CN103756917B - The preparation method of the protoplast of Chinese catalpa endogenetic fungus - Google Patents
The preparation method of the protoplast of Chinese catalpa endogenetic fungus Download PDFInfo
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Abstract
The invention provides a kind of preparation method of protoplast of Chinese catalpa endogenetic fungus; Said method comprising the steps of: step 1, mycelial preparation; Step 2, the preparation of protoplast; Step 3, protoplast purifying; Step 4, protoplast regeneration. Protoplast quantity prepared by the method for the invention is more, can reach 6 × 107Individual/milligram mycelia, the vigor of the protoplast of acquisition is higher, and regeneration rate can reach 1.2%. The present invention selects to cultivate the mycelium of 12-24h, and fungi is in fast growing period, and growth is fast, and cell membrane is easy to the cracking of enzyme, and the quantity of the protoplast discharging is many, is difficult for cracking, and vigor is high. Does the present invention choose 5～30mg/ml? Glucanex complex enzyme is as cell membrane lyases liquid, and lysis efficiency is high, and protoplast liberation quantity is many. The present invention selects KCl or NaCl as homeo-osmosis agent, has ensured that protoplast prepares the stable of quantity and protoplast.
Description
Technical field
The present invention relates to the field of biology of fungi, especially a kind of preparation method of protoplast 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. Interior lifeFungi can produce and the same or analogous bioactivator of host plant, has huge potential value, can becomeThe source of new type anticancer medicine. This seminar from juglans mandshurica separation and Extraction one strain there is the interior life of very strong anticancer vigorFungi, is numbered FSN002, and this bacterium of Preliminary Identification is Deuteromycotina, Hyphomycetes, and Moniliales, trichoderma, itsFermentation extract has very strong inhibitory action to HCC. This is a brand-new bacterial classification that this laboratory is exclusive, existsOpen in Chinese patent CN101487022, but the drawbacks limit such as the biological active matter quality of its generation is few, unstable its enterRow suitability for industrialized production, carries out thalline transformation by means such as bio-transformation, structure mutant to it, improves its anticancer vigorAnd study its anticancer mechanism, and can utilize better this fungus resource, be the base that carries out above research and build protoplastPlinth and key technology.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of system of protoplast of Chinese catalpa endogenetic fungusPreparation Method.
The present invention is achieved by the following technical solutions:
The preparation method who the invention provides a kind of protoplast of Chinese catalpa endogenetic fungus, said method comprising the steps of:
Step 1, mycelial preparation;
Step 2, the preparation of protoplast;
Step 3, protoplast purifying;
Step 4, protoplast regeneration.
Preferably, described mycelium preparation is specially: get the fungal spore that is placed in-80 DEG C of preservations, under room temperature, thaw,In aseptic operating platform, draw 200 μ l, add in the 250ml conical flask that 100ml liquid PDA culture medium is housed, putOn shaking table, 23～28 DEG C are rocked cultivation, cultivate hypha fermentation, collect mycelia with the aseptic metal filtration sieve of 400 order, putEnter in mycelia cleaning fluid and clean 2 times, with aseptic blotting paper suck dry moisture.
Preferably, described hypha fermentation condition of culture: be 23～28 DEG C in temperature, the rotating speed of shaking table is 100rpm, shakesRolling incubation time is 12～24h.
Preferably, described protoplasm structure is standby is specially: the agent of Glucanex enzyme homeo-osmosis is formulated as 5～30mg/mlEnzyme liquid, fully dissolve and carry out enzymolysis, for subsequent use with aseptic filter membrane filtration sterilization.
Preferably, described homeo-osmosis agent is 0.7～1mol/LKCl or NaCl solution.
Preferably, the condition of described enzymolysis is: be 28 DEG C in temperature, the rotating speed of shaking table is 100rpm, and the time is 4～8h.
Preferably, described protoplast purifying is specially: after mycelia enzymolysis, filter and remove mycelia with 4 layers of aseptic lens wiping paper,Filtrate is collected in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplast STC to preserveThe resuspended protoplast of liquid is preserved.
Preferably, described STC preserves consisting of of liquid: 1mol/L sorbierite, 0.01mol/LTris-HCl, 0.01Mol/L calcium chloride.
Preferably, described protoplast regeneration is specially: first in culture dish, spread the solid regenerated culture medium of one deck protoplast,For subsequent use after condensation, the semi-solid regeneration culture medium of preparation PDA, is placed under room temperature and is cooled to 40 DEG C, draws 10ml and cultivatesBase adds 100 μ L protoplast suspensions, mixes, and is directly poured on the original solid regenerated culture medium of culture dish, makesThe plate that is paved with form a solid thin layer, treat that culture medium solidifying is placed in 28 DEG C of incubators to cultivate 2～3d, visibleRegeneration bacterium colony.
Preferably, the solid regenerated culture medium of described protoplast is: add 0.004% deoxysodium cholate, 1mol/L sweetReveal the PDA solid medium of alcohol, 15g/L agar.
Compared with prior art, the present invention has following beneficial effect:
(1) the protoplast quantity that prepared by the method for the invention is more, can reach 6 × 107Individual/milligram mycelia, obtainsThe vigor of protoplast higher, regeneration rate can reach 1.2%.
(2) the hyphal cell wall construction difference of different cell ages, affects protoplast preparation, and the present invention selects to cultivate 12-24The mycelium of h, fungi is in fast growing period, and growth is fast, and cell membrane is easy to the cracking of Glucanex enzyme, dischargesThe quantity of protoplast is many, is difficult for cracking, and vigor is high.
(3) many, the complex structure of fungal cell wall composition, the preparation impact of the selection of cell membrane lyases on protoplastVery large, the present invention chooses 5～30mg/mlGlucanex complex enzyme as cell membrane lyases liquid, and lysis efficiency is high, formerRaw plastid burst size is many.
(4) kind of homeo-osmosis agent and the selection of concentration preparation, regeneration and the protoplast to protoplast is steadyFixed extremely important, the present invention selects 0.7～1mol/LKCl or NaCl as homeo-osmosis agent, has ensured protoplastStablizing of preparation quantity and protoplast.
(5) the present invention adopts and adds the high PDA of the oozing regeneration culture medium of bilayer of 0.004% deoxysodium cholate to carry out protoplastRegeneration, improved the regeneration rate of protoplast, and facilitated selecting of monoclonal thalline.
Brief description of the drawings
By reading the detailed description of non-limiting example being done with reference to the following drawings, other spy of the present inventionLevy, object and advantage will become more obvious:
Fig. 1 is the optical microscope image of protoplast prepared in embodiment 1;
Fig. 2 is the image that protoplast prepared in embodiment 1 carries out the fluorescence microscope of vigor assessment, if protoplastCan show that 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.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail. Following examples will contribute to the technology people of this areaMember further understands the present invention, but does not limit in any form the present invention. It should be pointed out that the common skill to this areaArt personnel, without departing from the inventive concept of the premise, can also make some distortion and improvement. These all belong toProtection scope of the present invention.
Embodiment 1
The present embodiment relates to a kind of preparation method of protoplast of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 DEG C of preservations, thaw under room temperature, in aseptic operating platformDraw 200 μ l and add in the 250ml conical flask that 100ml liquid PDA culture medium is housed, be placed on 100rpm shaking tableUpper, 25 DEG C are rocked cultivation, cultivate 12h, collect mycelia with the aseptic metal filtration sieve of 400 order, put into mycelia cleaning fluidClean 2 times, with aseptic blotting paper suck dry moisture;
(2) preparation of protoplast: Glucanex enzyme is formulated as 10mg/ml with the agent of 0.8mol/LKCl homeo-osmosisEnzyme liquid, for subsequent use with aseptic filter membrane filtration sterilization after fully dissolving. Mycelia is placed in to enzyme liquid in 28 DEG C, 100rpmRotating speed rocks enzymolysis 6h;
(3) protoplast purifying: after mycelia enzymolysis 6h, filter and remove mycelia with 4 layers of aseptic lens wiping paper, filtrate is receivedCollect in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplast STC to preserve liquid resuspended primaryPlastid is preserved, the optical microscope image of seeing Fig. 1 the present embodiment protoplast. Fig. 2 is the prepared plasm of the present embodimentBody carries out the image of the fluorescence microscope of vigor assessment, represents that it has vigor if protoplast can show fluorescence; By Fig. 1In can find out that protoplast quantity is many, size is more or less the same, mass ratio is higher.
The protoplast quantity that the present embodiment method obtains reaches 6 × 107Individual/milligram mycelia.
Embodiment 2
The operating procedure of Formation and regeneration method that the present embodiment relates to a kind of protoplast of Chinese catalpa endogenetic fungus is as follows:
(1) mycelium preparation: get the fungal spore that is placed in-80 DEG C of preservations, thaw under room temperature, in aseptic operating platformDraw 200 μ l and add in the 250ml conical flask that 100ml liquid PDA culture medium is housed, be placed on 100rpm shaking tableUpper, 25 DEG C are rocked cultivation, cultivate 24h, collect mycelia with the aseptic metal filtration sieve of 400 order, put into mycelia cleaning fluidClean 2 times, with aseptic blotting paper suck dry moisture;
(2) protoplast preparation: Glucanex enzyme is formulated as 5mg/ml with 0.7mol/LNaCl homeo-osmosis liquidEnzyme liquid, for subsequent use with aseptic filter membrane filtration sterilization after fully dissolving. Mycelia is placed in to enzyme liquid in 28 DEG C, 100rpmRotating speed rocks enzymolysis 6h;
(3) protoplast purifying: after mycelia enzymolysis 6h, filter and remove mycelia with 4 layers of aseptic lens wiping paper, by filtrateCollect in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplast STC to preserve liquid resuspendedProtoplast is preserved.
The protoplast quantity that the present embodiment method obtains reaches 2 × 107Individual/milligram mycelia.
Embodiment 3
The present embodiment relates to a kind of preparation of protoplast of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 DEG C of preservations, thaw under room temperature, in aseptic operating platformDraw 200 μ l and add in the 250ml conical flask that 100ml liquid PDA culture medium is housed, be placed on 100rpm shaking tableUpper, 25 DEG C are rocked cultivation hypha fermentation, cultivate 18h, collect mycelia with the aseptic metal filtration sieve of 400 order, put into myceliaIn cleaning fluid, clean 2 times, with aseptic blotting paper suck dry moisture;
(2) protoplast preparation: Glucanex enzyme is formulated as 20mg/ml with 1mol/LNaCl homeo-osmosis liquidEnzyme liquid, for subsequent use with aseptic filter membrane filtration sterilization after fully dissolving, mycelia is placed in to enzyme liquid in 28 DEG C, 100rpmRotating speed rocks enzymolysis 2h;
(3) protoplast purifying: after mycelia enzymolysis 2h, filter and remove mycelia with 4 layers of aseptic lens wiping paper, by filtrateCollect in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplast STC to preserve liquid resuspendedProtoplast is preserved.
The protoplast quantity that the present embodiment method obtains reaches 1.5 × 107Individual/milligram mycelia.
Embodiment 4
The present embodiment relates to a kind of preparation of protoplast of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 DEG C of preservations, thaw under room temperature, in aseptic operating platformDraw 200 μ l and add in the 250ml conical flask that 100ml liquid PDA culture medium is housed, be placed on 100rpm shaking tableUpper, 25 DEG C are rocked cultivation, cultivate 14h, collect mycelia with the aseptic metal filtration sieve of 400 order, put into mycelia cleaning fluidClean 2 times, with aseptic blotting paper suck dry moisture.
(2) protoplast preparation: Glucanex enzyme is formulated as 30mg/ml with 0.8mol/LKCl homeo-osmosis liquidEnzyme liquid, for subsequent use with aseptic filter membrane filtration sterilization after fully dissolving. Mycelia is placed in to enzyme liquid in 28 DEG C, 100rpmRotating speed rocks enzymolysis 8h.
(3) protoplast purifying: after mycelia enzymolysis 8h, filter and remove mycelia with 4 layers of aseptic lens wiping paper, by filtrateCollect in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplast STC to preserve liquid resuspendedProtoplast is preserved.
The present embodiment method obtains protoplast quantity and reaches 5 × 107Individual/milligram mycelia.
Embodiment 5
The present embodiment relates to the renovation process of the protoplast of Chinese catalpa endogenetic fungus prepared by aforementioned 1-4, is specially:
First in culture dish, spread the solid regenerated culture medium of one deck protoplast (protoplast that embodiment 1～4 obtains),For subsequent use after condensation, the semi-solid regeneration culture medium of preparation PDA (add 0.004% deoxysodium cholate, 1mol/L sweet mellow wine,The PDA solid medium of 15g/L agar), be placed under room temperature and be cooled to 40 DEG C, draw 10ml culture medium and add 100μ L protoplast suspension, mixes, and is directly poured on the original solid regenerated culture medium of culture dish, makes it to be paved with plate shapeBecome a solid thin layer. Treat that culture medium solidifying is placed in 28 DEG C of incubators and cultivate 2d.
The regeneration rate of the protoplast 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 protoplast of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 DEG C of preservations, thaw under room temperature, in aseptic operating platformDraw 200 μ l and add in the 250ml conical flask that 100ml liquid PDA culture medium is housed, be placed on 100rpm shaking tableUpper, 25 DEG C are rocked cultivation, cultivate 10h, collect mycelia with the aseptic metal filtration sieve of 400 order, put into mycelia cleaning fluidClean 2 times, with aseptic blotting paper suck dry moisture;
(2) protoplast preparation: Glucanex enzyme is formulated as 1mg/ml with 0.8mol/LKCl homeo-osmosis liquidEnzyme liquid, for subsequent use with aseptic filter membrane filtration sterilization after fully dissolving, mycelia is placed in to enzyme liquid in 28 DEG C, 100rpmRotating speed rocks enzymolysis 4h.
(3) protoplast purifying: after mycelia enzymolysis 4h, filter and remove mycelia with 4 layers of aseptic lens wiping paper, by filtrateCollect in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplast STC to preserve liquid resuspendedProtoplast is preserved.
The protoplast quantity that this comparative example 1 obtains only has 1.5 × 105Individual/milligram mycelia.
Comparative example 2
This comparative example relates to a kind of preparation method of protoplast of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 DEG C of preservations, thaw under room temperature, in aseptic operating platformDraw 200 μ l and add in the 250ml conical flask that 100ml liquid PDA culture medium is housed, be placed on 100rpm shaking tableUpper, 25 DEG C are rocked cultivation, cultivate 12h, collect mycelia with the aseptic metal filtration sieve of 400 order, put into mycelia cleaning fluidClean 2 times, with aseptic blotting paper suck dry moisture;
(2) protoplast preparation: Glucanex enzyme is formulated as 4mg/ml with 0.8mol/L sweet mellow wine homeo-osmosis liquidEnzyme liquid, for subsequent use with aseptic filter membrane filtration sterilization after fully dissolving. Mycelia is placed in to enzyme liquid in 28 DEG C, 100rpmRotating speed rocks enzymolysis 6h.
(3) protoplast purifying: after mycelia enzymolysis 6h, filter and remove mycelia with 4 layers of aseptic lens wiping paper, by filtrateCollect in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplast STC to preserve liquid resuspendedProtoplast is preserved.
The protoplast quantity that this comparative example 2 obtains only has 1.3 × 105Individual/milligram mycelia.
Comparative example 3
This comparative example relates to a kind of preparation method of protoplast of Chinese catalpa endogenetic fungus, comprises the steps:
(1) mycelium preparation: get the fungal spore that is placed in-80 DEG C of preservations, thaw under room temperature, in aseptic operating platformDraw 200 μ l and add in the 250ml conical flask that 100ml liquid PDA culture medium is housed, be placed on 100rpm shaking tableUpper, 25 DEG C are rocked cultivation, cultivate 10h, collect mycelia with the aseptic metal filtration sieve of 400 order, put into mycelia cleaning fluidClean 2 times, with aseptic blotting paper suck dry moisture;
(2) protoplast preparation: Glucanex enzyme is formulated as 3mg/ml with 0.6mol/LKCl homeo-osmosis liquidEnzyme liquid, for subsequent use with aseptic filter membrane filtration sterilization after fully dissolving. Mycelia is placed in to enzyme liquid in 28 DEG C, 100rpmRotating speed rocks enzymolysis 6h;
(3) protoplast purifying: after mycelia enzymolysis 6h, filter and remove mycelia with 4 layers of aseptic lens wiping paper, by filtrateCollect in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplast STC to preserve liquid resuspendedProtoplast is preserved.
The protoplast quantity that this comparative example 3 obtains only has 1 × 104Individual/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: the protoplast (protoplast that comparative example 1～3 obtains) that absorption 100 μ l have diluted is moltenLiquid, in the solid regenerated culture medium flat plate of PDA, is directly coated with evenly with spreading rod gently, and culture dish is placed in to 28 DEG C of incubatorsMiddle cultivation 2～3d left and right.
The regeneration rate of the protoplast that this comparative example 4 obtains only has 0.1%, as the picture of Fig. 4 protoplast regeneration.
In sum: protoplast quantity prepared by the method for the invention is more, can reach 6 × 107Individual/milligram mycelia,The vigor of the protoplast obtaining is higher, and regeneration rate can reach 1.2%. Select the hyphal cell wall construction difference of different cell ages,Affect protoplast preparation, the present invention selects to cultivate the mycelium of 12-24h, and fungi is in fast growing period, and growth is fast,Cell membrane is easy to the cracking of Glucanex enzyme, and the quantity of the protoplast discharging is many, is difficult for cracking, and vigor is high. VeryBacterial cell wall composition is many, complex structure, and the selection of cell membrane lyases is very large on the preparation impact of protoplast, the present inventionChoose 5～30mg/mlGlucanex complex enzyme as cell membrane lyases liquid, lysis efficiency is high, protoplast liberationQuantity is many. Stablizing of the kind of homeo-osmosis agent and the selection of concentration preparation, regeneration and the protoplast to protoplastExtremely important, the present invention selects 0.7～1mol/LKCl or NaCl as homeo-osmosis agent, has ensured protoplastStablizing of preparation quantity and protoplast. The present invention adopts the high PDA of the oozing regeneration of the bilayer training that adds 0.004% deoxysodium cholateFoster base carries out the regeneration of protoplast, has improved the regeneration rate of protoplast, and has facilitated selecting of monoclonal thalline.
By finding out in Fig. 1 that protoplast quantity is many, size is more or less the same, and mass ratio is higher, compares and finds out with Fig. 2Most of protoplast is stable, 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, bacterium colonyBe difficult for the overlapping qualification that is easy to separate.
Above specific embodiments of the invention are described. It will be appreciated that, the present invention is not limited toState specific implementations, those skilled in the art can make various distortion or amendment within the scope of the claims,This does not affect flesh and blood of the present invention.
Claims (4)
1. a preparation method for the protoplast of Chinese catalpa endogenetic fungus, is characterized in that, described method comprises following stepRapid:
Step 1, mycelial preparation;
Step 2, the preparation of protoplast;
Step 3, protoplast purifying;
Step 4, protoplast regeneration;
Described mycelium preparation is specially: get the fungal spore that is placed in-80 DEG C of preservations, under room temperature, thaw, aseptic behaviourDo to draw 200 μ l in platform, add in the 250ml conical flask that 100ml liquid PDA culture medium is housed, be placed on shaking table,23～28 DEG C are rocked cultivation, cultivate hypha fermentation, collect mycelia with the aseptic metal filtration sieve of 400 order, put into mycelia and cleanIn liquid, clean 2 times, with aseptic blotting paper suck dry moisture;
Described hypha fermentation condition of culture: be 23～28 DEG C in temperature, the rotating speed of shaking table is 100rpm, rocks while cultivationBetween be 12～24h;
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 dissolve and carry out enzymolysis, for subsequent use with aseptic filter membrane filtration sterilization;
Described protoplast regeneration is specially: first in culture dish, spread the solid regenerated culture medium of one deck protoplast, after condensationFor subsequent use, the semi-solid regeneration culture medium of preparation PDA, is placed under room temperature and is cooled to 40 DEG C, draws 10ml culture medium and adds100 μ L protoplast suspensions, mix, and are directly poured on the original solid regenerated culture medium of culture dish, make it to be paved with flatWare forms a solid thin layer, treats that culture medium solidifying is placed in 28 DEG C of incubators to cultivate 2～3d, the bacterium colony of regenerating as seen;
The solid regenerated culture medium of described protoplast is: add 0.004% deoxysodium cholate, 1mol/L sweet mellow wine,The PDA solid medium of 15g/L agar;
Described protoplast purifying is specially: after mycelia enzymolysis, filter and remove mycelia with 4 layers of aseptic lens wiping paper, filtrate is receivedCollect in centrifuge tube, the centrifugal 10min of 4000rpm, abandoning supernatant, adds protoplast STC to preserve liquid resuspended formerRaw plastid is preserved.
2. the preparation method of the protoplast of Chinese catalpa endogenetic fungus according to claim 1, is characterized in that, described inHomeo-osmosis agent is 0.7～1mol/LKCl or NaCl solution.
3. the preparation method of the protoplast of Chinese catalpa endogenetic fungus according to claim 1, is characterized in that, described inThe condition of enzymolysis is: be 28 DEG C in temperature, the rotating speed of shaking table is 100rpm, and the time is 4～8h.
4. the preparation method of the protoplast of Chinese catalpa endogenetic fungus as claimed in claim 1, is characterized in that, described inSTC preserves consisting of of liquid: 1mol/L sorbierite, 0.01mol/LTris-HCl, 0.01mol/L calcium chloride.
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| 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 |
| 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 |
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