罗马洋甘菊吉马烯A合成酶基因的克隆与表达分析

发布时间：2019-03-08 10:37

【摘要】：目的从罗马洋甘菊Chamaemelum nobile中克隆萜类化合物合成关键酶吉马烯A合成酶(germacrene A synthase,GAS)cDNA全长,并对其进行生物信息学和组织表达分析。方法基于前期对罗马洋甘菊转录组测序结果,设计特异性引物,利用反转录聚合酶链式反应(RT-PCR)扩增得到GAS的cDNA全长,利用生物信息学手段对其核苷酸和编码的氨基酸序列进行分析,并预测其编码的蛋白质的理化性质和功能。通过实时定量PCR技术(qRT-PCR)检测罗马洋甘菊GAS基因(CnGAS)在罗马洋甘菊不同组织的表达水平。结果扩增得到的罗马洋甘菊吉马烯A合成酶基因的cDNA全长为1 785 bp,命名为CnGAS(GenBank登录号为KU589283),包含1个1 683 bp的开放阅读框(ORF),编码561个氨基酸,预测的相对分子质量和等电点分别为6 470和5.21。CnGAS基因编码的氨基酸序列与其他植物的GAS蛋白高度同源,且含有DDxx D保守序列。系统进化树表明CnGAS基因与菊科植物的GAS基因的亲缘关系最近。qRT-PCR结果显示CnGAS基因在罗马洋甘菊各组织中均有表达,且花中表达量最高。结论从罗马洋甘菊中克隆得到CnGAS基因,分析了该基因在不同组织中的表达水平,为罗马洋甘菊萜类化合物的生物合成代谢途径的研究奠定了基础。
[Abstract]:Aim to clone the full length of (germacrene A synthase,GAS cDNA, a key enzyme of terpenoid synthesis from chamomile Chamaemelum nobile, and to analyze its bioinformatics and tissue expression. Methods the full length of GAS was amplified by reverse transcription polymerase chain reaction (RT-PCR) based on the sequencing results of the previous transcripts of chamomile Roman chamomile, and the specific primers were designed to amplify the full length of cDNA by reverse transcription polymerase chain reaction (RT PCR). The nucleotide and amino acid sequences were analyzed by bioinformatics, and the physicochemical properties and functions of the encoded proteins were predicted. Real-time quantitative PCR (qRT-PCR) was used to detect the expression level of GAS gene (CnGAS) in different tissues of chamomile Roman chamomile. Results the full length of cDNA was 1 785 bp, named CnGAS (GenBank accession number (KU589283), which contained an open reading frame (ORF) (ORF), encoding 561 amino acids (ORF) of 1 683 bp. The predicted molecular weight and isoelectric point were 6470 and 6470 respectively. The amino acid sequence encoded by the 5.21.CnGAS gene was highly homologous to the GAS protein of other plants and contained the conserved DDxx D sequence. Phylogenetic tree showed that CnGAS gene was closely related to GAS gene of Compositae. QRT-PCR results showed that CnGAS gene was expressed in all tissues of chamomile, and the highest expression was found in flowers. Conclusion CnGAS gene was cloned from chamomile and its expression level in different tissues was analyzed, which laid a foundation for the study of biosynthetic pathway of chamomile terpenoids in Roman chamomile.
【作者单位】：长江大学园艺园林学院;靶向抗肿瘤药物湖北省协同创新中心;荆楚理工学院化工与药学院;
【基金】：国家自然科学基金资助项目(31400603) 大学生创新创业训练计划项目(201207014)
【分类号】：R284

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