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WO1993008682A1 - Procedes d'obtention de graines de mais a haute teneur en methionine, et leurs utilisations - Google Patents

Procedes d'obtention de graines de mais a haute teneur en methionine, et leurs utilisations Download PDF

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Publication number
WO1993008682A1
WO1993008682A1 PCT/US1992/009433 US9209433W WO9308682A1 WO 1993008682 A1 WO1993008682 A1 WO 1993008682A1 US 9209433 W US9209433 W US 9209433W WO 9308682 A1 WO9308682 A1 WO 9308682A1
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Prior art keywords
maize
zein
nucleic acid
methionine
acid molecule
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PCT/US1992/009433
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English (en)
Inventor
Joachim Messing
Hans Fisher
Takashi Ueda
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State University Of New Jersey - Rutgers
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Publication of WO1993008682A1 publication Critical patent/WO1993008682A1/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
    • C12N15/8242—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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4684—Zea mays [maize]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • 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

Definitions

  • Maize fzea mays is a nutritious food substance commonly used for feeding livestock. However, it is deficient with respect to certain nutrients, one of which is the
  • Mature maize seeds contain very low levels of free amino acids and, therefore, most of their amino acids are 30 derived from.seeds as hydrolysates, either by germination to feed the growing seedling or by digestion with respect to human and livestock consumption (3).
  • proteins serve as a storage form of amino acids, and the
  • 35 genes encoding these proteins exercise control over the total balance of amino acids by their primary structure and by the level of their accumulation during seed development.
  • storage proteins As a sink for assimilated nitrogen produced during the photosynthetic period of the life cycle of the plant.
  • the different locations of synthesis and storage require an interesting signalling pathway and transport system between leaves, where photosynthesis occurs, and flowers, where storage proteins are synthesized after fertilization is successful.
  • the differential expression of storage proteins amplifies any imbalances of amino acid accumulation (for review see (4)).
  • An interesting example of such quantitative variability is the expression of the high-methionine storage protein gene of maize.
  • the majority of storage proteins in maize are extracted with ethanol and, when their amino acid composition is determined, one can readily recognize that certain essential amino acids are underrepresented (1, 2) . Tryptophan, lysine, and methionine are low, while leucine is very high.
  • the amino acid proline is also very abundant and, besides glutamine, contributes to the general name prolamine, a group also referred to as zeins in maize.
  • the loci operate by affecting the accumulation of a subset of storage proteins.
  • the best known regulatory locus is probably opaque-2 which affects lysine content (1) .
  • the opaque-2 gene has been cloned and its product, belonging to the leucine zipper family of transcription factors, has been shown to control transcription of a certain subset of zein genes by binding to the promoter region (5) .
  • a regulatory locus that acts on the amino acid balance without noticeable reduction of storage protein synthesis is Zprl0/(22) (7).
  • This locus causes the increase of the level of a minor zein protein that is rich in methionine residues.
  • the methionine accumulates mainly into two zein gene products—one of 15kDa and one of lOkDa relative molecular weight. Both proteins are encoded by single genes and are present in all standard inbreds looked at so far.
  • the 15kDa zein gene contains about 12% methionine codons, while the lOkDa zein gene contains about 23% methionine codons.
  • the regulatory gene Zprl0/(22) selectively causes the increased accumulation of the lOkDa zein protein during endosperm development.
  • Zeins are the alcohol-soluble fraction of storage proteins in maize .Zea mays.. They constitute more than 50% of the total endosperm proteins at seed maturity.
  • Zein ⁇ consist of a group of heterologous hydr ⁇ phobic proteins, which are classified according to their molecular weight, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) into subclasses with Mr of 27, 22, 19, 16, 15, and lOkDa (8, 9). Based on structural similarities, they are also classified into ⁇ -(22 and 19 kDa), ⁇ -(15kDa), (16 and 27kDa) , and f-(lOkDa) zeins (10).
  • Zeins are encoded by a complex ultigene family of over 100 gene members (Il ⁇ ls) and regulated in a tissue- and developmental stage- specific manner. Their expression is confined to triploid (3n) endosperm tissue and starts at a specific stage [around 12 days after pollination (DAP) ] during endosperm development (14) . Furthermore, the onset of elevated zein gene expression coincides with the genome amplification process starting at this particular stage in endosperm development (15) .
  • This invention provides a method of obtaining corn seeds or kernels having a methionine content of greater than 1.39 percent by weight of the total amino acid composition of the corn seeds or kernels. This invention also provides a method for providing greater than 36 percent of the methionine nutritional requirements of poultry * This invention further provides a method of improving the growth performance of poultry.
  • This invention further provides a corn plant resulting from a genetic cross comprising high zein protein-containing seeds. This invention further provides a recombinant nucleic acid molecule consisting essentially of (1) a sequence encoding a zein protein, (2) a sequence which when present in the molecule is capable of functioning as a promoter of transcription of the sequence encoding the zein protein, and (3) an exogenous sequence capable of
  • RNA samples were fractionated in a formaldehyde-agarose gel, transferred onto a filter, and hybridized to the zein-specific probes as indicated: 5 ⁇ g of total RNA from each maize tissue was analyzed (left) ; 20 ⁇ g of total RNA from maize tissue cultures was analyzed (right) .
  • RNA isolated from the maize tissues indicated, 2 ⁇ g was blotted on a filter in a slot-blot apparatus.
  • The-filter was hybridized to probes specific for the 10-, 15-, and 27-kDa zein genes and for 17S rDNA. Hybridization intensity on the autoradiogram was quantitated by densitometry.
  • SI nuclease mapping of the 10- and 27-kDa zein genes Diagrammatic representation of the 5' coding regions of the 10- and 27-kDa zein genes. The coding sequences of the two zein genes are indicated by solid boxes. The probes used for the 10- and 27-kDa zein genes are the 188-bp Avall-Banl fragment and the 343-bp Rsal-Hpall fragment, respectively. The 5' ends of the noncoding strands of these fragments were radiolabeled with 7 - 32 P- ATP. The sizes of the protected bands are also shown. The trinucleotide ATC found at the transcription initiation site for the two zein genes is indicated together with TATAA sequences and the ATG initiation codon.
  • SI nuclease mapping of the 10- and 27-kDa zein genes were fractionated on 6% polyacrylamide gels containing 8 M urea. Maxa and Gilbert (16) sequencing reactions of the probes were run along with the SI nuclease reaction products (not shown) . Higher counts of the SI nuclease reaction products for the RNA samples from endosperm culture were loaded in the gels for the comparison. The nucleotide sequences of the coding strands in the region of the protected bands and the location of the bands (arrows) are shown at the left margin of each figure.
  • pFFCAT contains the CAT gene coding sequence fused to the CaMV35S promoter with a duplicated enhancer and the CaMV35S terminator in pFF19 (17).
  • the pZ10(-1076/+42)CAT and pZ27(-1042/+61) contain l.l-kb 5 « flanking sequences of the 10- and 27-kDa zein genes, respectively. End points with respect to the cap sites are designated in parentheses.
  • Promoter-less CAT construct ⁇ -CAT was used as a negative control.
  • pFFGUS (17) containing the GUS coding sequences fused to the CaMV35S gene promoter and terminator, was cotransfected with each CAT construct to serve as an internal standard for electroporation.
  • Transient expression of chimeric genes in endosperm protoplasts CAT enzyme assay of transfected endosperm protoplasts. Lane 1, no plasmid DNA added; lane 2, 150 ⁇ g of promoter-less ⁇ -CAT added; lane 3, 150 ⁇ g of pZ10(- 1076/+42)CAT added; lane 4, 150 ⁇ g of pZ27(-1042/+61)CAT added; lane 5, 25 ⁇ g pFFCAT added. For each assay, 100 ⁇ g of protein extract was used.
  • RNA isolated from maize plant tissue and cultured cells.
  • Total RNA was isolated from endosperm (16 DAP) , root and leaf tissue of an A636 maize plant, as well as from A636 endosperm and leaf tissue- derived BMS cultures.
  • Five micrograms of RNA sample from each tissue were fractionated in a formaldehyde-agarose gel, transferred onto a filter, and hybridized to a ⁇ P- labelled 02 cDNA probe. It is demonstrated here that the endosperm-specific expression of the 02 gene is maintained in the A636 endosperm culture.
  • A A diagrammatic representation of chimeric constructs used to test the effect of the 02 overexpression on the two zein promoters; ⁇ -GUS, the promoter-less GUS reporte equipped with the CaMV35S terminator sequence (35ST) ; pZ 4GUS, the GUS-35ST placed under the regulation of th 0.9-kb 22-kDa Z-4 zein promoter; pZ27GUS, the GUS-35S sequence placed under the regulation of the 1.1-kb 27-kD zein promoter.
  • ⁇ -GUS the promoter-less GUS reporte equipped with the CaMV35S terminator sequence (35ST)
  • pZ 4GUS the GUS-35ST placed under the regulation of th 0.9-kb 22-kDa Z-4 zein promoter
  • pZ27GUS the GUS-35S sequence placed under the regulation of the 1.1-kb 27-kD zein promoter.
  • FIG. 1 A diagrammatic representation of the 02 overexpressio construgtg which aCS cotransfected with the zei promoter-GUS constructs shown in panel A; pFF02+, a ful length 02 cDNA placed under the regulation of the CaMV35 promoter with duplicated enhancer elements (pCaMV35S+) and its terminator (35ST) ; pFF02m, an internal deletio clone of the 02 cDNA (lacking the sequence encoding part of the 02 protein including the bZip domain) place under the regulation of the pCaMV35S+ and the 35ST.
  • pFF02+ a ful length 02 cDNA placed under the regulation of the CaMV35 promoter with duplicated enhancer elements (pCaMV35S+) and its terminator (35ST)
  • pFF02m an internal deletio clone of the 02 cDNA (lacking the sequence encoding part of the 02 protein including the bZip domain)
  • the non-specific GUS activit derived from the ⁇ -GUS construct was subtracted fro those derived from zein promoters for eac cotransfeetion.
  • ABA abscisic acid
  • Opaque-2 proteins it is also found that ABA can differentially regulate promoter function of zein genes.
  • exogenously added ABA 50 ⁇ M
  • the promoter activity of the CaMV35S promoter is not affected by ABA. It is speculated here that different classes of endogenous zein genes may respond differentially to the endogenous ABA level in the kernel which is known to increase during seed maturation.
  • lane 1 CAT enzyme purified from E. coli; lanes 2 and 3, no plasmid controls; lanes 4 and 5, promoter-less ⁇ -CAT; lanes 6 and 7, pZ27(2.0)CAT consisting of a CAT reporter placed under the regulation of the 2.0-kb 27-kDa zein promoter and CaMV35S terminator (35ST) ; pZ10(l.l)CAT consisting of the chimeric CAT reporter gene placed under the regulation of the 1.1-kb 10-kDa zein promoter; lanes 10 and 11, pFFCAT consisting of the chimeric CAT reporter gene placed under the regulation of the CaMV35S promoter with duplicated enhancer. Endosperm protoplasts transfected with each construct were cultured for two days in the absence (-) or presence (+) of 50 ⁇ M ABA before enzymatic assays were per ormed.
  • the plasmid pUMSOlO was deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession No. 68644.
  • This invention provides a method of obtaining corn seeds or kernels having a methionine content of greater than 1.39 percent by weight of the total amino acid composition of the corn seeds or kernels, which comprises crossing a paternal inbred corn line containing the Zprl0/(22) locus with a maternal inbred corn line lacking the Zprl0(22) locus and selecting for Fl hybrid seeds containing methionine at greater than 1.39 percent by weight of the total amino acid composition of the Fl hybrid seeds or kernels.
  • the paternal inbred corn line may be BSSS-53.
  • the maternal inbred corn line may be M017, 23 or 22.
  • This invention also provides a method for providing greater than 36 percent of the methionine nutritional requirements of poultry which comprises feeding the poultry corn having a methionine content of at least 1.39 percent by weight of the total amino acid composition of the corn.
  • the corn may have a methionine content of at least 3.8 percent.
  • the poultry may be chickens.
  • the corn may be in the form of seeds or kernels, or in the form of cornmeal.
  • This invention also provides a method of improving the growth performance of poultry which comprises feeding the poultry corn having a methionine content of at least 3.8 percent by weight of the total amino acid composition of the corn.
  • This invention further provides a corn plant resulting from a genetic cross comprising high zein protein- containing seeds having a methionine content of at least 1.39 percent by weight of the total amino acid composition of the seeds.
  • the seeds may have a methionine content of at least 3.8 percent.
  • the genetic cross may be a reciprocal cross between a ZprlO/(22)-containing maize inbred and a normal female maize inbred lacking ZprlO/(22) .
  • the ZprlO/(22)-containing inbred may be BSSS- 53.
  • the normal female maize inbred may be M017, 23 or 22.
  • This invention further provides a recombinant nucleic acid molecule consisting essentially of (1) a sequence encoding a zein protein, (2) a sequence which when present in the molecule is capable of functioning as a promoter of transcription of the sequence encoding the zein protein, and (3) an exogenous sequence capable of
  • an "exogenous sequence n means a nucleic acid sequence, from maize or any other source, which would not naturally be situated next to the zein protein-encoding sequence of the subject invention as it is so situated in the recombinant nucleic acid molecule of the subject invention.
  • One example of the recombinant nucleic acid molecule of the subject invention is a molecule having these three distinct, non-overlapping sequences: (1) a sequence encoding a zein protein; (2) a sequence which is capable of functioning as a promoter of transcription of the sequence encoding the zein protein, and (3) an exogenous sequence capable of (a) enhancing the functioning of the promoter sequence, (b) stabilizing the transcription product of the zein protein-encoding sequence, or (c) enhancing the translation of the transcription product of the zein protein-encoding sequence.
  • nucleic acid molecule of the subject invention is a molecule having these two distinct, non- overlapping sequences: (1) a sequence encoding a zein protein; and (2) a sequence which is capable of functioning as a promoter of transcription of the sequence encoding the zein protein, and which contains an exogenous sequence capable of (a) enhancing the functioning of the promoter sequence, (b) stabilizing the transcription product of the zein protein-encoding sequence, or (c) enhancing the translation of the transcription product of the zein protein-encoding sequence.
  • the exogenous sequence may contain a portion of, or all of, the sequence capable of functioning as a promoter.
  • the sequence encoding the zein protein may a naturally occurring zein gene.
  • the naturally occurri zein gene may be a maize zein gene, and this maize zei gene may be a high-methionine maize zein gene.
  • the hig methionine maize zein gene may be a lOkDa high-methioni maize zein gene.
  • the sequence encoding the 10-kDa high methionine maiz zein gene may be obtained by cleaving it from the plasmi pUM5010 using a suitable restriction enzyme.
  • the exogenous sequence may comprise an 02 binding region of a maize 22kDa promoter.
  • the exogenou sequence may be a multimer of 02-binding regions of maiz 22kDa promoters. Such multimers show an enhancing effec superior to that of monomers.
  • the multimer of 02-bindin regions of maize 22kD promoters may comprise 5 copies o the 02-binding region.
  • the recombinant molecule of th subject invention may be the molecule designated pUMSOlO
  • the 02-binding region may be obtained by cleaving it fro pUM5010 using a suitable restriction enzyme.
  • the exogenous sequence may comprise an AB regulatory element.
  • the exogenous sequence may be multimer of ABA regulatory elements. Such multimers sho an enhancing effect superior to that of monomers.
  • Th multimer of ABA regulatory elements may comprise 5 copies of the ABA regulatory element.
  • the exogenous sequence may comprise the 3 1 region of the B gene of the 27 Da maize gene.
  • the 3' region of the B gene of the 27kDa maize gene may be fused to the 3• end of the sequence encoding the zein protein.
  • This invention further provides a method of increasing the concentration of zein protein in a plant, which comprises treating the plant so as to incorporate the nucleic acid molecule of the subject invention into the genome of the plant.
  • the plant may be maize, rice, soybean, alfalfa, barley or wheat.
  • the incorporation of the nucleic acid molecule may comprise microinjecting the molecule into cells of the plant.
  • the incorporation of the nucleic acid molecule may comprise iring the molecule with a particle gun into cells of the plant.
  • the incorporation of the nucleic acid molecule may comprise contacting embryonic culture cells of the plant with the molecule.
  • This invention further provides a genetically engineered corn plant having high zein protein-containing seeds and a methionine content of at least 1.39 percent by weight of the total, amino acids present in the seeds comprising the recombinant nucleic acid of the subject invention.
  • the methionine content of the high zein protein- containing seeds may be at least 3.8 percent by weight of the total amino acids present in the seeds.
  • This invention further provides a method of determining whether an exogenous nucleic acid molecule will be expressed in maize endosperm tissue which comprises introducing the exogenous nucleic acid molecule into cultured maize endosperm protoplasts suspended in a suitable buffer; culturing the resulting maize endosperm protoplasts containing the exogenous nucleic acid molecule; and detecting expression of the nucleic acid molecule by the maize endosperm protoplasts so as to thereby determine whether the nucleic acid molecule will be expressed in maize endosperm tissue.
  • the exogenous nucleic acid molecule may be the recombinant nucleic acid molecule of the subject invention.
  • the introduction of the exogenous nucleic acid molecule may comprise performing electroporation on the protoplasts in the presence of the exogenous nucleic acid molecule.
  • the detection of expression may comprise isolating RNA from the cultured protoplasts containing the nucleic acid molecule; and determining the presence of RNA transcribed from the nucleic acid molecule in the RNA so isolated.
  • the detection of expression may comprise performing an enzyme assay on cultured protoplasts containing the nucleic acid molecule, wherein the nucleic acid molecule encodes an enzyme having detectable activity, and detecting this activity of the enzyme.
  • the enzyme may be chloramphenicol acetyltranferase (CAT) or ⁇ -glucuronidase (GUS) .
  • Thi ⁇ invention further provides a purified antibody specific for a maize high-methionine protein.
  • the antibody of the subject invention may be a monoclonal antibody, a murine antibody or a rabbit antibody.
  • the maize high- methionine protein may be the maize lOkDa high-methionine protein.
  • this invention provides a method of determining the level of high-methionine protein produced by a maize strain which comprises preparing a protein-containing sample from the maize strain; contacting the sample with the antibody of the subject invention under conditions such that the antibody complexes with any high-methionine protein present in the sample for which the antibody is specific; and determining the amount of high-methionine protein present in any resulting complex.
  • the inbred BSSS-53 (Maize stock Center, University of Illinois, Urbana) containing the ZprlO/(22) factor on chromosome 4 that influences the levels of the high methionine lOkDa zein protein in maize encoded by the Zpsl0/(22) locus on chromosome 9 was crossed reciprocally with a line that lacks the ZprlO/(22) product.
  • the amino acid composition of the prolamine fraction from progeny seeds of both reciprocal crosses and self-fed parents were determined.
  • the level of protein-bound methionine in the two hybrids shows a maternal influence rather than a simple dosage effect as one would expect for a nuclear endosperm gene.
  • Mature seeds contain very low levels of free amino acids and, therefore, most of their amino acids are derived from seeds as hydrolysates either by germination to feed the growing seedling or by digestion with respect to human and livestock consumption (3) .
  • proteins serve as a storage form of amino acids, and the g ⁇ ne ⁇ encoding these proteins exercise control over the total balance of amino acids by their primary structure and by the level of their accumulation during seed development.
  • storage proteins can think about storage proteins as a sink for assimilated nitrogen produced during the photosynthetic period of the life cycle of the plant.
  • the majority of storage proteins in maize are extracted with ethanol and, when their amino acid composition is determined, one can readily recognize that certain essential amino acids are underrepresented (1, 2). Tryptophan, lysine, and methionine are low, while leucine is very high.
  • the amino acid proline is also very abundant and, besides glut mine, contributes to the general name prolamine, a group also referred to as zeins in maize.
  • the loci operate by affecting the accumulation of a subset of storage proteins.
  • the best known regulatory locus is probably opaque-2 which affects lysine content (1). Although it was first though that this regulation was accomplished by increasing the accumulation of lysine-rich proteins, it turned out that the primary effect was the failure to express lysine-poor proteins.
  • the opaque-2 gene has been cloned and its product, belonging to the leucine zipper family of transcription factors, has been shown to control transcription of a certain subset of zein genes by binding to .the promoter region (5) .
  • a regulatory locus that acts on the amino acid balance without noticeable reduction of storage protein synthesis is ZprlO/(22) (7). This locus causes the increase of the level of a minor zein protein that is rich in methionine r ⁇ sidues.
  • the methionine accumulates mainly into t zein gene products, one of 15kDa and one of lOk relative molecular weight. Both proteins are encoded single genes and are present in all standard inbre looked at so far.
  • the 15 kDa zein gene contains abo
  • ZprlO/(22) selectively causes the increas accumulation of the lOkDa zein protein during endospe development.
  • BSSS-53 was used as a donor for ZprlO/(22) which has bee shown to be a single Mendelian trait located on the shor arm of chromosome 4 (7) and which is absent in inbre lines like M017 and W23 (Maize Stock Center, Universit of Illinois, Urbana) which were used as recipients Therefore, these recipients were treated as zprl0/(22) All inbreds used contain the locus expressing the lOkD zein gene.
  • the chicks were fed a highly methionine-deficient diet that derived its protein principally from isolated soy protein. This protein and the composition of the remainder of the diet have been previously shown to be deficient only in methionine (24-26) .
  • the National Research Council stated that the methionine requirement for chicks of the type used is 0.50% of the diet (27), and the control diet with zprlO/(22)/ZprlO/(22) corn provided only 0.18%.
  • the high-methionine corn diet provided double that amount, or 0.38%.
  • methionine in maize seeds appears to be a) variable among inbred lines, b) dependent on genetic factors that segregate as Mendelian factors, and c) unusually high in the inbred BSSS-53 because of the presence of at least two genetic factors in the same inbred.
  • prolamine fraction of mature seeds was extractedwith ethanol as described previously (22) ; b Amino acids are listed in the order of their abundance; only primary amines were determined which do not include proline; essential amino acids are underlined Z*ZprlO/(22) ; the methionine values are highlighted in bold. c Z»ZprlO/(22) the female * parent is contributing two doses to the triploid endosperm. *The amino a A Z%lcomposition of the prolamine faction was carried out by liquid phase hydrolysis as described by Metzler, et al. (23) and is expressed in percent of total amino acids. "Not detectable. b.
  • corn meal was used from the two reciprocal crosses that show the five-fold difference in methionine levels to set up two different feed rations (Materials and Methods, Part I).
  • Table II a soybean/corn composition was chosen that usually is supplemented with free methionine. Since it has been shown previously that this diet is complete except for methionine (24-26), the corn meal from the two reciprocal crosses was the only variable for the relative amounts of methionine. Even if other amino acid levels in the two corn meals differed, their margin had already been adequately covered by the soybean protein.
  • 'Peterson's Arbor Acre male chicks were used in a 14 day feeding trial starting with one-day-old animals in duplicate groups of five per treatment; 'the composition is expressed in percentage of the total diet; c the soy protein mixture contains 1.0 methionine and 0.6 cystine per 16% nitrogen; d corn meal was derived either from the ZprlO/(22)/ZprlO/(22)/+ or +/+/ZprlO(22) hybrids as described in Table I.
  • Body weight was determined in g after 14 days of growth starting with one-day-old-animals ranging in weight from 35-40 g; b Z-ZprlO/(22) ; the female parent is contributing two doses to the triploid endosperm; c average weight is given in g for each group of five chicks and the standard deviation for each average weight is given in parentheses; d average amount of food consumed is given in g for each group; the efficiency factor is the proportion of g weight gained per g of food consumed.
  • BSSS-53 as a source of lOkDa zein antigen and to prepare specific antibodies to the Zpsl0/(22) product because this inbred is likely to be devoid of lOkDa "filler proteins". If the synthesis of other zein proteins of lOkDa relative molecular weight with different methionine content can be derepressed in the appropriate genetic background, they could be further characterized by cDNA cloning. With respect to such differential expression of lOkDa zeins, it is interesting that earlier findings of Zpsl ⁇ /(22) mRNA levels also did not reflect a simple dosage pattern (28) .
  • DNA binding proteins can protect the DNA from methylation (39) , it is quite conceivable to think of DNA methylation as a footprint of coated DNA. Conversely, once methylation has occurred, the binding specificity of proteins to DNA may change in turn. If this is true, the sequence-specific DNA binding proteins have to be replenished after each mitosis and their binding races against the methylation process.
  • the high methionine phenotype is also an endosperm trait, and therefore subject to the same ambiguity between imprinting and the threshold potential of two copies introduced by the female gamete.
  • the unusually high levels of methionine in the hybrid with the maternal contribution of ZprlO/(22) are reminiscent of the general effect on heterosis observed in hybrid corn.
  • BSSS-53 the high methionine inbred line
  • ZprlO(22) because it regulates the expression of the lOkDa zein locus which is also called Zpsl0(22). Since these two loci are on different chromosomes and segregate as single Mendelian factors, ZprlO(22) encodes a product that acts in trans (7) .
  • RNA Overexpression of the RNA on the other hand could be regulated either on the transcriptional or post- transcriptional level. However, using nuclear run-off transcription experiments we found that post-tranmcriptional regulation is required for the increased steady state lOkDa RNA levels (28) .
  • pairs of the cis-acting sites of the mosaic gene ar matched with the corresponding trans-acting factors tha positively enhances lOkDa zein gene expression.
  • the term "pair” means a cis-acting element an its corresponding trans-acting factor. Except for on case, these relevant trans-acting factors are naturall produced during endosperm development of all commo inbreds. In these cases only the cis acting sites hav to be engineered.
  • the first pair is the opaque-2 gene product present i all elite lines and its cis-acting sites. Absence of th opaque-2 product, which is a leucine-zipper type of DN binding protein, causes the elimination of th transcription of a specific subset of zein genes, othe zein genes that lack the "opaque-2 box" are not affected Recently, it has been shown in a homologous expressio system (43) that introduction of such an "opaque-2 box into a gene, where it usually is absent, leads to specific increase in gene expression when the opaque- gene is expressed on a second plasmid. Furthermore, thi effect is more dramatic if tandem copies of th restriction fragment containing the "opaque-2 box" ar placed into the promoter region.
  • the trans-acting opaque-2 fails to sho increased expression in our homologous expression system
  • the 10 Da zein gene has a the same distance from the transcriptional start site sequence that differs from the canonical opaque-2 bindin site. Therefore, the lOkDa promoter also does not respond to the trans-acting opaque protein in the homologous expression system, which is in agreement with the observation that opaque-2 variants are not affected in their lOkDa zein levels.
  • a fivemer of the "opaque-2 box" into the right position of the promoter of the lOkDa zein gene should render the lOkDa promoter sensitive to opaque-2 regulation. This can be tested in the homologous expression system and should lead to enhanced transcription of lOkDa zein message in transgenic plants.
  • the second pair is ABA (abscisic acid) and an ABA regulatory element absent in the lOkDa, but present in the 27kDa zein gene.
  • the ABA regulatory element can be obtained by 1) isolating a genomic subclone containing the 27kDa zein gene containing the ABA regulatory element according to the method of Das and Messing (44), and 2) isolating from the subclone a 1,103-bp Pvul fragment containing the ABA regulatory element.
  • preliminary data were obtained showing differences in the response of the lOkDa and 27kDa zein promoters to increased ABA levels.
  • the third pair is related to post-transcriptional regulation.
  • ZprlO/(22) shows the importance of post-transcriptional regulation (7, 42).
  • it has two problems. First, it is absent in standard inbreds. Second, it is regulated by imprinting.
  • the 27 kDa zein gene In contrast to the ZprlO/(22) responsive element in the lOkDa zein gene, the 27 kDa zein gene possesses a responsive element that seems not to be subject to c segregating trans-acting factors. This became clear from dosage experiments described previously (44) .
  • the 27 Da zein gene is present in a tandem duplication in many inbred lines. This organization is also called the S allele, because homologous recombination products were found and isolated having the deletion of the duplication that differs in one retaining the first (A gene) or the other retaining the second (B gene) copy of the 27kDa zein gene (45, 46) .
  • the second gene copy is expressed at a higher level due to the accumulation of 2.5-fold more message.
  • the 3' untranslated regions of the lOkDa zein gene could be exchanged with the one from the 27kDa B gene, making the lOkDa message relatively more stable due to a trans-acting factor that does not segregate.
  • the fourth pair is the yeast GAL cis-acting element and the trans-acting factor GCN4, a non-conventional involving a heterologous expression system.
  • the yeast GAL cis- and trans-acting regulatory system can be introduced by cloning the cis-acting element into the lOkDa promoter region and the expression the trans-acting factor GCN4 under the lOkDa zein promoter from a different chromosomal location. In the latter case, the cis and trans-acting regulatory pair can actually be introduced into different transgenic plants and the overexpression tested in the hybrid.
  • III A Homolo g ous Expression System Tor cloned Zein genes.
  • Zeins are the alcohol-soluble fraction of storage proteins in maize (Zea mays.. They constitute more than 50% of the total endosperm proteins at seed maturity. Zeins consist of a group of heterologous hydrophobic proteins, which are classified according to their molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) into subclasses with Ms of 27, 22, 19, 16, 15, and 10kDa (8, 9). Based on structural similarities, they are also classified into - (22 and 19 kDa), ⁇ -(15kDa), 7 -(16 and 27kDa) , and 6- (lOkDa) zeins (10) .
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • Zeins are encoded by a complex multigene family of over 100 gene members (11-13) and regulated in a tissue- and developmental stage-specific manner. Their expression is confined to triploid (3n) endosperm tissue and starts at a specific stage [around 12 days after pollination (DAP) ] during endosperm development (14). Furthermore, the onset of elevated zein gene expression coincides with the genome amplification process starting at this particular stage in endosperm development (15) .
  • maize endosperm cells have been successfully cultured, and unlike many other cultured plant cells, they remain differentiated. They maintain the syntheses of starch (48) anthocyanins (49-51) , and zeins (52, 53) , which are characteristic of developing endosperm cells. As for the zein synthesis, accumulation in protein bodies has been observed in cultured maize endosperm cells as in endosperm cells of developing kernels, indicating the similarity in cellular processes between the two systems (52, 54). RNA slot-blot analysis confirmed transcription of the 27-kDa zein genes in short-term cultured endosperm cells (55) .
  • Such maize endosperm cultures have been initially characterized here for the expression of zein genes.
  • the regulation of 7 - and 6-class zein genes is of particular interest. Unlike ⁇ -zeins, which are encoded by a large multigene family of 25-50 gene members (12, 13, 56), 7 - and fi-zeins, together with ⁇ -zeins, are encoded by genes present in few copies (20, 44, 57, 58), which simplifies molecular analysis of their gene regulation.
  • the work presented here shows tissue-specific expression of genes encoding 10-, 15-, and 27-kDa zeins in cultured maize endosperm cells. Accurate transcription initiation of the 10- and 27-kDa zein genes was observed.
  • Endosperm tissue cultures were established from developing endosperm tissue (13 DAP) from maize inbred line A636 (Maize Stock Center, University of Illinois, Urbana) . Callus cultures were initiated from the excised endosperm tissues on a semisolid medium consisting of Murashige and Skoog (59) (MS) salts supplemented with 0.15 g/1 L-asparagine, 0.5 mg/1 thiamine HCI, 3% (w/v) sucrose, and 0.8% (w/v) Bacto agar, pH 5.8. One to two months after the culture initiation, calli proliferating on the surface of the explants were transferred into a liquid medium (the same medium as above, except that agar is omitted) .
  • MS Murashige and Skoog
  • Endosperm cell cultures were maintained thereafter in liquid suspension for more than 1 year, while being subcultured routinely every 7 days.
  • Suspension cultures derived from immature leaf tissues of germinating seedlings of maize inbred line Black Mexican Sweet (BMS) were used as a control. They were maintained in a liquid medium consisting of MS salts kept in the dark at 26 * C in a growth room.
  • A636 endosperm and BMS suspension cultures were shaken on horizontal shakers at 160 and 230 rpm, respectively.
  • the 10-kDa zein gene probe used was a 450-bp Ncol-Xbal fragment of cDNA clone plOkz-1 (constructed according to the method of Kirihara et al. (21)) from inbred line W22 (21).
  • the 15-kDa zein gene probe used was a 932-bp EcoRl-BamHl fragment of genomic subclone pGEMZ14 (constructed according to the method of Pederson et al.
  • the 27-kDa zein gene probe used was a 1.2-kb Sphl- Sall fragment (obtained according to the method of Das and Messing (44)) of a genomic subclone from inbred line W22 (61).
  • the 17S rDNA probe used was a 1.5-kb Sstl fragment of the M13 clone 6L-1 (constructed according to the method of Messing et al. (62)) (62) . All probes were labeled with ⁇ P-dCTP by nick-translation (63) . Hybridization intensities on the autoradiograms for the slot-blots were quantitated densitometrically with a Joyce-Loebl Chromoscan-3 densitometer at 530 nm.
  • a 1,238-bp Hindlll-Ban fragment was initially isolated, and the terminal phosphates were removed with calf intestine alkaline pho ⁇ phatase (CIP, Boehringer Mannheim) . It was 5' end-labeled with 7 - 32 P-ATP by T4 polynucleotide kinase. Subsequently, the radiolabeled fragment was digested with Avail and a 188-bp 5' end-labeled AvaII*-BanI fragment was purified by electrophoresis in an 8% polyacrylamide gel. The probe for the 27-kDa zein mRNA was prepared from a genomic subclone derived from inbred line W22.
  • a 474-bp Hpall fragment was isolated and 5' end-labeled with 7 - 32 P-ATP by T4 polynucleotide kinase. Subsequently, it was digested with Rsal and a 343-bp fragment purified by electrophoresis in an 8% polyacrylamide gel. Strand-specific probes (20,000 cpm) were precipitated with ethanol together with total RNA isolated from developing (16 DAP) endosperm tissue (25 ⁇ g) or from cultured endosperm cells (50-75 ⁇ g) .
  • the SI nuclease reaction was terminated and the nucleic acids were precipitated by the addition of 10 ⁇ l of 500 mM EDTA, 50 ⁇ l of 4 M ammonium acetate, 1 ⁇ l of tRNA (10 mg/ml) , and 1 ml of ethanol.
  • the products of the SI nuclease reactions were analyzed together with Maxam and Gilbert (16) sequencing reactions of the probes on 6% polyacrylamide gels containing 8 M urea.
  • Plasmid pFFCAT was used for construction of chimeric genes containing 5' flanking sequences of the 10- and 27- kDa zein genes.
  • pFFCAT contains a 777-bp Taql fragment of the CAT coding sequence (from -30 to +747 with respect to the ATG initiation codon) cloned into the Sail site of pF 19 (17) (Fig. 4A) .
  • the 5' flanking sequences of the 10-kDa zein gene were isolated from genomic subclone PG10BH7 (constructed according to the method of Kirihara et al.
  • a 1,103-bp Pvul fragment spanning from - 1042 to +61 (with respect to the cap site, ATC, as described in the Results- section, Part,i l) was isolated from a genomic subclone. The fragment was blunt-ended with T4 DNA polymerase and subcloned into the Hindlll and Xbal, and cloned into the Hindlll/Xbal sites of pFFCAT, replacing the CaMV35S promoter. It was designated as pZ27(-1042/+61) CAT (Fig. 4A) .
  • a promoter-less CAT construction ( ⁇ -CAT, Fig. 4A) was prepared by digesting pFFCAT with Hindlll and Smal. The protruding Hindlll end was made blunt by Klenow reaction and the plasmid was religated.
  • Protoplasts were isolated enzymatically from endosperm suspension cells. Approximately 20-30 g (fresh weight) of suspension cells was collected in sterile, 50-ml disposable tubes and washed once with CPW solution (65) containing 0.65 M D-mannitol.
  • Electroporation of endosperm protoplasts The isolated endosperm protoplasts were washed once with phosphate buffer saline (PBS) containing 0.65 M D- annitol and resuspended in the same buffer at a final density of 2-3 X 10 6 protoplasts/ml. The protoplasts were kept on ice until electroporation.
  • PBS phosphate buffer saline
  • the covalently closed circular plasmid DNA containing the chimeric gene construct was suspended in 500 ⁇ l of PBS containing 0.65 M D-mannitol in a 2.9-ml disposable spectrophotometer cuvette (Ultra-VU cuvettes-micro, Fisher) .
  • endosperm protoplast suspension Five hundred microliters of endosperm protoplast suspension was added to the cuvette and mixed thoroughly with the plasmid DNA suspension. While keeping the cuvette on ice, electroporation was carried out at 250 V, 600 ⁇ F using a stainless steel electrode with a gap width of 4.5 mm (PDS, Inc.). The electro-suspension culture medium was supplemented with 0.65 M.D-mannitol in a plastic petri dish. The dish was sealed with parafilm and incubated at 25*C in the dark.
  • transfected protoplasts were collected in a 15-ml conical disposable tube. Subsequently, they were resuspended in 400 ⁇ l of 250 mM TRIS (pH 7.0), 10 mM EDTA in a 1.5-ml microfuge tube and homogenized with a disposable pellet pestle (Kontes Scientific Glassware/Instruments) .
  • CAT Chloramphenicol acetvltransferase
  • GUS. enzvme assays At the end of a 44- to 48-h culture period, transfected protoplasts were collected in a 15-ml conical disposable tube. Subsequently, they were resuspended in 400 ⁇ l of 250 mM TRIS (pH 7.0), 10 mM EDTA in a 1.5-ml microfuge tube and homogenized with a disposable pellet pestle (Kontes Scientific Glassware/Instruments) .
  • the homogenate was spun down in a micro-centrifuge for 10 minutes at 4'C, and the protein concentration in the supernatant was determined using a BioRad Protein Assay Kit.
  • CAT and GUS assays were carried out according to the procedures described by Malmberg et al. (66) and Jefferson et al. (67), respectively.
  • the CAT activity was quantitated by measuring in a scintillation counter the radioactivity of the silica gel spots containing the u C-labeled chloramphenicol and acetylated forms.
  • GUS activity was determined fluorimetrically, using 4-methyl umbelliferyl glucuronide (MUG) as a substrate. Fluorescence was measured with a Perkin-Elmer Fluorescence Spectrometer (model LSD-3B) , with excitation at 365 nm and emission at 455 nm.
  • RNA blot analysis of total RNA isolated from different tissues of maize plants showed endosperm-specific expression of the genes encoding the 10-, 15-, and 27-kDa zeins (Fig. 1) .
  • Transcripts of these three zein genes were also detected in total RNA isolated from suspension culture cells derived from developing endosperm tissue. To rule out the possibility that the observed zein gene expression in cultured cells had arisen from aberrant gene regulation during tissue culture manipulation, the expression of these zein genes in BMS suspension culture cells derived from leaf tissue was examined.
  • slot blot Fig. 2
  • RNA blot analysis described previously (Fig. 1) a discrete major transcript was identified for the 10- 15-, and 27-kDa zein genes in both cultured endosper cells and developing endosperm tissues. The sizes o these transcripts appeared to be identical in bot systems, suggesting accuracy in transcription of zei genes in the cultured endosperm cells. To furthe analyze the accuracy of transcription, transcriptio initiation sites were determined for the 10- and 27-kDa zein genes by SI nuclease mapping.
  • the CaMV35S gene promoter yielded a high level of CAT-__-g.ene expression in transfected endosperm protoplasts at an input plasmid amount of 25 ⁇ g when assayed 48 hours after transfection.
  • levels, of CAT gene expression promoted by the 5' flanking sequences of the 10- and 27-kDa zein genes at this input plasmid level were much lower (data not shown) .
  • a higher amount of plasmid DNA was required per electroporation.
  • Electroporation with 150-200 ⁇ g of plasmid DNA resulted in easily detectable levels of CAT gene expression in transfected protoplasts (Fig. 4B) .
  • CAT gene expression driven by the 5' flanking sequences of these zein genes was significant when compared with the negative controls, where neither plasmid DNA nor the same amount of promoter-less ⁇ -CAT construction was electroporated (Fig. 4B) .
  • the promoter activity of the 5' flanking sequences was higher, by six- to seven-fold, for the 10-kDa zein gene than for.
  • Cultured maize endosperm cells are unique in that they remain differentiated rather than becoming “dedifferentiated” as do most cultured plant cells.
  • An endosperm tissue-specific characteristic maintained in these cultured endosperm cells is the synthesis of zein proteins (52, 53). Biochemical and cellular processes involved in zein synthesis and accumulation in protein bodies in cultured endosperm cells follow, to some extent, those taking place in developing endosperm tissue (52, 54).
  • the RNA analysis presented here reveals that genes encoding the 10-, 15-, and 27-kDa zeins are expressed in endosperm cultures of maize inbred line A636.
  • zein genes represents the maintenance of the differentiated state of explants rather than their reactivation during tissue culture manipulation, since these genes are not expressed in leaf tissue-derived BMS cultures. Furthermore, maintenance of accurate transcription of zein genes in endosperm cultures can be inferred from the following findings: 1) synthesis of a discrete major transcript for each zein gene; 2) sizes of the zein transcripts, which are identical to those in developing endosperm tissue; and 3) accurate transcription initiation sites for the 10- and 27-kDa zein genes.
  • Endosperm tissue in developing kernels consists of a population of heterogeneous cells with regard to their developmental stages (71) . It is known that active zein synthesis starts around 10-12 DAP, when cell division ceases for most endosperm cells (72) . Cessation of cell division occurs first in the cells present in the central part of the endosperm, while cells present at the region immediately beneath the aleurone (outer cell layer or subaleurone layer) remain meristematic (73, 74).
  • the l.l-kb 5* flanking sequences of the 10- and 27-kDa zein genes can promote expression of the CAT reporter gene in transfected endosperm culture protoplasts.
  • the levels of CAT gene expression promoted by the 5' flanking sequences of these two zein genes are much lower than that driven by the CaMV35S promoter with a duplicated enhancer element.
  • a large amount of plasmid DNA harboring these chimeric gene constructs is required in electroporation to obtain detectable levels of CAT activity, suggesting weak promoter activities of the 5' flanking sequences of these zein genes.
  • maize endosperm cultures offer at least the following two advantages as a homologous model system to study endosperm-specific gene regulation. The first is that these cultures can be maintained in a laboratory throughout the year under a defined environmental condition, which circumvents a long waiting period and a large field or greenhouse space required for obtaining endosperm tissues from maize plaints.
  • This method relies on the uptake of DNA into embryonic protoplasts isolated from embryogenic maize cell culture and the subsequent regeneration of whole plants. DNA uptake is induced by polyethylene glycol (PEG) or electroporation. A critical requirement for this method is the establishment of a highly regenerable maize cell culture. This method has been successfully used in the genetic transformation of maize plants. See, for example, Gordon-Kamm et al. (77).
  • This method utilizes the bombardment of intact cells and tissues with DNA-coated microprojectiles (78).
  • An advantage of this method is its application for intact cells, especially for meristematic cells, thus, eliminating a laborious step for plant regeneration from single protoplasts.
  • This method has been successfully used to generate transgenic maize plants. See, for example, From et al. (79-).
  • the Aorobacterium-mediated gene transfer method originally believed to be applicable only to dicotyledonous plant species, has been proven to be capable of transforming monocotyledonous plant species including maize.
  • This method relies on the ability of Agrobacterium tumefaciens. a soil bacterium, to transfer and integrate a part of DNA sequence (T-DNA) on its plasmid (Ti plasmid) into the plant genome.
  • T-DNA DNA sequence
  • Ti plasmid plasmid
  • Successful genetic transformation of maize plants can be achieved by the infection of meristematic cells at the shoot apex with Agrobacterium and subsequent development of whole plants. See, for example, Gould et al. (80). Materials and Methods
  • RNA isolated from maize plant tissue and cultured cells was analyzed by Northern blot analysis. Total RNA was isolated from endosperm (16 DAP) , root and leaf tissues of an A636 maize plant, as well as from A636 endosperm and leaf tissue-derived BMS cultures. Five micrograms of RNA sample from each tissue were fractionated in a formaldehyde-agarose gel, transferred onto a filter, and hybridized to a 32P-labelled 02 cDNA probe. The results from this experiment, shown in Figure 5, demonstrate that the endosperm-specific expression of the 02 gene is maintained in the A636 endosperm culture.
  • FIG. 6 panel A A diagrammatic representation of the 02 overexpression constructs which are cotransfected with the zein promoter-GUS constructs shown in Figure 6, panel A, is provided in Figure 6, panel B.
  • Relative GUS activities derived from the transiently transformed maize endosperm protoplasts which have been contransfected with the zein promoter-GUS and the 02 overexpression constructs are shown in Figure 6, panel C.

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Abstract

L'invention concerne des procédés d'obtention de graines de maïs ayant une teneur en méthionine supérieure à 1,39 % en poids de la composition d'amino-acide totale et qui peut constituer plus de 36 % des besoins en méthionine de l'alimentation des volailles. Le maïs à haute teneur en méthionine est obtenu en tant que descendant d'une lignée de maïs parentale sélectionnée pour produire du maïs contenant des niveaux élevés de zéine de 10 kDa riche en méthionine. Dans une variante, les graines de maïs à teneur élevée en méthionine sont produites par une plante de maïs qui a été transformée génétiquement avec une construction d'expression qui comprend un promoteur graine-fonctionnel effectivement lié à un gène codant une protéine de zéine riche en méthionine, une séquence d'ADN effectivement reliée et pouvant améliorer le fonctionnement du promoteur, une séquence d'ADN effectivement reliée pouvant stabiliser l'ARN messager du gène de zéine, et une séquence d'ADN effectivement reliée pouvant améliorer la translation de l'ARN messager du gène de zéine. L'invention concerne également un procédé permettant de déterminer si un gène introduit est exprimé dans les tissus de l'endosperme du maïs, et il fournit également un anticorps purifié spécifique pour une protéine de maïs à teneur élevée en méthionine.
PCT/US1992/009433 1991-11-05 1992-11-04 Procedes d'obtention de graines de mais a haute teneur en methionine, et leurs utilisations WO1993008682A1 (fr)

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US7064248B2 (en) 1990-01-22 2006-06-20 Dekalb Genetics Corp. Method of preparing fertile transgenic corn plants by microprojectile bombardment
US6960709B1 (en) 1993-08-25 2005-11-01 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
US7547820B2 (en) 1993-08-25 2009-06-16 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
US6169232B1 (en) 1997-07-15 2001-01-02 Dow Agrosciences Llc Nucleotide sequences of genes encoding sink protein and uses thereof for improving the nutritional quality of feeds
WO1999004024A3 (fr) * 1997-07-15 1999-04-22 Dow Agrosciences Llc Sequences nucleotidiques de genes codant des proteines pieges et leur utilisation pour ameliorer la qualite nutritive d'aliments pour animaux
US6905877B1 (en) 1997-12-10 2005-06-14 Pioneer Hi-Bred International, Inc. Compositions and methods for altering amino acid content of proteins
US6946589B1 (en) 1997-12-10 2005-09-20 Pioneer Hi-Bred International, Inc. Compositions and methods for altering amino acid content of proteins
WO1999029882A3 (fr) * 1997-12-10 1999-09-16 Pioneer Hi Bred Int Procede permettant de modifier la valeur nutritive d'une proteine vegetale, la proteine de stockage vegetatif, en modifiant la teneur en acides amines des proteines
US7053282B1 (en) 1998-02-09 2006-05-30 Pioneer Hi-Bred International, Inc. Alteration of amino acid compositions in seeds
WO1999040209A1 (fr) * 1998-02-09 1999-08-12 Pioneer Hi-Bred International, Inc. Modification de compositions d'acides amines dans des graines
EP1108009A4 (fr) * 1998-08-27 2004-07-14 Univ Rutgers Compositions et procedes permettant de produire une expression genique de niveau eleve propre a une graine dans le mais
US6849779B1 (en) 1998-08-27 2005-02-01 Rutgers, The State University Of New Jersey Method for producing high methionine corn seeds
EP1370130A4 (fr) * 2001-02-27 2004-08-04 Multigenesis Corp Procedes de transfert medie par l'arnm d'informations genetiques dans des plantes, et produits resultants
EP2216405A1 (fr) 2002-05-03 2010-08-11 Monsanto Technology LLC Promoteurs USP spécifiques de sémences pour l'expression de gènes dans des plantes
EP1445321A1 (fr) 2002-12-18 2004-08-11 Monsanto Technology LLC Promoteur spécifique de l'embryon de mais et procédés d'utilisation correspondants
EP2116607A1 (fr) 2003-03-28 2009-11-11 Monsanto Technology, LLC Nouveaux promoteurs végétaux pour une utilisation pendant le développement précoce des graines
EP2116606A1 (fr) 2003-03-28 2009-11-11 Monsanto Technology, LLC Nouveaux promoteurs végétaux pour une utilisation pendant le développement précoce des graines
US10154679B2 (en) 2004-02-03 2018-12-18 Cargill, Incorporated Protein concentrate and an aqueous stream containing water-soluble carbohydrates
WO2006089950A2 (fr) 2005-02-26 2006-08-31 Basf Plant Science Gmbh Cassettes d'expression destinees a une expression preferentielle de semences chez des plantes
US7902423B2 (en) 2005-04-20 2011-03-08 Basf Plant Science Gmbh Expression cassettes for seed-preferential expression that utilize the promoter from a flax tonoplast intrinsic protein gene
WO2006120197A2 (fr) 2005-05-10 2006-11-16 Basf Plant Science Gmbh Cassettes d'expression pour l'expression preferentielle de semence dans des plantes
US7790873B2 (en) 2005-05-10 2010-09-07 Basf Plant Science Gmbh Expression cassettes for seed-preferential expression in plants
WO2008099013A1 (fr) 2007-02-16 2008-08-21 Basf Plant Science Gmbh Séquences d'acides nucléiques pour la régulation de l'expression spécifique de l'embryon dans des plantes monocotyles
WO2011003901A1 (fr) 2009-07-10 2011-01-13 Basf Plant Science Company Gmbh Cassettes d'expression pour l'expression spécifiquement dans l'endosperme dans des plantes
WO2011067712A1 (fr) 2009-12-03 2011-06-09 Basf Plant Science Company Gmbh Cassette d'expression pour expression spécifique de l'embryon dans des plantes
EP3002332A2 (fr) 2009-12-03 2016-04-06 BASF Plant Science Company GmbH Cassettes d'expression pour expression spécifique à des embryons dans des plantes
RU2650764C2 (ru) * 2011-07-14 2018-04-17 Агридженетикс, Инк. Кукурузные продукты и способы их получения

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