Molecular Cloning Of The Inactive Y-type Genes For Glu-B1 In Common Wheat

The high-molecular-weight glutenin subunits (HMW-GSs) are a major class of storage proteins in common wheat (Triticurn aestivum L., AABBDD, 2n = 6x = 42). The bread-making quality of common wheat flour is directly influenced by the composition and quantity of HMW-GSs. Theoretically, in common wheat there are six genes for HMW-GSs. However, due to gene silencing, common wheat usually expresses three to five HMW-GSs. Genes coding for subunits 1Bx, 1Dx, and 1Dy are always expressed, subunits 1Ax and 1By are unexpressed in some cultivars, and subunit 1Ay is almost unexpressed. In previous studies, two different silencing mechanisms derived from the variation in the coding region of a Glu-1 allele (1Ax and 1Ay) of wheat have been reported. One is due to the insertion of transposon elements. The other is the presence of in-frame premature stop codons (pre-stop codons). However, to date, there is no report for the silent mechanisms underlying the silenced 1By gene. In this study, we try to reveal the silent mechanisms in the molecular level for 1By in two Chinese common wheat landraces. The results are as follows:1.Characterization and isolation of the inactive HMW-GS 1By geneGenomic PCR reaction was carried out by using degenerate primers specific for 1By and DNA fragments that represented the complete coding region sequences of the 1By genes were respectively obtained from Yunnan Hulled Wheat (AS332) and Tibetan Weedrace Wheat (AS908). The size of the amplified fragment was approximately between 1.9 to 2.0 kb. The DNA fragments amplified from the two wheat landraces were separately cloned into the pMD 18-T vector. Then, DNA sequencing was carried out by using overlapping sub-clones. The size of the inserts for both AS332 and AS908 was 1979 bp. The nucleotide sequences of AS332 and AS908 were deposited in GenBank under the accession numbers EF381741 and EF381742, respectively. Comparison analysis by using BLASTn in NCBI, it was found that the DNA sequence of the silent 1By genes showed the highest homology with that of previously published 1By9 (AY486485) in Chinese common wheat cultivar Xingkehan. Therefore, it was concluded that the inactive HMW-GS genes both for AS332 and AS908 were 1By9. 2. The sequence character of the inactive 1By HMW-GS geneThe length of the expressed 1By9 in cultivar Xingkehan was 1980bp (AY486485). In comparison with it, the inactive 1By9 in AS332 has a deletion ofnucleotide A at 955 bp downstream from the start sequence, while AS908 has a deletion of nucleotide A at 785 bp downstream from the start sequence. According to the expressed 1By9 sequences in Xingkehan, the primary structure of the translated 1By subunit contained a signal peptide (21 amino acid residues), an N-terminal domain (104 amino acid residues), a C-terminal domain (42 amino acid residues), and a central repetitive domain (491 amino acid residues). If the deletions of nucleotide A were ignored, the resulted hypothetic protein would also resemble the expressed 1By9 and contained the signal peptide, N- and C-terminal domains and central repetitive domain. However, the deletion ofnucleotide A in DNA sequences of 1By gene had led to frameshift mutation, thus the amino acid sequences were changed after the 320th amino acid locus in AS332 and 262th amino acid locus in AS908, respectively. In AS332, the frameshift mutation induced the presence of five in-frame premature stop codons at 1160 bp (TAG), 1178 bp (TAG), 1544 bp (TAG), 1670 bp (TAG) and 1688 bp (TAG), respectively. Besides above five in-frame premature stop codons, Asg08 had an extra in-frame premature stop codon at 780 bp (TAG).3. The molecular mechanism for the silence of 1By9The two wheat landraces are different in taxon, AS332 belongs to T. aestivum ssp.yunnanese King, while AS908 belongs to T. aesitvurn ssp.tibetanum Shao. They showed a difference on the deletion position of nucleotides. However, it is interesting that the both deletions resulted in frameshift mutations and produced the presence of stop codon TAG. The both deletions were taken place in trinucleotide CAA (CAA→CA). Different from previously reported mechanisms for silenced genes 1Ax and 1ay, which were derived from the insertion of transposon elements or presence of premature stop codon via base substitution of C→T transition in trinucleotides CAA or CAG; the silence of 1By9 was caused by premature stop codons via the deletion of base A in trinucleotide CAA, which resulted in frameshift mutation and indirectly produced several premature stop codons (TAG) at the downstream of the coding sequence. The amino acid sequences were early stopped by the premature stop codons. This is a new molecular mechanism for the silence of HMW-GS gene in common wheat.