CN102352349A - Method utilizing protoplasts to fuse and screen rich-selenium high-yield strains among different ganoderma varieties - Google Patents
Method utilizing protoplasts to fuse and screen rich-selenium high-yield strains among different ganoderma varieties Download PDFInfo
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Abstract
A method utilizing protoplasts to fuse and screen rich-selenium high-yield strains among different ganoderma varieties belongs to the technical field of biological husbandry. The method selects different ganodermas to produce strains, the ganoderma cultivation is carried out, the ganoderma strains with high ganoderma sporocarp yield and good product agronomic trait are selected as the high-yield fusion original strains; and the selenium-resistant experiment is utilized to screen the ganoderma strains with strong rich-selenium capability as the rich-selenium fusion original strains. The method determines one parent strain as the inactivation strain through the hyphal growth speed and form, pigmental secretion, protoplast regeneration rate, protoplast inactivation time and the like; then the fusant screening is carried out through the hyphal form and antagonism, so the operation simplicity and practicability are obtained. The method is adopted to fuse one ganoderma fusion strain which has the sporocarp selenium content and yield higher than those of the original strain, so a good economical benefit is created.
Description
Technical field
The present invention relates to merge the method for screening rich selenium superior strain through protoplastis between a kind of different lucidum variety, belong to the biological husbantry technical field.
Background technology
Glossy ganoderma is a Basidiomycetes polyporaceae ganoderma fungi, and what wherein present Application and Development was the widest is red sesame, and its sporophore then is one of main raw material that carries out deep processing.Present numerous research confirmed glossy ganoderma have regulate immune, anti-oxidant, anoxia, resist radiation, anti-chemotherapy, function of resisting myocardial ischemia and anti-aging effects and sedative effect, cardiac stimulant, regulating blood fat effect, hypoglycemic, effect such as relieving asthma, protect the liver, be widely used among the clinical treatment various diseases.And output Ganderma lucidum strain high, that biological character is excellent is to ensure that glossy ganoderma produces and promote the prerequisite of glossy ganoderma industry development; The most output of isolating Ganderma lucidum strain is not high from the field; Need through domestication and constantly seed selection; To improve the output and the production traits of glossy ganoderma, therefore, the research of the seed selection aspect of bacterial classification just seems particularly important.The efficient of nature seed selection is lower; And take the method effect of artificially breeding better; Through the years of researches practice; With fruiting body yield and economical character is that the strain improvement of major objective develops into induced mutations breeding, protoplast fusion breeding and the utilization Protocols in Molecular Biology carries out breeding etc. from initial selection breeding, cross-breeding, on the The Breeding of Edible Mushroom method, weeds out the old and bring forth the new, updates, and breeding efficiency improves constantly.It is few that the strain improvement of glossy ganoderma aspect research is at present carried out; Main report with ultraviolet mutagenesis seed selection glossy ganoderma, but its mutagenic and breeding efficient is not high, and novel bacterial output promotes not obvious; Effect is not remarkable; The inactivation times of the secretion of the speed of growth of the present invention through mycelia, form, pigment, protoplasts regenerated rate, protoplastis etc. carry out the fusion of protoplastis as confirming that wherein a strain parent is an inactivated strain through chemical process (PEG6000), carry out The Fusants Screen through mycelia form, antagonistic action then; Have simplicity, the practicality of operation, improved the efficient of edible fungus species seed selections such as glossy ganoderma.
The conventional strain selection of edible mushrooms can be divided into nature seed selection and artificially breeding, and artificially breeding is divided into selection by mutation, cross-breeding and protoplast fusion breeding again.The nature breeding is to utilize natural variation, chooses required useful variation.Selection by mutation is the DNA of utilization physics or chemical mutagen effect bacterial strain, makes its generation heritable variation and obtains the good character bacterial strain.Thereby cross-breeding is to obtain new proterties through suitable segmental exchange of parental set of chromosome of two parents and reorganization to bring out the breeding method with parents' good character.The nature seed selection just accumulates and utilize the useful variation that takes place under the natural condition, and is not only time-consuming but also require great effort.Although selection by mutation have speed fast, bring notable results, advantage such as method is easier, its genetic mutation is bigger, exists blindness unavoidably.Though cross-breeding has certain directional property, because edible mushrooms is that the polar fungi is arranged, because of the existence of not affine and cell walls makes cross-breeding receive certain restriction, process is slow.
It is a kind of cell engineering that new development is got up that protoplastis merges, and is an integral part of cytogamy.It is after utilizing enzyme process to divest cell walls, under certain physics or electrochemical conditions, makes the protoplastis of different inherited character bacterial strains produce fusion, makes both Gene Partial or all reorganization, thereby obtains fusant.It is a step of most critical in the protoplastis fusion process that the protoplastis The Fusants Screen is identified, also is the judgement of whole fusion process success or not.Usually the evaluation of edible mushrooms protoplastis can select for use auxotrophy, resistance sudden change, antimetabolic sudden change and fluorescent mark as detecting mark.But the working process more complicated, workload is bigger.These methods generally be between not belonging to together like the fusion between flat mushroom and mushroom etc., and existing report.And between glossy ganoderma belongs to together, find out these characteristics difficulty more, the inactivation times of the secretion of the speed of growth of present method through mycelia, form, pigment, protoplasts regenerated rate, protoplastis etc. are as confirming that wherein a strain parent is an inactivated strain.Carry out The Fusants Screen through mycelia form, antagonistic action then, have simplicity, the practicality of operation.
Do not see at present different lucidum varieties between relevant ganoderma merge the new bacterial strain of the rich selenium high yield of screening through protoplastis any report as yet.
Summary of the invention
The object of the invention merges the new bacterial strain of the rich selenium high yield of screening through different lucidum varieties between ganoderma through protoplastis.
Technical scheme of the present invention: different lucidum varieties merge the method for screening rich selenium superior strain through protoplastis between a kind of ganoderma, and its concrete steps are following:
1, the glossy ganoderma starting strain that merges with protoplastis of screening: selects 20 kinds of different glossy ganodermas production bacterial classifications, carry out cultivation of glossy ganoderma, select Ganoderma sporophore output is the highest, the product economical character is good ganoderma strain capable fusion starting strain as high yield; And through the strong ganoderma strain capable of the experiment screening of anti-selenium selenium rich ability, as the fusion starting strain of rich selenium; 20 kinds of different Ganderma lucidum strains are respectively the big glossy ganoderma of the U.S., Mount Taishan glossy ganoderma, South Korean Ganoderma, Xinzhou glossy ganoderma, Huizhou glossy ganoderma, Japanese red sesame, the flat sesame of Japan, the big glossy ganoderma in capital, sweet sesame, G-F1, G-F2, G-902, the red sesame of Korea S, G1, G2, G3, G6, G8, G9, G11;
Confirm that Huizhou glossy ganoderma is as the fusion starting strain of rich selenium with the fusion starting strain of the big glossy ganoderma of the U.S. as high yield.
2, the starting strain that filters out confirms that through the following aspects a wherein strain is an inactivated strain:
1. observe the speed of growth and mode of appearance and the mycelia pigment secretion situation of starting strain mycelia;
2. protoplasts regenerated situation;
3. the regeneration situation after the protoplastis deactivation is tested.
Confirm that Huizhou glossy ganoderma is an inactivated strain.
3, protoplastis merges: a strain glossy ganoderma protoplastis and another strain glossy ganoderma are through the deactivation protoplastis, and (PEG6000) carries out the fusion of protoplastis through chemical process.Process is:
PDA liquid nutrient medium: get juice after yam 200g boils, glucose 20g, MgSO
40.5g, KH
2PO
41g, agar 1.5g, water 1000mL, pH nature;
RM
4Regeneration culture medium: SANMALT-S 5g, glucose 10g, yeast powder 4g, N.F,USP MANNITOL 0.6mol/L, agar 5.5g, the pH nature, it is 1000mL that zero(ppm) water is settled to TV;
Homeo-osmosis agent: sucrose 0.6mol/L;
The preparation of enzyme liquid: the mass concentration 2% lywallzyme solution (lywallzyme is available from the Guangdong Microbes Inst) with 0.6mol/L sucrose homeo-osmosis agent dissolving preparation, use after 0.22 μ m filtering with microporous membrane degerming;
Fusogen is: 50mmolCaCl
230% PEG 6000 that solution is mixed with;
Fusion steps:
(A) mycelium is cultivated
Mycelium is cultivated: get the eugonic mycelia in PDA inclined-plane, be inoculated in the 100mL PDA liquid nutrient medium, put one deck granulated glass sphere in the bottom of triangular flask, 26～28 ℃ of lucifuges leave standstill and cultivated 2～4 days, shake triangular flask every day 2 times, break up mycelia with granulated glass sphere;
(B) protoplastis preparation
1., get the empty test tube after the sterilization, W1 weighs;
2., use the aperture 100 order copper screen filtration nutrient solutions after sterilizing, collection Ganoderma mycelium;
3., wash centrifugal mycelium 3 times with sterilized water and homeo-osmosis agent;
4., change the mycelium after centrifugal in the ready empty test tube over to the W2 that weighs, the wet mycelia weight W 2-W1 of calculating;
5., the ratio interpolation enzyme liquid that adds 1mL enzyme liquid in the wet mycelia of 100mg;
6., be enzymolysis 2.5 hours under 6.0 the condition at 30 ℃, pH, draw some enzymolysis solutions, microscopically is observed, and calculates the protoplastis number with blood counting chamber;
(C) protoplastis is refining
Filter enzymolysis solution with 8 layers of lens wiping paper, be total to 8mL gradation washing and filtering, get filtered liq with the homeo-osmosis agent; Centrifugal 10 minutes of 4000r/min removes supernatant, with homeo-osmosis agent 8mL centrifuge washing, gets the protoplastis of purifying; Protoplastis with homeo-osmosis agent 10mL dilution, is processed the protoplastis suspension, count with blood counting chamber;
(D) protoplastis deactivation
Get the refining glossy ganoderma protoplastis suspension 1mL that obtains of aforesaid method and carry out the inactivation treatment of 50 ℃, 55 ℃, 60 ℃ differing tempss and 1h, 1.5h, 2h different time; After the processing, coat on the regeneration culture medium, with comparing of not deactivation protoplastis;
(E) protoplast regeneration
To make with extra care the protoplastis suspension and be diluted to 10
5Individual mL
-1, draw protoplastis suspension 0.2mL and be coated in the RM4 regeneration culture medium solid plate, cultivate 8-10d for 26-28 ℃;
The calculating of regeneration rate: regeneration rate=[regeneration colony count/protoplastis sum] * 100%;
(F) protoplastis merges
Get the refining glossy ganoderma protoplastis suspension 1mL that obtains, mix with 1:1 with the glossy ganoderma protoplastis of deactivation, 4000rpm is centrifugal, and 10min removes supernatant, dropwise adds 1mL and uses 50mmolCaCl
230% PEG6000 that is mixed with handled 30 minutes for 30 ℃, and 4000rpm is centrifugal, and 10min removes supernatant, coated RM to the protoplastis suspension with the washing of 0.6M sucrose solution and centrifugal twice back of the bacterium of going out
4On the regeneration culture medium, 26-28 ℃ of constant temperature culture 8-10d.
4, merge the whole regenerating and culturing of liquid, select the bacterium colony of antagonism reaction.
Glossy ganoderma protoplastis suspension after the chemistry fusion is diluted to 10
-2Content is got 0.1mL and is coated on RM
4In the regeneration culture medium solid plate, cultivate 8-10d for 26-28 ℃, utilize the antagonistic action between different strains, pick out the bacterial strain of antagonism reaction.
5, the bacterium colony of picking out also need further to cultivate and with the antagonism reaction experiment of parent strain, through to mycelial growth rate, form and pigment secretion situation, and with parent's antagonism situation, determine whether to be new bacterial strain.
Parent strain and the inoculation of picking out are cultivated in the PDA liquid nutrient medium, pick out bacterial strain with the reaction of parent strain antagonism.
6, the bacterial strain that filters out; The picking mycelia goes on the PDA inclined-plane and cultivates; Carry out the liquid fermenting test then; Carry out the test of esterase isozyme electrophoresis with former bacterial strain simultaneously, select the big and different new fusant bacterial strain of liquid fermenting mycelial biomass, carry out cultivation of glossy ganoderma verification experimental verification Ganoderma sporophore output and selenium content then as the primary dcreening operation bacterial strain with former bacterial strain.
7, stabilization characteristics of genetics property checking: with 20 generations of bacterial strain continuous passage after the above-mentioned screening, carry out experiment in cultivation then, stable yield, the unconverted new fusion ganoderma strain capable that is used to produce of finally confirming as of proterties.
Obtain three strain fusant bacterial strains through this method; Utilize Ganoderma lucidum mycelium to add different selenium contents then at the PDA substratum; Growth conditions on the viewing plane substratum is judged the anti-selenium ability of bacterial strain; And ganoderma lucidum liquid fermenting organism output, the condition of Ganoderma sporophore high-yield selenium-rich bacterial strain primary dcreening operation is carried out the screening of glossy ganoderma fusant bacterial strain with the cultivation of glossy ganoderma sporophore ultimate capacity of certain scale as the ultimate criterion of multiple sieve.
Beneficial effect of the present invention:
The breeding of edible fungus species such as glossy ganoderma in the past adopts means such as cross-breeding and ultraviolet mutagenesis breeding to carry out usually; Have efficiency of inducing mutation low, be prone to cause shortcomings such as negative sudden change, inherited character instability; It is a kind of cell engineering that new development is got up that protoplastis merges, and is an integral part of cytogamy.It is after utilizing enzyme process to divest cell walls, under certain physics or electrochemical conditions, makes the protoplastis of different inherited character bacterial strains produce fusion, makes both Gene Partial or all reorganization, thereby obtains fusant.It is a step of most critical in the protoplastis fusion process that the protoplastis The Fusants Screen is identified, also is the judgement of whole fusion process success or not.Usually the evaluation of edible mushrooms protoplastis can select for use auxotrophy, resistance sudden change, antimetabolic sudden change and fluorescent mark as detecting mark.But the working process more complicated, workload is bigger.These methods generally be between not belonging to together like the fusion between flat mushroom and mushroom etc., and existing report.And between glossy ganoderma belongs to together, find out these characteristics difficulty more, the inactivation times of the secretion of the speed of growth of the present invention through mycelia, form, pigment, protoplasts regenerated rate, protoplastis etc. are as confirming that wherein a strain parent is an inactivated strain.Carry out The Fusants Screen through mycelia form, antagonistic action then, have simplicity, the practicality of operation.
Adopt the present invention to merge a strain sporophore selenium content, output to be higher than the glossy ganoderma fusant bacterial strain of former bacterial strain, can create good economic benefits.
Microbe-derived
:
Starting strain: the big glossy ganoderma of the U.S. is available from lucky gill fungus garden, Beijing Science and Technology Ltd., and Huizhou glossy ganoderma is available from Jinxiang, Shandong fungal studies institute.
Description of drawings
Fig. 1 picks out the bacterial strain of antagonism reaction.Pick out 93 strains.
Fig. 2 picks out the bacterial strain with the reaction of parent strain antagonism.Pick out 10 strains.
Fig. 3 isozyme collection of illustrative plates.Bacterial strain order from left to right is: contrast 1, the big glossy ganoderma of the U.S., contrast 2, Huizhou glossy ganoderma, 3, YG1,4, YG2,5, YG3,6, YG4,7, YG5,8, YG6,9, YG7,10, YG8,11, YG9,12, YG10.By figure find out 4, YG2 is best.
Embodiment
Through instance the present invention is further specified below: following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following instance.
Different lucidum varieties merge the method for screening the new bacterial strain of rich selenium high yield through protoplastis between ganoderma, the steps include:
1, the screening of starting strain
Introduce Ganderma lucidum strain 20 strains of different varieties from glossy ganoderma main producing region, domestic each province.Be inoculated on the PDA slant medium, 28 ℃ of lucifuges are cultivated and were carried out activation in 7 days.The activatory inoculation in the central authorities that PDA culture medium culturing ware is housed, the time that the record Ganoderma lucidum mycelium is sprouted, is measured its speed of growth, the form (mycelia thickness, dense) of observation mycelia.Then carry out the ganoderma lucidum liquid fermentation test, slant strains is seeded to (culture medium prescription as follows) in the liquid nutrient medium, and 28 ℃ of lucifuges of 140 rev/mins of rotary shaking tables were cultivated 7 days; Transfer then in fermention medium; 28 ℃ of lucifuges of 140 rev/mins of rotary shaking tables were cultivated 3 days, 4000 rev/mins centrifugal 5 minutes, collect mycelium; Dry to constant weight for 65 ℃, measure its quality and get the ganoderma lucidum liquid fermentation biomass.Simultaneously through adding 0.5%, 0.2%, 0.05% Sodium Selenite on the PDA slant medium, observation mycelial growth rate, the anti-selenium ability of mensuration different strains.Carry out the ganoderma lucidum liquid fermentation test then, add the mycelia selenium content after 0.5%, 0.2%, 0.05% Sodium Selenite is measured the different strains fermentation in the liquid nutrient medium.
Carry out the cultivation of glossy ganoderma simultaneously, its concrete process is: the slant strains of glossy ganoderma is inoculated in (the liquid culture based formulas is as follows) in the Ganoderma lucidum by submerged culture base, and 140 rev/mins of rotary shaking tables, 28 ℃ of lucifuges were cultivated 7 days; Inoculum size with 2% is inoculated in the glossy ganoderma solid medium after the sterilization, and 26 ℃ of lucifuges were cultivated about about 40 days, treat that mycelia is covered with the bacterium bag after; Remove to the fruiting room and carry out management of producing mushroom, relative air humidity 85%～95%, 30 ℃ of temperature; Certain scattered light is handled, often ventilation, and mycelia forms original hase; Be differentiated to form sporophore, wait Ganoderma sporophore cap edge by white commentaries on classics when yellow, be placed in kraft bag and collect Ganoderma spore powder on the Ganoderma sporophore.Manage simultaneously, gather when treating the Ganoderma sporophore fully matured, the output of statistics glossy ganoderma, the biological transformation ratio of calculating glossy ganoderma, the highest with Ganoderma sporophore output, the biological transformation ratio soprano is as the glossy ganoderma starting strain.Find through statistics that simultaneously the output of Ganoderma sporophore and Ganoderma lucidum mycelium form and the size of liquid fermenting living weight on the PDA plating medium has dependency.The sturdy dense and fast growth of mycelia, its Ganoderma sporophore output of the big more person of liquid fermenting living weight are high on the PDA plane.Test is last confirms that Huizhou glossy ganoderma is as the fusion starting strain of rich selenium with the fusion starting strain of the big glossy ganoderma of the U.S. as high yield.
2, the starting strain that filters out confirms that through the following aspects a wherein strain is that inactivated strain is used in experiment.Glossy ganoderma is an inactivated strain in final definite Huizhou.
(1) speed of growth of observation starting strain mycelia and mode of appearance and mycelia pigment secretion situation.
Glossy ganoderma is an inactivated strain in final definite Huizhou.(1. low temperature, short period of time deactivation can keep the activity of deactivation protoplastis.2. the speed of growth and mycelia pigment secretion can be used as fusant and screens preliminary foundation.）
| ? | The speed of growth of bacterial strain mycelia | Mode of appearance | The secretion of mycelia pigment |
| The big glossy ganoderma of the U.S. | Every day 0.72cm | Mycelia is dense | Do not have |
| Huizhou glossy ganoderma | Every day 0.51cm | Mycelia is carefully not dense | The mycelia secretion is yellow after 8 days |
(2) protoplasts regenerated situation
| ? | Recovery time | Regeneration rate |
| The big glossy ganoderma of the U.S. | The 4th day | 0.45% |
| Huizhou glossy ganoderma | The 6th day | 0.4% |
(3) the regeneration situation after the protoplastis deactivation experiment
| ? | 50℃ | 55℃ | 60℃ |
| The big glossy ganoderma of the U.S. | 3h | 2.5h | 2h |
| Huizhou glossy ganoderma | 2.5h | 2.5h | 2h |
3, protoplastis merges: a strain glossy ganoderma protoplastis and another strain glossy ganoderma are through the deactivation protoplastis, and (PEG6000) carries out the fusion of protoplastis through chemical process.
Instrument and equipment: whizzer (4000r/min), thermostat water bath, constant temperature water isolation type incubator, microscope, blood counting chamber, analytical balance (sensibility reciprocal is 0.01g).
Reagent and material: lywallzyme (purchasing), suction pipe (1mL, 5mL), copper screen cloth (diameter is 10cm, aperture 100 orders), 8 layers of lens wiping paper, funnel (diameter 8cm), pin type filter, 0.22 μ m millipore filtration in the Guangdong Microbes Inst.
PDA liquid nutrient medium: yam 200g (getting juice after boiling), glucose 20g, MgSO
40.5g, KH
2PO
41g, agar 1.5g, water 1000mL, pH nature.
RM
4Regeneration culture medium: SANMALT-S 5g, glucose 10g, yeast powder 4g, N.F,USP MANNITOL 0.6mol/L, agar 5.5g, the pH nature, it is 1000mL that zero(ppm) water is settled to TV.
Homeo-osmosis agent: sucrose 0.6mol/L.
The preparation of enzyme liquid: 2% lywallzyme, with 0.6mol/L sucrose homeo-osmosis agent dissolving, use after 0.22 μ m filtering with microporous membrane degerming.
Fusogen is: 50mmolCaCl
230% PEG 6000 that solution is mixed with.
(1) mycelium is cultivated
Mycelium is cultivated: get the eugonic mycelia in PDA inclined-plane, be inoculated in the 100mL PDA liquid nutrient medium and (put one deck granulated glass sphere in the bottom of triangular flask), 26～28 ℃ of lucifuges leave standstill and cultivated 2～4 days, shake triangular flask every day 2 times, break up mycelia with granulated glass sphere.(when the culture bacteria filament, static cultivation is superior to shaking culture, can obtain more protoplastis, because shaking culture can form a large amount of mycelium pellets, the inner mycelia of mycelium pellet is difficult to contact with enzyme, thereby it is lower to make that protoplastis prepares rate.And static cultivation can form cotton-shaped mycelia, has enlarged the contact area of mycelia and enzyme, makes that the protoplasm structure yield is higher, in culturing bottle, adds a certain amount of aseptic little granulated glass sphere in addition, shakes every day several times, and mycelia is broken up, and can improve the protoplast regeneration rate).
(2) protoplastis preparation
1, get empty test tube after the sterilization, W1 weighs.
2, filter nutrient solution with the copper screen cloth (diameter is 10cm, aperture 100 orders) after the sterilization, collect Ganoderma mycelium.
3, wash centrifugal mycelium 3 times with sterilized water and homeo-osmosis agent, each consumption is 8mL.
4, change the mycelium after centrifugal in the ready empty test tube over to, the W2 that weighs calculates wet mycelia weight W 2-W1
5, the ratio interpolation enzyme liquid that adds 1mL enzyme liquid in the wet mycelia of 100mg.
6, at 30 ℃, pH is an enzymolysis 2.5 hours under 6.0 the condition.Draw some enzymolysis solutions, microscopically is observed, and calculates the protoplastis number with blood counting chamber.
(3) protoplastis is refining
Filter enzymolysis solution with 8 layers of lens wiping paper, be total to 8mL gradation washing and filtering, get filtered liq with the homeo-osmosis agent.Centrifugal 10 minutes of 4000r/min removes supernatant, with homeo-osmosis agent 8mL centrifuge washing, gets the protoplastis of purifying.Protoplastis with homeo-osmosis agent 10mL dilution usefulness, is processed the protoplastis suspension, count with blood counting chamber.
(4) protoplastis deactivation
Get aforesaid method and make with extra care the inactivation treatment that the glossy ganoderma protoplastis suspension 1mL that obtains carries out differing temps (50 ℃, 55 ℃, 60 ℃) and time (1h, 1.5h, 2h).After the processing, coat on the regeneration culture medium, with comparing of not deactivation protoplastis.
(5) protoplast regeneration
To make with extra care the protoplastis suspension and be diluted to 10
5Individual mL
-1, draw protoplastis suspension 0.2mL and be coated on RM
4In the regeneration culture medium solid plate, cultivate 8-10d for 26-28 ℃.
The calculating of regeneration rate: regeneration rate=[regeneration colony count/protoplastis sum] * 100%.
(6) protoplastis merges
Get the refining glossy ganoderma protoplastis suspension 1mL that obtains, mix with 1:1 (V/V) with the glossy ganoderma protoplastis of deactivation, 4000rpm is centrifugal, and 10min removes supernatant, dropwise adds 1mL and uses 50mmolCaCl
230% PEG that is mixed with handled 30 minutes for 6000,30 ℃, and 4000rpm is centrifugal, and 10min removes supernatant, coated RM to the protoplastis suspension with the washing of 0.6M sucrose solution and centrifugal twice back of the bacterium of going out
4On the regeneration culture medium, 26-28 ℃ of constant temperature culture 8-10d.
4, merge the whole regenerating and culturing of liquid, select the bacterium colony of antagonism reaction
Glossy ganoderma protoplastis suspension after the chemistry fusion is diluted to 10
-2Content is got 0.1mL and is coated on RM
4In the regeneration culture medium solid plate, cultivate 8-10d for 26-28 ℃.Utilize the antagonistic action between different strains, pick out the bacterial strain of antagonism reaction.Pick out 93 strains (Fig. 1)
5, parent strain and the inoculation picked out are cultivated in the PDA liquid nutrient medium, picked out bacterial strain with the reaction of parent strain antagonism.Pick out 10 strains (Fig. 2)
6, the bacterial strain that filters out; The picking mycelia goes on the PDA inclined-plane and cultivates; Carry out the liquid fermenting test then; Carry out the test of esterase isozyme electrophoresis with former bacterial strain simultaneously, select the big and different new mutagenic strain of liquid fermenting mycelial biomass, carry out cultivation of glossy ganoderma verification experimental verification Ganoderma sporophore output and selenium content then as the primary dcreening operation bacterial strain with former bacterial strain.Pick out 2 strains (Fig. 3) and (find that through the esterase isozyme bands of a spectrum YG1, YG2 have different with starting strain.）
7, stabilization characteristics of genetics property checking: with 20 generations of bacterial strain continuous passage after the above-mentioned screening, carry out experiment in cultivation then, stable yield, the unconverted new fusion ganoderma strain capable that is used to produce of finally confirming as of proterties.(confirm that finally YG2 is for merging superior strain.）
Be used for the checking of stabilization characteristics of genetics property and carry out the culture medium prescription of experiment in cultivation:
PDA substratum: yam 200g liquor, glucose 20g, KH
2PO
410g, MgSO
45g, agar 20g, H
2O 1000mL.
Liquid nutrient medium: glucose 20g, Semen Maydis powder 20g, soybean cake powder 20g, KH
2PO
410g, MgSO
45g, VB 1100mg, H
2O 1000mL.
Fermention medium: glucose 20g, Semen Maydis powder 20g, soybean cake powder 20g, KH
2PO
410g, MgSO
45g, VB 1100mg, H
2O 1000mL.
The solid state cultivation substratum: cotton seed hulls 83%, wheat bran 15%, sucrose 1%, terra alba 1%, the content of moisture can be extruded 1～2 with the extruding of exerting oneself with hand and be advisable.
Claims (1)
1. different lucidum varieties merge the method for the rich selenium superior strain of screening through protoplastis between a ganoderma, it is characterized in that step is following:
(1), the glossy ganoderma starting strain that merges with protoplastis of screening: selects different glossy ganodermas to produce bacterial classification, carry out cultivation of glossy ganoderma, select the fusion starting strain of the good ganoderma strain capable of Ganoderma sporophore output height, product economical character as high yield; And through the strong ganoderma strain capable of the experiment screening of anti-selenium selenium rich ability, as the fusion starting strain of rich selenium;
(2), the starting strain that filters out confirms that through the following aspects a wherein strain is an inactivated strain:
1. observe the speed of growth and mode of appearance and the mycelia pigment secretion situation of starting strain mycelia;
2. protoplasts regenerated situation;
3. the regeneration situation after the protoplastis deactivation is tested;
Confirm that a strain glossy ganoderma is an inactivated strain;
(3), protoplastis merges: a strain glossy ganoderma protoplastis and another strain glossy ganoderma carry out the fusion of protoplastis through the deactivation protoplastis through chemical process PEG 6000; Process is:
PDA liquid nutrient medium: get juice after yam 200g boils, glucose 20g, MgSO
40.5g, KH
2PO
41g, agar 1.5g, water 1000mL, pH nature;
RM
4Regeneration culture medium: SANMALT-S 5g, glucose 10g, yeast powder 4g, N.F,USP MANNITOL 0.6mol/L, agar 5.5g, the pH nature, it is 1000mL that zero(ppm) water is settled to TV;
Homeo-osmosis agent: sucrose 0.6mol/L;
The preparation of enzyme liquid: the mass concentration 2% lywallzyme solution with 0.6mol/L sucrose homeo-osmosis agent dissolving preparation, use after 0.22 μ m filtering with microporous membrane degerming;
Fusogen is: 50mmolCaCl
230% PEG 6000 that solution is mixed with;
Fusion steps:
(A) mycelium is cultivated
Mycelium is cultivated: get the eugonic mycelia in PDA inclined-plane, be inoculated in the 100mLPDA liquid nutrient medium, put one deck granulated glass sphere in the bottom of triangular flask, 26～28 ℃ of lucifuges leave standstill and cultivated 2～4 days, shake triangular flask every day 2 times, break up mycelia with granulated glass sphere;
(B) protoplastis preparation
1., get the empty test tube after the sterilization, W1 weighs;
2., use the aperture 100 order copper screen filtration nutrient solutions after sterilizing, collection Ganoderma mycelium;
3., wash centrifugal mycelium 3 times with sterilized water and homeo-osmosis agent;
4., change the mycelium after centrifugal in the ready empty test tube over to the W2 that weighs, the wet mycelia weight W 2-W1 of calculating;
5., the ratio interpolation enzyme liquid that adds 1mL enzyme liquid in the wet mycelia of 100mg;
6., be enzymolysis 2.5 hours under 6.0 the condition at 30 ℃, pH, draw some enzymolysis solutions, microscopically is observed, and calculates the protoplastis number with blood counting chamber;
(C) protoplastis is refining
Filter enzymolysis solution with 8 layers of lens wiping paper, be total to 8mL gradation washing and filtering, get filtered liq with the homeo-osmosis agent; Centrifugal 10 minutes of 4000r/min removes supernatant, with homeo-osmosis agent 8mL centrifuge washing, gets the protoplastis of purifying; Protoplastis with homeo-osmosis agent 10mL dilution, is processed the protoplastis suspension, count with blood counting chamber;
(D) protoplastis deactivation
Get the refining glossy ganoderma protoplastis suspension 1mL that obtains of aforesaid method and carry out the inactivation treatment of 50 ℃, 55 ℃, 60 ℃ differing tempss and 1h, 1.5h, 2h different time; After the processing, coat RM
4On the regeneration culture medium, with comparing of not deactivation protoplastis;
(E) protoplast regeneration
To make with extra care the protoplastis suspension and be diluted to 10
5Individual mL
-1, draw protoplastis suspension 0.2mL and be coated on RM
4In the regeneration culture medium solid plate, cultivate 8-10d for 26-28 ℃;
The calculating of regeneration rate: regeneration rate=[regeneration colony count/protoplastis sum] * 100%;
(F) protoplastis merges
Get the refining glossy ganoderma protoplastis suspension 1mL that obtains, mix with 1:1 with the glossy ganoderma protoplastis of deactivation, 4000rpm is centrifugal, and 10min removes supernatant, dropwise adds 1mL and uses 50mmolCaCl
230% PEG that is mixed with handled 30 minutes for 6000,30 ℃, and 4000rpm is centrifugal, and 10min removes supernatant, coated RM to the protoplastis suspension with the washing of 0.6M sucrose solution and centrifugal twice back of the bacterium of going out
4On the regeneration culture medium, 26-28 ℃ of constant temperature culture 8-10d;
(4), merge the whole regenerating and culturing of liquid, select the bacterium colony of antagonism reaction:
Glossy ganoderma protoplastis suspension after the chemistry fusion is diluted to 10
-2Content is got 0.1mL and is coated on RM
4In the regeneration culture medium solid plate, cultivate 8-10d for 26-28 ℃, utilize the antagonistic action between different strains, pick out the bacterial strain of antagonism reaction;
(5), parent strain and the inoculation picked out are cultivated in the PDA liquid nutrient medium, pick out bacterial strain with the reaction of parent strain antagonism;
(6), the bacterial strain that filters out; The picking mycelia goes on the PDA inclined-plane and cultivates; Carry out the liquid fermenting test then; Carry out the test of esterase isozyme electrophoresis with former bacterial strain simultaneously, select the big and different fusant bacterial strain of liquid fermenting mycelial biomass, carry out cultivation of glossy ganoderma verification experimental verification Ganoderma sporophore output and selenium content then as the primary dcreening operation bacterial strain with former bacterial strain;
(7), stabilization characteristics of genetics property checking: with 20 generations of bacterial strain continuous passage after the above-mentioned screening, carry out experiment in cultivation then, stable yield, the unconverted fusion ganoderma strain capable that is used to produce of finally confirming as of proterties.
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| CN103374563B (en) * | 2012-04-13 | 2016-07-20 | 上海医药工业研究院 | A kind of method improveing 7-ACA producing strains |
| CN103609331A (en) * | 2013-11-18 | 2014-03-05 | 安徽农业大学 | Method for rejuvenating cordyceps militaris strains and increasing sporocarp yield by mating type |
| CN103609331B (en) * | 2013-11-18 | 2016-01-13 | 安徽农业大学 | Utilize mating type to cordyceps militaris link bacterial strain rejuvenation and the method improving fruiting body yield |
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| CN112715351B (en) * | 2021-01-18 | 2022-02-08 | 广西民族师范学院 | Rapid breeding method of phellinus igniarius |
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| CN114250153A (en) * | 2021-12-15 | 2022-03-29 | 江南大学 | High-selenium-resistance Ganoderma lucidum JNUSE-200 and selenium-rich fermentation strategy thereof |
| CN114250153B (en) * | 2021-12-15 | 2023-07-25 | 江南大学 | High selenium-resistant ganoderma Ganoderma lucidum JNUSE-200 and selenium-rich fermentation strategy thereof |
| CN114250183A (en) * | 2021-12-27 | 2022-03-29 | 陕西麦可罗生物科技有限公司 | Method for screening high-yield strains with multiple antibiotic B and multiple antibiotic L components |
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