title: Electrophoretic and chromatographic evaluation of transgenic barley expressing a bacterial dihydrodipicolinate synthase.
abstract: Nutritional quality of human and animal foodstuffs is determined by the content of essential amino acids. Barley is the fourth most important cereal of the world and the second most important cereal grown in the Czech Republic. Cereal grains such as barley contain insufficient levels of some essential amino acids, especially lysine. Dihydrodipicolinate synthase is the key enzyme involved in the regulatory step for lysine biosynthesis. Two constructs pBract214::sTPdapA and pBract214::mdapA containing the dapA gene from Escherichia coli coding for the bacterial dihydrodipicolinate synthase were used for transformation of barley. An Agrobacterium-mediated technique was used for transformation of immature embryos of spring barley cv. Golden Promise. Transgenic barley plants of the T0 and T1 generations were evaluated by PCR, real-time PCR, gel electrophoresis, and Western blot. Amino acid content was analyzed by HPLC after HCl hydrolysis. The lysine content in leaves of the T1 generation plant no. 5/5 was 50% higher than in wild-type plants; the lysine content in seeds of T2 generation plant no. 5/16 was 30% higher than in wild-type seeds of spring barley cv. Golden Promise.
authors.affiliation: Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Olomouc, Czech Republic.
authors.forename: Ludmila
authors.initials: L
authors.lastname: Ohnoutkova
authors.forename: Ondrej
authors.initials: O
authors.lastname: Zitka
authors.forename: Katarina
authors.initials: K
authors.lastname: Mrizova
authors.forename: Jana
authors.initials: J
authors.lastname: Vaskova
authors.forename: Petr
authors.initials: P
authors.lastname: Galuszka
authors.forename: Natalie
authors.initials: N
authors.lastname: Cernei
authors.forename: Mark A
authors.initials: MA
authors.lastname: Smedley
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Harwood
authors.forename: Vojtech
authors.initials: V
authors.lastname: Adam
authors.forename: Rene
authors.initials: R
authors.lastname: Kizek
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D001467:Hordeum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D006836:Hydro-Lyases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D008239:Lysine / Q000032:analysis / Q000378:metabolism; D018515:Plant Leaves / Q000737:chemistry; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase; D008239:Lysine