目的 克隆芍药萜类物质生物合成关键酶法呢基焦磷酸合酶(farnesyl pyrophosphate synthase,FPPS)cDNA全长序列并对其进行生物信息学分析。方法 根据芍药转录组测序数据,设计一对特异性PCR引物,应用RT-PCR技术扩增出芍药FPPS基因目标条带并克隆至pMD18-T载体上,菌落PCR和质粒PCR鉴定出阳性重组子后进行序列测定及序列同源性搜索、分子系统进化树构建、结构域搜索及3D结构预测等生物信息学分析。结果 测序结果表明芍药FPPS基因全长1315bp,含60 bp的5'-UTR、1050 bp的CDS和205 bp的3'-UTR,共编码349个氨基酸,GenBank登录号为KP708571;Blastn和Blastp分析结果显示芍药FPPS(KP708571)及其编码的蛋白(AKJ26301)在核苷酸水平和氨基酸水平上与多种植物的FPPS基因和FPPS蛋白具同源性;分子进化树分析结果表明芍药FPPS蛋白与其他物种FPPS蛋白的亲缘关系相对较远;预测芍药FPPS蛋白相对分子质量为40200,等电点为5.33,是一个定位于胞液、不含跨膜结构域、不含信号肽分子的亲水性、稳定蛋白;发现芍药FPPS含有底物结合位点、底物-Mg²⁺结合位点、催化位点、富含天冬氨酸位点1及富含天冬氨酸位点2共7个保守结构域;3D结构中α-螺旋所占比例高且α-螺旋之间由β-转角(loop)相连接;中央腔(central cavity)由大约10个核心α-螺旋排列而成。结论 首次从芍药中克隆了FPPS基因并对其进行了初步的生物信息学分析。

Objective To clone the full-length cDNA sequence which encodes one of the key enzymes of terpene biosynthesis, farnesyl pyrophosphate synthase (FPPS) and analyze the bioinformation. Methods Based on the transcriptome data of Paeonia lactiflora (Pl), a pair of specific PCR primers were designed. The objective PCR band of PlFPPS was successfully amplified using RT-PCR technique and then it was cloned to pMD18-T vector. After clone and plasmid PCR characterization, the recombinants were sequenced and then a series of bioinformatic analysis was carried out including sequence homology search, construction of molecular phylogenetic tree, domain search, and 3D structure prediction, etc. Results Sequencing results showed that the full-length PlFPPS was 1315 bp, which contained 60 bp 5'-UTR, 1050 bp CDS, and 205 bp 3'-UTR. It encoded 349 amino acids and the accession number of GenBank was KP708571. Blastn and Blastp analysis revealed that PlFPPS (KP708571) and its encoding protein (AKJ26301) had high homology with FPPS genes and proteins from several plants at the nucleotide and amino acid levels. Phylogenetic tree analysis indicated that the genetic relationship of PlFPPS with FPPS from other plants was relatively far. It predicted that the molecular weight and isoelectric point of PlFPPS were 40 200 and 5.33, which was a hydrophilic and stable protein, located in cytoplasm without transmembrane domain and signal peptide. It contained seven conservative domains like substrate binding pocket, substrate-Mg²⁺ binding site, catalytic site, aspartate-rich region 1, and aspartate-rich region 2. The proportion of α-helix in its 3D structure was high and the α-helix was linked by β-loop. The central cavity was composed of 10 core α-helixes. Conclusion The full-length cDNA sequence of FPPS is cloned firstly from P. lactiflora and the bioinformatic analysis is then carried out.

国家自然科学基金资助项目(U1204323);河南省科技厅国际合作项目(134300510052)