Cloning and molecular characterization of three novel LMW-i glutenin subunit genes from cultivated einkorn (Triticum monococcum L.)

Abstract

Three novel low molecular weight (LMW) glutenin subunits from cultivated einkorn (Triticum monococcum L., A(m)A(m), 2n = 2x = 14) were characterized by SDS-PAGE and molecular weights determined by MALDI-TOF-MS. Their coding genes were amplified and cloned with designed AS-PCR primers, revealing three complete gene sequences. All comprised upstream, open reading frame (ORF), downstream and no introns were present. The deduced amino acid sequences showed that all three genes, named as LMW-M1, LMW-M3 and LMW-M5, respectively, belonged to the LMW-i type subunits with the predicted molecular weight between 38.5206 and 38.7028 kDa. They showed high similarity with other LMW-i type genes from hexaploid bread wheats, but also displayed unique features. Particularly, LMW-M5 subunit contained an extra cysteine residue in the C-terminus except for eight conserved cysteines, which resulted from a single-nucleotide polymorphism (SNP) of the T-C transition, namely arginine --> cysteine substitution at position 242 from the N-terminal end. This is the first report that the LMW-i subunit contained nine cysteines residues that could result in a more highly cross-linked and more elastic glutenin suggesting that LMW-M5 gene may associates with good quality properties. In addition, a total of 25 SNPs and one insertions/deletions (InDels) were detected among three LMW-i genes, which could result in significant functional changes in polymer formation of gluten. It is anticipated that these SNPs could be used as reliable genetic markers during wheat quality improvement. The phylogenetic analysis indicated that LMW-i type genes apparently differed from LMW-m and LMW-s type genes and diverged early from the primitive LMW-GS gene family, at about 12.92 million years ago (MYA) while the differentiation of A(m) and A genomes was estimated at 3.98 MYA.

A Novel Chimeric Low-Molecular-Weight Glutenin Subunit Gene From the Wild Relatives of Wheat Aegilops kotschyi and Ae. juvenalis: Evolution at the Glu-3 Loci

“…With the LMW-GS gene group, three types of LMW-GS genes were divided into different subgroups but LMW-s-and LMW-m-type genes were shown to have higher homology. These results were consistent with their sequence characteristics and recent reports of An et al (2006) and Pei et al (2007). Particularly, the AjkLMW-i gene located on the U genome was clustered in the same subgroup with other LMW-m-type genes and shared 86% homology.…”

“…Since the previous reported LMW-GS genes were from different Triticum and Aegilops genomes ( Johal et al 2004;An et al 2006), there contained considerable SNP variations in the repetitive and C-terminal domains. A total of 20 SNPs resulted from point mutation, but indel variations were detected among the four cloned LMW-m-type genes, which scattered in the different domains, except the signal peptide and N-terminal domains (Table 1).…”

“…Additionally, a single locus could encode different types of LMW-GS genes; for example, both LMW-s and LMW-m subunit genes appeared to be present at the Glu-B3 locus in the common wheat cultivar Norin 61 (Ikeda et al 2006). The Glu-A3 locus could encode m-type (Lee et al 1999) and i-type LMW-GS (Cloutier et al 2001;Zhang et al 2004;An et al 2006;Li et al 2008). Consequently, it could be deduced that each Glu-3 locus might encode more than one type of LMW-GS.…”

“…The gene sizes vary, depending mainly on the number of repeats in the repetitive domain, normally ranging from 12 to 25 amino acid residues. The extensive allelic variation among both LMW-GS and HMW-GS genes are due mainly to single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) of repetitive units ( Johal et al 2004;Yan et al 2004;An et al 2006;Zhang et al 2006;Li et al 2008). It was speculated that the genetic mechanisms for these variations may result from unequal crossing over and slippage during replication and dot mutation (Anderson and Greene 1989;D'Ovidio et al 1996;An et al 2006).…”

Molecular cloning and characterization of five novel low molecular weight glutenin subunit genes from Tibetan wheat landraces (Triticum aestivum L.)

“…Generally, LMW-GS contain eight cysteine residues that are involved in forming intra-and inter-molecular disulfide bonds in the gluten macro polymers (GMP) . In LMW-m and LMW-s type subunits the first cysteine residue is present in the N-terminal domain and the other seven cysteine residues are in the C-terminus, but in LMW-i type subunits all eight cysteine residues are located in the C-terminus (Ikeda et al 2006;An et al 2006;Pei et al 2006).…”