A modified gene encoding a sulfur-rich maize seed storage protein, δ-zein, was introduced into white clover plants by Agrobacterium-mediated transformation. Expression of the gene was under the control of the double 35S promoter of cauliflower mosaic virus and the nopaline synthase gene transcription terminator. All of the transgenic plants expressing transgene-specific mRNA also accumulated δ-zein in their leaves. Levels of the HA epitope tagged δ-zein in the first fully expanded young leaves of different transgenic plants varied from 0.06 to 0.3% of total water-soluble protein. Expression of the protein was also detected in petioles, nodes, internodes, roots and seeds of the transgenic plants. N-terminal sequencing of the modified δ-zein from transgenic plants revealed that the protein is processed in white clover leaves as in maize seeds. All the transgenic plants expressing the δ-zein showed monogenic inheritance of the linked nptII gene conferring kanamycin resistance. The epitope tagged δ-zein is relatively stable in white clover leaves and in the highest expressing plants, its accumulation increased with increasing leaf age from 0.3 (youngest leaves) to 1.3% (oldest leaves) of total water-soluble protein. These results open up the possibility of using sulfur-rich and rumen-protected δ-zein to improve white clover forage quality.
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Allen GC, Hall Jr, G, Michalowski S, Newman W, Spiker S, Weissinger AK, Thompson WF: High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Plant Cell 8: 899–913 (1996).
Bagga S, Adams H, Kemp JD, Sengupta-Gopalan C: Accumulation of 15-kilodalton zein in novel protein bodies in transgenic tobacco. Plant Physiol 107: 13–23 (1995).
Bagga S, Adams HP, Rodriguez FD, Kemp JD, Sengupta-Gopalan C: Coexpression of the maize δ-zein and β-zein genes results in stable accumulation of δ-zein in endoplasmic reticulum-derived protein bodies formed by β-zein. Plant Cell 9: 1683–1696 (1997).
Baulcombe DC: RNA as a target and an initiator of posttranscriptional gene silencing in transgenic plants. Plant Mol Biol 32: 79–88 (1996).
Barry TN: Protein metabolism in growing lambs fed on fresh ryegrass (Lolium perenne)-white clover (Trifolium repens) pasture ad lib. Br J Nutr 46: 521–532 (1981).
Bhattacharyya MK, Stermer BA, Dixon RA: Reduced variation in transgene expression from a binary vector with selectable markers at the right and left T-DNA borders. Plant J 6: 957–968 (1994).
Benfey PN, Ren L, Chua, N-H: The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J 8: 2195–2202 (1989).
Bode W, Greyling HJ, Huber RJ, Otlewski J, Wilusz T: The refined 2.0 Å X-ray crystal structure of the complex formed between bovine β-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes. FEBS Lett 242: 285–292 (1989).
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Chem 72: 248–254 (1976).
Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction. Anal Biochem 162: 156–159 (1987).
Chomczynski P: One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal Biochem 201: 134–139 (1992).
Cobon DH, Suter GR, Connelly PT, Shephard RK, Hopkins PS: The residual effects of methionine supplementation on the wool growth performance of grazing sheep. Proc Aust Soc Anim Prod 17: 383 (1988).
Coleman CE, Herman EM, Takasaki K, Larkins BA: The maize γ-zein sequesters α-zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm. Plant Cell 8: 2335–2345 (1996).
Comai L, Moran P, Maslyar D: Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements. Plant Mol Biol 15: 373–381 (1990).
Croissant G, Melton B, Miller D, Kellog W: Amino acid composition of two-clone crosses of alfalfa, research report 12. New Mexico State University, Agricultural Experiment Station, Las Cruces, NM (1976).
Cruz-Alvarez M, Kirihara JA, Messing J: Post-transcriptional regulation of methionine content in maize kernels. Mol Gen Genet 225: 331–339 (1991).
Datla RSS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W: Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 211: 383–384 (1992).
Dove H, Robards GE: Effects of abomasal infusions of methionine on wool production of Merino wethers fed on lucerne and wheaten chaff. Aust J Agric Res 25: 945–956 (1974).
Dynan WS: Modularity in promoters and enhancers. Cell 48: 1–4 (1989).
Ealing PM, Hancock KR, White DWR: Expression of the pea albumin 1 gene in transgenic white clover and tobacco. Transgen Res 3: 344–354 (1994).
Esen A: Separation of alcohol-soluble proteins (zeins) from maize into three fractions by differential solubility. Plant Physiol 80: 623–627 (1986).
Esen A, Stetler DA: Immunocytochemical localization of δ-zein in the protein bodies of maize endosperm cells. Am J Bot 79: 243–248 (1992).
Frazer DL, Poppi DP, Frazer TJ: The effect of protein or amino acid supplementation on growth and body composition of lambs grazing white clover. In: Proceedings of the IV International Symposium on Nutrition of Herbivores, p. 22 (1991).
Geli MI, Torrent M, Ludevid D: Two structural domains mediate two sequential events in γ-zein targeting: protein endoplasmic reticulum retention and protein body formation. Plant Cell 6: 1911–1922 (1994).
Hancock KR, Ealing PM, White DWR: Identification of sulphur-rich proteins which resist rumen degradation and are hydrolysed rapidly by intestinal proteases. Br J Nutr 72: 855–863 (1994).
Hancock KR, Phillips LD, White DWR, Ealing PM: pPE1000: A versatile vector for the expression of epitope-tagged foreign proteins in transgenic plants. BioTechniques 22: 861–865 (1997).
Higgins TJV, Chandler PM, Randall PJ, Spencer D, Beach LR, Blugrove, RJ, Kortt AA, Inglis, AS: Gene structure, protein structure and regulation of the synthesis of a sulfur-rich protein in pea seeds. J Biol Chem 261: 11124–11130 (1986).
Junghans H, Metzlaff M: A simple and rapid method for the preparation of total plant DNA. Bio Techniques 8: 176 (1990).
Khan MRI, Ceriotti A, Tabe L, Aryan A, McNabb W, Moore A, Craig S, Spencer D, Higgins TJV: Accumulation of a sulphur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L.). Transgen Res 5: 179–185 (1996).
Kirihara JA, Hunsperger JP, Mahoney WC, Messing JW: Differential expression of a gene for a methionine-rich storage protein in maize. Mol Gen Genet 211: 477–484 (1988).
Kirihara JA, Petri JB, Messing J: Isolation and sequence of a gene encoding a methionine-rich 10-kDa zein protein from maize. Gene 71: 359–370 (1988).
Kolodziej PA, Young RA: Epitope tagging and protein surveillance. Meth Enzymol 194: 508–519 (1991).
Langlands JP: Efficiency of wool production of grazing sheep. 3. The use of sulphur-containing amino acids to stimulate wool growth. Aust J Exp Agric Anim Husb 10: 665–671 (1970).
Lending CR, Larkins BA: Changes in the zein composition of protein bodies during maize endosperm development. Plant Cell 1: 1011–1023 (1989).
McElroy D, Rothenberg M, Wu R: Structural characterisation of a rice actin gene. Plant Mol Biol 14: 163–171 (1990).
Masumura T, Shibata D, Hibino T, Kato T, Kawabe K, Takeba G, Tanaka K, Fujii S: cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds. Plant Mol Biol 12: 123–130 (1989).
Matsudaira P: Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262: 10035–10037 (1987).
Morgan WA, Cobon DH, Connelly PT: Effect of frequency, dose and site of administration of methionine on wool growth of wethers grazing low quality pastures. Proc Aust Soc Anim Prod 18: 531 (1990).
Narasimhulu SB, Deng X-b, Sarria R, Gelvin SB: Early transcription of Agrobacterium T-DNA genes in tobacco and maize. Plant Cell 8: 873–886 (1996).
Ni M, Cui D, Einstein J, Narasimhulu S, Vergara CE, Gelvin SB: Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes. Plant J 7: 661–676 (1995).
Ohtani T, Wallace JC, Thompson GA, Galili G, Larkins BA: Normal and lysine containing zeins are unstable in transgenic tobacco seeds. Plant Mol Biol 16: 117–128 (1990).
Phillips RL, McClure BA: Elevated protein-bound methionine in seeds of a maize line resistant to lysine plus threonine. Cereal Chem 62: 213–218 (1985).
Pickering FS, Reis PJ: Effects of abomasal supplements of methionine on wool growth of grazing sheep. Aust J Exp Agric 33: 7–12 (1993).
Rago R, Mitchen J, Wilding G: DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. Anal Biochem 191: 31–34 (1990).
Reis PJ: Effects of amino acids on the growth and properties of wool. In: Black JL, Reis PJ (eds) Physiological and Environmental Limitations to Wool Growth, pp. 223–242. University of New England Publishing Unit, Armidale, NSW, Australia (1979).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Schägger H, von Jagow G: Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368–379 (1987).
Schernthaner MA, Matzke MA, Matzke AJM: Endosperm-specific activity of a zein gene promoter in transgenic tobacco plants. EMBO J 7: 1249–1255 (1988).
Shewry PR, Napier JA, Tatham AS: Seed storage proteins: structures and biosynthesis. Plant Cell 7: 945–956 (1995).
Spencer D, Higgins TJV, Freer M, Dove H, Coombe JB: Monitoring the fate of dietary proteins in rumen fluid using gel electrophoresis. Br J Nutr 60: 141–147 (1988).
Thompson DJ: The nitrogen supplied by and the supplementation of fresh or grazed forage. In: Thompson DJ, Beever E, Gunn RG (eds) Forage Protein in Ruminant Animal Production, pp. 53–66. Occasional Publication No. 6, British Society of Animal Production (1982).
Towbin H, Stachelin T, Gordon J: Electrophoretic transfer from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 (1979).
Ulyatt MJ, Thomson DJ, Beever DE, Evans RT, Haines MJ: The digestion of perennial ryegrass (Lolium perenne cv. Melle) and white clover (Trifolium repens cv. Blanca) by grazing cattle. Br J Nutr 60: 137–149 (1988).
Vierstra, RD: Proteolysis in plants: mechanisms and functions. Plant Mol Biol 32: 275–302 (1996).
Voisey CR, White DWR, Dudas B, Appleby RD, Ealing PM, Scott AG: Agrobacterium-mediated transformation of white clover using direct shoot organogenesis. Plant Cell Rep 13: 309–314 (1994).
Wallace JC, Lopes MA, Paiva E, Larkins BA: New methods for extraction and quantification of zeins reveal a high content of γ-zein in modified opaque-2 maize. Plant Physiol 92: 191–196 (1990).
Wandelt CI, Khan MRI, Craig S, Schroeder HE, Spencer D, Higgins TJV: Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant J 2: 181–192.
White DWR, Voisey CR: Prolific direct plant regeneration from cotyledons of white clover. Plant Cell Rep 13: 303–308 (1994).
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Sharma, S.B., Hancock, K.R., Ealing, P.M. et al. Expression of a sulfur-rich maize seed storage protein, δ-zein, in white clover (shape Trifolium repens) to improve forage quality. Molecular Breeding 4, 435–448 (1998). https://doi.org/10.1023/A:1009656002068