Molecular characterization and functional properties of two novel x-type HMW-GS from wheat line CNU608 derived from Chinese Spring × Ae. caudata cross

Two novel HMW-GS, designed as 1Dx2s and 1Dx2f in the wheat line CNU608 derived from hybrids between Chinese Spring (CS) and Ae. caudata were identified. Quality analysis showed that the introgression of 1Dx2s and 1Dx2f subunits led to significant improvement of dough strength and breadmaking quality. The complete encoding sequences of 1Dx2s and 1Dx2f subunits were isolated and cloned. 1Dx2s and 1Dx2f genes encode 831 and 808 amino acid residues, respectively. Their presence and authenticity were confirmed through Escherichia coli expression, Western blotting and tandem mass spectrometry identification. The subunit 1Dx2s had an octapeptide deletion (ELQELQER) in the N-terminal domain compared to 1Dx2 while the subunit 1Dx2f was encoded by a chimeric gene originated from 1Dx2 and 1Cy genes. Particularly, 1Dx2f had an extra cysteine residue from 1Cy subunit at position 730 as well as longer repetitive domain and higher glutamine content, indicating its potential values for wheat gluten quality improvement. Both 1Dx2s and 1Dx2f subunits were simultaneously expressed in the derived line, suggesting that one x-type gene duplication event at the Glu-D1 locus occurred. A putative origin of both 1Dx2s and 1Dx2f genes indicated that they could be originated from one octapeptide deletion and two unequal cross-over events.