中国农业科学 ›› 2011, Vol. 44 ›› Issue (8): 1533-1542 .doi: 10.3864/j.issn.0578-1752.2011.08.001

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

华山新麦草α-醇溶蛋白基因的克隆及原核表达分析

王玉卿, 赵继新, 陈新宏, 将彦苗, 武军, 庞玉辉, 刘淑会   

  1. 西北农林科技大学农学院
     
  • 收稿日期:2010-08-20 修回日期:2010-10-21 出版日期:2011-04-15 发布日期:2011-04-15
  • 通讯作者: 赵继新

Cloning and Prokaryotic Expression of α-gliadin gene from Psathyrostachys huashanica

  • Received:2010-08-20 Revised:2010-10-21 Online:2011-04-15 Published:2011-04-15
  • Contact: Ji-Xin Zhao

摘要: 【研究目的】克隆华山新麦草(Psathyrostachys huashanica)的α-醇溶蛋白基因,并对其进行生物信息学分析,构建该基因的原核表达载体,在大肠杆菌中诱导表达融合蛋白。【方法】采用同源克隆法从华山新麦草基因组DNA中分离克隆出α-醇溶蛋白基因并进行序列分析,将克隆的华山新麦草α-醇溶蛋白基因Gli-Ns-5克隆到表达载体pET-28a (+)上,获得重组质粒pET28a-Gli-Ns转化大肠杆菌BL21 (DE3)并诱导表达。【结果】从华山新麦草基因组DNA中克隆了4个α-醇溶蛋白基因:Gli-Ns-2 (FJ713595)、Gli-Ns-3 (GQ139525)、Gli-Ns-4 (GQ139526)和Gli-Ns-5 (GQ139527)。序列分析表明,4条序列具有α-醇溶蛋白基因的典型结构特征,含有8个或9个半胱氨酸残基,序列FJ713595为假基因。利用所构建的大肠杆菌表达载体,经IPTG诱导,华山新麦草α-醇溶蛋白基因Gli-Ns-5(GQ139527)可在原核系统中特异性表达。Western-blot证实融合蛋白可成功表达。【结论】克隆了4个华山新麦草的α-醇溶蛋白基因序列,基因Gli-Ns-5(GQ139527)可在原核表达系统中成功表达,为小麦品质改良提供了新的候选基因。

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