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Cloning and Functional Analysis of a cDNA Encoding Ginkgo biloba Farnesyl Diphosphate Synthase
Kexuan Tang,Peng Wang,Zhihua Liao,Liang Guo,Wenchao Li,Min Chen,Yan Pi,Yifu Gong,Xiaofen Sun 한국분자세포생물학회 2004 Molecules and cells Vol.18 No.2
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.
Wang, Yechun,Guo, Binhui,Zhang, Fei,Yao, Hongyan,Miao, Zhiqi,Tang, Kexuan Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.6
The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR (designated as CgHMGR, GenBank accession number EF206343) from hazel (Corylus avellana L. Gasaway), a taxol-producing plant species. The full-length cDNA of CgHMGR was 2064 bp containing a 1704-bp ORF encoding 567 amino acids. Bioinformatic analyses revealed that the deduced CgHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of CgHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that CgHMGR belonged to a small gene family. Expression analysis revealed that CgHMGR expressed high in roots, and low in leaves and stems, and the expression of CgHMGR could be up-regulated by methyl jasmonate (MeJA). The functional color assay in Escherichia coli showed that CgHMGR could accelerate the biosynthesis of $\beta$-carotene, indicating that CgHMGR encoded a functional protein. The cloning, characterization and functional analysis of CgHMGR gene will enable us to further understand the role of CgHMGR involved in taxol biosynthetic pathway in C. avellana at molecular level.
Metabolic Engineering of Vitamin C Production in Arabidops
Ling Xiao,Ying Xiao,Zinan Wang,Hexin Tan,Kexuan Tang,Lei Zhang 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.4
Vitamin C (L-ascorbic acid, AsA) is a compound which provides major nutritional value, both for plants and humans. In this study, three distinct metabolic engineering strategies, including overexpression of biosynthesis enzyme, suppression of catabolism enzyme and switching the sub-cellular localization of compartment enzyme, were employed to enhance the production of AsA in Arabidopsis thaliana. The results showed that (1) overexpression of L-Galactose-1-P Phosphatase (GalPPase) enhanced AsA content to 3.96 μmol/g FW, which was 1.6-fold more than that in their wild-type (WT) counterparts; (2) RNAi suppression of ascorbate oxidase (AO) resulted in a significant increase of AsA accumulation (0.86 μmol/g FW) in apoplast; (3) both mitochondrion-target and none-target overexpression of L-Galactose dehydrogenase (GalDH) did not significantly promote AsA production compared with WT (1.96 μmol/g), however a dramatic enhancement was observed following infiltration with L-galactono-1, 4-lactone (L-GalL), both in transgenic and WT plants. The best line produced AsA with the content of 3.90 μmol/g FW, which was about 2-fold of that in the untreated control (1.99 μmol/g FW). This study provides new strategies including GalPPase overexpression, AO suppression as well as L-GalL feeding for modern breeding aimed at stimulating the AsA content in plants.
Gao, Shi,Lin, Juan,Liu, Xuefen,Deng, Zhongxiang,Li, Yingjun,Sun, Xiaofen,Tang, Kexuan Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.5
2C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MECPS, EC: 4.6.1.12) is the fifth enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate (MEP) pathway for ginkgolide biosynthesis. The full-length mecps cDNA sequence (designated as Gbmecps) was cloned and characterized for the first time from gymnosperm plant species, Ginkgo biloba, using RACE (rapid amplification of cDNA ends) technique. The full-length cDNA of Gbmecps was 874 bp containing a 720 bp open reading frame (ORF) encoding a peptide of 239 amino acids with a calculated molecular mass of 26.03 kDa and an isoelectric point of 8.83. Comparative and bioinformatic analyses revealed that GbMECPS showed extensive homology with MECPSs from other species and contained conserved residues owned by the MECPS protein family. Phylogenetic analysis indicated that GbMECPS was more ancient than other plant MECPSs. Tissue expression pattern analysis indicated that GbMECPS expressed the highest in roots, followed by in leaves, and the lowest in seeds. The color complementation assay indicated that GbMECPS could accelerate the accumulation of $\beta$-carotene. The cloning, characterization and functional analysis of GbMECPS will be helpful to understand more about the role of MECPS involved in the ginkgolides biosynthesis at the molecular level.
Kai, Guoyin,Zhao, Lingxia,Zhang, Lei,Li, Zhugang,Guo, Binhui,Zhao, Dongli,Sun, Xiaofen,Miao, Zhiqi,Tang, Kexuan Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.6
A full-length cDNA encoding taxadiene synthase (designated as TmTXS), which catalyzes the first committed step in the Taxol biosynthetic pathway, was isolated from young leaves of Taxus media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTXS had a 2586 bp open reading frame (ORF) encoding a protein of 862 amino acid residues. The deduced protein had isoelectric point (pI) of 5.32 and a calculated molecular weight of about 98 kDa, similar to previously cloned diterpene cyclases from other Taxus species such as T. brevifolia and T. chinenisis. Sequence comparison analysis showed that TmTXS had high similarity with other members of terpene synthase family of plant origin. Tissue expression pattern analysis revealed that TmTXS expressed strongly in leaves, weak in stems and no expression could be detected in fruits. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in Taxol biosynthetic pathway in different tissues of Taxus plants. Phylogenetic tree analysis showed that TmTXS had closest relationship with taxadiene synthase from T. baccata followed by those from T. chinenisis and T. brevifolia. Expression profiles revealed by RT-PCR under different chemical elicitor treatments such as methyl jasmonate (MJ), silver nitrate (SN) and ammonium ceric sulphate (ACS) were also compared for the first time, and the results revealed that expression of TmTXS was all induced by the tested three treatments and the induction effect by MJ was the strongest, implying that TmTXS was high elicitor responsive.
Molecular Cloning and Characterization of a Novel Stem-specific Gene from Camptotheca acuminata
Pi, Yan,Liao, Zhihua,Chai, Yourong,Zeng, Hainian,Wang, Peng,Gong, Yifu,Pang, Yongzhen,Sun, Xiaofen,Tang, Kexuan Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.1
In higher plants, P450s participate in the biosynthesis of many important secondary metabolites. Here we reported for the first time the isolation of a new cytochrome P450 cDNA that expressed in a stem-specific manner from Camptotheca acuminata (designated as CaSS), a native medicinal plant species in China, using RACE-PCR. The full-length cDNA of CaSS was 1735 bp long containing a 1530 bp open reading frame (ORF) encoding a polypeptide of 509 amino acids. Bioinformatic analysis revealed that CASS contained a heme-binding domain PFGXGRRXCX and showed homology to other plant cytochrome P450 monooxygenases and hydroxylases. Southern blotting analysis revealed that there was only one copy of the CaSS present in the genome of Camptotheca acuminata. Northern blotting analysis revealed that CaSS expressed, in a tissue-specific manner, highly in stem and lowly in root, leaf and flower. Our study suggests that CaSS is likely to be involved in the phenylpropanoid pathway.
Wang, Xinglong,Liu, Li,Liu, Sixiu,Sun, Xiaoqing,Deng, Zhongxiang,Pi, Yan,Sun, Xiaofen,Tang, Kexuan Korean Society for Biochemistry and Molecular Biol 2004 Journal of biochemistry and molecular biology Vol.37 No.5
A new CRT binding factor (CBF) gene designated Cbcbf25 was cloned from Capsella bursa-pastoris, a wild grass, by the rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbcbf25 was 898 bp with a 669 bp open reading frame (ORF) encoding a putative DRE/CRT (LTRE)-binding protein of 223 amino acids. The predicted CbCBF25 protein contained a potential nuclear localization signal (NLS) in its N-terminal region followed by an AP2 DNA-binding motif and a possible acidic activation domain in the C-terminal region. Bioinformatic analysis revealed that Cbcbf25 has a high level of similarity with other CBF genes like cbf1, cbf2, and cbf3 from Arabidopsis thaliana, and Bncbf5, Bncbf7, Bncbf16, and Bncbf17 from Brassica napus. A cold acclimation assay showed that Cbcbf25 was expressed immediately after cold triggering, but this expression was transient, suggesting that it concerns cold acclimation. Our study implies that Cbcbf25 is an analogue of other CBF genes and may participate in cold-response, by for example, controlling the expression of cold-regulated genes or increasing the freezing tolerance of plants.
Huang, Zhuoshi,Jiang, Keji,Pi, Yan,Hou, Rong,Liao, Zhihua,Cao, Ying,Han, Xu,Wang, Qian,Sun, Xiaofen,Tang, Kexuan Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.5
The enzyme squalene synthase (EC 2.5.1.21) catalyzes a reductive dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for the sterol and triterpene biosynthesis. A full-length cDNA encoding squalene synthase (designated as TcSqS) was isolated from Taxus cuspidata, a kind of important medicinal plants producing potent anti-cancer drug, taxol. The full-length cDNA of TcSqS was 1765 bp and contained a 1230 bp open reading frame (ORF) encoding a polypeptide of 409 amino acids. Bioinformatic analysis revealed that the deduced TcSqS protein had high similarity with other plant squalene synthases and a predicted crystal structure similar to other class I isoprenoid biosynthetic enzymes. Southern blot analysis revealed that there was one copy of TcSqS gene in the genome of T. cuspidata. Semi-quantitative RT-PCR analysis and northern blotting analysis showed that TcSqS expressed constitutively in all tested tissues, with the highest expression in roots. The promoter region of TcSqS was also isolated by genomic walking and analysis showed that several cis-acting elements were present in the promoter region. The results of treatment experiments by different signaling components including methyl-jasmonate, salicylic acid and gibberellin revealed that the TcSqS expression level of treated cells had a prominent diversity to that of control, which was consistent with the prediction results of TcSqS promoter region in the PlantCARE database.