关键词: 华山新麦草, α-醇溶蛋白基因, 基因克隆, 序列分析, 原核表达

Abstract: 【Objective】The present study aimed at cloning and analyzing the α-gliadin genes from P. huashanica and expressing it in E. coli.【Method】The α-gliadin genes were amplified from P. huashanica by AS-PCR and then the cloned gene Gli-Ns-5 was inserted into pET-28a (+). The recombinant plasmid pET28a-Gli-Ns was expressed in a prokaryotic expression system after its transformation into BL21 (DE3) pLysS host strain.【Result】Four novel α-glidain genes: Gli-Ns-2 (FJ713595), Gli-Ns-3 (GQ139525), Gli-Ns-4 (GQ139526) and Gli-Ns-5 (GQ139527) were isolated and characterized from the genomic DNA of P. huashanica. Molecular structure analysis revealed that these four genes had the typical structure of α-gliadin genes and contained 8 or 9 cysteine residues respectively. Two internal stop codons were identified in coding region of FJ713595, indicating that it was a pseudogene. The results of SDS-PAGE and Western-blot demonstrated that the fusion protein could express normally in the prokaryotic expression system.【Conclusion】The four α-gliadin genes of P. huashanica were cloned and the gene Gli-Ns-5 (GQ139527) was successfully expressed in E. coli. This study could provide new candidate genes for the wheat quality improvement.

Key words: Psathyrostachys huashanica, α-Gliadin, Gene cloning, Sequences analysis, Prokaryotic expression