caa a22 4500 467900604 CHVBK 20180406152816.0 cr unu---uuuuu 170328e20061201xx s 000 0 eng 10.1007/s00299-006-0191-9 doi (NATIONALLICENCE)springer-10.1007/s00299-006-0191-9 Targeted expression of redesigned and codon optimised synthetic gene leads to recrystallisation inhibition and reduced electrolyte leakage in spring wheat at sub-zero temperatures [Elektronische Daten] [Harjeet Khanna, Grant Daggard] Antifreeze proteins (AFPs) adsorb to ice crystals and inhibit their growth, leading to non-colligative freezing point depression. Crops like spring wheat, that are highly susceptible to frost damage, can potentially be made frost tolerant by expressing AFPs in the cytoplasm and apoplast where ice recrystallisation leads to cellular damage. The protein sequence for HPLC-6 α-helical antifreeze protein from winter flounder was rationally redesigned after removing the prosequences in the native protein. Wheat nuclear gene preferred amino acid codons were used to synthesize a recombinant antifreeze gene, rAFPI. Antifreeze protein was targeted to the apoplast using a Murine leader peptide sequence from the mAb24 light chain or retained in the endoplasmic reticulum using C-terminus KDEL sequence. The coding sequences were placed downstream of the rice Actin promoter and Actin-1 intron and upstream of the nopaline synthase terminator in the plant expression vectors. Transgenic wheat lines were generated through micro projectile bombardment of immature embryos of spring wheat cultivar Seri 82. Levels of antifreeze protein in the transgenic lines without any targeting peptide were low (0.06-0.07%). The apoplast-targeted protein reached a level of 1.61% of total soluble protein, 90% of which was present in the apoplast. ER-retained protein accumulated in the cells at levels up to 0.65% of total soluble proteins. Transgenic wheat line T-8 with apoplast-targeted antifreeze protein exhibited the highest levels of antifreeze activity and provided significant freezing protection even at temperatures as low as −7°C. Springer-Verlag, 2006 Antifreeze protein nationallicence Codon optimisation nationallicence Frost tolerance nationallicence Signal peptide nationallicence Transgenic nationallicence Wheat nationallicence Khanna Harjeet Centre for Systems Biology, Faculty of Sciences, University of Southern Queensland, 4350, Toowoomba, Qld., Australia aut Daggard Grant Centre for Systems Biology, Faculty of Sciences, University of Southern Queensland, 4350, Toowoomba, Qld., Australia aut Plant Cell Reports Springer-Verlag 25/12(2006-12-01), 1336-1346 0721-7714 25:12<1336 2006 25 299 https://doi.org/10.1007/s00299-006-0191-9 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00299-006-0191-9 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Khanna Harjeet Centre for Systems Biology, Faculty of Sciences, University of Southern Queensland, 4350, Toowoomba, Qld., Australia aut NATIONALLICENCE 700 1- Daggard Grant Centre for Systems Biology, Faculty of Sciences, University of Southern Queensland, 4350, Toowoomba, Qld., Australia aut NATIONALLICENCE 773 0- Plant Cell Reports Springer-Verlag 25/12(2006-12-01), 1336-1346 0721-7714 25:12<1336 2006 25 299 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer