CN110172412A - A kind of method for preparing protoplast of true pleurotus cornucopiae - Google Patents
A kind of method for preparing protoplast of true pleurotus cornucopiae Download PDFInfo
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- CN110172412A CN110172412A CN201910538167.6A CN201910538167A CN110172412A CN 110172412 A CN110172412 A CN 110172412A CN 201910538167 A CN201910538167 A CN 201910538167A CN 110172412 A CN110172412 A CN 110172412A
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Abstract
The present invention provides a kind of method for preparing protoplast of true pleurotus cornucopiae, change tradition and generates spherical mycelia extraction protoplast method equipped with PDB culture medium conical flask shaking table culture, the stationary culture of sample strip glass dregs, the culture mycelia of regular shaking flask and sterile nylon net filtration method, choose different mycelia cell ages, lywallzyme enzyme solution concentration, enzymolysis time, filter centrifugation rate and time etc., and according to protoplast diameter using G2 funnel filtering protoplast etc., 20ul drop is drawn on blood counting chamber, the microscopy under 40 times of optical microscopy, it counts, form a whole set of true pleurotus cornucopiae method for preparing protoplast.Using the method for the present invention, true pleurotus cornucopiae protoplast quantity reaches 1.0-2.2 × 107A/mL meets the research requirement of the molecular biology such as true pleurotus cornucopiae genetic transformation.
Description
Technical field
The invention belongs to the method for Microbial Breeding in bioengineering, in particular to prepared by the protoplast of a kind of true pleurotus cornucopiae
Method.
Background technique
Mushroom industry occupies an important position in agricultural economy, and " point grass Cheng Jin, turn waste into wealth " realizes circular economy,
It is Agricultural Sustainable Development.True pleurotus cornucopiae (Hypsizigus marmoreus), also known as spot jade gill fungus, beautiful gill fungus, white jade, seafood mushroom,
Letter happiness mushroom etc., taste is fresher than oyster mushroom, and matter is more tough than mushroom, and meat is thicker than sliding mushroom, has the fragrant unique taste of crab, belongs to mycota
(Fungi), Basidiomycota (Basidiomycotina), agaric guiding principle (Agaricomycetes), Agaricales
(Agaricales), from Zhe San section (Lyop Hyllaceae), beautiful gill fungus category (Hypsizigus), it is that north temperate zone is a kind of rare rare
Edible fungus, be suitable for north temperate zone spring and autumn and Winter Cultivation, NATURAL DISTRIBUTION is in Europe, Siberia, Japan and North America and other places.
Pilot study is carried out from key factor of the mycelia to the preparation of true pleurotus cornucopiae protoplast herein, to utilize
Protoplast High-efficient Genetic Transformation technology and Protoplast Fusion Technique cultivate Multifucntional bacterial strain and lay the foundation.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation methods of the protoplast of true pleurotus cornucopiae, make the system of its protoplast
Standby rate reaches 1.0-2.2 × 107A/mL.
To achieve the above object, the present invention adopts the following technical scheme:
The technical scheme is that
(1) it connects on healthy and strong true pleurotus cornucopiae mycelia to PDA thallus culture medium flat plate, 24 DEG C are protected from light culture 144h-240h.Selection growth is strong
Strong mycelia plate, connect mycelia in 150ml with glass dregs PDB fluid nutrient medium in, glass dregs size be 0.5-1cm ×
0.4 cm × 0.2cm is protected from light stationary culture 96h-120h, and after cultivating 48h, every 8h shakes conical flask 10min manually, cuts off new length
Mycelia.The 50ml that transfers after 120h contains the fluid nutrient medium of healthy and strong mycelia to 120ml with cullet slag PDB fluid nutrient medium
In, stationary culture 48h-144h again, every 8h is in 250rpm, 24 DEG C of shaking table culture 1h.
(2) in superclean bench, go out mycelia with two layers of sterile nylon filtered through gauze, with aseptic water washing, then use
It 0.6mol/L mannitol wash 2 times, is blotted with the filter paper of sterilizing.It is prepared with 0.6mol/L mannitol solution molten containing 1-4wt.%
The enzyme solution of wall enzyme, mycelia: enzyme solution is added in 1:10g/mL ratio equipped in mycelial centrifuge tube, and centrifuge tube is put into 30 DEG C
1-4h, every 30min are digested in water-bath, overturn shake up 20 times up and down.
(3) it in superclean bench, into centrifuge tube and is sealed with G2 funnel filtering crude extract, in supercentrifuge
It is centrifuged 10min under the conditions of 3000-6000rpm, outwells supernatant, draws the steady penetration enhancer of 5ml with liquid-transfering gun and is added in centrifuge tube,
Precipitating is mixed, repeats to be centrifuged 10min under the conditions of 3000-6000rpm, outwells supernatant, add the steady penetration enhancer of 1ml, precipitating is mixed.
20ul drop is drawn on blood counting chamber, the microscopy under 40 times of optical microscopy counts.Protoplast quantity reaches 1.0-
2.2× 107A/mL.
The method for preparing protoplast of the true pleurotus cornucopiae, optimal processing parameter is:
(1) it connects on healthy and strong true pleurotus cornucopiae mycelia to PDA thallus culture medium flat plate, 24 DEG C are protected from light culture 200h；Select robust growth
Mycelia plate connects mycelia in the 150mlPDB fluid nutrient medium with glass dregs, and glass dregs size is the cm of 0.5-1cm × 0.4
× 0.2cm is protected from light stationary culture 110h, and after cultivating 48h, every 8h shakes conical flask 10min manually, cuts off newly long mycelia；
The 50ml that transfers after 120h contains the fluid nutrient medium of healthy and strong mycelia into other 120ml band cullet slag PDB fluid nutrient medium, then
Secondary stationary culture 96h, every 8h are in 250rpm, 24 DEG C of shaking table culture 1h；
(2) in superclean bench, go out mycelia with two layers of sterile nylon filtered through gauze, with aseptic water washing, then use 0.6mol/
It L mannitol wash 2 times, is blotted with the filter paper of sterilizing, with the enzyme for the lywallzyme containing 2wt.% that 0.6mol/L mannitol solution is prepared
Liquid, mycelia: enzyme solution is added in 1:10g/mL ratio equipped in mycelial centrifuge tube, and centrifuge tube is put into 30 DEG C of water-baths
3h, every 30min are digested, overturns shake up 20 times up and down；
(3) it in superclean bench, into centrifuge tube and is sealed with G2 funnel filtering crude extract, in supercentrifuge 4000rpm
Under the conditions of be centrifuged 10min, outwell supernatant, with liquid-transfering gun draw the steady penetration enhancer of 5ml be added in centrifuge tube, will precipitating mix, weight
It is centrifuged 10min under the conditions of multiple 4000rpm, supernatant is outwelled, adds the steady penetration enhancer of 1ml, precipitating is mixed.20ul drop is drawn in hemocytometer
On number, the microscopy under 40 times of optical microscopy is counted.Protoplast quantity reaches 2.2 × 107A/mL or more.
Effect of the present invention is:
The method for preparing protoplast of the true pleurotus cornucopiae of the application changes traditional shaking table and shakes bacterium, and growing spherical mycelia is static gas wave refrigerator, glass
Glass slag cuts off mycelia, constantly grows the tender mycelia of children.Explore different cell ages, enzymolysis time, enzyme solution concentration, filter centrifugation rate and mistake
Some column key technology points such as instrument are filtered, the peculiar true pleurotus cornucopiae protoplast extracting method of extraction is formed, are solved true for a long time
Pleurotus cornucopiae protoplast prepares the low problem of recovery rate, is that the molecular biology researches such as true pleurotus cornucopiae genetic transformation are taken a firm foundation.
1. preparation process is simple, easy to operate；
2. the preparation rate of protoplast reaches 2.2 × 107A/mL or more, regeneration rate is up to 40%.
Detailed description of the invention
Influence of Fig. 1 difference cell age to protoplast.
Influence of Fig. 2 difference enzymolysis time to protoplast.
Influence of Fig. 3 difference enzyme concentration to protoplast.
Influence of Fig. 4 difference centrifugal speed to protoplast.
Specific embodiment
A kind of method for preparing protoplast of true pleurotus cornucopiae provides the method for being suitble to prepare true pleurotus cornucopiae protoplast quantity.
True pleurotus cornucopiae used in embodiment 1-5 is University Of Agriculture and Forestry In Fujian's genome and biotech research center preservation strain,
Strain number is Hy61.
Embodiment 1:
(1) it connects on healthy and strong true pleurotus cornucopiae mycelia to PDA thallus culture medium flat plate, 24 DEG C are protected from light culture 200h.Number is A1, A2, A3,
B1、B2、B3。
(2) the mycelia plate for selecting robust growth connects mycelia in PDB fluid nutrient medium of 3 bottles of 150ml with glass dregs
Number is A1, A2, A3 in 250ml conical flask, and glass dregs size is 0.5-1cm × 0.4 cm × 0.2cm, is protected from light stationary culture
110h, after cultivating 48h, every 8h shakes conical flask 10min manually, cuts off newly long mycelia, switching 50ml contains stalwartness after 120h
For liquid mycelia to other 120ml in cullet slag PDB fluid nutrient medium, cultivating 96h again, every 8h is placed in 250rpm, and 24
DEG C shaking table culture 1h；
The mycelia plate for selecting robust growth connects mycelia and trains in PDB liquid of 3 bottles of 150ml with micro glass beads (r=0.1cm)
It supports in base 250ml conical flask, number B1, B2, B3 are put into 250rpm, 24 DEG C of shaking table cultures, and switching 50ml contains strong after 120h
Strong liquid mycelia is into the 120mlPDB fluid nutrient medium further with micro glass beads, 250rpm, 24 DEG C of shaking table culture 72h.
(3) it in superclean bench, uses two layers of sterile nylon filtered through gauze to go out mycelia respectively the mycelia that 6 bottles are cultivated, uses
Aseptic water washing is then used 0.6mol/L mannitol wash 2 times, is blotted with the filter paper of sterilizing；With 0.6mol/L mannitol solution
The enzyme solution for the lywallzyme containing 2wt.% prepared, mycelia: enzyme solution is added in 1:10g/mL ratio equipped in mycelial centrifuge tube,
Enzyme solution is filtered with biofilter, ready-to-use, and centrifuge tube is put into 30 DEG C of water-baths and digests 3h, and every 30min is overturned up and down
It shakes up 20 times.
(4) it in superclean bench, into centrifuge tube and is sealed with G2 funnel filtering crude extract, in supercentrifuge
It is centrifuged 10min under the conditions of 4000rpm, outwells supernatant, draws the steady penetration enhancer of 5ml with liquid-transfering gun and is added in centrifuge tube, will precipitate
It mixes, repeats to be centrifuged 10min under the conditions of 4000rpm, outwell supernatant, add the steady penetration enhancer of 1ml, precipitating is mixed.Draw 20ul drop in
On blood counting chamber, the microscopy under 40 times of optical microscopy counts, the results are shown in Table 1.
Training method is screened when 1 protoplast of table obtains
It can be obtained by table 1, most suitable cultural hypha mode is the static gas wave refrigerator with glass dregs, periodically shakes up, cuts off mycelia.
Embodiment 2:
(1) it connects on healthy and strong true pleurotus cornucopiae mycelia to PDA thallus culture medium flat plate, 24 DEG C are protected from light culture 200h.Select robust growth
Mycelia plate, connecing mycelia, (glass dregs size is the cm of 0.5-1cm × 0.4 in PDB fluid nutrient medium of the 150ml with glass dregs
× 0.2cm), it is protected from light stationary culture 110h, after cultivating 48h, every 8h shakes conical flask 10min manually, cuts off newly long mycelia.
The 50ml that transfers after 120h contains healthy and strong liquid mycelia into other 120ml band cullet slag PDB fluid nutrient medium, number 1,2,3
For 24 hours, 72 h of cultural hypha of number 4,5,6 bacterium bottle, number 7,8,9 bacterium bottle cultural hypha 96h are numbered the cultural hypha of bacterium bottle
10, the cultural hypha 120h of 11,12 bacterium bottle, number 13,14,15 bacterium bottle cultural hypha 144h.The every 8h of every group of bacterium bottle in 250rpm,
24 DEG C of shaking table culture 1h.
(2) in superclean bench, go out mycelia with two layers of sterile nylon filtered through gauze, with aseptic water washing, then use
It 0.6mol/L mannitol wash 2 times, is blotted with the filter paper of sterilizing.The molten wall containing 2wt.% prepared with 0.6mol/L mannitol solution
The enzyme solution of enzyme, mycelia: enzyme solution is added in 1:10g/mL ratio equipped in mycelial centrifuge tube, enzyme solution biofilter mistake
Filter, it is ready-to-use, centrifuge tube is put into 30 DEG C of water-baths and digests 3h, every 30min is overturned shake up 20 times up and down.
(3) it in superclean bench, into centrifuge tube and is sealed with G2 funnel filtering crude extract, in supercentrifuge
It is centrifuged 10min under the conditions of 4000rpm, outwells supernatant, draws the steady penetration enhancer of 5ml with liquid-transfering gun and is added in centrifuge tube, will precipitate
It mixes, repeats to be centrifuged 10min under the conditions of 4000rpm, outwell supernatant, add the steady penetration enhancer of 1ml, precipitating is mixed.Draw 20ul drop in
On blood counting chamber, the microscopy under 40 times of optical microscopy is counted.
It can be obtained by Fig. 1, most suitable mycelia cell age is 96h.
Embodiment 3:
(1) it connects on healthy and strong true pleurotus cornucopiae mycelia to PDA thallus culture medium flat plate, 24 DEG C are protected from light culture 200h.Select robust growth
Mycelia plate, connecing mycelia, (glass dregs size is the cm of 0.5-1cm × 0.4 in PDB fluid nutrient medium of the 150ml with glass dregs
× 0.2cm), it is protected from light stationary culture 110h, after cultivating 48h, every 8h shakes conical flask 10min manually, cuts off newly long mycelia.
Containing healthy and strong liquid mycelia into other 120ml band cullet slag PDB fluid nutrient medium, stationary culture culture 96h again, often
8h is in 250rpm, 24 DEG C of shaking table culture 1h.
(2) in superclean bench, go out mycelia with two layers of sterile nylon filtered through gauze, with aseptic water washing, then use
It 0.6mol/L mannitol wash 2 times, is blotted with the filter paper of sterilizing.The molten wall containing 2wt.% prepared with 0.6mol/L mannitol solution
The enzyme solution of enzyme, mycelia: enzyme solution is added in 1:10g/mL ratio equipped in mycelial centrifuge tube, enzyme solution biofilter mistake
Filter, it is ready-to-use, 1,2,3 centrifuge tube of number is put into 30 DEG C of water-baths and digests 1.5h, number 4,5,6 centrifuge tube, 30 DEG C of water-baths
2h is digested in pot, 7,8,9 centrifuge tube of number, which is put into 30 DEG C of water-baths, digests 2.5h, and number, 10,11,12 centrifuge tubes are put into 30
3h is digested in DEG C water-bath, 13,14,15 centrifuge tube of number, which is put into 30 DEG C of water-baths, digests 3.5h, and number 16,17,18 is centrifuged
Pipe, which is put into 30 DEG C of water-baths, digests 4h, every group of every 30min, takes out gently to overturn shaking up up and down 20 times.
(3) it in superclean bench, into centrifuge tube and is sealed with G2 funnel filtering crude extract, in supercentrifuge
It is centrifuged 10min under the conditions of 4000rpm, outwells supernatant, draws the steady penetration enhancer of 5ml with liquid-transfering gun and is added in centrifuge tube, will precipitate
It mixes, repeats to be centrifuged 10min under the conditions of 4000rpm, outwell supernatant, add the steady penetration enhancer of 1ml, precipitating is mixed.Draw 20ul drop in
On blood counting chamber, the microscopy under 40 times of optical microscopy is counted.
It can be obtained by Fig. 2, most suitable enzymolysis time is 3h.
Embodiment 4:
(1) it connects on healthy and strong true pleurotus cornucopiae mycelia to PDA thallus culture medium flat plate, 24 DEG C are protected from light culture 200h.Select robust growth
Mycelia plate, connect mycelia in glass dregs 150mlPDB fluid nutrient medium in (glass dregs long 0.5-1cm × 0.4 cm ×
0.2cm), it is protected from light stationary culture 110h, after cultivating 48h, every 8h shakes conical flask 10min manually, cuts off newly long mycelia.120h
Contain healthy and strong liquid mycelia afterwards into other 120ml band cullet slag PDB fluid nutrient medium, again stationary culture 96h, every 8h
In 250rpm, 24 DEG C of shaking table culture 1h.
(2) in superclean bench, go out mycelia with two layers of sterile nylon filtered through gauze, with aseptic water washing, then use
It 0.6mol/L mannitol wash 2 times, is blotted with the filter paper of sterilizing.The enzyme containing lywallzyme prepared with 0.6mol/L mannitol solution
Liquid, mycelia: enzyme solution is added in 1:10g/mL ratio equipped in mycelial centrifuge tube, and enzyme solution is filtered with biofilter, existing
With current, number 1,2,3, the centrifuge tube number containing 1.5% enzyme solution is 4,5,6, the centrifuge tube number containing 2% enzyme solution is 7,
8,9, the centrifuge tube number containing 2.5% enzyme solution is 10,11,12, and the centrifuge tube number containing 3% enzyme solution is 13,14,15, will be from
Heart pipe, which is put into 30 DEG C of water-baths, digests 3h, and every 30min is overturned shake up 20 times up and down.
(3) it in superclean bench, into centrifuge tube and is sealed with G2 funnel filtering crude extract, in supercentrifuge
It is centrifuged 10min under the conditions of 4000rpm, outwells supernatant, draws the steady penetration enhancer of 5ml with liquid-transfering gun and is added in centrifuge tube, will precipitate
It mixes, repeats to be centrifuged 10min under the conditions of 4000rpm, outwell supernatant, add the steady penetration enhancer of 1ml, precipitating is mixed.Draw 20ul drop in
On blood counting chamber, the microscopy under 40 times of optical microscopy is counted.
It can be obtained by Fig. 3, most suitable enzyme solution concentration is 2%.
Embodiment 5:
(1) it connects on healthy and strong true pleurotus cornucopiae mycelia to PDA thallus culture medium flat plate, 24 DEG C are protected from light culture 200h.Select robust growth
Mycelia plate, connect mycelia in 150ml with glass dregs PDB fluid nutrient medium in (glass dregs long 0.5-1cm × 0.4 cm ×
0.2cm), it is protected from light stationary culture 110h, after cultivating 48h, every 8h shakes conical flask 10min manually, cuts off newly long mycelia.120h
Contain healthy and strong liquid mycelia afterwards into other 120ml band cullet slag PDB fluid nutrient medium, again stationary culture culture 96h, often
8h is in 250rpm, 24 DEG C of shaking table culture 1h.
(2) in superclean bench, go out mycelia with two layers of sterile nylon filtered through gauze, with aseptic water washing, then use
It 0.6mol/L mannitol wash 2 times, is blotted with the filter paper of sterilizing.The molten wall containing 2wt.% prepared with 0.6mol/L mannitol solution
The enzyme solution of enzyme, mycelia: enzyme solution is added in 1:10g/mL ratio equipped in mycelial centrifuge tube, enzyme solution biofilter mistake
Filter, it is ready-to-use, centrifuge tube is put into 30 DEG C of water-baths and digests 3h, every 30min is overturned shake up 20 times up and down.
(3) it in superclean bench, into centrifuge tube and is sealed with G2 funnel filtering crude extract, 1,2,3 centrifuge tube of number
It is centrifuged 10min under the conditions of supercentrifuge 3000rpm, is numbered, 4,5,6 centrifuge tubes are in supercentrifuge 4000rpm condition
Lower centrifugation 10min, 7,8,9 centrifuge tube of number are centrifuged 10min under the conditions of supercentrifuge 5000rpm, and number 1,2,3 is centrifuged
Pipe is centrifuged 10min under the conditions of supercentrifuge 6000rpm.Supernatant is outwelled, the steady penetration enhancer of 5ml is drawn with liquid-transfering gun and is added to
In centrifuge tube, precipitating is mixed, above-mentioned condition is centrifuged 10min, outwells supernatant, adds the steady penetration enhancer of 1ml, and precipitating is mixed.It draws
In on blood counting chamber, the microscopy under 40 times of optical microscopy counts 20ul drop.
Available from figure 4, most suitable centrifugation rate is 4000rpm/min.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (2)
1. a kind of method for preparing protoplast of true pleurotus cornucopiae, which comprises the following steps:
(1) it connects on healthy and strong true pleurotus cornucopiae mycelia to PDA thallus culture medium flat plate, 24 DEG C are protected from light culture 144h-240h；Selection growth is strong
Strong mycelia plate, connect mycelia in glass dregs 150mlPDB fluid nutrient medium in, glass dregs size be 0.5-1cm ×
0.4 cm × 0.2cm is protected from light stationary culture 96h-120h, and after cultivating 48h, every 8h shakes conical flask 10min manually, cuts off new length
Mycelia；The 50ml that transfers after 120h contains the fluid nutrient medium of healthy and strong mycelia to other 120ml with cullet slag PDB Liquid Culture
In base, stationary culture 48h-144h again, every 8h is in 250rpm, 24 DEG C of shaking table culture 1h；
(2) in superclean bench, go out mycelia with two layers of sterile nylon filtered through gauze, with aseptic water washing, then use 0.6mol/
L mannitol solution cleans 2 times, is blotted with the filter paper of sterilizing；The lywallzyme containing 1-4wt.% prepared with 0.6mol/L mannitol solution
Enzyme solution, mycelia: enzyme solution is added in 1:10g/mL ratio equipped in mycelial centrifuge tube, and centrifuge tube is put into 30 DEG C of water-baths
1-4h, every 30min are digested in pot, overturns shake up 20 times up and down；
(3) it in superclean bench, into centrifuge tube and is sealed with G2 funnel filtering crude extract, in supercentrifuge 3000-
It is centrifuged 10min under the conditions of 6000rpm, outwells supernatant, draws the steady penetration enhancer of 5ml with liquid-transfering gun and is added in centrifuge tube, will precipitate
It mixes, repeats to be centrifuged 10min under the conditions of 3000-6000rpm, outwell supernatant, add the steady penetration enhancer of 1ml, precipitating is mixed；It draws
In on blood counting chamber, the microscopy under 40 times of optical microscopy counts 20ul drop.
2. preparation method according to claim 1, it is characterised in that: the following steps are included:
(1) it connects on healthy and strong true pleurotus cornucopiae mycelia to PDA thallus culture medium flat plate, 24 DEG C are protected from light culture 200h；Select robust growth
Mycelia plate connects mycelia in the 150mlPDB fluid nutrient medium with glass dregs, and glass dregs size is the cm of 0.5-1cm × 0.4
× 0.2cm is protected from light stationary culture 110h, and after cultivating 48h, every 8h shakes conical flask 10min manually, cuts off newly long mycelia；
The 50ml that transfers after 120h contains the fluid nutrient medium of healthy and strong mycelia into other 120ml band cullet slag PDB fluid nutrient medium, then
Secondary stationary culture 96h, every 8h are in 250rpm, 24 DEG C of shaking table culture 1h；
(2) in superclean bench, go out mycelia with two layers of sterile nylon filtered through gauze, with aseptic water washing, then use 0.6mol/
It L mannitol wash 2 times, is blotted with the filter paper of sterilizing, with the enzyme for the lywallzyme containing 2wt.% that 0.6mol/L mannitol solution is prepared
Liquid, mycelia: enzyme solution is added in 1:10g/mL ratio equipped in mycelial centrifuge tube, and centrifuge tube is put into 30 DEG C of water-baths
3h, every 30min are digested, overturns shake up 20 times up and down；(3) in superclean bench, crude extract is filtered to centrifuge tube with G2 funnel
In and seal, be centrifuged 10min under the conditions of supercentrifuge 4000rpm, outwell supernatant, with liquid-transfering gun draw the steady penetration enhancer of 5ml
It is added in centrifuge tube, precipitating is mixed, repeat to be centrifuged 10min under the conditions of 4000rpm, outwell supernatant, add the steady penetration enhancer of 1ml, it will
Precipitating mixes；20ul drop is drawn on blood count, the microscopy under 40 times of optical microscopy counts.
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| CN110607241A (en) * | 2019-09-25 | 2019-12-24 | 上海市农业科学院 | Preparation method of hypsizigus marmoreus protoplast monokaryon |
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