WO2003104464A1 - Promoteur specifique de gametophyte femelle (zmea1) - Google Patents

Promoteur specifique de gametophyte femelle (zmea1) Download PDF

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WO2003104464A1
WO2003104464A1 PCT/EP2003/006037 EP0306037W WO03104464A1 WO 2003104464 A1 WO2003104464 A1 WO 2003104464A1 EP 0306037 W EP0306037 W EP 0306037W WO 03104464 A1 WO03104464 A1 WO 03104464A1
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seq
sequence
promoter
gene
zmeal
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Mihaela Marton
Simone Cordts
Thomas Dresselhaus
Jean Broadhvest
Michiel Van Lookeren Campagne
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Bayer CropScience NV
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Bayer Bioscience NV
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8233—Female-specific, e.g. pistil, ovule
    • 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
    • C12N15/8265—Transgene containment, e.g. gene dispersal
    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/829—Female sterility
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to sequences expressed selectively in the female gametophyte isolated from corn and to a promoter capable of directing transcription of an operably linked foreign DNA sequence selectively, preferably exclusively in the female gametophyte of plants.
  • the invention also relates to the use of chimeric genes comprising the promoter of the invention for the selective expression of foreign DNA sequences in the female gametophyte of plants. Plants comprising the chimeric genes of the invention, in which foreign DNA is selectively expressed in the female gametophyte are also provided.
  • the female gametophyte or embryo sac develops from a haploid megaspore which undergoes three subsequent mitotic divisions, resulting in eight nuclei. These are arranged in two groups of four at each end of the gametophyte. One nucleus from each group migrates to the center to form the central cell. The three remaining nuclei at the micropylar end become cellularized and organized as the egg apparatus, consisting of an egg-cell and two cellular synergids. The three nuclei at the chalazal end form separate cells called antipodals. This seven-celled structure (with eight nuclei) is called the embryo sac (polygonum type).
  • the pollen tube Upon reaching the embryo sac through the micropyle, the pollen tube penetrates one of the synergids, which subsequently starts to disintegrate allowing release of the two sperm cells.
  • One sperm cell releases its nucleus into the egg-cell producing a zygote which will then develop into an embryo.
  • the other sperm cell releases its nucleus into the central cell, which will then develop into the endosperm.
  • the FIE/FIS3 (fertilization independent endosperm) gene mutant allows endosperm development without fertilization, suggesting the wild-type protein functions to suppress transcription of genes in the female gametophyte prior to fertilization (Ohad et al. 1999).
  • the MEA/FIS1 gene is believed to act by reducing cell proliferation in the embryo (Goodrich, 1998).
  • the FIS1, FIS2 and FIS3 genes are believed to have a sequential regulatory role in the suppression of seed development genes (Luo et al. 1999).
  • a family of genes specifically expressed in the female gametophyte has been isolated from maize egg cells (ZmESl-4, Cordts et al. 2001). Expression of these genes was found to be high in the synergids, lower in the egg cell and central cell while the antipodal cells showed only weak expression.
  • the present invention relates to isolated nucleotide sequences specifically expressed in the female gametophyte of plants, preferably highly expressed in the egg cell and expressed at a lower level or not expressed in the synergids, which sequences can be used for the modulation of endogenous expression of female gametophytic genes and the identification of further genes expressed in the female gametophyte. Furthermore, the promoter identified in these sequences or parts thereof have an application in the direction of expression of heterologous genes in the female gametophyte for the production of plants with modified endosperm and embryo development.
  • a particular embodiment of the invention relates to the production of female sterile plants and of plants capable of apomictic propagation, which are of significant agricultural interest.
  • the present invention relates to isolated DNA sequences that are expressed in the female gametophyte of plants, preferably selectively in the egg apparatus.
  • a first aspect of the invention relates to promoters, capable of directing expression in the female gametophyte of plants, which can be used for expression of a heterologous DNA sequence in the female gametophyte, in particular in the female gametophyte of monocotyledonous plants, such as maize or rice.
  • a particular embodiment of the invention relates to the regulatory region of the ZmEAl gene of SEQ ID No.l, more specifically to the promoter, which directs expression specifically in the female gametophyte of plants.
  • the promoter of the ZmEAl gene is reduced to a shorter promoter sequence which is still capable of directing expression in the egg apparatus of the female gametophyte of plants.
  • the invention further relates to functional equivalents of the ZmEAl promoter, which are capable of directing selective expression of a heterologous DNA in the female gametophyte of plants more specifically of monocots, such as corn and rice, preferably expressed selectively in the egg apparatus.
  • Such functional equivalents can include but are not limited to: a) sequences hybridizing to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions. Such sequences can be isolated from different corn varieties, or from other plant species. Such functional equivalents preferably have 90% sequence identity with SEQ ID NO: 2. b) sequences which can be amplified using oligonucleotide primers comprising at least about 25, preferably at least about 50 or up to 100 consecutive nucleotides of SEQ ID NO:2 in a polymerase chain reaction.
  • Functional equivalents of the ZmEAl promoter can also be obtained by substitution, addition or deletion of nucleotides of the sequence of SEQ ID No. 2. They can be partly or completely synthesized.
  • the DNA sequences of the ZmEAl gene described herein are used for the cloning and isolation of female gametophyte specific genes, regulatory regions or coding regions from plants. More specifically these sequences can be used for the isolation of functional equivalents of the ZmEAl promoter from plants, preferably monocotyledonous plants, especially preferably cereal plants such as corn or rice.
  • the female gametophyte- specific promoter of the invention directs expression essentially in the egg apparatus, i.e. the egg cell and the synergids of the female gametophyte of plants.
  • the present invention further relates to chimeric genes comprising the ZmEAl promoter or a functional equivalent thereof which directs expression of a heterologous DNA in the female gametophyte.
  • the heterologous DNA is a DNA encoding an RNA or protein capable of modifying reproductive development.
  • the heterologous DNA is a DNA encoding an RNA or protein capable of modifying embryogenesis and/or endosperm development.
  • Such constructs can be applied in engineering female sterility, seedless fruit, embryoless seed, haploid or doubled haploid plants or plants capable of apomictic propagation.
  • a heterologous DNA can be a DNA encoding a cytotoxic molecule, whereby expression of the chimeric ZmEAl -heterologous DNA results in cell death of the female gametophytes.
  • the present invention also relates to plant cells or plants and seeds or tissues of plants comprising a chimeric gene according to the invention.
  • a method for modifying reproduction of a plant comprises introducing into a plant cell a chimeric gene comprising the ZmEAl promoter or a functional equivalent thereof operably linked to a heterologous DNA sequence which, upon expression, modifies the development of the egg apparatus, and growing the plant cell into a mature plant.
  • a method for modifying the development of the female gametophyte comprises modulating the endogenous expression of the ZmEAl gene or overexpression of a gene comprising he ZmEAl coding region.
  • the present invention relates to sequences expressed specifically in the female gametophyte of plants, more particularly to the ZmEAl gene and regulatory and coding sequences derived therefrom. More particularly the invention relates to the use of the female gametophyte-specific promoters, such as the ZmEAl promoter sequences derived therefrom to direct selective expression of a heterologous DNA in the female gametophyte of plants.
  • the term "gene” as used herein refers to any DNA sequence comprising several operably linked DNA fragments such as a promoter, a 5' untranslated region (the 5 'UTR), a coding region (which may or may not code for a protein), and an untranslated 3' region (3 'UTR) comprising a polyadenylation site.
  • the 5 'UTR, the coding region and the 3 'UTR are transcribed into an RNA of which, in the case of a protein encoding gene, the coding region is translated into a protein.
  • a gene may include additional DNA fragments such as, for example, introns.
  • the term 'regulatory region' as used herein refers to a DNA region which is involved in regulating the transcription, such as the specificity, timing or level, of a DNA sequence, such as, but not limited to, a DNA sequence encoding a protein.
  • the 5 'regulatory region is a region located upstream from a coding sequence which comprises the promoter and the 5' untranslated UTR.
  • the 3' regulatory region is a sequence downstream of the coding sequence comprising suitable termination signals (e.g. one or more polyadenylation signals).
  • the term 'promoter' refers to a DNA region, a sequence of which is recognized (directly or indirectly) by a DNA-dependent RNA polymerase during initiation of transcription and which includes the transcription initiation site, binding sites for transcription initiation factors and RNA polymerase.
  • the promoter may also comprise binding sites for other regulatory proteins, such as enhancers or inhibitors.
  • the term 'chimeric' when referring to a gene or DNA sequence is used to refer to the fact that the gene or DNA sequence comprises at least two functionally relevant DNA fragments (such as promoter, 5 'UTR, coding region, 3 'UTR, intron) that are not naturally associated with each other and/or originate, for example, from different sources.
  • Heterologous referring to a gene or DNA sequence with respect to a plant species is used to indicate that the gene or DNA sequence is not naturally found in that plant species, or is not naturally found in that genetic locus in that plant species.
  • An endogenous gene is a gene which is naturally found in a plant species.
  • Heterologous when referring to a parts of a gene (such as coding region, promoter, 3' end) is used to indicate that the heterologous part of the gene is not naturally found associated with at least one other part of that gene.
  • RNA which itself is biologically active (eg antisense RNA, ribozyme or other kind of interaction with a DNA, RNA or protein sequence) or which is translated into a biologically active protein or polypeptide.
  • biologically active eg antisense RNA, ribozyme or other kind of interaction with a DNA, RNA or protein sequence
  • 'female gametophyte specific expression' refers to expression of a DNA sequence predominantly, preferably exclusively in the female gametophyte, including in the egg apparatus of plants. Expression selectively in the egg apparatus is referred to as 'egg apparatus specific expression'.
  • An 'egg apparatus specific promoter' is a promoter capable of directing egg apparatus specific expression, i.e. expression predominantly, preferably exclusively in the egg apparatus (the egg-cell and the synergids) of the female gametophyte.
  • the female gametophyte specific promoter is characterized in that it is an egg apparatus specific promoter.
  • a particular embodiment of the present invention relates to a female gametophyte specific promoter, more specifically the promoter of the ZmEAl gene isolated from corn, which confers female gametophyte specific expression and egg apparatus specific expression in monocots, more specifically in corn.
  • the term 'functional equivalent of the ZmEAl promoter' as used herein refers the fact that the promoter is capable of directing female gametophyte specific expression of a gene.
  • a functional equivalent of the ZmEAl promoter is capable of directing egg apparatus specific expression.
  • a specific embodiment of the invention relates to promoters capable of directing female gametophyte specific expression in corn or rice. Such functional equivalents include 'shortened' ZmEAl promoters, i.e.
  • promoters which comprise only part of the sequence of SEQ ID NO:2, more particularly the fragments of about 1.5kbp, about l.Okbp and about 0.5kbp upstream from the coding region, for the ZmEAl protein as described herein. Most preferably, the fragments are of 1570 bp, 1014 bp, and 470 bp upstream from the coding region.
  • promoter sequences hybridizing to the nucleotide sequence of SEQ ID NO:2 under stringent conditions and which are capable of directing female gametophyte specific expression are herein considered as functional equivalents of the ZmEAl promoter.
  • Such functional equivalents can be isolated from different corn varieties, or from other plant species. They can also be obtained by substitution, addition or deletion of nucleotides of SEQ ID NO: 2. They can be partly or completely synthesized.
  • ZmEAl promoter comprise sequences which can be amplified using oligonucleotide primers comprising at least about 25, preferably at least about 50 or up to 100 consecutive nucleotides of SEQ ID NO:2 in a polymerase chain reaction.
  • a functional equivalent of the ZmEAl promoter can be isolated by using a cDNA of the transcript of the ZmEAl gene of SEQ ID NO:l or part thereof, as a probe to isolate the genomic DNA upstream of the nucleotide sequence corresponding to the nucleotide sequence of the cDNA.
  • Functional equivalents of the ZmEAl promoter can also be obtained by screening a cDNA library with oligonucleotides that are deduced from the amino acid sequence of the protein encoded by the ZmEAl gene.
  • a nested-PCR approach can also be used, whereby the oligonucleotides are used to amplify a fragment, which can serve as a probe to screen a cDNA library.
  • Other methods for obtaining functional equivalents of the ZmEAl promoter are based on hybridizations of DNA, cDNA, RNA or oligonucleotides deduced from the ZmEAl gene of SEQ ID NO:l. Such methods have been described and are known to the person skilled in the art.
  • Egg apparatus specific expression can be ascertained in different ways such as, but not limited to in situ hybridization, detection of GUS-expression after linkage of the promoter to the coding region of the gus gene, detection of cell-ablation after linkage of the promoter to a cytotoxic (or 'killer') gene and RNA detection.
  • Stringent hybridization conditions refers to the fact that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence.
  • Stringent hybridization conditions can for instance comprise the following steps: prehybridizing the filter for 1 to 2 hours at 42°C in 50% formamide, 5 X SSPE, 2 X Denhardt's reagent and 0.1% SDS, or for 1 to 2 hours at 68°C in 6 X SSC, 2 X Denhardt's reagent and 0.1% SDS, 3) adding the hybridization probe which has been labeled, 4) incubating for 16 to 24 hours, 5) washing the filter for 20 min.
  • the heterologous DNA of interest to which the female gametophyte specific promoter is linked in the chimeric gene of the present invention, can encode a protein or polypeptide or a biologically active RNA, such as an antisense RNA, a sense RNA, a ds-RNA (comprising both sense and antisense sequences so as to form double stranded RNA, as in WO99/53050) which can be used for posttranscriptional gene silencing of a target sequence.
  • a biologically active RNA such as an antisense RNA, a sense RNA, a ds-RNA (comprising both sense and antisense sequences so as to form double stranded RNA, as in WO99/53050) which can be used for posttranscriptional gene silencing of a target sequence.
  • the heterologous DNA of interest is a DNA sequence which encodes a biologically active RNA, or a protein or polypeptide which, when expressed in the female gametophyte, is capable of significantly disturbing the metabolism and/or functioning of the cells of the female gametophyte, more particularly the cells of the egg apparatus, so as to modulate embryogenesis and/or endosperm development.
  • DNA sequences encoding proteins modulating embryogenic and/or endosperm development include but are not limited to the coding sequence of the FIS-genes (Luo et al, 1999, above), LEC-genes (WO 99/67405; WO 01/70777), BBM gene (WO 00/75330), ZmES genes (Cordts et al, 2001, above; WO/0164924), the WUS gene (Zuo et al., 2002) and the coding sequence of genes directly or indirectly resulting in increased levels of hormones, such as cytokinin, auxin, ethylene and/or brassinosteroids.
  • hormones such as cytokinin, auxin, ethylene and/or brassinosteroids.
  • DNA suitable for causing death of the cells in which they are expressed include the DNA sequences encoding cytotoxic molecules, such as but not limited to the ribonucleases barnase or Rnase Tl, diptheria toxin A (as described, for instance in PCT patent publications WO 89/10396 and WO 91/02068 ).
  • the invention relates inter alia to methods for expressing a heterologous DNA of interest in the female gametophyte of plants, whereby the method comprises the following steps: introducing of the chimeric genes of the invention into plant cells so as to obtain stable integration in the genome of the plant cells and regeneration of the plant cells into plants.
  • Modulation of endogenous expression of the ZmEAl gene can be suppression of expression by using an antisense RNA of the ZmEAl gene of SEQ ID NO:l, or a ds- RNA (comprising both sense and antisense sequences so as to form double stranded RNA, as in WO99/53050) which can be used for posttranscriptional gene silencing of the ZmEAl gene or by cosuppression using a sense RNA.
  • Other methods of suppressing or eliminating gene expression (functional knock-outs) known in the art can also be envisaged.
  • the ZmEAl gene product can be over-expressed by introducing one or more additional copies of the ZmEAl gene in the genome of the plant.
  • the coding sequence of the ZmEAl gene of the present invention can be used in the development of plants with modified embryogenesis and/or endosperm development.
  • the coding sequence of the ZmEAl gene can be placed under control of a heterologous promoter in order to direct expression in cells not naturally expressing the ZmEAl protein and/or to overexpress the ZmEAl coding region in the female gametophyte.
  • the ZmEAl coding region is the region encoding the protein of SEQ ID NO:3, most preferably it corresponds to the coding region of SEQ ID NO:l.
  • Introduction of a foreign DNA into a plant cell can be obtained by conventional transformation methods described in the art. Such methods include but are not limited to Agrobacterium mediated transformation (US 6,074,877, Hiei et al., 1997), microprojectile bombardment (as described, for example by Chen et al., 1994; Casas et al., 1995; Christou, 1997, Finer et al., 1999, Vasil et al. 1999), direct DNA uptake into protoplasts (as described, for example by De Block et al.
  • Operably linking the DNA of interest to a female gametophyte specific promoter according to the invention can also be achieved by replacing the DNA naturally associated with the female gametophyte specific promoter by homologous recombination with the gene of interest, provided that the DNA of interest comprises a homology region with the DNA normally associated with the female gametophyte specific promoter.
  • Such methods have been described in the art (eg US 5,744,336).
  • SEQ ID NO: 1 nucleotide sequence of the ZmEAl gene isolated from corn SEQ ID NO:2 nucleotide sequence of a ZmEAl promoter SEQ ID NO:3 deduced amino acid sequence of the ZmEAl protein encoded by SEQ ID NO: 1
  • Cells of the embryo sac were mechanically isolated with glass needles from ovule tissues treated with a cell wall degrading enzyme mixture and transferred using a hydraulic microcapillary system according to Kranz et al. (1991).
  • In vitro zygotes were generated after fusing isolated gametes by a short electric pulse and cultivated as described (Kranz and Lorz, 1993).
  • In vivo zygotes were isolated as described by Cordts et al. (2001). The cells were collected and stored in 200 nl each at -80°C until usage.
  • 70 clones were selected which produced strong signals after hybridization with the egg cell cDNA population, but no or weak signals with cDNA populations of in vitro zygotes and seedlings, respectively. All 70 clones were fully sequenced. Partial cDNAs of the ZmEAl gene were represented 17 times among these clones varying in length from 356-438 bp. All clones contained a 3' UTR (untranslated region) which varied in length due to different polyA sites used to terminate transcription. In order to isolate the full length cDNA of ZmEAl, cDNA was generated from in vivo zygotes, 16 to 18 hours after fertilization.
  • Poly(A) + mRNA isolation out of 9 zygotes was performed using the Dynabeads® mRNA DIRECTTM Micro Kit (Dynal). Immediately after isolation, poly(A) + mRNA was used for first strand cDNA synthesis. Reverse transcription and cDNA amplification by long distance PCR (LD PCR) was performed using the SMARTTM cDNA synthesis Kit (Clontech, Palo Alto) according to the user manual. Quality of obtained cDNA was checked by separating 5 ⁇ l of PCR reactions on a 0,8% agarose gel. After gel electrophoresis, cDNA was blotted onto Hybond NXTM nylon membrane (Amersham) and hybridized with a GAPDH-specific probe.
  • LD PCR long distance PCR
  • the missing 5'-end was amplified from 10 ng/ul of this cDNA using a gene specific primer and a universal primer mix (UPM- a mixture of two specific race primers detailed below) in a standard PCR reaction with the following profile: 2 min 94°C followed by 5 cycles: 30 sec 94°C, 3 min 72°C; 5 cycles: 30 sec 94°C, 30 sec 70°C, 3 min 72°C; 20 cycles: 30 sec 94°C, 30 sec 68°C, 3 min 72°C and a final extension for 10 min at 72°C.
  • PCR products were cloned and sequenced.
  • RACE primers Long primer: 5'-GTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'
  • the cDNA of ZmEAl is 534 bp in length with the largest open reading frame encoding 94 amino acids (position: 1571 - 1855 in SEQ ID NO:l).
  • the 5' UTR is 107 bp (5' UTR: position 1464 - 1570 SEQ ID NO:l ) in length, calculated from the putative start point of transcription at position 1464 as analyzed by Single Cell RT- PCR and excluding the ATG at position 1571-1573.
  • the 3' UTR is 142 bp (3' UTR: position 1856 - 1997).
  • the sequence of the full-length cDNA is given in SEQ ID NO: 7.
  • the amino acid sequence of ZmEAl is given in SEQ ID NO: 3.
  • the ZmEAl gene encodes a very hydrophobic peptide and contains no introns. Homology searches revealed that ZmEAl does not match to any other ESTs in public data bases (February 2002).
  • Genomic DNA from leaf material of Zea mays inbred line A188 was used to construct Genome Walker - "libraries", based on the Universal Genome WalkerTM Kit protocol (Clontech, Palo Alto). Separate aliquots of DNA were completely digested with 6 different restriction enzymes that leave blunt ends (EcoRN, Dral, Hindi, PvuII, Seal and Smal). Each batch of digested genomic D ⁇ A was then ligated separately to the GenomeWalker Adaptor (Adaptor 2R: 5'-
  • Tissue in situ hybridization was performed according to Cordts et al. (2001) to investigate the expression of ZmEAl in ovules at maturity.
  • the in situ hybridization experiments were made with ovule tissues embedded in BMM (butyl-methyl methacrylate).
  • BMM butyl-methyl methacrylate
  • a partial cDNAs of the ZmEAl gene was used as a hybridization probe. Strong signals were detected in the cytoplasm of synergids and egg cell close to the micropyle region. Signals in nucellus cells, integuments or ovary tissues were never observed (Cordts 2000).
  • total RNA and poly(A) + RNA Northern blot analyses were performed.
  • the investigated tissues included immature male and female inflorescences of different developmental stages, immature and mature ovaries, kernels at different developmental stages, immature and mature embryos, embryogenic and non-embryogenic suspension cultures, different seedling stages, light and dark green leaves, internodes, meristematic leaf base, root tips and roots without tips. A signal was not obtained in any sample tested.
  • RT-PCR was performed according to Cordts et al. (2001) and applied to prove absense of ZmEAl messenger RNA in most of the tissues investigated by Northern blot analysis and in addition with nodes, scutellum tissue, immature and mature pollen, microspores at the 2 nucleus stage, immature cob, immature and mature ovules.
  • RT-PCR with single cells of the embryosac before and after fertilization was performed as described by Cordts et al.
  • RT1 5'-AGCGCCCGCTGTCCATTCAT-3', SEQ ID NO:14
  • Qbr 5'- ACGACGATCACTTGCTCACAG-3'
  • SEQ ID NO:15 A maize GAPDH gene was used as a control for the success of the RT-PCR using the forward primer Gapl: 5'- AGGGTGGTGCCAAGAAGGTTG-3' (SEQ ID NO: 16) and the reverse primer Gap2: 5'-GTAGCCCCACTCGTTGTCGTA-3' (SEQ ID NO: 17), as described by Richert et al. (1996).
  • the data obtained from the Northern blot analysis were confirmed.
  • genomic DNA was digested with restriction enzymes and resolved on 0.8 % agarose gels. DNA was transferred to Hybond N membranes (Amersham Pharmacia Biotech) with 0.4 M NaOH. Blots were hybridized overnight with radioactive probes derived from a partial ZmEAl cDNA and prepared by Prime- It Random Primer Labelling Kit (Stratagene) in QuickHyb buffer (Stratagene) containing 100 ⁇ g/ml salmon sperm DNA. Filters were washed with decreasing concentrations of SSC with a final wash at 65 °C in 0.2 x SSC / 0.1% SDS.
  • ZmEAl maps on chromosome 7 L between the molecular markers isc(b32B) (119.3) and bnl8.39 (124.8). ZmEAl is not co-localized with known phenotypic markers.
  • Example 4 Generation of constructs for rice transformation
  • ZE-Bbs CTCACTCACGAAGACGACATGCAGAATTCAGCGTC
  • the ZmEAl ::GUS T-DNAs are designed as follows: LB- 3'nos ⁇ BAR ⁇ cab22L ⁇ p35S2 ⁇ >ZmEAlpromoter>GUS>3'nos-RB
  • the constructs are transformed into rice cells, followed by regeneration and analysis of GUS-expression. Based hereon it can be demonstrated that the 1.57 kbp upstream of the transcription start codon is sufficient to drive cell specific expression in the female gametophyte of rice.
  • Example 5 Use of the ZmEAl promoter to engineer embryo sac lethality Constructs are made to engineer embryo sac lethality in rice by placing a DNA encoding a cytotoxic molecule under the control of the ZmEAl promoter.
  • a construct comprising the coding region of the barnase gene (Hartley et al., 1988) and the prophylactic barstar (WO 96/26283) was made for introduction into rice.
  • the "full-length" ZmEAl promoter (1.57 kbp) was amplified using the following primers: ZE-Bbs: CTCACTCACGAAGACGACATGCAGAATTCAGCGTC
  • the ZmEAl promoter was cloned in front of the BARNASE coding sequence.
  • the prophylactic BARSTAR under the control of the 35S promoter was included in the T-
  • the final T-DNA construct was designed as follows:
  • Transformation of the ZmEAl -Barnase construct into plant cells can result in plants in which female gametophyte development is disturbed, due to the expression of the barnase gene in the egg apparatus.
  • Use of the prophylactic barstar construct is expected to increase transformation efficiency and can be of interest to counter-act possible non-specific expression of the ZmEAl promoter.
  • the specific transgenic trait and the ZmEAl "cytotoxic gene construct can be transferred together into the genome of the plants as a single piece of DNA or as two separate events. In the latter, the two events should have complete genetic and or physical linkage.

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Abstract

L'invention concerne des séquences isolées du blé qui sont exprimées sélectivement dans le gamétophyte femelle, plus particulièrement dans l'appareil de l'oeuf, et un promoteur capable de diriger sélectivement la transcription d'une séquence d'ADN étrangère liée fonctionnellement dans le gamétophyte femelle de plantes.
PCT/EP2003/006037 2002-06-06 2003-06-05 Promoteur specifique de gametophyte femelle (zmea1) Ceased WO2003104464A1 (fr)

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AU2003246404A AU2003246404A1 (en) 2002-06-06 2003-06-05 Female gametophyte specific promoter (zmea1)

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EP02077299 2002-06-06
EP02077299.2 2002-06-06

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007092992A1 (fr) * 2006-02-13 2007-08-23 Adelaide Research & Innovation Pty. Ltd. Sequences de controle transcriptionnel de cellule œuf de plante
WO2008052285A1 (fr) * 2006-11-03 2008-05-08 Adelaide Research & Innovation Pty Ltd Séquences de régulation transcriptionnelle
EP4210475A4 (fr) * 2020-09-10 2025-03-05 Monsanto Technology LLC Augmentation d'édition de gène et d'événements d'intégration dirigés sur le site à l'aide de promoteurs méiotiques et de lignée germinale

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0412911A1 (fr) * 1989-08-10 1991-02-13 Plant Genetic Systems, N.V. Plantes à fleurs modifiées
WO2001021785A2 (fr) * 1999-09-20 2001-03-29 Cambia Elements du controle de la transcription de la megagametophyte et utilisations de ceux-ci
WO2001064924A1 (fr) * 2000-03-02 2001-09-07 Südwestdeutsche Saatzucht Genes specifiques du sac embryonnaire

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0412911A1 (fr) * 1989-08-10 1991-02-13 Plant Genetic Systems, N.V. Plantes à fleurs modifiées
WO2001021785A2 (fr) * 1999-09-20 2001-03-29 Cambia Elements du controle de la transcription de la megagametophyte et utilisations de ceux-ci
WO2001064924A1 (fr) * 2000-03-02 2001-09-07 Südwestdeutsche Saatzucht Genes specifiques du sac embryonnaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CORDTS SIMONE ET AL: "ZmES genes encode peptides with structural homology to defensins and are specifically expressed in the female gametophyte of maize", PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 25, no. 1, January 2001 (2001-01-01), pages 103 - 114, XP002174129, ISSN: 0960-7412 *
DROUAUD JAN ET AL: "A Brassica napus skp1-like gene promoter drives GUS expression in Arabidopsis thaliana male and female gametophytes.", SEXUAL PLANT REPRODUCTION, vol. 13, no. 1, July 2000 (2000-07-01), pages 29 - 35, XP002253404, ISSN: 0934-0882 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007092992A1 (fr) * 2006-02-13 2007-08-23 Adelaide Research & Innovation Pty. Ltd. Sequences de controle transcriptionnel de cellule œuf de plante
EP1989306A4 (fr) * 2006-02-13 2009-09-23 Adelaide Res & Innovation Pty Séquences de contrôle transcriptionnel de cellule uf de plante
EP2405010A1 (fr) * 2006-02-13 2012-01-11 Adelaide Research & Innovation Pty Ltd. Séquences de contrôle transcriptionnel de cellule d'oeuf de plante
US8173864B2 (en) 2006-02-13 2012-05-08 Adelaide Research & Innovation Pty Ltd Plant egg cell transcriptional control sequences
AU2007215376B2 (en) * 2006-02-13 2012-09-06 Adelaide Research & Innovation Pty. Ltd. Plant egg cell transcriptional control sequences
US9139839B2 (en) 2006-02-13 2015-09-22 Adelaide Research & Innovation Pty Ltd Plant egg cell transcriptional control sequences
WO2008052285A1 (fr) * 2006-11-03 2008-05-08 Adelaide Research & Innovation Pty Ltd Séquences de régulation transcriptionnelle
EP4210475A4 (fr) * 2020-09-10 2025-03-05 Monsanto Technology LLC Augmentation d'édition de gène et d'événements d'intégration dirigés sur le site à l'aide de promoteurs méiotiques et de lignée germinale
US12545920B2 (en) 2020-09-10 2026-02-10 Monsanto Technology Llc Increasing gene editing and site-directed integration events utilizing meiotic and germline promoters

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