ABSTRACT
Brachypodium distachyon, is a new model plant for most cereal crops while gliadin is a class of wheat storage proteins related with wheat quality attributes. In the published B. distachyon genome sequence databases, no gliadin gene is found. In the current study, a number of gliadin genes in B. distachyon were isolated, which is contradictory to the results of genome sequencing projects. In our study, the B. distachyon seeds were found to have no gliadin protein expression by gel electrophoresis, reversed-phase high-performance liquid chromatography and Western blotting analysis. However, Southern blotting revealed a presence of more than ten copies of α-gliadin coding genes in B. distachyon. By means of AS-PCR amplification, four novel full-ORF α-gliadin genes, and 26 pseudogenes with at least one stop codon as well as their promoter regions were cloned and sequenced from different Brachypodium accessions. Sequence analysis revealed a few of single-nucleotide polymorphisms among these genes. Most pseudogenes were resulted from a C to T change, leading to the generation of TAG or TAA in-frame stop codon. To compare both the full-ORFs and the pseudogenes among Triticum and Triticum-related species, their structural characteristics were analyzed. Based on the four T cell stimulatory toxic epitopes and two ployglutamine domains, Aegilops, Triticum, and Brachypodium species were found to be more closely related. The phylogenetic analysis further revealed that B. distachyon was more closely related to Aegilops tauschii, Aegilops umbellulata, and the A or D genome of Triticum aestivum. The α-gliadin genes were able to express successfully in E. coli using the functional T7 promoter. The relative and absolute quantification of the transcripts of α-gliadin genes in wheat was much higher than that in B. distachyon. The abundant pseudogenes may affect the transcriptional and/or posttranscriptional level of the α-gliadin in B. distachyon.
Subject(s)
Brachypodium/genetics , Genome, Plant , Gliadin/genetics , Phylogeny , Amino Acid Sequence , Blotting, Southern , Epitopes , Escherichia coli/genetics , Gene Expression Regulation, Plant , Gliadin/isolation & purification , Gliadin/metabolism , Molecular Sequence Data , Multigene Family , Mutation , Open Reading Frames , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Pseudogenes , Seeds/genetics , Seeds/growth & development , Triticum/geneticsABSTRACT
Fifteen novel α-gliadin genes were cloned and sequenced from Triticum and related Aegilops genomes by allele-specific polymerase chain reaction (AS-PCR). Sequence comparison displayed high diversities in the α-gliadin gene family. Four toxic epitopes and glutamine residues in the two polyglutamine domains facilitated these α-gliadins to be assigned to specific chromosomes. Five representative α-gliadin genes were successfully expressed in Escherichia coli, and their amount reached a maximum after 4 h induced by isopropyl-ß-D-thiogalactoside (IPTG), indicating a high level of expression under the control of T7 promoter. The transcriptional expression of α-gliadin genes during grain development detected by quantitative real-time polymerase chain reaction (qRT-PCR) showed a similar up-down regulation pattern in different genotypes. A neighbor-joining tree constructed with both full-open reading frame (ORF) α-gliadin genes and pseudogenes further revealed the origin and phylogenetic relationships among Triticum and related Aegilops genomes. The evolutionary analysis demonstrated that α-gliadin genes evolved mainly by synonymous substitutions under strong purifying selection during the evolutionary process.
