Cloning, expression, and evolutionary analysis of alpha-gliadin genes from Triticum and Aegilops genomes.

摘要

Fifteen novel alpha-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 alpha-gliadin gene family. Four toxic epitopes and glutamine residues in the two polyglutamine domains facilitated these alpha-gliadins to be assigned to specific chromosomes. Five representative alpha-gliadin genes were successfully expressed in Escherichia coli, and their amount reached a maximum after 4 h induced by isopropyl- beta-D-thiogalactoside (IPTG), indicating a high level of expression under the control of T₇ promoter. The transcriptional expression of alpha-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) alpha-gliadin genes and pseudogenes further revealed the origin and phylogenetic relationships among Triticum and related Aegilops genomes. The evolutionary analysis demonstrated that alpha-gliadin genes evolved mainly by synonymous substitutions under strong purifying selection during the evolutionary process