Fanguo Chen

Variation and potential value in wheat breeding of low-molecular-weight glutenin subunit genes cloned by genomic and RT-PCR in a derivative of somatic introgression between common wheat and Agropyron elongatum

In order to exploit the composition, origination and possible relationship with quality, 26 low-molecular-weight glutenin subunit (LMW-GS) open reading frames (ORFs) were cloned and sequenced from a derivative of an asymmetric somatic hybrid between wheat and Agropyron elongatum and parent wheat. Most of these (23) were of LMW-m type. One sequence derived from the selected derivative has a unique N-terminal peptide sequence NGRLDASL. Most of the deduced peptide sequences have eight Cys residues, but two have nine and two others ten. Reverse transcriptase polymerase chain reaction (RT-PCR) results show the LMW-GS genes that can express in the endosperm of the derivative. Lmwgh-15 from the derivative has four extra deduced Cys residues and comprises 14.6% of the total expression sequences, which may play an important role in wheat flour quality. A phylogenetic analysis, based on both deoxyribonucleic acid (DNA) and peptide sequences, indicated that most of derivative’s LMW-GS genes were inherited from the receptor wheat, but a few could be traced to the donor A. elongatum. There was evidence for both chimerism and mutation in a number of the derivative’s LMW-GS genes. We conclude that somatic hybridization is able not only to introduce alien genes but also to generate novel LMW-GS alleles.

Molecular cloning and variation of ω-gliadin genes from a somatic hybrid introgression line II-12 and parents (Triticum aestivum cv. Jinan 177 and Agropyron elongatum)

Gluten proteins, including gliadins and glutenins, together are responsible in large part for the end-use quality of the flour, have been extensively studied (Shewry and Tatham 1997; Shewry et al. 2003). The gliadins are monomeric polypeptides, classified into α-, β-, γandωtypes (Shewry et al. 2003). The ω-gliadin genes are encoded by the Gli-1 loci on the short arm of chromosome 1 which is tightly linked to Glu-3 loci, at which the LMW-GS are encoded (Tatham and Shewry 1995). There are also a few ω-gliadins encoded by ‘selfish genes’ at the Gli-A3, Gli-A5, Gli-B5 and Gli-A6 loci (Shewry et al. 2003). The number of ω-gliadins present in the hexaploid wheat endosperm ranges from 15 to 18 (Sabelli and Shewry 1991), and have been classified, according to the first three peptide residues present in the mature protein into ARQ/E-, KEL-, SRLor TRQ-types. ARQ/ E-type shares a 19-residue putative signal peptide, followed by a 10–11-residue N-terminal domain, a large repetitive region (encompassing 90–96% of the protein), and a 10– 11-residue C-terminal domain (Hsia and Anderson 2001). ω-Gliadins are deficient in both cysteine and methionine, and are therefore ‘sulphur poor’ polypeptides (Tatham and Shewry 1995). However, rather few ω-gliadin genes are represented (Hsia and Anderson 2001; Masoudi-Nejad et al. 2002; Matsuo et al. 2005; Hassania et al. 2008), possibly because their coding sequence includes a large repetitive domain, which hampers cloning. Further analysis ofω-gliadins at the DNA level would provide more information to define the evolution and function of this gene family (Hassania et al. 2008).

Molecular characterization of LMW-GS genes from a somatic hybrid introgression line II-12 between Triticum aestivum and Agropyron elongatum in relation to quick evolution

In order to exploit the evolution and find novel low-molecular-weight glutenin subunit (LMW-GS) for improvement of common wheat quality, thirteen variants from a somatic hybrid introgression line II-12 between Triticum aestivum cv. Jinan 177 (JN177) and Agropyron elongatum were characterized via genomic PCR. Four clones were pseudogenes because they contained an internal stop codon. The remaining nine variants contained intact open reading frames (ORFs). Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously. However, they have some unique modifications in the structures. For example, EU292737 contains not only an extra Cys residue in the C-terminal domain but also a long repetitive domain. Both EU159511 and EU292738 start their first Cys residue in the N-terminal repetitive domain, but not in the N-conserved domain traditionally. These structural alterations may have positive contributions to wheat flour quality. The results of phylogeny showed that most LMW-GS variances from II-12 were homologous to those from parent JN177 and other wheat lines. The reason for quick evolution of LMW-GS in II-12 was discussed.

The Brachypodium distachyon methionine sulfoxide reductase gene family

The methionine sulfoxide reductases (MSRs) are a group of thiol-dependent enzymes able to catalyze the conversion of methionine sulfoxide to methionine. Although some plant MSRs are known to act as protectants against various abiotic stresses, their activity in the model grass species Brachypodium distachyon has not been characterized as yet. Here, six B. distachyon MSR (BdMSR) genes have been isolated; they generate eight distinct cDNAs, since two of them (BdMSRB1 and -B5) produce a pair of alternatively spliced messages. The genes were transcribed in the root, culm, leaf and during various stages of caryopsis development. Those induced by abiotic stress (salinity, drought, low temperature, CdCl2, H2O2 and abscisic acid) harbored known stress-responsive cis elements in their promoter sequences. The heterologous expression of five of the BdMSRs (-A2, -A4, -B1.1, -B3 and -B5.1) in yeast revealed that their products gave a measure of protection against salinity, mannitol and oxidative stress. Substrate specificity analysis revealed that BdMSRB1.1 could reduce free Met-R-SO to Met. The enzymatic activities of BdMSRA4, -B1.1 and -B5.1 in transformed yeast under salt treatment have checked and increased obviously resulting in reducing more Met-SO to Met including the peptide and the free types under salt stress than those in control.

Wheat methionine sulfoxide reductase genes and their response to abiotic stress

The wheat cultivar Shanrong no. 3 (cv. SR3) tolerates both salinity and drought stress more effectively than does its progenitor cultivar Jinan 177 (cv. JN177). When the cultivars are subjected to stress, a number of genes encoding methionine sulfoxide reductase (MSRs) are known to be upregulated in SR3. Here, a set of 12 full length Triticum aestivum MSR (TaMSR) cDNAs have been isolated from cv. SR3. The genes were transcribed in the wheat root, stem, and leaf in plants sampled at various developmental stages. Those induced by salinity and drought harbored known stress-responsive cis elements in their promoter region. The constitutive expression in Arabidopsis thaliana of four MSRs which were induced by salt and drought in microarray assay showed that the product of one (TaMSRA2) heightened the plant’s tolerance to NaCl, methylviologen (MV), and abscisic acid, that of the second (TaMSRA5) enhanced salinity tolerance, that of the third (TaMSRB1.1) increased tolerance to salinity, MV and H2O2, and that of the fourth (TaMSRB5.1) increased tolerance to both salinity and mannitol. The effect of the presence in A. thaliana of TaMSRB1.1 was to suppress the accumulation of reactive oxygen species and to increase the intracellular content of soluble sugars.