甘薯肉桂酸-4-羟化酶基因克隆及序列分析

肉桂酸-4-羟化酶(Cinnamic acid-4-hydroxylase,C4H,EC 1.14.13.11)是苯丙烷途径中第二步反应酶,同时也是花色素苷前体生物合成途径中关键酶。该研究根据植物C4H的同源序列设计引物,通过RTPCR结合RACE的方法,在紫色甘薯中获得了与其相应的C4H基因,命名为Ib C4H(Gen Bank登录号GQ373157)。结果表明:(1)序列分析表明Ib C4H长1 668 bp,编码505个氨基酸,该氨基酸序列与其c DNA序列与Ib C4H蛋白与马铃薯C4H蛋白序列最为接近,与苹果、黑莓、大阿米芹、油菜一致性很高,均在70%以上。(2)二级结构预测表明α-螺旋和无规则卷曲是Ib C4H蛋白最大量的结构元件,而延伸链则散布于整个蛋白中。(3)三维结构建模预测,Ib C4H蛋白具备细胞色素P450氧和铁离子结合位点等典型的C4H结构。该研究结果为进一步了解花色素苷生物合成途径中的作用奠定了基础,也为花青素生物合成分子机理和代谢调控提供了靶位点和理论参考。

Molecular cloning and sequence analysis of acid-4-hydroxylase gene from sweet potato

(Cinnamic acid-4-hydroxylase, C4H,EC 1.14.13.11)is the second key enzyme involved in the biosynthetic pathway of phenylpropanoid and precursors of anthocyanin biosynthesis. Based on cDNA sequence conserved domain of C4H,a pair of primers were designed and used to amplify a fragment of IbC4H gene(GenBank accession: GQ373157)by RT-PCR from sweet potato using RT-PCR and RACE technique. A 1 668 bp full-length cDNA sequence was obtained. Analysis of C4H cDNA indicated that it encoded a peptide containing 505 amino acids and the sequence comparison with the C4H gene of potatoes, apples, blackberries, ammi majus and rapes showed that identity was all above 70%. The predicted secondary structure demonstrated that alpha helix and random coil were the most important structural conformation. However, extended chain distributed in the whole protein. The predicted tertiary structure demonstrated that IbC4H had binding sites of cytochrome P450 with oxygen and iron. The study will be helpful to understand more about the roles involved in anthocyanin biosynthesis at the molecular level and provides a candidate genes for metabolic engineering of anthocyanin biosynthesis pathway in purple-fleshed sweetpotato.