CN104130946A - Trichoderma reesei strain and preparation method thereof - Google Patents
Trichoderma reesei strain and preparation method thereof Download PDFInfo
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Abstract
Belonging to the field of biotechnologies, the invention in particular relates to a cellulase high-yielding strain constructed through a protoplast fusion technology, and a method for preparation, separation, fusion, regeneration and screening of a Trichoderma reesei protoplast. The method includes: taking three Trichoderma reesei as starting strains, carrying out PDA medium activation and culture, hydrolyzing cell walls by snailase, lywallzyme and the like, conducting oscillation under a constant temperature, preparing a protoplast, performing protoplast fusion, and then carrying out screening by a cellulose-containing panel, thus finally obtaining the Trichoderma reesei JL16 strain with high yield of cellulose. The cellulase production capacity of the strain is enhanced by 25% than that of the starting strains. The Trichoderma reesei JL16 strain provided by the invention can be used for industrial production. And the protoplast preparation, separation, fusion, regeneration and screening method put forward for the Trichoderma reesei has strong application prospects.
Description
Technical field
The invention belongs to biological technical field, the Li Shi trichoderma strain of the High Cellulase Production that is specifically related to build by Protoplast Fusion Technique, and the mould preparation method of Li Shi wood.
Technical background
For the production of cellulase generally come from mould, be relatively typically Trichoderma.Li Shi wood is mould is of paramount importance one in Trichoderma, and is used widely in cellulose degradation field.Obtain the first strain Li Shi wood from separation till now mould, Li Shi wooden mould the experienced mutagenesis of many wheels, screening, obtained comprising MCG-77, QM9414, numerous superior strains such as RUTC-30.
In industrial production, generally use the production of cellulose enzymes such as fungi, wherein foremost is that Li Shi wood is mould, the wooden mould feature that cellulase zymogram is complete, vigor is high that has of Li Shi.Since the sixties in 20th century, people are taking wild strain T.reesei QM6a as starting strain, carried out a large amount of selection by mutation work, wherein QM9414, RutC30 and MCG77 etc. can great expression inscribe and exoglucanases, are the main bacteria seed of domestic and international cellulase production.
By using the method for protoplast fusion can obtain the Li Shi trichoderma strain of High Cellulase Production.Protoplast fusion refers to that by artificial method the good character bacterial strain that conventional mutagenesis is obtained focuses in a bacterial strain by protoplast fusion.At present, the main patent that obtains strain excellent by Protoplast Fusion Technique comprises CN201210301323.5, CN201010210226.6, CN201010174196.8, CN200880132218.5 etc., these protoplast fusions mainly adopt " biparental inactivate method ", working method still needs further simplification, and induced mutation rate also needs further raising.The present invention selects three strain cellulase high-yields to merge, thereby improves the overall activity of cellulase system.
Summary of the invention
One of the object of the invention is by the mould protoplast fusion of many strains Li Shi wood, obtains a strain cellulase high-yield.
Two of the object of the invention is to set up the Li Shi mould mutagenesis of wood and screening method.
Protoplast fusion is the random restructuring of genomic level in itself, its general step is, obtain multiple gain mutant bacterial strains by classical method for mutation breeding, namely multifarious genomic library, then, these mutant strains are prepared into protoplastis, pass through protoplast fusion, carry out the random restructuring of full genomic level, screening obtains superior strain.
The present invention carries out protoplast fusion to QM9414, RutC30 and MCG77, by screening, obtains cellulase high-yield.Meanwhile, set up the screening method of the mould protoplastis preparation of Li Shi wood, separation, fusion, regeneration and superior strain.The High Cellulase Production (the mould JL16 of Li Shi wood) that the present invention builds, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 22nd, 2014, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution address, deposit number is CGMCC No.8807, and Classification And Nomenclature is Li Shi wood mould (Trichoderma reesei).This bacterial strain cellulase yield is high, and genetic stability is good.
A kind of Li Shi trichoderma strain of request protection of the present invention, is that the deposit number that is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 22nd, 2014 is the bacterial strain of CGMCC No.8807, and the name of bacterial strain is called the mould JL16 of Li Shi wood.
The mould JL16 of described Li Shi wood, be adopt three strain cellulase high-yields by protoplastis prepare, the preparation process structure of protoplastis separation, protoplast fusion, protoplast regeneration and screening; Three described strain cellulase high-yields are Li Shi Trichoderma QM9414, Li Shi Trichoderma MCG77 and Li Shi Trichoderma RutC-30.
The preparation method's of the mould JL16 of Li Shi wood of the present invention technical scheme is as described below.
A preparation method for Li Shi trichoderma strain, has the preparation of protoplastis, fusion, the regeneration of protoplastis and the process of screening of protoplastis;
The preparation of described protoplastis, is that Li Shi Trichoderma QM9414, Li Shi Trichoderma MCG77 and Li Shi Trichoderma RutC-30 are seeded in respectively to potato dextrose agar (PDA) substratum, obtains the mycelia that contains the mould spore of ripe Li Shi wood; The mould spore suspension of three strain Li Shi wood is seeded in seed culture medium, obtains containing the mycelial Li Shi wood of uniting mould; By the citrate buffer solution constant volume of pH6.5 for mycelium, centrifugal, again add citrate buffer solution and mix after abandoning supernatant; Join respectively again enzymolysis cell walls in wall mixed enzyme solution, obtain the solution containing protoplastis; Finally be isolated to the mould protoplastis of Li Shi wood of white precipitate;
The fusion of described protoplastis, blows three kinds of white precipitate protoplastiss moltenly together with fusogen, room temperature leaves standstill 10～60min; Described fusogen, is made up of the material of pressing row weight proportion: 20%～80% polyoxyethylene glycol (PEG4000), 0.01～0.05M CaCl
2with 0.2～1.0M sorbyl alcohol;
The regeneration of described protoplastis, is by the dilution of the protoplastis suspension having merged NaCl solution gradient, gets diluent and is coated on protoplast regeneration substratum, sees light cultivation 96h, keeps relative humidity to be greater than 60%, until grow transparent circle for 28 DEG C; Described regeneration culture medium, is made up of the material of following weight proportion: KH
2pO
43～5g/L, NaNO
32.6～3.0g/L, urea 0.5～1.0g/L, FeSO
47H
2o 7.5～10mg/L, MnSO
4h
2o 2.5～3.0mg/L, ZnSO
47H
2o 3.6～5.0mg/L, CoCl
26H
2o 3.7～5.0mg/L, MgSO
47H
2o 0.5～1.0g/L, CaCl
20.5～1.0g/L, Sodium desoxycholate 2～5g/L, peptone 10～15g/L, NaCl 35～50g/L, agar 16～20g/L, ball milling Mierocrystalline cellulose 20g/L;
Described screening, is that the bacterial strain one that picking transparent circle diameter/colony diameter is large encircles to glycerine, obtains the mould JL16 of Li Shi wood and preserves at-80 DEG C.
In the preparation of protoplastis, citrate buffer solution is made up of the material of following weight proportion: in every liter, contain 0.1M citric acid solution 70ml, 0.1M sodium citrate solution 930ml, NaCl 0.6mol.
In the preparation of protoplastis, go wall mixed enzyme solution by lywallzyme, helicase, cellulase, add pH6.5 citrate buffer solution to form, each component content is lywallzyme 2～8mg/ml, helicase 2～8mg/ml, cellulase 2～8mg/ml.
In the preparation of protoplastis, separating the tripping device using is the centrifuge tube by a 5～50ml, filling up absorbent cotton apart from 1～10cm place, bottom, thickness is about 1～5cm, rifle head is inserted in bottom, soak absorbent cotton with citrate buffer solution, add the bacterium liquid after enzymolysis at tripping device top, centrifugal 8～the 10min of 1000～3000rpm, bottom liquid is the steady sepage that contains protoplastis, take out the absorbent cotton in tripping device, by in separation unit bottoms liquid subpackage to 1～10ml centrifuge tube, centrifugal 12～the 15min of 3000～8000rpm, after removing supernatant, the residual white precipitate in bottom is protoplastis.
At the ball milling Mierocrystalline cellulose described in the regeneration of protoplastis, it is the granulated glass sphere Mierocrystalline cellulose that oscillation treatment obtains for 48 hours under 200rpm through diameter 2～4mm.
The mould protoplastis preparation of Li Shi wood provided by the invention, the method that separates and merge, operation steps is described below more specifically:
1. preparation three parents' the mould spore suspension of Li Shi wood
Three strain Li Shi trichoderma strain QM9414, MCG77, RutC-30 mono-in picking glycerine cryopreservation tube encircles respectively, be seeded in and contain on the solid inclined-plane that 50ml potato dextrose agar (PDA) substratum makes, at 28 DEG C of temperature, cultivation 72h, obtain the mycelia that contains the mould spore of ripe Li Shi wood, add 2ml sterile distilled water, with the vortex 2min that vibrates, obtain the spore liquid that contains spore, this spore liquid is carried out to doubling dilution, and count under the microscope, make the ripe mould spore concentration of Li Shi wood 1 × 10
6the mould spore suspension of Li Shi wood of/ml.
Described PDA substratum is made up of the material of following weight proportion: potato: 200g/L (boil 30min and filter); Glucose: 20g/L; Agar: 20g/L.
2. preparation three parents' mycelia suspension
Respectively the mould spore suspension of Li Shi wood of three strain 1ml is seeded in 20ml seed culture medium, controls shaking speed is 200rpm, 28 DEG C of temperature, and cultivation 72h, falls substratum by 8 layers of filtered through gauze, obtains containing the mycelial Li Shi wood of uniting mould.
Mycelium is imported in the centrifuge tube of 50ml, be settled to 15ml with the citrate buffer solution of pH6.5,5000 turn 4 DEG C of centrifugal 10min, again add citrate buffer solution to 15ml after discarding supernatant, repeat 3 times, last, and mycelium is mixed in damping fluid.
The citrate buffer solution of described pH6.5 is made up of the material of following weight proportion: in every liter, contain: 0.1M citric acid solution 70ml, 0.1M sodium citrate solution 930ml, NaCl 0.6mol.
3. go the making of wall enzyme
Get the EP pipe of three 5ml, the lywallzyme, helicase, the cellulase that add respectively, and add respectively pH6.5 citrate buffer solution, fully dissolve.
Filter respectively three kinds of enzyme liquid with the filter membrane that aperture is 0.2 μ m also admixed together, the mixed enzyme solution finally obtaining, content is lywallzyme 2～8mg/ml, helicase 2～8mg/ml, cellulase 2～8mg/ml.
4. enzymolysis cell walls
Three strain bacterium are respectively got the mycelia suspension of 3ml, and add respectively the mixed enzyme solution of 1ml, 35 DEG C of slight vibration enzymolysis of temperature 3 hours, per half hour one microscopy observation.
5. the separation of three parents' protoplastis and fusion
Enzymolysis goes after wall, gets the centrifuge tube of a 5～50ml, is filling up absorbent cotton apart from 1～10cm place, bottom, and thickness is about 1～5cm, and rifle head is inserted in bottom, forms homemade protoplastis tripping device (as Fig. 1).Soak the absorbent cotton in tripping device with the citrate buffer solution of pH6.5, add respectively enzymolysis to remove three kinds of bacterium liquid after wall at tripping device top, centrifugal 8～the 10min of 1000～3000rpm, after centrifugal, take out absorbent cotton with tweezers, the citrate buffer solution that separation unit bottoms is contained to protoplastis divides and is filled in 1～10ml centrifuge tube, centrifugal 12～the 15min of 3000～8000rpm, after removing supernatant, the residual white precipitate in bottom is protoplastis, add at once 400 μ l fusogens that the white precipitate of 3 kinds of bacterial strains is blown molten together, final room temperature leaves standstill 10～60min, and microscopy is observed.
Described fusogen is made up of the material of following weight proportion: 20%～80% polyoxyethylene glycol (PEG4000); 0.01～0.05M CaCl
2; 0.2～1.0M sorbyl alcohol.
The present invention provides again regeneration, the screening method of cellulase high-yield, and concrete operation step is as follows:
1. the regeneration of protoplastis
The protoplastis suspension having merged is diluted to 100 times with the NaCl solution gradient of concentration 0.6mol/L, get 0.1ml diluent and be coated on protoplast regeneration substratum, see light cultivation 96h, keep relative humidity to be greater than 60%, until grow transparent circle for 28 DEG C.
Described regeneration culture medium is made up of the material of following weight proportion: KH
2pO
4: 3～5g/L; NaNO
3: 2.6～3.0g/L; Urea: 0.5～1.0g/L; FeSO
47H
2o:7.5～10mg/L; MnSO
4h
2o:2.5～3.0mg/L; ZnSO
47H
2o:3.6～5.0mg/L; CoCl
26H
2o:3.7～5.0mg/L; MgSO
47H
2o:0.5～1.0g/L; CaCl
2: 0.5～1.0g/L; Sodium desoxycholate: 2～5g/L; Peptone: 10～15g/L; NaCl:35～50g/L; Agar: 16～20g/L; Ball milling Mierocrystalline cellulose 20g/L (ball milling Mierocrystalline cellulose is used the granulated glass sphere 200rpm oscillation treatment 48h of 100g diameter 3mm left and right in advance).
2. screening and the detection method of the Li Shi trichoderma strain of High Cellulase Production:
The 6 strain bacterium colonies one that picking transparent circle diameter/colony diameter is large encircle to 1ml deionized water, counting under the microscope, and being diluted in right amount bacteria concentration is 1 × 10
6/ ml, gets 1ml and adds in fermented liquid, 28～32 DEG C of cultivation 96～144h of 200rpm, after get the fermented liquid of 1ml, 8000rpm is centrifugal, and 8～10min gets supernatant, measures enzyme and lives, the enzyme work of finding JL16 bacterial strain is high apparently higher than three strain original strains, and JL16 bacterial strain is mutant strain.
In the Erlenmeyer flask of each 250ml, contain the fermented liquid of 100ml, described fermented liquid is made up of the material of following weight proportion: peptone 3～5g/L, yeast extract 0.5～1.0g/L, KH
2pO
43～5g/L, (NH
4)
2sO
42～5g/L, urea 0.3～0.5g/L, MgSO
40.3～0.5g/L, CaCO
35～10g/L, gas explosion stalk 30～40g/L, regulate pH to 5.5.
The measuring method that enzyme is lived: the doubling of 1*6cm filter paper bar is rolled and put into test tube bottom, add 1ml citrate buffer solution, note: damping fluid will soak into filter paper completely, 50 DEG C of insulations of water-bath, add the enzyme liquid of suitable dilution, 50 DEG C of 60min reactions.Add 3ml DNS, after fully mixing, put into boiling water bath 5min, as make typical curve, add 3mlDNS simultaneously.Blank: 1.5ml citrate buffer solution, enzyme sky: 1.0ml citrate buffer solution+0.5ml enzyme liquid, filter paper blank: 1.5ml citrate buffer solution+filter paper, draw glucose typical curve, the glucose content of calculation sample, be that 2mg/ml place finds out corresponding enzyme concn at sample glucose content, with the i.e. vigor of cellulase for this reason of 0.37/ enzyme concn.
The present invention selects three strain cellulase high-yields to merge, obtain the cellulase high-yield Li Shi mould JL16 of wood (Trichoderma reesei JL16), thereby improve the overall activity of cellulase system, the energy force rate starting strain of cellulase-producing has improved 25%, can be for industrial production.In addition, the protoplastis preparation, separation, fusion, regeneration and the screening method thereof that the present invention is directed to the mould proposition of Li Shi wood have very strong application prospect.
Brief description of the drawings
Fig. 1 is protoplastis tripping device.The protoplastis tripping device accompanying drawing providing, helps related personnel to implement the present invention.
Embodiment
Embodiment 1: mould protoplastis preparation and the fusion of Li Shi wood under optimum condition
1. preparation three parents' the mould spore suspension of Li Shi wood
Li Shi Trichoderma QM9414 and Li Shi Trichoderma MCG77 buy in ATCC (American Type Culture Collection), and Li Shi Trichoderma RutC-30 buys in CGMCC (China General Microbiological Culture Collection Center).Three strain Li Shi trichoderma strain QM9414, MCG77, RutC-30 mono-in picking glycerine cryopreservation tube encircles respectively, be seeded in and contain on the solid inclined-plane that 50ml potato dextrose agar (PDA) substratum makes, at 28 DEG C of temperature, cultivation 72h, obtain the mycelia that contains the mould spore of ripe Li Shi wood, add 2ml sterile distilled water, with the vortex 2min that vibrates, obtain the spore liquid that contains spore, this spore liquid is carried out to gradient dilution, and count under the microscope, make the ripe mould spore concentration of Li Shi wood 1 × 10
6the mould spore suspension of Li Shi wood of/ml;
Described PDA substratum is made up of the material of following weight proportion: potato: 200g/L (boil 30min and filter); Glucose: 20g/L; Agar: 20g/L.
2. preparation three parents' mycelia suspension
Respectively the mould spore suspension of Li Shi wood of three strain 1ml is seeded in 20ml seed culture medium, controls shaking speed is 200rpm, and 28 DEG C of cultivation 72h of temperature, fall substratum by 8 layers of filtered through gauze, obtains containing the mycelial Li Shi wood of uniting mould.
Mycelium is imported in the centrifuge tube of 50ml, be settled to 15ml with the citrate buffer solution of pH6.5,5000 turn 4 DEG C of centrifugal 10min, again add citrate buffer solution to 15ml after discarding supernatant, repeat 3 times, last, after mycelium is mixed in damping fluid.
3. go the making of wall enzyme
The EP pipe of getting three 5ml, adds respectively lywallzyme, helicase, the cellulase of 144mg, and adds respectively the pH6.5 citrate buffer solution of 3ml, fully dissolves.
Filter respectively three kinds of enzyme liquid with the filter membrane that aperture is 0.2 μ m, obtain three kinds of enzyme liquid of about 1.5ml left and right, respectively get 1ml admixed together, finally obtain the mixed enzyme solution of 3ml, content is lywallzyme 4mg/ml, helicase 4mg/ml, cellulase 4mg/ml.
4. enzymolysis cell walls
Three strain bacterium are respectively got the mycelia suspension of 3ml, and add respectively the mixed enzyme solution of 1ml, 35 DEG C of slight vibration enzymolysis of temperature 3 hours, per half hour one microscopy observation, the enzyme liquid concentration of final liquid is lywallzyme 4mg/ml, helicase 4mg/ml, cellulase 4mg/ml.
5. the separation of protoplastis and fusion
Enzymolysis goes after wall, gets the centrifuge tube of three 15ml, is filling up absorbent cotton apart from 4cm place, bottom, and thickness is about 1cm, and 1ml rifle head is inserted in bottom, forms protoplastis tripping device.Soak the absorbent cotton in tripping device with the citrate buffer solution of pH6.5, add respectively three kinds of bacterium liquid after enzymolysis at tripping device top, the centrifugal 10min of 1000rpm, centrifugal after, take out absorbent cotton with tweezers, by separation unit bottoms liquid subpackage to 1ml centrifuge tube, the centrifugal 15min of 4000rpm, removes the residual white precipitate in bottom after supernatant and is protoplastis, adds at once 400 μ l fusogens that the white precipitate of 3 kinds of bacterial strains is blown molten together, final room temperature leaves standstill 30min, and microscopy is observed.
Described fusogen is made up of the material of following weight proportion: 50%PEG4000; 0.01M CaCl
2; 0.6M sorbyl alcohol.
Embodiment 2: the screening method of the cellulase high-yield under optimum condition
1. the regeneration of protoplastis
The protoplastis suspension having merged is diluted to 100 times with the NaCl solution gradient of concentration 0.6mol/L, get 0.1ml diluent and be coated on protoplast regeneration substratum, see light cultivation 96h, keep relative humidity to be greater than 60%, until grow transparent circle for 28 DEG C.
Described regeneration culture medium is made up of the material of following weight proportion:: KH
2pO
4: 3g/L; NaNO
3: 2.6g/L; Urea: 0.5g/L; FeSO
47H
2o:7.5mg/L; MnSO
4h
2o:2.5mg/L; ZnSO
47H
2o:3.6mg/L; CoCl
26H
2o:3.7mg/L; MgSO
47H
2o:0.5g/L; CaCl
2: 0.5g/L; Sodium desoxycholate: 2g/L; Peptone: 10g/L; NaCl:35g/L; Agar: 16g/L; Ball milling Mierocrystalline cellulose 20g/L (ball milling Mierocrystalline cellulose is used the granulated glass sphere 200rpm oscillation treatment 48h of 100g diameter 3mm left and right in advance).
2. the screening method of High Cellulase Production Li Shi trichoderma strain
The 6 strain bacterium colonies one that picking transparent circle diameter/colony diameter is large encircle to 1ml deionized water, counting under the microscope, and being diluted in right amount bacteria concentration is 1 × 10
6/ ml, gets 1ml and adds in fermented liquid, and 200rpm turns 28 DEG C and cultivates 144h, after get the fermented liquid of 1ml, 8000rpm is centrifugal, and 10min gets supernatant, measures enzyme and lives, the enzyme work of finding JL16 bacterial strain is high apparently higher than three strain original strains, and cellulose enzyme activity improves 25%, and JL16 bacterial strain is mutant strain.
In the Erlenmeyer flask of each 250ml, contain the fermented liquid of 100ml, described fermented liquid is made up of the material of following weight proportion: peptone: 3g/L; Yeast extract: 0.5g/L; KH
2pO
4: 3g/L; (NH
4) 2SO
4: 2g/L; Urea: 0.3g/L; MgSO
4: 0.3g/L; CaCO
3: 5g/L; Gas explosion stalk: 30g/L; Regulate pH to 5.5.
The screening method of the protoplastis preparation that embodiment 3: Li Shi wood is mould and fusion, cellulase high-yield
On the basis of optimum condition, within the scope of the disclosed preparation condition of summary of the invention, all can implement the present invention, obtain the bacterial strain of the mould JL16 of Li Shi wood.
Embodiment 4: the Li Shi mould JL16 bacterial strain of wood being made by embodiment 1,2 and the beta-glucosidase enzyme of three strain original strains are lived and protein yield comparison
1. the extraction of fermentation broth sample
Superior strain JL16 and the three strain original strains one of the growth of picking inclined-plane encircle to 1ml deionized water respectively, counting under the microscope, and being diluted in right amount bacteria concentration is 1 × 10
6/ ml, gets 1ml and adds in fermented liquid, 200rpm turn 28 DEG C cultivate 144h, after get appropriate fermented liquid, 8000rpm is centrifugal, and 10min gets supernatant, supernatant is preserved in-20 DEG C of refrigerators.
In the Erlenmeyer flask of each 250ml, contain the fermented liquid of 100ml, described fermented liquid is made up of the material of following weight proportion: peptone: 3g/L; Yeast extract: 0.5g/L; KH
2pO
4: 3g/L; (NH
4) 2SO
4: 2g/L; Urea: 0.3g/L; MgSO
4: 0.3g/L; CaCO
3: 5g/L; Gas explosion stalk: 30g/L; Regulate pH to 5.5.
2. the measuring method of beta-glucosidase and albumen
1.. the mensuration that beta-glucosidase enzyme is lived: pH4.8 suitably dilutes enzyme liquid with 50mM citrate buffer solution, get 1ml enzyme liquid+1ml 15mmol/L cellobiose, 50 DEG C of reaction 30min, then boiling water bath 5min, cooling rapidly, survey its glucose content by Reagent kit of glucose.By measuring, the beta-glucosidase enzyme of JL16 bacterial strain is lived and is improved 15%.
2.. the mensuration of protein content: the BSA solution of preparation Xylene Brilliant Cyanine G G-250 reagent and 1mg/ml, and to prepare BSA concentration be that the BSA standardized solution of 0～1mg/ml is some, get respectively the enzyme liquid 0.1ml of BSA standardized solution and suitably dilution in 15ml test tube, add 5ml Xylene Brilliant Cyanine G G-250 reagent fully to mix, after 5 minutes, survey OD595.By measuring, the protein yield of JL16 bacterial strain improves 12%.
Claims (7)
1. a Li Shi trichoderma strain, is characterized in that, is that the deposit number that is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 22nd, 2014 is the bacterial strain of CGMCC No.8807, and the name of bacterial strain is called the mould JL16 of Li Shi wood.
2. Li Shi trichoderma strain as claimed in claim 1, it is characterized in that, described Li Shi trichoderma strain JL6, adopt three strain cellulase high-yields by protoplastis prepare, the preparation process structure of protoplastis separation, protoplast fusion, protoplast regeneration and screening; Three described strain cellulase high-yields are Li Shi Trichoderma QM9414, Li Shi Trichoderma MCG77 and Li Shi Trichoderma RutC-30.
3. a preparation method for the Li Shi trichoderma strain of claim 1, has the preparation of protoplastis, fusion, the regeneration of protoplastis and the process of screening of protoplastis;
The preparation of described protoplastis, is that Li Shi Trichoderma QM9414, Li Shi Trichoderma MCG77 and Li Shi Trichoderma RutC-30 are seeded in respectively to potato dextrose agar, obtains the mycelia that contains the mould spore of ripe Li Shi wood; The mould spore suspension of three strain Li Shi wood is seeded in seed culture medium, obtains containing the mycelial Li Shi wood of uniting mould; By the citrate buffer solution constant volume of pH6.5 for mycelium, centrifugal, again add citrate buffer solution and mix after abandoning supernatant; Join respectively again enzymolysis cell walls in wall mixed enzyme solution, obtain the solution containing protoplastis; Finally be isolated to the mould protoplastis of Li Shi wood of white precipitate;
The fusion of described protoplastis, blows three kinds of white precipitate protoplastiss moltenly together with fusogen, room temperature leaves standstill 10～60min; Described fusogen, is made up of the material of pressing row weight proportion: 20%～80% polyoxyethylene glycol, 0.01～0.05M CaCl
2with 0.2～1.0M sorbyl alcohol;
The regeneration of described protoplastis, is by the dilution of the protoplastis suspension having merged NaCl solution gradient, gets diluent and is coated on protoplast regeneration substratum, sees light cultivation 96h, keeps relative humidity to be greater than 60%, until grow transparent circle for 28 DEG C; Described regeneration culture medium, is made up of the material of following weight proportion: KH
2pO
43～5g/L, NaNO
32.6～3.0g/L, urea 0.5～1.0g/L, FeSO
47H
2o7.5～10mg/L, MnSO
4h
2o2.5～3.0mg/L, ZnSO
47H
2o3.6～5.0mg/L, CoCl
26H
2o3.7～5.0mg/L, MgSO
47H
2o0.5～1.0g/L, CaCl
20.5～1.0g/L, Sodium desoxycholate 2～5g/L, peptone 10～15g/L, NaCl35～50g/L, agar 16～20g/L, ball milling Mierocrystalline cellulose 20g/L;
Described screening, is that the bacterial strain one that picking transparent circle diameter/colony diameter is large encircles to glycerine, obtains the mould JL16 of Li Shi wood and preserves at-80 DEG C.
4. the preparation method of Li Shi trichoderma strain as claimed in claim 3, it is characterized in that, in the preparation of protoplastis, described citrate buffer solution, is made up of the material of following weight proportion: in every liter, contain 0.1M citric acid solution 70ml, 0.1M sodium citrate solution 930ml, NaCl0.6mol.
5. the preparation method of Li Shi trichoderma strain as claimed in claim 3, it is characterized in that, in the preparation of protoplastis, the described wall mixed enzyme solution that goes, by lywallzyme, helicase, cellulase, add pH6.5 citrate buffer solution to form, each component content is lywallzyme 2～8mg/ml, helicase 2～8mg/ml, cellulase 2～8mg/ml.
6. the preparation method of Li Shi trichoderma strain as claimed in claim 3, it is characterized in that, in the preparation of protoplastis, described separation, the tripping device using is the centrifuge tube by a 5～50ml, filling up absorbent cotton apart from 1～10cm place, bottom, thickness is about 1～5cm, rifle head is inserted in bottom, soak absorbent cotton with citrate buffer solution, add the bacterium liquid after enzymolysis at tripping device top, centrifugal 8～the 10min of 1000～3000rpm, bottom liquid is the steady sepage that contains protoplastis, take out the absorbent cotton in tripping device, by in separation unit bottoms liquid subpackage to 1～10ml centrifuge tube, centrifugal 12～the 15min of 3000～8000rpm, after removing supernatant, the residual white precipitate in bottom is protoplastis.
7. the preparation method of Li Shi trichoderma strain as claimed in claim 3, is characterized in that, in the regeneration of protoplastis, described ball milling Mierocrystalline cellulose, is the granulated glass sphere Mierocrystalline cellulose that oscillation treatment obtains for 48 hours under 200rpm through diameter 2～4mm.
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| CN105039183A (en) * | 2015-08-28 | 2015-11-11 | 广东省微生物研究所 | Dichotomomyces cejpii FS110 protoplast and preparation and conversion method thereof |
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| CN104673780A (en) * | 2015-04-02 | 2015-06-03 | 天津市畜牧兽医研究所 | Pleurotus ostreatus protoplast and saccharomyces cerevisiae protoplast fusion method |
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| CN105039183A (en) * | 2015-08-28 | 2015-11-11 | 广东省微生物研究所 | Dichotomomyces cejpii FS110 protoplast and preparation and conversion method thereof |
| CN105039183B (en) * | 2015-08-28 | 2018-06-19 | 广东省微生物研究所 | A kind of angstrom moral bacterium FS110 protoplasts and preparation method thereof and method for transformation |
| CN107502624A (en) * | 2017-09-08 | 2017-12-22 | 上海市农业科学院 | A kind of method that hickory chick genetic transformation is carried out using electric shocking method |
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