罗汉果法呢基焦磷酸合成酶基因的克隆及其序列分析

Cloning and sequence analysis of Farnesyl pyrophosphate synthase gene in Siraitia grosvenorii

目的对罗汉果甜苷V生物合成途径的关键酶法呢基焦磷酸合成酶(Farnesyl pyrophosphate synthase,FPPS)基因的全长cDNA序列进行克隆,为研究罗汉果甜苷V生物合成与基因调控奠定基础。方法根据植物FPPS基因保守功能域设计简并引物,通过PCR和RACE方法克隆罗汉果FPPS基因的全长cDNA。结果获得罗汉果FPPS(SgFPPS)基因的全长cDNA序列共1 354个核苷酸,包含一个1 026核苷酸的开放阅读框架(open reading frame,ORF),cDNA编码的蛋白包含342个氨基酸,推断蛋白质相对分子质量为3.92×104。NCBI Blastx结果显示,SgFPPS基因编码的蛋白与苹果树来源FPPS具有最高同源性,氨基酸一致度达85.1%。SgFPPS具有异戊烯基转移酶的5个典型保守功能域。进化树分析结果显示,SgFPPS基因与苹果树的FPPS具有较近的亲缘关系。结论首次克隆SgFPPS基因的全长cDNA序列,为分析SgFPPS基因表达特性及其在罗汉果甜苷V生物合成中的功能奠定基础。

Objective To clone the full length cDNA encoding Farnesyl pyrophosphate synthase（FPPS） gene associated with mogroside V biosynthesis pathway in Siraitia grosvenorii and to provide basis for further studies on biosynthesis and gene regulation of mogroside V.Methods Degenerate primers were designed based on the conserved functional domains found in FPPS.A full-length cDNA of S.grosvenorii FPPS（designated as SgFPPS gene） was cloned by the polymerase chain reaction（PCR） and rapid amplification of cDNA ends（RACE）.Results The full length cDNA of SgFPPS composed of 1 354 nucleotides was obtained.The open reading frame（ORF） of SgFPPS is 1 026 bp in length,corresponding to a predicted polypeptide of 342 amino acid residues with a molecular mass of 3.92 × 104.The deduced SgFPPS amino acid sequence exhibited 85.1% identity to the FPPSs of Malus x domestica.The predicted SgFPPS shared five conserved functional domains involved in the typical prenyltransferase with the FPPSs of varied species.Phylogenetic analysis on the amino acid sequence of SgFPPS with those of other plants showed that SgFPPS was closely related to M.x domestica.Conclusion The full length cDNA of SgFPPS is cloned and reported for the first time.This work lays a foundation for studying the gene expression pattern and regulatory functions of SgFPPS in mogroside V biosynthesis.