ABSTRACT
As a secondary metabolite, sesquiterpenes are not only have important functions in plant defense and signaling, but also play potential roles in basic materials for pharmaceuticals, cosmetic and flavor. As a traditional Chinese herbal medicine, Senecio scandens exhibits effects of anti-inflammatory and immunosuppressive, as well as invigorating the blood and removing extravasated blood. Over 600 sesquiterpenes with diverse structures were isolated from S. scandens and related species in the same genus. To characterize sesquiterpenes synthesis, two FPS genes(SsFPS1 and SsFPS2) were identified in S. scandens through transcriptomic analysis. Bioinformatic analysis showed that both SsFPSs have conserved motifs for FPS function. Both SsFPSs exhibited constitutive gene expression in S. scandens tissues and SsFPS2 accumulated higher transcript in leaves and roots than SsFPS1. Meanwhile consistent with constitutive sesquiterpene accumulation in S.scandens tissues, most of these sesquiterpenes were detected in leaves and roots more than stems and flowers. Recombinant expression through Escherichia coli metabolic engineering, SsFPS1 or SsFPS2 was co-transformed with ZmTPS11(maize β-macrocarpene synthase) into BL21 competent cells. The results showed that the content of β-macrocarpene was increased by co-transformation with SsFPSs. It is demonstrated that SsFPS1 and SsFPS2 catalyzed E,E-FPP formation and provided FPP precursor for downstream sesquiterpene synthases. Characterization of SsFPSs provided the foundation for the exploration of biosynthesis of sesquiterpenoid with diverse structures and potential pharmaceutical values in S.scandens, and provide an important theoretical basis for the development of S. scandens abundant resources.
Subject(s)
Cloning, Molecular , Gene Expression Profiling , Geranyltranstransferase , Medicine, Chinese Traditional , Senecio/genetics , SesquiterpenesABSTRACT
Objective: To clone the full-length cDNA encoding farnesyl pyrophosphate synthase (FPPS) from Atractylodes lancea and analyze its expression. Methods: The full-length cDNA of FPPS in A. lancea was cloned via homology-based cloning and rapid amplification of cDNA ends approach. Also, the characterization of gene was revealed by bioinformatic analysis. The expression of FPPS was determined by qRT-PCR while the content of sesquiterpenes of rhizome in different growth stages in A. lancea was measured by GC-MS. Meanwhile the correlation between them was analyzed. Results: The full-length cDNA (1320 bp) of FPPS gene was obtained (AlFPPS, GenBank accession number KX443242), with an open reading frame of 1029 bp, encoding 342 amino acids. The deduced AlFPPS protein sequence contained five conserved motifs, two of which is full of Asp (DDXXD). qRT-PCR and GC-MS results showed a significant positive correlation between the content of sesquiterpenes and AlFPPS expression level. Conclusion: It can be primarily deduced that AlFPPS gene should be an important control point in the biosynthetic pathway of sesquiterpenes in A. lancea. This work provides scientific basis for clarifying the biosynthetic pathway of sesquiterpenes and application of biological engineering.
ABSTRACT
Objective: To obtain the distribution and functional activity of CpG islands in promoters of FPS, SS, and SE from Eleutherococcus Senticosus. Methods: Based on the promoter sequence of FPS, SS and SE, CpG islands were predicted by using EMBOSS and Li Lab. The functional verification of transformed Arabidopsis thaliana was mediated by Agrobacterium tumefaciens by using GUS and pCAMBIA1301 plasmid as the reporter gene and expression vector respectively. Results: Two CpG islands were found in FPS and SS promoter with the lengths of 520 bp, 218 bp and 108 bp, 103 bp, and three CpG islands in SE promoter in 290 bp, 119 bp and 149bp. The promoters of FPS, SS and SE all had promoter activities at different level, in which SS promoter was the highest one. Conclusion: The functional verification and distributions of CpG islands in the promoters area of FPS, SS, and SE were reported in this research at first time, which established the foundation for the methylation analysis of FPS, SS, and SE and the further studying of the mechanism expression regulation in E. Senticosus.
ABSTRACT
Objective: To clone and analyze the coding region of Farnesyl diphosphate synthase (FPS) gene PcFPS1 from Phlegmariurus carinatus. Methods: According to the acquired transcriptome dataset of P. carinatus, one transcript coding FPS was obtained. The coding region sequence was obtained using RT-PCR method, and the physicochemical properties, protein secondary structure, and three-dimensional structure of PcFPS1 protein were predictively analyzed. Results: The cloned PcFPS1 gene contained a 1 119-bp coding region for encoding a predicted protein of 372 amino acids with high homology (70%) to FPS gene in Picea abies. PcFPS1 contained almost no transmembrane region and had the conserved domain of terpenoid cylases, without signal peptide. Conclusion: This study clones and analyzes the FPS gene from P. carinatus which is obtained for the first time. The results will provide a foundation for exploring the function of PcFPS1 in terpene and sterol biosynthesis of plants in Huperziaceae.
ABSTRACT
Glycyrrhizin is the major bioactive compound in Glycyrrhiza uralensis. Farnesyl-diphosphate synthase (FPS) is one of the major enzymes involved in the glycyrrhizin biosynthetic. We cloned the full length of FPS coding sequence based on the transcriptome data of G. uralensis. The FPS gene of G. uralensis (GlyurFPS) is 1029 bp, coding 342 amino acid. The homology between the sequence of its protein and that of the FPS in Cicer arietinum, Medicago sativa, Medicago truncatula, Glycine max, Lotus japonicus was of 94%, 94%, 93%, 93% and 92%, respectively. Plant FPS gene is very conservative, therefore the phylogenetic tree is same with the plant classification consistent.