CN1197959C - Ephedra crown-gall nodule cell ZHA-2 CGMCC NO.0521 and its inducing method - Google Patents
Ephedra crown-gall nodule cell ZHA-2 CGMCC NO.0521 and its inducing method Download PDFInfo
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- CN1197959C CN1197959C CN 01131006 CN01131006A CN1197959C CN 1197959 C CN1197959 C CN 1197959C CN 01131006 CN01131006 CN 01131006 CN 01131006 A CN01131006 A CN 01131006A CN 1197959 C CN1197959 C CN 1197959C
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Abstract
The present invention relates to an ephedra crown-gall tumor cell ZHA-2 CGMCC NO. 0521 generated by ephedra by leading agricultural bacillus into ephedra through inducement, and an inducing method thereof. The induced ephedra crown-gall tumor cell ZHA-2 is preserved in the Chinese Microbe Preservation Center on 11st of December in 2000, and is induced by culturing strains on a cane sugar agar medium containing a small quantity of hormone at a temperature of about 23 to 32 DEG C for 10 to 15 days, and the form of the ephedra crown-gall tumor cell presents an irregular similarly-spherical block shape; the appearance is from light yellow to dark green; the diameter is from 0.5mm to 15mm. In the inducing method, the Ti-plasmids of the agricultural bacillus are used as carriers in the ephedra, and the ephedra crown-gall tumor cell (Ephedra) ZHA-2 CGMCC NO. 0521 is induced by leading exogenous genes into culture solutions. The ephedra crown-gall tumor cell can be subcultured in a culture medium containing 1.0 to 5.0% of sugar for 5 to 60 days at a temperature of 23 to 32 DEG C, and the proliferation quantity of the obtained ephedra crown-gall tumor cell is 2 to 8 times of the proliferation quantity during inoculation. The content of ephedrine is from 0.1 to 25 wt%.
Description
Technical field
The present invention relates to utilize biotechnology to induce Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO.0521 from ephedra.More specifically, the present invention relates to, induce ephedra to produce Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO.0521 and induction method thereof by Agrobacterium is imported ephedra.
Background technology
Mainly contain ephedrine, pseudoephedrine, L-N N-Methylephedrine, d-N methyl pseudoephedrine, L-demethyl ephedrine, d-demethyl pseudoephedrine etc. in the Chinese ephedra plant.Herba Ephedrae is a kind of herbal medicine commonly used, has sweating, relievings asthma, effect such as dispel the wind.Ephedrine is the same with suprarenin can exciting sympathetic nerves, but ephedrine can be oral, and long action time, and this point can not for suprarenin institute.Ephedrine can make bronchiectasis, and nasal mucosa shrinks, and makes elevation of blood pressure, clinically is used for specific treatment cough, asthma, spring fever and Whooping cough etc., and can lower the pain of cramp disease, also can make the mydriasis medicine.
As everyone knows, realize that in bio-reactor it is an important directions of biotechnology research in recent years that natural plant cells is cultivated.Yet up to now, it is also not high that reactor is cultivated targeted activity material (as the many crude drug active ingredients) content that is produced, for improving output, the botanist has done a large amount of breedings and the research of gene regulating, and biochemical engineering investigator is then seeking from aspects such as culture process and breaking through.
The researchist of India, the U.S. and Britain utilizes ephedra cell to cultivate and produces ephedrine by Chinese ephedra plant evoked callus.Studied ambient conditionss such as illumination condition, the various compositions that change substratum and ratio are produced ephedrine to ephedra cell influence.The content of ephedra cell culture epheday intermedia alkali is 0.17～0.6%.
The callus culture thing is loose, cell tissue particulate diameter does not wait, greatly about 2～200 μ m, in the process of utilizing cell culture production secondary metabolite, special in bio-reactor in the process of culturing plants cells produce meta-bolites, nutrient solution and cell culture are not easily separated.In addition, the synthetic often contradiction of the growth of callus culture thing and meta-bolites.
And general crown-gall nodule cell is lumps, and crown-gall nodule cell tissue particle diameter is bigger.The crown-gall nodule cell interior has the differentiation of cell or tissue, so its ability that produces secondary metabolite will be higher than the dispersive cell, simultaneously thisly organizes and energy for growth faster arranged.
Summary of the invention
The objective of the invention is to induce Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2CGMCC NO.0521 by the Chinese ephedra plant; The culture that utilizes Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO.0521 to obtain can significantly improve the content of biomass and culture epheday intermedia alkali, two of purpose is to overcome in the process that has culturing plants cells produce meta-bolites in bio-reactor, nutrient solution and cell culture are not easily separated, the synthetic often shortcoming of contradiction of the growth of callus culture thing and meta-bolites, thus a kind of Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO.0521 and inductive method thereof are provided.
Chinese ephedra crown-gall nodule cell provided by the invention (Ephedra) ZHA-2 is deposited in Chinese microbial preservation center (it abbreviates CGMCC as) on December 11st, 2000, and preserving number is CGMCC NO.0521.
The microbiological property of Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO.0521:
(1) ecological characteristic of Chinese ephedra crown-gall nodule cell
Containing on the sucrose nutrient agar of a small amount of hormone, cultivated this bacterial strain 10～15 days in about 23～32 ℃, Chinese ephedra crown-gall nodule cellular form presents the spherical lumps of irregular class, and apparent is light yellow to deep green, and diameter is at 0.5～15 millimeter.
(2) physiological and biochemical property
The growth of Chinese ephedra crown-gall nodule cell can utilize multiple glycogen, and suitable culture temperature is 23～32 ℃, is higher than 40 ℃ of cell tissue poor growths until death; Suitable growth pH value is 5.0～6.0; There is certain tissue differentiation structure Chinese ephedra crown-gall nodule cell tissue inside.Under the condition of illumination and unglazed photograph, all can produce ephedrine.Peroxidase isozyme is the activity sign of Chinese ephedra crown gall tumor cell growth, and the phenylalanine transaminase is the key enzyme of synthetic ephedrine, itself and ephedrine synthetic proportional.
The invention provides and utilize foreign gene to import the ephedra cell, produce the method for Chinese ephedra crown-gall nodule cell.Ephedra used among the present invention has: ephedra equisetina Ephedra equisetina Bunge, ephedra sinica Ephedra sinica Stapf, epheday intermedia Ephedra intermedia Schrenk, Ephedra przewalskii Ephedraprzewalskii Stapf; Horsetail Beefwood Casuarina Adans, and Herba Ephedrae monospermae, blue branch Chinese ephedra, sea grape, thin sub-Chinese ephedra etc.
The present invention utilizes the Agrobacterium Ti-plasmids as carrier, foreign gene is imported ephedra induce the step of Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO.0521 to carry out in the following order:
(1) the Ephedra plant is cleaned distilled water flushing three times with detergent solution; With sterilizing agent sterilization 10～30 minutes, clean the Ephedra plant that obtains sterilizing with sterilized distilled water again; Described sterilizing agent is as 1-5% clorox or 10～15% hydrogen peroxide (meter) by volume, or 0.1～1.0% mercuric chloride aqueous solution (by weight/the volume ratio meter);
(2) the sterilized Ephedra plant of above-mentioned steps (1) is cut into 0.3～1.0 centimetre of length, puts into to be equipped with and dilute 5～20 times of (OD
600Be 0.6～0.8) the shaking in the bottle of nutrient solution of Agrobacterium, will shaking bottle, to be placed on rotating speed be co-cultivation 5～60 minutes on the 150rpm shaking table, culture temperature is 23～32 ℃, obtains by the Ephedra plant of agroinfection;
(3) the Ephedra plant by agroinfection of above-mentioned steps (2) is placed on to contain in the antibiotic substratum of 0.5～5.0wt% cultivated 5～20 days, culture temperature is 23～32 ℃, grows Chinese ephedra crown-gall nodule cell on the Ephedra plant gradually; Described microbiotic comprises carbenicillin, paraxin, sulphuric acid kanamycin etc.;
(4) the Chinese ephedra crown-gall nodule cell that above-mentioned steps (3) is obtained downcuts from the plant of its growth, is transferred in the substratum of step (3) of new preparation to cultivate 10～20 days, and culture temperature is 23～32 ℃, obtains the Chinese ephedra crown-gall nodule cell of succeeding transfer culture;
In order to improve the content of ephedrine, can also comprise following continuation cultural method:
The Chinese ephedra crown-gall nodule cell that above-mentioned steps (4) is obtained was placed in the substratum of step (3) cultured continuously 10～60 days, and culture temperature is 23～32 ℃, obtains containing the Chinese ephedra crown-gall nodule cell culture of ephedrine 0.1～25wt%.
The substratum (step (3)-(5) used substratum) of wherein said cultivation Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521 is composed as follows:
| Saltpetre | 1～20mmol/L | Vitamins C | 0.01～0.5mmol/L |
| SODIUM PHOSPHATE, MONOBASIC | 0.5～3.0mmol/L | Vitamin B12 | 0.01～10mmol/L |
| Sal epsom | 1.0～5.0mmol/L | Nicotinic acid | 0.1～10mmol/L |
| Sodium Tetraborate | 0.001～0.0075mmol/L | Pyridoxine hydrochloride | 0.01～2.0mmol/L |
| Sodium sulfate | 0.5～4.0mmol/L | The 6-vitamin | 0.01～2.0mmol/L |
| Calcium chloride | 0.3～5.0mmol/L | 6-chaff aminoadenine | 0.01～0.5mmol/L |
| Zinc sulfate | 0.01～0.5mmol/L | Indolylacetic acid | 0.01～0.1mmol/L |
| Copper sulfate | 0.001～0.2mmol/L | 2,4 dichlorophenoxyacetic acid | 0.01～0.6mmol/L |
| Iron edetate | 0.1～3.5mmol/L | Glucose | 0.5～5.0％ |
| Cobalt chloride | 0.001～0.2mmol/L | Sucrose | 0.5～5.0％ |
In order to improve the content of biomass and ephedrine in the Chinese ephedra crown-gall nodule cell cultivation process, can also in the substratum of described cultivation Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCCNO 0521, add 0.1～100mmol/L phenylalanine.
In order to improve the content of biomass and ephedrine in the Chinese ephedra crown-gall nodule cell cultivation process, can also in the substratum of described cultivation Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521, add 0.01～150mmol/L sodium formiate.
In order to improve the content of biomass and ephedrine in the Chinese ephedra crown-gall nodule cell cultivation process, can also in the substratum of described cultivation Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521, add 0.1～50mmol/L Serine.
In order to improve the content of biomass and ephedrine in the Chinese ephedra crown-gall nodule cell cultivation process, can also in the substratum of described cultivation Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521, add 0.05～150mmol/L methionine(Met).
In order to improve the content of biomass and ephedrine in the Chinese ephedra crown-gall nodule cell cultivation process, can also in the substratum of described cultivation Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521, add 0.01～200mmol/L tyrosine.
In order to improve the content of biomass and ephedrine in the Chinese ephedra crown-gall nodule cell cultivation process, can also in the substratum of described cultivation Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521, add 0.025～250mmol/L tryptophane.
In order to improve the content of biomass and ephedrine in the Chinese ephedra crown-gall nodule cell cultivation process, can also in the substratum of described cultivation Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521, add 1.0～50% volume ratio fungal fermented filtrates.
In order to improve the content of biomass and ephedrine in the Chinese ephedra crown-gall nodule cell cultivation process, (Ephedra) ZHA-2 CGMCC NO 0521 can also add 0.01～300mmol/L phenylformic acid in the substratum of described cultivation Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521.
The invention has the advantages that: the present invention utilizes the Agrobacterium Ti-plasmids as carrier, and foreign gene is imported ephedra, produces Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521.The product of gained is that Chinese ephedra crown-gall nodule cell is a kind spheric cell group, and diameter is generally at 5～15 millimeters, and much larger than general plant callus cell, when suspension culture, nutrient solution is clarifying, and Chinese ephedra crown-gall nodule cell separates with nutrient solution easily.And Chinese ephedra crown-gall nodule cell interior has the differentiation of cell or tissue, and energy for growth is faster arranged.Import foreign gene, changed the pathways metabolism of cell epheday intermedia alkali, so its ability that produces secondary metabolite to be higher than callus cell, solve cell growth and meta-bolites synthetic contradiction.With throughput and the productive rate of further raising ephedrine in cellular process, be convenient in bio-reactor, cultivate and produce ephedrine.
Induction method of the present invention is optimized the growth conditions of cell, through metabolic regulation, obtains the Chinese ephedra crown-gall nodule clone of high ephedrine produced thereby, when the proliferative amount of Chinese ephedra crown-gall nodule cell is subculture 2～8 times, and the content of ephedrine is 0.1～25.0wt%; This Chinese ephedra crown-gall nodule cell can carry out suitability for industrialized production, and the ephedrine produced thereby of making a living provides a kind of new raw material approach.
Embodiment
Embodiment 1
Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521, induce according to the following steps:
1) ephedra equisetina or ephedra sinica belong to plant and clean distilled water flushing three times with detergent solution; With 2% (weight/volume) clorox sterilization 30 minutes, clean with sterilized distilled water, be cut to 0.5 centimetre of length, the Ephedra plant that obtains sterilizing;
2) with sterilized Ephedra plant, with the OD that has diluted 20 times
600Be 0.6 Agrobacterium nutrient solution co-cultivation 30 minutes, culture temperature is 25 ℃, obtains infecting the Ephedra plant of Agrobacterium;
3) plant that above-mentioned steps 2 has been infected Agrobacterium is placed in the growth medium that contains the 2Wt% kantlex and cultivated 15 days, and culture temperature is 25 ℃, grows Chinese ephedra crown-gall nodule cell on the plant gradually;
4) above-mentioned resulting Chinese ephedra crown-gall nodule cell is downcut from the plant of its growth, be transferred in the substratum of new preparation and cultivated 15 days, culture temperature is 25 ℃, obtains the Chinese ephedra crown-gall nodule cell of succeeding transfer culture;
5) with above-mentioned resulting Chinese ephedra crown-gall nodule cell cultured continuously 60 days, culture temperature was 25 ℃, obtains containing the Chinese ephedra crown-gall nodule cell culture of ephedrine 0.1～25wt%.
The substratum of used cultivation Chinese ephedra crown-gall nodule cell (Ephedra) ZHA-2 CGMCC NO 0521 is formed among the embodiment 1:
| Saltpetre | 5mmol/L | Vitamins C | 0.01mmol/L |
| SODIUM PHOSPHATE, MONOBASIC | 0.5mmol/L | Vitamin B12 | 0.02mmol/L |
| Sal epsom | 1.25mmol/L | Nicotinic acid | 0.1mmol/L |
| Sodium Tetraborate | 0.001mmol/L | Pyridoxine hydrochloride | 0.025mmol/L |
| Sodium sulfate | 0.5mmol/L | Vitamin | 0.025mmol/L |
| Calcium chloride | 0.3mmol/L | 6-chaff aminoadenine | 0.01mmol/L |
| Zinc sulfate | 0.1mmol/L | Indolylacetic acid | 0.01mmol/L |
| Copper sulfate | 0.0025mmol/L | 2,4 dichlorophenoxyacetic acid | 0.02mmol/L |
| Iron edetate | 0.1mmol/L | Glucose | 0.5％ |
| Cobalt chloride | 0.0025mmol/L | Sucrose | 3.0％ |
Embodiment 2,3, and 4,5,6,7 and example 8
Chinese ephedra crown-gall nodule cell (Ephedra) the ZHA-2 CGMCC NO.0521 that is cultivated among the embodiment 2-8 with induce process and inductive condition identical with embodiment 1, but used Chinese ephedra plant can be divided and classified as: ephedra equisetina, ephedra sinica, epheday intermedia, Ephedra przewalskii, Horsetail Beefwood, Herba Ephedrae monospermae, blue branch Chinese ephedra, sea grape, thin sub-Chinese ephedra, the substratum of Chinese ephedra crown-gall nodule cell induction is formed different, and the substratum composition is listed as follows:
Embodiment 9,10,11,12,13,14, Chinese ephedra crown-gall nodule cell (Ephedra) the ZHA-2 CGMCC NO.0521 that is cultivated in 15 and 16 is identical with embodiment 1, and Chinese ephedra crown-gall nodule cell induces process and inductive condition described also identical with embodiment 1 by the Ephedra plant, but the composition difference of the substratum of Chinese ephedra crown-gall nodule cell induction is described as follows:
Embodiment 9
On the method basis of embodiment 1, also the 40mmol/L phenylalanine will be added in the composition of substratum again.Owing to add phenylalanine, improved the inductivity of Chinese ephedra crown-gall nodule cell and the productivity of ephedrine.
Embodiment 10
On the basis of embodiment 9, also the 1.0mmol/L sodium formiate will be added in the composition of substratum again.Owing to add sodium formiate, improved the inductivity of Chinese ephedra crown-gall nodule cell and the productivity of ephedrine.
Embodiment 11
On the basis of embodiment 10, also the 5.0mmol/L Serine will be added in the composition of substratum again.Owing to add Serine, improved the inductivity of Chinese ephedra crown-gall nodule cell and the productivity of ephedrine.
Embodiment 12
On the basis of embodiment 11, also the 30.0mmol/L methionine(Met) will be added in the composition of substratum again.Owing to add methionine(Met), improved the inductivity of Chinese ephedra crown-gall nodule cell and the productivity of ephedrine.
Embodiment 13
On the basis of embodiment 12, also the 8.5mmol/L tryptophane will be added in the composition of substratum again.Owing to add tryptophane, improved the inductivity of Chinese ephedra crown-gall nodule cell and the productivity of ephedrine.
Embodiment 14
On the basis of embodiment 13, also 22.5mmol/L tyrosine will be added in the composition of substratum again.Owing to add tyrosine, improved the inductivity of Chinese ephedra crown-gall nodule cell and the productivity of ephedrine.
Embodiment 15
On the basis of embodiment 14, also 5.6% volume ratio fungal fermented filtrate will be added in the composition of substratum again.Owing to add fungal fermented filtrate, improved the inductivity of Chinese ephedra crown-gall nodule cell and the productivity of ephedrine.
Embodiment 16
On the basis of embodiment 15, also the 2.0mmol/L phenylformic acid will be added in the composition of substratum again.Owing to add phenylformic acid, improved the inductivity of Chinese ephedra crown-gall nodule cell and the productivity of ephedrine.
Claims (1)
1. Chinese ephedra crown-gall nodule cell ZHA-2 CGMCC NO 0521, this Chinese ephedra crown-gall nodule cell ZHA-2 CGMCCNO 0521 has following microbiological property:
(1) ecological characteristic of Chinese ephedra crown-gall nodule cell:
Containing on the sucrose nutrient agar of a small amount of hormone, cultivated this bacterial strain 10～15 days in about 23～32 ℃, Chinese ephedra crown-gall nodule cellular form presents the spherical lumps of irregular class, and apparent is light yellow to deep green, and diameter is at 0.5～15 millimeter;
(2) physiological and biochemical property:
The growth of Chinese ephedra crown-gall nodule cell can utilize multiple glycogen, and culture temperature is 23～32 ℃, is higher than 40 ℃ of cell tissue poor growths until death; Suitable growth pH is 5.0～6.0; There is certain tissue differentiation structure Chinese ephedra crown-gall nodule cell tissue inside; Under the condition of illumination and unglazed photograph, all can produce ephedrine; Peroxidase isozyme is the activity sign of Chinese ephedra crown gall tumor cell growth, and the phenylalanine transaminase is the key enzyme of synthetic ephedrine, itself and ephedrine synthetic proportional.
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| CN 01131006 CN1197959C (en) | 2001-08-30 | 2001-08-30 | Ephedra crown-gall nodule cell ZHA-2 CGMCC NO.0521 and its inducing method |
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| CN106070316A (en) * | 2016-06-02 | 2016-11-09 | 湖南农业大学 | The disinfectant of field Boehmeria nivea leaves callus regeneration, preparation method and Quick disinfection method |
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