Trifolium As Promoteurs specifiques aux tissus issus de plantes
GB2418917B (en) * 2004-09-30 2010-04-21 Malaysian Palm Oil Board Novel B-type gene from oil palm
GB2418917A (en) * 2004-09-30 2006-04-12 Malaysian Palm Oil Board B-type MADS box genes from oil palm
US7550579B2 (en) 2005-04-29 2009-06-23 Pioneer Hi-Bred International, Inc. Pericarp-preferred regulatory element
US7851614B2 (en) 2005-04-29 2010-12-14 Pioneer Hi-Bred International, Inc. Terminator from Zea mays lipid transfer protein 1 gene
US7897746B2 (en) 2005-04-29 2011-03-01 Pioneer Hi-Bred International, Inc. Pericarp-preferred promoter from maize lipid transfer protein gene
WO2019204373A1 (fr) * 2018-04-18 2019-10-24 Pioneer Hi-Bred International, Inc. Protéines de mads box et amélioration de caractéristiques agronomiques dans des plantes
US12077766B2 (en) 2018-04-18 2024-09-03 Pioneer Hi-Bred International, Inc. MADS box proteins and improving agronomic characteristics in plants
US12234470B2 (en) 2018-04-18 2025-02-25 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6
US12371702B2 (en) 2018-04-18 2025-07-29 Pioneer Hi-Bred International, Inc. Improving agronomic characteristics in maize by modification of endogenous mads box transcription factors
CN113429465A (zh) * 2021-05-24 2021-09-24 哈尔滨学院 一种桑黄MADS-box类转录因子PbMADS1及其编码基因与应用
CN114058630A (zh) * 2021-11-25 2022-02-18 仲恺农业工程学院 荔枝MADS-box转录因子LcMADS1及其在抑制植物器官脱落中的应用
CN114058630B (zh) * 2021-11-25 2022-06-07 仲恺农业工程学院 荔枝MADS-box转录因子LcMADS1及其在抑制植物器官脱落中的应用

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