枸骨IcFPS1基因的克隆、表达及生物信息学分析

法尼基焦磷酸合酶(farnesyl diphosphate synthase,FPS)是三萜皂苷生物合途径的一个关键酶,为研究FPS基因在枸骨中的功能,该研究采用PCR技术将一个FPS基因的cDNA序列从枸骨叶中分离出来,并命名为IcFPS1。结果表明:根据测序结果分析发现扩增获得的IcFPS1基因cDNA长度为1 591 bp,包含一个完整的开放阅读框,大小为1 029 bp。通过序列分析发现枸骨IcFPS1基因编码342个氨基酸,分子量和等电点分别为39.58 kDa和5.18。通过理化性质预测分析发现IcFPS1蛋白不含信号肽,不含有跨膜区域,该IcFPS1蛋白为亲水性蛋白质。通过序列多重比对发现IcFPS1蛋白质与其他植物的FPS蛋白质高度同源,有共同的保守区域和氨基酸序列,其中与西洋参FPS序列的相似性高达89%。通过系统进化树分析发现枸骨FPS蛋白与同属于被子植物的五加科植物FPS蛋白亲缘关系较近,说明FPS基因在进化过程中相对比较保守。根据蛋白调控网络预测分析结果发现该蛋白可能与IPP1、IPP2、GGPS3、GGPS6和ERA1相互作用,参与类异戊二烯的合成代谢过程。通过实时荧光定量PCR分析发现IcFPS1基因在枸骨各个组织部位中均有表达,其中在枸骨根中表达量最高,在茎和雌花中表达量最低。

Cloning, expression and bioinformatics analysis of IcFPS1 gene from Ilex cornuta

Farnesyl diphosphate synthase(FPS)is a key enzyme in the biosynthesis of triterpene saponins, it can promote the biosynthesis triterpenoid saponins in plants. In order to study the function of FPS gene in Ilex cornuta, we isolated a FPS gene from Ilex cornuta, and carried out bioinformatics analysis and expression analysis. In this study, PCR was used to isolate the cDNA sequence of a FPS gene from the leaves of I. cornuta, and the gene was named as IcFPS1. DNA sequencing results showed that the amplified cDNA of IcFPS1 gene was 1 591 bp. Sequencing analysis revealed the IcFPS1 gene contained a complete open reading frame, and the open reading frame was 1 029 bp in length, encoding 342 amino acids. The molecular weight and isoelectric point of predicted IcFPS1 protein were 39.58 kDa and 5.18, respectively. Physical and chemical properties analysis revealed that IcFPS1 protein was a hydrophilic protein, and it did not contain a signal peptide, and had no transmembrane region of the IcFPS1 protein. The multiple alignment of FPS sequences with BLASTP and Align X revealed that the IcFPS1 protein was highly homologous to other known FPS proteins from different plant species, and they had common conserved regions and amino acid sequences. The similarity between the IcFPS1 and the Panax quinquefolius FPS sequence was as high as 89%. Phylogenetic tree analysis showed that the FPS protein of the Ilex cornuta was closely related to FPS proteins of the Araliaceae family belonging to the angiosperm. These results indicate that the FPS gene is conserved during evolution. Protein-protein interaction network analysis showed that the IcFPS1 protein is involved in the synthesis pathway of isoprenoids, which may be the same as IPP1, IPP2, GGPS3, GGPS6 and ERA1 proteins. Real-time quantitative PCR analysis showed that IcFPS1 gene was expressed in all tested tissues of Ilex cornuta, while the expression level of IcFPS1 gene in different tissues was different. IcFPS1 gene had the highest expression in roots, but was less expressed in the stems and female flowers of Ilex cornuta.