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.     

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.     

Isolation and characterization of a 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene from Taxus media

2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) synthase (MECS, EC: 4.6.1.12) is the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and further Taxol biosynthesis. The full-length MECS cDNA sequence (GenBank accession number DQ286391) was cloned and characterized for the first time from Taxus media, using the Rapid Amplification of cDNA Ends (RACE) technique. The full-length cDNA of Tmmecs was 1081 bp containing a 741 bp open reading frame (ORF) encoding a peptide of 247 amino acids with a calculated molecular mass of 26.1 kDa and an isoelectric point of 8.97. Comparative and bioinformatic analyses revealed that TmMECS had extensive homology with MECSs from other plant species. Phylogenetic analysis indicated that TmMECS was more ancient than other plant MECSs. Southern blot analysis revealed that Tmmecs belonged to a small gene family. Tissue expression pattern analysis indicated that Tmmecs expressed constitutively in all tissues including roots, stems and leaves. The cloning and characterization of Tmmecs will be helpful to understand more about the role of MECS involved in the Taxol biosynthesis at the molecular level. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 6, pp. 1013–1020. The article was submitted by the authors in English.     

Cloning and characterization of the Rubisco activase gene from <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam

A full-length cDNA of Rubisco activase (IBrcaI) was cloned from sweet potato (Ipomoea batatas (L.) Lam) using Rapid-Amplification of cDNA Ends (RACE). IBrcaI contains a 1,347 bp open reading frame encoding a protein of 439 amino acids. The sequence alignment of multiple Rubisco activase genes from sweet potato and other plants showed high homology at two previously described ATP-binding sites. Western blot analysis indicated that there are two Rubisco activase proteins in sweet potato. Expression of IBrcaI was only detected in leaves. In the 14 h light and 10 h dark photoperiods, maximal and minimal IBrcaI mRNA expression levels were detected at 8:00 in the morning and at midnight, respectively.     

Molecular characterization and expression analysis of a gene encoding for farnesyl diphosphate synthase from Euphorbia pekinensis Rupr

The root of Euphorbia pekinensis as a traditional herbal medicine has been recorded in Chinese pharmacopoeias for the treatment of oedema, gonorrhea, migraine and wart cures. In this work, we reported on the cDNA cloning and characterization of a novel farnesyl diphosphate synthase (FPS) from E. pekinensis. The full-length cDNA named EpFPS (Genbank Accession Number FJ755465) contained 1431 bp with an open reading frame of 1029 bp encoding a polypeptie of 342 amino acids. The deduced amino acid sequence of the EpFPS named EpFPS exhibited a high homology with other plant FPSs, and contained five conserved domains. Phylogenetic analysis showed that EpFPS belonged to the plant FPS group. Southern blot analysis revealed that there exists a small FPS gene family in E. pekinensis. Expression pattern analysis revealed that EpFPS expressed strongly in root, weak in leaf and stem. In callus, expression of EpFPS gene and biosynthesis of triterpenoids were strongly induced by Methyl jasmonate and slightly induced by Salicylic acid. Functional complementation of EpFPS in an ergosterol auxotrophic yeast strain indicated that the cloned cDNA encoded a functional farnesyl diphosphate synthase.     

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.     

Fruit carotenoid‐deficient mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis

Isoprenoids consist of a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP). In plants, IDP is synthesized in the cytoplasm from mevalonic acid via the MVA pathway, and in plastids from 2‐C‐methyl‐d ‐erythritol‐4‐phosphate through the MEP pathway. The enzyme IDP isomerase (IDI) catalyzes the interconversion between IDP and DMADP. Most plants contain two IDI enzymes, the functions of which are characteristically compartmentalized in the cells. Carotenoids are isoprenoids that play essential roles in photosynthesis and provide colors to flowers and fruits. They are synthesized in the plastids via the MEP pathway. Fruits of Solanum lycopersicum (tomato) accumulate high levels of the red carotene lycopene. We have identified mutations in tomato that reduce overall carotenoid accumulation in fruits. Four alleles of a locus named FRUIT CAROTENOID DEFICIENT 1 (fcd1) were characterized. Map‐based cloning of fcd1 indicated that this gene encodes the plastidial enzyme IDI1. Lack of IDI1 reduced the concentration of carotenoids in fruits, flowers and cotyledons, but not in mature leaves. These results indicate that the plastidial IDI plays an important function in carotenoid biosynthesis, thus highlighting its role in optimizing the ratio between IDP and DMADP as precursors for different downstream isoprenoid pathways.     

Characterization,expression profiling,and functional identification of a gene encoding geranylgeranyl diphosphate synthase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes including tanshinone. In this study, a full-length cDNA encoding GGPPS was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmGGPPS (GenBank Accession No. FJ643617). The full-length cDNA of SmGGPPS was 1,234 bp containing a 1,092 bp open reading frame (ORF) encoding a polypeptide of 364 amino acids. Analysis of SmGGPPS genomic DNA revealed that it contained 2 exons and 1 intron. Bioinformatics analyses revealed that the deduced SmGGPPS had extensive homology with other plant GGPPSs contained all 5 conserved domains and functional aspartate-rich motifs of the prenyltransferases. Molecular modeling showed that SmGGPPS is a new GGPPS with a spatial structure similar to other plant GGPPSs. Phylogenetic tree analysis indicated that SmGGPPS belongs to the plant GGPPS super-family and has the closest relationship with GGPPS from Nicotiana attenuate. The functional identification in Escherichia coli showed that SmGGPPS could accelerate the biosynthesis of carotenoid, demonstrating that SmGGPPS encoded a functional protein. Expression pattern analysis implied that SmGGPPS expressed higher in leaves and roots, weaker in stems. The expression of SmGGPPS could be up-regulated by Salicylic acid (SA) in leaves and inhibited by methyl jasmonate (MeJA) in 3 tested tissues, suggesting that SmGGPPS was elicitor-responsive. This work will be helpful to understand more about the role of SmGGPPS involved in the tanshinones biosynthesis pathway and metabolic engineering to improve tanshiones production in S. miltiorrhiza.     

甘薯叶片超氧化物歧化酶基因克隆及测序

以甘薯（Ｉｐｏｍｏｅａｂａｔａｔａｓ（Ｌ．）Ｐｏｉｒ）叶为材料提取植物总ＲＮＡ,经反转录后,利用多聚酶链式反应技术,扩增并克隆超氧化物歧化酶基因的ｃＤＮＡ,并进行测序分析。该序列全长４８２ｂｐ,其读码框编码１５２个氨基酸,与国外文献报道的甘薯块根ＳＯＤ基因的ｃＤＮＡ序列相比,具有９９％的同源性。     

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.     

Molecular cloning and characterization of a flavonoid 3′-hydroxylase gene from purple-fleshed sweet potato (Ipomoea batatas)

Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is an important enzyme which determines the hydroxylation pattern of anthocyanins. In this study, the full-length cDNA and genomic DNA of F3′H were isolated and characterized from the purple-fleshed sweet potato (Ipomoea batatas). IbF3’′H was 1,789 bp containing a 1,554 bp open reading frame (ORF) encoding 518 amino acids. Comparative and bioinformatic analysis revealed that IbF3′H was highly homologous with F3′Hs from other plant species. Conserved domain search revealed that IbF3′H was a cytochrome P450 dependent enzyme. Three F3′H-specific motifs (V₇₅VVAAS₈₀, G₄₂₇GEK₄₃₀ and V₄₃₃DVKG₄₃₇) were conserved in IbF3′H. Phylogenetic analysis revealed that IbF3′H was clustered into the same subgroup with the homologues from I. purpurea, I. tricolor and I. nil. There were multiple copies of the IbF3′H gene in the genome of I. batatas. IbF3′H was constitutively expressed in all tested tissues including fibrous roots, thick roots, storage roots, stems and leaves. During storage root formation, IbF3′H was expressed most abundantly in the storage roots, suggesting that the anthocyanin biosynthesis is also active in the under-ground organs. IbF3′H expression was associated with anthocyanin accumulation in five different sweet potato cultivars tested. Complementative analysis implied that the full-length cDNA of IbF3′H could encode a functional protein and had a special catalytic activity of flavonoid 3′-hydroxylase.     

Isolation and characterization of a new mannose-binding lectin gene from<Emphasis Type="Italic">Taxus media</Emphasis>

In this paper, we report the cloning and characterization of the first mannose-binding lectin gene from a gymnosperm plant species,Taxus media. The full-length cDNA ofT. media agglutinin (TMA) consisted of 676 bp and contained a 432 bp open reading frame (ORF) encoding a 144 amino acid protein. Comparative analysis showed that TMA had high homology with many previously reported plant mannose-binding lectins and thattma encoded a precursor lectin with a 26-aa signal peptide. Molecular modelling revealed that TMA was a new mannosebinding lectin with three typical mannose-binding boxes like lectins from species of angiosperms. Tissue expression pattern analyses revealed thattma is expressed in a tissue-specific manner in leaves and stems, but not in fruits and roots. Phylogenetic tree analyses showed that TMA belonged to the structurally and evolutionarily closely related monocot mannose-binding lectin superfamily. This study provides useful information to understand the molecular evolution of plant lectins.     

Isolation and characterization of a 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene from Taxus media

2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) synthase (MECS, EC: 4.6.1.12) is the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and further Taxol biosynthesis. The full-length MECS cDNA sequence (GenBank accession number DQ286391) was cloned and characterized for the first time from Taxus media, using Rapid Amplification of cDNA Ends (RACE) technique. The full-length cDNA of Tmmecs was 1081 bp containing a 741 bp open reading frame (ORF) encoding a peptide of 247 amino acids with a calculated molecular mass of 26.1 kDa and an isoelectric point of 8.97. Comparative and bioinformatic analyses revealed that TmMECS had extensive homology with MECSs from other plant species. Phylogenetic analysis indicated that TmMECS was more ancient than other plant MECSs. Southern blot analysis revealed that Tmmecs belonged to a small gene family. Tissue expression pattern analysis indicated that Tmmecs expressed constitutively in all tissues including roots, stems and leaves. The cloning and characterization of Tmmecs will be helpful to understand more about the role of MECS involved in the Taxol biosynthesis at the molecular level.     

Molecular Cloning of ADP-Glucose Pyrophosphorylase Large Subunit cDNA from Oncidium

A full-length cDNA for ADP-glucose pyrophosphorylase large subunit (AGPL) was isolated from tropical epiphytic orchid Oncidium hybrid Goldiana. The cDNA was 1754 bp in length with an open reading frame of 1551 bp encoding 517 amino acids. The deduced amino acid sequence showed 73 % identity with those of potato isoform 3 (AGPL3) and Arabidopsis thaliana isoform 1 (AGPL1), 71 % identity with that of barley isoform BLPL. RT-PCR analysis showed that AGPL was expressed in mature leaf, immature leaf, developing inflorescence and flower of Oncidium. No expression was detected in roots.     

A Novel Cytoplasmic Isopentenyl Diphosphate Isomerase Gene from Tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>): Cloning,Expression, and Color Complementation

Isopentenyl diphosphate isomerase (IPI; EC5.3.3.2) catalyzes isomerization between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), both of which are essential precursors for terpenoid biosynthesis. A novel gene encoding isopentenyl diphosphate isomerase (designated as SlIPI) was isolated from tomato based on tentative consensus (TC183769) and unigene SGN-U569721 sequences. The SlIPI cDNA contained a 708-bp open reading frame (ORF) encoding a 235-amino-acid protein. The deduced SlIPI protein had an isoelectric point of 5.06 and molecular weight of about 27.18 kDa. Amino acid sequence comparison analysis showed 83–95% similarity to IPIs from other plant species. Phylogenetic analysis revealed that SlIPI had the closest relationship to IPI from Nicotiana tabacum. The SlIPI was likely to be localized in cytoplasm; while, SlIPI2 contained a chloroplast transit peptide. A three dimensional structure modeling revealed that the structure of SlIPI was similar to that of SlIPI2. Tissue expression analysis indicated that SlIPI was constitutively expressed, with the highest expression level detected in the root. Heterologous expression of the recombinant SlIPI in engineered Escherichia coli resulted in the production and accumulation of carotenoid in E. coli, thus confirming that the SlIPI was a functional gene.