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Method for producing vindoline

A technology of Wendolin and polyploidy, applied in plant cells, fermentation, etc., can solve the problems of failure to realize industrial production, and achieve the effect of short cultivation period and low production cost

Inactive Publication Date: 2010-08-11
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the 1960s, foreign countries began to study periwinkle tissue culture to produce indole alkaloids. From the end of the 1970s, the research of producing vinca alkaloids with this has become a hot spot in the field of bioengineering, but so far, it has not been able to produce indole alkaloids. The two anti-cancer alkaloids obtained from suspension culture cells can only produce amarine, serpentine and vinblastine, and industrial production has not been realized

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1, utilize periwinkle cell large-scale culture to produce Wenduolin

[0017] 1. Culture of periwinkle cell line:

[0018] 1) Obtaining periwinkle callus

[0019] Use the young stems of periwinkle as explants, sterilize the surface with 70% ethanol for 30-50 seconds, put them into 5-15% sodium hypochlorite solution for secondary sterilization for 10-20 minutes, and then rinse them with sterile water three times. Cut the young stems into 2 mm long stems in the ultra-clean workbench, inoculate them into the dedifferentiation medium, and cultivate them under 2000 lx, 12-16 hours of light, and room temperature of about 25 degrees for 3-4 weeks to obtain white cured stems. damage tissue. Then put the callus in step 2) and subculture for more than 3 times in the subculture acclimation medium, and at the same time screen to obtain a cell line with fast growth rate, soft texture and stable growth amount.

[0020] Preparation of dedifferentiation medium: in MS mediu...

Embodiment 2

[0034] Embodiment 2, utilize periwinkle cell large-scale culture to produce Wenduolin

[0035] 1. Culture of periwinkle cell line:

[0036] 1) Obtaining periwinkle callus

[0037] Use the internodes of periwinkle as explants, sterilize the surface with 70% ethanol for 30-50 seconds, put them into 5-15% sodium hypochlorite solution for secondary sterilization for 10-20 minutes, and then rinse them with sterile water three times. Cut the internodes into 2 mm long stems and inoculate them into the dedifferentiation medium in an ultra-clean workbench, and culture them at 2000 lx, 12-16 hours of light, and room temperature of about 25 degrees for 3-4 weeks to obtain white calluses organize. Then put the callus in step 2) and subculture for more than 3 times in the subculture acclimation medium, and at the same time screen to obtain a cell line with fast growth rate, soft texture and stable growth amount.

[0038] Preparation of dedifferentiation medium: in MS medium, add indole ...

Embodiment 3

[0052] Embodiment 3, utilize periwinkle cell large-scale culture to produce Wenduolin

[0053] 1. Culture of periwinkle cell line:

[0054] 1) Obtaining periwinkle callus

[0055] Use the leaves of periwinkle as explants, sterilize the surface with 70% ethanol for 30-50 seconds, put them into 5-15% sodium hypochlorite solution for secondary sterilization for 10-20 minutes, then rinse them with sterile water three times, take them out and put them in In the ultra-clean workbench, cut the leaf veins into small pieces of 0.5 cm square and inoculate them in the dedifferentiation medium, and cultivate them at 2000 lx, 12-16 hours of light, and at room temperature of about 25 degrees for 3-4 weeks, and then white callus can be obtained. organize. Then put the callus in step 2) and subculture for more than 3 times in the subculture acclimation medium, and at the same time screen to obtain a cell line with fast growth rate, soft texture and stable growth amount.

[0056] Preparatio...

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PUM

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Abstract

The invention discloses a method for producing vindoline. The method comprises: inoculating a periwinkle polyploid cell strain in a synthetic medium, and obtaining a cell rich in vindoline by cultivating the strain for 3 to 7 days at the temperature between 20 and 28DEG C; and the synthetic medium is a liquid culture medium obtained by adding 0 to 4mg / L of plant cell auxin, 0 to 4mg / L of plant cytokinin, 30 to 60g / L of sucrose, 10 to 40mu g / L of acetyl coenzyme A, 0.5 to 1.5mu mol / L of Benzotriazole methl, 5 to 30mu mol / L of tranylcypromine, 10 to 40mu l / L of acetic anhydride and 10 to 40mg / Lof dithiothreitol in a minimal medium. The method using periwinkle cells to cultivate and produce vindoline can stably realize the industrialized production of vindoline, and the production cost is rather low, the culture period is short, and the production is insusceptible to the natural environment and weather, so the year-round production can be realized.

Description

technical field [0001] The invention relates to a method for producing ventolin, in particular to a method for producing ventolin by using large-scale cultivation of plant cells and metabolic regulation technology. Background technique [0002] Periwinkle (Catharanthus roseus (L) G. Don) is a genus with rich alkaloid content in Apocynaceae. Vinca alkaloids mainly include: Aspicospermatan type such as Vindoline (Vindoline) etc., Ibogan type such as Vinblastine (Catharanthine) etc., Corynanthean type such as Ajmalicine (Ajmalicine) and Serpentine (Serpentine) etc. and Bisindoles type such as Vinblastine and Vincristine etc. In the mid-1950s, some scholars found that the extract of periwinkle plants also had the effect of lowering white blood cells when studying the hypoglycemic effect of periwinkle plants. In 1958, Canadian Noble and Beer jointly isolated this active ingredient and named it Vinblastine. (vinblastine) and prepared its sulfate. Subsequently, Lilly Company of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04C12P17/18
Inventor 郭志刚刘云
Owner TSINGHUA UNIV
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