Characterization Of An Expressed Triticum Monococcum Glu-Aly Gene Containing A Premature Termination Codon In Its C-terminal Coding Region

T.monococcum ssp.monococcum(AmAm,2n=2x=14)contain lots of valuable genes that can broaden the genetic bases of wheat and play an important role in improving the quality of wheat.High molecular weight glutenin subunits(HMW-GSs)have been regarded as the most important wheat seed storage proteins,the concentration and composition of glutenin in wheat flour determine its end-use quality.The Glu-Aly allele is always silent in common wheat.However,a majority of active lAy genes existed in many species of diploid and tetraploid wheat.Premature termination codon(PTC)plays a crucial role in the silence of Glu-Aly gene.In this study,lAy8.3 gene of Triticum monococcum L.ssp.monococcum accession 10-1 was characterized by SDS-PAGE,gene cloning,3'RACE,bacterial expression and MALDI-TOF/MS.We sought to characterize the expressed gene containing a PTC and examined its expression in vivo and in vitro.The main results are as follows:1.SDS-PAGE analysis indicated that the T.monococcum ssp.monococcum accession 10-1 expressed an active y-type subunit with the electrophoretic mobility between that of the 1Bx7 and 1By8 proteins of the common wheat cultivar Chinese Spring,which was different from the previously identified 1Ay genes.Based on its electrophoretic mobility,the y-type subunit was designated as 1Ay8.3.2.The active y-type HMW-GS allele termed 1Ay8.3 was cloned and sequenced.The sequence of lAy8.3 was deposited in GenBank(No.KU207221).Compared with previously reported active 1Ay subunits,a PTC of TAG was present at position 627 of the amino acid sequence in its C-terminal coding region.The PTC was also found at the same position as in its corresponding cDNA sequences.In order to analyze the effects of the PTC on the transcriptional level of 1Ay8.3,the PTC at the same position as in the genomic DNA and corresponding cDNA sequences.And we didn't find the PTC TAG was substituted by some unknown amio acids and the 'new 1Ay8.3 transcripts' which have no PTC TAG at the same position are nonexistent,confirming its normal transcription.3.Considering the presence of the premature stop codon in the C-terminal domain of the 1Ay8.3 gene,the bacterial expression experiments were conducted using two pairs of primers combinations.The 1Ay8.3 full length sequence with PTC expressed protein in E.coli with the same electrophoretic mobility as the protein from N-terminal to terminate at PTC TAG in C-terminal domain by the 1Ay8.3 partial sequence,but whose electrophoretic mobility was slightly faster than the lAy8.3 subunit in the seeds.Further,the 1Ay8.3 mutant protein was successfully expressed in E.coli.Although its expression product showed no obvious size difference compared with that of the 1Ay8.3 subunit in the seeds,it had slower electrophoretic mobility than the expressed wild-type sequence containing the TAG PTC.4.On the basis of MALDI-TOF/MS and Nano LC-MS/MS analysis,the expression products from seeds and E.coli,including the wild-type TAG PTC and its mutant,were identified as 1Ay,since the examined peptides from E.coli are specific to the Glu-1 protein.MALDI-TOF/MS analysis also showed that the glutenin proteins expressed in seeds and from the 1Ay8.3 mutant in E.coli exhibited a similar size of approximately 70 kDa,which is higher than that of 1Ay8.3 expressed in E.coli(approx.68 kDa).This also indicated that the 1Ay8.3 expressed in E.coli was a truncated Glu-Aly protein.5.To study the condition of expression of 1Ay8.3 gene with a PTC in vivo,PA7-yellow fluorescent protein(YFP)vector was used to transfected transiently of leaf protoplasts.To construct the lAy649-YFP fusion sequence,for which the complete cDNA of 1Ay8.3 contains a PTC and signal peptide sequence without double stop codons.And then the construction of lAy649-YFP fusion sequence was transiently transformed in 10-1 leaf protoplast cells.After transient transformation of leaf protoplasts and 12h of incubation in the dark,we observed the green fluorescence signals for 1Ay649-YFP by using epifluorescence microscope.The expression of 1Ay649-YFP having the PTC suggested that the PTC translation termination suppression existed in vivo.