EP1831376A1 - Qualite de grain amelioree grace a l'expression modifiee de proteines de la semence - Google Patents

Qualite de grain amelioree grace a l'expression modifiee de proteines de la semence

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Publication number
EP1831376A1
EP1831376A1 EP04815600A EP04815600A EP1831376A1 EP 1831376 A1 EP1831376 A1 EP 1831376A1 EP 04815600 A EP04815600 A EP 04815600A EP 04815600 A EP04815600 A EP 04815600A EP 1831376 A1 EP1831376 A1 EP 1831376A1
Authority
EP
European Patent Office
Prior art keywords
zein
plant
protein
grain
alpha
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04815600A
Other languages
German (de)
English (en)
Inventor
Rudolf Jung
Wang-Nan Hu
Robert B. Meeley
Vincent J.H. Sewalt
Ramesh Nair
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pioneer Hi Bred International Inc
Original Assignee
Pioneer Hi Bred International Inc
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Filing date
Publication date
Application filed by Pioneer Hi Bred International Inc filed Critical Pioneer Hi Bred International Inc
Priority to EP08013040.4A priority Critical patent/EP2003205B1/fr
Publication of EP1831376A1 publication Critical patent/EP1831376A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243Phenotypically 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
    • C12N15/8251Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants

Definitions

  • the invention relates to the field of plant molecular biology and the use of genetic modification to improve the quality of crop plants, more particularly to methods for improving the nutritional value of grain and the efficiency of grain processing.
  • the present invention is directed to compositions and methods for altering the levels of seed proteins in plant seed, particularly reducing the levels of gamma-zein proteins in maize and the levels of kafarin in sorghum. Modification of seed protein composition causes changes in the physical and/or chemical properties of the grain.
  • Corn is a major crop used as a human food source, an animal feed, and as a source of carbohydrate, oil, protein and fiber. It is principally used as an energy source in animal feeds. Most corn grain is handled as a commodity, since many of the industrial and animal feed requirements for corn can be met by common varieties of field corn which are widely grown and produced in volume. However, there exists at present a growing market for corn with special end-use properties which are not met by corn grain of standard composition. Sorghum (Sorghum bicolor), one of the most important staple crops in
  • Africa represents the fifth most important cereal crop in the world. It is the only viable food grain for many of the world's most food insecure people, and can make critically important contributions to nutrition of families and children affected by AIDS and other pandemics.
  • the invention is directed to the alteration of protein composition and levels in plant seed, resulting in grain with increased digestibility, increased energy availability, improved amino acid composition, increased response to feed processing, improved silage quality, increased efficiency of wet or dry milling, and decreased anti-nutritional properties.
  • the claimed sequences encode proteins preferentially expressed during seed development.
  • “grain” means the mature kernel produced by commercial growers for purposes other than growing or reproducing the species
  • “seed” means the mature kernel used for growing or reproducing the species.
  • “grain”, “seed”, and “kernel”, will be used interchangeably.
  • the invention is directed to altering seed hardness, decreasing seed caloric value for use in diet foods and other food for human use, pet food, increasing the antioxidant properties of seed, and taking advantage of the metal chelating properties of the corn legumin 1 protein to purify other polypeptides of interest and to increase iron and zinc content and bioavailability in the grain.
  • compositions of the invention comprise sequences encoding maize seed proteins and variants and fragments thereof.
  • Methods of the invention involve increasing or inhibiting a seed protein by such means as, but are not limited to, transgenic expression, antisense suppression, co-suppression methods including: RNA interference, gene activation or suppression using transcription factors and/or repressors, mutagenesis including transposon tagging, directed and site- specific mutagenesis, chromosome engineering (see Nobrega et.
  • Transgenic plants producing seeds and grain with altered seed protein content are also provided.
  • the modified seed and grain of the invention can also be obtained by breeding with transgenic plants, by breeding between independent transgenic events, by breeding of plants with one or more alleles (including mutant alleles) of genes encoding abundant seed proteins and by breeding of transgenic plants with plants with one or more alleles (including mutant alleles) of genes encoding abundant seed proteins. Breeding, including introgression of transgenic and mutant loci into elite breeding germplasm and adaptation (improvement) of breeding germplasm to the expression of transgenes and mutant alleles, can be facilitated by methods such as by marker assisted selected breeding.
  • the 50 kD gamma-zein of the instant invention maps to chromosome 7, bin 7.03, the 18 kD alpha-globulin to chromosome 6, bin 6.05, and the 50 kD legumin 1 to chromosome 6, Bin 6.01.
  • This information as well as the map location for 15kD beta zein, for 16kD and 27kD gamma-zein, the map location of members of the alpha-zein gene families, the map location for the 1OkD and 18kD delta zeins (see Table 1 in Woo et al., 2001 , Plant Cell 13:2297-2317) enables one of skill in the art to employ these map locations to generate improved maize lines with altered seed protein profiles and levels.
  • sequences of the invention can be used to identify and isolate similar sequences in other plants based on sequence homology or sequence identity.
  • the maize sequences can be used to modulate expression in sorghum.
  • Sorghum grain has a nutritional profile similar to corn and other cereals
  • the proteins of the present invention have been designated for the purposes of this invention as "abundant seed proteins".
  • a single species that is a polypeptide encoded by a specific gene
  • a group of similar species that are protein family members with similar molecular properties like size
  • proteins make up 1% or more by dry weight of the total protein of seed and a single protein species or a group of similar species can be visualized by commonly used protein analytical methods such as gel electrophoresis and detection of proteins by staining of protein bands with Coomassie Blue or by liquid chromatography and detection of protein peaks by means of a UV detector (ref: Walker, JM 1 (2002) The Protein Protocol Handbook, second edition, Humana Press, Totowa, New Jersey).
  • these proteins are "abundant” in the majority of maize lines (hereafter referred to simply as maize) they may be of low abundance or even absent in specific maize lines or can be transgenically manipulated to become suppressed. Additionally, these proteins may originally not be “abundant” in grain from wild-type maize but be structurally related to proteins found abundantly in seeds of other plant species. For example, legumins are abundant proteins in legumes and rice but corn legumin is a protein of lower abundance in grain from common maize. Thus we refer to corn legumin as an "abundant seed protein". Traditionally "abundant seed proteins” as defined herein have also been called “seed storage proteins” as they are the major source for nitrogen and amino acids to provide nutrients for seedling growth and development.
  • the major seed proteins are the alpha-zeins such as the 19 kDa and 22kDa alpha-zeins, and the gamma-zeins such as the 27kDa gamma-zein protein.
  • Zeins are typically prolamins; that is they are typically characterized by being extractable in 70% ETOH and a reducing agent (see Woo et al., 2001 , Plant Cell 13:2297-2317, and Shewry and Casey (eds.) (1999) Seed Proteins 141-157, Academic Publishers, Dordrecht.j.
  • a zein can also be identified phylogenetically through the use of sequence analysis.
  • the alpha-zeins are a family of related proteins that typically comprise 10- 50% of the total protein (based on dry weight) in the grain, i.e. they are major seed proteins. This protein family is further comprised of two 19kD alpha-zein protein subfamilies and one 22kD alpha-zein protein subfamily (Woo et al).
  • the chromosomal loci (genomic sequence) of the alpha-zein gene subfamilies have been sequenced in their entirety for a common maize inbred line and are known to the art (ref. Song R., Messing J, (2002) Plant Physiol, Vol. 130, pp. 1626-1635, Song R, Llaca V, Linton E, Messing J. (2001 ) Genome Res. 11 , pp. 1817-25).
  • the gamma-zeins are a family of related proteins that typically make up 10- 15% of the total seed proteins (based on dry weight). The structure and characteristics of this family are exemplified by the 16kD gamma-zein, the 27 kD gamma-zein - which are major seed proteins - and the 5OkDa gamma-zein - a minor seed protein.
  • the 15kD beta-zein is a minor seed protein and belongs to this family as well (Woo et al).
  • Non-zein abundant seed proteins in maize include the corn legumins and alpha-globulins.
  • the corn legumins and the corn alpha-globulins are minor seed proteins in maize; the name designation of both proteins are based on their phylogentic relationship to seed proteins from other species (Woo et al).
  • Seed proteins have been traditionally characterized based on solubility characteristics (Shewry and Casey (eds.) (1999) Seed Proteins, 141-157, Academic Publishers, Dordrecht). Thus most seed proteins are either extractable in aqueous alcoholic solutions (prolamins), extractive in aqueous solutions of low ionic strength (albumins), or extractable in aqueous solutions of high ionic strength (globulins).
  • seed proteins by extraction methods are well known in the art (Shewry and Casey (1999)). However it is also common to designate seed proteins with unknown extraction characteristics as globulins, albumins, or prolamins if they are phylogenetically or sequence-related to proteins that have originally been classified based on extraction experiments. Therefore, it is a common practice to name seed proteins based on their phylogenetic association rather then their extraction properties. The name of a seed protein gene may therefore not reflect the properties of the encoded protein in a strict sense.
  • zein proteins are down regulated in combination with over expression of non-zein proteins to produce a synergistic effect of increased digestibility of cereal grain.
  • the present invention provides isolated nucleic acid molecules comprising a nucleotide sequence encoding a maize protein, designated herein as the 50 kD gamma-zein, having the amino acid sequence shown in SEQ ID NO:2. Further provided is a polypeptide having an amino acid sequence encoded by the nucleic acid molecules described herein, for example that set forth in SEQ ID NO:1 and fragments and variants thereof.
  • the present invention also provides isolated nucleic acid molecules comprising a nucleotide sequence encoding a maize protein, herein designated as the 18 kD alpha-globulin, having the amino acid sequence shown in SEQ ID NO:4. Further provided is a polypeptide having an amino acid sequence encoded by the nucleic acid molecules described herein, for example that set forth in SEQ ID NO:3, and fragments and variants thereof.
  • the present invention also provides isolated nucleic acid molecules comprising a nucleotide sequence encoding a maize protein, herein designated as the 50 kD legumin 1 protein, having the amino acid sequence shown in SEQ ID NO:6. Further provided is a polypeptide having an amino-acid sequence encoded by the nucleic acid molecules described herein, for example that set forth in SEQ ID NO:5, and and fragments and variants thereof.
  • the present invention also provides isolated nucleic acid molecules comprising a nucleotide sequence encoding a sorghum protein, herein designated as the sorghum bicolor 50 kD legumin 1 protein, having the amino acid sequence shown in SEQ ID NO:23. Further provided is a polypeptide having an amino-acid sequence encoded by the nucleic acid molecules described herein, for example that set forth in SEQ ID NO:22, and fragments and variants thereof. The present invention also provides isolated nucleic acid molecules comprising a nucleotide sequence encoding a sugar cane protein, herein designated as the Saccharum officinale 50 kD legumin 1 protein, having the amino acid sequence shown in SEQ ID NO:25. Further provided is a polypeptide having an amino-acid sequence encoded by the nucleic acid molecules described herein, for example that set forth in SEQ ID NO:24, and fragments and variants thereof.
  • a plasmid containing the nucleotide sequence encoding the 50 kD gamma- zein protein was deposited with the Patent Depository of the American Type Culture Collection (ATCC), Manassas, Virginia, on July 26, 2000 and assigned Patent-Deposit No. PTA-2272.
  • a plasmid containing the nucleotide sequence encoding the 18 kD alpha-globulin protein was deposited with the Patent Depository of the American Type Culture Collection (ATCC), Manassas, Virginia, on July 26, 2000 and assigned Patent Deposit No. PTA-2274.
  • a plasmid containing the nucleotide sequence encoding the 50 kD legumin 1 protein was deposited with the Patent Depository of the American Type Culture Collection (ATCC), Manassas, Virginia, on July 26, 2000 and assigned Patent Deposit No. PTA-2273. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • a comparison of the amino acid content of cereal grains shows that 18 kD alpha-globulin is an excellent source of tryptophan and methionine for amino acid balance in all cereals and that 50 kD corn legumin 1 is an excellent source of methionine for all cereals and a good source of lysine and tryptophan for the amino acid balance of most cereals.
  • the present invention also provides isolated nucleotide sequences comprising transcriptional units for gene over -expression and gene-suppression that have been used either as single units or in combination as multiple units to transform plant cells.
  • biologically active means a protein that folds, assemble and interacts with other proteins, is available as a nitrogen source for seed germination and accumulates (ie: synthesis exceeds deposition) during seed development.
  • the invention encompasses isolated or substantially purified nucleic acid or protein compositions.
  • An "isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the nucleic acid molecule or protein as found in its naturally occurring environment.
  • an isolated or purified nucleic acid molecule or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3 1 ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide-sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%,-(by dry weight) of contaminating protein.
  • culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
  • Fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention.
  • fragment is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby.
  • Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the targeted gene.
  • fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity.
  • fragments may be used to inhibit the expression of a targeted gene product of interest.
  • fragments of a nucleotide sequence may range from at least about 10 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence-encoding native alpha- zein proteins, native-gamma-zein proteins, native delta-zein proteins, the native 50 kD gamma-zein, the 18 kD alpha-globulin, or the 50 kD legumin 1 protein of the invention.
  • fragments of a nucleotide sequence that are useful for generating cells, tissues or plants transiently or permanently suppressing a gene or genes may not encode fragment proteins retaining biological activity. Fragments may be in sense or antisense or reverse orientation or a combination thereof.
  • fragments of such nucleotide sequence may range from at least about 10 nucleotides, at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding native alpha-zein proteins, native gamma-zein proteins, native delta-zein proteins, the 50 kD gamma-zein, the 18 kD alpha-globulin, or the 50 kD legumin 1 protein of the invention.
  • Fragments of the maize nucleotide sequences of the invention that encode a biologically active portion of the 50 kD gamma-zein protein, the 18 kD alpha-globulin protein, or the 50 kD legumin 1 protein of the invention, respectively, will encode at least 15, 25, 30, 50, 100, 150, or 200 contiguous amino acids, or up to the total number of amino acids present in the full-length 50 kD gamma-zein protein, the 18 kD alpha-globulin protein, or the 50 kD legumin 1 protein of the invention (for example, 295 amino acids for SEQ ID NO:1 ; 206 amino acids for SEQ ID NO:3; and 483 amino acids for SEQ ID NO:5). Fragments of SEQ ID NO:1 , SEQ ID NO:3, and SEQ ID NO:5 that are useful as hybridization probes or PCR primers need not encode a biologically
  • a fragment of SEQ ID NO:1 , SEQ ID NO:3, or SEQ ID NO:5 may encode a biologically active portion of a prolamin or globulin protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below or it may be used to inhibit the expression of the protein.
  • a biologically active portion of the 50 kD gamma-zein protein, the 18 kD alpha- globulin protein, or the 50 kD legumin 1 protein of the invention can be prepared by isolating a portion of the disclosed nucleotide sequence that codes for a portion of the 50 kD gamma-zein protein, the 18 kD alpha-globulin protein, or the 50 kD legumin 1 protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the prolamin protein.
  • Nucleic acid molecules that are fragments of SEQ ID NO:1 comprise at least 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1100 nucleotides, or up to the number of nucleotides present in the full-length gamma-zein cDNA (for example, 1129 nucleotides for SEQ ID NO:1 ).
  • Nucleic acid molecules that are fragments of SEQ ID NO:3 comprise at least 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, or 900 nucleotides, or up to the number of nucleotides present in the full-length alpha-globulin cDNA (for example, 950 nucleotides for SEQ ID NO:3).
  • Nucleic acid molecules that are fragments of SEQ ID NO:5 comprise at least 40, 50,-75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 1000, 1200, 1400, or 1600 nucleotides, or up to the number of nucleotides present in the full-length legumin 1 cDNA (for example, 1679 nucleotides for SEQ ID NO:5).
  • variants is intended substantially similar sequences.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of 50 kD gamma-zein protein, the 18 kD alpha-globulin protein, or the 50 kD legumin 1 protein of the invention.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site- directed mutagenesis but which still-encode a 50 kD gamma-zein protein, an 18 kD alpha-globulin protein, or an 50 kD legumin 1 protein.
  • variants of a particular nucleotide sequence of the invention will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to that particular nucleotide sequence over a length of 20, 30, 50, or 100 nucleotides or less, as determined by sequence alignment programs described elsewhere herein using default parameters.
  • variant protein is intended a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N- terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variant proteins encompassed by the present invention are biologically active, that is they continue to possess all or some of the activity of the native proteins of the invention as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • Biologically active variants of the native 50 kD gamma-zein protein, the 18 kD alpha-globulin protein, or the 50 kD legumin 1 protein of the invention will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the amino-acid sequence for the native protein over a length of 10, 30, 50, or 100 amino acid residues or less as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • the proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of the 50 kD gamma-zein protein, the 18 kD alpha-globulin protein, or the 50 kD legumin 1 protein can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art.
  • genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
  • proteins of the invention encompass both naturally occurring variant proteins as well as variations and modified forms thereof. Such variants will continue to be biologically active as defined herein.
  • the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See EP Patent Application Publication No. 75,444.
  • Variant nucleotide sequences and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different alpha-zein, delta-zein, beta-zein, gamma-zein, alpha-globulin, or legumin protein coding sequences can be manipulated to create a new alpha-zein, delta-zein, beta-zein, gamma-zein, alpha-globulin, or legumin protein possessing the desired properties.
  • libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • sequence motifs encoding a domain of interest may be shuffled between 50 kD gamma-zein coding sequence, the 18 kD alpha-globulin coding sequence, or the 50 kD legumin 1 protein coding sequence of the invention and other known gene coding sequences to obtain a new coding sequence for a protein with an improved property of interest.
  • Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl.
  • nucleotide sequences of the invention and known abundant corn seed proteins can be used to isolate corresponding sequences from other plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequence set forth herein. Sequences isolated based on their sequence identity to known abundant corn seed proteins and the entire 50 kD gamma-zein, 18 kD alpha- globulin, or 50 kD legumin 1 sequences set forth herein or to fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. By "orthologs" is intended genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species.
  • oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest.
  • Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also lnnis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); lnnis and Gelfand, eds.
  • PCR PCR Strategies
  • cDNA fragments i.e., genomic or cDNA libraries
  • the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker.
  • probes for hybridization can be made by labeling synthetic oligonucleotides based on, for example, the 50 kD gamma-zein-sequence of the invention.
  • Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
  • the entire 50 kD gamma-zein, 18 kD alpha-globulin, or 50 kD legumin 1 sequence disclosed herein, or one or more portions thereof may be used as a probe capable of specifically hybridizing to corresponding seed protein sequences and messenger RNAs.
  • probes include sequences that are unique among the seed protein sequences of the invention and are preferably at least about 40 nucleotides in length.
  • Such probes may be used to amplify corresponding gamma-zein, alpha-globulin, and legumin 1 sequences from a chosen plant by PCR.
  • Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
  • Hybridization of such sequences may be carried out under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background).
  • Stringent conditions are sequence-dependent and will be different in different circumstances.
  • target sequences that are 100% complementary to the probe can be identified (homologous probing).
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 0 C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 0 C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCI, 1 % SDS at 37°C, and a wash in 0.5X to 1X SSC at 55 to 6O 0 C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in 0.1 X SSC at 60 to 65 0 C. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem.
  • T m 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe.
  • T m is reduced by about 1 0 C for each 1 % of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10 0 C.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
  • isolated sequences that encode polypeptides that function as a seed protein and which hybridize under stringent conditions to the 50 kD gamma-zein, the 18 kD alpha-globulin protein, or the 50 kD legumin 1 sequence disclosed herein, or to fragments thereof, are encompassed by the present invention.
  • sequences will be at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homologous with the disclosed sequence.
  • sequence identity of sequences may range, sharing at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity.
  • sequence relationships between two or more nucleic acids or polynucleotides are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • a gap penalty is typically introduced and is subtracted from the number of matches.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters.
  • the CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237- 244 (1988); Higgins et al. (1989) CABIOS 5: 151 -153; Corpet et al.
  • Gapped BLAST in BLAST 2.0
  • PSI-BLAST in BLAST 2.0
  • PSI-BLAST in BLAST 2.0
  • sequence identity/similarity values provided herein refer to the value obtained using GAP version 10 using the following parameters: % identity using GAP Weight of 50 and Length Weight of 3; % similarity using Gap Weight of 12 and Length Weight of 4, or any equivalent program, aligned over the full length of the sequence.
  • equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
  • GAP uses the algorithm of Needleman and Wunsch (1970) J. MoI. Biol.
  • GAP finds the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively.
  • the default gap creation penalty is 50 while the default gap extension penalty is 3.
  • the gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200.
  • the gap creation and gap extension penalties can be 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
  • GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity.
  • the Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment.
  • Percent Identity is the percent of the symbols that actually match.
  • Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored.
  • a similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
  • the scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters.
  • sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters.
  • Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.
  • nucleotide sequences are substantially identical if two molecules hybridize to each other under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • stringent conditions encompass temperatures in the range of about 1 0 C to about 20 0 C lower than the T m , depending upon the desired degree of stringency as otherwise qualified herein.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identity in the context of a peptide indicates that a peptide comprises a sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the reference sequence over a specified comparison window. Alignment can be conducted using the homology alignment algorithm of Needleman and Wunsch (1970) J. MoI. Biol. 48:443-453.
  • Peptides that are "substantially similar" comprise a sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity or sequence similarity to the reference sequence over a specified comparison window. In this case residue positions that are not identical instead differ by conservative amino acid changes.
  • the 50 kD gamma-zein nucleotide sequence cloned from a maize endosperm cDNA library (Example 1 ) and disclosed in the present invention displays sequence similarity to the other two described corn gamma-zein genes, 27 kD gamma-zein (represented herein by GenBank Accession No. P04706) and 16 kD gamma-zein (represented herein by GenBank Accession No. AAA33523).
  • the 50 kD gamma-zein was named due to its apparent molecular weight by migration in SDS-PAGE.
  • This cDNA encodes a 295 amino acid protein and also shows sequence similarity to seed proteins of other plant species.
  • wheat alpha-gliadin (GenBank -ID:TAU51305, Accession No. U51305).
  • the 50 kD gamma-zein DNA sequences isolated from different inbred lines showed an unusually low level of polymorphism. Only one single nucleotide polymorphism (SNP) (a 3 bp insertion) was detected along the entire cDNA sequence from DNA isolated from the inbred lines Mo17 and B73.
  • SNP single nucleotide polymorphism
  • the 50 kD gamma-zein gene has been located on chromosome 7, bin 7.03.
  • the 18 kD alpha-globulin nucleotide sequence was also cloned from a maize endosperm cDNA library (Example 5) and is disclosed in the present invention (SEQ ID NO:3).
  • the 18 kD alpha-globulin was named due to its similarity to a rice seed globulin (rice alpha-globulin, GenBank Accession No.
  • This cDNA encodes a 206 amino acid protein. Unlike the case of the
  • the 18 kD alpha-globulin cDNA shows sequence similarity to seed proteins from other cereals including rice, wheat, and oats and distantly to maize proteins with the conserved domain pfam00234.11
  • AAI family plant lipid transfer proteins and seed storage proteins
  • the disulfide- bonding pattern varies between members.
  • Polypeptides belonging to this protein family contain three regions with conserved cysteine residues.
  • Members of this gene family from other plant species e.g. puroindulin from wheat, GenBank Account No. gi_509109
  • Corn alpha-globulin and rice alpha-globulin (Account No. JC4784) are 46% identical on the amino acid level, but are 80% -100% identical in the cysteine domains.
  • SPLDACRQVLDRQLTG The first of these regions (SPLDACRQVLDRQLTG) is 100% identical between rice and corn over 16 aa residues.
  • the Cys residue followed by an Arg residue is conserved also in other members of this protein family found in other plant species.
  • CQQLQDVSRECRCAAIR 100% identical between rice and corn over 18 aa residues.
  • the two consecutive cysteine residues followed by 9 amino acids and a CysArgCys tripeptide are conserved in other members of this protein family.
  • the 50 kD legumin 1 nucleotide sequence was also cloned from a maize endosperm cDNA library (Example 8) and is disclosed in the present invention (SEQ ID NO:5).
  • the 50 kD legumin 1 was named due to its similarity to 11 S globulins found in other plant species: the so-called legumins.
  • the 50 kD legumin 1 appears to be encoded by a single gene in the maize genome. It belongs to the 11 S globulin superfamily and is closely related to legumins from other cereals (also called glutenins in rice and wheat or globulins in oat) and dicot plants.
  • the 50 kD legumin polypeptide sequence is missing the evolutionary conserved 11S globulin pro-protein proteolytic site (Asn-Gly bond between the acidic chain and basic chain legumin regions) which makes it unique among the legumin protein superfamily.
  • This cDNA encodes a 483 amino acid protein with a predicted N- terminal endoplasmic reticulum import signal peptide of 36 amino acids.
  • the 50 kD legumin 1 DNA sequences isolated from different inbred lines showed a considerable level of polymorphism.
  • the 50 kD legumin 1 gene has been mapped to chromosome 6, Bin 6.01.
  • the chromosomal location of the genes corresponding to the known maize seed proteins and the three cDNA's of the present invention are known (see Woo et al, et seq) or have been determined as stated above. Knowing the map position of a gene is important and useful if it correlates with a trait, as is the case for the encoded polypeptides of the present invention. Certain alleles of these genes can, for instance, have an impact on seed hardness, starch extractability, energy availability, etc as is described in detail infra. Considerable knowledge has been accumulated regarding the so called Quantitative Trait Loci (QTL). Linkage of a gene to a QTL is of significance regarding the impact of this gene on the corresponding trait. Further, the map position can be used for marker assisted breeding, which is a very economical and time saving way to introduce alleles into elite germplasm. Alternatively, SNP's can also be used to screen a wide variety of germplasm for advantageous alleles.
  • QTL Quantitative Trait Loci
  • the 50 kD gamma-zein protein of the present invention displays a high cysteine content and is therefore predicted to have a high number of disulfide bonds or high "disulfide status", as is observed for the other gamma-zein proteins.
  • disulfide status is intended the portion of cysteine residues within a protein that participate in disulfide bonds or disulfide bridges. Such disulfide bonds can be formed between the sulfur of a first cysteine residue and the sulfur of a second cysteine residue.
  • first and second cysteine residues can occur as part of a single polypeptide chain, or alternatively, can occur on separate polypeptide chains referred to herein as "inter-molecular disulfide bonds".
  • disulfide status is used in reference to a seed or part thereof, the “disulfide status” of such a seed or part thereof is the total disulfide status of the proteins therein.
  • the disulfide-rich, gamma-zein protein fraction in corn has been implicated as a major determinant of the poor amino acid content of this grain which contributes to its low nutrient content.
  • this gamma-zein fraction of corn endosperm it can also be a significant contributor to the wet-milling properties of corn grain.
  • intermolecular disulfide bridges of the gamma-zeins are also important for the formation and maintenance of protein bodies.
  • These protein bodies contribute to the physical properties of the grain that also affect the wet-milling process.
  • chemical reductants are required to break protein disulfide bonds to maximize starch yield and quality (Hoseney, R.C. (1994), Principles of Cereal Science and Tech., (Ed.2)).
  • the use in wet mills of odorous chemical such as sulfur dioxide and bisulfite requires extensive precautions and poses significant environmental problems.
  • a decrease in the number of protein bodies can also be expected to improve the efficiency of the wet-milling process.
  • Zein proteins interact during formation of protein bodies (through intermolecular disulfide bonds and hydropobic interactions), and these interactions are important for the formation of proteolytically stable complexes.
  • a decrease in the expression of two or three gamma-zein genes can be expected to have an additive effect on the reduction of protein bodies resulting in a corresponding improvement in wet-milling properties.
  • the wet-milling properties of the corn grain of the present invention can be analyzed using a small-scale simulated wet-milling process incorporating or leaving out a reducing agent (bisulfite) in the steep water as used by Eckhoff et al., (1996, Cereal Chem. 73:54-57).
  • a reducing agent bisulfite
  • reducing agents can increase the dry matter digestibility of sorghum and corn and, thus, improve their feed properties.
  • This result is supported by the results of data from in vitro digestibility assays described in the present invention (Examples 2-4) that demonstrate that reducing agents increase the dry matter digestibility or energy availability of corn. See also: Hamaker, B. R., et al., 1987, Improving the in vitro protein digestibility of sorghum with reducing agents, Proc. Natl. Acad. Sci. USA 84:626-628.
  • energy availability means the degree to which energy- rendering nutrients are available to the animal, often referred to as energy conversion (ratio of metabolizable energy content to gross energy content).
  • energy conversion ratio of metabolizable energy content to gross energy content.
  • excreta are collected by standard methodology (e.g., Sibbald, I. R., Poultry Science, 58(5):1325-29 (1979); McNab and Blair, British Poultry Science 29(4):697-708 (1988)).
  • Energy availability is largely determined by food or feed digestibility in the gastro-intestinal tract, although other factors such as absorption and metabolic utilization also influence energy availability.
  • Digestibility is defined herein as the fraction of the feed or food that is not excreted as feces. Digestibility is a component of energy availability. It can be further defined as digestibility of specific constituents (such as carbohydrates or protein) by determining the concentration of these constituents in the foodstuff and in the excreta. Digestibility can be estimated using in vitro assays, which is routinely done to screen large numbers of different food ingredients and plant varieties. In vitro techniques, including assays with rumen inocula and/or enzymes for ruminant livestock (e.g.
  • the 18 kD alpha-globulin can also be expected to have a high number of disulfide bonds and to participate in intermolecular protein cross-linking. For this reason, over-expression of the 18 kD alpha-globulin protein can be predicted to increase seed hardness. The ability to confer seed hardness is particularly useful in the case of soft kernel phenotypes that are induced by mutation or transgenic polypeptides. An increase in the levels of the 18 kD alpha-globulin can be used as a method for improving the dry-milling properties of soft kernel phenotypes.
  • the 18 kD alpha-globulin protein In addition to its high cysteine content, the 18 kD alpha-globulin protein also possesses a relatively high percentage of the essential amino acids tryptophan (4.6% by weight, cysteine (5.1% by weight), and methionine (3.9% by weight). For this reason, transgenic over-expression of the 18 kD alpha-globulin protein can be expected to significantly increase the percentage of tryptophan and sulfur- containing amino acids in corn grain and, thus, increase the nutritional value of the grain.
  • the "nutritional value" of a feed or food is defined as the ability of that feed or food to provide nutrients to animals or humans.
  • the nutritional value is determined by 3 factors: concentration of nutrients (protein & amino acids, energy, minerals, vitamins, etc.), their physiological availability during the processes of digestion, absorption and metabolism, and the absence (or presence) of anti- nutritional compounds.
  • concentration of nutrients protein & amino acids, energy, minerals, vitamins, etc.
  • the 50 kD legumin 1 protein Similar to the 18 kD alpha-globulin, the 50 kD legumin 1 protein also possesses a relatively high percentage of essential amino acids. This protein contains 6.7%, 0.7%, 2.2%, 1.1 %, 3.6%, and 2.7% by weight of lysine, tryptophan, methionine, cysteine, isoleucine, and threonine, respectively. For this reason, transgenic over-expression of the 50 kD legumin 1 protein can also be expected to increase the nutritional value of the grain.
  • the 50 kD legumin 1 protein is assembled differently than other legumin polypeptides.
  • the 50 kD legumin 1 protein is not cleaved into acidic and basic chains. Instead this legumin assembles into 9S polypeptide primers (presumably in the endoplasmic reticulum) and does not undergo assembly into 11S globulin hexamers.
  • the assembly properties of this 50 kD legumin 1 polypeptide could contribute to unique food processing properties of protein extracts from seed expressing this protein.
  • the 5OkD legumin 1 polypeptide could be ectopically expressed in soybean seed and protein isolates from corresponding soybean seed display altered functionalities such as solubility under acidic conditions, improved water-holding capacity and the like.
  • Another feature of the 50 kD legumin 1 polypeptide is a string of histidine residues that can function as a metal binding site. Native 50 kD legumin 1 polypeptide binds with high affinity to nickel chelation columns. This property can be used to purify corn legumin 1 in bulk from complex protein mixtures and to purify other polypeptides of interest through the production of fusion proteins.
  • the metal chelation properties of the 50 kD legumin 1 polypeptide could also be of importance for bio-remediation or food health (antioxidant) applications. Additionally, in cereal grain such as maize or sorghum transgenically overexpressing the 50 kD legumin 1 polypeptide, the Zn and Fe chelating properties of the 50 kD legumin 1 polypeptide may result in an increased concentrations of Zn and Fe in the grain and in increased bio-availability of these micro-nutrient from the diet.
  • the over-expression of the 50 kD corn legumin 1 protein unexpectedly also resulted in a significant increase of grain digestibility (See Example 11 ).
  • Endosperm from transgenic corn grain over-expressing corn either alpha-globulin or corn legumin was investigated by immuno- Electron Microscopy (EM). Both transgenic proteins were found to accumulate in non-zein storage organelles, which appear greatly enhanced in number and in size in the transgenic endosperm samples, compared to control EM images obtained from endosperm from non-transgenic corn.
  • EM Immun- Electron Microscopy
  • the alpha-gliadin and gamma-gliadin from wheat have also been identified as major allergens (Maruyama et al. (1998) Eur. J. Biochem. 256:604. For these reasons the methods of the present invention are also directed to the elimination or the reduction of the levels of at least one seed protein in wheat, barley, oats, or rye to produce a grain with eliminated or reduced anti-nutritional or allergenic properties.
  • compositions and methods of the invention are useful for modulating the levels of at least one seed protein in seeds.
  • modulate is defined herein as an increase or decrease in the level of a seed protein within seed of a genetically manipulated plant relative to the level of that protein in seed from the corresponding wild-type plant (i.e., a plant not genetically manipulated in accordance with the methods of the present invention).
  • inhibitor refers to any decrease in the expression or function of a target gene product, including any relative decrement in expression or function up to and including complete abrogation of expression or function of the target gene product.
  • expression refers to the biosynthesis of that gene product, including the transcription and/or translation of the gene product.
  • Inhibition of expression or function of a target gene product can be in the context of a comparison between any two plants, for example, expression or function of a target gene product in a genetically altered plant versus the expression or function of that target gene product in a corresponding wild-type plant.
  • inhibition of expression or function of the target gene product can be in the context of a comparison between plant cells, organelles, organs, tissues, or plant parts within the same plant or between plants, and includes comparisons between developmental or temporal stages within the same plant or between plants.
  • Any method or composition that down-regulates expression of a target gene product, either at the level of transcription or translation, or down-regulates functional activity of the target gene product can be used to achieve inhibition of expression or function of the target gene product.
  • inhibitory sequence encompasses any polynucleotide or polypeptide sequence that is capable of inhibiting the expression of a target gene product, for example, at the level of transcription or translation, or which is capable of inhibiting the function of a target gene product.
  • inhibitory sequences include, but are not limited to, full-length polynucleotide or polypeptide sequences, truncated polynucleotide or polypeptide sequences, fragments of polynucleotide or polypeptide sequences, variants of polynucleotide or polypeptide sequences, sense-oriented nucleotide sequences, antisense-oriented nucleotide sequences, the complement of a sense- or antisense-oriented nucleotide sequence, inverted regions of nucleotide sequences, hairpins of nucleotide sequences, double-stranded nucleotide sequences, single-stranded nucleotide sequences, combinations thereof, and the like.
  • polynucleotide sequence includes sequences of RNA, DNA, chemically modified nucleic acids, nucleic acid analogs, combinations thereof, and the like.
  • Inhibitory sequences are designated herein by the name of the target gene product.
  • an "27 kD gamma zein inhibitory sequence” would refer to an inhibitory sequence that is capable of inhibiting the expression of 27 kD gamma zein, for example, at the level of transcription and/or translation, or which is capable of inhibiting the function of 27 kD gamma zein.
  • the phrase "capable of inhibiting" is used in the context of a polynucleotide inhibitory sequence, it is intended to mean that the inhibitory sequence itself exerts the inhibitory effect; or, where the inhibitory sequence encodes an inhibitory nucleotide molecule (for example, hairpin RNA, miRNA, or double-stranded RNA polynucleotides), or encodes an inhibitory polypeptide (i.e., a polypeptide that inhibits expression or function of the target gene product), following its transcription (for example, in the case of an inhibitory sequence encoding a hairpin RNA, miRNA, or double-stranded RNA polynucleotide) or its transcription and translation (in the case of an inhibitory sequence encoding an inhibitory polypeptide), the transcribed or translated product, respectively, exerts the inhibitory effect on the target gene product (i.e., inhibits expression or function of the target gene product).
  • an inhibitory nucleotide molecule for example, hairpin RNA, miRNA,
  • the terms “increase, “ “increased, “ and “increasing” in the context of the methods of the present invention refer to any increase in the expression or function of a gene product, including any relative increment in expression or function.
  • increases in the expression or function of a gene product of interest i.e., a target gene product
  • increases in the expression or function of the target gene product can be in the context of a comparison between plant cells, organelles, organs, tissues, or plant parts within the same plant or between plants, and includes comparisons between developmental or temporal stages within the same plant or between plants. Any method or composition that up-regulates expression of a target gene product, either at the level of transcription or translation, or up-regulates functional activity of the target gene product can be used to achieve increased expression or function of the target gene product.
  • a method that increases oil production in a plant can be any method that increases the total oil content or the percent oil content of that plant relative to that observed in another plant, for example a comparison between a genetically modified plant and a corresponding wild-type plant, any method that increases oil content of a cell, organelle, organ, tissue, or plant part relative to a different cell, organelle, organ, tissue, or plant part within the same plant or between two plants, or any method that increases oil content of a cell, organelle, organ, tissue, plant part, or whole plant relative to that observed during different developmental or temporal stages within the same plant or another plant.
  • methods are particularly directed to reducing the level of zein proteins, such as, but not limited to, 16 kD, the 27 kD protein and the 50 kD gamma-zein proteins to improve the nutritional value and industrial use of grain.
  • Another embodiment is directed to the reduction or elimination of the alpha-zein of maize.
  • the levels of alpha-zeins and of gamma-zeins are reduced in maize grain resulting in an increase in digestibility.
  • Yet another embodiment is directed to the reduction or elimination of the alpha-, beta-, and gamma-gliadins of wheat, barley, rye, and oats to eliminate or ameliorate the anti- nutritional or allergenic effects of these proteins.
  • the levels of the alpha-globulin protein or the corn legumin 1 protein in plant seed are modulated to affect the nutritional value, or the hardness of the seed.
  • Another embodiment is directed to the reduction of the major zein seed proteins and the concurrent increase of the levels of the alpha-globulin protein or the corn legumin 1 protein to incrementally or synergistically improve the grain digestibility.
  • Other embodiments of the invention include methods directed to screening for particular plant phenotypes based on antibodies specific for the polypeptides of the invention, or using SNP's of the nucleotide sequences of the invention.
  • Reduction of the level of the 16 kD, the 27 kD protein or the 50 kD gamma- zein proteins in plant seed can be used to improve the nutritional value and industrial use of such grain.
  • the methods of the invention can be useful for producing grain that is more rapidly and extensively digested than grain with normal/wild-type gamma-zein protein levels.
  • inhibition of gamma-zein genes can be used to increase the nutritional value of seed, particularly by increasing the energy availability of seed.
  • Reduction in the gamma-zein levels in such seed can be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and up to 100%.
  • Energy availability can be improved by at least 3%, 6%, 9%, 12%, 15%, 20% and greater.
  • Reduction of the level of alpha-zein proteins in plant seed can be used to improve the nutritional value and industrial use of such grain.
  • the methods of the invention are also useful for producing grain that is more rapidly and extensively digested than grain with normal gamma-zein protein levels.
  • Inhibition of alpha-zein genes can be used to increase the nutritional value of seed, particularly by increasing the energy availability of seed.
  • Reduction in the alpha-zein levels in such seed can be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and up to 100%.
  • Energy availability can be improved by at least 3%, 6%, 9%, 12%, 15%, 20% and greater.
  • the combination of inhibiting either, or both, alpha- or gamma- zein proteins, with over-expressing either, or both, 18kD alpha-globulin or 5OkD corn legumin 1 leads to an increase in grain digestibility greater than inhibition of alpha- and gamma-zein proteins alone or increasing levels of 18kD alpha-globulin and/or 5OkD corn legumin 1 alone.
  • Methods of the invention are also directed to the reduction or elimination of the expression of one or more specific prolamin-like proteins in the grain of wheat, barley, oats, and rye that are known to give rise to anti-nutritional peptides.
  • These proteins include, but are not limited to, the alpha-, beta-, and gamma-gliadins of wheat. Grain and grain products possessing reduced levels of these proteins would not possess such negative characteristics as inducing coeliac disease or stimulating an allergic response.
  • modifications made to the grain by the present invention typically do not compromise grain handling properties with respect to mechanical damage: taking into account that grain handling procedures are adapted to specific properties of the modified grain.
  • Mechanical damage to grain is a well- described phenomenon (e.g., McKenzie, B.A., Am Soc Ag Engineers (No: 85- 3510): 10pp, 1985) that contributes to dust in elevators and livestock facilities, and which may increase susceptibility to pests. Grain damage can be quantified and assessed by objective measures (e.g., Gregory, J. M., et al., Am Soc. Ag.
  • the invention also encompasses modulation of an 18 kD alpha-globulin protein or a corn legumin 1 protein to affect the nutritional value and/or the hardness of plant seed.
  • a decrease in or an elimination of the expression of at least one of these proteins results in seed with decreased nutritional value.
  • Such grain has applications for use in diet food products.
  • an increase in the levels of these proteins in plant seed would result in an increase in the nutritional value of the seed.
  • the levels of the maize 18 kD alpha-globulin protein can be increased in maize seed, resulting in seed that can be predicted to possess at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, and up to a 300% increase in tryptophan and sulfur-containing amino acids relative to grain of wild-type plants.
  • the level of the corn legumin 1 protein can be similarly increased in maize seed to increase the level of essential amino acids in the grain. Food products and feed based on such seed will have a higher nutritional value based on the increased levels of essential amino acids.
  • an increase in the level of the 18 kD alpha-globulin protein in plant seed can be predicted to result in grain possessing altered hardness. This is due to an increase in non-zein protein accumulated in non-zein storage organelles relative to grain from wild-type plants, and has applications for improving the dry-milling properties of such modified grain.
  • Introduction of this trait into corn plants with inferior kernel phenotypes, particularly inferior kernel phenotypes induced by the introduction of other transgenic polypeptides including, but not limited to, hordothionin 12 (US Patent No. 5,990,389), can ameliorate or eliminate the undesirable dry-milling properties of such grain by altering seed hardness.
  • the levels of the 50 kD legumin 1 polypeptide are increased in cereal grain for the purpose of increasing the metal chelating properties of the grain.
  • the unique string of histidine residues present in the 50 kD legumin 1 polypeptide function as a metal chelating site. Products produced from such grain could be used for bio-remediation, in food health (antioxidant) applications, or in biofortification (increase of zinc and iron bioavailability).
  • Methods are provided for modulating the level of at least one seed protein in plant seed including, but not limited to seed proteins such as: zeins, such as the 50 kD gamma-zein (SEQ ID NO:2), ), the 27 kD gamma-zein (Accession No.
  • the 16 kD gamma-zein (Accession No. AAA33523), the15 kD beta-zein (Accession No. P06673) the delta zeins (Woo, et al) the alpha-zeins (Song et al, 2002, 2001 ), and the like, globulins, such as the 18 kD alpha-globulin (SEQ ID NO:4), legumins such as the corn legumin 1 (SEQ ID NO:6), the kafarins, the alpha-, beta-, and gamma-gliadins and the like.
  • globulins such as the 18 kD alpha-globulin (SEQ ID NO:4)
  • legumins such as the corn legumin 1 (SEQ ID NO:6)
  • the kafarins the alpha-, beta-, and gamma-gliadins and the like.
  • the methods of the invention comprise either increasing or decreasing the level of a target gene product.
  • Methods for inhibiting gene expression are well known in the art. Although any method know in the art for reducing the level of protein in a plant could be used, possible methods for reducing protein include, but are not limited to, homology-dependent gene silencing, antisense technology, co-suppression including, for example, RNA interference (RNAi), micro RNA and the like, site-specific recombination, site- specific integration, mutagenesis including transposon tagging, and biosynthetic competition, homologous recombination, and gene targeting, alone or in combination.
  • RNAi RNA interference
  • micro RNA micro RNA
  • site-specific recombination site-specific integration
  • mutagenesis including transposon tagging
  • biosynthetic competition homologous recombination
  • gene targeting alone or in combination.
  • the level of at least one seed protein may be increased, decreased, or eliminated entirely as described below.
  • Methods of the invention can be utilized to alter the level of any seed protein found within a particular plant species, including but not limited to, the alpha-, beta-, delta-, gamma-zeins of maize, and alpha-globulins of maize, the legumin 1 and other seed proteins of maize, rice and sorghum, and the alpha-, beta-, and gamma-gliadins of wheat, barley, rye, and oats.
  • the nucleotide sequences for use in the methods of the present invention are provided in transcriptional units with for transcription in the plant of interest.
  • a transcriptional unit is comprised generally of a promoter and a nucleotide sequence operably linked in the 3' direction of the promoter, optionally with a terminator.
  • operably linked is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • the expression cassette will include 5 1 and 3' regulatory sequences operably linked to at least one of the sequences of the invention.
  • operably linked means that the nucleotide sequences being linked are contiguous and, where necessary to join two or more protein coding regions, contiguous and in the same reading frame.
  • the encoded polypeptide is herein defined as a "heterologous polypeptide” or a “chimeric polypeptide” or a "fusion polypeptide”.
  • the cassette may additionally contain at least one additional coding sequence to be co-transformed into the organism.
  • the additional coding sequence(s) can be provided on multiple expression cassettes.
  • the methods of transgenic expression can be used to increase the level of at least one seed protein in grain.
  • the methods of transgenic expression comprise transforming a plant cell with at least one expression cassette comprising a promoter that drives expression in the plant operably linked to at least one nucleotide sequence encoding a seed protein.
  • Methods for expressing transgenic genes in plants are well known in the art.
  • nucleotide sequences for use in the methods of the invention are provided in transcriptional units as co-supression cassettes for transcription in the plant of interest.
  • Transcription units can contain coding and/or non-coding regions of the genes of interest. Additionally, transcription units can contain promoter sequences with or without coding or non-coding regions.
  • the co-suppression cassette may include 5' (but not necessarily 3') regulatory sequences, operably linked to at least one of the sequences of the invention.
  • Co- supression cassettes used in the methods of the invention can comprise sequences of the invention in so-called "inverted repeat" structures.
  • the cassette may additionally contain a second copy of the fragment in opposite direction to form an inverted repeat structure: opposing arms of the structure may or may not be interrupted by any nucleotide sequence related or unrelated to the nucleotide sequences of the invention, (see Fiers et al. US Pat No: 6,506,559).
  • the transcriptional units are linked to be co-transformed into the organism.
  • additional transcriptional units can be provided on multiple over- expression and co-suppression cassettes.
  • the methods of transgenic co-suppression can be used to reduce or eliminate the level of at least one seed protein in grain.
  • One method of transgenic co-suppression comprise transforming a plant cell with at least one transcriptional unit containing an expression cassette comprising a promoter that drives transcription in the plant operably linked to at least one nucleotide sequence transcript in the sense orientation encoding at least a portion of the seed protein of interest.
  • Methods for suppressing gene expression in plants using nucleotide sequences in the sense orientation are known in the art.
  • the methods generally involve transforming plants with a DNA construct comprising a promoter that drives transcription in a plant operably linked to at least a portion of a nucleotide sequence that corresponds to the transcript of the endogenous gene.
  • nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, at least about 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity over the entire length of the sequence.
  • portions, rather than the entire nucleotide sequence, of the polynucleotides may be used to disrupt the expression of the target gene product.
  • sequences of at least 10, 15, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200 nucleotides, or greater may be used. See U.S. Patent Nos. 5,283,184 and 5,034,323; herein incorporated by reference.
  • the endogenous gene targeted for co-suppression may be a gene encoding any seed protein that accumulates as a seed protein in the plant species of interest, including, but not limited to, the seed genes noted above.
  • the endogenous gene targeted for co-suppression is the 50 kD gamma-zein gene disclosed herein
  • co-suppression is achieved using an expression cassette comprising the 50 kD gamma-zein gene sequence, or variant or fragment thereof.
  • RNA interference and promoter silencing involve the silencing of a targeted gene by spliced hairpin RNA's and similar methods also called RNA interference and promoter silencing (see Smith et al. (2000) Nature 407:319-320, Waterhouse and Helliwell (2003)) Nat. Rev. Genet. 4:29-38; Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964; Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk et al. (2002) Plant Phystiol.
  • co- suppression is used to collectively designate gene silencing methods based on mechanisms involving the expression of sense RNA molecules, aberrant RNA molecules, double-stranded RNA molecules, micro RNA molecules and the like.
  • the expression cassette for co-suppression may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA.
  • the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene.
  • it is the loop region that determines the specificity of the RNA interference.
  • inhibition of the expression of a protein of interest may be obtained by RNA interference by expression of a gene encoding a micro RNA (miRNA).
  • miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNA are highly efficient at inhibiting the expression of endogenous genes. See, for example Javier et al. (2003) Nature 425: 257-263, herein incorporated by reference.
  • the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene.
  • the miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence).
  • miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants.
  • the polynucleotide to be introduced into the plant comprises an inhibitory sequence that encodes a zinc finger protein that binds to a gene encoding a protein of the invention resulting in reduced expression of the gene.
  • the zinc finger protein binds to a regulatory region of a zein gene, a legumin gene or a globulin gene.
  • the zinc finger protein binds to a messenger RNA encoding a seed protein and prevents its translation.
  • the methods of antisense suppression comprise transforming a plant cell with at least one expression cassette comprising a promoter that drives expression in the plant cell operably linked to at least one nucleotide sequence that is antisense to a nucleotide sequence transcript of such a gamma-zein gene.
  • antisense suppression is intended the use of nucleotide sequences that are antisense to nucleotide sequence transcripts of endogenous plant genes to suppress the expression of those genes in the plant.
  • the methods generally involve transforming plants with a DNA construct comprising a promoter that drives expression in a plant operably linked to at least a portion of a nucleotide sequence that is antisense to the transcript of the endogenous gene.
  • Antisense nucleotides are constructed to hybridize with the corresponding mRNA. Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA.
  • antisense constructions having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the corresponding antisense sequences may be used.
  • portions, rather than the entire nucleotide sequence, of the antisense nucleotides may be used to disrupt the expression of the target gene.
  • sequences of at least 10 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be used.
  • Methods for transposon tagging can be used to reduce or eliminate the level of at least one seed protein in grain.
  • the methods of transposon tagging comprise insertion of a transposon within an endogenous plant seed gene to reduce or eliminate expression of the seed protein.
  • mutagenesis such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted (for examples of these methods see Ohshima et al. (1998) Virology 243:472-481 ; Okubara et al. (1994) Genetics 137:867-874; Quesada et al. (2000) Genetics 154:421-436.
  • TILLING Targeting Induced Local Lesions In Genomes
  • a denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention (see McCallum et al. (2000) Nat. Biotechnol. 18:455-457).
  • the coding sequence of the 27kD gamma-zein protein can be replaced by the coding sequence of the 18 kD alpha-globulin resulting in suppression of 27kD gamma-zein protein expression and in over-expression of the alpha-globulin protein.
  • the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of a protein of interest.
  • the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of one or more proteins. This method is described, for example, in U.S. Patent No. 4,987,071 , herein incorporated by reference.
  • the polynucleotide comprises an inhibitory sequence that encodes an antibody that binds to at least one isoform of a seed protein, and reduces the level of the seed protein.
  • the binding of the antibody results in increased turnover of the antibody-antigen complex by cellular quality control mechanisms.
  • the expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald (2003) Nature Biotech. 21 :35-36, incorporated herein by reference.
  • Methods of biosynthetic competition with other high-sulfur-containing proteins are used to reduce the levels of at least one seed protein in plant seed.
  • the methods of biosynthetic competition comprise transforming plant cells with at least one expression cassette comprising a promoter that drives expression in the plant cell operably linked to at least one nucleotide sequence encoding a protein selected from the group consisting of delta-zeins, hordothionin 12, and other naturally occurring or engineered high-sulfur-containing proteins.
  • the competing protein may possess a high lysine content in addition to a high sulfur content to further increase the nutritional value of the grain.
  • Biosynthetic competition of seed proteins with other sulfur-rich proteins occurs naturally. This natural process can be manipulated to reduce the levels of certain seed proteins, because the synthesis of some seed proteins is transcriptionally and/or translationally controlled by the nitrogen and/or sulfur supply in the developing seed.
  • the expression of recombinant polypeptides, including the ectopic (transgenic) expression of seed proteins or other high-sulfur-, high-nitrogen-containing proteins can have a substantial impact on intracellular nitrogen and sulfur pools.
  • the expression of these proteins can result in suppression of the expression of other seed proteins such as, for example, the high-sulfur containing gamma-zein proteins.
  • Plant transformants containing a desired genetic modification as a result of any of the above described methods resulting in increased, decreased or eliminated expression of the seed protein of the invention can be selected by various methods known in the art. These methods include, but are not limited to, methods such as SDS-PAGE analysis, immunoblotting using antibodies which bind to the seed protein of interest, single nucleotide polymorphism (SNP) analysis, or assaying for the products of a reporter or marker gene, and the like.
  • SNP single nucleotide polymorphism
  • Another embodiment is directed to the screening of transgenic maize plants for specific phenotypic traits conferred by the expression, or lack thereof, of known corn seed proteins and the 50 kD gamma-zein, the 18 kD alpha-globulin, or the 50 kD legumin 1 polypeptides of the invention.
  • the specific phenotypic traits for which this method finds use include, but are not limited to, all of those traits listed herein.
  • Maize lines can be screened for a particular phenotypic trait conferred by the presence or absence of known com seed proteins and the 50 kD gamma-zein, the 18 kD alpha-globulin, or the 50 kD legumin 1 protein using an antibody that binds selectively to one of these polypeptides.
  • tissue from the maize line of interest is contacted with an antibody that selectively binds the seed- protein polypeptide for which the screen is designed.
  • recombinant DNA techniques known in the can be used to produce an expression cassette encoding a heterologous polypeptide consisting of the 50 kD legumin 1 polypeptide or a fragment thereof fused to a polypeptide of interest.
  • the expression cassette can be introduced into either a eucaryotic or a bacterial host cell and the protein expressed in the host cell.
  • the protein can then be isolated from the cells by an appropriate purification scheme using standard metal chelating column techniques such as high affinity nickel chelating columns that are commercially available.
  • the legumin 1 nucleotide sequence can be fused to either the N-terminus or C-terminus of the nucleotide sequence encoding the polypeptide of interest.
  • a plant is genetically manipulated to have a suppressed or increased level of one or more seed proteins in seed and/or to ectopically express one or more seed or other high-sulfur, high-lysine-containing protein.
  • each of the respective coding sequences for such proteins can be operably linked to a promoter and then joined together in a single continuous fragment of DNA comprising a multigenic expression cassette.
  • Such a multigenic expression cassette can be used to transform a plant to produce the desired outcome utilizing any of the methods of the invention including sense and antisense suppression and biosynthetic competition.
  • Transgenic plants resulting from any or a combination of methods including any method to modulate protein levels can be selected by standard methods available in the art. These methods include, but are not limited to, methods such as immunoblotting using antibodies which bind to the proteins of interest, SNP analysis, or assaying for the products of a reporter or marker gene, and the like. Then, all of the desired coding sequences and/or transposon tagged sequences can be brought together into a single plant through one or more rounds of cross pollination utilizing the previously selected transformed plants as parents.
  • the nucleotide sequences for use in the methods of the present invention are provided in expression cassettes for transcription in the plant of interest.
  • Such expression cassettes are provided with a plurality of restriction sites for insertion of the 50 kD gamma-zein, the 18 kD alpha-globulin, the 50 kD legumin 1 sequence or any other sequence of the present invention to be placed under the transcriptional regulation of the regulatory regions.
  • the expression cassettes may additionally contain selectable marker genes.
  • the expression cassette can include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, any seed protein sequence of the invention, and optionally, a transcriptional and translational termination region functional in plants.
  • the transcriptional initiation region may be native or analogous or foreign or heterologous to the plant host.
  • the promoter may be the natural sequence or alternatively a synthetic sequence.
  • “foreign” is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced.
  • a gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • a gene comprises fragments of at least two independent transcripts that are linked in a single transcription unit.
  • the native promoter sequences may be used. Such constructs would alter expression levels of the proteins in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.
  • the promoter sequence may be used to alter expression.
  • the promoter (or fragments thereof) of 27 kD gamma-zein can modulate expression of the native 27 kD gamma-zein protein or other closely related proteins.
  • Use of a termination region is not necessary for proper transcription of plant genes but may be used as part of an expression construct. The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991 ) MoI. Gen. Genet. 262:141-144; Proudfoot (1991 ) Cell 64:671-674; Sanfacon et al. (1991 ) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91 :151- 158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987). Nucleic Acid Res. 15:9627-9639.
  • the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831 , and 5,436,391 , and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • the expression cassettes may additionally contain 5 1 leader sequences in the expression cassette construct.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al.
  • EMCV leader Engelphalomyocarditis 5' noncoding region
  • potyvirus leaders for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):23
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • the expression cassette will comprise a selectable marker gene for the selection of transformed cells.
  • Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase Il (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton
  • nucleotide constructs are not intended to limit the present invention to nucleotide constructs comprising DNA.
  • nucleotide constructs particularly polynucleotides and oligonucleotides, comprised of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein.
  • nucleotide constructs of the present invention encompass all nucleotide constructs that can be employed in the methods of the present invention for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues.
  • the nucleotide constructs of the invention also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
  • the methods of the invention may employ a nucleotide construct that is capable of directing, in a transformed plant, the expression of at least one protein, or at least one RNA, such as, for example, an antisense RNA that is complementary to at least a portion of an mRNA.
  • the methods of the invention may employ a nucleotide construct that is not capable of directing, in a transformed plant, the expression of a protein or an RNA.
  • methods of the present invention do not depend on the incorporation of the entire nucleotide construct into the genome, only that the plant or cell thereof is altered as a result of the introduction of the nucleotide construct into a cell.
  • the genome may be altered following the introduction of the nucleotide construct into a cell.
  • the nucleotide construct, or any part thereof may incorporate into the genome of the plant.
  • Alterations to the genome of the present invention include, but are not limited to, additions, deletions, and substitutions of nucleotides in the genome.
  • nucleotide constructs of the invention also encompass nucleotide constructs that may be employed in methods for altering or mutating a genomic nucleotide sequence in an organism, including, but not limited to, chimeric vectors, chimeric mutational vectors, chimeric repair vectors, mixed-duplex oligonucleotides, self-complementary chimeric oligonucleotides, and recombinogenic oligonucleobases.
  • nucleotide constructs and methods of use such as, for example, chimeraplasty
  • Chimeraplasty involves the use of such nucleotide constructs to introduce site-specific changes into the sequence of genomic DNA within an organism. See U.S. Patent Nos. 5,565,350; 5,731 ,181 ; 5,756,325; 5,760,012; 5,795,972; and 5,871 ,984; all of which are herein incorporated by reference. See also, WO 98/49350, WO
  • a number of promoters can be used in the practice of the invention.
  • the promoters can be selected based on the desired outcome.
  • the nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants, more preferably a promoter functional during seed development.
  • Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171 ); ubiquitin (Christensen et al. (1989) Plant MoI. Biol. 12:619-632 and Christensen et al. (1992) Plant MoI. Biol. 18:675- 689); pEMU (Last et al. (1991 ) Theor. Appl. Genet.
  • Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-Ia promoter, which is activated by salicylic acid.
  • promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991 ) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNeINs et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991 ) MoI. Gen. Genet. 227:229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156), herein incorporated by reference.
  • Tissue-preferred promoters can be utilized to target enhanced protein expression within a particular plant tissue.
  • Tissue-preferred promoters include, but are not limited to: Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) MoI. Gen Genet. 254(3):337-343; Russell et'al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331 -1341 ; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol.
  • seed-specific promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10:108, herein incorporated by reference.
  • seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kD zein); and milps (myo-inositol-1 -phosphate synthase; see US Pat. No 6,225,529 herein incorporated by reference).
  • the 27kD gamma-zein is a preferred endosperm- specific promoter.
  • Glb-1 is a preferred embryo-specific promoter.
  • seed-specific promoters include, but are not limited to, bean ⁇ -phaseolin, napin, ⁇ - conglycinin, soybean lectin, cruciferin, and the like.
  • seed-specific promoters include, but are not limited to, maize 15 kD zein, 22 kD zein, 27 kD zein, 1OkD delta-zein, waxy, shrunken 1 , shrunken 2, globulin 1 , etc.
  • nucleic acid sequences of the present invention can be combined with any combination of polynucleotide sequences of interest or mutations in order to create plants with a desired phenotype.
  • the polynucleotides of the present invention can be combined with any other polynucleotides of the present invention, such as any combination of SEQ ID NOS: 1 , 3, 5, or with other seed storage protein genes or variants or fragments thereof such as: zeins, fatty acid desaturases, lysine ketoglutarate, led, or Agp.
  • the combinations generated can also include multiple copies of any one of the polynucleotides of interest.
  • polynucleotides or mutations of the present invention can also be combined with any other gene or combination of genes to produce plants with a variety of desired trait combinations including, but not limited to, traits desirable for animal feed such as high oil genes (e.g., U.S. Patent No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S. Patent Nos. 5,990,389; 5,885,801 ; 5,885,802; 5,703,409 and 6,800,726); high lysine (Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122); and high methionine proteins (Pedersen et al.
  • traits desirable for animal feed such as high oil genes (e.g., U.S. Patent No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S. Patent Nos. 5,990,389; 5,885,
  • polynucleotides of the present invention can also be combined with traits desirable for insect, disease or herbicide resistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Patent Nos.
  • acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations
  • inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene)
  • traits desirable for processing or process products such as high oil (e.g., U.S. Patent No. 6,232,529 ); modified oils (e.g., fatty acid desaturase genes (U.S. Patent No.
  • modified starches e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)
  • polymers or bioplastics e.g., U.S. patent No. 5.602,321 ; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bactehol. 170:5837- 5847) facilitate expression of polyhydroxyalkanoates (PHAs)), the disclosures of which are herein incorporated by reference.
  • PHAs polyhydroxyalkanoates
  • the traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes.
  • the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters.
  • Methods of the invention can be utilized to alter the level of at lease one seed protein in seed from any plant species of interest.
  • Plants of particular interest include grain plants that provide seeds of interest including grain seeds such as corn, wheat, barley, rice, sorghum, rye, oats, etc.
  • the present invention may be used for many plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., S. napus, B. rapa, B.
  • juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower ⁇ Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), oats, and barley.
  • millet e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)),
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include, but are not limited to: microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (Townsend et al., U.S. Patent No. 5,563,055; Zhao et al., U.S. Patent No.
  • the methods of the invention involve introducing a nucleotide construct into a plant.
  • introducing is intended presenting to the plant the nucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant.
  • the methods of the invention do not depend on a particular method for introducing a nucleotide construct to a plant, only that the nucleotide construct gains access to the interior of at least one cell of the plant.
  • Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
  • stable transformation is intended that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof.
  • transient transformation is intended that a nucleotide construct introduced into a plant does not integrate into the genome of the plant.
  • the nucleotide constructs of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that the protein of interest of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing nucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos. 5,889,191 , 5,889,190, 5,866,785, 5,589,367 and 5,316,931 ; herein incorporated by reference.
  • the cells that have been transformed may be grown into plants in accordance with conventional ways, under plant forming conditions. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
  • the desired genetically altered trait can be bred into other plant lines possessing desirable agronomic characteristics using conventional breeding methods (see Example 3) and/or top-cross technology.
  • the top-cross method is taught in US Pat. No. 5,704,160 herein incorporated in its entirety by reference. Methods for cross pollinating plants are well known to those skilled in the art, and are generally accomplished by allowing the pollen of one plant, the pollen donor, to pollinate a flower of a second plant, the pollen recipient, and then allowing the fertilized eggs in the pollinated flower to mature into seeds.
  • Progeny containing the entire complement of heterologous coding sequences of the two parental plants can be selected from all of the progeny by standard methods available in the art as described infra for selecting transformed plants. If necessary, the selected progeny can be used as either the pollen donor or pollen recipient in a subsequent cross pollination.
  • This invention allows for the improvement of grain properties such as increased digestibility/nutrient availability, nutritional value, silage quality, and efficiency of wet or dry milling in maize strains already possessing other desirable characteristics.
  • Corn grain with reduced gamma-zein protein content offers the following advantages: First, ground corn grain with a reduced gamma-zein protein content offer increased energy availability and protein digestibility to monogastric livestock (see Example 2). "Monogastric animals” include but are not limited to: pigs, poultry, horses, dogs, cats, rabbits and rodents. It can be deduced from analysis of the in vitro experimental data provided herein that the corn grain from maize genetically altered to contain reduced gamma-zein protein levels will have a 5% increase in metabolizable energy for poultry and pigs. Using this assumption the following replacement value can be assigned to the high energy availability trait in grain resulting from the reduction of gamma-zein protein.
  • Grain is fed to ruminants in minimally processed form, and the rigid protein structure of corn endosperm has been shown to constitute a large impediment to microbial digestion in the rumen, which can be partly overcome by predigestion with protease (McAllister et al. (1993) J. Anim. Sci. 77:205-212).
  • a reduced gamma-zein protein content imparts a similar or even larger improvement to ruminal digestion of whole corn.
  • corn grain with reduced levels of gamma-zein proteins has an increased response to feed processing.
  • the nutrient availability from whole corn grain can be increased by extensive processing (steam-flaking or extrusion) resulting in starch gelatinization and protein disulfide bond reduction (Blackwood and Richardson, 1994).
  • the response to processing is sometimes lower than expected.
  • the heat and/or shearing force applied during processing causes rearrangements of protein disulfide bonds, which may partly counteract the improvement in digestibility resulting from starch gelatinization.
  • the response to steam-flaking of corn and sorghum grain is negatively correlated with protein disulfide content (Blackwood and Richardson, 1994).
  • corn grain with a lower gamma-zein protein content has improved silage quality for dairy cattle, especially for silage harvested at late maturity.
  • silage is harvested at earlier maturity than grain, a certain degree of dry- down (and protein disulfide formation) has already occurred by the time the crop is ensiled, especially under dry and hot conditions.
  • Our work has shown that pretreatment with a reducing agent of immature, dough-stage corn kernels, sampled at silage maturity, resulted in drastically improved in vitro digestibility, a strong indication that the protein disulfide imposed barriers to digestion had already been established, (data in Example 2).
  • the digestibility of the "yellow portion" of corn silage can be expected to be higher for grain with a reduced gamma-zein protein content. Increased digestibility will be especially notable in the case of silage made from mature corn and for high-yielding dairy cows in which high passage rates do not allow for extensive ruminal digestion.
  • corn grain with a reduced gamma-zein protein content will have an increased efficiency of wet milling. An increase in wet-milling efficiency and starch recovery can be expected due to the lower disulfide content of grain with reduced gamma-zein protein content.
  • Efficiencies in the processes of wet milling also include reduced steeping time and/or reduced need for chemical reductants such as sulfur dioxide and sodium bisulfite. The use of fewer chemicals will improve wet-milling economics and reduce environmental pollution.
  • Maize lines (transgenic and transposon-mutagenized) have been developed with increased or decreased levels of specific endosperm proteins. For several of the obtained lines, experimental evidence indicates that the introduced changes result in improved grain properties.
  • Gamma-Zein A 50 kD gamma-zein nucleotide sequence was cloned from a maize endosperm cDNA library (mid and late development). Based on EST numbers 50 kD gamma-zein transcripts are relatively abundant (compared to other seed protein transcripts) and represent approximately 0.5% of the endosperm mRNA during mid development. A large variation in the abundance of 50 kD gamma-zein transcripts has been observed between different inbred lines (transcript profiling results). The 50 kD gamma-zein gene has been located on chromosome 7, bin
  • the 5OkD gamma-zein cDNA sequences isolated from different inbred lines show an unusually low level of polymorphism. Only one SNP (a 3bp insertion) was detected along the entire cDNA sequence from DNA isolated from the inbred lines
  • the 50-kD gamma-zein transformation event described herein was one of various high-digestibility events produced.
  • the event was generated with a construct containing the 27kD gamma-zein promoter, 50 kD gamma-zein ORF in sense orientation, and 27-kD gamma zein terminator using particle bombardment. It was found to be reduced in all known gamma zein proteins, i.e., 50 kD-, 27 kD-, and 16 kD gamma-zein. Protein gel & 50 kD gamma-zein Western blots of segregating CS50 events were performed to confirm co-suppression. The kernel phenotype of the transgenic seed was normal (i.e., vitreous).
  • Segregating kernels from transgenic corn co-suppressed in 50 kD gamma- zein were ground to a fine meal and subjected to the monogastric in vitro digestibility assay as described in Example 2 to determine Enzyme Digestible Dry Matter (EDDM).
  • EDDM of 50 kD gamma-zein co-suppressed grain was improved by 3.0 percentage units.
  • DTT dithiothreietol
  • Example 2 In Vitro Enzyme Digestible Dry Matter (EDDM) Assay.
  • Corn grain was ground in a micro Wiley Mill (Thomas Scientific, Swedesboro, NJ) through a 1 mm screen; 0.5 g of ground corn sample was placed in a pre-weighed nylon bag (50 micron pore size) and heat sealed. Approximately 40 bags were placed in an incubation bottle with 2L of 0.2M phosphate buffer (pH 2.0) containing pepsin (0.25 mg/ml). Samples were incubated in a Daisy Il incubator (ANKOM Technology, Fairport, NY) at 39°C for 2 hours. After 2 hours, samples were placed in a mesh bag and washed for 2 minutes with cold water in a washer (Whirlpool) using delicate cycle.
  • a micro Wiley Mill Thiomas Scientific, Swedesboro, NJ
  • Approximately 40 bags were placed in an incubation bottle with 2L of 0.2M phosphate buffer (pH 2.0) containing pepsin (0.25 mg/ml). Samples were incubated in a Daisy Il incubator (ANKOM Technology, Fairport
  • EDDM enzyme digestible dry matter digestibility
  • Example 3 Transgenic Co-suppression of 27 kD Gamma-Zein.
  • a second event was generated with a construct containing the CZ19B1 promoter (US Pat No:6,225,529), 27 kD gamma-zein ORF in sense orientation, and the 27-kD gamma-zein terminator using Agrobacterium-mediated transformation (see Example 12). It also was found to be reduced in 27 kD-, and 16 kD-gamma-zein.
  • the kernel phenotype of the transgenic seed was normal (i.e., vitreous).
  • Assays for seed hardness are well known in the art and include such methods as those used in the present invention, described in Pomeranz et a/. Cereal Chemistry 61 (2): 147-150 (1984), herein incorporated in its entirety by reference. In essence, breaking susceptibility and grain hardness are measured by density, near-infrared reflectance or average particle size of ground material. The three measure of hardness are highly, linearly, and positively correlated provided the maize samples are homogenous in terms of starch composition (such as waxy, regular or high amylose), and in protein, oil, and ash content.
  • starch composition such as waxy, regular or high amylose
  • the co-suppression trait was shown to be dominant.
  • Various normal and transgenic maize lines, as well as commercial hybrids, were pollinated with pollen from the gamma-zein co-suppressing events with the result of total suppression of gamma-zein protein in the hemizygous endosperm as determined by SDS-PAGE and immunoblotting. Therefore, the gamma-zein gene co-suppression trait can be introduced into specific pollinators (i.e., high oil corn) using conventional methods and/or the top-cross technology found in US Patent No. 5,704,160.
  • T 3 -segregating grain was phenotyped for gamma-zein protein levels and were divided into two samples, one with wild-type gamma-zein protein levels and a second with reduced gamma-zein protein levels (less than 10% of wild-type). Ground corn from both samples was subjected to an in vitro energy availability assay.
  • Phenotyped kernel samples (those with normal levels of 27 kD gamma-zein protein and those with low levels of 27 kD gamma-zein protein) from segregating ears from the same events were analyzed using a small-scale simulated wet-milling process incorporating or leaving out a reducing agent (bisulfite) in the steep water (Eckhoff et al., (1996) Cereal Chem. 73:54-57). Similar to the digestibility assay, the reductant had a lesser impact on starch extractability in grain containing low levels of 27 kD gamma-zein protein compared to wild-type grain.
  • a reducing agent bisulfite
  • Coarsely ground mature grain samples were weighed into pre-tared nylon bags (4 replicates each). The bags were sealed and placed in the rumen of a fistulated steer for 18hrs, then washed to remove microbial mass, ovendried, and weighed. Ruminal digestibility of the low gamma-zein grain was on average, 9% higher than the control top-cross and 5% higher than that of the grain parent. (See also: Nocek, J. E. 1988. In situ and other methods to estimate ruminal protein and energy digestibility. J. Dairy Sci. 71 :2051 -2069).
  • Immature kernels of various wild-type inbreds & hybrids sampled at various stages of seed development and maturation, consistently respond to DTT pretreatment in the monogastric in vitro assay when sampled 1 month after pollination or later.
  • the improvement in digestibility with DTT (20% at the 4 hour timepoint) points at a consistent inhibitory role of protein disulfide bonds on digestibility of wild-type kernels from about 28 DAP onwards. From these results one can conclude that kernels harvested at dough stage or silage maturity (approximately 40-45 DAP) would benefit from reduced gamma- zein levels.
  • the same low gamma-zein topcross was also compared with the control topcross in a pig in vivo digestion trial. Metabolizable Energy content of the low gamma zein topcross amounted to 3646 kcal/kg, 73 kcal (or 2%) higher than the control topcross. Protein digestibility was improved from 75.8 to 79.8% for the low gamma-zein topcross. This represents a 4% improvement in protein digestibility, and a 15% reduction in nitrogen excretion into the environment.
  • Example 4 Suppression of 27 kD Gamma-Zein through Interruption of the 27 kD Gamma-Zein Gene by Transposon Tagging.
  • a maize line containing a Mu-insertion in the 27kD gamma-zein coding region was isolated using the method of US Patent No. 5,962,764.
  • Pool screening was initiated with 27kD gamma-zein primers 296 (SEQ ID NO:11 ) and 170 (SEQ ID NO:12) in combination with Mu TIR primer 9242 (SEQ ID NO: 13). Pools were selected for fragment sizing based on signal intensity and reproducibility. Bands were detected in fifteen of the sixteen pools selected for fragment sizing with primer 170.
  • An individual master plate was constructed with the nine best pools for which bands were detected.
  • the nine pools were selected based on the putative insertion location of mutator in the gene and possibly the promoter region.
  • the material originating from this source was advanced genetically, predominately via backcrossing strategies, to create material suitable for feeding trials, energy availability studies, and product development applications. Grain from progeny of this TUSC27 line was tested in the in vitro digestibility assay (EDDM) with essentially similar results as observed with the 27 kD gamma-zein gene co-suppressed lines (see Example 3). Seed from this progeny is represented by, but not limited to, ATCC Dep. No:PTA-6323. Four F2 hybrid grain lines containing TUSC27 were assayed for dry matter digestibility (EDDM) using the procedure of Example 2 against four wild-type lines.
  • EDDM in vitro digestibility assay
  • the trait was semi-dominant rather than recessive: that is the 27 kD gamma-zein level in endosperm showed a strong gene-dosage effect.
  • Example 5 Suppression of 27 kD Gamma-Zein by Over-Expression of High- Sulfur Proteins through Competition for Biosynthetically Available Pools of Sulfur Amino Acids.
  • Transgenic plants expressing the 18 kD delta-zein protein or the engineered high-lysine, high-sulfur protein hordothionin 12 (US Patent 5,990,389) in the endosperm showed an 80% decrease in gamma-zein protein levels, possibly due to limitations of free sulfur-amino acid pools. Seed from these events were tested essentially under the same conditions as seed from gamma-zein gene co- suppressing events (see Example 2) using the in vitro digestibility assay in both the presence and absence of disulfide reducing agents. The results obtained were similar to those described in the previous two Examples. The reduced levels of gamma-zein protein had a large positive impact on dry matter digestibility in the absence of DTT.
  • Plants expressing high-sulfur protein showed 6% improvement in EDDM dry matter digestibility.
  • the improvement in digestibility in this case is not as high as in CS27 (example 3). This could be due to the higher residual level of gamma zein in high-sulfur protein plants as compared to CS27.
  • Maize plants ectopically expressing 18 kD delta-zein protein or hordothionin 12 protein in corn endosperm were both produced using the top-cross technology. Comparable results were also obtained using hemizygous seed from top-crossed elite inbreds and hybrids with hordothionin 12 corn as the male parent. Grain from these plants showed improved digestibility, and therefore improved energy availability.
  • Example 6 Cloning of a Novel Maize 18 kD Alpha-globulin.
  • W tryptophane residues
  • Example 7 Transgenic Expression of 18 kD Alpha-globulin in Maize.
  • the cDNA encoding the maize 18 kD alpha-globulin was placed under the control of the strong endosperm specific 27kD gamma-zein promoter and introduced into maize plants by Agrobacteri ⁇ m-med ⁇ a ⁇ ed transformation.
  • Several transgenic events were identified that had increased levels alpha-globulin protein as demonstrated by SDS-PAGE and staining of gels with Coomassie blue.
  • a prominent band was visible at a molecular weight corresponding to the 18 kD protein extracted from transgenic seed, but absent from protein extracted similarly from wild type seed.
  • the seed of transformants and progeny overexpressing 18kD alpha-globulin is phenotypically normal (vitreous).
  • the identity of the polypeptide migrating at 18 kD in the polyacrylamide gel was confirmed by immune blotting using 18 kD alpha-globulin protein specific antibodies.
  • the 18 kD alpha-globulin protein accumulated to levels of between 2-5% of the SDS-sample buffer (60 mM Tris, pH 6.8, 100 mM DTT, 2% SDS) extractable seed protein. Seed expressing these amounts of omega zein protein contained 0.162% tryptophan per dry weight
  • Example 8 Preparation of Maize 18 kD Alpha-globulin-Specific Antibodies.
  • antibodies for 18 kD alpha-globulin polypeptides were produced by injecting female New Zealand white rabbits (Bethyl Laboratory, Montgomery, Tex.) six times with homogenized polyacrylamide gel slices containing 100 micrograms of PAGE purified alpha-globulin polypeptide.
  • the alpha-globulin polypeptide was purified by sub-cloning into a pET28 vector resulting in an insert encoding a His-tag fusion of the alpha-globulin polypeptide.
  • the fusion protein was expressed in E. coli BL21 (DE3) cells and purified from the lysate by Nickel chelation chromatography. The denatured purified fusion protein was used for immunization.
  • Example 9 Cloning and Sequencing of Maize, Sorghum and Sugarcane 50 kD Legumin 1 Protein.
  • a 50 kD legumin 1 nucleotide sequence was cloned from a maize endosperm cDNA library (mid and late development). Based on EST numbers 50 kD legumin 1 transcripts are relatively abundant (compared to other seed protein transcripts) and represent approximately 0.5% of the endosperm mRNA during mid development. The 50 kD legumin 1 DNA sequences isolated from different inbred lines showed a considerable level of polymorphism. The 50 kD legumin 1 gene has been mapped to chromosome 6, Bin 6.01.
  • Example 10 Preparation of Maize 50 kD Legumin 1-Specific Antibodies. Antibodies to this protein were prepared essentially as described for the 18 kD alpha-globulin polypeptide.
  • Example 11 Transgenic Expression of 50 kD Legumin 1 in Maize.
  • Additional copies of the 50 kD legumin 1 cDNA under control of the strong endosperm specific 27kD gamma-zein promoter were introduced into transgenic corn plants.
  • Several maize lines were identified that over-express the 50 kD legumin 1 protein. Over-expression was demonstrated by SDS-PAGE and staining of the gels with Coomassie blue. A prominent band was visible at 50 kD in protein extracted from transgenic seed but absent in protein from wild type seed. The identity of the polypeptide band was confirmed to be the 50 kD legumin 1 protein by immune blotting using the 50 kD legumin 1 protein specific antibodies.
  • the 50 kD legumin 1 protein In the seed of transgenic maize plants over-expressing the 50 kD legumin 1 protein, this protein accumulates to levels of between 2-5% of the SDS-sample buffer (60 mM Tris, pH 6.8, 100 mM DTT 1 2% SDS) extractable seed protein. The seed over-expressing the 50 kD legumin 1 protein showed a normal (vitreous) phenotype. In addition to overexpression of the 50 kD legumin 1 , independent transformants were also obtained in which the legumin 1 gene was silenced as evidenced by reduced protein level using immune blotting. These events were also silenced for the 27 kD gamma-zein, by apparent promoter-induced silencing.
  • SDS-sample buffer 60 mM Tris, pH 6.8, 100 mM DTT 1 2% SDS
  • Example 12 Agrobacterium-Me ⁇ iated Transformation of Maize.
  • a nucleotide sequence encoding a protein of the present invention was operably linked to either the 27 kD gamma-zein promoter or the maize CZ19B1 promoter, and the method of Zhao was employed (U.S. Patent No. 5,981 ,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos were isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the nucleotide sequence of interest to at least one cell of at least one of the immature embryos (step 1: the infection step).
  • step 2 the co- cultivation step
  • step 3 the immature embryos were incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants.
  • step 3 resting step
  • the immature embryos were cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells.
  • step 4 the selection step
  • step 4 the selection step
  • step 5 the regeneration step
  • Example 13 Agrobacterium-Med ⁇ ated Transformation of Sorghum.
  • Example 14 Transformation of Maize Embryos by Particle Bombardment.
  • Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing the nucleotide sequence encoding a protein of the present invention operably linked to a selected promoter plus a plasmid containing the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to the herbicide Bialaphos. Transformation is performed as follows. Preparation of Target Tissue The ears are surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5-cm target zone in preparation for bombardment. Preparation of DNA
  • a plasmid vector comprising the nucleotide sequence encoding a protein of the present invention operably linked to a promoter is made.
  • This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 ⁇ m (average diameter) tungsten pellets using a CaCI 2 precipitation procedure as follows:
  • Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer.
  • the final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes.
  • the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 ⁇ l 100% ethanol is added to the final tungsten particle pellet.
  • the tungsten/DNA particles are briefly sonicated and 10 ⁇ l spotted onto the center of each macrocarher and allowed to dry about 2 minutes before bombardment.
  • sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
  • the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection- resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established.
  • Plants are then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for the desired phenotypic trait.
  • Bombardment medium comprises 4.0 g/l N6 basal salts (SIGMA C- 1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511 ), 0.5 mg/l thiamine HCI, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H 2 O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H 2 O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).
  • Selection medium comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511 ), 0.5 mg/l thiamine HCI, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H 2 O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H 2 O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos(both added after sterilizing the medium and cooling to room temperature).
  • Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO
  • Hormone-free medium comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/I MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H 2 O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D- I H 2 O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-I H 2 O), sterilized and cooled to 60 0 C.
  • Example 15 Transformation of Rice Embryogenic Callus by Bombardment.
  • Embryogenic callus cultures derived from the scutellum of germinating seeds serve as the source material for transformation experiments. This material is generated by germinating sterile rice seeds on a callus initiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-D and 10 ⁇ M AgNO 3 ) in the dark at 27-28°C.
  • Embryogenic callus proliferating from the scutellum of the embryos is then transferred to CM media (N6 salts, Nitsch and Nitsch vitamins, 1 mg/1 2,4-D, Chu et al., 1985, Sci. Sinica 18:659-668).
  • CM media N6 salts, Nitsch and Nitsch vitamins, 1 mg/1 2,4-D, Chu et al., 1985, Sci. Sinica 18:659-668.
  • Callus cultures are maintained on CM by routine sub-culture at two week intervals and used for transformation within 10 weeks of
  • Callus is prepared for transformation by subculturing 0.5-1.0 mm pieces approximately 1 mm apart, arranged in a circular area of about 4 cm in diameter, in the center of a circle of Whatman #541 paper placed on CM media. The plates with callus are incubated in the dark at 27-28 C for 3-5 days. Prior to bombardment, the filters with callus are transferred to CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr. in the dark. The petri dish lids are then left ajar for 20-45 minutes in a sterile hood to allow moisture on tissue to dissipate.
  • Circular plasmid DNA from two different plasmids one containing the selectable marker for rice transformation and one containing the nucleotide of the invention are co-precipitated onto the surface of gold particles.
  • a total of 10 ⁇ g of DNA at a 2:1 ratio of trait:selectable marker DNAs is added to a 50 ⁇ l aliquot of gold particles resuspended at a concentration of 60 mg ml- 1 .
  • Calcium chloride (50 ⁇ l of a 2.5 M solution) and spermidine (20 ⁇ l of a 0.1 M solution) are then added to the gold-DNA suspension as the tube is vortexing for 3 min.
  • the gold particles are centrifuged in a microfuge for 1 sec and the supernatant removed.
  • the gold particles are then washed twice with 1 ml of absolute ethanol and then resuspended in 50 ⁇ l of absolute ethanol and sonicated (bath sonicator) for one second to disperse the gold particles.
  • the gold suspension is incubated at -70 C for five minutes and sonicated (bath sonicator) if needed to disperse the particles.
  • Six ⁇ l of the DNA-coated gold particles are then loaded onto mylar macrocarrier disks and the ethanol is allowed to evaporate.
  • a petri dish containing the tissue is placed in the chamber of the PDS-1000/He.
  • the air in the chamber is then evacuated to a vacuum of 28-29 inches Hg.
  • the macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1080-1100 psi.
  • the tissue is placed approximately 8 cm from the stopping screen and the callus is bombarded two times. Five to seven plates of tissue are bombarded in this way with the DNA-coated gold particles. Following bombardment, the callus tissue is transferred to CM media without supplemental sorbitol or mannitol.
  • SM media CM medium containing 50 mg/l hygromycin.
  • callus tissue is transferred from plates to sterile 50 ml conical tubes and weighed. Molten top-agar at 4O 0 C is added using 2.5 ml of top agar/100 mg of callus.
  • Callus clumps are broken into fragments of less than 2 mm diameter by repeated dispensing through a 10 ml pipet. Three ml aliquots of the callus suspension are plated onto fresh SM media and the plates incubated in the dark for 4 weeks at 27-28°C. After 4 weeks, transgenic callus events are identified, transferred to fresh SM plates and grown for an additional 2 weeks in the dark at 27-28°C.
  • RM1 media MS salts, Nitsch and Nitsch vitamins, 2% sucrose, 3% sorbitol, 0.4% gelrite + 50 ppm hyg B
  • RM2 media MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 0.4% gelrite + 50 ppm hyg B
  • Plants are transferred from RM3 to 4" pots containing Metro mix 350 after 2-3 weeks, when sufficient root and shoot growth has occurred. Plants are grown using a 12 hr/12 hr light/dark cycle using -30/18°C day/night temperature regimen.
  • Example 16 Breeding crosses made with transgenic or mutant high-digestible- grain corn lines.
  • a transgenic line that segregated for co-suppressed 4-coumarate ligase (4CL) was planted and the segregating progeny was either self-fertilized or pollinated with the transgenic low gamma-zein line.
  • Ground grain samples were subjected to a two-stage /n-wfro-mimicking small intestinal digestion procedure as described in Example 2 followed by an additional step that included viscozyme that mimics large-intestinal fermentation (Boisen and Fernandez, 1997, Animal Feed Science and Technology 68:277-286).
  • Example 17 Isolation of Kafirin sequences from Sorghum.
  • Kafirin fragments for RNAi cassette construction are obtained by PCR amplification from kafirin cDNA clones.
  • a sorghum cDNA library from developing endosperm (20 days after pollination) is constructed and EST sequences are obtained from 1000 randomly selected cDNA clones.
  • the EST sequences are clustered into EST contigs and analyzed to determine the complete transcript sequences and the relative expression levels of kafirin genes.
  • Example 18 Modulating Seed Proteins in Sorghum.
  • a plant transformation vector for the delivery of the two tandem- assembled RNAi gene suppression cassettes are constructed. Each step of vector construction is performed by standard DNA analysis techniques (See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor,
  • Aqrobacterium-mediated transformation of sorghum is performed by the method described in Example 12.
  • Biolistic transformation is done by co-bombarding minimal concentrations of linearized transgene fragments and the PMI selectable marker cassettes. This strategy has been successfully used to minimize DNA rearrangements in transgenic plants (Loc, et al., 2002; Breitler, et.al., 2002) and reduces the risk of trait loss due to transgene silencing.
  • the PMI system (see above) addresses concerns often associated with transgenic crops by avoiding herbicide resistance for selection.
  • Event analysis has two major components: 1 ) PCR for trait gene copy number, absence of vector backbone DNA, herbicide gene elimination, and Southern for rearrangement analysis; and 2) digestibility, seed protein and micronutrient analysis.
  • the marker gene typically at least 50% of the events segregate for the marker gene. Segregation and elimination of the marker gene are assayed by PCR of 50 segregating T2 plants. Seedlings that contain only the trait genes are transferred to pots for propagation. The seeds harvested from these marker-free plants are used for event analysis and trait gene expression analysis.
  • the trait efficacy of the remaining events are assessed by protein expression analysis (protein electrophoresis, immune blotting) and by grain composition analysis. Altered expression of kafirin genes can easiest be assayed in stained protein gels. Zein-antibodies that cross-react with corresponding kafirins are used.
  • Grain samples are further evaluated for grain quality characteristics (hardness, grain moisture, test weight) and grain yield. The outcome of this analysis is the selection of 5 events (per transformation experiment) for breeding and field release.
  • Expression cassettes were made comprising a chimeric maize alpha-zein fusion of polynucleotide fragments from the coding regions of 19KD alpha-zein clone D1 (563 bp), 19KD alpha-zein clone B1 (536 bp), and 22KD alpha-zein (610 bp) (SEQ ID NO: 16).
  • the cassette included a selectable marker gene such as PAT (Wohlleben et al (1988) Gene 70:25-37, or BAR for resistance to
  • Basta/phosphinothricin were constructed.
  • the polynucleotides were operably linked to the CZ19B1 promoter to direct expression to maize endosperm.
  • the construction of such expression cassettes is well known to those of skill in the art in light of the present disclosure. See, for example, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, Laboratory Press, Plainview, NY; Gelvin et al. Plant Molecular Biology Manual (1990); each incorporated herein in its entirety by reference.
  • Corn was transformed as described in Example 14 using particle bombardment. Seeds from 30 transgenic events were analyzed for protein profiles by SDS-PAGE and more than 20% of the events showed segregating kernels with suppressed (>90% suppression of the alpha-zein protein when compared to non-transgenic control seed) alpha- zein protein levels (designated as CS-AZ). One event showed suppression of only the 19kD alpha-zein (designated as CS19). Total alpha-zein suppression (CS-AZ) resulted in improved 4 hr EEDM by 7 percentage units. Similarly, 19 kD alpha-zein suppression (CS19) resulted in improved 4 hr EDDM by 4.9 percentage units.
  • Example 20 Combination of over-expressed corn legumin and co-suppressed 27kD gamma-zein.
  • a construct is made that links in tandem a selectable marker gene, a corn legumin over-expression cassette and a 27KD gamma-zein co-suppression cassette.
  • the selectable marker gene consists of PAT for resistance to Basta/phosphinothricin.
  • the corn legumin coding sequence is placed under the transcriptional control of the maize GZ-W64A promoter and terminator (SEQ ID NOs:26 and 27) (Reina, M. et al, (1990), Nucleic Acids Res., 18:6426) to drive expression in maize endosperm as described in Example 11.
  • the 27KD gamma- zein co-suppression cassette contains the CZ19B1 promoter, and an inverted repeat with 27 kD gamma-zein cDNA fragments as described in Example 3.
  • the entire construct of linked cassettes is transformed into maize by Agrobacterium- mediated transformation and resulting TO plants are pollinated by a non-transgenic male parent corn plant.
  • Seed from transgenic events are analyzed for their seed protein profile by SDS-PAGE and Coomassie-staining of gels.
  • SDS-PAGE SDS-PAGE and Coomassie-staining of gels.
  • 90% of events indicate a 1 :1 segregation of wildtype seed protein profiles and altered seed protein profile phenotypes.
  • transgenic maize events generated with this construct are found to contain segregating kernels that are reduced in 27 kD-, and 16 kD-gamma-zein by at least 50% (dry weight; i.e.: "low gamma-zein phenotype") and about 30% of the transgenic events are found to contain segregating kernels that are increased in corn legumin by at least 200% (dry weight; i.e.: " high corn legumin protein phenotype") in addition to the reduced gamma-zein protein phenotype.
  • the kernel phenotype of the transgenic seed is normal (i.e., vitreous).
  • Segregating seed from low gamma-zein/ high corn legumin protein transgenic events are analyzed by SDS-PAGE for their seed protein phenotypes and sorted into two batches of 50 seed each.
  • One batch contains seed with wild type seed protein phenotype (controls) and the second batch contains seed with low gamma- zein/ high corn legumin protein phenotypes.
  • the seed of both batches are ground and analyzed by the EDDM assay. The analysis shows improved EDDM digestibility of the seed sample with low gamma-zein/ high corn legumin protein phenotypes compared to the seed sample with the wild type seed protein phenotype.
  • the EDDM improvement in the sample derived from low gamma- zein/ high corn legumin seed is greater than in seed samples with only a low 27kD gamma-zein content (Example 3) or in seed samples with only a high corn legumin protein content (Example 11); showing a partially additive effect of the suppression of gamma-zein and the increased expression of corn legumin on grain digestibility.
  • Example 21 Combination of over-expressed corn alpha-globulin and co- suppressed 27kD gamma-zein.
  • a construct comprising a tandem-linked corn alpha-globulin over- expression cassette, a 27KD gamma-zein co-supression cassette, and selectable marker cassette.
  • the selectable marker cassette consists of the ubiquitin promoter, PAT selectable marker (for resistance to Basta/phosphinothhcin) and PINII terminator.
  • the corn alpha-globulin coding sequence (SEQ ID NO:3) is placed under the transcriptional control of the maize GZ-W64A promoter and terminator (SEQ ID NOs:26 and 27) to drive expression in maize endosperm as described in Example 11.
  • the 27KD gamma-zein co-suppression cassette contains the CZ19B1 promoter, and an inverted repeat with 27 kD gamma-zein cDNA fragments as described in Example 3.
  • the entire construct of linked cassettes is transformed into maize by Agrobacterium-mediated transformation and resulting TO plants are pollinated by a nontransgenic male parent corn plant.
  • Seed from transgenic events are analyzed for their seed protein profile by SDS-PAGE and Coomassie-staining of gels.
  • the protein profiles of kernels in about 90% of events indicate a 1 :1 segregation of wildtype seed protein profiles to altered seed protein profile phenotypes.
  • transgenic maize events generated with this construct are found to contain segregating kernels that are reduced in 27 kD-, and 16 kD-gamma-zein by at least 50% (dry weight; e.g.: "low gamma-zein phenotype") and about 30% of the transgenic events are found to contain segregating kernels that are increased in corn alpha-globulin by at least 500% (dry weight; e.g.: " high corn alpha-globulin protein phenotype") in addition to the reduced gamma-zein protein phenotype.
  • the kernel phenotype of the transgenic seed is normal (i.e., vitreous).
  • Segregating seed from low gamma-zein/ high corn alpha-globulin protein transgenic events are analyzed by SDS-PAGE for their seed protein phenotypes and sorted into two batches of 50 seed each.
  • One batch contains seed with wild type seed protein phenotype (controls) and the second batch contains seed with low gamma-zein/ high corn alpha-globulin protein phenotypes.
  • the seed of both batches are ground and analyzed by the EDDM assay.
  • the analysis shows improved EDDM digestibility of the sample with seed with low gamma-zein/ high corn alpha-globulin protein phenotypes compared to the EDDM digestibility of the wild type seed sample.
  • the EDDM improvement in the low gamma-zein/ high corn alpha-globulin seed is greater than in seed samples with only a low 27kD gamma-zein content (Example 3), or in seed samples with only a high corn alpha-globulin protein content (Example 7), demonstrating a partially additive effect on grain digestibility of the suppression of gamma-zein and the increased expression of corn alpha-globulin.
  • Example 22 Transgenic Over-Expression of Combination of Alpha-Globulins and Corn Legumin 1.
  • a construct is made comprising a tandem-linked corn alpha-globulin over- expression cassette, a corn legumin over-expression cassette and selectable- marker cassette.
  • the selectable marker cassette consists of the ubiquitin promoter, PAT selectable marker (for resistance to Basta/phosphinothricin) and PINII terminator.
  • the corn alpha-globulin coding sequence is placed under the transcriptional control of the maize floury2 22kD alpha-zein promoter and terminator to drive expression in maize endosperm.
  • the corn legumin over- expression cassette contains the corn legumin coding sequence placed under the transcriptional control of the maize GZ-W64A promoter and terminator to drive expression in maize endosperm as described in Example 11.
  • the entire construct of linked cassettes is transformed into maize by Agrobacterium-mediated transformation and resulting TO plants are pollinated by a nontransgenic male parent corn plant.
  • Seed from transgenic events are analyzed for their seed protein profile by SDS-PAGE and Coomassie-staining of gels.
  • the protein profiles of kernels in about 90% of events indicate a 1 :1 segregation of wild-type seed protein profiles to altered seed protein profile phenotypes.
  • About 50% of the transgenic maize events generated with this construct are found to contain segregating kernels that are increased in corn alpha-globulin by at least 500% (dry weight; e.g.: "high corn alpha-globulin protein phenotype") and in corn legumin by at least 200% (dry weight; e.g.: "high corn legumin phenotype”).
  • the kernel phenotype of the transgenic seed is normal (i.e., vitreous).
  • Segregating seed from high corn legumin/ high corn alpha-globulin protein transgenic events are analyzed by SDS-PAGE for their seed protein phenotypes and sorted into two batches of 50 seed each.
  • One batch contains seed with wild type seed protein phenotype (controls) and the second batch contains seed with high corn legumin/ high corn alpha-globulin protein phenotypes.
  • the seed of both batches are ground and analyzed by the EDDM assay. The analysis shows improved EDDM digestibility of the seed sample with high corn legumin/ high corn alpha-globulin protein phenotypes compared to the EDDM digestibility of the seed sample with wild- type seed protein phenotype.
  • the EDDM improvement in the sample derived from high corn legumin/ high corn alpha- globulin seed is greater than in samples from seed with only a high corn legumin content (Example 11 ) or in samples from seed with only a high corn alpha-globulin protein content (Example 7), demonstrating a partially additive effect of the increased expression of corn legumin and the increased expression of corn alpha- globulin on grain digestibility.
  • Example 23 Down Regulation of 27kD gamma-zein and alpha-zeins.
  • a construct is made comprising a tandem-linked selectable marker cassette, and a 27KD gamma-zein/ 22kD alpha-zein/ 19kD alpha-zein clone B/ 19kD alpha-zein clone D co-supression cassette.
  • the selectable marker cassette consists of the ubiquitin promoter, PAT selectable marker (for resistance to Basta/phosphinothhcin) and PINII terminator.
  • the co-suppression cassette contains the CZ19B1 promoter and an inverted repeat with fragments of the 27 kD gamma-zein cDNA, the 19kD alpha-zein B1 cDNA, the 19kD alpha-zein D1 cDNA and the 22kD alpha-zein 1 cDNA (SEQ ID NO: 17).
  • the transcriptional - fusion is arranged as an inverted repeat around a spliceable ADH 1 INTRON 1 to effect silencing of all genes and genes highly similar to genes represented in the fusion.
  • Seed from transgenic events are analyzed for their seed protein profile by SDS-PAGE and Coomassie-staining of gels.
  • the protein profiles of kernels in about 90% of events indicate a 1 :1 segregation of wild-type seed protein profiles to altered seed protein profile phenotypes.
  • About 90% of the transgenic maize events generated with this construct are found to contain segregating kernels that are reduced in 27 kD- and 16 kD-gamma-zein, all 22kD alpha-zein proteins, and all 19kD alpha-zein proteins. These zeins are suppressed by at least 90% (dry weight; e.g.: "low gamma-zein/low alpha-zein phenotype") in about 90% of the altered seed protein events.
  • Segregating seed from low gamma-zein/ low alpha- zein transgenic events are analyzed by SDS-PAGE for their seed protein phenotypes and sorted into two batches of 50 seed each.
  • One batch contains seed with wild type seed protein phenotype (controls) and the second batch contains seed with low gamma-zein/ low alpha-zein protein phenotypes.
  • the seed of both batches are ground and analyzed by the EDDM assay.
  • the analysis shows improved EDDM digestibility of the seed sample with low gamma-zein/ low alpha-zein protein phenotypes compared to the wild-type seed sample.
  • the EDDM improvement in the low gamma-zein/ low alpha- zein seeds sample is greater than in seed samples with only a low 27kD gamma- zein content (Example 3) or in seed samples with only a low alpha-zein protein content (Example 19); demonstrating a partially additive effect of the suppression of gamma-zein and the suppression of alpha-zeins on grain digestibility.
  • Example 24 Combined Down-regulation of 27kD and 16kD gamma-zein, 22kD and 19kD alpha-zein and over-expression of alpha-globulin, 15kD beta-zein, and 18kD delta zein.
  • a stacked construct (SEQ ID NO: 17) was constructed which included the following expression cassettes: floury2 promoter:: 18KD alpha-globulin coding sequence: :floury2 terminator; ZM-LEG1A PRO (the promoter from the maize legumin gene): HSZ (high sulfur zein) coding sequence::ZM-LEG1 terminator; GZ- W64A Pro::15KD Beta Zein coding sequence: :GZ-W64A terminator; and the CZ19B1 promoter driving a transcriptional fusion of fragments of 27KD gamma- zein, both 19KD alpha-zeins, 22KD alpha-zein, and Lysine Ketoglutarate Reductase (LKR; Arruda P, et al, (2000), Trends Plant Sci.
  • LLR Lysine Ketoglutarate Reductase
  • the transcriptional fusion was arranged as an inverted repeat around a spliceable ADH1 INTRON1 to effect silencing of all five genes represented in the fusion.
  • the entire construct of linked cassettes was transformed into maize by Agrobacterium-mediated transformation. 361 TO plants derived from 252 independent events were obtained and pollinated by a nontransgenic male parent corn plant. From those 361 ears, 302 ears contained more than 50 kernels per ear.
  • the identity of the 15kD protein as the 15kD beta-zein, of the 18kD protein as the 18kD corn alpha-globulin and 18kD delta-zein proteins and of the 5OkD protein as the 5OkD corn legumin was confirmed by immuno-blotting using beta- zein, alpha- globulin, delta-zein and corn legumin specific antibodies (Woo et a!) with seed extracts from 9 selected events. Moreover, the suppression of LKR was also confirmed in these events by immuno-blotting. These results demonstrated a very high rate of combined efficacy of the co-suppression cassette and the linked over-expression cassettes.
  • Segregating seed from 9 events showing the low gamma-zein/ low alpha- zein/ high corn legumin /high alpha-globulin/ high 18kD delta-zein/ high beta-zein phenotype were analyzed by SDS-PAGE for their seed protein phenotypes. Each event was sorted into two batches of 50 seeds each. The seed weights of each pair of batches were determined and showed no significant difference.
  • Meal was prepared from each sample (nine events, two batches each) and analyzed for its amino acid composition and protein content. For all events, the average protein content was reduced in the altered seed protein phenotype samples by 10% (dry weight), the lysine content was increased by >70% (dry weight) and the tryptophane content was increased by >60% (dry weight).
  • Meal was prepared from each of the pairs of batches of six events and analyzed for EDDM at the 6hr. time point.
  • the altered seed protein phenotype samples showed, on average, an improvement of 9.8 percentage points compared to the segregating control samples.

Abstract

La présente invention concerne des compositions et des procédés pour modifier les taux de protéines de la semence dans les graines de céréales. L'invention concerne la modification des taux de protéines de la semence dans le grain de la plante, avec pour résultat un grain présentant une meilleure digestibilité, une meilleure disponibilité des nutriments, une composition en acides aminés et une valeur nutritionnelle améliorées, une meilleure réponse au traitement de l'aliment, une qualité d'ensilage améliorée et une plus grande efficacité de mouture à l'état humide.
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AU2004326206A1 (en) 2006-07-06
WO2006071219A1 (fr) 2006-07-06
MX2007007948A (es) 2007-09-11
EP2003205A2 (fr) 2008-12-17
EP2003205A3 (fr) 2009-05-13
AU2004326206B2 (en) 2011-03-17
CA2592894C (fr) 2013-08-27
CA2592894A1 (fr) 2006-07-06
EP2003205B1 (fr) 2013-05-01

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