紫苏4-羟苯基丙酮酸双加氧酶基因片段的克隆及表达分析

Molecular cloning and expression analysis of 4-Hydroxyphenyl pyruvate dioxygenase gene fragment from Perilla frutescens

为获得紫苏迷迭香酸合成途径旁路中的4-羟苯基丙酮酸双加氧酶(HPPD)基因,采用同源克隆的方法,根据已报道的其他植物的HPPD基因序列设计合成简并引物,克隆得到紫苏HPPD基因片段,命名为PerHPPD-1,该片段长663 bp,编码221个氨基酸。通过氨基酸比对分析发现,其氨基酸序列与丹参和彩叶草的HPPD基因片段一致性分别为66.36%和77.93%,系统进化树分析又进一步表明该片段与唇形科植物的亲缘关系最近。荧光实时定量PCR检测结果显示,PerHPPD-1基因在紫苏根、茎、叶中均有表达,在叶中表达量最高,其次为茎、根。激素(赤霉素、茉莉酸甲酯、脱落酸)处理可在一定程度上上调PerHPPD-1在叶中的表达水平。

To obtain 4-Hydroxyphenyl pyruvate dioxygenase （HPPD） gene involved in the bypath of rosmarinic acid （RA） biosynthesis pathway from Perilla frutescens, we designed degenerate primers according to parallel analysis of the amino acid sequence of HPPD genes from other species and successfully cloned the fragment of HPPD gene by homology cloning method. The fragment of HPPD designated as PerHPPD-1 was 663 bp, encoded 221 amino acids. Sequence alignment revealed that the deduced amino acid sequence of PerHPPD-1 was 66.36, 77.93% identical to Salvia miltiorrhiza, Coleus blume. Phylogenetie tree analysis showed that PerHPPD-1 had closest relationship with Lamiaeeae plants. The quantitative fluorescence Real-Time PCR analysis showed that the constitutive expression of PerHPPD-1 in leaf was much higher than that in stem and root. Further expression analysis indicated that the hormone, including Gibberellins, MeJA, ABA treatments could up-regulate the PerHPPD-1 level in leaves.