научная статья по теме SCREENING OF TAXOL-PRODUCING ENDOPHYTIC FUNGI FROM TAXUS CHINENSIS VAR. MAIREI Химия

Текст научной статьи на тему «SCREENING OF TAXOL-PRODUCING ENDOPHYTIC FUNGI FROM TAXUS CHINENSIS VAR. MAIREI»

ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2007, том 43, № 4, с. 490-494

УДК 582.28

SCREENING OF TAXOL-PRODUCING ENDOPHYTIC FUNGI FROM Taxus chinensis var. mairei © 2007 г. X. Zhou*, Z. Wang**, K. Jiang**, Y. Wei**, J. Lin**, X. Sun**, K. Tang***

*Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU- Nottingham Plant Biotechnology

R&D Center, Shanghai Jiao Tong University, Shanghai 200030, China; e-mail: khang1@yahoo.com **State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU- Nottingham Plant Biotechnology

R&D Center, Fudan University, Shanghai 200433, China Received June 30, 2006

A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the tax-ol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive strains, separated by column chromatography and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compounds-producing endophytic fungi which can improve prominently screening efficiency.

Since 1993, observations of taxol-producing fungi, Taxomyces andreanae and Pestalotiopsis microspora, have been reported, demonstrating that organisms other than Taxus sp. could produce taxol [1-3]. A worldwide scientific effort to isolate endophytes and study their natural products is now underway [4]. In the past few decades, the scientists had succeeded in isolation of endophytic tax-ol-producing fungi from various yew and its related plants [5-8]. In the process of separation and selection of taxol-producing fungi, many of the scientists used the similar method to isolate fungi. The initial step was that endophytic fungus was successfully separated from plant materials. Then, the taxol-producing fungi were identified among the separated endophytic fungus. Finally, the isolation and characterization of bioactive substances from fungal cultures were done using bioassay guided fractionation and spectroscopic methods. However, extraction and analysis of bioactive substance was a complex physical and chemical process, which included dealing and analysis of fungal sample by various means, and finally detected the final product in the fungal cultures [9-11].

The primarily separation of endophytic fungus from plant materials were comparatively simple operational process, but the detected processes of taxol-producing en-dophytic fungus were the laborious and uneconomic. Consequently, the objectives of this study were to develop an efficient protocol for screening taxol-producing fungi from diverse endophytic fungus with the ultimate purpose of reducing workloads. In order to explore a simple and efficient protocol for screening of taxol-producing endophytic fungi, the taxus taxadiene synthase gene-specific PCR primers were used to screen 38 diverse isolates. This research laid a foundation for further development of

new molecular markers in screening of taxol-producing fungi.

MATERIALS AND METHODS

Sources of yew materials. In this study, we used the yew, Taxus chinensis var. mairei, collected at Jinyun Mountain, Chongqing, Southwest China, grown at an altitude ofabout 800-1000 m.

Preparation of media. The following media were prepared and used in the experiment: 1. Solid medium: the reformative potato dextrose agar (PDA), containing (g l-1): potato - 200, dextrose - 20, MgSO4 ■ 7H2O - 1.5, KH2PO4 - 2.5, VBi - 10 mg l-1, agar - 20, and Czapek medium that composed (g l-1): NaNO3 - 2.0, K2HPO4 -1.0, MgSO4- 7H2O - 0.5, FeSO4 - 0.01, sucrose - 30, agar - 20.0, were used in primary colony isolation of endophytic fungi. 2. Liquid medium: seed medium was prepared with (g l-1) glucose - 40, peptone - 10.0, sodium acetate - 1.0, sodium benzoate - 100 mg l-1, and distilled water of 1.0 l, with natural pH value, and sterilized at 121°C for 20 min. Fermentation medium was prepared as in literature [12]. 3. The modified YPS (yeast extract, peptone and sucrose) liquid medium, containing (g l-1): sucrose -20.0, peptone - 20.0, yeast extract - 5.0, MgSO4 - 1.0, KH2PO4 - 1.0, pH 6.8, were used cultured mycelia for extracting DNA.

Chemicals. The reference chemical substance of taxol, 10-Deacetyl Baccatin III (10-DAB) and Baccatin III are purchased from Sigma (USA). Other chemicals used were reagent grade.

Isolation and identification of endophytic fungi.

The plant materials included the bark pieces, branch nods

and seeds. The microorganisms were isolated as follows: the yew bark was cut into pieces (0.5 x 0.5 x 0.5 cm); the branch was cut into 0.5 cm, the seeds were kept in origin status. The plant materials were designed to be treated with different disinfectants, 75% (v/v) ethanol, or 2.5% sodium hypochlorite, or 0.1% mercuric chloride, and then washed with sterilized water.

The outer bark of the sample was removed with a sterilized sharp blade. Small pieces of inner bark, short burl of branch nods and seed were placed on the surface of PDA medium in Petri plates. The three materials were planted in Petri plates, respectively. There are four pieces planting in every plate. The primary bourgeoning rates from origin materials were calculated as the ratio of the primary growing colony of endophytic fungi to the planting numbers of origin materials.

After several days, fungi were observed growing from the inner bark fragments in the plates. Individual hyphal tips of the various fungi were removed from the agar plates and placed on new PDA medium, and incubated at 25°C for at least 2 weeks. Each fungal culture was checked for purity and transferred to another agar plate by the hyphal tip method [3, 6]. Fungi identification was based on the morphology of the fungal colony, the mechanism of spore production and the characteristics of the spores [13, 14].

Primary screening based on PCR amplification. The DNA extraction from endophytic fungi was as follow programs: 2 agar blocks (ф 9 mm) were obtained from the margins of actively growing hypha and inoculated into a 250 ml Erlenmeyer flask containing 50 ml of YPS liquid medium. Cultures were maintained on a rotary shaker at 150 rpm at 25°C for 36 h and harvested by centrifuged at 5000 g for 15 min. After washed twice with asepsis water, the mycelia were collected by centrifuged at 3000 g for 15 min. 1-2 g of mycelia were ground into powder in liquid nitrogen (N2) using a pre-chilled mortar and pestle. DNA was then isolated from mycelia following the cetyl-trimethyl ammonium bromide method as described by literature [15]. The quality and concentration of DNA samples were examined by agarose gel electrophoresis and spectrophotometer analysis.

Subsequent step was screening oftaxol-producing fungi based on PCR amplification. Based on the conserved sequence of the ts (Gene Bank: AY364469), the specific primers TsF (5'-CAAACC CATGTCGAAT-TGAGAAG-3') and TsR (5'-CAAGTTTGCATA-CACTCTGGAATCT-3') were designed and synthesized. The PCR amplification was performed in a PTC-100TM programmable (MJ Research, INC) for 32 cycles (94°C for 50 s, 55°C for 1 min, 72°C for 80 s) followed by extension for 7 min at 72°C. The PCR products were purified using Gel Extraction Mini Kit (Watson, China), ligated to pMD18-T vectors (TaKaRa, China), transformed into E. coli strain DH5a and then sequenced with DYEnamic Direct dGTP Sequencing Kit (Amersham Pharmacia, England) and a 373A DNA sequencer.

Determination of taxol-producing fungi by HPLC-MS. The fermentation procedure was described in reference [12]. The fungus was inoculated into 500 ml Erlen-

ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ

meyer flask containing 100 ml liquid medium and cultured with 150 rpm shaking at 25°C for about 15 days. Then the mycelia were harvested by filtration and refrigeration-dried at room temperature.

Dried mycelia of various fungal strains were crushed and extracted with ethanol for 3 times. The fermentation liquid was extracted with ethyl acetate for 3 times. The ethanol extract and the ethyl acetate extracts were concentrated using reduced pressure method, respectively. The 2 kinds of liquid so formed were suspended in ethanol, respectively. The taxol and its related compounds were detected using High Performance Liquid Chromatography coupled with Mass Spectrometry (HPLC-MS).

The separation conditions for liquid chromatography were as follows: an RP-C18 column was used; the mobile phase was composed of methyl alcohol (A) and water (B) with binary solvent-delivery gradient elution (0-35 min, 50%-100% A, 50%-0% B, volume fraction); the sample size was 10 ^l, the flow rate was 0.6 ml/min and the UV absorbance detection was set at 230 nm. The post-column effluent was directly detected under UV lights and mass spectrometry detector. Flow out substance through UV detector entered in electro-spray ionization room.

The mass spectrometry conditions were as followings, the determination mode was scan mode; ion-dipole was positives ion. Ionization mode was Electro Spray Ioniza-tion (ESI); molecular weight distribution was from 50 to 2300 Da. Ions of monitor had H+, Na+ and K+ under 350°C of drying gas temperature respectively. Nebulizer gas flow rate was 10 l/min; the voltage of collision-induced dissociation was 70 V and the capillary voltage was 3.0 kV.

RESULTS AND DISCUSSION

Isolation and identification of fungi isolates. A total of 38 isolates were isolated from Taxus chinensis var. mairei. The results of the prim

Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.

Показать целиком