Molecular Cloning Of LMW-GS Genes In Wheat Related Species And In Vitro Small-scale Testing Of HMW-GS Functions

Wheat seed storage proteins, especially high- and low-molecular-weight glutenin subunits, are importat for processing-quality of wheat flour. At present, relatively fewer studies for the gene isolation of low-molecular-weight glutenin subunits (LMW-GS) and the functional small-scal testing of single glutenin subunits have been carried out. This work included two parts: isolation and cloning of LMW-GS genes from wheat related species and small-scale functional identification of 8 major high molecular weight glutenin subunits (HMW-GS). The main results obtained were as the followings:· Cloning, characterization and molecular evolutional analysis of LMW-GSgenesLMW-GSs in Aegilops tauschii were identified by SDS-PAGE and mass spectrometry (MS). A pair of AS-PCR primers for LMW-GS genes was used to amplify the LMW-GS genes from Triticum dicoccoides and Aegilops tauschii. Single amplified band with about 900bp from three Aegilops tauschii and two amplified bands with lOOObp from Triticum dicoccoides were obtained, and the amplified products were cloned and sequenced. The complete nucleotide sequences of four LMW-GS genes had 1021bp, 918bp, 921bp and 918bp, respectively. All four genes were complete, and there was a stop code in the coding sequence of the LMW-Y21 gene, indicating that LMW-Y21 was a pseudogene. Frist amino acid of the deduced mature proteins of LMW-Y21 was isoleucine residues, and therefore it belonged to LMW-i type glutenin subunit whereas LMW-T132-1, LMW-T128 and LMW-T121 with methionine residues as frist amino acid of the deduced mature proteins were LMW-m type glutenins. Three LMW-m type glutenin gene sequences showed a clear structural organization comparing with other six LMW-GS DNA sequences from Genebank. They are all composed of signal pepetide, domain I, domain II, domain III, domain IV and domain V. The deduced protein sequences of LMW-Y21 gene possessed five parts without domain I, with a typical structural organization of LMW-i type glutenin subunit. The expected number of eight cysteine residues of LMW-Y21 subunit was all located in the C-terminal region. One of the eight cysteine residues in LMW-T132-1 LMW-T128 and LMW-T121 was located at the fifth amino acid in N-terminal region and the...