Taxus kok-saghyz for producing paclitaxel, method for improving yield of paclitaxel and precursor substances thereof in Taxus kok-saghyz and application

Abstract

The invention provides kok-saghyz for producing paclitaxel, a method for improving the yield of paclitaxel and precursor substances thereof in kok-saghyz and application, and belongs to the technical field of planting, and the kok-saghyz for producing paclitaxel is obtained by planting kok-saghyz. According to the method, kok-saghyz is planted under the condition of phosphorus stress, so that the yield of paclitaxel and a precursor substance of paclitaxel in kok-saghyz is increased; according to the application, the 10-deacetylbaccatin III and / or the paclitaxel is obtained by extracting the 10-deacetylbaccatin III and / or the paclitaxel from the kok-saghyz. According to the method, taxol in kok-saghyz is detected, it is found that the kok-saghyz contains a certain amount of taxol and other related components, and it is further found that by applying a phosphorus-containing culture solution to kok-saghyz, the content of taxol and the content of taxol precursor baccatin in kok-saghyz are greatly increased; and a solid theoretical foundation and technical support are laid for replacing the kok-saghyz with the existing taxol extraction source plant.

Description

Technical Field

[0001] The present invention belongs to the technical field of planting, and particularly relates to a Taraxacum kok-saghyz producing paclitaxel, a method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz, and an application thereof. Background Art

[0002] Taraxacum kok-saghyz ( Taraxacum kok-saghyz ) is a plant of the genus Taraxacum. Its roots can produce a natural rubber, so it is considered as an alternative crop to rubber trees. Taraxacum kok-saghyz is also an edible plant. Its leaves can be eaten or used as livestock feed. In addition, the roots of Taraxacum kok-saghyz, like most dandelion plants, have medicinal value and can clear heat and detoxify, disperse swelling and dissipate nodules. In recent years, the anti-tumor effect of dandelion has also been widely explored, but its anti-tumor mechanism is not very clear. Whether Taraxacum kok-saghyz, as a kind of dandelion, has anti-tumor medicinal value has not been explored. As one of the most successful anti-cancer drugs in the world, the demand for paclitaxel far exceeds the supply.

[0003] At present, the source of paclitaxel mainly comes from the extraction of Taxus and related plant materials. However, the extraction efficiency is low. And because the growth cycle of Taxus and other trees is relatively long, the extraction of paclitaxel causes great damage to the survival of Taxus. On the other hand, paclitaxel can be synthesized by chemical methods, but it still highly depends on the supply of the precursor substance baccatin extracted from Taxus or related cell cultures. Therefore, it is urgent to find and develop a new alternative plant rich in paclitaxel and increase its paclitaxel yield to meet the growing market demand for paclitaxel in the world. Summary of the Invention

[0004] In view of the above problems, the present invention provides a Taraxacum kok-saghyz producing paclitaxel, a method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz, and an application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows: A Taraxacum kok-saghyz producing paclitaxel, which is obtained by planting Taraxacum kok-saghyz; The Taraxacum kok-saghyz producing paclitaxel produces paclitaxel.

[0006] Further, the Taraxacum kok-saghyz is planted under phosphorus stress conditions.

[0007] Further, the phosphorus content in the planting environment of the Taraxacum kok-saghyz is ≥0 mg, preferably ≥1.3 mg, and most preferably ≥8 mg.

[0008] An application of the above Taraxacum kok-saghyz producing paclitaxel, which is to extract 10-deacetylbaccatin III and / or paclitaxel from the Taraxacum kok-saghyz producing paclitaxel to obtain 10-deacetylbaccatin III and / or paclitaxel.

[0009] A method for increasing the yield of taxol and its precursor substances in Taraxacum kok-saghyz, the method is to plant Taraxacum kok-saghyz under the condition of phosphorus stress to increase the yield of taxol and its precursor substances in Taraxacum kok-saghyz; The precursor substance is 10-deacetylbaccatin III.

[0010] Furthermore, the method is to plant Taraxacum kok-saghyz and apply phosphate fertilizer to increase the yield of taxol and its precursor substances in Taraxacum kok-saghyz.

[0011] Furthermore, the amount of phosphate fertilizer applied per plant per day is ≥0 mg, preferably ≥1.3 mg, and most preferably ≥8 mg; The application of phosphate fertilizer starts after Taraxacum kok-saghyz germinates and grows for 0.5 - 2 months; The types of phosphate fertilizers include but are not limited to: at least one of phosphorus-containing culture solution, potassium dihydrogen phosphate, and phosphate fertilizer.

[0012] Furthermore, the method is to plant Taraxacum kok-saghyz in soil applied with phosphate fertilizer to increase the yield of taxol and its precursor substances in Taraxacum kok-saghyz.

[0013] Furthermore, the soil applied with phosphate fertilizer is to apply ≥0 mg of phosphate fertilizer in the soil, preferably ≥1.3 mg, and most preferably ≥8 mg.

[0014] The types of phosphate fertilizers include but are not limited to: at least one of phosphorus-containing culture solution, potassium dihydrogen phosphate, and phosphate fertilizer.

[0015] An application of Taraxacum kok-saghyz, the application is to extract 10-deacetylbaccatin III and / or taxol from Taraxacum kok-saghyz obtained by the above method to obtain 10-deacetylbaccatin III and / or taxol.

[0016] The beneficial effects of a taxol-producing Taraxacum kok-saghyz, a method for increasing the yield of taxol and its precursor substances in Taraxacum kok-saghyz, and the application of the present invention are as follows: By detecting taxol in Taraxacum kok-saghyz, the present invention finds that Taraxacum kok-saghyz contains a certain amount of taxol and other related components. Further, by exploring the influence of phosphorus element content on the production of taxol by Taraxacum kok-saghyz, it is found that when the phosphorus content in the planting environment (such as the culture medium) is low, the taxol content in Taraxacum kok-saghyz is also very low, while when the phosphorus content in the planting environment (such as the culture medium) increases, the taxol content in Taraxacum kok-saghyz increases significantly; finally, by applying a phosphorus-containing culture solution to Taraxacum kok-saghyz cultivated in soil, the contents of taxol and its precursor baccatin in Taraxacum kok-saghyz are greatly increased, laying a solid theoretical foundation and technical support for Taraxacum kok-saghyz to become an alternative to existing taxol extraction source plants. Brief Description of the Drawings

[0017] Figure 1In Example 1 of the present invention, Taraxacum kok-saghyz plants grown for 16 days in a phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate and a phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate; among them, on the left is the Taraxacum kok-saghyz planted in the phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate, and on the right is the Taraxacum kok-saghyz planted in the phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate; the overall plant of the Taraxacum kok-saghyz on the left is slightly smaller, with shorter roots, and at the same time, the color of the stem and leaf parts and the roots shows a purplish red color, possibly containing anthocyanins; the overall plant of the Taraxacum kok-saghyz on the right is larger, with longer roots, and the color of the stem and leaf parts and the roots is greener; Figure 2 It is the content of inorganic phosphorus in the Taraxacum kok-saghyz plants grown in a phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate and a phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate in Example 1 of the present invention; among them, the content of inorganic phosphorus in the Taraxacum kok-saghyz plants cultured in the phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly higher than that in the Taraxacum kok-saghyz plants cultured in the phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate; Figure 3 It is the content of paclitaxel in the Taraxacum kok-saghyz plants grown in a phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate and a phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate in Example 1 of the present invention; among them, the content of paclitaxel in the Taraxacum kok-saghyz plants cultured in the phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly higher than that in the Taraxacum kok-saghyz plants cultured in the phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate; Figure 4 It is the content of anthocyanins in the Taraxacum kok-saghyz plants grown in a phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate and a phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate in Example 1 of the present invention; among them, the content of anthocyanins in the Taraxacum kok-saghyz plants cultured in the phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly lower than that in the Taraxacum kok-saghyz plants cultured in the phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate; Figure 5 It is the phenotype of 2-month-old Taraxacum kok-saghyz seedlings grown in soil irrigated with a phosphorus-free 1 / 2 MS culture solution containing 10 μM potassium dihydrogen phosphate and a phosphorus-free 1 / 2 MS culture solution containing 600 μM potassium dihydrogen phosphate in Example 2 of the present invention; among them, the leaves of the Taraxacum kok-saghyz irrigated with the phosphorus-free 1 / 2 MS culture solution containing 600 μM potassium dihydrogen phosphate for 1 month are significantly larger than those of the Taraxacum kok-saghyz treated with the phosphorus-free 1 / 2 MS culture solution containing 10 μM potassium dihydrogen phosphate for 1 month; Figure 6It is the contents of 10 - deacetylbaccatin III and paclitaxel in Taraxacum kok - saghyz Rodin plants grown in soil irrigated with a phosphorus - free 1 / 2MS culture medium containing 10 μM potassium dihydrogen phosphate and a phosphorus - free 1 / 2MS culture medium containing 600 μM potassium dihydrogen phosphate in Example 2 of the present invention. Among them, the contents of 10 - deacetylbaccatin III and paclitaxel in Taraxacum kok - saghyz Rodin plants watered with the phosphorus - free 1 / 2MS culture medium containing 600 μM potassium dihydrogen phosphate for 1 month are significantly higher than those in Taraxacum kok - saghyz Rodin plants watered with the phosphorus - free 1 / 2MS culture medium containing 10 μM potassium dihydrogen phosphate for 1 month. Detailed implementation manners

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0019] The phosphorus - free 1 / 2MS medium was purchased from Beijing Coolaber Technology Co., Ltd., and potassium dihydrogen phosphate was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0020] Example 1 Effect of planting environment on the yields of paclitaxel and precursor substances in Taraxacum kok - saghyz Rodin In this example, Taraxacum kok - saghyz Rodin seeds were planted in culture media with different phosphorus concentrations to investigate the effect of phosphorus content on the yields of paclitaxel and precursor substances in Taraxacum kok - saghyz Rodin, as follows: Take a phosphorus - free 1 / 2MS medium, and add 10 μM and 600 μM potassium dihydrogen phosphate respectively for standby; Take Taraxacum kok - saghyz Rodin seeds, disinfect them with 20% 84 disinfectant for 15 minutes, and rinse them 6 times with distilled water. Sow the obtained disinfected seeds in a phosphorus - free 1 / 2MS medium containing 10 μM potassium dihydrogen phosphate and a phosphorus - free 1 / 2MS medium containing 600 μM potassium dihydrogen phosphate respectively, place them in an incubator at 26 °C (light 16 h / dark 8 h), take pictures after culturing for 16 days, as Figure 1 shown, and then take samples as test tissues respectively to detect the contents of inorganic phosphorus, anthocyanin and paclitaxel in them. The inorganic phosphorus contents in Taraxacum kok - saghyz Rodin plants grown in culture media with different phosphorus concentrations are shown in Figure 2 , the paclitaxel contents are shown in Figure 3 , and the anthocyanin contents are shown in Figure 4 . It can be seen from Figure 1 that the overall plants of Taraxacum kok - saghyz Rodin cultured in the phosphorus - free 1 / 2MS medium containing 10 μM potassium dihydrogen phosphate are slightly smaller, with shorter roots. At the same time, the colors of the stems, leaves and roots show purplish red, and may contain anthocyanin. While the overall plants of Taraxacum kok - saghyz Rodin cultured in the phosphorus - free 1 / 2MS medium containing 600 μM potassium dihydrogen phosphate are larger, with longer roots, and the colors of the stems, leaves and roots are greener. FromFigure 2 It can be seen that the inorganic phosphorus content in Taraxacum kok-saghyz Rodin cultured in a phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly higher than that in Taraxacum kok-saghyz Rodin cultured in a phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate. From Figure 3 It can be seen that Taraxacum kok-saghyz Rodin contains paclitaxel, and the paclitaxel content in Taraxacum kok-saghyz Rodin cultured in a phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly higher than that in Taraxacum kok-saghyz Rodin cultured in a phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate, indicating that increasing the phosphorus addition amount in the medium can significantly increase the paclitaxel content in Taraxacum kok-saghyz Rodin. At the same time, from Figure 4 It can be seen that the anthocyanin content in Taraxacum kok-saghyz Rodin cultured in a phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly lower than that in Taraxacum kok-saghyz Rodin cultured in a phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate.

[0021] In summary, it can be seen that by increasing the phosphorus content in the planting environment (such as applying phosphate fertilizer), the inorganic phosphorus content and paclitaxel content in the planted Taraxacum kok-saghyz Rodin can be effectively increased, while the anthocyanin content is reduced.

[0022] Among them, the detection method for the inorganic phosphorus content is as follows: Take potassium dihydrogen phosphate and add purified water to prepare an inorganic phosphorus standard solution with a concentration of 1 mmol / L.

[0023] Take 0.1 g of the tissue to be tested, add 1 mL of distilled water, homogenize thoroughly on ice, and then centrifuge at 4 °C and 10000 rpm for 10 min. Take the supernatant as the test solution.

[0024] Respectively take the inorganic phosphorus standard solution and the test solution, detect the absorbance of inorganic phosphorus in them by the molybdenum blue method, calculate the inorganic phosphorus content in the test solution by the external standard single-point method, and then calculate the inorganic phosphorus content in the tissue to be tested.

[0025] The detection method for the paclitaxel content is as follows: Take paclitaxel and add methanol to prepare standard curve solutions with final concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, and 10000 ng / mL.

[0026] Respectively perform liquid chromatography-tandem mass spectrometry detection on each standard solution, obtain the chromatogram corresponding to each standard solution and the paclitaxel peak area in the chromatogram, and use the concentration of paclitaxel in each standard solution and its corresponding paclitaxel peak area to draw the standard curve of paclitaxel.

[0027] Place the tissue to be tested in liquid nitrogen for grinding and crushing. Accurately weigh 1 g of the obtained sample, add 10 mL of methanol, ultrasonicate in an ice-water bath for 30 min, centrifuge at 11000 rpm for 30 min to obtain a supernatant and a precipitate; add 5 mL of methanol to the precipitate for extraction again, combine the supernatants, add 70 mg of C18 (octadecylsilane-bonded silica gel) and 40 mg of GCB (graphitized carbon black), centrifuge at 4 °C and 5000 rpm for 10 min, take the supernatant, dry it under nitrogen, then add 400 μL of methanol for reconstitution, and filter through a 0.22 μm organic phase filter membrane to obtain the test solution.

[0028] Take the test solution for liquid chromatography-tandem mass spectrometry detection to obtain the chromatogram of the test solution and the peak area of paclitaxel contained therein. Substitute the peak area of paclitaxel into the standard curve of paclitaxel to calculate the content of paclitaxel in the test solution, and then calculate the content of paclitaxel in the tissue to be tested.

[0029] Among them, the chromatographic conditions for liquid chromatography-tandem mass spectrometry detection are as follows: The chromatographic column is waters ACQUITY UPLC BEH HSS T3 1.8um 2.1╳100mm; The column temperature is 30 °C; Mobile phase A is an aqueous solution of 0.1% formic acid, and mobile phase B is acetonitrile; The flow rate is 0.3 mL / min; The injection volume is 1 μL; The elution mode of liquid chromatography is gradient elution, and the specific elution program is as follows: 0 - 2 min, 99% mobile phase A, 1% mobile phase B; 2 - 6 min, 99% → 0% mobile phase A, 1% → 100% mobile phase B; 6 - 8 min, 0% mobile phase A, 100% mobile phase B; 8 - 8.1 min, 0% → 99% mobile phase A, 100% → 1% mobile phase B; 8.1 - 10 min, 99% mobile phase A, 1% mobile phase B.

[0030] The mass spectrometry conditions are as follows: The capillary voltage is 3 kV, the source compensation electrophoresis is 50 V, the ion source temperature is 150 °C, the desolvation temperature is 500 °C, the cone hole gas flow is 150 L / Hr, and the desolvation gas flow is 1000 L / Hr.

[0031] The polarity of the detected paclitaxel is +, the parent ion (m / z) is 854.37, and the daughter ions (m / z) are 286.2 / 509.1, the declustering voltage is 10 V, and the collision energy is 18 / 17 V. Among them, Represent the quantitative ions.

[0032] The detection method for the total anthocyanin content is as follows: Determination principle: Anthocyanins are red in acidic solutions, and the depth of their color is proportional to the concentration of anthocyanins. The absorption peak wavelength of the anthocyanin acidic solution is 530 nm, and the molar extinction coefficient is 4.62×10 4 , so the spectrophotometric method can be used to determine its content. However, chlorophyll often exists in some extracts, interfering with the determination. Therefore, it is necessary to simultaneously measure the optical density values of the extract at wavelengths of 620 nm (soluble sugar) and 650 nm (absorption value of chlorophyll), and accurately calculate the optical density value of anthocyanins using the Greey formula before calculating the content of anthocyanins.

[0033] Experimental steps: a. Sample extraction Weigh 0.05 - 1 g of the tissue to be tested and place it in a centrifuge tube. Add 10 mL of 0.1 mol / L hydrochloric acid ethanol solution, tighten the tube mouth, and extract in a water bath at 60 °C for 30 min. Pour the extract into a 25 mL volumetric flask, then add another 5 mL of 0.1 mol / L hydrochloric acid ethanol solution and extract for 15 min. Pour the extract into the 25 mL volumetric flask, and then add another 5 mL of 0.1 mol / L hydrochloric acid ethanol solution and extract for 15 min. Pour the extract into the 25 mL volumetric flask. The total extraction time is 1 h. Rinse the residue and make up the volume to 25 mL to obtain the test solution.

[0034] b. Content determination Using 0.1 mol / L hydrochloric acid ethanol solution as the reference solution, take the test solution and measure the optical density values of the extract at wavelengths of 530 nm, 620 nm, and 650 nm with a spectrophotometer, calculate the optical density value of anthocyanins, and then calculate the content of anthocyanins.

[0035] Example 2 Effect of planting environment on the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz In this example, different amounts of phosphate fertilizer were added during the process of planting Taraxacum kok-saghyz seeds to investigate the effect of phosphate fertilizer on the yields of paclitaxel and its precursor substances, as follows: Take phosphorus-free 1 / 2MS culture solution and add 10 μM and 600 μM potassium dihydrogen phosphate respectively for standby; Directly sow Taraxacum kok-saghyz seeds in moist soil. After germination and growth for 1 month, continuously irrigate and culture with 10 μM potassium dihydrogen phosphate phosphorus-free 1 / 2MS culture solution and 600 μM potassium dihydrogen phosphate phosphorus-free 1 / 2MS culture solution for 1 month respectively. Irrigate 100 mL per plant per day, and the daily irrigation amount per plant is 60 μmol (equivalent to 8 mg of potassium dihydrogen phosphate), and take pictures as Figure 5As shown, the roots and above-ground parts were separately taken to detect the contents of 10-deacetylbaccatin III and paclitaxel, and the results are as Figure 6 shown.

[0036] It can be Figure 5 seen that the leaves of Taraxacum kok-saghyz Rodin irrigated with a phosphorus-free 1 / 2MS culture solution containing 600 μM potassium dihydrogen phosphate for 1 month were significantly larger than those of Taraxacum kok-saghyz Rodin treated with a phosphorus-free 1 / 2MS culture solution containing 10 μM potassium dihydrogen phosphate for 1 month.

[0037] It can be Figure 6 seen that the contents of 10-deacetylbaccatin III and paclitaxel in Taraxacum kok-saghyz Rodin irrigated with a phosphorus-free 1 / 2MS culture solution containing 600 μM potassium dihydrogen phosphate for 1 month were significantly higher than those of Taraxacum kok-saghyz Rodin irrigated with a phosphorus-free 1 / 2MS culture solution containing 10 μM potassium dihydrogen phosphate for 1 month, indicating that the application of phosphate fertilizer can increase the contents of 10-deacetylbaccatin III and paclitaxel in Taraxacum kok-saghyz Rodin.

[0038] In summary, it can be seen that during the cultivation of Taraxacum kok-saghyz Rodin, increasing the application rate of phosphate fertilizer can promote the growth of Taraxacum kok-saghyz Rodin and increase the contents of 10-deacetylbaccatin III and paclitaxel in Taraxacum kok-saghyz Rodin.

[0039] Among them, the detection method for the content of paclitaxel is the same as that in Example 1; The detection method for the content of 10-deacetylbaccatin III is as follows: 10-deacetylbaccatin III was taken and added to methanol to prepare standard curve solutions with final concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, and 10000 ng / mL.

[0040] Each standard solution was separately subjected to liquid chromatography-tandem mass spectrometry detection to obtain the chromatogram corresponding to each standard solution and the peak area of 10-deacetylbaccatin III in the chromatogram, and a standard curve of 10-deacetylbaccatin III was plotted using the concentration of 10-deacetylbaccatin III in each standard solution and its corresponding peak area of 10-deacetylbaccatin III.

[0041] Place the tissue to be tested in liquid nitrogen for grinding and pulverization. Accurately weigh 1 g of the obtained sample, add 10 mL of methanol, ultrasonicate in an ice-water bath for 30 min, centrifuge at 11000 rpm for 30 min to obtain a supernatant and a precipitate; add 5 mL of methanol to the precipitate for extraction again, combine the supernatants, add 70 mg of C18 (octadecylsilyl-bonded silica gel) and 40 mg of GCB (graphitized carbon black), centrifuge at 4 °C and 5000 rpm for 10 min, take the supernatant, dry it under nitrogen, then add 400 μL of methanol for reconstitution, and filter through a 0.22 μm organic phase filter membrane to obtain the test solution.

[0042] Take the test solution for liquid chromatography-tandem mass spectrometry detection to obtain the chromatogram of the test solution and the peak area of 10-deacetylbaccatin III contained therein. Substitute the peak area of 10-deacetylbaccatin III into the standard curve of 10-deacetylbaccatin III to calculate the content of 10-deacetylbaccatin III in the test solution, and then calculate the content of 10-deacetylbaccatin III in the tissue to be tested.

[0043] Among them, the chromatographic conditions for liquid chromatography-tandem mass spectrometry detection are as follows: The chromatographic column is waters ACQUITY UPLC BEH HSS T3 1.8um 2.1╳100mm; The column temperature is 30 °C; Mobile phase A is an aqueous solution of 0.1% formic acid, and mobile phase B is acetonitrile; The flow rate is 0.3 mL / min; The injection volume is 1 μL; The elution mode of liquid chromatography is gradient elution, and the specific elution program is as follows: 0 - 2 min, 99% mobile phase A, 1% mobile phase B; 2 - 6 min, 99% → 0% mobile phase A, 1% → 100% mobile phase B; 6 - 8 min, 0% mobile phase A, 100% mobile phase B; 8 - 8.1 min, 0% → 99% mobile phase A, 100% → 1% mobile phase B; 8.1 - 10 min, 99% mobile phase A, 1% mobile phase B.

[0044] The mass spectrometry conditions are as follows: The capillary voltage is 3 kV, the source compensation electrophoresis is 50 V, the ion source temperature is 150 °C, the desolvation temperature is 500 °C, the cone gas flow is 150 L / Hr, and the desolvation gas flow is 1000 L / Hr.

[0045] The detected 10-deacetylbaccatin III has a polarity of +, a parent ion (m / z) of 545.2, and a daughter ion (m / z) of 121.2 / 527.1, and the declustering voltage is 40 V, and the collision energy is 20 / 10 V. Among them, represents the quantitative ion.

[0046] Example 3 A method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz This example is a method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, specifically as follows: I. Method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz Take Taraxacum kok-saghyz seeds and directly sow them in moist soil. After germination and growth for 1 month, apply 8 mg of potassium dihydrogen phosphate to each plant daily to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and taxol.

[0047] II. Application By separately extracting 10-deacetylbaccatin III and taxol in Taraxacum kok-saghyz, 10-deacetylbaccatin III and taxol can be obtained.

[0048] Among them, the method for extracting taxol can be to place Taraxacum kok-saghyz in a flask, add an appropriate amount of methanol, cover the flask with a stopper, place it on a constant temperature water bath, soak at 70 ± 5 °C for 12 to 20 hours, filter the soaking solution into an Erlenmeyer flask, wash the residue with methanol more than three times until the washing solution is colorless, combine the extraction solutions, and blow the extraction solutions to near dryness under nitrogen blowing conditions to obtain the product.

[0049] The method for extracting 10-deacetylbaccatin III and taxol can also be to take Taraxacum kok-saghyz, add an appropriate amount of methanol, perform ultrasonic treatment in an ice-water bath, centrifuge to obtain a supernatant and a precipitate; repeat the extraction of the precipitate with an appropriate amount of methanol once, combine the supernatants, add C18 (octadecylsilyl-bonded silica gel) and GCB (graphitized carbon black), centrifuge, take the supernatant, and dry it under nitrogen blowing to obtain the product.

[0050] Similarly, 10-deacetylbaccatin III and / or taxol can also be obtained by other extraction methods.

[0051] Example 4 A method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz This example is a method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, specifically as follows: I. Method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz Sow the seeds of Taraxacum kok-saghyz directly in moist soil. After germination and growth for 0.5 months, apply 0.1 mg of potassium dihydrogen phosphate per plant per day to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and paclitaxel.

[0052] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0053] Example 5 A method for improving the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application This example is a method for improving the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is specifically as follows: I. Method for improving the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz Sow the seeds of Taraxacum kok-saghyz directly in moist soil. After germination and growth for 2 months, apply 4 mg of potassium dihydrogen phosphate per plant per day to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and paclitaxel.

[0054] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0055] Example 6 A method for improving the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application This example is a method for improving the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is specifically as follows: I. Method for improving the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz Sow the seeds of Taraxacum kok-saghyz directly in moist soil. After germination and growth for 1 month, apply 136 mg of potassium dihydrogen phosphate per plant per day to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and paclitaxel.

[0056] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0057] Example 7 A method for improving the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application This example is a method for improving the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is specifically as follows: I. Method for improving the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz Sow the seeds of Taraxacum kok-saghyz directly in moist soil. After germination and growth for 1 month, apply 1.3 mg of potassium dihydrogen phosphate per plant per day to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and paclitaxel.

[0058] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0059] Example 8 A method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application This example is a method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is specifically as follows: I. Method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz Sow the seeds of Taraxacum kok-saghyz directly in moist soil. After germination and growth for 1 month, apply 50 mg of phosphate fertilizer per plant per day to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and paclitaxel.

[0060] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0061] Example 9 A method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application This example is a method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is specifically as follows: I. Method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz Sow the seeds of Taraxacum kok-saghyz directly in moist soil. After germination and growth for 1 month, apply 100 mg of phosphorus-containing culture solution per plant per day to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and paclitaxel.

[0062] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0063] Example 10 A method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application This example is a method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is specifically as follows: I. Method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz Sow the seeds of Taraxacum kok-saghyz directly in moist soil. After germination and growth for 1 month, apply 16 mg of phosphorus-containing culture solution per plant per day to obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.

[0064] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0065] Example 11 A method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application This example is a method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is as follows: I. Method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz Apply potassium dihydrogen phosphate equivalent to 0.1 mg per plant in moist soil, and then sow the seeds of Taraxacum kok-saghyz directly to obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.

[0066] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0067] Example 12 A method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application This example is a method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is as follows: I. Method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz Apply potassium dihydrogen phosphate equivalent to 8 mg per plant in moist soil, and then sow the seeds of Taraxacum kok-saghyz directly to obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.

[0068] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0069] Example 13 A method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application This example is a method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is as follows: I. Method for increasing the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz Apply 136 mg of potassium dihydrogen phosphate per plant in moist soil, then sow the Taraxacum kok-saghyz seeds directly to obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.

[0070] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0071] Example 14 A Method and Application for Increasing the Yields of Paclitaxel and Its Precursors in Taraxacum kok-saghyz This example is a method and application for increasing the yields of paclitaxel and its precursors in Taraxacum kok-saghyz, which are as follows: I. Method for Increasing the Yields of Paclitaxel and Its Precursors in Taraxacum kok-saghyz Apply 50 mg of phosphorus-containing culture solution per plant in moist soil, then sow the Taraxacum kok-saghyz seeds directly to obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.

[0072] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0073] Example 15 A Method and Application for Increasing the Yields of Paclitaxel and Its Precursors in Taraxacum kok-saghyz This example is a method and application for increasing the yields of paclitaxel and its precursors in Taraxacum kok-saghyz, which are as follows: I. Method for Increasing the Yields of Paclitaxel and Its Precursors in Taraxacum kok-saghyz Apply 100 mg of phosphorus-containing culture solution per plant in moist soil, then sow the Taraxacum kok-saghyz seeds directly to obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.

[0074] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0075] Example 16 A Method and Application for Increasing the Yields of Paclitaxel and Its Precursors in Taraxacum kok-saghyz This example is a method and application for increasing the yields of paclitaxel and its precursors in Taraxacum kok-saghyz, which are as follows: I. Method for Increasing the Yields of Paclitaxel and Its Precursors in Taraxacum kok-saghyz Apply 4 mg of potassium dihydrogen phosphate per plant in moist soil, then directly sow the seeds of Taraxacum kok-saghyz Rodin to obtain Taraxacum kok-saghyz Rodin with relatively high contents of 10-deacetylbaccatin III and paclitaxel.

[0076] II. Application Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz Rodin respectively to obtain 10-deacetylbaccatin III and paclitaxel.

[0077] Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A rubber grass producing paclitaxel, characterized in that: The paclitaxel-producing rubber grass is obtained by planting rubber grass; The taxol-producing rubber grass produces taxol.

2. The paclitaxel-producing rubber grass according to claim 1, characterized in that: The rubber grass is planted under phosphorus stress conditions.

3. The paclitaxel-producing rubber grass according to claim 1 or 2, characterized in that: The phosphorus content in the planting environment of the rubber grass is 0-136 mg.

4. An application of the rubber grass producing paclitaxel according to any one of claims 1 to 3, characterized in that: The application is to obtain 10-deacetylbaccatin III and / or paclitaxel by extracting 10-deacetylbaccatin III and / or paclitaxel from the paclitaxel-producing rubber grass.

5. A method for increasing the yield of paclitaxel and its precursors in rubber grass, characterized in that: The method comprises planting rubber grass under the condition of phosphorus stress to increase the yield of paclitaxel and its precursor substances in the rubber grass.

6. The method for increasing the yield of paclitaxel and its precursors in rubber grass according to claim 5, characterized in that: The method comprises planting rubber grass and applying phosphorus fertilizer to increase the yield of paclitaxel and its precursor substances in the rubber grass.

7. The method for increasing the yield of paclitaxel and its precursors in rubber grass according to claim 6, characterized in that: The amount of phosphorus fertilizer applied per plant per day is 0~136 mg.

8. The method for increasing the yield of paclitaxel and its precursors in rubber grass according to claim 5, characterized in that: The method comprises planting rubber grass in soil with phosphorus fertilizer to increase the yield of paclitaxel and its precursor substances in the rubber grass.

9. The method for increasing the yield of paclitaxel and its precursor in rubber grass according to claim 8, characterized in that: Soil fertilized with phosphorus fertilizer means that 0-136 mg of phosphorus fertilizer is applied to the soil.

10. An application of rubber grass, characterized in that: The application is to extract 10-deacetylbaccatin III and / or paclitaxel from the rubber grass obtained by the method of any one of claims 5-9 to obtain 10-deacetylbaccatin III and / or paclitaxel.

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