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利用转录组测序获得的表达序列信息,结合RACE技术,从桂花(Osmanthus fragrans)花瓣中克隆到两个参与苯丙烷代谢第2步反应的肉桂酸–4–羟基化酶(C4H)基因。其ORF全长分别为1 518 bp和1 611 bp,各自编码505和536个氨基酸,命名为OfC4H1和Of C4H2(登录号分别为KR861466、KF254842)。系统进化树表明,OfC4H1与矮牵牛中的PhC4H1、PhC4H2等C4H蛋白聚为一类,属于ClassⅠ类型;OfC4H2与金银花中的LjC4H等属于ClassⅡ类型。进一步的C4H蛋白序列多重比较发现,OfC4H1和OfC4H2蛋白具有该基因家族特有的保守结构域,在这些保守结构域中存在区别两类C4H蛋白的典型特征。利用Real-time PCR比较分析了OfC4H1和OfC4H2基因的时空表达模式,结果显示,Of C4H1在花瓣中的表达量最高;OfC4H2在花瓣中的表达量较低,在花梗、雌蕊和幼叶等组织中表达量较高。构建原核表达载体pET6xHN-C4Hs,转化Transetta(DE3)大肠杆菌并诱导表达的结果表明,目的蛋白能在原核表达系统中顺利表达,而且与预期大小一致,但因溶解度较低无法进行酶活性分析。
Two cinnamic acid-4-hydroxylase (C4H) genes involved in the second-step reaction of phenylpropanoid were cloned from the petals of Osmanthus fragrans by using the RACE technique and the expressed sequence information obtained by transcriptome sequencing. The ORFs were 1 518 bp and 1 611 bp in length, encoding 505 and 536 amino acids, respectively, and were named OfC4H1 and Of C4H2 (accession numbers KR861466 and KF254842, respectively). The phylogenetic tree showed that OfC4H1 and C4 petunia cells such as PhC4H1, PhC4H2 clustered into a class, which belongs to the ClassⅠ type; OfC4H2 and honeysuckle LjC4H belong Class Ⅱ type. Further C4H protein sequence multiple comparisons revealed that OfC4H1 and OfC4H2 proteins have conserved domains unique to this gene family, and there are typical features that distinguish these two types of C4H proteins in these conserved domains. The temporal and spatial expression patterns of OfC4H1 and OfC4H2 genes were analyzed by Real-time PCR. The results showed that Of C4H1 was the highest expressed in the petals and OfC4H2 was lower in the petals. In the peduncle, pistil and young leaves, Higher expression. The prokaryotic expression vector pET6xHN-C4Hs was transformed into Escherichia coli Transetta (DE3) to induce expression in E.coli. The result showed that the target protein was successfully expressed in the prokaryotic expression system and was consistent with the expected size. However, the enzyme activity analysis was impossible because of its low solubility.