title: A biotechnological approach to improving the nutritive value of alfalfa.
abstract: The postruminal supply of the sulfur-containing amino acids, methionine and cysteine, has been reported to be a major limitation to wool growth in sheep. We aim to improve the protein quality of forage for ruminants by introducing into alfalfa chimeric genes encoding a ruminally stable, sulfur amino acid-rich protein from sunflower seeds. Four gene constructs were transferred to Australian commercial cultivars of alfalfa using Agrobacterium tumefaciens-mediated transformation and selection with phosphinothricin (PPT). Modification of the sunflower seed albumin protein-coding region by addition of the coding information for an endoplasmic reticulum (ER) retention signal was found to greatly increase the level to which the sulfur amino acid-rich protein accumulated in the leaves of transgenic alfalfa plants. The Cauliflower Mosaic Virus (CaMV) 35S promoter and two light-regulated plant gene promoter regions were compared for their ability to direct high-level expression of the introduced genes in alfalfa leaves. The highest expression of sunflower seed albumin was found in transformants bearing a gene incorporating the promoter from the Arabidopsis thaliana ats1A gene, which encodes the ribulose bisphosphate carboxylase small subunit. The highest level of sunflower seed albumin found in transgenic alfalfa leaves was estimated to constitute .1% of soluble leaf protein. This level of accumulation of the foreign protein would be predicted to supply an extra 40 mg of sulfur amino acids daily to sheep fed the modified forage. Published studies in which wool growth rates were significantly increased employed supplementation of approximately 1 to 2 g of sulfur amino acids daily.
authors.affiliation: CSIRO Division of Plant Industry, Canberra, ACT, Australia.
authors.forename: L M
authors.initials: LM
authors.lastname: Tabe
authors.forename: T
authors.initials: T
authors.lastname: Wardley-Richardson
authors.forename: A
authors.initials: A
authors.lastname: Ceriotti
authors.forename: A
authors.initials: A
authors.lastname: Aryan
authors.forename: W
authors.initials: W
authors.lastname: McNabb
authors.forename: A
authors.initials: A
authors.lastname: Moore
authors.forename: T J
authors.initials: TJ
authors.lastname: Higgins
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000418:Albumins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000603:Amino Acids, Sulfur / Q000008:administration & dosage; D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods* / Q000592:standards; D002417:Cattle; D018744:DNA, Plant / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D018014:Gene Transfer Techniques; D006368:Helianthus / Q000737:chemistry; D000455:Medicago sativa / Q000737:chemistry* / Q000235:genetics / Q000592:standards; D008969:Molecular Sequence Data; D009753:Nutritive Value; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D030821:Plants, Genetically Modified / Q000737:chemistry* / Q000235:genetics; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012418:Ruminants; D012639:Seeds / Q000737:chemistry; D012756:Sheep; D014026:Nicotiana / Q000235:genetics
chemical_list: D000418:Albumins; D000603:Amino Acids, Sulfur; D018744:DNA, Plant; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins