Isolation and expression of a new high molecular weight glutenin subunit gene at the Glu-D-1-2 locus from Aegilops tauschii
Two new y-type HMW-GSs in Ae. tauschii, 1Dy12.1*t and 1Dy12.2t with the mobility order of 1Dy12.2t > 1Dy12.1*t > 1Dy12.1t>1Dy12, were identified by both SDS-PAGE and MALDI-TOF-MS. Molecular cloning and sequencing showed that the genes encoding subunits 1Dy12.1*t and 1Dy12.2t had identical nucleotide acid sequences with 1,947 bp encoding a mature protein of 627 residues. Their deduced molecular weights were 67,347.6 Da, satisfactorily corresponding to that of 1Dy12.2t subunit determined by MALDI-TOF-MS (67,015.7 Da), but was significantly smaller than that of the the 1Dy12.1*t subunit (68,577.1 Da). Both subunits showed high similarities to 1Dy10, suggesting that they could have a positive effect on bread-making quality. Interestingly, the expressed protein of the cloned ORF from accessions TD87 and TD130 in E. coli co-migrated with subunit 1Dy12.2t, but moved slightly faster than 1Dy12.1*t on SDS-PAGE. The expressed protein in transgenic tobacco seeds, however, had the same mobility as the 1Dy12.1*t subunit, as confirmed by both SDS-PAGE and Western blotting. Although direct evidence of phosphoprotein could not be obtained by specific staining method, certain types of post-translational modifications (PTMs) of the 1Dy12.1*t subunit could not be excluded. We believe PTMs might be responsible for the molecular weight difference between the subunits 1Dy12.1*t and 1Dy12.2t.
KeywordsAegilops tauschii common wheat HMW-GS SDS-PAGE MALDI-TOF-MS protein post-translational modifications
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