青稞OMT1基因克隆及原核表达分析

Molecular Cloning and Prokaryotic Expression of OMT1 Gene From Hulless Barley

类黄酮O-甲基转移酶(FOMT)是一种在植物甲基化黄酮代谢合成中有着重要作用的酶,但在中国青藏高原的特色作物青稞中研究较少。为了更好了解该酶及编码基因在青稞中的生理功能及作用,以高总黄酮青稞品系94-19-1为材料,通过RT-PCR扩增、克隆得到青稞OMT1基因的ORF序列。结果表明,该基因ORF长1 071 bp,编码356个氨基酸,预测蛋白分子量为38.65 KDa,等电点为5.33。序列比对分析发现,所克隆的基因与NCBI数据库收录的大麦Hv OMT1基因有99.44%的一致性,氨基酸序列存在5个残基差异,编码的蛋白序列具有OMT蛋白家族典型的保守结构域。进一步将该基因连接到原核表达载体转化大肠杆菌,最终成功将该基因所编码的蛋白在大肠杆菌诱导表达,并利用亲和层析柱分离技术,将该表达蛋白进行了纯化。这为进一步研究该酶蛋白功能和选育高品质青稞品种奠定了基础。

Flavonoids O-methyltransferase （FOMT） is a critical enzyme in plant methoxy-flavonoid biosynthesis and metabolic pathway. However, little knowledge of the coding enzyme gene was available in hulless barley which is a characteristic cereal crop in Qinghai-Tibet plateau. In order to understand the physiological function and the role of FOMT and related coding gene in hulless barley, OMT1 gene of hulless barley was cloned by using RT-PCR from hulless barley CV 94 - 19 - 1. The results showed that the open reading frame was 1 071 bp encodes 356 amino acids, the molecular weight of the protein was 38.65KDa with the theoretical isoelectric point of 5.33. The comparison analysis of the sequences indicated that the similarity between the OMT1 gene sequence and the HvOMT1 sequence from NCBI database, was 99.44%, and also five amino acid residues were differential between amino acid sequences. The encoding protein sequence possessed typical conserved domains of OMT protein family. The encoding protein of the OMT1 gene was successfully expressed in E. coli after ligation of OMT1 gene to prokaryotic expressive vector. And the purification of the expressive protein was carried out though affinity chromatography. The results would lay the foundation for further studying the function of the enzyme protein and selection of high-quality hulless barley cultivars.