CN1186443C - Snow lotus cell series with stable high productive flavone for mass plant cell culture and its establishing method - Google Patents
Snow lotus cell series with stable high productive flavone for mass plant cell culture and its establishing method Download PDFInfo
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Abstract
The present invention establishes a method for a stable high yield snow lotus cell line of flavone as secondary metabolites and a culture technique thereof so that wild snow lotuses do no need to be dug. Snow lotus calluses are mutated by methods of 60Co-gamma ray irradiation, temperature mutation, ultraviolet light ray irradiation, etc. so as to increase the flavone content in snow lotus cells. When the three mutation methods are used for processing the original snow lotus cell line, a stable high yield snow lotus cell line TUIP-8 is obtained, and the flavone content is larger than 12% and is 20 to 30 times as high as natural plants. Due to the stability of genes for cell growth and flavone synthesis, the TUIP-8 realizes the continuous passage of 50 generations, and the cell culture density and the flavone content are always respectively maintained at the level of 20g (dry weight)/L and 12%. The cell line has strong adaptation capability, grows in the environment of 18 DEG C to 30 DEG C and has the optimum culture temperature range of 20 DEG C to 25 DEG C.
Description
Technical field
The present invention relates to be used for the saussurea involucrata cell line and the establishment method thereof of the stable high yield flavones of extensive culture plant cell, more specifically say so and utilize biotechnology, make the callus producer sudden change of saussurea involucrata by induced mutations, obtain to stablize the saussurea involucrata cell line and the establishment method thereof of high yield saussurea involucrata flavones, belong to bioengineering field and modern Chinese traditional medicine field.
Background technology
With the technology that the vegetable cell large-scale cultivation method is produced useful secondary substance, become an important component part of the later contemporary biotechnology of continue microbial fermentation technology and animal cell culture technology.Contain materials such as various special secondary metabolites such as alkaloid, steroidal class, terpene, flavonoid in many medicinal plants, have many important pharmaceutical uses, but, because many secondary metabolites are difficult to synthetic, and natural resource are very deficient, degradation reason under the artificial culture variety and quality brings very big restriction for clinical use and Chinese patent medicine manufacturing.Therefore, by a large amount of natural resources of Chinese medicinal materials problems that solve of cultivating of medicinal plant cell, an important research field of the modernization of Chinese medicine beyond doubt.In fact, the shikonin that antitumor drug taxol, vincaleucoblastine, vincristine(VCR), antisepsis and anti-inflammation are used etc. are cultivated production in a large number by vegetable cell greatly.Though the domestic plant cell cultures of having studied is a lot of, as Ramulus et folium taxi cuspidatae, Herba Ephedrae, Radix Rhodiolae, the Radix Astragali, Radix Panacis Quinquefolii, foxglove, the bark of eucommia, pseudo-ginseng or the like, but a large amount of culture techniques of vegetable cell are obviously slow than microorganism and animal cell culture technical development, the domestic a large amount of culture techniques of vegetable cell that also do not adopt are produced the project that medicine has been realized industrialization, and its major cause is: (1) is difficult to obtain the clone of high and stable yields.The content of purpose secondary metabolite all is lower than natural plant in many inductive plant cell, and clone is easily degenerated and lost the ability of synthesizing the purpose product gradually in the continuous passage culturing process; (2) the serious hysteresis of China's vegetable cell large scale culturing technology.
Saussurea medusa (Saussurea medusa Maxim) is a composite family phoenix hair Chrysanthemum plant, is a kind of famous and precious vegetable drug, originates between the high mountain gravel of 4800～5100 meters of height above sea level on ground such as China Gansu, Qinghai, Tibet, Xinjiang.Have effects such as dispelling cold and removing dampness, tonifying yang and enriching blood, relieving inflammation and relaxing pain, among the people be used for the treatment of rheumatic arthritis, impotence, menoxenia, tongue clothing not down, high mountain inadaptation etc.Clinical and pharmacological research proves that saussurea involucrata delays senility and treats diseases such as cerebral arteriosclerosis, ishemic stroke recently.Herba Saussureae Involueratae extract acacetin (jaceosidin) and dinatin (hisidulin) are to treatment cancer, anti-inflammatory and anti-ageingly have a tangible effect.Herba Saussureae Involueratae injection and saussurea involucrata rheumatism capsule with the saussurea involucrata development have good effect to the treatment rheumatism.But the artificial culture of saussurea involucrata fails; because predatoriness is excavated; the natural snow lotus resource is seriously deficient; and artificial chemosynthesis effective medicinal ingredients wherein can't be realized at present; therefore; for protecting national rare plant, satisfy clinical demand, a method very likely is to produce the useful secondary metabolite of saussurea involucrata by the large scale culturing of vegetable cell.
Saussurea involucrata cell line patent (the application number 00123740.3 of the Zhao Dexiu researcher of Institute of Botany, Chinese Academy of Sciences application, publication number CN1340611A) have the ability of cell growth characteristics preferably and higher biosynthesizing flavones, but still come with some shortcomings: (1) flavones content is also lower; (2) especially relatively poor to environment to the variation of temperature adaptive faculty; (3) not too be fit to cultivate, and this can limit its industrialization with the bio-reactor large scale and high density.(4) do not investigate the genetic stability of cell.The present invention then adopts several mutagenesis means, the saussurea involucrata cell line of the stable high yield saussurea involucrata flavones cultivated of having obtained infinitely to go down to posterity.
Summary of the invention
The purpose of this invention is to provide the saussurea involucrata cell line TUIP-8 that stablizes high yield secondary metabolite flavones, its preserving number is CGMCC No.0855.
Another object of the present invention provides the establishment method of this clone.
For achieving the above object, the present invention is by the following technical solutions:
Stablize the saussurea involucrata cell line TUIP-8 of high yield secondary metabolite flavones, and in (No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, 100080, phone: 010-62542758), preservation day is on December 16th, 2002, preserving number is CGMCC No.0855, the classification called after Saussurea medusa purple clone of suggestion.This saussurea involucrata cell line has following feature: color is an intense violet color, compact structure, and quality is harder, flavones content is about 12% of a dry cell weight, the culture density of cell can reach that 20g (dry weight)/more than the L, yield of flavone is generally 2.5g/L, and culture cycle was generally 7～10 days.
A kind of method of setting up the saussurea involucrata cell line of stablizing high yield secondary metabolite flavones, this method may further comprise the steps:
(1) natural plant callus inducing and cultivating;
(2) screening of callus obtains original saussurea involucrata cell line;
(3) induced mutations;
(4) screening of high and stable yields clone, go down to posterity;
(5) cultivation of high yield clone.
Described induced mutations comprises
60The mutagenesis of Co-gammairradiation, temperature mutagenesis and ultraviolet mutagenesis.Original saussurea involucrata cell line is adopted
60The mutagenesis of Co-gammairradiation is to obtain the higher saussurea involucrata cell line of yield of flavone; Right
60The mutagenic obtained saussurea involucrata cell line of Co-gammairradiation is adopted temperature mutagenesis and is promptly cultivated with high temperature and low temperature, and its gene is undergone mutation, and adapting to the needs of wide temperature range growth, and improves the ability of the synthetic secondary metabolite flavones of cell biological; Several saussurea involucrata cells are adopted the ultraviolet light irradiation, its gene is undergone mutation, to improve the ability of the synthetic secondary metabolite flavones of cell biological.
Advantage of the present invention is: the present invention is by artificial mutation, makes the callus producer sudden change of saussurea involucrata, and the saussurea involucrata cell line of the stable high yield saussurea involucrata flavones cultivated of obtaining infinitely to go down to posterity.This clone (1) yield of flavone is big, and flavones content accounts for about 12% of dry cell weight, the culture density height, and greater than 20g (dry weight)/L substratum, culture cycle is short, is generally 10d.(2) stability high, can infinitely go down to posterity.(3) growth temperature range broad, well-grown in 18～30 ℃ of scopes.(4) do not exist two-phase to cultivate, in whole culture cycle, can not make cell injury.(5) well-grown in reactor is fit to commercial scale reactor culturing plants cell and realizes industrialization.Therefore cultivate this saussurea involucrata cell at certain environment low suspension, can prepare a large amount of saussurea involucrata cell cultures as the saussurea involucrata medicinal raw material to replace excavating of natural snow lotus, protected country to treasure resources of medicinal plant, satisfied the clinical application demand simultaneously.
Description of drawings
Fig. 1 is a various dose
60The cell growth and the biosynthetic performance chart of flavones of the new clone that Co-gammairradiation saussurea involucrata cell line RP-1 obtains
Fig. 2 is the cell growth and the biosynthetic performance chart of flavones of the saussurea involucrata cell line of different mutafacient system acquisitions
Fig. 3 is growth of TUIP-8 saussurea involucrata cell line continuous passage cultured cells and the biosynthetic stability diagram of flavones
Fig. 4 is the influence figure of temperature to the growth of TUIP-8 saussurea involucrata cell line cell
Embodiment
Embodiment 1, the foundation of stablizing the saussurea involucrata cell line of high yield secondary metabolite flavones
One, natural plant callus inducing and cultivating
Get the bud or the tender stem of natural Saussurea medusa and do explant, fully rinse well, in 75% ethanol, soak 30s, wash repeatedly 5 times with autoclaved deionized water again, be placed on autoclaved Xinhua filter paper and blot deionized water with tap water.With aseptic operation scissors or sterile razor blade saussurea involucrata bud or the tender stem of handling well is cut into the long segment of 2～3mm, in MS (prescription sees Table 1), add on 0.2mg/L-0.4mg/L BA (benzyladenine), 1mg/L-4mg/L NAA (naa), 0.8g/L agar and the 40g/L-50g/L sucrose solids substratum and grow, 25 ℃ of illumination cultivation, about 14d induces callus.
Table 1:MS culture medium prescription
| Constituent concentration (mg/L) | Constituent concentration (mg/L) | Composition P concentration (mg/L) |
| NH 4NO 3 1650 KNO 3 1900 KH 2PO 4 170 MgSO 4·7H 2O 370 CaCl 2 339.2 FeSO 4·7H 2O 27.9 Na 2-EDTA 37.3 | MnSO 4·4H 2O 22.3 ZnSO 4·7H 2O 8.6 H3BO3 6.2 KI 0.83 Na 2MoO 4·2H 2O 0.25 CuSO 4·5H 2O 0.025 CoCl 2·6H 2O 0.025 | Glycine 2 vitamin B1 0.4 pyridoxine hydrochloride B6 0.5 nicotinic acid 0.5 |
Two, the screening of callus, the acquisition of original saussurea involucrata cell line
1, select color the darkest, be in bright colors, the quality densification is harder is organized in succeeding transfer culture on the solid medium, culture condition: temperature is 25 ℃, is 50～100 μ mol/ (m with illumination intensity
2.s) fluorescent lamp irradiation, every 15～20d subculture once;
2, it is the darkest to select color with appearance method from solid medium, the purple callus that is in bright colors shakes suspension culture in the bottle in the 300mL triangle that the 50mL liquid nutrient medium is housed, liquid nutrient medium is that MS adds suitable plant growth hormones and 40g/L sucrose, inoculum size is about fresh weight 3g/ bottle, shaking speed 150r/min, 6～7d changes liquid 30mL, it is purple black to select color behind the 12d again from liquid nutrient medium, color is bright and new, the cell mass of quality densification is transferred on the solid medium and is cultivated, the size of the cell mass of inoculation is about 3mm, it is vivid to select the color and luster homogeneous behind 4～5d, the cell mass that the harder cell lump of quality densification is divided into the about 1～2mm of size continues succeeding transfer culture 12～15d on solid medium, selects cell afterwards again and changes in the liquid and cultivate.So by solid culture to fluid suspension culture again to the solid culture repeated screening repeatedly, the just original saussurea involucrata cell line RP-1 of the purple that available matter is stable.Adopt flavones content in the determined by ultraviolet spectrophotometry RP-1 cell, its flavones content is about 4.34% ± 0.21% (wt%) of dry cell weight, and the flavones content in the natural snow lotus flower generally has only 0.4～0.5wt%.
Three, induced mutations
1,
60The mutagenesis of Co-gammairradiation
Adopt 2.5Gy respectively, 5Gy, 10Gy, the dosage of 20Gy and 40Gy, irradiation speed are 0.9Gy/min's
60The Co-gammairradiation RP-1 clone of 10d of on solid medium, having grown, and carry out succeeding transfer culture, according to the described method of step 2 by solid culture to fluid suspension culture again to the clone of solid culture repeated screening stable in properties.The result as shown in Figure 1, wherein the IRP-5 clone flavones content that screens through 40Gy irradiation is the highest, is about 6% of dry cell weight, flavones content is that RP-1 has improved nearly 40% than initiating cell.
2, temperature mutagenesis
Will
60The high yield flavones clone IRP-5 that the Co-gammairradiation obtains places 37 ℃ to cultivate 20h down, and carry out succeeding transfer culture, according to the clone of the described method repeated screening of step 2 stable in properties.Again the high yield clone that obtains is placed 15 ℃ and cultivate 24h down, by screening with quadrat method.The result as shown in Figure 2, the culture density of the clone TIP-33 that obtains in shaking bottle can reach 18.6 ± 2g (dry weight)/L, flavones content is about 10%, is about 20 times of natural plant.
3, ultraviolet mutagenesis
(1) will
60It is that 30min is shone at 30cm place under the ultraviolet lamp of 20W that the high yield flavones clone IRP-5 that the Co-gammairradiation obtains places apart from power, clone according to the described method repeated screening of step 2 stable in properties, the UIP-17 clone that obtains, its flavones content brings up to 8.8% from 5.96%.
(2) will
60It is irradiation 30min in 30cm place under the ultraviolet lamp of 20W that the mutagenic obtained high yield flavones clone TIP-33 of mutagenesis of Co-gammairradiation and temperature places apart from power.According to the clone of the described method repeated screening of step 2 stable in properties, the result as shown in Figure 2, the flavones content of the atropurpureus saussurea involucrata cell line TUIP-8 that obtains brings up to 12.3% from 8.8%.
Four, the screening of high and stable yields clone, go down to posterity
Saussurea involucrata cell line TUIP-8 is under 25 ℃ of conditions, shake a bottle suspension culture with the 300mL triangle, liquid amount is 50mL, inoculum density is about 3～4g (dry weight)/L, 6～7d changes liquid 30mL, go down to posterity once every 10～12d, the result as shown in Figure 3, the continuous passage suspension culture is after 50 generations, the growth characteristics of cell and flavones biosynthesizing characteristic remain unchanged, culture density in shaking bottle maintains 19g (dry weight)/L always, and flavones content maintains 12% always, and the clone TUIP-8 that shows acquisition is can infinitely the go down to posterity saussurea involucrata cell line of character homogeneous of the high and stable yields of cultivating of a strain.
Five, the cultivation of high yield clone
Shake suspension culture high yield clone in the bottle at triangle: the 300mL triangle shakes a bottle suspension culture, and liquid amount is 50mL, and inoculum density is about 3～4g (dry weight)/L, and substratum is that MS adds suitable plant growth hormones and 40g/L sucrose, shaking speed 150r/min.If culture temperature is 25 ℃, then 6d～7d changes liquid 30mL, goes down to posterity once or direct harvested cell every 10d～12d.The cell of results is extracted behind the moisture available 95% ethanolic soln leaching flavones wherein, also can 60 ℃～65 ℃ down oven dry preserve, the cell of oven dry can leach flavones wherein with 70% ethanolic soln.With rotatory evaporator ethanol evaporation and water, just can obtain the flavones crude product.
This saussurea involucrata cell line has following advantage:
(1) yield of flavone is big, culture density is high, culture cycle is short.
(2) stability high, can infinitely go down to posterity.
(3) there is not more ubiquitous two-phase cultivation in the culture plant cell in this saussurea involucrata cell line, be that growth phase and induction phase are cultivated, cell can not sustain damage in whole culture cycle, thereby realizes that easily vegetable cell cultured continuously and results are to improve the throughput of reactor.
(4) growth temperature range broad, well-grown in 18～30 ℃ of scopes.
(5) well-grown in reactor is fit to commercial scale reactor culturing plants cell and realizes industrialization.
Therefore cultivate this saussurea involucrata cell at certain environment low suspension, can prepare a large amount of saussurea involucrata cell cultures as the saussurea involucrata medicinal raw material to replace excavating of natural snow lotus, protected country to treasure resources of medicinal plant.From this cell culture, can extract the main active ingredient flavonoid compound of high-load saussurea involucrata, be used to research and develop into various treatments and Chinese medicine for health care, cultivate medicinal ingredientss such as producing the saussurea involucrata flavonoid compounds and realize that industrialization haves laid a good foundation for a large amount of.
Embodiment 2, stablize the shake-flask culture of high yield saussurea involucrata flavones saussurea involucrata cell line TUIP-8
Stablize high yield saussurea involucrata flavones saussurea involucrata cell line TUIP-8 through 37 ℃ of high temperature and 15 ℃ of low temperature mutagenesis, having obtained can be in the ability of the good growth of 18～30 ℃ of wide temperature range.As table 2 and shown in Figure 4, under 30 ℃ of environment, to grow, specific cell growth rate is very fast, but the cell mass water content is big, finally cover with the whole maximum culture density that shakes bottle and can reach 16.9g (DW)/L, but flavones content is lower.And in 18 ℃ of environment, cultivate, cell poor growth and densification, culture cycle is longer, and the cell mass water content is little, and quality is harder, and (25.6g (DW)/L), the flavones content in the cell has decline slightly therefore can to reach higher culture density.Comprehensive various factors, our general setting culture temperature in culturing process is 20～25 ℃, and under this environment, the cell speed of growth is moderate, and culture density is generally 20g (dry weight)/L, and flavones content is about 11%～13%.
The saussurea involucrata cell line TUIP-8 density height that uses present method to cultivate, yield of flavone is big.Carry out suspension culture in shaking bottle, cell yield is 20 ± 2g/L (DW), and cell total flavones turnout is 2.3g/L～2.5g/L, and culture cycle is 7～12d.
Table 2 temperature is to growth of suspension culture TUIP-8 saussurea involucrata cell and the influence of flavones synthetic
The optimum incubation time of the maximum culture density flavones content of temperature specific growth rate yield of flavone
(℃) (d
-1) (g(DW)/L) (％) (mg/L) (d)
*
18 0.168 25.6 10.15 2598.4 16
25 0.221 19.8 12.23 2421.5 12
30 0.266 16.9 8.86 1497.3 8
*When cell inoculation density is about 3～4g (DW)/L.
Embodiment 3, the reactor cultivation of stablizing high yield saussurea involucrata flavones saussurea involucrata cell line TUIP-8
In 7.5L Biostat CT sterilization stirring type bioreactor on the throne (German B.Braun company), cultivate, inoculum density is 2.5g (dry weight)/L, culture temperature is 25 ℃, pH is controlled at 4.8～5.6, dissolved oxygen (DO) is controlled at 30%～60%, adopt the gas distributor oxygen supply of direct aeration, mixing speed is 60r/min～90r/min, substratum is the MS substratum, add 0.2mg/L-0.4mg/L BA (benzyladenine), 1mg/L-4mg/L NAA (naa) and 40g/L-50g/L sucrose, liquid amount is 6L, be cultured to the 7th day and change liquid 4L,, be full of fresh saussurea involucrata cell in the whole reactor to the following jar harvested cell of 12d～14d, the stacking volume of cell is near 100%, be that cell almost grows to the substratum liquid level, but more than the harvested cell 130g (dry weight), culture density is generally the 21g/L working volume, flavones content utilizes this reactor once to cultivate and can obtain more than the flavones 14g greater than 11%.
Claims (1)
1, stablize the saussurea involucrata cell line TUIP-8 of high yield secondary metabolite flavones, its preserving number is CGMCCNo.0855.
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Families Citing this family (8)
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| CN101961357B (en) * | 2010-10-18 | 2011-11-02 | 天津金鹏源辐照技术有限公司 | Method for improving extraction rate of total flavonoid in spices by using radiation technique |
| CN105755087B (en) * | 2016-03-22 | 2019-05-28 | 何伟 | The method of marigold cell culture production free lutein |
| CN106718931B (en) * | 2017-01-19 | 2019-07-23 | 重庆市风景园林科学研究院 | The method for carrying out succulent breeding using bioreactor |
| CN106754633A (en) * | 2017-02-14 | 2017-05-31 | 天津艾赛博生物技术有限公司 | A kind of regulation and control method of pilot scale culture Herba Saussureae Involueratae cell high yield flavones |
| CN106867958A (en) * | 2017-02-14 | 2017-06-20 | 天津艾赛博生物技术有限公司 | A kind of technique of extensive squamous subculture Herba Saussureae Involueratae cell |
| CN107646685A (en) * | 2017-10-30 | 2018-02-02 | 江苏凤谷生物有限公司 | A kind of method for industrializing Herba Saussureae Involueratae culture of the large-scale production rich in flavones |
| CN108504623A (en) * | 2018-04-28 | 2018-09-07 | 大连普瑞康生物技术有限公司 | A kind of medusa-snow lotus cell line for generating flavone with high output and preparation method thereof |
| CN110800611A (en) * | 2019-11-29 | 2020-02-18 | 杭州职业技术学院 | Plant tissue culture method and device and production method of metabolite |
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