Yanzhen Zhang

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

Two new x-type high-molecular-weight glutenin subunits with similar size to 1Dx5, designated 1Dx5*t and 1Dx5.1*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*t consists of 2481 bp encoding a mature protein of 827 residues with deduced Mr of 85,782 Da whereas 1Dx5.1*t comprises 2526 bp encoding 842 residues with Mr of 87,663 Da. The deduced Mrs of both genes were consistent with those determined by MALDI-TOF-MS. Molecular structure analysis showed that the repeat motifs of 1Dx5*t were correspondingly closer to the consensus compared to 1Dx5.1*t and 1Dx5 subunits. A total of 11 SNPs (3 in 1Dx5*t and 8 in 1Dx5.1*t) and two indels in 1Dx5*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*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*t, 1Dx2t, 1Dx1.6t, 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.

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

Four LMW-m and one novel chimeric (between LMW-i and LMW-m types) low-molecular-weight glutenin subunit (LMW-GS) genes from Aegilops neglecta (UUMM), Ae. kotschyi (UUSS), and Ae. juvenalis (DDMMUU) were isolated and characterized. Sequence structures showed that the 4 LMW-m-type genes, assigned to the M genome of Ae. neglecta, displayed a high homology with those from hexaploid common wheat. The novel chimeric gene, designed as AjkLMW-i, was isolated from both Ae. kotschyi and Ae. juvenalis and shown to be located on the U genome. Phylogentic analysis demonstrated that it had higher identity to the LMW-m-type than the LMW-i-type genes. A total of 20 single nucleotide polymorphisms (SNPs) were detected among the 4 LMW-m genes, with 13 of these being nonsynonymous SNPs that resulted in amino acid substitutions in the deduced mature proteins. Phylogenetic analysis demonstrated that it had higher identity to the LMW-m-type than the LMW-i-type genes. The divergence time estimation showed that the M and D genomes were closely related and diverged at 5.42 million years ago (MYA) while the differentiation between the U and A genomes was 6.82 MYA. We propose that, in addition to homologous recombination, an illegitimate recombination event on the U genome may have occurred 6.38 MYA and resulted in the generation of the chimeric gene AjkLMW-i, which may be an important genetic mechanism for the origin and evolution of LMW-GS Glu-3 alleles as well as other prolamin genes.

Comparative proteome analysis of glutenin synthesis and accumulation in developing grains between superior and poor quality bread wheat cultivars

BACKGROUND Wheat glutenins are the major determinants of wheat quality. In this study, grains at the development stage from three wheat cultivars (Jimai 20, Jin 411 and Zhoumai 16) with different bread-making quality were harvested based on thermal times from 150 °C(d) to 750 °C(d) , and were used to investigate glutenin accumulation patterns and their relationships with wheat quality. RESULTS High and low molecular weight glutenin subunits (HMW-GSs and LMW-GSs) were synthesised concurrently. No obvious correlations between HMW/LMW glutenin ratios and dough property were observed. Accumulation levels of HMW-GSs and LMW-GSs as well as 1Bx13 + 1By16 and 1Dx4 + 1Dy12 subunits were higher in superior gluten quality cultivar Jimain 20 than in poor quality cultivar Jing 411 and Zhoumai 16. According to the results of two-dimensional gel electrophoresis, six types of accumulation patterns in LMW-GSs were identified and classified. The possible relationships between individual LMW-GSs and gluten quality were established. CONCLUSION The high accumulation level of HMW-GSs and LMW-GSs as well as 1Bx13 + 1By16 and 1Dx4 + 1Dy12 subunits contributed to the superior gluten quality of Jimai 20. Two highly expressed and 16 specifically expressed LMW glutenin subunits in Jimain 20 had positive effects on dough quality, while 17 specifically expressed subunits in Zhoumai 16 and Jing 411 appeared to have negative effects on gluten quality.

Molecular cloning and characterization of four novel LMW glutenin subunit genes from Aegilops longissima, Triticum dicoccoides and T. zhukovskyi.

This paper reports cloning and characterisation of four novel low-molecular-weight glutenin subunit (LMW-GS) genes (designated as TzLMW-m2, TzLMW-m1, TdLMW-m1 and AlLMW-m2) from the genomic DNA of Triticum dicoccoides, T. zhukovskyi and Aegilops longissima. The coding regions of TzLMW-m2, TzLMW-m1, TdLMW-m1 and AlLMW-m2 were 1056 bp, 903 bp, 1056 bp and 1050 bp in length, encoding 350, 300, 350 and 348 amino acid residues, respectively. The deduced amino acid sequences showed that the four novel genes were classified as LMW-m types and the comparison results indicated that the four genes had a more similar structure and a higher level of homology with the LMW-m genes than the LMW-s and -i types genes. However, the first cysteine residues positions of TzLMW-m2, TdLMW-m1 and AlLMW-m2 were different from the others. Moreover, AlLMW-m2, TdLMW-m1 and TzLMW-m2 all possessed a longer repetitive domain, which was considered to be associated with good quality of wheat. The secondary structure prediction revealed that the content of beta-strand in AlLMW-m2 and TdLMW-m1 exceeded the positive control, suggesting that AlLMW-m2 and TdLMW-m1 should be considered as candidate genes that may have positive effect on dough quality. In order to investigate the evolutionary relationship of the novel genes with the other LMW-GSs, a phylogenetic tree was constructed. The results lead to a speculation that AlLMW-m2, TdLMW-m1 and TzLMW-m2 may be the middle types during the evolution of LMW-m and LMW-s.

Characterization and comparative analysis of three low molecular weight glutenin C-subunit genes isolated from Aegilops tauschii

Three low molecular weight glutenin subunit (LMW-GS) genes from T121, T128 and T132 accessions of Aegilops tauschii (DD, 2n = 2x = 14) were amplified using allelic-specific PCR primers. The amplified products with a size of about 900 bp were cloned and sequenced. Three complete coding sequences of LMW-GS with 918 bp, 921 bp and 918 bp were obtained and named as LMW-T121, LMW-T128, LMW-T132, respectively. Each gene contained a complete open reading frame and had no introns. The deduced amino acid sequences showed that all belonged to LMW-m type subunit with a predicted molecular weight of about 32 kDa, corresponding to the size of LMW C-subunits. All three subunits possessed eight cysteine residues and had greater homology with previously characterized LMW-m subunits from bread wheat and related species than LMW-s or LMW-i sequences. Some amino acid substitutions and insertion/deletion variations among the sequences were detected. The corresponding three C-subunits in seed endosperm encoded by LMW-T121, LM...

Rapid separation and characterization of grain water-soluble proteins in bread wheat cultivars (Triticum aestivum L.) by capillary electrophoresis

Water-soluble (WS) proteins in wheat grain are considered to represent the suite of biologically active enzymes and enzyme inhibitors in the grain. In this study, a rapid capillary electrophoresis (CE) method for WS protein separations was developed using untreated fused-silica columns and an acidic phosphate-glycine buffer system. In order to optimize the resolution and reproducibility of CE separation, different protein extraction methods, organic modifiers in phosphate-glycine buffer and capillary electrophoresis conditions, including capillary length and inner diameter (ID), operating temperature, performance voltages, sample injection times, etc., were investigated. High resolution and reproducibility of WS proteins were achieved using 20% ethanol as the extracting buffer. The optimal condition to separate these proteins was 50 μm ID × 31.5 cm (26.5 cm to the detector) capillary at 11.0 kV and 35°C. The optimum buffer was 0.1 M phosphate-glycine (pH 2.5) containing 20% acetonitrile (ACN) and 0.05% hy...

Characterization of monoclonal antibodies specific to wheat glutenin subunits and their correlation with quality parameters

Immunochemical methods are very useful in predicting the quality of wheat and differentiating alleles. In order to prepare appropriate monoclonal antibodies, HMW-GS 1Bx13 and 1By16 from spelt wheat were used as antigens to immunize BALB/C mice. Four monoclonal antibodies (mAbs) were obtained and designated 24231, 24245, 14588 and 14587, respectively. Results of Western blot showed that mAbs 24231 and 24245 prepared against 1Bx13 bound only to LMW-GS. The mAb 14588 prepared against 1By16 bound strongly to LMW-GS, but weakly to 1By and some 1Dy type HMW-GS. The mAb 14587 prepared against 1By16 bound only to 1Dx HMW-GS. The results of indirect ELISA and statistical analysis showed that correlations between mAb 24231 and development time and stability were significantly (P < 0.05) and highly significantly (P < 0.01) negative, respectively, whereas those of mAb 24245 with development time and extensibility were highly significantly (P < 0.01) and significantly (P < 0.05) negative, respectively. Significantly (...