Sub-cellular localization and overexpressing analysis of hydroxylase gene TcCYP725A22 of Taxus chinensis / 生物工程学报

The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725A22 (GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane. Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725A22 is likely involved in the biosynthetic pathway of taxol.

Molecular cloning and characterization of 1-deoxy-D-xylulose 5-phosphate synthase gene from Taxus chinensis / 中草药

Objective To obatin the key enzymes of 1-deoxy-D-xylulose 5-phosphate synthase (DXS) in taxol biosynthetic pathway from Taxus chinensis (TcDXS), and carry out the bioinformatics analysis, tissue profile, subcellular localization, and functional complementation assay. Methods RACE technologies were used to obtain the full length cDNA of TcDXS for the bioinformatics analysis. Semi-quantitative PCR was used to detect the gene expression levels in different parts of T. chinensis. The localization and function of TcDXS were carried out by subcellular localization and functional complementation assay. Results The full-length cDNA of TcDXS was 3 031 bp containing a coding sequence of 2 229 bp encoding a 742-amino-acid residues which was predicted to have a molecular weight of 79 400 and an isoelectric point of 7.99. The qRT-PCR results showed that the highest expression level of TcDXS was detected in petioles and followed by leaves and barks. However, the expression of TcDXS was very low in roots and stems. What’s more, functional complementation assay results showed that the E. coli, co-transformed with PAC-BETA and pTrcTcDXS, was changed to orange. Conclusion TcDXS was considered to play an essential role in the control of taxol biosynthesis and provided a target for the metabolic engineering of taxol production and plant molecular breeding in T. chinensis.

Taxus chinensis ameliorates diabetic nephropathy through down-regulating TGF-β1/Smad pathway / 中国天然药物

Diabetic nephropathy (DN) is one of the common microvascular complications of diabetes mellitus. Renal fibrosis is closely related to the deterioration of renal function. The present study aimed to investigate protective effect of Taxus chinensis on high-fat diet/streptozotocin-induced DN in rats and explore the underlying mechanism of action. The rat DN model was established via feeding high fat diet for 4 weeks and subsequently injecting streptozotocin (30 mg·kg body weight) intraperitoneally. The rats with blood glucose levels higher than 16.8 mmol·L were selected for experiments. The DN rats were treated with Taxus chinensis orally (0.32, 0.64, and 1.28 g·kg) once a day for 8 weeks. Taxus chinensis significantly improved the renal damage, which was indicated by the decreases in 24-h urinary albumin excretion rate, blood serum creatinine, and blood urea nitrogen. Histopathological examination confirmed the protective effect of Taxus chinensis. The thickness of glomerular basement membrane was reduced, and proliferation of mesangial cells and podocytes cells and increase in mesangial matrix were attenuated. Further experiments showed that Taxus chinensis treatment down-regulated the expression of TGF-β1 and α-SMA, inhibited phosphorylation of Smad2 and Smad3. These results demonstrated that Taxus chinensis alleviated renal injuries in DN rats, which may be associated with suppressing TGF-β1/Smad signaling pathway.

Taxus chinensis ameliorates diabetic nephropathy through down-regulating TGF-β1/Smad pathway / 中国天然药物

Diabetic nephropathy (DN) is one of the common microvascular complications of diabetes mellitus. Renal fibrosis is closely related to the deterioration of renal function. The present study aimed to investigate protective effect of Taxus chinensis on high-fat diet/streptozotocin-induced DN in rats and explore the underlying mechanism of action. The rat DN model was established via feeding high fat diet for 4 weeks and subsequently injecting streptozotocin (30 mg·kg body weight) intraperitoneally. The rats with blood glucose levels higher than 16.8 mmol·L were selected for experiments. The DN rats were treated with Taxus chinensis orally (0.32, 0.64, and 1.28 g·kg) once a day for 8 weeks. Taxus chinensis significantly improved the renal damage, which was indicated by the decreases in 24-h urinary albumin excretion rate, blood serum creatinine, and blood urea nitrogen. Histopathological examination confirmed the protective effect of Taxus chinensis. The thickness of glomerular basement membrane was reduced, and proliferation of mesangial cells and podocytes cells and increase in mesangial matrix were attenuated. Further experiments showed that Taxus chinensis treatment down-regulated the expression of TGF-β1 and α-SMA, inhibited phosphorylation of Smad2 and Smad3. These results demonstrated that Taxus chinensis alleviated renal injuries in DN rats, which may be associated with suppressing TGF-β1/Smad signaling pathway.

Genetic diversity of taxus chinensis var. mairei from fujian based on rapd markers / 中草药

Objective To reveal the genetic diversity of provenances and populations of Taxus chinensis var. mairei in Fujian province from 23 orgins, providing certain theoretical foundation for the preservation and protection. Methods RAPD molecular marker technology was used to study the genetic diversity of T. chinensis var. mairei in Fujian province. Results The number of observed allele was 1.974 4, the number of effective alleles was 1.603 8, the average value of the Nei’s genetic diversity was 0.351 3 and the Shannon’s information index was 0.522 8. And there existed more accurate information on genetic relationship, diversity among different geographical provenances. The genetic distance of those 23 species ranged from 0.104 to 0.620 7, the genetic coefficients of 23 varieties ranged from 0.401 7 to 0.786 3. The genetic distance of seven T. chinensis var. mairei of different populations ranged from 0.021 0 to 0.379 4, the genetic coefficients ranged from 0.684 3 to 0.979 2. According to Nei’s method calculation of T. chinensis var. mairei, those seven different populations’ genetic diversity was Dst = 0.108 8, differentiation index Gst = 0.369 0, and gene flow coefficient Nm = 0.855 0. 36.9% of the total genetic variation existed among populations, the variation within populations was only 63.1%, and analysis showed high differentiation existed in provenances. Conclusion There is a certain heritable variation among different provenances of T. chinensis var. mairei, but there still exists more accurate information and high similarity coefficients among different populations.

Content Comparison of Taxol in Different Parts of Taxus Madia and Taxus Chinensis var. Mairei / 中国药师

Objective:To compare the taxol content in different parts of Taxus madia and Taxus chinensis var. mairei to provide reference for the future researches. Methods:The active ingredients were extracted by a mixed solvent (50% acetone and 50% ethyl acetate) , the target compound taxol was separated by a neutral Al2 O3 solid phase column, and then the taxol content was analyzed and compared among the different parts of Taxus madia and Taxus chinensis var. mairei by HPLC. Results:Taxol in velamen of Taxus ma-dia had the highest content among all the samples, which was 0. 0439% and 88 times as high as that in the bark of Taxus chinensis var. mairei. Conclusion:Among all the samples of Taxus madia and Taxus chinensis var. mairei, velamen of Taxus madia has the highest taxol content.

Isolation of endophytic fungi from Taxus chinensis var. mairei Cheng et L. K and its antimicrobial activity / 中国药学杂志

OBJECTIVE: To isolate the endophytic fungi from medicinal plant Taxus chinensis var. mairei Cheng et L. K in Guangxi Zhuang autonomous region, and investigate its antimicrobial activity. METHODS: Endophytic fungi were isolated by the tissue separation method. Staphylococcus aureus, Escherichia coli, and Candida albicans were used as the indicators for human pathogenic microbes. Antimicrobial tests of the isolated endophytic fungi were carried out by agar block and disc diffusion. RESULTS: Among 34 strains of endophytic fungi isolated from roots, stems, and leaves of T. chinensis var. mairei, eight strains (23.53% of the total isolated strains) showed antimicrobial activity against one or more of three human pathogenic microbes. CONCLUSION: Diverse antimicrobial natural products exist in endophytic fungi from T. chinensis var. mairei, which can be the resources for new antimicrobial products.

Cloning and expression analysis of TcAPLs in Taxus chinensis / 中草药

Objective: To clone altered phloem development (APL) genes from Taxus chinensis and reveal their potential regulatory role in tissues regeneration after bark girdling by investigating the expression profiles of these APLs. Methods: The full-length three APL genes were isolated using reverse transcription-polymerase chain reaction (RT-PCR) and were named as TcAPL1, TcAPL2, and TcAPL3, respectively. The expression profiles of these genes in different tissues and at different regeneration stages after bark girdling were analyzed by semi-quantitative RT-PCR and quantitative real-time PCR (qRT-PCR), respectively. Results: Phylogenetic tree analysis suggested that TcAPL1 and TcAPL2 could be clustered together with APL protein of Morus notabilis, which are closest in genetic relationship; TcAPL3 could be clustered with APL proteins of another big independent branch. The analysis of gene expression patterns in different tissues showed that the transcript abundances of TcAPL1 and TcAPL2 were mainly expressed in the roots, stems, leaves, and phloem with cambium; While TcAPL3 was higher in the leaves than that in the roots and xylem with cambium. Through analysis of the expression patterns in regeneration tissues after bark girdling, the mRNA expressions of TcAPL1 and TcAPL2 showed a up-down trend in the following periods and were found to decrease notably at 36 d after bark girdling, while the expression of TcAPL3 was repressed at all stages after bark girdling. Conclusion: In this study, three TcAPLs genes are cloned from T. chinensis, and their expressions are regulated in the regeneration processes after bark girdling. Our results demonstrate that APL might play a regulatory role in tissue regeneration after bark girdling in T. chinensis.

Tissue culture and analysis on taxane diterpenoids in callus of Taxus chinensis var. mairei / 中草药

Objective: To investigate some factors which could influence the tissue culture of callus from Taxus chinensis var. mairei such as the culturing callus time, callus induction rate, callus development rate, growth rate, and browning degree. Then, the five taxane diterpenoids including paclitaxel in callus were studied and compared. Methods: The factors which influence the T. chinensis var. mairei callus induction and growth rate were studied by single factor analysis and orthogonal test design. The five taxane diterpenoids including paclitaxel in callus from different sources were determined by HPLC analysis. The weighted score for each of the above factors mentioned would be taken account to optimise the tissue culture conditions. Results: Among the explants, the stems intacted leaves would exhibit the best ability of dedifferentiation to form callus. The enrichment dark culture was conducive to paclitaxel and other five kinds of terpene constituents. The most optimum culture medium of the callus induction was B5 + 2, 4-D 3.0 mg/L + 6-BA 1.0 mg/L + NAA 1.0 mg/L + KT 0.5 mg/L. Conclusion: The callus culture conditions for T. chinensis var. mairei have indicated greater association with different types of the explantation, nutrient medium, and light illumination. The five kinds of enrichment taxane paclitaxel diterpenoids would be significantly affected by the type and concentration of the plant growth regulators. This study has shown some references value for T. chinensis var. mairei tissue culture and for the screening of high-yielding cell lines.

Isolation, Identification and Bioactivity Assays of Endophytic Bacteria Associated with Taxus chinensis / 微生物学通报

Two endophytic-bacteria isolates of G18 and F19 were isolated from the stem of Taxus chinensis. The G18 and F19 were respectively classified into Psudomonas sp. and Stenotrophomonas sp. based on biological characteristics and 16S rDNA sequence analysis. The bioactivity analysis showed that the fermented broths of the G18 and F19 exhibited antagonistic activities against three pathogenic bacteria, and had good antagonistic effectiveness to Verticillium dahliae and Colletotrichum gloeosporioides, respectively. The G18 can degrade salicylic acid, and the F19 can do dichlorvos.

Studies on chemical constituents of planted Taxus mairei (III) / 药学学报

From the chloroform-soluble fraction of the ethanol extracts of the whole plant of Taxus chinensis var. mairei (Lemee et Levl), four compounds were isolated by using repeated column chromatography on silica gel and Sephadex LH-20. Based on spectroscopic data (UV, IR, ESI-MS, 1H NMR and 13C NMR), the compounds were identified as taxamairin K (1), 2α, 4α-dideacetoxy-7β-benzoyloxy-5β,20-epoxy-9α,10β,13α,15-tetrahydroxy-11(15→1)abeotaxa-11-ene (2), 7β-xylosyl-taxol (3), 10-deacetoxy-7-xylosyl-taxol (4). Among them, taxamairin K is a new compound.

Anti-tumor activity of extracts from cultured cells of Taxus chinensis on cancer cell lines SMMC-7721 and HEp-2 / 中国药理学通报

AIM To analyze the toxicity and inhibitory mechanism of extracts from cultured cells (F 4 cell line) of Taxus chinensis on cancer cell lines SMMC 7721 and HEp 2. METHODS MTT assay for cell viability and flow cytometry for cell cycle analysis. RESULTS IC 50 of SMMC 7721 and HEp 2 were 0 161 4 g DCW?L -1 and 0 275 6 g DCW?L -1 respectively,tumor cells in G 2～M stage all increased with higher concentration and longer incubation of extracts from Taxus chinensis cells. CONCLUSION Extracts from cultured cells of Taxus chinensis could have cytotoxic effect on SMMC 7721 and HEp 2 and could induce apoptosis of both two cancer cells.

Extract from cultured cells of Taxus chinensis on proliferation inhibition and apoptosis induction of tumor cells / 中草药

Object To study the inhibitory effect and mechanism of extract from cultured cell of Taxus chinensis (Pilg.) Rehd. on hepatocarcinoma cell line SMMC 7721. Methods Trypan blue stain assay was used to determine tumor cell growth curve, flow cytometry and Giemsa staining were used to analyze cell cycle and cell apoptosis. Results Inhibitory effect happened on tumor cell line SMMC 7721, G 2/M stage of tumor cells increased with the addition of T. chinensis cell extract by carrene and apoptosis happened. Conclusion The extracts from cultured cell of T. chinensis have inhibitory effect on SMMC 7721 cells and can further induce cell apoptosis.

Cell metabolism of Taxus chinensis var. mairei in suspension cultures treated with methyl jasmonate / 中草药

Object To study the physiological changes of Taxus chinensis var. mairei (Lemee et L?vl.) Cheng et L. K. Fu in the case of methyl jasmonate (MJ). Methods TTC assay, soluble protein measurement and enzyme analysis were used. Results It was observed that MJ inhibited Taxus cell growth in the view of primary metabolism. MJ induced phenylalanine ammonia lyase(PAL) activity while it inhibited polyphenoloxidase (PPO) activity. Extracellular phenolic content after addition of MJ increased to the maximum at the three days than that of the control group. Conclusion It was demonstrated that MJ induced the transition of Taxus cell from primary metabolism to secondary metabolism. This is favorable for secondary metabolism of Taxus cells. It is important to study the physiology of Taxus cells for revealing the mechanism of MJ.

Effect of explant source from different maternal tree and medium composition on callus growth and paclitaxel yield of Taxus chinensis var. mairei / 中草药

Objective To establish and optimize the technology and method of producing large quantity and high-paclitaxe yield callus of 〖WTBX〗Taxus chinensis var. mairei. Methods Wild maternal tree grown in Lingchuan County of Shanxi Province and cultivated tree grown in Xi′an were used as explant source. And the optimum maternal tree for explant cutting, optimum explant type, basic medium, composition and concentration of growth regulators in medium and so on, which were factors of affecting on callus induction, growth and paclitaxe yield, were examined in a series order. Results The juvenile stem segments were the optimum explants because of their earlier and higher rate callus induction than that of other explants. Medium Y5: MS+2,4-D 4.0 mg/L+KIN 1.0 mg/L or medium B5 Ⅲ: B5+2,4-D 3.0 mg/L+KIN 0.1 mg/L+Phe 0.1 mol/L was confirmed optimum callus induction medium in which callus induction rate had reached to 100%. In callus subculture medium, lower concentration of 2,4-D (0.5—3 mg/L) always increased callus growth, but higher concentration of 2,4-D (8 mg/L) reduced callus growth. When 2,4-D concentration was suitable, callus grown on B5 medium displayed lighter browning and faster tissue growth than that on MS medium. Further more, HPLC analysis confirmed that the paclitaxel yield in callus grown on medium MSⅢ was highest and had reached 0.004% of callus dry weight. In a general condition, the level of paclitaxel in calli derived from juvenile stems of wild maternal tree was higher than that in calli initiated from cultivated maternal tree's juvenile stems. Conclusion The optimization sequence of obtaining a large quantity and high-paclitaxe yield callus of T. chinensis var. mairei are dividing juvenile stem segments from wild maternal tree in May and culturing on medium Y5 or B5 Ⅲ for callus induction. After the calli having been subcultured on the same medium for 8—10 generations, one or two generations are recultured on medium MSⅢ. Finally, the calli with more paclitaxel are obtained by extracting paclitaxel out of it.

Effects of fed-batch carbohydrate, nitrogen, phosphorus on growth of Taxus chinensis var. mairei cell during suspension culture / 中草药

Object To solve the problem that Taxus chinensis var. mairei (Lemee et L?vl.) Cheng et L. K.Fu grows slowly. Methods The consumption of phosphorus during the cell suspension culture and the effect of fed-batch carbohydrate, nitrogen and phosphorus on cell growth and living-cell activity were assayed. Results The carbohydrate was exhausted in the middle phase of suspension culture, dificiency of carbohydrate led to inhibition of cell growth in the late culture. Conclusion Compared with the control group, the cell growth rate and the cell density in the fed-batch carbohydrate group were increased significantly, and the cell growth rate was up to 83%.