白木香法呢基焦磷合酶基因AsFPS1的克隆及表达分析

Cloning and expression analysis of farnesyl pyrophosphate synthase from Aquilaria sinensis

法呢基焦磷酸合酶(FPS)是倍半萜代谢途径中的关键限速酶之一。本研究在454高通量测序已获得的cDNA序列基础上,PCR扩增其开放阅读框并测序验证;提取三年生白木香根、茎、叶的总RNA及健康和伤害处理的愈伤组织的总RNA,以反转录成的单链cDNA为模板进行荧光定量PCR,检测AsFPS1基因的组织表达特异性及对伤害处理的反应。结果表明,扩增到的AsFPS1基因全长1 342 bp,开放阅读框1 029 bp,编码342个氨基酸。组织表达分析的结果显示,AsFPS1基因主要在根和茎中表达,叶中的表达量较低;该基因同时受物理伤害和结香液处理的诱导,分别在6,12 h达到最高表达水平。通过AsFPS1基因全长cDNA的克隆和表达分析,为后续研究其生物功能及沉香倍半萜合成机制奠定了基础。

Farnesyl diphosphate synthase （FPS） is one of the key rate-limiting enzymes in the sesquiterpene metabolic path- ways. In this study, the open reading frame （ORF） of FPS was cloned by PCR based on the transcript sequence of AsFPS1 from the Aquilaria sinensis transcriptome database and sequenced. Total RNA was extracted from the root, stem and leaves of three-year-old A. sinensis, and from healthy and wounded A. sinensis calli, and then reverse-transcribed into single-stranded cDNA as a template for real-time PCR, to detect the expression specificity of AsFPS1 in different tissues and its expression profile in responding to different treatments. The result showed that the full length of AsFPS1 was 1 342 bp with the ORF 1 029 bp, encoding 342 amino acids. Tissue expression analysis indicated that AsFPS1 was mainly expressed in root and stem, and was lower in leaves. Inducible-experiments showed that the genes was induced by mechanical wound as well as chemical liquid induction, and reached the highest expression level at 6 h and 12 h, respectively. The full-length cDNA clone of AsFPS1 and its expression patterns analysis will provide a foundation for follow-up study on its biological function and agarwood sesquiterpene biosynthesis mechanism.