Molecular cloning and expression analysis of a squalene synthase gene from a medicinal plant, Euphorbia pekinensis Rupr.

Euphorbia pekinensis Rupr., which is also known as a medicinal plant, produces a large amount of alkaloids, phytosterols and triterpenes. In this study, we reported on the cDNA cloning and characterization of a novel squalene synthase (SQS) from E. pekinensis. Squalene synthase catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to produce squalene (SQ), the first committed precursor for sterol and triterpene biosynthesis. The full length cDNA named EpSQS (Genbank Accession Number JX509735) contained 1,614 bp with an open reading frame of 1,236 bp encoding a polypeptide of 411 amino acids. The deduced amino acid sequence of the EpSQS named EpSQS exhibited a high homology with other plant SQSs, and contained a single domain surrounded by helices. Phylogenetic analysis showed that EpSQS belonged to the plant SQS kingdom. Tissue expression analysis revealed that EpSQS expressed strongly in roots, weakly in stems and leaves, implying that EpSQS was a constitutive expression gene. The recombinant protein was expressed in Escherichia coli and detected by SDS-PAGE and western blot. The high performance liquid chromatography (HPLC) analysis showed that EpSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene.     

Molecular cloning and functional analysis of an organ-specific expressing gene coding for farnesyl diphosphate synthase from <Emphasis Type="Italic">Michelia chapensis</Emphasis> Dandy

Farnesyl diphosphate synthase (FPS; EC 2.5.1.1/EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate, a key intermediate in the biosynthesis of sesquiterpenes. This present study described the cloning and characterization of a cDNA encoding FPS from leaves of Michelia chapensis Dandy (designated as McFPS, GenBank accession number: GQ214406) for the first time. McFPS was 1,432 bp and contained an open reading frame (ORF) of 1,056 bp, encoding a protein of 351 amino acids with a calculated molecular mass of 40.52 kDa. Bioinformatic analysis revealed that the deduced McFPS had high homology with FPSs from other plant species. Phylogenetic tree analysis indicated that McFPS belonged to the plant FPS group and had the closest relationship with FPS from Chimonanthus praecox. Southern blot analysis revealed that there were at most two copies of McFPS gene existed in M. chapensis genome. The organ expression pattern analysis showed that McFPS expressed strongly only in leaves, and there were no expression in stems and roots, implying that McFPS was an organ-specific expressing gene. Functional complementation of McFPS in a FPS-deficient yeast strain demonstrated that cloned cDNA encoded a farnesyl diphosphate synthase.     

A New Farnesyl Diphosphate Synthase Gene from Taxus media Rehder： Cloning, Characterization and Functional Complementation

Farnesyl dlphosphate synthase （FPS; EC 2.5.1.10） catalyzes the production of 15-carbon farnesyl dlphosphate which Is a branch-point Intermediate for many terpenoids. This reaction Is considered to be a ratelimiting step In terpenold biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl dlphosphate synthase from a gymnosperm plant species, Taxus media Rehder, designated as TmFPS1. The full-length cDNA of TmFPS1 （GenBank accession number： AY461811） was 1 464 bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptlde with a calculated molecular weight of 40.3 kDa and a theoretical pl of 5.07. Biolnformatlc analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetlc analysis showed that farnesyl dlphosphate synthases can be divided Into two groups： one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homologybased structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature Is the arrangement of 13 core helices around a large central cavity In which the catalytic reaction takes place. Our blolnformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPSgene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, Including the needles, stems and roots of T. media. Subsequently, functional complementatlon with TmFPS1 in a FPS-deflclent mutant yeast demonstrated that TmFPS1 did encode farnesyl dlphosphate synthase, which rescued the yeast mutant. This study will be helpful In future Investigations aiming at understanding the detailed role of FPS In terpenold biosynthesis flux control at the molecular genetic level.     

Molecular characterization of ginseng farnesyl diphosphate synthase gene and its up-regulation by methyl jasmonate

We isolated a gene encoding for farnesyl diphosphate synthase (FPS) from Panax ginseng, a species that produces a large quantity of triterpene saponins such as ginsenosides. The deduced amino acid sequence of PgFPS was 77, 84 and 95 % identical to those of Arabidopsis, Hevea, and Centella. Southern blot analysis indicated that P. ginseng contained more than two genes encoding for FPS. When the cDNA of PgFPS was expressed in Escherichia coli, the recombinant enzyme, purified with a His-tag column, was found to possess FPS activity. When cultures of ginseng hairy root were treated with 0.1 mM methyl jasmonate (MJ), PgFPS mRNA was detected within 12 h of the treatment, and achieved maximum after 24 h. Also FPS activity in the hairy root cultures after 12 h of MJ treatment was higher than that of the control.     

Cloning and functional analysis of a cDNA encoding Ginkgo biloba farnesyl diphosphate synthase

Farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2. 5.1.10) catalyzes the synthesis of farnesyl diphosphate, and provides precursor for biosynthesis of sesquiterpene and isoprenoids containing more than 15 isoprene units in Ginkgo biloba. Here we report the cloning, characterization and functional analysis of a new cDNA encoding FPS from G. biloba. The full-length cDNA (designated GbFPS) had 1731 bp with an open reading frame of 1170 bp encoding a polypeptide of 390 amino acids. The deduced GbFPS was similar to other known FPSs and contained all the conserved regions of trans-prenyl chain-elongating enzymes. Structural modeling showed that GbFPS had the typical structure of FPS, the most prominent feature of which is the arrangement of 13 core helices around a large central cavity. Southern blot analysis revealed a small FPS gene family in G. biloba. Expression analysis indicated that GbFPS expression was high in roots and leaves, and low in stems. Functional complementation of GbFPS in an FPS-deficient strain confirmed that GbFPS mediates farnesyl diphosphate biosynthesis.     

Cloning and expression of a farnesyl diphosphate synthase in Centella asiatica (L.) Urban

A cDNA encoding farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2.5.1.10) was isolated from Centella asiacita (L.) Urban, using degenerate primers based on two highly conserved domains. A full-length cDNA clone was subsequently isolated by rapid amplification of cDNA ends (RACE) PCR. The sequence of the CaFPS (C. asiatica farnesyl diphosphate synthase) cDNA contains an open reading frame of 1029 nucleotides encoding 343 amino acids with a molecular mass of 39.6 kDa. The deduced CaFPS amino acid sequence exhibits 84, 79, and 72%, identity to the FPSs of Artemisia annua, Arabidopsis thaliana, and Oryza sativa, respectively. Southern blot analysis suggested that the C. asiatica genome contains only one FPS gene. An artificially expressed soluble form of the CaFPS was identified by SDS-PAGE. It had high specific activity and produced farnesyl diphosphate as the major isoprenoid.     

Spatial and temporal patterns of GUS expression directed by 5′ regions of the Arabidopsis thaliana farnesyl diphosphate synthase genes FPS1 and FPS2

Farnesyl diphosphate synthase (FPS), the enzyme that catalyses the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), is considered a regulatory enzyme of plant isoprenoid biosynthesis. The promoter regions of the FPS1 and FPS2 genes controlling the expression of isoforms FPS1S and FPS2, respectively, were fused to the -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana plants. The FPS1S:GUS gene is widely expressed in all plant tissues throughout development, thus supporting a role for FPS1S in the synthesis of isoprenoids serving basic plant cell functions. In contrast, the FPS2:GUS gene shows a pattern of expression restricted to specific organs at particular stages of development. The highest levels of GUS activity are detected in flowers, especially in pollen grains, from the early stages of flower development. After pollination, much lower levels of GUS activity are detected in the rest of floral organs, with the exception of the ovary valves, which remain unstained throughout flower development. GUS activity is also detected in developing and mature seeds. In roots, GUS expression is primarily detected at sites of lateral root initiation and in junctions between primary and secondary roots. No GUS activity is detected in root apical meristems. GUS expression is also observed in junctions between primary and secondary stems. Overall, the pattern of expression of FPS2:GUS suggests a role for FPS2 in the synthesis of particular isoprenoids with specialized functions. Functional FPS2 gene promoter deletion analysis in transfected protoplasts and transgenic A. thaliana plants indicate that all the cis-acting elements required to establish the full pattern of expression of the FPS2 gene are contained in a short region extending from positions –111 to +65. The potential regulatory role of specific sequences within this region is discussed.     

Cloning of an Arabidopsis thaliana cDNA coding for farnesyl diphosphate synthase by functional complementation in yeast

A cDNA encoding farnesyl diphosphate synthase, an enzyme that synthesizes C15 isoprenoid diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, was cloned from an Arabidopsis thaliana cDNA library by complementation of a mutant of Saccharomyces cerevisiae deficient in this enzyme. The A. thaliana cDNA was also able to complement the lethal phenotype of the erg20 deletion yeast mutant. As deduced from the full-length 1.22 kb cDNA nucleotide sequence, the polypeptide contains 343 amino acids and has a relative molecular mass of 39689. The predicted amino acid sequence presents about 50% identity with the yeast, rat and human FPP synthases. Southern blot analyses indicate that A. thaliana probably contains a single gene for farnesyl diphosphate synthase.     

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。     

Amorpha-4,11-diene synthase of Artemisia annua: cDNA isolation and bacterial expression of a terpene synthase involved in artemisinin biosynthesis

Artemisia annua, an indigenous plant to Korea, contains an antimalarial sesquiterpene, artemisinin. The first committed step of artemisinin biosynthesis is the cyclization of farnesyl diphosphate by a sesquiterpene synthase to produce an amorphane-type ring system. The aims of this research were to molecularly clone and express amorpha-4,11-diene synthase for metabolic engineering. PCR amplification of genomic DNA with a pair of primers, designed from the conserved regions of sesquiterpene synthases of several plants, produced a 184-bp DNA fragment. This fragment was used in Northern blot analysis as a probe, showing approximately 2.2 kb of a single band. Its sequence information was used to produce 2106 bp of a full-length cDNA sequence including 1641 bp of open reading frame for 546 amino acids (kcs12) through a rapid amplification of cDNA ends (RACE). The deduced amino acid sequence displayed 36% identity with 5-epi-aristolochene synthase of Nicotiana tabacum. A soluble fraction of Escherichia coli harboring kcs12 catalyzed the cyclization of farnesyl diphosphate to produce a sesquiterpene, which was identified through GC-MS analysis as amorpha-4,11-diene.     

Molecular cloning and tissue expression of an insect farnesyl diphosphate synthase.

The enzyme farnesyl-diphosphate synthase (FPS, EC2.5.1.1/EC2.5.1.10), which has been shown to play a key role in isoprenoid biosynthesis, catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and di-methylallyl diphosphate. Insects do not synthesize cholesterol de novo, rather farnesyl diphosphate leads to the formation of nonsterol isoprenoids, which are essential for insect development and reproduction. In this paper, we describe the characterization of one FPS from the moth Agrotis ipsilon, the first insect FPS to be reported. An homologous probe was obtained through a nested PCR strategy using degenerate primers designed from the conserved domains of FPS from other organisms. The complete cDNA clone was isolated by PCR screening of a brain cDNA library by using homologous primers deduced from the probe. Analysis of the nucleotide sequence revealed that the cDNA encodes a polypeptide of 412 amino acids (Mr = 47 170), which shares regions similar to the FPS of other organisms, but exhibits singularities such as an extra N-terminal extension of approximately 70 amino acid residues. Using an RNase protection assay, a protected fragment corresponding to the region encoding the FPS catalytic site was found in brain, ovary, fat body and corpora allata samples, but not in muscle. FPS is overexpressed in the corpora allata, the endocrine gland that produces the juvenile hormones. These hormones are specific to insects and play a crucial role in regulating insect physiology.     

Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua

A cDNA encoding farnesyl diphosphate (FPP) synthase (FPPS) has been cloned from a cDNA library of Artemisia annua. The sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with a calculated molecular weight of 39 420 kDa. The deduced aa sequence of the cDNA was highly similar to FPPS from other plants, yeast and mammals, and contained the two conserved domains found in polyprenyl synthases including FPPS, geranylgeranyl diphosphate synthases and hexaprenyl diphosphate synthases. The expression of the cDNA in Escherichia coli showed enzyme activity for FPPS in vitro.     

Cloning and characterization of farnesyl diphosphate synthase from the rubber-producing mushroom Lactarius chrysorrheus

Farnesyl diphosphate is involved in rubber biosynthesis as an initiating substrate for both polyprenol and mushroom rubber. So far, we have isolated the cDNA of a farnesyl diphosphate synthase (FPS) for the first time from a rare rubber-producing mushroom, Lactarius chrysorrheus, by the degenerate RT-PCR technique based on sequence information of FPS genes from fungi and yeasts. The open reading frame was clarified to encode a protein of 381 amino acid residues with a calculated molecular weight of 42.9 kDa. The deduced amino acid sequence of L. chrysorrheus FPS showed about 50% identity with those of other fungi and yeasts as well as plants. We expressed the cDNA of L. chrysorrheus FPS in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein. The purified obtained protein showed FPS activity in which geranyl diphosphate (GPP) served as primary substrate, with a 2.4-fold higher k(cat)/K(m) value for GPP than for dimethylallyl diphosphate (DMAPP).     

巴西橡胶树鲨烯合酶基因的克隆与序列分析

鲨烯合酶(SQS )是植物甾醇和三萜化合物生物合成途径中的关键酶。以巴西橡胶树为试验材料,提取胶乳总 RNA,利用 RT-PCR 以及 RACE 的方法克隆橡胶树鲨烯合酶 cDNA 编码区片段,并进行序列分析。结果表明：橡胶树鲨烯合酶 cDNA 编码区为1239 bp,编码413个氨基酸,命名为 HbSQS 。荧光定量分析表明鲨烯合酶基因在不同组织里表达水平存在明显差异,且受乙烯调控。     

Isolation and characterization of farnesyl diphosphate synthase from the cotton boll weevil,Anthonomus grandis

Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full‐length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three‐dimensional structure of coleopteran FPPS was determined and compared to the X‐ray crystal structure of avian FPPS. The α‐helical fold is conserved in AgFPPS and the size of the active site cavity is consistent with the enzyme being a FPPS. © 2009 Wiley Periodicals, Inc.     

Molecular cloning and functional expression analysis of a new gene encoding geranylgeranyl diphosphate synthase from hazel (<Emphasis Type="Italic">Corylus avellana</Emphasis> L. Gasaway)

Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes such as taxol. Herein, a full-length cDNA encoding GGPPS (designated as CgGGPPS) was cloned and characterized from hazel (Corylus avellana L. Gasaway), a taxol-producing angiosperms. The full-length cDNA of CgGGPPS was 1515 bp with a 1122 bp open reading frame (ORF) encoding a 373 amino acid polypeptide. The CgGGPPS genomic DNA sequence was also obtained, revealing CgGGPPS gene was not interrupted by an intron. Southern blot analysis indicated that CgGGPPS belonged to a small gene family. Tissue expression pattern analysis indicated that CgGGPPS expressed the highest in leaves. RT–PCR analysis indicated that CgGGPPS expression could be induced by exogenous methyl jasmonate acid. Furthermore, carotenoid accumulation was observed in Escherichia coli carrying pACCAR25ΔcrtE plasmid carrying CgGGPPS. The result revealed that cDNA encoded a functional GGPP synthase.     

法呢基焦磷酸(FPP)的生物合成及其分子调控

法呢基焦磷酸合酶作为异戊二烯途径中的重要调节酶,是许多萜类物质的合成前体。FPS的cDNA克隆在许多生物体中也已得到了分离并进行了表达特性研究。从FPP的生物合成途径入手,对FPP生物学特性、FPS酶基因调控的相关信息进行了综述,同时对FPS在基因工程方面的应用进行了展望。     

Candidate genes involved in tanshinone biosynthesis in hairy roots of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> revealed by cDNA microarray

Salvia miltiorrhiza is a valuable Chinese herb (Danshen) that is widely used in traditional Chinese medicine. Diterpene quinones, known as tanshinones, are the main bioactive components of S. miltiorrhiza; however, there is only limited information regarding the molecular mechanisms underlying secondary metabolism in this plant. We used cDNA microarray analysis to identify changes in the gene expression profile at different stages of hairy root development in S. miltiorrhiza. A total of 203 genes were singled out from 4,354 cDNA clones on the microarray, and 114 unique differentially expressed cDNA clones were identified: six genes differentially expressed in 45-day hairy root compared with 30-day hairy root; 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root; and 12 genes unstably expressed at different stages. Among the 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root, a total of 57 genes were up-regulated, and 26 genes represent 29 metabolism-related enzymes. Copalyl diphosphate synthase, which catalyzes the conversion of the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate to copalyl diphosphate, was up-regulated 6.63 fold, and another six genes involved in tanshinone biosynthesis and eight candidate P450 genes were also differentially expressed. These data provide new insights for further identification of the enzymes involved in tanshinone biosynthesis.     

Cloning and characterization of squalene synthase gene from <Emphasis Type="Italic">Fusarium fujikuroi</Emphasis> (Saw.) Wr.

The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5′-untranslated region (UTR), 3′-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS–PAGE and western blot. GC–MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.     

Overexpression of Farnesyl Diphosphate Synthase in Arabidopsis Mitochondria Triggers Light-dependent Lesion Formation and Alters Cytokinin Homeostasis

To investigate the role of mitochondrial farnesyl diphosphate synthase (FPS) in plant isoprenoid biosynthesis we characterized transgenic Arabidopsis thaliana plants overexpressing FPS1L isoform. This overexpressed protein was properly targeted to mitochondria yielding a mature and active form of the enzyme of 40 kDa. Leaves from transgenic plants grown under continuous light exhibited symptoms of chlorosis and cell death correlating to H₂O₂ accumulation, and leaves detached from the same plants displayed accelerated senescence. Overexpression of FPS in mitochondria also led to altered leaf cytokinin profile, with a reduction in the contents of physiologically active trans-zeatin- and isopentenyladenine-type cytokinins and their corresponding riboside monophosphates as well as enhanced levels of cis-zeatin 7-glucoside and storage cytokinin O-glucosides. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase did not prevent chlorosis in plants overexpressing FPS1L, but did rescue accelerated senescence of detached leaves and restored wild-type levels of cytokinins. We propose that the overexpression of FPS1L leads to an enhanced uptake and metabolism of mevalonic acid-derived isopentenyl diphosphate and/or dimethylallyl diphosphate by mitochondria, thereby altering cytokinin homeostasis and causing a mitochondrial dysfunction that renders plants more sensitive to the oxidative stress induced by continuous light.