青稞OMT1基因克隆及原核表达分析

发布时间：2018-03-24 20:03

  本文选题：青稞　切入点：类黄酮O-甲基转移酶　出处：《核农学报》2017年12期

【摘要】：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。
[Abstract]:Flavonoid O-methyltransferase (FOMT) is an enzyme that plays an important role in the metabolism and synthesis of plant methylated flavonoids. In order to better understand the physiological function and function of the enzyme and coding gene in highland barley, the high flavonoid highland barley line 94-19-1 was used as material and amplified by RT-PCR. The ORF sequence of OMT1 gene of highland barley was cloned. The results showed that the ORF length of the gene was 1071 BP, encoding 356 amino acids, the molecular weight of predicted protein was 38.65 KDaand the isoelectric point was 5.33. The cloned gene was 99.44% consistent with that of barley Hv OMT1 gene collected in NCBI database, and the amino acid sequence was different from that of barley Hv OMT1 gene. The encoded protein sequence has the typical conserved domain of OMT protein family. The gene was further ligated into prokaryotic expression vector and transformed into Escherichia coli. Finally, the protein encoded by the gene was successfully induced to express in E. coli. The expressed protein was purified by affinity chromatography, which laid a foundation for further study on the function of the enzyme and breeding of highland barley varieties with high quality.
【作者单位】： 四川农业大学农学院/作物遗传育种学系大麦青稞研究中心;
【基金】：国家现代农业产业技术体系(大麦青稞体系)建设专项基金(CARS-05)
【分类号】：Q943.2;S512.3
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本文编号：1659819