阅读说明：本技术 一种可提取获得巨大戟醇的续随子愈伤组织细胞悬浮培养方法 (Euphorbia lathyris callus cell suspension culture method capable of extracting ingenol ) 是由 李丕睿 陈雨 冯煦 黄佳楠 刘飞 王碧 印敏 单宇 王奇志 管福琴 田梅 徐曙 于 2019-09-07 设计创作，主要内容包括：本发明提供了一种可提取获得巨大戟醇的续随子愈伤组织细胞悬浮培养的方法,包括以下步骤：1)将续随子无菌苗的根部作为外植体,接入愈伤组织诱导培养基中进行培养,获得愈伤组织；2)愈伤组织切分成小块接入继代培养基中进行继代培养；3)将愈伤组织夹碎接种到悬浮培养的液体培养基中,进行振荡培养；4)悬浮培养更新培养液进行继代培养；5)收集悬浮细胞为原料,经分离制备可获得巨大戟醇和续随子二萜醇。应用本方法获得的续随子悬浮细胞增殖速度快,可稳定地从悬浮细胞中提取获得巨大戟醇和续随子二萜醇,为续随子规模化细胞培养及巨大戟醇生物合成工业化生产提供技术支撑,具有重要的应用价值。(The invention provides a method for suspension culture of euphorbia lathyris callus cells capable of extracting and obtaining ingenol, which comprises the following steps: 1) taking the root of the euphorbia lathyris aseptic seedling as an explant, inoculating the root into a callus induction culture medium for culture to obtain callus; 2) cutting the callus into small pieces, and inoculating the small pieces into a subculture medium for subculture; 3) crushing the callus, inoculating the crushed callus into a liquid culture medium for suspension culture, and performing shaking culture; 4) performing subculture on the suspension culture renewal culture solution; 5) collecting suspension cells as raw materials, and separating to obtain ingenol and euphorbia lathyris diterpene alcohol. The euphorbia lathyris suspension cell obtained by the method has high proliferation speed, can stably extract and obtain ingenol and euphorbia lathyris diterpene alcohol from the suspension cell, provides technical support for scale cell culture of euphorbia lathyris and industrial production of euphorbia lathyris alcohol biosynthesis, and has important application value.)

1. A euphorbia lathyris callus cell suspension culture method capable of extracting ingenol is characterized by comprising the following steps:

1) Taking the root of the euphorbia lathyris aseptic seedling as an explant, cutting the root into 1-1.5 cm long, inoculating the root into a callus induction culture medium for culture, wherein the culture temperature is 24-26 ℃, the illumination intensity is 1800-2000 lx, and the illumination time is 12 h/d. Inducing callus for 20-25 days;

2) selecting light yellow loose callus, cutting into 0.5cm ² small blocks, inoculating into a subculture medium for subculture, subculturing once every 20-25 d, and continuously subculturing for 4-5 times to obtain the callus with vigorous growth, small particles and looseness, wherein the subculture conditions of the callus are the same as the induction culture conditions;

3) The callus of the subculture is crushed and inoculated into a cell suspension culture medium, the inoculation amount is 50-60 g/L, the shaking culture condition is that the rotating speed is 100-120 r/min, the culture temperature is 24-26 ℃, the illumination intensity is 1800-2000 lx, and the illumination time is 12 h/d;

4) And (5) carrying out subculture on the suspension culture solution every 12-14 days, wherein the subculture condition is unchanged.

5) collecting suspension cells as raw materials, and extracting and separating to obtain ingenol and euphorbia lathyris diterpenoid alcohol.

2. the method for culturing euphorbia lathyris callus cell suspension capable of extracting ingenol according to claim 1, wherein the culture method comprises the following steps: the callus induction culture medium in the step 1) is a solid culture medium, and the formula of the solid culture medium is as follows: MS culture medium +1.5 mg/L2, 4-D +0.5 mg/L6-BA +30g/L sucrose +7g/L agar, and pH value is 5.8.

3. The method for culturing euphorbia lathyris callus cell suspension capable of extracting ingenol according to claim 1, wherein the culture method comprises the following steps: the callus subculture medium in the step 2) is a solid medium, and the formula of the solid medium is as follows: MS culture medium +1.0 mg/L2, 4-D +0.3 mg/L6-BA +40g/L sucrose +8g/L agar, and pH value is 5.8.

4. The method for culturing euphorbia lathyris callus cell suspension capable of extracting ingenol according to claim 1, wherein the culture method comprises the following steps: the callus cell suspension culture medium in the step 3) is a liquid culture medium, and the formula of the liquid culture medium is as follows: MS culture medium +1.0 mg/L2, 4-D +0.3 mg/L6-BA +200mg/L hydrolyzed casein +40g/L sucrose, and pH value is 5.8.

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a euphorbia lathyris callus cell suspension culture method capable of extracting and obtaining ingenol.

background

Euphorbia lathyris (Euphorbia lathyris L.) is a biennial herb plant of Euphorbiaceae (Euphorbiaceae), the original Europe is widely distributed in China, the oil content of seeds of Euphorbia lathyris is up to 45 percent, and the Euphorbia lathyris has the potential of replacing petroleum as a new energy source and is an ideal energy source plant. Meanwhile, the dried seeds (moleplant seed) of euphorbia lathyris is a traditional Chinese medicine in China, and has the effects of breaking blood and resolving masses, and expelling water and reducing swelling. Modern medical research finds that the euphorbia lathyris extract also has the effects of resisting tumors, cancers, whitening and the like. In recent years, researchers at home and abroad discover pharmacological active ingredients such as anticancer and anti-AIDS from euphorbia lathyris, wherein ingenol, an ingenol diterpene compound, has important value and is a raw material for producing ingenol methyl butenoate by industrial semisynthesis. However, ingenol is only distributed in trace amounts in a few euphorbia plants such as euphorbia lathyris, the yield of extraction and separation from euphorbia lathyris is only 0.03%, which is about 17 times lower than the yield of direct separation of artemisinin from plants, so the cost of semi-synthesis of finally obtaining ingenol mebutate is also high. In 2013, LEO pharmaceutical company combined with the American Stipropus research institute researches a 14-step total synthesis method of ingenol, but the method depends on a chiral catalyst, and the yield is only about 1%, so that the total synthesis process is difficult, the cost is high, and the industrial production is difficult.

The plant cell suspension culture is to suspend the plant callus cell mass in a liquid culture medium for oscillation culture, so that the small cell mass is always in a dispersed suspension state, and the small cell mass is favorably and uniformly contacted with a culture solution. The cell suspension culture has the advantages of rapid growth, large propagation scale, uniform and controllable products and the like, and is widely applied to the industrial production of plant secondary metabolites. In the process of plant cell suspension culture, the composition of a culture medium and culture conditions are important factors influencing the cell growth and metabolism, and in order to obtain specific cell metabolites, establishing a proper stable cell suspension culture system is important basic work. At present, the suspension culture technology of euphorbia lathyris callus cells is not perfect enough, and the synthesis regulation effect on secondary metabolites is not ideal, so that the cell suspension culture method capable of stably extracting and obtaining target products is researched by adjusting and optimizing the ratio of each component in a culture medium when euphorbia lathyris cells and other secondary metabolites are produced by suspension culture.

Disclosure of Invention

The invention aims to provide a method for suspension culture of euphorbia lathyris callus cells capable of extracting and obtaining ingenol.

In order to achieve the purpose, the invention provides the following technical scheme:

A euphorbia lathyris callus cell suspension culture method capable of extracting and obtaining ingenol comprises the following steps of 1) taking the root of euphorbia lathyris aseptic seedlings as explants, cutting the roots into 1-1.5 cm long, inoculating the euphorbia lathyris callus into a callus induction culture medium for culture, wherein the culture temperature is 24-26 ℃, the illumination intensity is 1800-2000 lx, and the illumination time is 12 h/d.20-25 d to induce callus, 2) selecting light yellow loose callus, cutting the callus into 0.5cm ² small blocks, inoculating the small blocks into a subculture medium for subculture, subculturing every 20-25 d for 4-5 times of continuous subculture, obtaining callus with vigorous growth, small and loose terpene, wherein the subculturing condition of the callus is the same as the induction culture condition, 3) clamping and inoculating the subculturing callus into a cell suspension culture medium, wherein the inoculation amount is 50-60 g/L, the rotation speed is 100-120 r/min, the culture temperature is 24-26 ℃, the illumination intensity is 2000 x, the subculturing liquid is 12 x, and the euphorbia lathyris are prepared and the raw materials are subjected to suspension culture for 14 h and are separated and cultured in suspension culture medium for 14-5 d.

The callus induction culture medium in the step 1) is a solid culture medium, and the formula of the solid culture medium is as follows: MS culture medium +1.5 mg/L2, 4-D +0.5 mg/L6-BA +30g/L sucrose +7g/L agar, and pH value is 5.8.

The callus subculture medium in the step 2) is a solid medium, and the formula of the solid medium is as follows: MS culture medium +1.0 mg/L2, 4-D +0.3 mg/L6-BA +40g/L sucrose +8g/L agar, and pH value is 5.8.

The callus cell suspension culture medium in the step 3) is a liquid culture medium, and the formula of the liquid culture medium is as follows: MS culture medium +1.0 mg/L2, 4-D +0.3 mg/L6-BA +200mg/L hydrolyzed casein +40g/L sucrose, and pH value is 5.8.

The suspension cells in the step 4) are used as raw materials, and ingenol and euphorbia lathyris diterpenoid alcohol can be prepared by extraction and separation.

The invention has the beneficial effects that: the invention provides a euphorbia lathyris callus cell suspension culture method capable of extracting and obtaining euphorbia lathyris alcohol.

Detailed Description

The present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.