WO2000012681A1 - Compositions et procedes permettant de produire une expression genique de niveau eleve propre a une graine dans le mais - Google Patents

Compositions et procedes permettant de produire une expression genique de niveau eleve propre a une graine dans le mais Download PDF

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WO2000012681A1
WO2000012681A1 PCT/US1999/020308 US9920308W WO0012681A1 WO 2000012681 A1 WO2000012681 A1 WO 2000012681A1 US 9920308 W US9920308 W US 9920308W WO 0012681 A1 WO0012681 A1 WO 0012681A1
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gene
kda
zein
promoter
utr
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PCT/US1999/020308
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English (en)
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Joachim Messing
Jinsheng Lai
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Rutgers, The State University Of New Jersey
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Priority to EP99945499A priority Critical patent/EP1108009A4/fr
Priority to AU58089/99A priority patent/AU5808999A/en
Priority to US09/763,329 priority patent/US6849779B1/en
Publication of WO2000012681A1 publication Critical patent/WO2000012681A1/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)
    • C12N15/8216—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
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm
    • 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
    • C07K14/425—Zeins
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8253—Methionine or cysteine

Definitions

  • This invention relates to agricultural molecular biology to improve the nutritional quality of maize and other cereal crops.
  • this invention provides a consistently highly expressed zein gene that produces a high methionine seed storage protein.
  • zeins The major seed storage proteins of maize are referred to as zeins .
  • zeins In normal maize genotypes, zeins constitute 50-60% of the total endosperm protein at maturity. Zeins are a heterologous group of proteins that can be classified by sequence homology and size (reviewed in Ueda, T. and Messing, J., 1993) .
  • the zeins are the largest subgroup (zein-1) , encoded by about 65 genes, and soluble in ethanol under nonreducing conditions.
  • zein-2 Most of these have a molecular weight of 19 kDa, with one subfamily having a molecular weight of 22 kDa.
  • the other subgroup (zein-2) consists of the ⁇ , ⁇ and ⁇ zeins that are soluble in ethanol under reducing conditions. They differ in amino acid composition and sequence homologies .
  • ⁇ zein gene encoding a 15 kDa zein has been cloned. This zein was found to encode a protein with a moderate level of methionine (11%) . Two cloned ⁇ zeins of 16 and 27 kDa molecular weight were found to be very high in proline. Two ⁇ zeins have been cloned, encoding 10- and 18- kDa proteins rich in methionine (22%, Anderson Kirihara et al . , 1988, and 28%, Swarup et al . , 1995) .
  • zeins are regulated at the transcriptional and post-transcriptional level. Differences in regulation occur in a subfamily- specific manner. For instance, opaque-2 (o2) variants prevent the transcriptional activation of 22 -kDa zein genes. Also, as described in more detail below, the dzrl locus regulates the accumulation of 10 -kDa ⁇ zein mRNA (Cruz-Alvarez et al . , 1991; Schickler et al . , 1993). Like many cereal storage proteins, zeins are deficient in lysine, tryptophan and methionine.
  • corn meals used in animal feeds are supplemented with legume (mainly soy) meals to increase the levels of lysine.
  • legume meals mainly soy
  • the corn-legume mixture is still deficient in methionine, and processed methionine is often added as a supplement to this mixture.
  • methionine level is limited in cereal-legume mixtures, comprising human diets in many third-world communities. Supplementation of cereal- legume mixtures with processed methionine is costly (estimated at about one billion dollars for the U.S. feed business), and in many instances, infeasible.
  • One approach to producing animal feed with an increased methionine content is to genetically engineer the feed plant (e.g., maize or soybeans) to produce or retain more methionine. Since composition and differential accumulation of various storage proteins, rather than amino acid biosynthesis, is the limiting step, it is the seed proteins themselves that must be engineered, either for altered composition or for enhanced expression.
  • the feed plant e.g., maize or soybeans
  • U.S. Patent No. 5,508,468 to Lundquist et al discloses a fertile hybrid transgenic maize plant regenerated from immature embryos of a cross between A188 and B73, transformed with a chimeric 10-kDa zein gene controlled by the promoter for a 27-kDa zein gene. If such a plant is crossed with Mol7 or any other variety carrying the dominant negative allele of dzrl , any overexpression of the 10-kDa zein transgene (or native gene) that might be seen in the parent will be reduced or lost in the progeny, due to the presence of the dominant negative dzrl allele.
  • the presence of the negative dominant dzrl allele is detrimental to the use of a 10-kDa zein gene for increasing methionine content in maize or any other plant. It would be of agronomic and economic significance, then, to identify the mechanism (s) by which the negative allele functions, and to devise methods and biological molecules to circumvent or alleviate such function. On the other hand, circumstances can be envisioned by which the negative function is desirable. Certain gene products that are highly expressed throughout plant development should be specifically reduced during seed maturation and therefore prevented from entering the food chain. Such an example might be the Bacillus thuringiensis insecticidal protein, which is needed for insect damage protection but not in the seed flour.
  • a DNA construct that encodes a ⁇ - zein which is expressed regardless of the presence of a dominant negative dzrl allele in the genome.
  • the construct comprises a ⁇ -zein coding sequence operably linked to a promoter and to a sequence encoding a modified 3 ⁇ untranslated region (UTR) , the 3 ' UTR being modified so as to be devoid of binding sites for a dzrl negative regulatory protein.
  • UTR 3 ⁇ untranslated region
  • the modified 3 ' UTR is produced by replacing the sequence encoding the dzrl binding site-containing 3 'UTR with a heterologous sequence encoding a 3 ' UTR devoid of those binding sites (e.g., the 3' UTR-encoding sequence from a CaMV 35S gene) .
  • the modified 3' UTR is produced by site-directed mutagenesis of sequences encoding the binding sites, so as to destroy the binding sites without affecting the other regulatory functions of the 3 ' UTR .
  • the DNA construct contains a coding region encoding a ⁇ -zein selected from the group consisting of a 10 kDa zein and an 18 kDa zein.
  • the promoter preferably is a seed-specific promoter, and may be selected from the group consisting of a 27 kDa zein gene promoter, a 27 kDa (02) zein gene promoter, a 10 kDa zein gene promoter and an 18 kDa zein gene promoter.
  • a vector for transforming a plant cell which comprises the DNA construct described above.
  • a plant cell transformed with that vector, and a fertile, transgenic plant regenerated from the transformed cell are also provided.
  • a chimeric gene encoding a 10 kDa zein comprises a 10 kDa zein coding region operably linked at its 5' end to a promoter, and to its 3' end to a heterologous 3' UTR.
  • the promoter is selected from the group consisting of a 27 kDa zein gene promoter, a 27 kDa (02) zein gene promoter, a 10 kDa zein gene promoter and an 18 kDa zein gene promoter.
  • the chimeric gene comprises a 10 kDa zein coding region operably linked to a 27 kDa zein gene promoter (or a 27 kDa (02) promoter) and a CaMV 35S gene 3' UTR.
  • a vector comprising such a chimeric gene is exemplified herein by plasmid pJM2710.
  • a fertile transgenic corn plant which expresses this chimeric gene is also provided.
  • a method of making high methionine corn seeds comprises the steps of (a) producing a fertile transgenic corn plant expressing the DNA constructs or chimeric genes described above; (b) growing the plant; and (c) harvesting seeds from the plant. Because such plants consistently express the 10 kDa zein, which has a high methionine content, seeds produced therefrom will be consistently enriched in methionine, as compared with equivalent non-transgenic plants, or as compared with transgenic plants expressing a ⁇ -zein that is negatively regulated by the dzrl regulatory protein. Moreover, the 10 kDa zein produced in such plants appears to be stabilized in protein bodies .
  • an isolated nucleic acid comprising a 3 ' untranslated region of a 10-kDa zein gene is provided, along with the use of the nucleic acid for dzrl-mediated negative regulation of a coding sequence to which it is operably linked.
  • the 3' UTR comprises SEQ ID N0:1.
  • the coding region codes for a gene product that is undesirable in the seeds (e.g., Bt insecticidal protein).
  • Figure 1 Schematic diagram showing a proposed mechanism by which dzrl could function in an allele- specific manner.
  • dzrl+BSSS53 refers to the dzrl allele carried on the HM phenotype variety BSSS53;
  • dzrl+Mol7 refers to the negative dominant dzrl allele carried on variety Mol7.
  • FIG. 1 Construction of plasmids pJM2710 and pJM2710(O2).
  • the chimeric gene comprises the 5' regulatory sequences of the 27-kDa zein gene (Z27 promoter or Z27 (02) promoter) , the coding region of the 10-kDa zein gene (Z10 coding) and the 3' untranslated region of the CaMV 35S gene (35S poly A) .
  • the chimeric gene is inserted into plasmid pUC119.
  • FIG. 3 Southern blot analysis of transgenic maize plants. Genomic DNA was isolated from leaf tissue and subjected to Southern blot analysis as described in Example 3. Lanes 1-5 represent samples of Basta- resistant plants; lanes 6 and 7 represent the non- transgenic parents; lane 8 is a size marker with a 1.6 kb band and lane 9 shows the restricted plasmid DNA prior to transformation. All transgenic plants show the diagnostic 1.4 kb fragment derived from the bar gene.
  • FIG. 4 Northern blot of RNA isolated from immature endosperm of parental and transgenic plant lines.
  • Lane 1, BSSS53 18 days after pollination (DAP) ; lane 2, Mol7 (18 DAP); lane 3, transgenic line (18 DAP); lane 4, BSSS53 X transgenic line (18 DAP) ; lane 5, Mol7 X transgenic line (15 DAP); lane 6, Mol7 X transgenic line (18 DAP) ; lane 7, Mol7 X transgenic line (21 DAP) ; lane 8, Mol7 X transgenic line (24 DAP) ; lane 9, Mol7 X BSSS53 (15 DAP); lane 10, Mol7 X BSSS53 (18 DAP); lane 11, Mol7 X BSSS53 (21 DAP); lane 12, Mol7 X BSSS53 (24 DAP) but degraded .
  • FIG. 1 Western blot showing accumulation of 10-kDa zein in transgenic and parental maize lines. After regeneration of fertile transgenic plants, they were selfed and grown to maturity. Zeins were extracted from seeds following standard protocols, separated by SDS PAGE, blotted to a filter and analyzed with a zein antibody as described previously (Chaudhuri & Messing, 1994) . Samples were loaded in the following order: lane 1, transgenic parent; lane 2, Mol7; lane 3, BSSS53; lane 4, Mol7 X BSSS53; lane 5, BSSS53 X Mol7; lane 6, Mol7 X transgenic line; lane 7, transgenic line X Mol7.
  • FIG. Histogram showing transcription rates of 10-kDa zein gene driven by 27-kDa (Construct #1) or 27-kDa (02) (Construct #2) promoter.
  • Labeled RNA was hybridized to one of three probes, corresponding to (a) the endogenous 10-kDa zein gene (int 10-kDa) , (b) the chimeric 10-kDa zein gene 3 'UTR transgene (ext 10-kDa) or (c) as a control, the 15 -kDa zein gene (15 -kDa) .
  • FIG. 7 Western blot showing accumulation of 10-kDa zein in parental and transgenic plants.
  • Lane 1 A654 harboring a null mutation of the internal 10-kDa zein gene; lane 2, Mol7; lane 3, BSSS53; lane 4, hybrid parental line used for the transformation; lanes 5 and 6, transgenic plants transformed with a gene controlled by construct #1; lanes 7-9, transgenic plants transformed with a gene controlled by construct #2.
  • Figure 8 Histogram showing results of a feeding trial of infant chickens .
  • Groups of chicks were fed (1) inbred corn without methionine supplement (control) ; or (2) inbred corn with methionine supplement (black bars) ; or (3) transgenic corn (white bars) .
  • Results are expressed as percent weight gain of the test groups (2 and 3) over the control group.
  • isolated nucleic acid refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived.
  • the "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote .
  • An "isolated nucleic acid molecule” may also comprise a cDNA molecule.
  • isolated nucleic acid primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form (the term “substantially pure” is defined below) .
  • isolated protein or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure” form.
  • substantially pure refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like) .
  • immunologically specific refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic molecules.
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single- stranded nucleic acids of non-complementary sequence.
  • promoter region refers to the 5 ' regulatory regions of a gene, including promoters, leader sequences and, optionally, enhancers. This term is used interchangeably with the term “5' regulatory region.”
  • 3'UTR or "3' untranslated region” refers to the transcribed portion of a gene following the stop codon.
  • heterologous 3' UTR refers to a 3 ' UTR from a source other than the 3 ' UTR that occurs naturally in a gene.
  • reporter gene refers to genetic sequences which may be operably linked to a promoter region forming a transgene, such that expression of the reporter gene coding region is regulated by the promoter and expression of the transgene is readily assayed.
  • selectable marker gene refers to a gene product that when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell or plant.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements
  • promoters e.g. promoters, enhancers, and termination elements
  • termination elements e.g. promoters, enhancers, and termination elements
  • DNA construct refers to genetic sequence used to transform plants and generate progeny transgenic plants. These constructs may be administered to plants in a viral or plasmid vector.
  • the biolistic process of transformation is preferred for practice of the present invention.
  • Other methods of delivery such as Agrobacterium T-DNA mediated transformation and transformation using electroporation are also contemplated to be within the scope of the present invention.
  • the transforming DNA may be prepared according to standard protocols such as those set forth in "Current Protocols in Molecular Biology", eds . Frederick M. Ausubel et al . , John Wiley & Sons, 1995.
  • genotype refers to the individual genetic background of each maize variety.
  • the genotype of each variant e.g. BSSS53 in respect to seed methionine levels can be determined by its "HM or high methionine phenotype.”
  • the HM phenotype is recognized by the levels of 10-kDa zein mRNA in immature maize seeds and the 10-kDa zein protein in mature seeds.
  • DNA finger printing methods can be used to correlate the molecular basis of a genotype with its corresponding phenotype. Two different genotypes that map to the same chromosomal location are referred to as "alleles.” To distinguish between different dzrl alleles, the name of each variant is added (e.g dzrl+BSSS53) .
  • a solution has been found to achieve genotype-independent overexpression of the 10-kDa zein gene.
  • this invention modifies the 3 'UTR of a gene, e.g., by replacing it. Therefore, the mRNA can no longer be negatively regulated by trans-acting factors present in many elite lines of corn.
  • novel DNA constructs for transforming plants with a 10-kDa zein gene have been made, which circumvent the function of the dzrl negative dominant allele.
  • Transgenic plants comprising these novel constructs consistently express the 10-kDa transgene, even in the presence of the negative dominant dzrl allele.
  • the lower level of expression of the 10-kDa zein gene in maize variety Mol7 which carries a negative dominant dzrl allele, is due to mRNA accumulation rather than transcription. It has been discovered in accordance with this invention that, unexpectedly, drzl influences the accumulation of the 10- kDa mRNA by presumably interacting with the 3' UTR of the mRNA, rather than the 5' regulatory sequences, as is usually the case, since modification of the 3 'UTR in fertile transgenic corn plants leads to increased levels of 10-kDa zein protein.
  • dzrl factor causes the 10-kDa mRNA to accumulate at different levels
  • dzrl encodes an RNA-binding protein. If this is the case, then negative dominance by dzrl+Mol7 over dzrl+BSSS53 might imply that such a RNA-binding protein dimerizes prior binding to its target site.
  • dzrl and dzsl O are both transcribed during endosperm development .
  • the gene product of dzrl+BSSS53 would dimerize and bind to the 10- kDa zein mRNA, which would lead to increased accumulation of this mRNA.
  • negative dominance indicates that the dzrl gene product possess at least two domains - one for binding to the 10-kDa mRNA and one for the protein- protein interaction leading to dimerization.
  • Negative dominant allele-encoded homodimers, as well as heterodimers carrying at least one negative-dominant encoded subunit, would act as negative regulators of the 10-kDa zein gene, at the post-transcription level, by virtue of their interaction with the 10-kDa zein mRNA.
  • dzrl Since dzrl has not been cloned yet, it is as yet unknown whether it encodes a RNA-binding protein, and its biochemical properties have not been tested.
  • cloning of dzrl is not a prerequisite for manipulating its function. Since we believe that its target is the 10-kDa mRNA, we can modify the primary sequence of the 10-kDa mRNA by modifying the 10-kDa gene. Such a synthetic 10-kDa zein gene is then introduced into the corn genome by DNA transformation methods and tested for its expression in different genotypes including those that carry a negative dominant allele of dzrl . Since the specific site(s) within the 10-kDa mRNA sequence recognized by the dzrl factor was unknown prior to the present invention, we selected to modify the 3 'UTR of the 10-kDa mRNA as our first preference.
  • the 10 kDa zein gene has been completely modified by replacing the 3' UTR (SEQ ID NO:l) with the equivalent region from the CAMV 35S transcript which has been tested in maize protoplasts before (Wu et al . , 1994) .
  • a preferred construct of this type is shown in Figure 2 and as SEQ ID NO: 4 at the end of the specification.
  • the coding region of the 10-kDa zein gene (SEQ ID NO:2) was operably linked to a 5 ' regulatory sequence of a 27-kDa zein gene (an exemplary sequence of which is SEQ ID NO:3), and to the 3' UTR of the CaMV 35S transcript.
  • the chimeric gene was inserted into a plasmid that uses the maize ubiquitin promoter and the nos 3 ' UTR to express the bar gene, the expression of which confers resistance to the herbicide, "Basta" (From et al . , 1990; Gordon- Kam et al . , 1990; Christiansen and Quail, 1996).
  • a plasmid of this type has been used to transform immature embryos of an A188 X B73 hybrid, using the particle bombardment process.
  • A188 demonstrates low expression of its endogenous 10-kDa zein gene, while B73 exhibits moderately low expression of the gene.
  • a non-transformed A188 (paternal) X B73 (maternal) hybrid should show low accumulation of the endogenous 10-kDa zein.
  • Phosphinotricin-resistant calli have been regenerated into fertile plants, which have either been selfed or backcrossed and grown to maturity.
  • Transgenic plants differ in their transgenes in three ways.
  • the 27-kDa zein promoter is known to be a strong seed-specific promoter that is in contrast to the 22 -kDa zein promoter not under the control of the b-zip transcription activator Opaque-2 (02) .
  • the 27-kDa zein promoter has a sequence motif in the same position as the 22 -kDa promoter that resembles the recognition sequence for 02 except for two nucleotides.
  • site- directed mutagenesis we have shown that by repairing these two nucleotides 02 can bind to the 27-kDa promoter, which we call the 27-kDa (02) promoter (Ueda et al . , 1992) .
  • the chimeric 10-kDa zein genes of the present invention enable consistent expression of the 10- kDa zein in any transgenic plant, regardless of its dzrl allelic composition, by virtue of one critical feature: the native 3' UTR (SEQ ID NO:l) has been modified so that is not the target for dzrl regulation. This sequence is modified either by replacing it with another 3 ' UTR or by oligonucleotide site-direct mutagenesis in order to generate chimeric 10-kDa zein genes that are consistently highly expressed in transgenic plants containing them, and progeny thereof. Moreover, transgenic plants expressing these genes produce a predictable and stable amount of 10-kDa zein protein, essentially independent of position effect and transgene copy number.
  • dzrl is known to regulate only dzslO , it may be discovered that the product of this gene also exerts a regulatory effect on other mRNAs, via their 3' UTR target sequences. Accordingly, chimeras of these genes, wherein dzrl targets are modified to be a non-target, can also be constructed, and are expected to exhibit the same consistent levels of expression. Alternatively, chimeras containing the dzrl target can be used to down-regulate gene expression. This will be useful in instances where it is desired that a transgene is expressed in other parts of the plant, but not the one entering the food chain. Such an instance may occur if the transgene is a regulatory gene .
  • Chimeric 10-kDa zein genes of the present invention comprise a 10-kDa coding region, operably linked to native or synthetic 5' regulatory sequences, and modified 3' regulatory region.
  • the coding region may comprise any high-methionine zein encoding sequence.
  • the coding region of the 10-kDa zein described by Anderson Kirihara et al . (1988) is used.
  • the 18 -kDa zein coding region described by Swarup et al . (1995) is used.
  • coding regions from other genes discovered to be regulated by dzrl may be used.
  • any suitable 5 ' regulatory region may be used in the chimeric 10-kDa zein gene.
  • a seed-specific promoter is preferred.
  • the 27-kDa zein promoter is used.
  • the 27- kDa (02) promoter is used.
  • the native 10-kDa promoter is used.
  • Other useful promoters include, but are not limited to maize ubiquitin gene promoters, rice actin promoters, maize Adh 1 promoter, rice or maize tubulin ( Tub A, B or C) promoters, and alfalfa His 3 promoter.
  • the promoter may be an inducible promoter or one that drives constitutive expression of the gene.
  • any suitable, non-native, 3' UTR may be used in the chimeric 10-kDa zein gene.
  • the 3' UTR from the cauliflower mosaic virus 35S gene is used.
  • the native 3' UTR may be used, but it must be modified (e.g., by site directed mutagenesis) such that the dzrl binding sites are removed or replaced, without altering the other regulatory features of the 3' UTR.
  • Transgenic plants can be generated using standard plant transformation methods known to those skilled in the art. These include, but are not limited to, biolistic DNA delivery (i.e., particle bombardment, Agrobacterium vectors, PEG treatment of protoplasts, UV laser microbeam, gemini virus vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions with microbeads coated with the transforming DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like.
  • biolistic DNA delivery i.e., particle bombardment, Agrobacterium vectors, PEG treatment of protoplasts, UV laser microbeam, gemini virus vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions with microbeads coated with the transforming DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like.
  • biolistic DNA delivery i.e., particle bombardment, Agrobacterium vectors,
  • the method of transformation depends upon the plant to be transformed.
  • the biolistic DNA delivery method is useful for nuclear transformation of monocotyledenous plants, such as maize, and is preferred for practice of the present invention. Transformation of maize immature embryos using the biolistic method is described in detail in Example 2.
  • Agrobacterium vectors particularly superbinary vectors such as described by Ishida et al . (Nature Biotechnology 14:745-750, 1996) are used for transformation of plant nuclei.
  • the chimeric gene is linked to a nuclear drug or herbicide resistance marker, such as hygromycin resistance or "Basta" resistance.
  • Biolistic transformation of plant nuclei is accomplished according to the following general procedure:
  • the gene is inserted into a selected vector
  • transgenic maize plants are produced that express high quantities of the 10-kDa zein seed storage protein.
  • This protein contains a high proportion of methionine codons (23%) .
  • Overexpression of this protein in maize seeds increases the capture of free methionine during plant maturation, which otherwise would be lost.
  • Transgenic plants of the present invention are superior to natural high-methionine variants, such as BSSS53 because they consistently express the 10-kDa transgene regardless of the dzrl allelic composition of the variety.
  • combinations of natural HM variants with other germplasms produces a suppression of the high methionine phenotype, rendering the natural variant unreliable for use in commercial corn.
  • the fertile, chimeric 10-kDa zein transgenic plants of this invention provide a distinct agronomic advantage over HM variants presently available.
  • this invention also provides a 3 ' negative regulatory target of the dzrl gene product, as exemplified by SEQ ID NO:l. This sequence is expected to be useful for influencing gene expression by negative dominance or once the dzrl gene is cloned by modified dzrl factors.
  • One particularly attractive application for the 3 ' negative regulatory target of the dzrl gene product is seed-specific suppression of gene expression, where such suppression would be considered desirable.
  • insect-resistant transgenic plants are currently being engineered by transforming the plants with the Bt gene, encoding the Bacillus thuringiensis insect toxin.
  • Such a construct would then be subject to dominant negative regulation by dzrl in seeds of the transgenic plants.
  • the mRNA encoding the Bt protein would be degraded in the endosperm (but not in the other plant parts) and the seeds would remain largely free of Bt toxin.
  • EXAMPLE 1 Construction of Plasmid pJM2710 The 27-kDa zein promoter was made by cloning of the 1103 bp Pvul fragment of the 5' flanking sequence of the 27-kDa zein genomic clone, stretching from position - 1042 to +61 in respect to the transcriptional start site of the gene as described before (Ueda, T., Messing, J. 1991, Ueda, T. et al , 1994) .
  • the 10-kDa zein coding region was made by cutting the 10-kDa genomic clone plOH3 from maize inbred line BSSS53 (Anderson Kirihara, J., Petri, J.
  • This fragment was inserted into the pFF plasmid together with the 203 bp CaMV 35S 3' polyA sequence (Timmermans, M. , Maliga P., Vieira J. , Messing J. , 1990, Journal of Biotechnology) .
  • the resulting plasmid pJM2710 contains three restriction fragments flanked by Hindlll sites: the 27-kDa promoter (1103 bp) , the 10-kDa coding region (465 bp) , and the 35S 3'UTR (203 bp) .
  • the particle samples were coated with 50 Fl of a (50mg/ml) 1 mm gold particle suspension containing 5-10 Fg purified plasmid DNA and 20 Fl of 0.1 M freshly prepared spermidine and 50 Fl of 2.5 M CaCl .
  • the DNA-coated particles were precipitated in ethanol, then washed three times and finally resuspended in 30 Fl of anhydrous ethanol .
  • Six Fl of a particle suspension were loaded on a macro carrier.
  • the membrane rupture pressure was set at 1,300 psi and a 15 mm petri dish with 20 to 30 embryos were put into the chamber 9 cm from the retaining screen and shot twice .
  • the embryos were transferred to fresh N6 medium with 3 Fg/mL Bialaphos for selection and then kept subcultured every two weeks. After two to three month of selection, resistant calli were grown either for further propagation or regeneration.
  • Plants were regenerated by placing the resistant calli on regeneration medium (MS or N6 , with 2,4-D) under light condition. Multiple plantlets were regenerated from each independent transgenic callus and either selfed, backcrossed to their nontransgenic parents, or outcrossed to another inbred line.
  • MS or N6 regeneration medium
  • 2,4-D 2,4-D
  • Second generation analysis of the transgene was performed by following the herbicide tolerance conferred by the selectable marker gene. Expression of this gene was analyzed by applying the herbicide Basta TX (2% v/v, with 0.1% Triton X-100) on small leaf sections. Herbicide resistance was scored six days after application. The resistant plants have leaves as vigorous as untreated plants while the susceptible plants show yellowish and friable leaves. Segregation of transgenic and nontransgenic plants occurs in an outcross with A654 at about 1:1 and in a sib-cross at about 3:1. EXAMPLE 6 Elimination of Negative Regulation
  • Figure 5 shows the relative level of 10-kDa protein produced. As can be seen, the transgenic parent exhibited high level expression of the 10-kDa protein, while Mol7, which carries the negative dominant dzrl allele produced significantly less 10-kDa zein protein.
  • BSSS53 which lacks the negative dominant allele and is a natural overexpresser of the 10-kDa zein gene, exhibited 10-kDa zein protein production similar to the transgenic parent.
  • the hybrid line when Mol7 was used as a female parent with BSSS53, exhibited a reduction of expression to the level observed for the Mol7 line.
  • BSSSS53 was used as the female parent, the negative dominant effect was not seen because of genomic imprinting (Chaudhuri and Messing, 1994) and protein levels were comparable to those displayed by the transgenic parent and the BSSS53 line.
  • 10-kDa protein expression levels were high regardless of the direction of the cross, as predicted from the Northern blot analysis described above.
  • Transcription run-on assays were performed with two constructs that differed by two nucleotides in position -225 and -226, where two T's were replaced by two C's (construct #1 bold and underlined) .
  • the sequence TCCACAGTAGA (part of construct #2, SEQ ID NO: 6) is the canonical binding site for Opaque-2 (02) .
  • the upstream TGTAAG motif (also bolded and underlined) is the so called prolamin box (PB) which is present in all zein promoters and believed to be a cis-acting site for a general transcription factor (Ueda et al . , 1994).
  • PB prolamin box
  • Construct #1 (SEQ ID NO: 5) (27-kDa zein promoter region, -349 to -217) :
  • Construct #2 (SEQ ID NO: 6) (27-kDa (02) zein promoter region, -248 to -217) : 5 ' -TCAAGCTAAATCTAATTCGTTCCACGTAGAT-3 ' Transcription rates of transgenic plants that were either selfed or crossed with Mol7 as the female were measured at 18 days after pollination when zein gene expression is very high. Nuclei were isolated and labeled as described previously (Cruz-Alvarez et al . , 1991; Schickler et al . , 1993) .
  • Results are shown in Figure 7.
  • A654 a line carrying a null mutation of the internal 10-kDa zein gene, showed no 10-kDa zein production.
  • Mol7 which carries the negative dominant dzrl allele
  • the hybrid parental line that was used for the transformation displayed minimal 10-kDa zein production.
  • the BSSS53 that lacks ' the negative dominant allele and is a natural overexpresser of the 10-kDa zein gene produced a significant amount of the protein, as did transgenic plants transformed with genes controlled by either construct #1 or construct #2.

Abstract

L'invention concerne de nouvelles structures d'ADN codant pour des protéines de zéine à méthionine élevée, dont l'expression est négativement régulée par la protéine de régulation dzr1. La structure de l'invention comprend une région de codage d'δ-zéine liée de manière fonctionnelle à un promoteur, et à une région 3' non traduite (UTR) modifiée de façon à être dépourvue de tout site de liaison pour la protéine de régulation dzr1. La totalité de 3' URT est, de préférence, remplacée par une séquence hétérologue qui ne contient aucun site de liaison dzr1. L'invention concerne également des plants de maïs transgénique comprenant les structures d'ADN de l'invention. Ces plants produisent uniformément des graines de maïs à méthionine élevée.
PCT/US1999/020308 1998-08-27 1999-08-25 Compositions et procedes permettant de produire une expression genique de niveau eleve propre a une graine dans le mais WO2000012681A1 (fr)

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EP99945499A EP1108009A4 (fr) 1998-08-27 1999-08-25 Compositions et procedes permettant de produire une expression genique de niveau eleve propre a une graine dans le mais
AU58089/99A AU5808999A (en) 1998-08-27 1999-08-25 Compositions and methods for producing high-level seed-specific gene expression in corn
US09/763,329 US6849779B1 (en) 1998-08-27 1999-08-25 Method for producing high methionine corn seeds

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CHAUDHURI ET AL.: "Allele-Specific Parental Imprinting of dzr1, a Posttranscriptional Regulator of Zein Accumulation", PROC. NATL. ACAD. SCI. USA, vol. 91, May 1994 (1994-05-01), pages 4867 - 4871, XP002922755 *
CRUZ-ALVAREZ ET AL.: "Post-transcriptional Regulation of Methionine Content in Maize Kernels", MOL. GEN. GENET., vol. 225, 1991, pages 331 - 339, XP002922756 *
KIRIHARA ET AL.: "Isolation and Sequence of a Gene Encoding a Methionine-Rich 10-kDa Zein Protein from Maize", GENE, vol. 71, 1988, pages 359 - 370, XP002070068 *
PIETRZAK ET AL.: "Expression in Plants of Two Bacterial Antibiotic Resistance Genes after Protoplast Transformation with a New Plant Expression Vector", NUCL. ACIDS RES., vol. 14, no. 14, 1986, pages 5857 - 5868, XP002092881 *
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SWARUP ET AL.: "Determinants of the High-methionine Trait in Wild and Exotic Germplasm May have Escaped Selection During Early Cultivation of Maize", THE PLANT JOURNAL, vol. 8, no. 3, 1995, pages 359 - 368, XP002922757 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9226515B2 (en) 2004-02-03 2016-01-05 Cargill, Incorporated Protein concentrate and an aqueous stream containing water-soluble carbohydrates
US10154679B2 (en) 2004-02-03 2018-12-18 Cargill, Incorporated Protein concentrate and an aqueous stream containing water-soluble carbohydrates
WO2005086667A2 (fr) 2004-02-27 2005-09-22 The Dow Chemical Company Production de peptides dans des cellules de plantes avec un rendement eleve
EP3388521A1 (fr) 2004-02-27 2018-10-17 Dow AgroSciences LLC Production de peptides dans des cellules de plantes avec un rendement eleve

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