College of Life Science, Capital Normal University, 100048, Beijing, China.
J Appl Genet. 2013 May;54(2):157-67. doi: 10.1007/s13353-013-0139-z. Epub 2013 Mar 1.
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-β-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.
通过等位基因特异性聚合酶链反应（AS-PCR），从小麦和相关的山羊草基因组中克隆并测序了 15 个新型α-醇溶蛋白基因。序列比较显示α-醇溶蛋白基因家族具有高度多样性。两个多谷氨酰胺区的四个毒性表位和谷氨酰胺残基有助于将这些α-醇溶蛋白分配到特定的染色体上。成功地在大肠杆菌中表达了 5 个代表性的α-醇溶蛋白基因，在异丙基-β-D-硫代半乳糖苷（IPTG）诱导 4 小时后达到最大量，表明在 T7 启动子的控制下具有高水平的表达。通过实时定量聚合酶链反应（qRT-PCR）检测到谷物发育过程中α-醇溶蛋白基因的转录表达，不同基因型呈现出相似的上调-下调调节模式。用全长开放阅读框（ORF）α-醇溶蛋白基因和假基因构建的邻接树进一步揭示了小麦和相关山羊草基因组的起源和系统发育关系。进化分析表明，在进化过程中，α-醇溶蛋白基因主要通过同义替换进化，受到强烈的纯化选择。
