Anfei Li

Cloning and Expression Analysis of Wheat Pheophorbide a Oxygenase Gene TaPaO

Pheophorbide a oxygenase (PaO) plays an important role in plant chlorophyll degradation. In this study, a novel gene, designated as TaPaO, was isolated from hexaploid wheat (Triticum aestivum). TaPaO was predicted to encode a protein with 523 amino acids. The encoded protein contained the typical Rieske (2Fe-2S) and mononuclear iron-binding domains as well as the C-terminus CxxC motif. Amino acid sequence alignment revealed that TaPaO shared a high degree of identity with other higher plant PaO proteins. The result of expression analysis indicated that TaPaO was expressed in roots, stems and leaves, and the expression level of TaPaO was obviously higher in leaves than in stems and roots. Quantitative real-time PCR showed that the expression of TaPaO was upregulated by abscisic acid (ABA) and methyl jasmonate (MeJA), but not by salicylic acid (SA) and gibberellic acid (GA3). Also, TaPaO could be induced by wounding, low temperature, and high salinity. Furthermore, the transcripts of TaPaO increased after infection with Puccinia recondita f. sp. tritici. However, the expression of TaPaO was not affected obviously after infection with Puccinia striiformis f. sp. tritici. These results suggest that TaPaO may be involved in plant defense responses to various stresses.

Phylogenetic relationships of Thinopyrum and Triticum species revealed by SCoT and CDDP markers

To evaluate the phylogenetic relationships among Thinopyrum species and Triticum species, 7 accessions of Thinopyrum species (2 Th. bessarabicum, 1 Th. elongatum, 2 Th. intermedium and 2. Th. ponticum), 11 accessions of Triticum species (2 Aeglips tauschii, 1 T. monococcum, 1 T. turgidum, 2 T. timopheevii and 5 T. aestivum) together with Hordeum vulgare cv. Golden were analysed using 17 SCoT and 10 CDDP markers. The mean number of observed alleles was 8.5 and 6.6 among the species for SCoT and CDDP markers, respectively. Based on the genetic data produced by the SCoT and CDDP markers, cluster analysis among Thinopyrum species, Triticum species and Hordeum was performed to generate dendrograms. The genetic relationships among Thinopyrum species, Triticum species and H. vulgare revealed by the SCoT markers were in agreement with the results of the CDDP markers. On both dendrograms, these species formed three clusters. The results indicated that Thinopyrum species and Triticum species were the most closely related, whereas H. vulgare was relatively distant from both genera. In addition, seven markers, i.e. SCoT 9, SCoT 31, SCoT 34, WRKY-R1, WRKY-R2, MADS-1 and MADS-4 were developed for monitoring introgression of Thinopyrum chromosomes or chromosome segments into Triticum species.

Cloning and characterization of novel fast ω-gliadin genes in Triticum monococcum

Fast ω-gliadin is the major allergen to wheat-dependent exercise-induced anaphylaxis (WDEIA). It is generally accepted that the proteins were expressed inGli-B1 loci located on the short arm of chromosome 1B in wheat. The knowledge of the orthologous genes in A and D genomes is still limited. In this study, the novel fastω-gliadin genes were cloned from genome A of diploid wheat Triticum monococcum. The deduced amino acid sequences of N-terminal domain showed that they referred to TRQ-type ω-gliadins. This type of ωgliadins have been identified in genome A by using mass spectroscopy and amino acid sequencing in previous studies (DuPont et al. 2004), but their coding sequences were lacking. Further analysis of IgE-binding epitopes suggested that the TRQ-typeω-gliadins have potential to trigger WDEIA. Wheat is one of the most important sources of food in the human diet. In the kernels of wheat, gliadins are the main components of the gluten. Traditionally, gliadins are further classified into α-gliadins, γ-gliadins and ω-gliadins on the basis of their mobility in electrophoresis at low pH (Wan et al. 2014). The ω-gliadins are minor components among wheat prolamins and they have not been studied well due to difficulties in cloning their genes caused by highly repetitive sequences (Anderson et al. 2009). In common wheat, most of the ω-gliadins were encoded by the Gli-A1, Gli-B1 and Gli-D1 on the short arm of chromosomes 1A, 1B and 1D, respectively (Pogna et al. 1990; Shewry et al. 1995; DuPont et al. 2000, 2004). Theω-gliadins have been categorized into ω1,ω2 andω5 types (DuPont et al. 2004). Theω5-gliadin, also referred as fastω-gliadin, are encoded byGli-B1 and the proteins can be distinguished by the amino acid sequences at the N-terminal domain, which are Ser-Arg-Leu (SRL) in ω5-gliadin, Ala-Arg-Gln (ARQ) or Lys-Glu-Leu (KEL)

Modification of a novel x-type high-molecular-weight glutenin subunit gene from Aegilops markgrafii to improve dough strength of wheat flour

Abstract. High-molecular-weight glutenin subunits (HMW-GS) in bread wheat are major determinants of dough viscoelastic properties and the end-use quality of wheat flour. Cysteine residues, which form intermolecular disulphide bonds in HMW-GS, could improve the strength of gluten. To our knowledge, the number and position of cysteine residues in HMW-GS are conserved between wheat (Triticum aestivum) and Aegilops markgrafii. In the present study, we modified a gene (1Cx1.1) from Ae. markgrafii for an HMW-GS that possessed the typical structure and conserved number of cysteines. Site-directed mutagenesis was carried out in 1Cx1.1 to investigate how the position of cysteine residues in HMW-GS affects the mixing properties of dough. Six HMW-GS containing an extra cysteine residue were expressed in Escherichia coli, and the proteins were purified at sufficient scale for incorporation into flour to test dough quality. There were large differences in dough property among samples containing different modified subunits. Cysteine substituting in the N-terminal or repetitive-domain of HMW-GS could significantly improve dough quality. The results showed that the strategy was useful for providing genetic resources for gene engineering, and hence could be valuable for improving the processing quality of wheat.

Molecular characterization of the IgE-binding epitopes in the fast ω-gliadins of Triticeae in relation to wheat-dependent, exercise-induced anaphylaxis.

Fast ω-gliadins were minor components of wheat storage proteins but a major antigen triggering allergy to wheat. Sixty-six novel full-length fast ω-gliadin genes with unique characteristics were cloned and sequenced from wheat and its relative species using a PCR-based strategy. Their coding regions ranged from 177bp to 987bp in length and encoded 4.28kDa to 37.56kDa proteins. On the base of first three deduced amino acids at the N-terminal, these genes could be classified into the six subclasses of SRL-, TRQ-, GRL-, NRL-, SRP- and SRM-type ω-gliadin genes. Compared by multiple alignments, these genes were significantly different from each other, due to the insertion or deletion at the repetitive domain. An analysis of the IgE-binding epitopes of the 66 deduced fast ω-gliadins demonstrated that they contained 0-24 IgE-binding epitopes. The phylogenetic tree demonstrated that the fast ω-gliadins and slow ω-gliadins were separated into two groups and their divergence time was 21.64millionyears ago. Sequence data of the fast ω-gliadin genes assist in the study of the origins and evolutions of the different types of ω-gliadins while also providing a basis for the synthesis of monoclonal antibodies to detect wheat antigen content.