目的 克隆枸骨Ilex cornuta三萜皂苷生物合成途径中关键酶法尼基焦磷酸合酶（farnesyl diphosphate synthase，FPS）基因，对其进行组织特异性表达分析，构建原核表达载体并进行重组蛋白诱导表达，探究其参与调控三萜皂苷生物合成的功能。方法 结合枸骨转录组数据设计特异性引物，采用PCR技术从枸骨叶中扩增得到了IcFPS2基因的cDNA序列，对其进行生物信息学分析；通过实时荧光定量进一步分析其组织表达特异性，构建原核表达载体pET32a-IcFPS2 ，并转化至大肠杆菌E.coli BL21（DE3）pLysS感受态细胞，经异丙基-β-D-硫代半乳糖苷（IPTG）诱导重组蛋白的表达。结果 IcFPS2基因长1259 bp，开放阅读框为1050 bp，编码350个氨基酸，其蛋白质相对分子质量和等电点分别为40 200、5.54。氨基酸序列比对分析表明枸骨与栀子、杜仲、甘草、绞股蓝、竹节参的FPS氨基酸序列具有较高的同源性。系统进化树分析显示，IcFPS2蛋白与西洋参、人参、竹节参FPS蛋白聚为一支，表明枸骨FPS蛋白可能与双子叶植物五加科FPS蛋白在功能上较为接近。实时荧光定量PCR分析表明，IcFPS2基因在根中的表达量最高，其次是叶，而后是雄花和茎，该基因在雌花中表达水平最低。原核表达分析结果显示，构建的IcFPS2-pET-32a载体能在大肠杆菌BL21（DE3）中成功表达，SDS-PAGE结果显示，诱导的重组表达蛋白相对分子质量在45 000左右，与预测的IcFPS2蛋白大小基本一致。结论 通过对IcFPS2基因的全长cDNA克隆与生物信息学分析、组织表达特异性分析和原核表达载体的构建，为后续进一步研究法尼基焦磷酸合酶基因在枸骨三萜皂苷生物合成途径的功能供科学依据。

Objective To clone the key gene of chalcone synthase farnesyl diphosphate synthase (FPS) in triterpenoid saponins biosynthetic pathway of Ilex cornuta, analyze tissue-specific expression of the chalcone synthase, construct the prokaryotic expression vector and induce the recombinant protein to express, explore its function in regulating the biosynthesis of triterpenoid saponins. Methods Based on the transcriptome data of I. cornuta in the previous study, the full-length cDNA of IcFPS2 was cloned by PCR from the leaves of I. cornuta and bioinformatics analysis was performed. The qPCR was used to further analyze the tissue-specific expression of IcFPS2. The prokaryotic expression vector pET32a-IcFPS2 was constructed, transformed into BL21 (DE3) pLysS competent cells and the expression of recombinant protein was induced by IPTG. Results The size of IcFPS2 gene was 1259 bp, containing an open reading frame (ORF) of 1050 bp and encoding 350 amino acids, its protein molecular weight and isoelectric point are 40 200 and 5.54, respectively. Amino acid sequence alignment analysis showed that FPS amino acid sequences of I. cornuta had high homology with those of Gardenia jasminoides, Eucommia ulmoides, Glycyrrhiza uralensis, Gynostemma pentaphyllum and Panax japonicus. Phylogenetic tree analysis showed that the IcFPS2 protein clustered with the FPS proteins of Panax quinquefolius, P. ginseng and P. japonicus, suggesting that the FPS protein of I. cornuta may be functionally close to the FPS protein of the dicotyledonous plant Pentaphyllaceae. Real-time fluorescence quantitative PCR analysis showed that the expression of IcFPS2 gene was highest in roots, followed by leaves, and then male flowers and stems, and the gene was expressed at the lowest level in female flowers. The results of the prokaryotic expression analysis showed that the constructed pET32a-IcFPS2 vector could be successfully expressed in E. coli BL21 (DE3), and the SDS-PAGE results showed that the induced recombinantly expressed protein was around 45 000, which was basically consistent with the predicted IcFPS2 protein size. Conclusion The full-length cDNA cloning and bioinformatics analysis of IcFPS2 gene, tissue-specific expression analysis and prokaryotic expression vector construction were used to provide scientific basis for further research on the function of farnesyl pyrophosphate synthase gene in triterpenoid saponins biosynthetic pathway of I. cornuta.

R286.12

国家自然科学基金资助项目(31500546)