Identification and characterization of a novel Ag. intermedium HMW-GS gene from T. Aestivum-Ag. intermedium addition lines TAI-I series

An unusually small Ag. intermedium HMW-GS was identified from T. aestivum-Ag. intermedium addition lines TAI-I series through SDS-PAGE and genealogical analyses. Subsequently, the gene encoding the glutenin subunit, named Glu-1Aix1, was isolated using PCR and confirmed by Mass Spectrometry, bacterial expression and Western Blot experiments. Sequence analysis indicated that the complete ORF of Glu-1Aix1 was 1770 bp, and its deduced protein possessed not only the primary structure of known HMW-GSs but also the following notable characteristics. First, the subunit may represent a 'hybrid' sort between x- and y-types of HMW-GSs. Second, there were two additional cysteine residues and two tandem duplications of tri-peptide modules present in its repetitive domain. Considering the novel characteristics of the target protein, their developmental mechanisms and potential values in affecting the processing properties were discussed. In addition to above results, the 5′ flanking sequence (−924 bp relative to the start codon) of Glu-1Aix1 ORF was isolated using genomic PCR. Nucleotide sequence comparisons revealed that the 5′ flanking region shared the basal conservative domains of HMW-GS gene promoters studied so far, indicative of its transcriptional activity. Furthermore, combined with the cytogenetical results published previously, evolutionary biology of the genome containing Glu-1Aix1 was investigated.