紫苏羟苯基丙酮酸还原酶基因片段的克隆及表达分析

Molecular Cloning and Expression Analysis of Hydroxyphenylpyruvate Reductase Gene Fragment in Perilla frutescens

为了克隆紫苏迷迭香酸生物合成途径中的羟苯基丙酮酸还原酶基因(Hydroxy phenylpyruvate reductase gene,HPPR),通过已报道的其他物种的HPPR基因序列设计特异性引物,采用同源克隆的方法成功克隆得到了紫苏HPPR基因片段,并命名为PfHPPR(GenBank登录号:HM152567.1),该片段长为426bp,共编码142个氨基酸残基。根据蛋白比对结果,PfHPPR基因编码的氨基酸序列与彩叶草、鼠尾草和丹参的一致性分别为92%、93%和92%。采用半定量RT-PCR法分析该基因在紫苏叶中的表达最高,茎次之,根中相对较弱。内源性植物激素信号分子及外界刺激对PfHPPR表达量影响的实验表明PfHPPR的表达受脱落酸、水杨酸和UV-B信号调控途经的影响。

The purpose of this paper was to obtain hydroxyphenylpyruvate reductase gene involved in rosmarinic acid （RA） biosynthesis pathway from Perilla frutescens. Gene special primers were designed based on sequences blast of HPPR genes from other species, and the fragment of HPPR gene in Perilla frutescens was successfully cloned by homology cloning method. The gene fragment, designated as PfHPPR （GenBank accession No. HM152567.1）, was 426 bp encoding 142 amino acid protein. Sequence alignment revealed that, the deduced amino acid sequence of PfHPPR was 92%, 93% and 92% identical to Coleus blumei, Salvia officinalis and Salvia miltiorrhiza, respectively. The PfHPPR expressed in all the tested tissues but at different levels with the highest in leaf, the second in stem and the lowest in root using the semi-quantitative RT-PCR analysis. Further expression analysis reveals that, the PfHPPR transcript levels might be influenced by the signaling components and stimulus such as ABA, SA and UV-B signaling pathways.