青钱柳法呢基焦磷酸合成酶基因的克隆及功能研究

Molecular Cloning and Functional Analysis of a Gene Encoding Farnesyl Diphosphate Synthase from Cyclocarya paliurus

青钱柳是集药用、材用和观赏等多种价值于一身的珍贵树种。法呢基焦磷酸合成酶(FPS)催化=牛儿基焦磷酸(GPP)与异戊烯基焦磷酸(IPP)缩合成法呢基焦磷酸(FPP),FPP是植物次生代谢产物倍半萜,三萜,甾醇等的前体。本研究通过RACE方法首次从青钱柳中扩增了法呢基焦磷酸合成酶的全长cDNA序列,序列命名为CpF-PS(Genbank登录号为GU121224),序列长度为1 420 bp,包含1 029 bp的开放阅读框,编码342个氨基酸残基,预测蛋白分子量为39.60 kDa。通过BlASTP分析,推断的青钱柳FPS蛋白序列与木本棉(Gossypium arboreum)(CAA72793.1)、橡胶树(Hevea brasiliensis)(BAF98301)等的FPS蛋白相似度较高。蛋白质保守区、特征区以及进化树分析初步证实扩增到的全长cDNA序列为青钱柳的FPS基因。将该基因连入酵母表达载体并转入麦角甾醇缺陷型酵母菌株CC25(MATa/MATalpha,deltaERG20/+),发现该基因可弥补营养缺陷使得CC25菌株在高温中正常生长,证明所得到的青钱柳CpFPS基因编码的蛋白是有功能的蛋白。

Cyclocarya paliurus is a medicinal,timber and ornamental tree.Farnesyl diphosphate synthase（FPS;EC2.5.1.1/EC2.5.1.10） catalyses 1′-4 condensation of the 5-carbon isoprenoid compounds isopentenyl diphosphate（IPP） and 10-carbon geranyl diphosphate（GPP） to form the 15-carbon product farnesyl diphosphate（FPP）.FPP is the common precursor of sesquiterpenes,tritepenes,steroids,etc.cDNA cloning and characterization of a novel FPS from C.paliurus were described in this present study.The full-length cDNA named CpFPS（Genbank Accession Number GU121224） contained 1420bp with an open reading frame of 1029bp encoding a polypeptide of 342 amino acids with a calculated molecular mass of 39.60kDa.Through sequence analysis by BlASTP online,the resulting protein showed high homology to the FPS of Gossypium arboretum（CAA72793） and Hevea brasiliensis（BAF98301）.Deduced amino acid sequence was also analyzed by biological software and the data presented the existence of conserved and functional domains of FPS.The functional analysis of CpFPS was performed by using the sterol-auxotrophic yeast strain CC25.The result indicated that the cloned cDNA of CpFPS encoding a functional FPS in C.paliurus.