鸭梨多酚氧化酶基因cDNA全长的克隆和生物信息学分析

Cloning and Bioinformatics Analysis for the Full Length cDNA Sequence of PPO Gene in Yali

为了从分子水平深入了解鸭梨多酚氧化酶结构特点,基于已知的鸭梨多酚氧化酶基因cDNA片段序列设计引物,以鸭梨果实总mRNA为模板,对该基因cDNA5’端和3’端未知序列进行了RACE扩增,最终获得鸭梨多酚氧化酶基因cDNA全长序列。该序列长度为2126bp,开放性读码框位于136～1917bp之间,可编码593个氨基酸残基。对该基因所翻译的氨基酸序列进行生物信息学分析,结果表明基因编码的多酚氧化酶属酪氨酸酶超级家族成员,三级空间结构为可溶性球状蛋白,不具备跨膜结构,在细胞中的定位应该位于类囊体腔中。这些生物信息的获得将为今后应用生物技术开展鸭梨多酚氧化酶的调控,从而抑制鸭梨果实褐变提供十分重要的参考资料。

In order to investigate the molecular structures of polyphenol oxidase （PPO） in Yali, the author designed series of primers based on the known cDNA fragment of PPO gene in Yali to clone the unknown 3’ and 5’cDNA terminal sequence for this gene by using RACE method. With the total RNA extracted from fruit of Yali as the template, the author finally obtained the full length cDNA sequence of PPO gene in Yali, which was 2126 bp in length, and has a open reading frame between 136-1917 bp, in which 276 predicted amino acid residues were encoded. Research the deduce amino acids of PPO gene from Yali by bioinformatics analysis, it showed that the PPO in Yali was a typical hydrophilic soluble protein which belonged to tyrosinase super family, and it did not contain distinct transmembrane domain in tertiary structure, so the Yali PPO should located in thylakoid lume of plastid. That information laid a good foundation for regulating the enzyme activity of PPO in Yali by biological technology and breeding a new cultivar of pear with browning resistance in the future.