Academic journal article Genetics

Novel X-Type High-Molecular-Weight Glutenin Genes from Aegilops Tauschii and Their Implications on the Wheat Origin and Evolution Mechanism of Glu-D1-1 Proteins

Academic journal article Genetics

Novel X-Type High-Molecular-Weight Glutenin Genes from Aegilops Tauschii and Their Implications on the Wheat Origin and Evolution Mechanism of Glu-D1-1 Proteins

Article excerpt

ABSTRACT

Two new x-type high-molecular-weight glutenin subunits with similar size to 1Dx5, designated 1Dx5*^sup t^ and 1Dx5.1*^sup t^ in Aegilops tauschii, were identified by SDS-PAGE, RP-HPLC, and MALDI-TOF-MS. The coding sequences were isolated by AS-PCR and the complete ORFs were obtained. Allele 1Dx5*^sup t^ consists of 2481 bp encoding a mature protein of 827 residues with deduced M^sub r^ of 85,782 Da whereas 1Dx5.1*^sup t^ comprises 2526 bp encoding 842 residues with M^sub r^ of 87,663 Da. The deduced M^sub r^'s of both genes were consistent with those determined by MALDI-TOF-MS. Molecular structure analysis showed that the repeat motifs of 1Dx5*^sup t^ were correspondingly closer to the consensus compared to 1Dx5.1*^sup t^ and 1Dx5 subunits. A total of 11 SNPs (3 in 1Dx5*^sup t^ and 8 in 1Dx5.1*^sup t^) and two indels in 1Dx5*^sup t^ were identified, among which 8 SNPs were due to C-T or A-G transitions (an average of 73%). Expression of the cloned ORFs and N-terminal sequencing confirmed the authenticities of the two genes. Interestingly, several hybrid clones of 1Dx5*^sup t^ expressed a slightly smaller protein relative to the authentic subunit present in seed proteins; this was confirmed to result from a deletion of 180 bp through illegitimate recombination as well as an in-frame stop codon. Network analysis demonstrated that 1Dx5*^sup t^, 1Dx2^sup t^, 1Dx1.6^sup t^, and 1Dx2.2* represent a root within a network and correspond to the common ancestors of the other Glu-D-1-1 alleles in an associated star-like phylogeny, suggesting that there were at least four independent origins of hexaploid wheat. In addition to unequal homologous recombination, duplication and deletion of large fragments occurring in Glu-D-1-1 alleles were attributed to illegitimate recombination.

WHEAT is the most important grain crop in the world, with total annual yields of almost 600 million tonnes (SHEWRY et al. 2001). The seed storage proteins, mainly including gliadins and glutenins that initially deposit in ER-derived protein bodies, have the ability to form gluten polymers linked by disulfide bonds, and these are among the largest protein molecules in nature (WRIGLEY 1996). Gliadins are mono-meric and confer dough tractility whereas glutenins are polymeric and consist of high-molecular-weight (HMW) and low-molecular-weight (LMW) subunits, which contribute to the visco-elasticity of dough (SHEWRY et al. 1992). Although the HMW glutenin subunits (HMW-GSs) compose only 8-10% of the total extractable flour protein, they play an important role in flour-processing quality due to network formation in dough by gluten polymerization, allowing wheat flour to be processed into bread, pasta, noodles, and a range of other food products (SHEWRY et al. 1992; SHEWRY and HALFORD 2002; MA et al. 2005).

The HMW subunits are encoded by the Glu-1 loci located on the long arms of chromosomes 1A, 1B, and 1D, and each locus consists of two closely linked genes, designated x- and y-types, with higher and lower molecular weights at two tightly linked loci, Glu-1-1 and Glu-1-2, respectively (PAYNE 1987). In general, the number of cysteine residues is four in x-type and seven in y-type subunits; repetitive motifs with hexapeptides and nanopeptides are present in both, and tripeptides are present only in x-type subunits (SHEWRY et al. 1992). Consequently, three loci encoding up to six HMW-GSs are present in hexaploid bread or common wheat (Triticum aestivum, AABBDD). However, silencing of specific genes leads to variation in the number of subunits from three to five while allelic variation in the subunits encoded by active genes results in proteins with different electrophoretic mobilities (PAYNE 1987; SHEWRY et al. 2001).

It is generally accepted that Aegilops tauschii (2n=2x= 14, DD) is the D-genome donor of hexaploid wheat, which is presumed to have arisen from interspecific hybridization between T. dicoccum (AABB) and Ae. tau-schii, with subsequent chromosome doubling, in southwestern Asia 8000-12,000 years ago (MCFADDEN and SEARS 1946a,b; DVORAK et al. …

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