EP1141347A1 - Co-expression des proteines - Google Patents

Co-expression des proteines

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Publication number
EP1141347A1
EP1141347A1 EP99918663A EP99918663A EP1141347A1 EP 1141347 A1 EP1141347 A1 EP 1141347A1 EP 99918663 A EP99918663 A EP 99918663A EP 99918663 A EP99918663 A EP 99918663A EP 1141347 A1 EP1141347 A1 EP 1141347A1
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EP
European Patent Office
Prior art keywords
zein
protein
plant
proteins
lokd
Prior art date
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EP99918663A
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German (de)
English (en)
Inventor
Suman Bagga
Champa Sengupta-Gopalan
John D. Kemp
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Arrowhead Center Inc
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Arrowhead Center Inc
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Publication of EP1141347A1 publication Critical patent/EP1141347A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8254—Tryptophan or lysine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C07K14/425—Zeins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8253—Methionine or cysteine

Definitions

  • Alfalfa (Medicago sativa L.) is considered to be the most important cultivated forage crop in the world (Hanson et al, 1988; Michaud et al, 1988) and is often referred to as "Queen of the forage crops" because it is widely grown, has a superb balance of vitamins and minerals, is high yielding, is an excellent source of biological nitrogen fixation, and it serves as an attractive nectar source for honeybees (Barnes et al, 1988; Smoliak and Bjorge, 1983). Alfalfa has been bred for years for both forage quality and plant performance.
  • alfalfa and other leguminous forage crops are high in protein, these plants are deficient in the sulfur amino acids (S-amino acids), methionine and cysteine (Kaldy et al, 1979). It has been shown that wool growth in sheep is limited by the availability of S-amino acids. Similarly, milk production by dairy animals is affected by the deficiency of S-amino acids in plants. Efforts to use conventional plant breeding and cell selection techniques to increase the S-amino acid content of alfalfa have met with little or no success.
  • a genetic engineering approach to improve the amino acid balance of alfalfa and other forage crops would be to introduce into these plants genes encoding proteins high in methionine driven by a strong constitutive promoter or a leaf promoter.
  • the foreign proteins should contain about 15 to 25% of S- amino acids and constitute 5 to 10% of the total leaf protein.
  • the digestibility of S-amino acid containing proteins by the rumen bacteria and the stomach enzymes is also an extremely critical issue in regard to providing a suitable forage crop for ruminant animals, but is often overlooked.
  • the S-amino acid rich protein should be relatively resistant to degradation in the rumen (first stomach) of the ruminant animals and should be assimilated in the lower gastrointestinal tract.
  • Zeins are a group of alcohol soluble proteins that are synthesized during endosperm development in corn and constitute 50% of the total protein in mature seeds (Lee et al, 1976).
  • the zeins can be divided into four groups, the ⁇ , ⁇ , ⁇ and ⁇ , based on their solubility (Larkins et al, 1989).
  • the zeins can also be separated by size into groups.
  • the ⁇ zeins, which is the most abundant class, are made up of the 22kD and 19kD zeins; the central region of these proteins consists of repetitive peptides of about 20 amino acid residues (Argos, 1982).
  • the ⁇ zeins comprise the 15kD zein which contains less proline and glutamine than the ⁇ zeins.
  • the ⁇ zeins include the 27kD and 16kD class and are very rich in proline (25%).
  • the ⁇ zeins are a relatively minor class consisting of the lOkD zein (Kirihara et al, 1988). All the zein classes are structurally unique. The repeat regions in the ⁇ and ⁇ zeins probably have a major role in the packing of protein bodies. Zeins, in general, contain extremely low levels of the essential amino acids lysine, tryptophan and to a lesser extent methionine. The 15kD and 1 OkD zeins, however, are distinguished by their extremely high methionine content (10% and 22.5%, respectively) (Giannaza et al, 1977).
  • the zeins are synthesized on the rough endoplasmic reticulum (RER) and they aggregate into protein bodies directly in the RER (Larkins and Hurkman, 1978). Based on the analysis of the zein composition of developing protein bodies in corn endosperm, Lending and Larkins ( 1989), have proposed a descriptive model for the pattern of zein deposition during protein body formation in corn endosperm: The ⁇ and ⁇ zeins are the first to start accumulating within the RER. Subsequently, ⁇ zeins begin to accumulate as locules within the ⁇ and ⁇ zeins.
  • Mutations in maize affect the expression of the different zein genes. Changes in zein gene expression in turn have direct impact on the amino acid composition of the seeds. Seeds of plants homozygous for the recessive mutation opaque-2, have increased levels of lysine compared to the wild-type seeds (Misra et al, 1972). The increase in lysine is due to the reduced expression of the 22kD ⁇ zeins (Langridge et al., 1983). The inbred line BSSS-53 has 30% higher level of seed methionine compared to other inbred lines. This increase in methionine content is because of a two-fold increase in the level of the lOkD zein (Phillips and McClure 1985).
  • Proteins that accumulate in the endoplasmicreticulum are known to have the amino acid sequence Lys(his) Asp Glu Leu (K(H)DEL) near their carboxy terminal end which prevents them from exiting into the Golgi (Pelham, 1990).
  • the zeins and other prolamines lack this sequence.
  • a cognate of the 70-kD heat shock protein, BiP which functions as a molecular chaperone has been shown to be involved in the formation of prolamine protein body formation in rice endosperm (Li et al, 1993b).
  • BiP The involvement of BiP in the formation of zein protein bodies is based on the fact that BiP accumulates to high levels in the ER and on the abnormal protein bodies of some of the zein regulatory mutants of corn (Boston et al., 1991 ; Zhang & Boston, 1992). Overall, however, the mechanisms of zein targeting and assembly in protein bodies are poorly understood and it is not known whether inter- and intra-molecular interactions play a key role in protein body formation. Abe et al. (1991), have suggested that the cytoskeleton plays a role in the biogenesis of zein protein bodies.
  • Figures 1A-1B show a steady-state accumulation pattern of the 15kD zein protein in leaves of transgenic L. japonicus (panel A) and alfalfa Regen SY (panel B).
  • 70% ethanol (EtOH) soluble protein equivalent to 50 ⁇ g of the phosphate buffered saline soluble fraction
  • Figure 1C shows a diagrammatic representation of the 15kD zein gene construct.
  • Figures 2A-2B show a steady-state accumulation pattern of the lOkD zein protein in leaves of transgenic L.
  • Figure 2C shows a diagrammatic representation of the lOkD zein gene construct.
  • Figures 3A-3C show a steady-state accumulation pattern of the 1 OkD zein in transgenic tobacco.
  • FIG. 3 A Diagrammatic representation of pMlOZ.
  • the construct consists of the CaMV 35 S promoter fused at the Bglll site to a 470 bp Bglll-Xhol fragment containing the coding region of the lOkD zein gene. This is followed by the NOS 3' terminator of pMON316 (Rogers et al., 1987).
  • Figure 3B Analysisofdifferentindependenttransformantsforthe accumulation of the
  • EtOH soluble protein extracted from the leaves was subjected to SDS PAGE, transferred to nitrocellulose and followed by immunoblot analysis using the lOkD zein antibodies.
  • Lanes labeled 1 through 7 contain samples from the leaves of different transformants while lanes 1 and 2 (under control), are leaf samples from nontransformed tobacco plants.
  • FIG. 3C Analysis of different plant organs (transformant 6) for the accumulation of the lOkD zein.
  • EtOH soluble fractions (equivalent to 50 ⁇ g of PBS soluble protein) from the various plant parts indicated, along with 2 ⁇ g of corn seed protein were subjected to SDS PAGE followed by western analysis using the lOkD zein antibodies.
  • UT and Mat stand for untransformed and mature seeds, respectively.
  • Molecular weight standards were included in the gels and the size of two of the relevant markers is indicated.
  • Figure 4 shows the fate of lOkD zein in germinating seeds/seedlings of transgenic tobacco. Seeds of a lOkD zein plant were sterilized and allowed to germinate under sterile conditions and at defined times as indicated in the Figure, seeds/seedlings were harvested and the EtOH soluble fraction extracted. EtOH soluble fraction equivalent of 50 ⁇ g of PBS soluble protein was then subjected to SDS PAGE followed by western analysis using the lOkD zein antibody. The position of the lOkD zein on the gel is indicated, and DAG stands for days after germination.
  • FIG. 5A-5D Ultrastructureand Immunogold localization of 1 OkD zein in transgenic tobacco leaves.
  • Figure 5A Regions of two mesophyll cells showing several lOkD zein protein bodies (indicated by arrowheads) in a lOkD zein transformant.
  • Figure 5B Higher magnification of lOkD zein protein bodies (indicated by arrowheads).
  • Figure 5C Structureof a 15kD zein protein body in the mesophyll cell of a 15kD zein transformant.
  • Figure 5D Immunolocalization of lOkD zein in the leaf cells of a lOkD zein transformant with the 5 nm gold particles (indicated by arrowheads).
  • Figure 6A-6D Comparison of lOkD and 15kD zein levels in the leaves and seeds of the lOkD, 15kD and 10kD/15kD zein plants.
  • FIG. 6A EtOH soluble fraction from the leaves (equivalent to 10 ⁇ g of PBS soluble protein) and seeds (equivalentto 50 ⁇ g ofPBS soluble protein) of the lOkD and 10kD/15kDzein plants were subjected to SDS PAGE followed by western analysis using the lOkD zein antibodies.
  • Figure 6B Quantification of band intensity from Figure 6A using the Bio Image Intelligent Quantifier.
  • FIG. 6C EtOH soluble fraction from the leaves and seeds (equivalentto 50 ⁇ g of PBS soluble protein) of the lOkD and 10kD/15kDzein plants were subjected to SDS PAGE followed by western analysis using the 15kD zein antibodies.
  • Figure 6D Quantification of band intensity from Figure 6C using the Bio Image Intelligent Quantifier.
  • Figure 7A-7D Subcellular localization of the lOkD and 15kD zeins in leaves of the 10kD/15kD zein plants.
  • Figure 7A Conventionallyfixed and stained sections of leaves from a 10kD/15kDzein plant. Arrowheads point to the protein bodies formed in the cytoplasm.
  • Figure 7B Immunolocalization of the lOkD zein protein using mouse anti- 1 OkD zein antibody diluted 1:50 followed by 10 nm diameter gold-conjugated goal anti-mouse IgG.
  • Figure 7C Co-immunolocalization of the lOkD and 15kD zeins using the mouse anti- 1 OkD zein antibody and the rabbit anti- 15kD zein antibodies followed by 10 nm diameter gold conjugated goat anti-mouse IgG and 5 nm gold conjugated goat anti rabbit IgG.
  • Figure 7D A higher magnification of a region showing double-labeling from panel C.
  • the arrowheads point to the 10 nm gold particles while the arrows point to the 5 nm gold particles.
  • Figure 8. Analysis of the accumulation pattern of BiP in the ⁇ -, ⁇ -, and ⁇ -/ ⁇ -zein tobacco transformants.
  • Phosphate buffered saline soluble extract 100 ⁇ g of protein
  • leaves of a control NT
  • ⁇ -zein, ⁇ -zein and ⁇ -/ ⁇ -zein plants were subjected to SDS PAGE followed by western blotting using an antibody raised to BiP from maize.
  • a positive control lane containing purified BiP (1 ⁇ g) was included in the gel.
  • the lower panel represents the results of band quantification using the Intelligent Quantifier.
  • Figure 9A represents SEQ ID NO: 5.
  • Figure 9B represent SEQ ID NO: 6.
  • Figure 10 is an overview of the construction of the Z10/OCI plasmid which was transformed into tobacco.
  • Figures 11A and 11B are western blots of extracted samples of transformed tobacco plants showing a positive immunoreaction with both the Z10 (14A) and OCI (14B) antibodies.
  • SEQ ID NO: 1 represents primer PA which was used to amplify a Z10 fragment from plasmid prep (993) Z10 pMON 316/DH56.
  • SEQ ID NO: 2 represents primer PB which was used to amplify a Z10 fragment from plasmid prep (993) Z10 pMON 316/DH5 ⁇ .
  • SEQ ID NO: 3 represents primer PC which was used to amplify a OC- 1 fragment from plasmid (809) OclpSP73/DH5 ⁇ .
  • SEQ ID NO: 4 represents primer PD which was used to amplify a OC- 1 fragment from plasmid (809) OclpSP73/DH5 ⁇ .
  • SEQ ID NO: 5 represents a Z10 fragment as illustrated in Figure 9A.
  • SEQ ID NO: 6 represents an OCI fragment as illustrated in Figure 9B.
  • the present invention relates to the co-expression of proteins that results in a greater accumulation in a cell of one of the proteins when compared to the levels of the same protein when expressed alone.
  • the proteins are expressed in prokaryotic or eukaryotic cells.
  • One or both of the proteins are accumulated in the cell at higher levels when expressed together than when the proteins are expressed alone in a cell.
  • Standard, routine genetic engineering techniques are employed to (i) isolate the appropriate DNA encoding desired proteins including regulatory sequences, (ii) transform a target cell and, in the case of plants, to regenerate a transgenic plant.
  • the transgenic cells are grown under conditions sufficient to result in the accumulation of one or both proteins at high levels.
  • the proteins accumulated in the cell exhibit increased stability and resistance to degradation.
  • the subject invention also concerns plants and plant tissues that are capable of expressing high levels of stable proteins which are localized as protein bodies within the plant cell. Specifically exemplified are plants co-expressing both the 15kD and lOkD zein proteins. Transformed plants co-expressing the 15kD and 1 OkD zein proteins are useful for providing forage crops containing increased levels of sulfur containing amino acids, such as methionine, in the diet of animals that normally feed on such crops.
  • plants or plant tissues containing novel heterotypic protein bodies elevated in one or more other essential amino acids e.g., arginine, histidine, leucine, isoleucine, lysine, phenylalanine, threonine, tryptophan, tyrosine and valine.
  • protein bodies which result in the enhanced accumulation of normally unstable proteins in plant tissue.
  • the subject invention also concerns plants or plant tissue comprising rumin stable protein bodies which contain other proteinaceousmaterial, for example, an antigenic determinant capable of eliciting an immune response, a proteinaceous drug, pesticide or antimicrobial peptide.
  • Heterologous proteins can be expressed in plants transformed with the storage proteins of the subject invention which can act as a "carrier protein,” whereby the proteins coalesce and accumulate in the cell as a protein body.
  • a rumin stable protein body is provided in a plant or plant tissue as a fusion protein expressed in the cell comprising a zein protein and a heterologous protein or peptide.
  • the subject invention concerns plants and plant tissues that are capable of expressing high levels of stable storage proteins which are localized as protein bodies within the plant cell.
  • Plants contemplated within the scope of the invention include forage crop plants, including, for example, alfalfa, clover, corn silage, sorghum and other leguminous crops, transformed to express the proteins of the invention.
  • plants for human consumption which have been transformed to express proteins that enhance the protein quality of the plant for improved nutrition.
  • plants expressing proteins containing high levels of S-amino acids, such as methionine and cysteine are expressed in the plant or plant tissue.
  • the zein protein expressed is the 15kD or 1 OkD zein protein.
  • both the 15kD and lOkD zein proteins are co-expressed in the plant or plant tissue. Plants having genotypes carrying the lOkD and 15kD zein genes were sexually crossed to create hybrids carrying both constructs.
  • the zein proteins expressed in the transformed plants are resistant to rumin degradation and, therefore, are useful for providing nutritionally important amino acids that can be digested in the stomach and absorbed by the ruminant animal because of the protein's capacity to "by-pass" the rumin.
  • plants or plant tissue comprising rumin stable protein bodies which contain other proteinaceous material, for example, an antigenic determinant capable of eliciting an immune response, a proteinaceous drug, pesticide or antimicrobial peptide.
  • Heterologous and endogenous proteins and synthetic peptides having essential amino acids can be expressed in plants transformed with the storage proteins of the subject invention which can act as a "carrier protein," whereby the proteins coalesce and accumulate in the cell as a protein body.
  • a rumin stable protein body is expressed in a plant or plant tissue as a fusion protein comprising a zein protein and a heterologous protein or peptide.
  • the fusion protein can be designed to yield the heterologous protein portion by cleavage with a selected enzyme or under certain physiological conditions.
  • the zein protein expressed as part of the fusion protein is the 15kD or lOkD zein protein. More preferably, both the 15kD and lOkD zein proteins are co-expressed in the plant or plant tissue comprising the fusion protein.
  • the subject invention also pertains to a rumin stable protein body. Rumin stable protein bodies of the invention are not subject to digestion by rumin bacteria in the rumin of an animal but can be digested proteolytic enzymes of an animal's stomach.
  • a rumin stable protein body of the present invention can be prepared which contains heterologous proteinaceous material in addition to the rumin stable protein.
  • an antigenic determinant capable of eliciting an immune response a proteinaceous drug, pesticide or antimicrobial peptide.
  • Rumin stable protein bodies can be isolated from plants that have been transformed with polynucleotide molecules encoding the desired rumin stable proteins. Plant cells expressing the polynucleotide molecules encoding the desired rumin stable proteins can be readily selected and regenerated into plants or plant tissue using standard techniques known in the art.
  • a storage protein gene is co-expressed in a cell with a second protein gene whereby the second protein is accumulated in the cell at a level that is higher than when the second gene is expressed alone in the cell, i.e., in the absence of the storage protein gene.
  • the storage protein gene is a seed storage protein gene
  • the target cell is a plant cell
  • the second protein gene can be any gene encoding a desirable protein.
  • Regulatory sequences employed with the protein genes are readily chosen by one of ordinary skill in the art based on a variety of factors, such as, for example, i) the specific protein genes employed, ii) the target cell to be transformed, iii) the plant tissue where expression/accumulation is desired, iv) the particular plant (monocot, dicot, etc) species to be transformed, etc.
  • a constitutive promoter may be chosen (e.g., CaMV 35S, ubiquitin, etc) or a tissue specific promoter may be employed that will express at high levels in specific tissues (seeds, green tissues, etc).
  • a 15 kD zein protein gene is employed with a second protein gene in a plant cell that results in accumulation of the second protein in the plant cell.
  • the genes can be contained on a single expression cassette and inserted into the plant genome employing standard transformation and regeneration techniques. Alternatively, the protein genes can be inserted into a plant cell genome independently in separate expression cassettes and transgenic plants can be regenerated therefrom.
  • the protein genes can be inserted into separate plant cells and regenerated into fertile, transgenic plants each containing one of the protein genes. These transgenic plants can then be cross fertilized employing standard plant breeding techniques to result in a cross that contains both the 15 kD zein protein gene and the second protein gene wherein the second protein is accumulated in one or more plant tissues.
  • alfalfa, tobacco or other plant cells are transformed with a 15 kD zein protein gene and a 10 kD zein protein (second protein) wherein both genes are driven by a constitutive promoter.
  • Fertile, transgenic plants containing both gene constructs are regenerated. Progeny plants are grown and the 10 kD zein protein is accumulated in green tissue at level of 5 to 10 times or more when compared to the accumulation level of the 10 kD protein when expressed alone.
  • the present invention encompasses novel protein bodies formed as a result of expressing a storage protein gene and a second protein gene in green plant tissues.
  • the novel protein body comprises a 15kD zein protein and a second protein.
  • the protein body is typically located in leaf tissue.
  • the novel protein body is located in leaf tissue and comprises a 15 kD zein protein and a 10 kD zein protein.
  • the subject invention also concerns a method for increasingthe forage quality of a plant comprising transforming a plant or plant tissue with a polynucleotide molecule that encodes a storage protein of the present invention.
  • Methods for transforming plants and selecting for expression of the transformed genotype are known in the art.
  • the polynucleotide encodes a zein protein which is expressed in the plant or plant tissue. More preferably, the zein protein expressed is the 15kD or lOkD zein protein. Most preferably, both the 15kD and 1 OkD zein proteins are co-expressed in the transformed plant or plant tissue.
  • Transgenic plants can be readily prepared from the transformed plant or plant tissue using standard techniques known in the art.
  • the subject invention also concerns methods for increasing the stability and storage of a heterologous protein in a plant or plant tissue.
  • Heterologous proteins can be expressed in plants transformed with the storage proteins of the subject invention which can act as a "carrier protein," whereby the proteins coalesce and accumulate in the cell as a protein body.
  • a plant is transformed with a polynucleotide molecule that encodes a fusion protein comprising a storage protein of the present invention operably linked with a heterologous protein or peptide.
  • the zein proteins of the present invention include not only those proteins having the same amino acid sequence as found in nature, including allelic variants, but also includes those variant zein proteins having conservative amino acid substitutions, additions and deletions in the protein sequence, as long as the variant protein retains substantially the same relevant biological activity as the native zein protein.
  • the skilled artisan, having the benefit of the teachings disclosed herein, can readily determine whether a variant protein retains the substantially the same biological activity as the non-modified protein.
  • Plasmid pMZEHOk containing the lOkD zein cDNA isolated from a corn endosperm cDNA library was a gift from Dr. J. Messing.
  • a 470bp EcoRl/Xbal fragment containing the entire coding region was removed from pUC 119 and cloned into the EcoRl and Xbal sites of pSP73.
  • the stop codon for the 1 OkD zein is contained within the Xbal site.
  • the lOkD zein gene was then recovered as a Bglll/Xhol fragment and inserted into the BglH and Xhol sites in the polylinker of pMON316 (Rogers et al, 1987).
  • the translation terminator following the stop codon of the lOkD zein is the NOS terminator.
  • the resulting plasmid was called pMIOZ (Fig 1 A). Plasmid pMEZ is as described by Bagga et al. (1995).
  • the plasmid pMIOZ was mobilized from E. coli DH5 ⁇ into the Agrobacterium tumefaciens receptor strain pTiT37ASE by triparental mating (Rogers et al, 1987). Nicotiana tabacum cv Xanthi was transformed by the leaf disc procedure (Horsch et al., 1987). Transformants were selected and regenerated on MS media containing 100 ⁇ g of kanamycin/ml shoots appeared within 4-6 weeks after inoculation. The shoots were rooted on the same media without hormones and transferred to the soil.
  • Plant tissues were ground and extracted in phosphate buffered saline (PBS) and centrifuged. The supernatant was used for protein determination using the Bradford assay (BIO RAD). The pellet from centrifugation was incubated in 70% ethanol containing 1% of mercaptoethanolat 65 °C for 30 minutes to extract the zein proteins. For western analysis, the EtOH-extractable fraction equivalent of a known amount of PBS soluble protein extract was subjected to SDS-PAGE (Laemmli, 1970), followed by electroblotting onto nitrocellulose membrane.
  • PBS phosphate buffered saline
  • the membrane was blocked for 1 to 2 hrs with 1% BSA in Tris-buffered saline containing 0.05% Tween 20 (TBST), followed by overnight incubation in the same solution containing the appropriate antibodies.
  • TST Tris-buffered saline containing 0.05% Tween 20
  • the lOkD zein monoclonal antibodies were provided by DEKALB Genetics Corporation, the 1 OkD zein polyclonal antibodies by Dr. J. Messing and the 15kD zein polyclonal antibodies by Dr. B. Larkins.
  • the protein bands reacting with the antibodies were made visible by using an alkaline phosphatase-linked second antibody (goat antibody or rabbit IgG in case of the polyclonal antibody or mouse IgG in the case of the monoclonal antibody) and the substrates, nitrobluetetrazolium and 5-bromo-4-chloro-3-indolyl- phosphate according to the manufacturer's instructions (Promega).
  • the tissue was dehydrated in EtOH and embedded in either Spurr's resin or LR White resin and polymerizedat 50°C. The remaining steps were all done at room temperature.
  • the different resins were purchased from Electron Microscopy Sciences, Ft. Washington, PA.
  • Silver sections on nickel grids were first incubated in a blocking solution of 10 mM Tris-saline containing 1% BSA (Sigma), 0.05% Tween 20 (Sigma), and 15 mM NaCl. This buffer mixture was used in all of the remaining steps.
  • 5 to 20% normal serum from the animal source of the antibody was added to the blocking solution in order to reduce non-specific staining.
  • Immunolabeling with the lOkD zein antibodies The grids of sections from the 1 OkD and 1 OkD/15kD zein crosses were drained and incubated with the monoclonal antibody to 1 OkD zein diluted 1 :50 in buffer for 45 min to 4 h. Controls were incubated in nonimmune mouse IgG. They were then washed in the buffer and placed in gold-labeled, goat anti-mouse IgG with a diameter of 10 nm (Sigma) diluted 1:50 in buffer for 45 min.
  • the grids were incubated with the polyclonal rabbit anti- 1 OkD zein diluted 1 :1000 in buffer for 60 min. They were then washed and incubated in a solution of gold- conjugated anti-rabbit IgG with a diameter of 5 nm, diluted 1 :50 for 60 min. Since the polyclonal lOkD zein antibody cross-reacted with the 15kD zein, in case of the 10kD/15kD zein cross, the grids were incubated with the lOkD zein monoclonal antibody followed by gold- conjugated anti-mouse IgG with a diameter of 10 nm. The grids were washed in Tris-Saline containing Tween 20 and 1% BSA, followed by double-distilled water. The grids were then examined either unstained or lightly poststained in uranyl acetate and lead citrate.
  • Double-labeling with the 1 OkD and 15kD zein antibodies The grids were incubated in rabbit anti- 15kD zein diluted to 1:100 for 45 to 60 min. The grids were washed and incubated in gold-conjugated goat anti-rabbit IgG with a diameter of 5 nm, diluted 1 :50 for 45-60 min. The grids were then thoroughly washed in the buffer and then incubated with mouse anti- 1 OkD zein diluted 1:50 in buffer for 45 to 60 min. They were then washed in buffer followed by distilled water and incubated in gold-conjugated goal anti-mouse IgG with a diameter of 10 nm diluted 1 :50 in buffer for 45 min. The grids were washed in Tris-Saline containing Tween 20 and 1% BSA, followed by double-distilled water. The grids were then examined either unstained or lightly poststained in uranyl acetate and lead citrate.
  • Example 1 Accelulation of the 15kD and 1 OkD zeins in vegetativetissuesofZ. r ⁇ pow/ctAS' and alfalfa
  • Example 2 Accumulation of zein protein in vegetative tissues of tobacco transformants Tobacco plants were transformed with the gene construct pMl 0Z, consisting of the lOkD zein gene driven by the CaMV 35S promoter (Figure 3 A). Leaves from seven randomly picked independent transformants were subjected to western analysis to measure the accumulation of 1 OkD zein ( Figure 3B). All of the transformants showed two immunoreactive proteins, one of which co-migrated with the lOkD zein from corn seeds (position indicated by arrow) and the other as a 29kD band. The latter did not co-migrate with the higher molecular weight immunoreactive band seen in corn seeds (Figure 3C).
  • the 29kD immunoreactive band obtained from the leaves of the transgenic plants probably represents an aggregate of the lOkD zein and another endogenous leaf protein.
  • the 20kD immunoreactive protein band in the corn seed may represent an aggregate of the lOkD zein with an endogenous corn protein.
  • the accumulation of the 1 OkD and the 29kD immunoreactive bands differed by about 10 fold among the different transformants.
  • Transformant 4 showed almost negligible level of both the immunoreactive bands. Differences in the amount of accumulation of the lOkD zein in the various transformants can probably be attributed to position effect or the number of copies of the integrated gene.
  • Example 3 Stability of zein in germinating tobacco seeds
  • Example 4 Immunolocalization of the 15kD and lOkD zein proteins in novel protein bodies in transgenic plants
  • protein bodies very different from the 15kD zein protein bodies (Figure 5C) were seen in the leaves of the 1 OkD zein plants.
  • the protein bodies in the 1 OkD zein plants appeared very osmophilic, the osmophilia being concentrated along the circumference of the bodies. In some cross sections, the osmophilia appeared to radiate in discrete spokes from a central hub (Figure 5B).
  • the protein bodies seen in the 15kD zein plants did not exhibit this extreme osmophilia (Figure 5C).
  • the 1 OkD zein protein bodies were found to be associated with the ER but in most cases because of the large size of the bodies the ER membranes appeared disjointed. Based on immunolocalization, the lOkD zein was found to be evenly distributed in these unique protein bodies, suggesting that they result from assembly of the lOkD zein (Figure 5D).
  • Example 5 Simultaneous accumulation of the 15kD and lOkD zeins in transformants expressing both genes
  • Sexual crosses were made between tobacco transformants expressing either the 15kD or 1 OkD zeins in order to determine if these proteins interact with one another and affect protein accumulation in the plant cell. Seeds from these crosses were germinated on 200 ⁇ g/ml of kanamycin and seedlings expressing both genes were selected based on positive PCR using both 15kD and 1 OkD zein gene-specificprimers. Protein extracts from the leaves of two independent plants expressing both genes and their respective parents were analyzed by immunoblotting using both 15kDand 1 OkD zein antibodies.
  • 15kD zein equal amounts of the protein extracts from the leaves and seeds of one of the lOkD zein plants, a 15kD zein plant and the corresponding 1 OkD/15kD zein cross was analyzed by western analysis followed by quantitation of the immunoreactive bands using an Intelligent Quantifier (Biolmage) ( Figure 6).
  • This quantitative analysis shoed that the 15kD zein levels in both the seeds and the leaves of the 1 OkD/15kD zein plants were essentially similar to those in the 15kD zein parent plant ( Figure 6C, 6D).
  • Example 6 1 OkD zein protein is localized in the same ER-derived protein bodies as the 15kD zein in plants co-expressing the 1 OkD and 15kD zein genes
  • Multiple copies of the 15kD and lOkD genes can be introduced in plants as a method of increasing the total leaf content of S-amino acid containing proteins.
  • gene constructs driven by the SSU promoter and the mannopine synthase promoter can be used to avoid any potential problems of co-suppression. Isogenic populations carrying either the lOkD, the 15kD, or both the lOkD and the 15kD zein constructs can be sexually developed.
  • Trends in zein dosage effects on expression "per se” in alfalfa, interactions between constructs, and the influence of each construct on plant development, forage quality and yield can be determined based on general comparisons of the average number of zein constructs expected within a population. Genetically defined genotypes from three populations carrying from one to four copies of either the lOkD or 15kD construct or one to two copies of both the lOkD and 15kD constructs can also be examined. The most direct approach to sexually increase zein copy number among regenerated somaclones would be to either self-pollinate individual regenerates or to intercross them. Intercrossing regenerants, however, is genetically equivalent to selling in this case. To minimize the confounding effects of inbreeding depression a series of hybrid populations can be developed to examine the influence of zein constructs on forage yield and quality of alfalfa.
  • ruminant animals In ruminant animals, food is first acted upon by microorganisms inhabiting the first stomach (rumen) of the animal.
  • the cellulose in plant material is digested by the inhabiting microorganisms since these animals do not produce cellulase on their own.
  • These microorganisms are also capable of breaking down plant proteins and utilizing the released amino acids for their own growth.
  • These ruminant microbes are subsequently digested as they pass through rest of the digestive tract of the animal, thereby providing an important source of protein and other nutrients.
  • a large part of these proteins are deminated in the nitrogen excreted in the urine. Not only does this reduce the nutritional quality of the feed material, it also results in excess nitrogenous environmental pollution.
  • Amino acid supplements e.g., methionine or lysine
  • Amino acid supplements are also subject to substantial degradation in the rumin and, thus, various rumin-protectedamino acid supplements have been developed.
  • Plant tissue was processed using mortar and pestle. Samples were placed into DACRON polyester bags (pore size 52 um). Approximately .3 g of each sample was kept for comparison purposes and the remaining amount was incubated inside the rumin of a cannulatedHolsteincow for a period of 12 hours. Bags were then removed from the rumin, washed and dried in a 60 degree C oven overnight. The 15kD zein and 10KD zein proteins were monitored immunologically (Western blotting) and by staining procedures. Ribulose biphosphate carboxylase which is highly degradable by ruminal bacteria (Nugent et al. , 1983) was mon itored and used as a internal control.
  • Example 9 Induction of BiP in transgenic plants expressing the zein genes
  • BiP an endogenous plant protein
  • zein protein bodies in transgenic tobacco plants.
  • protein samples from leaves of ⁇ -zein, ⁇ - zein and ⁇ -/ ⁇ -zein plants were subjected to quantitative western analysis using a corn BiP antibody (Zhang and Boston, 1992).
  • the immunoreactive bands were then analyzed using the Intelligent Quantifier.
  • the lower panel in Figure 8 is the graphical representation of the relative intensity of the immunoreactive bands. All three transgenic plants showed a significantly higher level of BiP accumulation when compared to the control; the levels of BiP were more or less similar in the three transformants. Thus, the synthesis of the zein proteins in the transgenic plants induces the synthesis or stable accumulation of BiP.
  • Example 10 Expression and Stabilization of Proteins for the Purpose of Plant Pest Control
  • Z 10/OCT consisting of a 1 OkD zein gene (Z 10) fused in frame to the front of an oryzacystatin I (OCI) protease inhibitor gene was designed to enhance the stability and effectiveness of OCI in transgenic plants for use in the control of plant pests.
  • the chimeric gene includes both the coding and signal peptide regions of the 1 OkD zein and the coding region of the OCI, and is controlled by the constitutive CaMV 355 promoter.
  • Example 11 Construction of Transgenic Plants Expressingthe 10 kD ZeimOCI Fusion Protein
  • the lOkDsplOkDzeinOC-1 (Z10/OCI) fusion was constructed using the spliced overlapping extension (SOE) technique (Horton, R.M. et al. , 1989).
  • SOE spliced overlapping extension
  • This technique utilizes the polymerase chain reaction (PCR) to splice together two separate fragments.
  • the PCR was first used to amplify the ZIO fragment from plasmid prep (993) Z10pMON316/DH5 ⁇ using primers: SEQ ID NO: 1 PA (CTACAAGATCTGATATCATCGATG) and
  • SEQ ID NO: 2 PB (GACATGGATCCGAATGCAGCAC) to produce a product approximately 500 base pairs (bp) in length.
  • the OC-1 fragment was amplified from plasmid (809) OclpSP73/DH5 ⁇ using primers:
  • SEQ ID NO: 3 PC (GTGCTGCATTCGGATCCATGTCG) and SEQ ID NO: 4 PD (CCGGTACCCTTAAATCGATGC) to produce a product of 322 bp fragment.
  • the two PCR products were combined in equal concentrations and used as the template for the second PCR reaction.
  • the primers, PA and PD were used to produce a fragment of 800 bp.
  • the bolded and larger nucleotides in the primer sequence PA, PB, and PC represent the sequences taken from the Z10 fragment.
  • the smaller nucleotides in primer sequence PB, PC, and PD represent the sequence taken from Ocl.
  • the bolded nucleotides are from the Z 10 sequence and the smaller nucleotides are from the Ocl sequence.
  • the primer sequences in Figure 9 are underlined and the primer name is in bold above or below the primer sequence.
  • the underlined primer sequences PB and PD in Figure 9 are the reverse complement of the PB and PD sequence listing above. The orientation of the primer is indicated by arrows.
  • Example 12 Tobacco Leaf Disc Transformation
  • the construct needed to be subcloned into plasmid pGG.
  • Both the ZlO/OClNPF-1 and pGG were digested with Bgl II and Kpn I, ligated, and transformed into DH5 ⁇ . The presence of the insert was confirmed with PCR.
  • Figure 10 is an overview of the construction of Z10/OC1.
  • the plasmid pGG-2 is a derivative of pMON316 (Rogers et al, 1987) with the addition of the AMV coat protein to enhance translation (Sutton, D.W. et al, 1992).
  • Triparental matings with pTiT37SE were used to co- integrate pGG-2 containing the Z10/OC1 insert.
  • Tobacco leaf discs were transformed by the method of Horschet ⁇ /. (Horsch et ⁇ /., 1985).
  • When individual tobacco shoots were visible they were transferred to Murashige and Skoog media (Murashige, T. and Skoog, F., 1962) containing clafaran (500 uf/ml) and kanamycin (100 ug ml). The individual plants were then tested for protein expression by Western blot.
  • Figures 11 A and 11 B are Western blots showing positive reaction with both the Z 10 and OCI antibodies.

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Abstract

La présente invention concerne des matériaux et des procédés pour des plantes transformées et pour des tissus de plantes qui permettent d'exprimer de fortes teneurs en protéines stables qui sont localisées sous forme de corps protéiques dans la cellule de la plante. L'invention concerne des plantes transformées co-exprimant des fortes concentrations de protéines de zéine 15kD et 10 kD qui s'accumulent sous forme de taux élevés de corps protéiques dans le tissu végétatif de la plante. Les plantes transformées co-exprimant les protéines de zéine 15kD et 10kD permettent de constituer des cultures fourragères contenant des taux accrûs d'acides aminés à base de soufre, comme la méthionine, dans l'alimentation des animaux nourris par ces cultures. L'invention concerne également des plantes ou des tissus de plantes transformés comprenant des corps protéiques stables qui renferment un matériau protéinique hétérologue. Selon un mode de réalisation, un corps protéique stable est exprimé dans une plante ou un tissu de plante sous forme d'une protéine de fusion comprenant une protéine de zéine et une protéine ou un peptide à liaison opérationnelle. Les corps protéiques selon l'invention résiste à la dégradation par l'environnement ou par la digestion du ruminant.
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