关键词: 陕253, α-醇溶蛋白, 融合蛋白, 原核表达, 品质效应

Abstract:

【Objective】 An α-gliadin gene, with an additional cysteine residue, was cloned and expressed in E. coli system. The target protein was purified by Ni+-NTA column, and then integrated into the control flour to understand the potential quality of this α-gliadin gene. 【Method】 A DNA fragment with 1 243 bp (GenBank accession GQ891685)was cloned from Shaan 253 by primers designed according to conserved domains of known α-gliadin genes. Meanwhile, the gene expression vector pET32a-S253-Agli was constructed. Fusion protein was induced in the host strain E. coli BL21(DE3)by IPTG. In order to determine its processing quality of subunit S253-Agli, the expressed product was purified and recovered by affinity chromatography and low temperature cryodesiccation, and then integrated into the control flour utilization of 4 g Micro-doughlab. 【Result】 Sequence analysis showed that the DNA fragment of this gene contained a complete 900 bp coding region and some of regulatory elements. In the deduced amino acid sequences, an extra cysteine replacement of serine at position 6, which may form an intermolecular disulfide bond, was caused by single base conversion of C/G. The fusion proteins, induced by IPTG, were confirmed by SDS-PAGE and Western-blot and mainly existed as inclusion body. The farinograph results suggested that the purified subunit S253-Agli has negative effects on flour quality because of its reduction of main parameters, including development time and stability time, although some of farinograph parameters increased significantly, such as width of curve and mixing tolerance index.【Conclusion】 It was suggested that the potential quality of α-gliadin protein encoded by gene GQ891685 increased the elasticity but decreased the strength of wheat flours.

Key words: Shaan 253, α-gliadin, fusion protein, prokaryotic expression, quality potential