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Molecule identification method for taxol-producing endophytic fungi in yew
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A technology for endophytic fungi and paclitaxel, which is applied in the field of molecular biology and can solve the problems of many reagents, large workload, and complicated extraction process of paclitaxel.
Inactive Publication Date: 2012-08-29
SHANGHAI JIAOTONG UNIV
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However, there are still some defects to be overcome urgently in the above-mentioned work flow of adopting the TLC method for the initial confirmation of paclitaxel-producing bacteria, such as: large fermentation volume; complicated paclitaxel extraction process; many reagents for solvent extraction; Extraction, decompression concentration and other steps; and the sensitivity of TLC detection is low, in the case of less paclitaxel content in the strain, TLC is not easy to detect, etc.
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Embodiment 1
[0033] PCR detection of paclitaxel produced by endophytic fungi isolated from Taxus media
[0034] 1. Culture of mycelia
[0035] Put the isolated and purified endophytic fungus slant strains stored in a refrigerator at 4°C for activation at 28°C for 24 hours, pick the hyphae of the strains from the slant test tubes and connect them to the PDA solid medium plate, and culture them at a constant temperature of 28°C for 2-3 days. Inoculate 1cm with an inoculation shovel 2 The left and right plate bacteria were connected to the YPS liquid medium and mashed. Culture at 28°C with shaking at 120rpm.
[0036] 2. Mycelium Preparation
[0037] Centrifuge the cultured mycelium at 10,000×g for 10 min, remove the supernatant, then add PBS buffer solution to wash the precipitate for 3 times, and finally grind the washed mycelium into powder with liquid nitrogen.
[0038] 3. Mycelia Genomic DNA Extraction
[0039] Add 65°C preheated extraction buffer to 1g of the material, incubate in a...
Embodiment 2
[0073] Using HPLC-MS to further analyze whether endophytic fungi produce paclitaxel
[0074] 1. Experimental method:
[0075] (1) Extraction and separation of paclitaxel and taxanes
[0076] Inoculate endophytic fungi (preserved on PDA slant) isolated from Taxus chinensis in 250mL Erlenmeyer flask seed solution, shake at 25°C for 3 days, transfer to 500mL Erlenmeyer flask fermentation broth at 10% inoculum size, shake at 25°C After culturing for 15 days, mycelium and supernatant were obtained by centrifugation. The fermentation broth was centrifuged, and the supernatant was rotatably evaporated at 55°C to one tenth of its volume, then an equal volume of ethyl acetate was added for extraction for 3 to 5 hours; after precipitation, an equal volume of ethyl acetate was added for extraction for 3 to 5 hours; the supernatant and Each precipitate was extracted 3 times, and the ester layers were combined respectively. The ester layer was rotary evaporated to dryness at 45°C, respe...
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Abstract
The invention discloses a molecular identification method in the molecular biology technical field. The detection method comprises the following steps that: endophytic fungi are separated from yew and are preserved and cultivated; mycelium of the cultivated endophytic fungi is scrubbed, precipitated and ground into powder; DNA of the endophytic fungi is extracted and purified; PCR augmentation, electrophoretic detection of a PCR product and the determination of a special gene sequence are performed; whether a strain produces taxol is distinguished by the fact that whether a special band with the size of 650bp can be augmented in the endophytic fungi; and the reliability of the result is further confirmed through a chemical detection program. The method performs simple, quick and accurate preliminary identification that whether the special gene sequence produces the taxol to the endophytic fungi, which lays the foundation for further research and development and use.
Description
technical field [0001] The invention relates to a molecular identification method in the field of molecular biology, in particular to a molecular identification method for paclitaxel-producing endophytic fungi in Taxus chinensis. Background technique [0002] Paclitaxel (Pacitaxel, trade name taxel) is the most promising anticancer drug after doxorubicin and cisplatin. It is a diterpene compound with four rings of A, B, C and D as the core skeleton. Potent cytotoxin. At present, paclitaxel is mainly derived from the bark of the yew plant (Taxus), and its content is only 0.01%-0.05%. Due to excessive logging, yew resources are decreasing day by day, and have been listed as protected plants by the country. Therefore, seeking new resources of paclitaxel or its substitutes has become the focus of current research. According to reports, Montana State University plant pathologist Strobel and chemist Stierle reported for the first time in 1993 that an endophytic fungus that could...
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