Isolation and Identification of a Taxol-producing Endophytic Fungus Identified from Taxus media

[Objective] The aim was to isolate and identify a taxol-producing endophytic fungus from Taxus media. [Method] 32 strains of endophytic fungi were identified form the inner bark of T. media,and their fermentation products were detected by high performance liquid chromatography (HPLC). [Result] Through the screening,a strain of taxol-producing endophytic fungi M57 was obtained,which could produce 45-50 μg/L of taxol,and M57 was defined as Rhizopus sp. through morphological observation and 18S rDNA sequence analysis. [Conclusion] The finding of Rhizopus sp. M57 provided a promising strain for producing taxol with taxol-producing fungi fermentation process.

A new 3, 11-cyclotaxane diterpenoid from Taxus media Rehd. cv. Hicksii

The aim of current study was to investigate the chemical constituents of the needles of Taxus media. The isolation and purification of components were achieved by a series of chromatography including silica gel, Sephadex LH-20, MCI and reversed- phase HPLC. Their structures were indentified based on 1D, 2D NMR, and mass spectral analysis. Seven taxane diterpenoids were isolated and identified as ll3,2tt,9ct,1013-tetrahydroxy-5ct-cinnamoyloxy-3,11-cyclotaxa-4（20）-en-13-one （1）, 2it, 7[3, 9ct, 1013, 13tt-pentaacetoxytaxa-4（20）, 11-dien-Sct-ol （2）, 2-deacetoxy-5-decinnamoyltaxinine J （3）, taxuspine F （4）, taxuspinanane K （5）, taxchin A （6）, and baccatin IV （7）. Among them, 1 is a new compound and compounds 2, 5, 6 and 7 are isolated from Taxus media for the first time.

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.

Taxoids from the Needles of Taxus×media Hicksii

A new taxane diterpenoid, named 7-deacetoxy taxinine J, together with ten knowncompounds were isolated from the needles of Taxus × media Hickiii. The structures of allcompounds were elucidated and identified by spectroscopic methods. T'he structure of 7-deacetoxytaxinine J was further confirmed by X-ray crystallographic analysis. The structure of the knowncompound, taxinine E, was also revised.

Smashing Tissue Extraction and HPLC Determination of Paclitaxel and 10-Deacetylbaccatin from Taxus x media

Objective To optimize the extraction technology of Taxus x media by using the contents of Paclitaxel and 10-deacetylbaccatin(10-DAB) ,two representative active diterpene alkaloids of taxane type from T.x media,as evaluation standard.Methods The smashing tissue extraction(STE) of Paclitaxel and 10-DAB from T.x media,was investigated by comparing with ultrasonic extraction(UE) which was one of the modern technologies of extraction.Results STE was more efficient than UE,and the contents of 10-DAB and Paclitaxel in the extracts obtained by STE were higher than those by UE.Conclusion STE is a fast,high-performance,and energy-saving technology for the extraction of diterpene alkaloids of taxane type.STE also provides a simple,component-safe,workable,and highly efficient method for the extraction of active natural product.

基质含沙量和移栽时间对曼地亚红豆杉幼苗生长影响

为提高曼地亚红豆杉(Taxus×media Rehd.)钵生商品苗的移栽植成活率和种苗质量,在大田培育曼地亚红豆杉幼苗。选用曼地亚红豆杉大棚苗床扦插苗,采用在同一时间移栽在不同含沙量的基质中和在不同时间将苗木移栽在含沙量相同的基质中2种方法,研究基质含沙量和移栽时间对曼地亚红豆杉幼苗成活率和生长量的影响。结果表明:在本田土和鸡粪混合基质配方(体积比)中添加10%、30%、50%沙子和不添加沙子这4种基质培育曼地亚红豆杉苗木,以及2月、3月、4月、10月、11月、12月这6个月中移栽曼地亚红豆杉大棚苗床扦插苗,其成活率和生长量均达到极显著差异;经多重比较,成活率和生长量最好的配方是添加了30%沙子的基质,最佳移栽时间是2月和12月。由此可见,利用繁育基地本田土作基质原材料,添加30%沙子的配方基质,保水性和透气性均达到适中水平;曼地亚红豆杉大棚苗床扦插苗移栽应选择在2月和12月进行;可在本区域推广应用。