CN105925600A - Method for genetic transformation of volvariella volvacea by using electric shock method - Google Patents
Method for genetic transformation of volvariella volvacea by using electric shock method Download PDFInfo
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Abstract
The invention relates to a method for genetic transformation of volvariella volvacea by using an electric shock method. The method comprises the following steps: determining the minimum tolerance concentration of a volvariella volvacea mycelium to hygromycin, culturing the volvariella volvacea mycelium in a PDA (Potato Dextrose Agar) culture medium, degrading the cell wall of a volvariella volvacea mycelium cell by utilizing a lywallzyme and collecting the volvariella volvacea mycelium; putting a constructed exogenous recombinant carrier plasmid and the volvariella volvacea mycelium into an electric shock cup, finishing the electric shock process under the action of different applied electric fields, coating the volvariella volvacea mycelium after electric shock in a regeneration culture medium, performing resistance screening on the culture medium containing the hygromycin and molecularly detecting whether a normally-growing monoclone is a positive clone or not. The invention provides a novel method for volvariella volvacea molecular biology research, opens up a convenient and economic way for the establishment of a volvariella volvacea bioreactor simultaneously and provides a technical support and a guarantee for effectively solving the difficulties in the production and the storage of volvariella volvacea.
Description
Technical field
The invention belongs to Volvariella volvacea (Bull.Ex Franch.) Singer． bioengineering field, particularly to a kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method.
Background technology
Volvariella volvacea (Bull.Ex Franch.) Singer． (Volvariella volvacea) is a kind of important common edible fungi, and its Origin of cultivation, in China, has long cultivation
Training history, has the title of " China mushroom " in the world.Volvariella volvacea (Bull.Ex Franch.) Singer． is a kind of to originate in the S of the torrid zone and areas of high temperature and rainfall, subtropical zone
Fungus, is grown in the substrate of fibre rich, such as Caulis et Folium Oryzae, Banana Leaf etc. more.Belong to Basidiomycetes, Agaricales, Amanitaceae,
Volvariella volvacea (Bull.Ex Franch.) Singer． belongs to.The cultivation of Volvariella volvacea (Bull.Ex Franch.) Singer． is regional based on Asia, provinces as all in coastal area of southeastern China and country in Southeast Asia: Singapore, west, Malaysia
Sub-etc..Volvariella volvacea (Bull.Ex Franch.) Singer． has abundant economic worth: (1) is of high nutritive value, containing multiple essential amino acid--serine in Volvariella volvacea (Bull.Ex Franch.) Singer．,
Alanine, glycine etc.；(2) medical value is high, containing multivitamin, often eats and can promote health, have fall
Cholesterol, blood pressure lowering and antitumor etc. act on.(3) can the multiple decomposition such as eccrine fiber element enzyme, hemicellulase, lignoenzyme
Enzyme, therefore can be at multiple cellulose, the grown on matrix of lignin.And with short production cycle, the most more Volvariella volvacea (Bull.Ex Franch.) Singer． produces and carries
For convenient, add economic benefit.
Being weak to freezing yet with Volvariella volvacea (Bull.Ex Franch.) Singer．, under Conventional cryogenic freezing conditions (4 DEG C), its mycelia can occur self-dissolving and dead；
Sporophore also can liquefy, rot.This characteristic seriously limits the culture presevation of Volvariella volvacea (Bull.Ex Franch.) Singer． and puerperal is fresh-keeping.Volvariella volvacea (Bull.Ex Franch.) Singer． is homothallism
Fungus, life cycle is complicated, and without clamp connection between mycelia, hybrid selects to lack genetic marker, and biological transformation ratio is low, Volvariella volvacea (Bull.Ex Franch.) Singer．
Therefore cross-breeding becomes the most difficult.The development of technique for gene engineering, provides new approach for solving this difficult problem, i.e. passes through
Set up efficient Volvariella volvacea (Bull.Ex Franch.) Singer． genetic conversion system, the gene with improvement Volvariella volvacea (Bull.Ex Franch.) Singer． technique economical character is proceeded in Volvariella volvacea (Bull.Ex Franch.) Singer．, it is thus achieved that normal
Expression, the desirable strain of inheritance stability.Thus in Volvariella volvacea (Bull.Ex Franch.) Singer．, import anti-freezing protein gene can effectively solve that Volvariella volvacea (Bull.Ex Franch.) Singer． is the most low temperature resistant asks
Topic.
The method being currently used for Volvariella volvacea (Bull.Ex Franch.) Singer． genetic transformation has multiple, such as PEG method, particle bombardment, Agrobacterium_mediated method etc..King
Outstanding persons etc. use PEG method hygromycin gene to be proceeded in the protoplast of Volvariella volvacea (Bull.Ex Franch.) Singer．, through resistance screening, have obtained having tide mould
The Volvariella volvacea (Bull.Ex Franch.) Singer． transformant of element resistance.Being mainly characterized by of PEG method is easy and simple to handle, and result is stable, reproducible, it is not necessary to expensive sets
Standby, the damage to protoplast is relatively small, but aberration rate is high, and conversion ratio also ratio is relatively low, and generally 10-5-10-6.Guo Li
Fine jade etc. with plasmid pCAMBIA1301 as expression vector, the experimental condition of optimized gene marksmanship, establish Volvariella volvacea (Bull.Ex Franch.) Singer． particle bombardment lose
Pass transformation system, and prove that exogenous gene gus can stable in the mycelium of transgenic Volvariella volvacea (Bull.Ex Franch.) Singer． express；And antifreeze protein is encoded
Gene (AFP) is integrated into Volvariella volvacea (Bull.Ex Franch.) Singer． genome.Bombardment-Mediated Transformation feature is can not to be limited by protoplast preparation and regeneration issues
System, and can be directly used for transformed bacteria filament, uredospore, sporophore.But the method there is also some problems, as transformation frequency is low,
Foreign DNA integration mechanism is unclear, it is more difficult to obtain the transformant etc. of stable heredity.Wang Jie etc. proceed to anti-cold with agrobacterium-mediated transformation
Freeze protein gene AFP, explore different transformation receptor, Agrobacterium kind, the concentration of acetosyringone, the temperature co-cultured and
The impact on transformation efficiency of the factors such as time, result of the test shows that AFP gene has been integrated in the genome of Volvariella volvacea (Bull.Ex Franch.) Singer．.Agrobacterium turns
The feature of change method is to convert complete cell, and transformation efficiency is high, and transformant is stable, but host range is narrower.
Electric shocking method genetic transforming method has easy to operate, and the advantage that flow process is simple and input cost is low, the most in multiple eating
Bacterium is succeeded application, but there is not yet the relevant report that electric shocking method genetic transforming method is applied in Volvariella volvacea (Bull.Ex Franch.) Singer． both at home and abroad at present.
Summary of the invention
The technical problem to be solved is to provide a kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method, and the method is that Volvariella volvacea (Bull.Ex Franch.) Singer． molecule is raw
The research of thing provides new method, opens the convenient and approach of economy, for efficient solution for setting up Volvariella volvacea (Bull.Ex Franch.) Singer． bioreactor simultaneously
A difficult problem during certainly Volvariella volvacea (Bull.Ex Franch.) Singer． produces and stores provides technical support and guarantee.
A kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method of the present invention, including:
(1) the Volvariella volvacea (Bull.Ex Franch.) Singer． mycelia minimum tolerable concentration to hygromycin is measured；
(2) Volvariella volvacea (Bull.Ex Franch.) Singer． strain is inoculated in liquid PDA culture medium, 32 DEG C of quiescent culture 3～5 days, obtains Volvariella volvacea (Bull.Ex Franch.) Singer． mycelia；By Volvariella volvacea (Bull.Ex Franch.) Singer．
Mycelia is filtered, lywallzyme enzyme liquid enzymolysis, centrifugal, adds electroporation buffer and makes mycelia liquid；
(3) in electric shock cup, add electroporation buffer and mycelia liquid, add the piping and druming mixing of external source transfer vector plasmid, put in ice quiet
Put；By high-voltage pulse electric shock conversion instrument electric shock, voltage is 800V, stands on ice；Renewal cultivation subsequently, adds liquid regeneration
PDA culture medium, shaking table cultivates 1～2h；
(4) the mycelia liquid after step of learning from else's experience (3) conversion processing is coated without on the regeneration PDA plate of hygromycin, 32 DEG C of cultivations,
Cut the mycelia of new growth, proceed to, on the regeneration PDA plate containing minimum tolerable concentration hygromycin, screen；Choosing can
The truffle of continued growth, proceeds to, in the liquid PDA culture medium containing minimum tolerable concentration hygromycin, in 32 DEG C of cultivations, be turned
Beggar；Extracting transformant genomic DNA genome, carry out PCR Molecular Detection, Successful amplification goes out exogenous gene and then turns for the positive
Beggar.
The minimum tolerable concentration range of choice in described step (1) is 2.5～22.5ug/ml.
Consisting of of liquid PDA culture medium in described step (2): Rhizoma Solani tuber osi 200g/L, glucose 20g/L.
The preparation method of the lywallzyme enzyme liquid in described step (2) is: take 0.03g lywallzyme, molten with 0.6M mannitol solution
Solving, be settled to 2mL, crude enzyme liquid is centrifuged 15min at 3000r/min, takes supernatant after 0.22 μm filtering with microporous membrane is degerming
Use；Enzymolysis time is 3～5h.
Centrifugal being specially in described step (2) is centrifuged 5～10min under 2000～4000rpm, then uses homeo-osmosis agent
Centrifuge washing 2 times.
Described homeo-osmosis agent is 0.6mol/L mannitol solution.
Electroporation buffer in described step (2) and (3) consist of mannitol 0.6mol/L, HEPES 1mmol/L.
Liquid regeneration PDA culture medium in described step (3) consist of Rhizoma Solani tuber osi 200g/L, glucose 20g/L, manna
Alcohol 0.6mol/L.
In described step (4) regeneration PDA plate consist of Rhizoma Solani tuber osi 200g/L, glucose 20g/L, mannitol 0.6mol/L,
Agar 1.5%.
Electric shocking method is successfully applied to the genetic transformation of Volvariella volvacea (Bull.Ex Franch.) Singer． by the present invention first, overcomes via Particle Bombardment Transformation process complicated, and cost is high
Shortcoming, and the feature that Agrobacterium-mediated Transformation is unstable.Electric shocking method genetic conversion system be established as grinding of Volvariella volvacea (Bull.Ex Franch.) Singer． molecular biology
The new method of providing is provided, opens the convenient and approach of economy for setting up Volvariella volvacea (Bull.Ex Franch.) Singer． bioreactor simultaneously, raw for effectively solving Volvariella volvacea (Bull.Ex Franch.) Singer．
A difficult problem in producing and storing provides technical support and guarantee.
Beneficial effect
(1) flow process of the present invention is simple, workable, it is not required that operator has the operating experience of abundant molecular biology experiment；
(2) present invention is compared with other genetic transforming methods, with low cost, it is not necessary to use commercial reagents box or commission business, converts
Reagent needed for during is all biological chemical reagent routinely, and the genetic transformation cost completing a sample only needs 10 yuan,
Far below the conventional genetic transforming method Gene Knock-out Mice (50 yuan/sample) in current Volvariella volvacea (Bull.Ex Franch.) Singer．；
(3) present invention is that the genetic transformation of Volvariella volvacea (Bull.Ex Franch.) Singer． establishes new method, has opened up Volvariella volvacea (Bull.Ex Franch.) Singer． exogenous gene and has proceeded to cultivate with low temperature resistant Volvariella volvacea (Bull.Ex Franch.) Singer．
New way, produce and a difficult problem in storage provides technical support and guarantee for effectively solving Volvariella volvacea (Bull.Ex Franch.) Singer．.
Accompanying drawing explanation
Fig. 1 is the resistance straw mushroom bacterial V23 sensitivity testing to hygromycin of the embodiment of the present invention 1；Wherein, the hygromycin of B-I culture medium
Concentration is respectively 5,7.5,10,12.5,15,17.5,20,22.5ug/ml, A be to compare without hygromycin；
Fig. 2 be the embodiment of the present invention 1 Volvariella volvacea (Bull.Ex Franch.) Singer． genetic transformation subgenom DNA in block the amplification situation of that gene；
Fig. 3 is that the recombinant vector pCAMBIA1300::AFP of the embodiment of the present invention 2 builds collection of illustrative plates；
Fig. 4 be the embodiment of the present invention 2 Volvariella volvacea (Bull.Ex Franch.) Singer． genetic transformation subgenom in the amplification situation of external source Gene A FP；
Fig. 5 is K cryogenic treatment original strain and the place converting mycelia containing transformant pCAMBIA1300::AFP in the embodiment of the present invention 2
Reason situation.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments be merely to illustrate the present invention and not
For limiting the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the application appended claims limited range equally.
Embodiment 1
1) resistance straw mushroom bacterial V23 sensitivity testing to hygromycin: preparation containing 2.5,5,7.5,10,12.5,15,17.5,20,
The PDA plate of 22.5ug/ml hygromycin, each concentration processes 3 times and repeats.Afterwards on super-clean bench, each flat board accesses limit
The strain of the square shape of a length of 5mm, to be not added with the PDA plate of hygromycin as comparison.Inoculation is placed on the constant temperature training of 32 DEG C
Supporting in case and cultivate, every day observes and record.The speed of growth of Volvariella volvacea (Bull.Ex Franch.) Singer． V23 is affected very big by hygromycin, and when 15ug/ml, mycelia is raw
Long extremely slow, stop growing during 20ug/ml, and during 22.5ug/ml, mycelia does not grows.Therefore select hygromycin resisting as Volvariella volvacea (Bull.Ex Franch.) Singer．
Property screening reagent, and determine that the V23 optimal screening concentration on PDA plate is 20ug/ml.
2) cultivation of Volvariella volvacea (Bull.Ex Franch.) Singer． V23 mycelia: strain is inoculated in the liquid PDA culture medium (Rhizoma Solani tuber osi 200g/L, glucose 20g/L) of 100mL
In, 32 DEG C of quiescent culture 3 days.
3) prepare enzymolysis mycelia: the preparation of enzyme liquid: weigh 0.03g lywallzyme, dissolve with 0.6M mannitol solution, constant volume to 2mL,
Crude enzyme liquid is centrifuged 15min at 3000r/min, takes supernatant and uses (now with the current) after 0.22 μm filtering with microporous membrane is degerming.
Take cultured mycelia, by the filtered through gauze of two layers of sterilizing, then use aseptic water washing three times.With aseptic paper suck dry moisture rear overhang
Floating in the enzyme liquid prepared in right amount, 32 DEG C, 100rpm vibrates enzymolysis；After enzymolysis 3 hours, 4000rpm is centrifuged 5min.
With homeo-osmosis agent (0.6mol/L mannitol solution) centrifuge washing 2 times, add 500 μ L electroporation buffer (mannitol
0.6mol/L, HEPES 1mmol/L) make mycelia liquid, finally count with blood counting chamber.Each process sets 5 repetitions.
4) convert: pure water rinsing electric shock cup, use soaked in absolute ethyl alcohol 1-2h, superclean bench is placed 1h and dries up.In electric shock cup
Adding electroporation buffer and the mycelia liquid of 150ul of 100ul, the binary vector pCAMBIA1301 plasmid adding 20ul blows
Play mixing, put into 10min in ice.By high-voltage pulse electric shock conversion instrument electric shock, voltage is 800V, places 10min on ice.Extensive
Multiple cultivation, adds liquid regeneration culture medium 1ml, and shaking table is cultivated 1h (32 DEG C, 170rpm).
5) screening of electric shocking method transformant: take the Volvariella volvacea (Bull.Ex Franch.) Singer． bacterium solution after 100ul conversion processing and coat the regeneration PDA plate (horse without hygromycin
Bell potato 200g/L, glucose 20g/L, mannitol 0.6mol/L, agar 1.5%) on, put into 32 DEG C of cultivations, after one week, cut
The mycelia of new growth, proceeds on the regeneration PDA plate of 20ug/ml hygromycin, continues screening.After one week, it is chosen at 20ug/ml
On the regeneration PDA plate of hygromycin, the truffle of continued growth, proceeds in 20ug/ml hygromycin PDA fluid medium, in 32 DEG C of trainings
Support.In energy liquid medium within, the truffle of growth is considered transformant.
6) Molecular Identification of positive transformant: to cultivating by the mycelium of normal growth in the resistant panel containing hygromycin,
Extract genomic DNA genome, it is carried out PCR Molecular Detection (detection primer: F 5`
-TGCAGGAATCGGCACGAGGAA-3`, R 5`-GACTTTCATGGCTTAATTAGC-3`.PCR expands body
System is 20ul, 10 × Taq Buffer 2 μ L, dNTPs Mixture (each 10mmol/L) 0.3 μ L, and upstream and downstream primer (10 μm ol/L) is each
0.6 μ L, Taq (5U/ μ L) 0.2 μ L, template DNA 2 μ L, ddH2O mends to 20 μ L.PCR reaction condition is: 95 DEG C of denaturations
5min；95 DEG C of degeneration 30s, 56 DEG C of annealing 30s, 72 DEG C extend 30s, totally 35 circulations；72 DEG C extend 10min, 4 DEG C of forever),
It is then positive transformant that Successful amplification goes out that gene of external source gene card, and amplification sees Fig. 2.
Embodiment 2
1) SacI and KpnI utilized on carrier pCAMBIA1300 in multiple clone site is built, by the anti-freeze gene AFP in Radix Dauci Sativae
It is building up in binary vector pCAMBIA1300 form recombinant vector pCAMBIA1300::AFP, vector construction collection of illustrative plates such as Fig. 3 institute
Show.
2) strain is inoculated in the liquid PDA culture medium of 100mL, 32 DEG C of quiescent culture 2 days.
3) enzymolysis solution collocation method is with embodiment 1.Cultured mycelia, by the filtered through gauze of two layers of sterilizing, then uses aseptic water washing
Three times.With being suspended in after aseptic paper suck dry moisture in the enzyme liquid prepared in right amount, 32 DEG C, 100rpm vibrates enzymolysis；Enzymolysis 5
After hour, 2000rpm is centrifuged 10min.With homeo-osmosis agent centrifuge washing 2 times, add 500 μ L electroporation buffer and make
Mycelia liquid, finally counts with blood counting chamber.Each process sets 5 repetitions.
4) convert: pure water rinsing electric shock cup, use soaked in absolute ethyl alcohol 1.5h, superclean bench is placed 1h and dries up.In electric shock cup
Add electroporation buffer and the mycelia liquid of 150ul of 100ul, add the piping and druming mixing of 20ul plasmid, put into 10min in ice.
By high-voltage pulse electric shock conversion instrument electric shock, voltage is 800V, places 10min on ice.Renewal cultivation, adds liquid regeneration and cultivates
Base 1ml, shaking table is cultivated 2h (32 DEG C, 170rpm).
5) screening of electric shocking method transformant: take the Volvariella volvacea (Bull.Ex Franch.) Singer． bacterium solution after 100ul conversion processing and coat without on the regeneration PDA plate of hygromycin,
Put into 32 DEG C of cultivations, after one week, cut the mycelia of new growth, proceed on the regeneration PDA plate of 20ug/ml hygromycin, continue sieve
Choosing.After one week, it is chosen at the truffle of continued growth on the regeneration PDA plate of 20ug/ml hygromycin, proceeds to 20ug/ml hygromycin
In PDA fluid medium, in 32 DEG C of cultivations.In energy liquid medium within, the truffle of growth is considered transformant.
6) Molecular Identification of positive transformant: to cultivating by the mycelium of normal growth in the resistant panel containing hygromycin,
Extract genomic DNA genome, it is carried out PCR Molecular Detection (detection primer: F 5`
-TGCAGGAATCGGCACGAGGAA-3`, R 5`-GACTTTCATGGCTTAATTAGC-3`.PCR expands body
System is 20ul, 10 × Taq Buffer 2 μ L, dNTPs Mixture (each 10mmol/L) 0.3 μ L, and upstream and downstream primer (10 μm ol/L) is each
0.6 μ L, Taq (5U/ μ L) 0.2 μ L, template DNA 2 μ L, ddH2O mends to 20 μ L.PCR reaction condition is: 95 DEG C of denaturations
5min；95 DEG C of degeneration 30s, 56 DEG C of annealing 30s, 72 DEG C extend 30s, totally 35 circulations；72 DEG C extend 10min, 4 DEG C of forever),
It is then positive transformant that Successful amplification goes out external source Gene A FP gene, and expanding effect is as shown in Figure 4.
7) the low temperature tolerant comparative test of Volvariella volvacea (Bull.Ex Franch.) Singer． transformant mycelia: cultivate positive transformant, 4 DEG C of incubators of transposition after 3 days, divide for 32 DEG C
Other places are managed 1,2,3,4,5,6,7,8,9,10 days, place 32 DEG C of incubator renewal cultivations, observe mycelial after cultivation
Growing state, as shown in Figure 5.Transformant shows stronger low temperature tolerant ability (table 1) than comparison.To impinging upon 4 DEG C of low temperature
Coerce down and be only capable of 2d of surviving, and transformant is resistant to 4 DEG C of low temperature stress of 3-6d.
The different transformant of table 1 and to the survival natural law impinged upon under 4 DEG C of low temperature stress
Claims (9)
1. a Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method, including:
(1) the Volvariella volvacea (Bull.Ex Franch.) Singer． mycelia minimum tolerable concentration to hygromycin is measured；
(2) Volvariella volvacea (Bull.Ex Franch.) Singer． strain is inoculated in liquid PDA culture medium, 32 DEG C of quiescent culture 3～5 days, obtains Volvariella volvacea (Bull.Ex Franch.) Singer． mycelia；By Volvariella volvacea (Bull.Ex Franch.) Singer．
Mycelia is filtered, lywallzyme enzyme liquid enzymolysis, centrifugal, adds electroporation buffer and makes mycelia liquid；
(3) in electric shock cup, add electroporation buffer and mycelia liquid, add the piping and druming mixing of external source transfer vector plasmid, put in ice quiet
Put；By high-voltage pulse electric shock conversion instrument electric shock, voltage is 800V, stands on ice；Renewal cultivation subsequently, adds liquid regeneration
PDA culture medium, shaking table cultivates 1～2h；
(4) the mycelia liquid after step of learning from else's experience (3) conversion processing is coated without on the regeneration PDA plate of hygromycin, 32 DEG C of cultivations,
Cut the mycelia of new growth, proceed to, on the regeneration PDA plate containing minimum tolerable concentration hygromycin, screen；Choosing can
The truffle of continued growth, proceeds to, in the liquid PDA culture medium containing minimum tolerable concentration hygromycin, in 32 DEG C of cultivations, be turned
Beggar；Extracting transformant genomic DNA genome, carry out PCR Molecular Detection, Successful amplification goes out exogenous gene and then turns for the positive
Beggar.
A kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method the most according to claim 1, it is characterised in that: in described step (1)
The minimum tolerable concentration range of choice is 2.5～22.5ug/ml.
A kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method the most according to claim 1, it is characterised in that: in described step (2)
Consisting of of liquid PDA culture medium: Rhizoma Solani tuber osi 200g/L, glucose 20g/L.
A kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method the most according to claim 1, it is characterised in that: in described step (2)
The preparation method of lywallzyme enzyme liquid is: take 0.03g lywallzyme, dissolves with 0.6M mannitol solution, is settled to 2mL, crude enzyme liquid
It is centrifuged 15min at 3000r/min, takes supernatant and use after 0.22 μm filtering with microporous membrane is degerming；Enzymolysis time is 3～5h.
A kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method the most according to claim 1, it is characterised in that: in described step (2)
Centrifugal being specially is centrifuged 5～10min under 2000～4000rpm, then uses homeo-osmosis agent centrifuge washing 2 times.
A kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method the most according to claim 5, it is characterised in that: described homeo-osmosis agent is
0.6mol/L mannitol solution.
A kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method the most according to claim 1, it is characterised in that: described step (2) and (3)
In electroporation buffer consist of mannitol 0.6mol/L, HEPES 1mmol/L.
A kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method the most according to claim 1, it is characterised in that: in described step (3)
Liquid regeneration PDA culture medium consist of Rhizoma Solani tuber osi 200g/L, glucose 20g/L, mannitol 0.6mol/L.
A kind of Volvariella volvacea (Bull.Ex Franch.) Singer． electric shocking method genetic transforming method the most according to claim 1, it is characterised in that: in described step (4)
Regenerate PDA plate consists of Rhizoma Solani tuber osi 200g/L, glucose 20g/L, mannitol 0.6mol/L, agar 1.5%.
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| CN106148411A (en) * | 2016-09-09 | 2016-11-23 | 福州大学 | Use external source single stranded RNA method of marking protein in aspergillus nidulans resting spore |
| CN106191122A (en) * | 2016-09-09 | 2016-12-07 | 福州大学 | A kind of method of marking protein in fusarium moniliforme resting spore |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148412A (en) * | 2016-09-09 | 2016-11-23 | 福州大学 | A kind of method not sprouting spore transfer outside the pale of civilization source ssRNA in aspergillus flavus |
| CN106148411A (en) * | 2016-09-09 | 2016-11-23 | 福州大学 | Use external source single stranded RNA method of marking protein in aspergillus nidulans resting spore |
| CN106191122A (en) * | 2016-09-09 | 2016-12-07 | 福州大学 | A kind of method of marking protein in fusarium moniliforme resting spore |
| CN106191098A (en) * | 2016-09-09 | 2016-12-07 | 福州大学 | Foreign DNA is allowed to pass through the method that volume branch Mucor does not sprouts spore cell wall and cell membrane |
| CN106244631A (en) * | 2016-09-09 | 2016-12-21 | 福州大学 | Use external source single stranded RNA method of marking protein in monascus resting spore |
| CN106399379A (en) * | 2016-09-09 | 2017-02-15 | 福州大学 | Method for expressing protein in mucor racemosus hypnospore |
| 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 |
| CN108467870A (en) * | 2018-04-08 | 2018-08-31 | 上海市农业科学院 | A kind of method of mushroom genetic transformation |
| CN109370921A (en) * | 2018-12-13 | 2019-02-22 | 福建农林大学 | A kind of straw mushroom mycelia rejuvenation method |
| CN111423359A (en) * | 2020-03-23 | 2020-07-17 | 上海国森生物科技有限公司 | Straw mushroom UBEV2 inhibitor and screening method and application thereof |
| CN114410690A (en) * | 2021-11-25 | 2022-04-29 | 广东省农业科学院蔬菜研究所 | Anti-carboxin straw mushroom and genetic transformation method thereof |
| CN114410690B (en) * | 2021-11-25 | 2022-11-01 | 广东省农业科学院蔬菜研究所 | Anti-carboxin straw mushroom and genetic transformation method thereof |
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