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  • 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
• • 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

Definitions

• the present invention relates to transgenic plants that have elevated levels of sulfur-containing amino acids. More specifically, the invention relates to the production of transgenic plants that contain a DNA segment with a coding sequence corresponding to that of a gene for a sulfur-rich rice prolamin or sunflower 2S albumin protein.
• Monocot plants also contain seed storage proteins.
• the predominant storage proteins in most of the agronomically important cereals are the prolamins .
• Rice and oats are unusual cereal crops, in that the major storage protein is an US-type globulin, and only small amounts of prolamin proteins are found.
• Fewer monocot genes have been introduced successfully into dicot plants, for several reasons. There are definite differences in seed composition, gene regulation and post transciptional processing of storage proteins in dicots and monocots. In particular, monocot promoters typically do not function well in dicots. Other concerns relate to the stability of the sulfur-rich gene in the seed, and possible affects of the introduced gene on normal development of the plant. Altenbach et al . , TIBTECH 8: 156 (1990) .
• a method for increasing the content of sulfur-containing amino acids in an animal feed preferably without supplementation, which comprises steps of (i) providing seeds from a plurality of plants, at least some of which plants contain a DNA molecule comprised of a nucleotide sequence corresponding to a sulfur-rich rice prolamin gene or sulfur-rich sunflower 2S albumin gene, operably linked to a promoter to effect expression of the nucleotide sequence by the plants; and (ii) processing the seeds into animal feed.
• the method results in a feed product that has not been supplemented by addition of sulf r-containing amino acids and that contains soybean or corn meal obtained from seed of transgenic plants.
• the present invention allows for the production of transgenic plants that bear seeds characterized by a higher content of sulfur-containing amino acids, relative to plants that lack the heterologous DNA in question.
• an enhanced sulfur content in soybean ( Glycine max) or corn (Zea mays) is achieved, without significant allergenicity problems, by transforming soybean oi corn material with a heterologous DNA segment that has a nucleotide sequence corresponding to one or more copies of a gene for a rice ⁇ Oryza sativa L) 10 kDa or 16 kDa prolamin or a sunflower (Helianthus annus L) 2S albumin protein, which are rich in sulfur.
• Sulfur-rich sunflower 2S albumin proteins are described, by example, by Lilley et al . , "Isolation and Primary Structure for a Novel Methionine-rich Protein from Sunflowerseeds (Helianthus annus . L) , " in PROCEEDINGS OF THE WORLD CONGRESS ON VEGETABLE PROTEIN UTILIZATION IN HUMAN FOODS AND ANIMAL FEEDSTUFFS 497-502 (1989) , and by Kortt et al . , Phytoche istry 29: 2805 (1990) , the respective contents of which are incorporated herein by reference. Eight proteins, denoted sunflower albumins (SFA) 1 to 8, are identified in the 2S albumin fraction.
• SFA sunflower albumins
• SFA-7 and SFA-8 are sulfur-rich. They contain about eight mol per cent cysteine and 16 mol per cent methionine. SFA-8 is present in greater amounts than SFA-7, and for this reason is preferred.
• the nucleotide sequence of SFA-8 is shown in Figure 1.
• a DNA molecule comprising a transformation/expression vector is engineered to include a sequence from one of the sulfur- rich sunflower 2S albumin genes or from the 10 kDa or 16 kDa sulfur-rich rice prolamin gene. Isolation and cloning of these genes by standard methodology is described in Lilley et al . , Kortt et al . and Matsumura et al . , supra .
• the rice prolamin genes first may be modified to reflect preferred codon usage in dicots, prior to introduction into soybean.
• the sulfur-rich sunflower 2S albumin genes and the 10 kDa or 16 kDa sulfur-rich rice prolamin gene of the present invention can be isolated from a natural source or synthesized from a known sequence.
• the sequence may be obtained by making a cDNA from mRNA.
• the sequence may also be derived from a genomic DNA sequence.
• the sequence may be subcloned in a vector of choice .
• the methodologies used would include identification of the gene by hybridization with probes, PCR, probe/primer/synthetic gene synthesis, sequencing, molecular cloning and other techniques which are well-known to those skilled in molecular biology.
• the vector also could contain additional sequences that are necessary to allow for the eventual integration of the vector into a chromosome.
• promoters are the promotor for the small subunit of ribulose-1, 5-bis- phosphate carboxylase, promoters from tumor-inducing plasmids of Agrobacterium tu efaciens, such as the nopaline synthase and octopine synthase promoters, and viral promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S promoters or the figwort mosaic virus 35S promoter.
• the promoter can be constitutive or inducible. - 7 -
• seed tissue-preferred or seed tissue-specific promoters that is, promoters that drive high expression of the heterologous DNA segment in seed tissue where control of genes that are involved in seed metabolism is desired, and little or no expression in other parts of the plant. Manufacture of sulfur-rich proteins in other parts of the plant needlessly expends the plant's energy.
• seed tissue- preferred or seed tissue-specific promoters include the soybean promoter of ⁇ -conglycinin, also known as the 7S protein, which drives seed-directed transcription (Bray, Planta 172: 364 (1987)) and seed-directed promoters from the zein genes of maize endosperm (Pedersen et al . , Cell 29: 1015 (1982)) .
• Dicot promoters are preferred for use in soybeans, and a particularly preferred promoter is the bean phaseolin promoter.
• one or more enhancers are useful in the invention to increase transcrip ion of the introduced DNA segment.
• the enhancer or enhancer-like element can be inserted into the promoter to provide higher levels of transcription.
• enhancers include inter alia , viral enhancers like those within the 35S promoter, as shown by Odell et al . , Plant Mol . Biol . 10: 263-72 (1988) , and an enhancer from an opine gene as described by Fromm et al . , Plant Cell 1: 977 (1989) .
• Selectable marker genes in physical proximity to the introduced DNA segment, are used to allow transformed cells to be recovered by either positive genetic selection or screening.
• the selectable marker genes also allow for maintaining selection pressure on a transgenic plant population, to ensure that the introduced DNA segment, and its controlling promoters and enhancers, are retained by the transgenic plant.
• Many of the commonly used positive selectable marker genes for plant transformation have been isolated from bacteria and code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide.
• Other positive selection marker genes encode an altered target which is insensitive to the inhibitor.
• a preferred selection marker gene for plant transformation is the neomycin phosphotransferase II (nptll) gene, isolated from Tn5, which confers resistance to kanamycin when placed under the control of plant regulatory signals. Fraley et al . , Proc . Nat ' l Acad . Sci . USA 30: 4803 (1983) .
• Another useful selectable marker is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al . , Plan Mol . Biol . 5: 299 (1985) .
• Additional positive selectable markers genes of bacterial origin that confer resistance to antibiotics include gentamicin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3' -adenyl transferase and the bleomycin resistance determinant. Hayford et al . , Plant Physiol .
• genes for plant transformation are not of bacterial origin. These genes include mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al . , Soma ti c Cell Mol . Genet . 13: 67 (1987) ; Shah et al . , Science 233: 478 (1986) ; Charest et al . , Plant Cell Rep . 8: 643 (1990) .
• Another class of useful marker genes for plant transformation with the DNA sequence requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantitate or visualize the spatial pattern of expression of the DNA sequence in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include ⁇ -glucuronidase (GUS) , jS-galactosidase, luciferase, and chloramphenicol acetyltransferase. Jefferson, Plant Mol . Biol . Rep . 5: 387 (1987) ; Teeri et al .
• GUS ⁇ -glucuronidase
• jS-galactosidase luciferase
• chloramphenicol acetyltransferase Jefferson, Plant Mol . Biol . Rep . 5:
• the vector also contains a gene encoding a selection marker which is functionally linked to promoters that control transcription initiation.
• a disarmed Ti- plasmid is used as a vector for foreign DNA sequences.
• an expression vector containing a sulfur-rich 2S albumin sunflower gene or sulfur-rich 10 kDa or 16 kDa rice prolamin gene can be introduced into protoplasts; into intact tissues, such as immature embryos and meristems; into callus cultures or into isolated cells.
• expression vectors are inserted into intact tissues, particularly explants derived from hypocotyl or cotyledonary nodes of a germinated seed.
• an explant is a piece of tissue that is taken from a donor plant and is capable of producing callus in culture.
• Hypocotyl tissue is that portion of the stem of a plant embryo or seedling below the cotyledons and above the root .
• a cotyledon is an embryonic leaf
• a cotyledonary node is that part of the seedling between the embryonic axis and the cotyledons which botanically defines the division of the hypocotyl and the epicotyl , or embryonic shoot .
• General methods of culturing plant tissues are provided, for example, by Miki et al . , "Procedures for Introducing Foreign DNA into Plants," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY 67-88 (CRC Press 1993) .
• a preferred method for introducing an expression vector into plant tissue is direct infection or co- cultivation of plant tissue with A . tumefaciens that contains an expression vector with the gene of interest and associated regulatory elements. Horsch et al . , Science 227: 1229 (1985) .
• a preferred expression vector is the vector pARC12 (pl830) , a plasmid which is part of a binary Ti plasmid system of A . tumefaciens and which contains nopaline synthase/neomycin phosphotransferase II (NPTII) as a promoter and selectable marker for transformed plant cells.
• NPTII nopaline synthase/neomycin phosphotransferase II
• a suitable model for demonstrating transformation of a plant by the sulfur-rich sunflower 2S albumin and sulfur-rich rice prolamin genes is Agrobacterium-mediated transformation of the tobacco plant, Nicotiana tabacum .
• an Agrobacteriu strain confirmed to contain the construct is used to inoculate wounded tobacco plants to generate the transgenic events.
• Agrojbac ⁇ eriuiT? is used to transfer an expression vector to soybean, induction of the virulence (vir) genes in Agrobacterium leads to enhanced transformation.
• Cultured soybean cells lack or have a limiting amount of the necessary signal molecules to initiate the transformation process, and induction of the vir genes is necessary to achieving successful transformation.
• Various physical parameters can be used to induce the vir genes.
• Various compounds can be used, individually or in combination, to induce the vir gene. Exemplary of such compounds are phenolic compounds such as acetosyringone, ⁇ -hydroxyacetosyringone, acetovanillone, syringaldehyde, syringic acid and sinapinic acid.
• the shoot population thus obtained then is screened, by means of a nonlethal enrichment assay, to identify either chimeric sectors that will contribute to germline transmission, or non-sectored, periclinal chimeras that will by definition transmit to progeny. Increased time in culture, under selection, enhances the prospects for sectoral-to-periclinal conversions, and also selects for Ll-to-L2 conversions which, through a shift in position, ultimately contribute to the germline.
• transgenic plants are established from transformed explants by conventional techniques known to the skilled artisan. A preferred technique is cultivation of transformed explants in liquid counterselection medium, followed by transfer to solidified selection medium.
• Seed from transgenic plants according to the invention contain significant levels of sulfur-containing amino acids, particularly methionine.
• Expression of the sulfur-rich genes was accompanied by a concomitant decrease in trypsin and chymotrypsin inhibitor activities.
• These endogenous inhibitors are relatively rich in sulfur-containing amino acids, and the overexpression of the introduced genes may be depleting the normal free sulfur pools in the seed, with the result that sulfur is being scavenged from endogenous proteins.
• the tran genic plants appear to accumulate the heterologous proteins at the expense of certain endogenous proteins to maintain a homeostatic condition. The changes produce no apparent effect on the viability of the transgenic plant or on the total seed protein.
• tumefaciens is a preferred vector
• other types of vectors can be used for transformation by procedures such as direct gene transfer, as described, for example, in PCT application WO 85/01856 and in European application 0 275 069; in vi tro protoplast transformation, which is the subject of U.S. patent No. 4,684,611, for instance; plant virus- mediated transformation, illustrated in European application 0 67 553 and U.S. patent No. 4,407,956; and liposome-mediated transformation according to U.S. patent No. 4,536,475, among other disclosures.
• Direct transfer methods also may be employed, such as microprojectile- mediated delivery, DNA injection, and electroporation. See, for example, Gruber et al . and Miki et al . , both cited above, and Klein et al . , Bio/Technology 10: 268 (1992) .
• Tobacco plants (Ni cotiana tabacum) var. Xanthii, were cultured from 1 cm apical or axillary explants on OMS, according to Murashige et al . , Physiologia Plantarum, 15:473 (1962) , in a Magenta Box (Magenta Corp., Chicago, 111.) at 27°C with a 16 hour light/8 hour dark photoperiod. The first four fully expanded leaves below the apex (leaves 3, 4, 5 or 6) were removed. Leaves were placed, one at a time, in a 100 mm petri dish with several Whatman #1 filter discs, 7cm, soaked with liquid medium. A sterile, sharp, #2 cork bore (0.5 cm i.d.) was used to punch leaf discs, avoiding the midrib and veins. Discs were held on filter paper at 100% humidity in a separate petri dish with Whatman filters and liquid medium.
• the explants were moved to a liquid counterselection medium, a medium having the same basic composition as that used during cocultivation, but with the addition of cefotaxime, 500 ⁇ g/ml and vancomycin, 100 ⁇ g/ml.
• the discs were washed with gentle continuous gyratory shaking for 3-6 hours with at least one change of liquid medium.
• the discs then were placed on solid medium, a medium having the same basic composition as that used during cocultivation, but with the addition of vancomycin, 100 ⁇ g/ml and carbenicillin, 500 ⁇ /ml .
• Excess liquid was evaporated before plating ten to twelve discs per 100 mm plate.
• the explants were cultures for 3 days at 28°C in low light .
• Discs were transferred to solid selection medium, a medium having the same basic composition as that used during cocultivation, but with the addition of vancomycin, 100 ⁇ g/ml; carbenicillin, ⁇ 00 ⁇ g/ml; and kanamycin, 100 ⁇ g/ml.
• the discs were cultured at 26°C in high light with 16 hour light/8 hour dark photoperiod.
• Counterselection medium consisted of B4 salts (G5893) , 3.2 gm/L; sucrose, 2.0% w/v; BAP, 5.0 ⁇ M; IBA, 0.5 ⁇ M; vancomycin, 200 ⁇ /ml ; cefotaxime, 500 ⁇ g/ml and was buffered to pH 5.7 with MES, 3 mM.
• Ten explants were washed in each petri dish with constant, slow gyratory agitation at room temperature for four days. Counterselection medium was replaced four times.
• explants were washed with liquid medium on the gyratory shaker. This time the wash was conducted overnight in counterselection medium containing kanamycin sulfate, 50 ⁇ g/ml. The following day explants were picked to agarose solidified selection medium. Again they were embedded in the medium, adaxial side down, and cultured as before for another two weeks. - 19 -
• Elongation medium consisted of B5 salts (G5893) , 3.2 gm/L; sucrose, 2.0% w/v; IBA, 3.3 ⁇ M; gibberellic acid, 1.7 ⁇ M; vancomycin, 100 ⁇ g/ml; cefotaxime, 30 ⁇ g/ml; timentin, 30 ⁇ g/ml and was buffered to pH 5.7 with MES, 3.0 mM. Elongation medium was solidified with gelrite, 0.2% w/v.
• Total amino acid composition can be determined by acid hydrolysis of tobacco or soybean meal by standard protocols. Seed proteins also can be analyzed for trypsin inhibitory activity according to the previously described protocols of Kollipara et al . , J. Agri cul . Food Chem . 40:2356 (1992) . Similarly chymotrypsin inhibitory assays can be done according to Geiger, Chymotrypsin. In "Methods of Enzymatic Analysis," pp. 99-109 (1984) .

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• 1997
  • 1997-04-24 WO PCT/US1997/006180 patent/WO1997041239A2/en not_active Application Discontinuation
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