CN103756917A - Preparation method of protoplasts of catalpa bungei endophytic fungi - Google Patents
Preparation method of protoplasts of catalpa bungei endophytic fungi Download PDFInfo
- Publication number
- CN103756917A CN103756917A CN201410016834.1A CN201410016834A CN103756917A CN 103756917 A CN103756917 A CN 103756917A CN 201410016834 A CN201410016834 A CN 201410016834A CN 103756917 A CN103756917 A CN 103756917A
- Authority
- CN
- China
- Prior art keywords
- protoplastis
- preparation
- mycelia
- endogenetic fungus
- chinese catalpa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a preparation method of protoplasts of catalpa bungei endophytic fungi. The method comprises the following steps: 1, preparing a mycelium; 2, preparing protoplasts; 3, purifying the protoplasts; 4 regenerating the protoplasts. The protoplasts prepared by using the method disclosed by the invention are large in quantity, and can be up to 6*10<7>/mg; the obtained protoplasts are high in activity; the regeneration rate can be up to 1.2%. The mycelium which is cultivated for 12-24 hours is selected, the fungi are in a rapid growth period, and fast in growth, dissociation of enzyme is facilitated by a cell wall; the released protoplasts are large in quantity, not easy to split, and high in activity. 5-30mg/ml of Glucanex compound enzyme is selected as cell wall cracking enzyme liquid, the cracking efficiency is high, and the related protoplasts are large in quantity. KCl or NaCl is selected as an osmotic pressure stabilizer, and the preparation quantity and the stability of the protoplasts are ensured.
Description
Technical field
The present invention relates to the field of biology of fungi, especially a kind of preparation method of protoplastis of Chinese catalpa endogenetic fungus.
Background technology
Endogenetic fungus refers to that those lives are that plant tissue inside can't make plant produce the fungi of obvious disease.Endogenetic fungus can produce and the same or analogous biologically active substance of host plant, has huge potential value, can become the source of new type anticancer medicine.This seminar from juglans mandshurica separation and Extraction one strain there is the endogenetic fungus of very strong anticancer vigor, be numbered FSN002, this bacterium of preliminary evaluation is Deuteromycotina, hyphomycetes, Moniliales, trichoderma, its fermented extracted liquid has very strong restraining effect to liver cancer cell.This is a brand-new bacterial classification that this laboratory is exclusive, open in Chinese patent CN101487022, but it carries out suitability for industrialized production the drawbacks limit such as the bio-active substance quality of its generation is few, unstable, by means such as bio-transformation, structure mutant, it is carried out to thalline transformation, improve its anticancer vigor and study its anticancer mechanism, can utilize better this fungus resource, be basis and the gordian technique of carrying out above research and build protoplastis.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of preparation method of protoplastis of Chinese catalpa endogenetic fungus.
The present invention is achieved by the following technical solutions:
The preparation method who the invention provides a kind of protoplastis of Chinese catalpa endogenetic fungus, said method comprising the steps of:
Step 1, mycelial preparation;
Step 2, the preparation of protoplastis;
Step 3, protoplastis purifying;
Step 4, protoplast regeneration.
Preferably, described mycelium preparation is specially: get the fungal spore that is placed in-80 ℃ of preservations, under room temperature, thaw, in aseptic operating platform, draw 200 μ l, add in the 250ml Erlenmeyer flask that 100ml liquid PDA substratum is housed, be placed on shaking table, 23～28 ℃ are rocked cultivation, cultivate hypha fermentation, with the aseptic metal filtration sieve of 400 order collection mycelia, put into mycelia scavenging solution and clean 2 times, with aseptic thieving paper suck dry moisture.
Preferably, described hypha fermentation culture condition: be 23～28 ℃ in temperature, the rotating speed of shaking table is 100rpm, rocking incubation time is 12～24h.
Preferably, described protoplasm structure is standby is specially: the agent of Glucanex enzyme homeo-osmosis is formulated as the enzyme liquid of 5～30mg/ml, fully dissolves and carries out enzymolysis, standby with aseptic filter membrane filtration sterilization.
Preferably, described homeo-osmosis agent is 0.7～1mol/L KCl or NaCl solution.
Preferably, the condition of described enzymolysis is: in temperature, be 28 ℃, the rotating speed of shaking table is 100rpm, and the time is 4～8h.
Preferably, described protoplastis purifying is specially: after mycelia enzymolysis, with 4 layers of aseptic lens wiping paper, filter and remove mycelia, by filtrate collection in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplastis STC to preserve the resuspended protoplastis of liquid and preserves.
Preferably, described STC preserves consisting of of liquid: 1mol/L sorbyl alcohol, 0.01mol/L Tris-HCl, 0.01mol/L calcium chloride.
Preferably, described protoplast regeneration is specially: first in culture dish, spread the solid regenerated substratum of one deck protoplastis, standby after condensation, the semi-solid regeneration culture medium of preparation PDA, is placed under room temperature and is cooled to 40 ℃, draw 10ml substratum and add 100 μ L protoplastis suspensions, mix, be directly poured on the original solid regenerated substratum of culture dish, make it to be paved with plate and form a solid thin layer, treat that culture medium solidifying is placed in 28 ℃ of incubators and cultivate 2～3d, the bacterium colony of regenerating as seen.
Preferably, the solid regenerated substratum of described protoplastis is: the PDA solid medium that adds 0.004% sodium deoxycholate, 1mol/L N.F,USP MANNITOL, 15g/L agar.
Compared with prior art, the present invention has following beneficial effect:
(1) the protoplastis quantity that prepared by the method for the invention is more, can reach 6 × 10
7individual/milligram mycelia, the vigor of the protoplastis of acquisition is higher, and regeneration rate can reach 1.2%.
(2) the hyphal cell wall construction difference of different cell ages, affects protoplastis preparation, and the present invention selects to cultivate the mycelium of 12-24h, fungi is in fast growing period, and growth is fast, and cell walls is easy to the cracking of Glucanex enzyme, the quantity of the protoplastis discharging is many, is difficult for cracking, and vigor is high.
(3) many, the complex structure of fungal cell wall composition, the selection of cell walls lyase is very large on the preparation impact of protoplastis, the present invention chooses 5～30mg/ml Glucanex prozyme as cell walls lyase liquid, and lysis efficiency is high, and protoplast liberation quantity is many.
(4) kind of homeo-osmosis agent and the selection of concentration preparation, regeneration and the protoplastis to protoplastis stablize extremely important, the present invention selects 0.7～1mol/L KCl or NaCl as homeo-osmosis agent, has guaranteed that protoplastis prepares the stable of quantity and protoplastis.
(5) the present invention adopts and adds the high PDA of the oozing regeneration culture medium of bilayer of 0.004% sodium deoxycholate to carry out the regeneration of protoplastis, has improved the regeneration rate of protoplastis, and has facilitated selecting of mono-clonal thalline.
Accompanying drawing explanation
By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:
Fig. 1 is the optical microscope image of protoplastis prepared in embodiment 1;
Fig. 2 is the image that protoplastis prepared in embodiment 1 carries out the fluorescent microscope of vigor assessment, if protoplastis can show fluorescence, represents that it has vigor;
Fig. 3 is the picture that the method in employing embodiment 5 is carried out protoplast regeneration;
Fig. 4 is the picture that the method in employing comparative example 4 is carried out protoplast regeneration.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art further to understand the present invention, but not limit in any form the present invention.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make some distortion and improvement.These all belong to protection scope of the present invention.
embodiment 1
The present embodiment relates to a kind of preparation method of protoplastis of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 ℃ of preservations, under room temperature, thaw, in aseptic operating platform, drawing 200 μ l adds in the 250ml Erlenmeyer flask that 100ml liquid PDA substratum is housed, be placed on 100rpm shaking table, 25 ℃ are rocked cultivation, cultivate 12h, with the aseptic metal filtration sieve of 400 order collection mycelia, put into mycelia scavenging solution and clean 2 times, with aseptic thieving paper suck dry moisture;
(2) preparation of protoplastis: Glucanex enzyme is formulated as the enzyme liquid of 10mg/ml with the agent of 0.8mol/L KCl homeo-osmosis, standby with aseptic filter membrane filtration sterilization after fully dissolving.Mycelia is placed in to enzyme liquid in 28 ℃, and 100rpm rotating speed rocks enzymolysis 6h;
(3) protoplastis purifying: after mycelia enzymolysis 6h, with 4 layers of aseptic lens wiping paper, filter and remove mycelia, by filtrate collection in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, add protoplastis STC to preserve the resuspended protoplastis of liquid and preserve, the optical microscope image of seeing Fig. 1 the present embodiment protoplastis.Fig. 2 is the image that the prepared protoplastis of the present embodiment carries out the fluorescent microscope of vigor assessment, if protoplastis can show fluorescence, represents that it has vigor; By finding out in Fig. 1 that protoplastis quantity is many, size is more or less the same, and mass ratio is higher.
The protoplastis quantity that the present embodiment method obtains reaches 6 × 10
7individual/milligram mycelia.
embodiment 2
The operation steps of Formation and regeneration method that the present embodiment relates to a kind of protoplastis of Chinese catalpa endogenetic fungus is as follows:
(1) mycelium preparation: get the fungal spore that is placed in-80 ℃ of preservations, under room temperature, thaw, in aseptic operating platform, drawing 200 μ l adds in the 250ml Erlenmeyer flask that 100ml liquid PDA substratum is housed, be placed on 100rpm shaking table, 25 ℃ are rocked cultivation, cultivate 24h, with the aseptic metal filtration sieve of 400 order collection mycelia, put into mycelia scavenging solution and clean 2 times, with aseptic thieving paper suck dry moisture;
(2) protoplastis preparation: Glucanex enzyme is formulated as the enzyme liquid of 5mg/ml with 0.7mol/L NaCl homeo-osmosis liquid, standby with aseptic filter membrane filtration sterilization after fully dissolving.Mycelia is placed in to enzyme liquid in 28 ℃, and 100rpm rotating speed rocks enzymolysis 6h;
(3) protoplastis purifying: after mycelia enzymolysis 6h, with 4 layers of aseptic lens wiping paper, filter and remove mycelia, by filtrate collection in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplastis STC to preserve the resuspended protoplastis of liquid and preserves.
The protoplastis quantity that the present embodiment method obtains reaches 2 × 10
7individual/milligram mycelia.
embodiment 3
The present embodiment relates to a kind of preparation of protoplastis of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 ℃ of preservations, under room temperature, thaw, in aseptic operating platform, drawing 200 μ l adds in the 250ml Erlenmeyer flask that 100ml liquid PDA substratum is housed, be placed on 100rpm shaking table, 25 ℃ are rocked cultivation hypha fermentation, cultivate 18h, with the aseptic metal filtration sieve of 400 order collection mycelia, put into mycelia scavenging solution and clean 2 times, with aseptic thieving paper suck dry moisture;
(2) protoplastis preparation: Glucanex enzyme is formulated as the enzyme liquid of 20mg/ml with 1mol/L NaCl homeo-osmosis liquid, standby with aseptic filter membrane filtration sterilization after fully dissolving, mycelia is placed in to enzyme liquid in 28 ℃, 100rpm rotating speed rocks enzymolysis 2h;
(3) protoplastis purifying: after mycelia enzymolysis 2h, with 4 layers of aseptic lens wiping paper, filter and remove mycelia, by filtrate collection in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplastis STC to preserve the resuspended protoplastis of liquid and preserves.
The protoplastis quantity that the present embodiment method obtains reaches 1.5 × 10
7individual/milligram mycelia.
embodiment 4
The present embodiment relates to a kind of preparation of protoplastis of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 ℃ of preservations, under room temperature, thaw, in aseptic operating platform, drawing 200 μ l adds in the 250ml Erlenmeyer flask that 100ml liquid PDA substratum is housed, be placed on 100rpm shaking table, 25 ℃ are rocked cultivation, cultivate 14h, with the aseptic metal filtration sieve of 400 order collection mycelia, put into mycelia scavenging solution and clean 2 times, with aseptic thieving paper suck dry moisture.
(2) protoplastis preparation: Glucanex enzyme is formulated as the enzyme liquid of 30mg/ml with 0.8mol/L KCl homeo-osmosis liquid, standby with aseptic filter membrane filtration sterilization after fully dissolving.Mycelia is placed in to enzyme liquid in 28 ℃, and 100rpm rotating speed rocks enzymolysis 8h.
(3) protoplastis purifying: after mycelia enzymolysis 8h, with 4 layers of aseptic lens wiping paper, filter and remove mycelia, by filtrate collection in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplastis STC to preserve the resuspended protoplastis of liquid and preserves.
The present embodiment method obtains protoplastis quantity and reaches 5 × 10
7individual/milligram mycelia.
embodiment 5
The present embodiment relates to the renovation process of the protoplastis of Chinese catalpa endogenetic fungus prepared by aforementioned 1-4, is specially:
First in culture dish, spread the solid regenerated substratum of one deck protoplastis (protoplastis that embodiment 1～4 obtains), standby after condensation, the semi-solid regeneration culture medium of preparation PDA (adding the PDA solid medium of 0.004% sodium deoxycholate, 1mol/L N.F,USP MANNITOL, 15g/L agar), be placed under room temperature and be cooled to 40 ℃, draw 10ml substratum and add 100 μ L protoplastis suspensions, mix, directly be poured on the original solid regenerated substratum of culture dish, make it to be paved with plate and form a solid thin layer.Treat that culture medium solidifying is placed in 28 ℃ of incubators and cultivate 2d.
The regeneration rate of the protoplastis of the present embodiment Chinese catalpa endogenetic fungus is 1.2%, sees the picture of Fig. 3 protoplast regeneration.
comparative example 1
This comparative example relates to a kind of preparation method of protoplastis of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 ℃ of preservations, under room temperature, thaw, in aseptic operating platform, drawing 200 μ l adds in the 250ml Erlenmeyer flask that 100ml liquid PDA substratum is housed, be placed on 100rpm shaking table, 25 ℃ are rocked cultivation, cultivate 10h, with the aseptic metal filtration sieve of 400 order collection mycelia, put into mycelia scavenging solution and clean 2 times, with aseptic thieving paper suck dry moisture;
(2) protoplastis preparation: Glucanex enzyme is formulated as the enzyme liquid of 1mg/ml with 0.8mol/L KCl homeo-osmosis liquid, standby with aseptic filter membrane filtration sterilization after fully dissolving, mycelia is placed in to enzyme liquid in 28 ℃, 100rpm rotating speed rocks enzymolysis 4h.
(3) protoplastis purifying: after mycelia enzymolysis 4h, with 4 layers of aseptic lens wiping paper, filter and remove mycelia, by filtrate collection in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplastis STC to preserve the resuspended protoplastis of liquid and preserves.
The protoplastis quantity that this comparative example 1 obtains only has 1.5 × 10
5individual/milligram mycelia.
comparative example 2
This comparative example relates to a kind of preparation method of protoplastis of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 ℃ of preservations, under room temperature, thaw, in aseptic operating platform, drawing 200 μ l adds in the 250ml Erlenmeyer flask that 100ml liquid PDA substratum is housed, be placed on 100rpm shaking table, 25 ℃ are rocked cultivation, cultivate 12h, with the aseptic metal filtration sieve of 400 order collection mycelia, put into mycelia scavenging solution and clean 2 times, with aseptic thieving paper suck dry moisture;
(2) protoplastis preparation: Glucanex enzyme is formulated as the enzyme liquid of 4mg/ml with 0.8mol/L N.F,USP MANNITOL homeo-osmosis liquid, standby with aseptic filter membrane filtration sterilization after fully dissolving.Mycelia is placed in to enzyme liquid in 28 ℃, and 100rpm rotating speed rocks enzymolysis 6h.
(3) protoplastis purifying: after mycelia enzymolysis 6h, with 4 layers of aseptic lens wiping paper, filter and remove mycelia, by filtrate collection in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplastis STC to preserve the resuspended protoplastis of liquid and preserves.
The protoplastis quantity that this comparative example 2 obtains only has 1.3 × 10
5individual/milligram mycelia.
comparative example 3
This comparative example relates to a kind of preparation method of protoplastis of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 ℃ of preservations, under room temperature, thaw, in aseptic operating platform, drawing 200 μ l adds in the 250ml Erlenmeyer flask that 100ml liquid PDA substratum is housed, be placed on 100rpm shaking table, 25 ℃ are rocked cultivation, cultivate 10h, with the aseptic metal filtration sieve of 400 order collection mycelia, put into mycelia scavenging solution and clean 2 times, with aseptic thieving paper suck dry moisture;
(2) protoplastis preparation: Glucanex enzyme is formulated as the enzyme liquid of 3mg/ml with 0.6mol/L KCl homeo-osmosis liquid, standby with aseptic filter membrane filtration sterilization after fully dissolving.Mycelia is placed in to enzyme liquid in 28 ℃, and 100rpm rotating speed rocks enzymolysis 6h;
(3) protoplastis purifying: after mycelia enzymolysis 6h, with 4 layers of aseptic lens wiping paper, filter and remove mycelia, by filtrate collection in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplastis STC to preserve the resuspended protoplastis of liquid and preserves.
The protoplastis quantity that this comparative example 3 obtains only has 1 × 10
4individual/milligram mycelia.
comparative example 4
This comparative example relates to the protoplast regeneration method of aforementioned comparative example 1～3 Chinese catalpa endogenetic fungus, is specially:
Protoplast regeneration: protoplastis (protoplastis that comparative example 1～3 obtains) solution that absorption 100 μ l have diluted is in the solid regenerated culture medium flat plate of PDA, directly with spreading rod, be coated with gently evenly, culture dish be placed in to 28 ℃ of incubators and cultivate 2～3d left and right.
The regeneration rate of the protoplastis that this comparative example 4 obtains only has 0.1%, as the picture of Fig. 4 protoplast regeneration.
In sum: protoplastis quantity prepared by the method for the invention is more, can reach 6 × 10
7individual/milligram mycelia, the vigor of the protoplastis of acquisition is higher, and regeneration rate can reach 1.2%.Select the hyphal cell wall construction difference of different cell ages, affect protoplastis preparation, the present invention selects to cultivate the mycelium of 12-24h, fungi is in fast growing period, and growth is fast, and cell walls is easy to the cracking of Glucanex enzyme, the quantity of the protoplastis discharging is many, is difficult for cracking, and vigor is high.Fungal cell wall composition is many, complex structure, and the selection of cell walls lyase is very large on the preparation impact of protoplastis, and the present invention chooses 5～30mg/ml Glucanex prozyme as cell walls lyase liquid, and lysis efficiency is high, and protoplast liberation quantity is many.Stablizing of the kind of homeo-osmosis agent and the selection of concentration preparation, regeneration and the protoplastis to protoplastis is extremely important, the present invention selects 0.7～1mol/L KCl or NaCl as homeo-osmosis agent, has guaranteed that protoplastis prepares the stable of quantity and protoplastis.The present invention adopts and adds the high PDA of the oozing regeneration culture medium of bilayer of 0.004% sodium deoxycholate to carry out the regeneration of protoplastis, has improved the regeneration rate of protoplastis, and has facilitated selecting of mono-clonal thalline.
By finding out in Fig. 1 that protoplastis quantity is many, size is more or less the same, and mass ratio is higher, compares find out that most of protoplastis is stable with Fig. 2, and vigor is high;
By Fig. 3, Fig. 4, both relatively can find out that the protoplast regeneration rate of employing renovation process of the present invention is high, and bacterium colony is difficult for the overlapping isolation identification that is easy to.
Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or modification within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. a preparation method for the protoplastis of Chinese catalpa endogenetic fungus, is characterized in that, said method comprising the steps of:
Step 1, mycelial preparation;
Step 2, the preparation of protoplastis;
Step 3, protoplastis purifying;
Step 4, protoplast regeneration.
2. the preparation method of the protoplastis of Chinese catalpa endogenetic fungus as claimed in claim 1, it is characterized in that, described mycelium preparation is specially: get the fungal spore that is placed in-80 ℃ of preservations, under room temperature, thaw, in aseptic operating platform, draw 200 μ l, add in the 250ml Erlenmeyer flask that 100ml liquid PDA substratum is housed, be placed on shaking table, 23～28 ℃ are rocked cultivation, cultivate hypha fermentation, with the aseptic metal filtration sieve of 400 order, collect mycelia, put into mycelia scavenging solution and clean 2 times, with aseptic thieving paper suck dry moisture.
3. the preparation method of the protoplastis of Chinese catalpa endogenetic fungus according to claim 1, is characterized in that, described hypha fermentation culture condition: in temperature, be 23～28 ℃, the rotating speed of shaking table is 100rpm, and rocking incubation time is 12～24h.
4. the preparation method of the protoplastis of Chinese catalpa endogenetic fungus as claimed in claim 1, it is characterized in that, described protoplasm structure is standby to be specially: the agent of Glucanex enzyme homeo-osmosis is formulated as the enzyme liquid of 5～30mg/ml, fully dissolves and carries out enzymolysis, standby with aseptic filter membrane filtration sterilization.
5. Chinese catalpa endogenetic fungus protoplastis preparation according to claim 4 and renovation process, is characterized in that, described homeo-osmosis agent is 0.7～1mol/L KCl or NaCl solution.
6. Chinese catalpa endogenetic fungus protoplastis preparation according to claim 4 and renovation process, is characterized in that, the condition of described enzymolysis is: in temperature, be 28 ℃, the rotating speed of shaking table is 100rpm, and the time is 4～8h.
7. the preparation method of the protoplastis of Chinese catalpa endogenetic fungus as claimed in claim 1, it is characterized in that, described protoplastis purifying is specially: after mycelia enzymolysis, with 4 layers of aseptic lens wiping paper, filter and remove mycelia, by filtrate collection in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplastis STC to preserve the resuspended protoplastis of liquid and preserves.
8. the preparation method of the protoplastis of Chinese catalpa endogenetic fungus as claimed in claim 7, is characterized in that, described STC preserves consisting of of liquid: 1mol/L sorbyl alcohol, 0.01mol/L Tris-HCl, 0.01mol/L calcium chloride.
9. the preparation method of the protoplastis of Chinese catalpa endogenetic fungus as claimed in claim 1, it is characterized in that, described protoplast regeneration is specially: first in culture dish, spread the solid regenerated substratum of one deck protoplastis, standby after condensation, the semi-solid regeneration culture medium of preparation PDA, be placed under room temperature and be cooled to 40 ℃, draw 10ml substratum and add 100 μ L protoplastis suspensions, mix, directly be poured on the original solid regenerated substratum of culture dish, make it to be paved with plate and form a solid thin layer, treat that culture medium solidifying is placed in 28 ℃ of incubators and cultivate 2～3d, i.e. visible regeneration bacterium colony.
10. the preparation method of the protoplastis of Chinese catalpa endogenetic fungus as claimed in claim 1, is characterized in that, the solid regenerated substratum of described protoplastis is: the PDA solid medium that adds 0.004% sodium deoxycholate, 1mol/L N.F,USP MANNITOL, 15g/L agar.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410016834.1A CN103756917B (en) | 2014-01-14 | 2014-01-14 | The preparation method of the protoplast of Chinese catalpa endogenetic fungus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410016834.1A CN103756917B (en) | 2014-01-14 | 2014-01-14 | The preparation method of the protoplast of Chinese catalpa endogenetic fungus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103756917A true CN103756917A (en) | 2014-04-30 |
| CN103756917B CN103756917B (en) | 2016-05-04 |
Family
ID=50524245
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410016834.1A Active CN103756917B (en) | 2014-01-14 | 2014-01-14 | The preparation method of the protoplast of Chinese catalpa endogenetic fungus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103756917B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104387425A (en) * | 2014-10-11 | 2015-03-04 | 上海交通大学 | Method for condensation and separation of catalpa bungei essence Qiulingsu in catalpa bungei endophytic fungus broth and use thereof |
| CN107058132A (en) * | 2017-05-11 | 2017-08-18 | 中国农业科学院植物保护研究所 | The preparation method of T contraversa protoplast |
| CN107904178A (en) * | 2017-12-12 | 2018-04-13 | 福建农林大学 | A kind of method for preparing protoplast for not strangling bacterium |
| CN109136113A (en) * | 2018-11-13 | 2019-01-04 | 广西南亚热带农业科学研究所 | A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method |
| CN113913411A (en) * | 2021-12-01 | 2022-01-11 | 北京林业大学 | Composition for extracting fungal protoplast from plant and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1188802A (en) * | 1998-01-14 | 1998-07-29 | 中山大学 | Method for external source gene inversion of Ganoderma Lucidum |
| CN1358840A (en) * | 2001-10-11 | 2002-07-17 | 李刚 | Method for transferring expogenous gene into glossy |
| CN1511943A (en) * | 2002-12-30 | 2004-07-14 | 黑龙江大学 | Engineering strain and its preparing method, and method for preparing taxol thereof |
| CN101487022A (en) * | 2008-07-31 | 2009-07-22 | 上海交通大学 | Preparation of fermentation liquor for inhibiting liver cancer cell growth |
-
2014
- 2014-01-14 CN CN201410016834.1A patent/CN103756917B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1188802A (en) * | 1998-01-14 | 1998-07-29 | 中山大学 | Method for external source gene inversion of Ganoderma Lucidum |
| CN1358840A (en) * | 2001-10-11 | 2002-07-17 | 李刚 | Method for transferring expogenous gene into glossy |
| CN1511943A (en) * | 2002-12-30 | 2004-07-14 | 黑龙江大学 | Engineering strain and its preparing method, and method for preparing taxol thereof |
| CN101487022A (en) * | 2008-07-31 | 2009-07-22 | 上海交通大学 | Preparation of fermentation liquor for inhibiting liver cancer cell growth |
Non-Patent Citations (2)
| Title |
|---|
| 吴运帷等: "一株楸树内生真菌的鉴定及其发酵产物抗肿瘤特性研究", 《东北农业大学学报》, 30 June 2010 (2010-06-30), pages 90 - 93 * |
| 周选围等: "红豆杉内生真菌发酵培养基和原生质体制备酶系统的筛选", 《应用与环境生物学报》, 25 April 2006 (2006-04-25), pages 176 - 181 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104387425A (en) * | 2014-10-11 | 2015-03-04 | 上海交通大学 | Method for condensation and separation of catalpa bungei essence Qiulingsu in catalpa bungei endophytic fungus broth and use thereof |
| CN107058132A (en) * | 2017-05-11 | 2017-08-18 | 中国农业科学院植物保护研究所 | The preparation method of T contraversa protoplast |
| CN107904178A (en) * | 2017-12-12 | 2018-04-13 | 福建农林大学 | A kind of method for preparing protoplast for not strangling bacterium |
| CN107904178B (en) * | 2017-12-12 | 2021-06-01 | 福建农林大学 | Preparation method of protoplast of basidiomycetes |
| CN109136113A (en) * | 2018-11-13 | 2019-01-04 | 广西南亚热带农业科学研究所 | A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method |
| CN113913411A (en) * | 2021-12-01 | 2022-01-11 | 北京林业大学 | Composition for extracting fungal protoplast from plant and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103756917B (en) | 2016-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Villena et al. | Structural analysis of biofilms and pellets of Aspergillus niger by confocal laser scanning microscopy and cryo scanning electron microscopy | |
| CN103756917A (en) | Preparation method of protoplasts of catalpa bungei endophytic fungi | |
| Overy et al. | An assessment of natural product discovery from marine (sensu strictu) and marine-derived fungi | |
| Hameş-Kocabaş et al. | Isolation strategies of marine-derived actinomycetes from sponge and sediment samples | |
| Moharrer et al. | Biological synthesis of silver nanoparticles by Aspergillus flavus, isolated from soil of Ahar copper mine | |
| Zhang et al. | Survival analyses of symbionts isolated from Endocarpon pusillum Hedwig to desiccation and starvation stress | |
| CN103436450A (en) | Alkaline-tolerant and halophilic aspergillus strain and application thereof in environmental management | |
| CN110250210B (en) | Optimal DSE strain for promoting corn seed soaking and rooting | |
| CN102586116B (en) | Common chlorella as well as culturing method and application thereof | |
| CN109762757B (en) | High-yield coenzyme Q10 rhodobacter sphaeroides and mutation breeding and application thereof | |
| CN102864087B (en) | Phaffia rhodozyma strain with high yield of natural astaxanthin as well as breeding method and application thereof | |
| CN105969670B (en) | Phellinus igniarius strain and breeding method thereof | |
| CN103602591B (en) | A kind of schizochytrium limacinum and the method for the production of docosahexaenoic acid grease | |
| CN101988035A (en) | Method for screening high-lipid content mutant microalgae strain | |
| Vurro et al. | Optimization of the production of herbicidal toxins by the fungus Ascochyta caulina | |
| CN101773050B (en) | Method for rejuvenating and detoxifying edible fungal strain by using protoplast regeneration technology | |
| CN105018352A (en) | Fungal strain capable of producing kojic acid and preparation method of fungal strain | |
| JPWO2019225171A1 (en) | Tomato pathogenic fungus detection device and detection method using it | |
| CN105063012B (en) | A kind of preparation of yeasty fusant and rapid screening method | |
| CN105087390A (en) | High-yield itaconic acid strains and high-throughput screening method thereof based on deep-hole plate | |
| CN109825495B (en) | Method for high-throughput screening of monascus pigment high-yield strains | |
| CN101948772B (en) | High-efficiency strain for degrading polycyclic aromatic hydrocarbon and construction method and application thereof | |
| US10822583B2 (en) | Lipid biosynthesis and abiotic stress resilience in photosynthetic organisms | |
| CN101701230B (en) | Method for improving output of anticancer anthraquinone compound generated by Fusarium proliferatum via utilizing different vaccination ways | |
| CN106957839A (en) | A kind of method that immobilized yeast prepares cigarette Flos Rosae Rugosae extract |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200327 Address after: Room 105G, No. 199 GuoShoujing Road (Biological and Medical Innovation Building), Pudong New Area Free Trade Pilot Area, Shanghai, 201203 Co-patentee after: Tang Kexuan Patentee after: Shanghai Jiaotong University Intellectual Property Management Co., Ltd. Address before: 200240 Dongchuan Road, Shanghai, No. 800, No. Patentee before: SHANGHAI JIAO TONG University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200827 Address after: 611134 No.301, building 11, No.399, Xuehai Road, Wenjiang District, Chengdu City, Sichuan Province Patentee after: Chengdu Shangjiao Zhiyuan Biotechnology Co., Ltd Address before: Room 105G, No. 199 GuoShoujing Road (Biological and Medical Innovation Building), Pudong New Area Free Trade Pilot Area, Shanghai, 201203 Co-patentee before: Tang Kexuan Patentee before: Shanghai Jiaotong University Intellectual Property Management Co.,Ltd. |