The effect of La, Ce was firstly tested on growth of the Taxus(T.) cuspidata cell suspensions, biosynthesis and release of taxol. The results indicate that the growth pattern of T.cuspidata cells is altered si...The effect of La, Ce was firstly tested on growth of the Taxus(T.) cuspidata cell suspensions, biosynthesis and release of taxol. The results indicate that the growth pattern of T.cuspidata cells is altered significantly by adding high concentration of rare earth in the medium. The lag and exponential phases of cell growth are shortened, the stationary phase disappears and the biomass fluctuates periodically during the decline phase. The rare earth compounds added in the exponential phase obviously increase the taxol biosyntheis and release yields of T.cuspidata cells, and the supplement of carbon source in the medium containing rare earth is also favorable to taxol biosynthesis.展开更多
The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nu...The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nuclear DNA (nDNA) into oligonucleosomal fragments(DNA laddering) was a characteristic of apoptosis, which involved cell shrinkage, condensation of cytoplasm and tracheary elements differentiation. Terminal deoxynucleotidy transferase-mediated dUTP nick end in situ labeling (TUNEL) assay of Taxus spp. cells showed that fungal extractive or abiotic elicltors (Ce4+, Taxol, H2O2) induced TUNEL positive. Also, the increase of the apoptotic cell ratio was accompanied by the increase of secondary metabolites (especially Taxol). These results suggest that apoptosis may have some coincidence with biosynthesis of Taxol. The implication of apoptosis for the production of secondary metabolites in plant cell cultures is discussed.展开更多
Conditions have been established for the callus initiation and subculture of T chinensis. The calliwere induced by the explants cultured first on the medium MS supplemented with 1 .0 mg/L 2,4-D, 2g/L CH, and 25g/L suc...Conditions have been established for the callus initiation and subculture of T chinensis. The calliwere induced by the explants cultured first on the medium MS supplemented with 1 .0 mg/L 2,4-D, 2g/L CH, and 25g/L sucrose, then on The medium f MS+1 .0 mg/L NAA+0.5 mg/L BA+2 g/L CH+25 g/L sucrose. When the callus was Subcultured and tamed several times, it could grow fast and stable on the medium : MS+0.2mg/L 2,4-D+0.5mg/L NAA+0.5 mg/L BA+2 g/LCH+25 g/L sucrose. The contamination of explants was a result of endophytic microbes of T Chinensis. This could be avoided by adopting the tender shoots 3-5 cm long collected in early spring as the source of explants. The browning of the Cultures could be prevented and controlled by means of the selection of a suitable explants, hormonal regime in the medium, culture methods and the use of antioxidants.展开更多
In this study to screen for stable, high Taxolproducing cell lines（CL5, CL12, and CL21） of Taxus cuspidata, stem tissues were used to induce calli, which were then subcultured nine times to establish suspension cell...In this study to screen for stable, high Taxolproducing cell lines（CL5, CL12, and CL21） of Taxus cuspidata, stem tissues were used to induce calli, which were then subcultured nine times to establish suspension cell cultures. From 97 cell lines obtained from conditioned cultures, 10 cell lines with high Taxol content were selected. Stability analyses on solid and liquid B5 media were then used to obtain lines that stably produced high levels of Taxol. Fresh biomass and Taxol production of the ninth generation became stable. Taxol content of selected CL5, CL12, and CL21 samples was 0.0448, 0.0477, and0.0428% of dry mass（DW）, respectively. Proliferation of CL5, CL12 and CL21 was 346.3, 382.5, and 409.2%,respectively. From work over about 2 years, the three cell lines appear suitable for mass production of Taxol,promoting the industrialisation and commercial-scale production of Taxol using cell culture.展开更多
The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of 'partition' and 'bifurcation' were o...The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of 'partition' and 'bifurcation' were observed in studying the dynamic effect of Ce4+ on soluble protein synthesis and cell activity. That is, Ce4+ of low concentration improves the soluble protein synthetic strength and cell activity, while Ce4+ of high concentration is harmful to protein synthesis and cell activity. In addition, Ce4+ of appropriate concentration enhances taxol synthesis.展开更多
Taxol(Paclitaxel)is a diterpene from Taxus species and has been used in treatment of various kinds of cancers.Geranylgeranyl diphosphate synthase(GGPPS)catalyzes the formation of geranylgeranyl diphosphate(GGPP,the co...Taxol(Paclitaxel)is a diterpene from Taxus species and has been used in treatment of various kinds of cancers.Geranylgeranyl diphosphate synthase(GGPPS)catalyzes the formation of geranylgeranyl diphosphate(GGPP,the common precursor for diterpenes and plays a key role in taxol biosynthesis.Here we report a functional GGPPS gene from Taxus chinensis(designated TcGGPPS).TcGGPPS is an intron free gene and has a 1,182-bp open reading frame encoding a polypeptide of 393 amino acid residues with a calculated molecular mass of 42.63 kDa and an isoelectric point of 5.58.The catalytic activity of TcGGPPS for production of GGPP was verified by a color enhancement assay in the Escherichia coli cells harboring plasmid pAC-BETA.Multiple sequence alignment indicates that TcGGPPS is a little different in sequence from the functional GGPPS genes from other Taxus species such as T.canadensis,T.media and T.wallichiana,which are almost identical to each other.Protein structure prediction by using bioinformatics reveals that TcGGPPS consists of 52.2%α-helix,10.9%extended strand,8.4%β-turn and 28.5%random coil,and has a three-dimensional structure highly similar to the structurally known Sinapis alba GGPPS.In silicon predictions also demonstrate that TcGGPPS has a plastid-targeting peptide at the N-terminus,suggesting it is responsible for the synthesis of GGPP in plastids.展开更多
文摘The effect of La, Ce was firstly tested on growth of the Taxus(T.) cuspidata cell suspensions, biosynthesis and release of taxol. The results indicate that the growth pattern of T.cuspidata cells is altered significantly by adding high concentration of rare earth in the medium. The lag and exponential phases of cell growth are shortened, the stationary phase disappears and the biomass fluctuates periodically during the decline phase. The rare earth compounds added in the exponential phase obviously increase the taxol biosyntheis and release yields of T.cuspidata cells, and the supplement of carbon source in the medium containing rare earth is also favorable to taxol biosynthesis.
文摘The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nuclear DNA (nDNA) into oligonucleosomal fragments(DNA laddering) was a characteristic of apoptosis, which involved cell shrinkage, condensation of cytoplasm and tracheary elements differentiation. Terminal deoxynucleotidy transferase-mediated dUTP nick end in situ labeling (TUNEL) assay of Taxus spp. cells showed that fungal extractive or abiotic elicltors (Ce4+, Taxol, H2O2) induced TUNEL positive. Also, the increase of the apoptotic cell ratio was accompanied by the increase of secondary metabolites (especially Taxol). These results suggest that apoptosis may have some coincidence with biosynthesis of Taxol. The implication of apoptosis for the production of secondary metabolites in plant cell cultures is discussed.
文摘Conditions have been established for the callus initiation and subculture of T chinensis. The calliwere induced by the explants cultured first on the medium MS supplemented with 1 .0 mg/L 2,4-D, 2g/L CH, and 25g/L sucrose, then on The medium f MS+1 .0 mg/L NAA+0.5 mg/L BA+2 g/L CH+25 g/L sucrose. When the callus was Subcultured and tamed several times, it could grow fast and stable on the medium : MS+0.2mg/L 2,4-D+0.5mg/L NAA+0.5 mg/L BA+2 g/LCH+25 g/L sucrose. The contamination of explants was a result of endophytic microbes of T Chinensis. This could be avoided by adopting the tender shoots 3-5 cm long collected in early spring as the source of explants. The browning of the Cultures could be prevented and controlled by means of the selection of a suitable explants, hormonal regime in the medium, culture methods and the use of antioxidants.
基金supported by the ‘‘12th Five Year Plan’’ National Science and Technology in Rural Area(Nos.2013AA103005-04 and 2012AA10A506-04)Changchun City Science and Technology Development Program(No.2014174)+1 种基金Changchun City Science and Technology Support Program(No.2014NK002)Graduate Innovation Fund of Jilin University(No.2016172)
文摘In this study to screen for stable, high Taxolproducing cell lines（CL5, CL12, and CL21） of Taxus cuspidata, stem tissues were used to induce calli, which were then subcultured nine times to establish suspension cell cultures. From 97 cell lines obtained from conditioned cultures, 10 cell lines with high Taxol content were selected. Stability analyses on solid and liquid B5 media were then used to obtain lines that stably produced high levels of Taxol. Fresh biomass and Taxol production of the ninth generation became stable. Taxol content of selected CL5, CL12, and CL21 samples was 0.0448, 0.0477, and0.0428% of dry mass（DW）, respectively. Proliferation of CL5, CL12 and CL21 was 346.3, 382.5, and 409.2%,respectively. From work over about 2 years, the three cell lines appear suitable for mass production of Taxol,promoting the industrialisation and commercial-scale production of Taxol using cell culture.
文摘The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of 'partition' and 'bifurcation' were observed in studying the dynamic effect of Ce4+ on soluble protein synthesis and cell activity. That is, Ce4+ of low concentration improves the soluble protein synthetic strength and cell activity, while Ce4+ of high concentration is harmful to protein synthesis and cell activity. In addition, Ce4+ of appropriate concentration enhances taxol synthesis.
文摘Taxol(Paclitaxel)is a diterpene from Taxus species and has been used in treatment of various kinds of cancers.Geranylgeranyl diphosphate synthase(GGPPS)catalyzes the formation of geranylgeranyl diphosphate(GGPP,the common precursor for diterpenes and plays a key role in taxol biosynthesis.Here we report a functional GGPPS gene from Taxus chinensis(designated TcGGPPS).TcGGPPS is an intron free gene and has a 1,182-bp open reading frame encoding a polypeptide of 393 amino acid residues with a calculated molecular mass of 42.63 kDa and an isoelectric point of 5.58.The catalytic activity of TcGGPPS for production of GGPP was verified by a color enhancement assay in the Escherichia coli cells harboring plasmid pAC-BETA.Multiple sequence alignment indicates that TcGGPPS is a little different in sequence from the functional GGPPS genes from other Taxus species such as T.canadensis,T.media and T.wallichiana,which are almost identical to each other.Protein structure prediction by using bioinformatics reveals that TcGGPPS consists of 52.2%α-helix,10.9%extended strand,8.4%β-turn and 28.5%random coil,and has a three-dimensional structure highly similar to the structurally known Sinapis alba GGPPS.In silicon predictions also demonstrate that TcGGPPS has a plastid-targeting peptide at the N-terminus,suggesting it is responsible for the synthesis of GGPP in plastids.
