CN106754433B - A kind of homeo-osmosis agent and application thereof - Google Patents
A kind of homeo-osmosis agent and application thereof Download PDFInfo
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Abstract
The present invention relates to a kind of compositions, wherein including: osmotic pressure maintains agent, moisturizer and pH buffer, and the pH value of the composition is 6.5~7.5.The composition has protective effect as homeo-osmosis agent, to protoplast, can prevent protoplast from inactivating.When using the composition homeo-osmosis agent, ARTP mutagenesis is carried out to protoplast, can be improved mutation efficiency.The invention further relates to a kind of methods using the present composition as homeo-osmosis agent mutagenesis protoplast.
Description
Technical field
The invention belongs to field of biotechnology, it is related to a kind of composition and application thereof.The composition is as homeo-osmosis
Agent has protective effect to protoplast, can prevent protoplast from inactivating.The invention further relates to a kind of application the composition works
For the method for homeo-osmosis agent mutagenesis protoplast.
Background technique
Protoplast Mutation is directly to handle protoplast with method of mutagenesis, and by breeding regeneration culture, therefrom breeding is obtained
Obtain the process of mutant strain.Since protoplast does not have cell-wall barriers, mutagens are easily accessible the probability to morph into the cell
More greatly, the time is shorter, more efficient.
Atmospheric pressure at room plasma (Atmospheric Room Temperature Plasma, abbreviation ARTP) mutagenesis is
A kind of new and effective new approaches of physical mutagenesis.ARTP induced-mutation technique uses atmospheric pressure radio-frequency glow discharge plasma source, excitation
Plasma jet with high concentration active particle acts on nucleic acid, protein, cell overall structure etc., can make microorganism
Cell wall, the structure of film and permeability changes, and cause gene damage, and then send out microbial gene sequences and its metabolism network
Raw significant changes eventually lead to microorganism and generate mutation.Different method of mutagenesis are evaluated according to DNA damage intensity and mutation rate size
Superiority and inferiority, discovery maximum sudden change rate be positively correlated with corresponding DNA damage intensity, ARTP can cause between more AT-GC
Mutation.DNA damage intensity, the mutation rate of ARTP induced-mutation technique are apparently higher than other classic mutagenesis methods.ARTP mutation breeding skill
Art is widely used in bacterium, fungi, actinomyces, mould, algae, macro fungi, plant and zooblast.
When ARTP induced-mutation technique directly acts on protoplast, homeo-osmosis agent plays important role.Firstly, seeping
Pressure stabilizer plays vital protective effect to the protoplast after removal cell wall thoroughly, prevents protoplast from inactivating.Its
Secondary, homeo-osmosis agent provides sample treatment environment when protoplast ARTP mutagenesis, the effect to ARTP high energy active ion
The power of ability has certain influence.
Influence of the composition of homeo-osmosis agent when ARTP is acted on to mutation effect is: ARTP mutagenesis, it is desirable that sample
Layer is thin, dispersion is uniform, and treating capacity is small (L grades of μ), in order to ARTP plasma jet concentration, efficiently acts on mutant materials, much
Less than the quantity of sample handling of 5mL in traditional plasm method of mutagenesis.With the extension of action time, moisture evaporation in micro system
Protoplast stability can be affected greatly, osmotic pressure increases, and the protoplast after losing protection may cause and can not draw
The property rescued shrinkage, dehydration inactivation.Therefore, under ARTP action condition, the stability of micro system directly influences ARTP to plasm
The function and effect of body.
The stability for how guaranteeing protoplast in micro system makes ARTP plasma preferably be applied to protoplast
Material becomes this field technical issues that need to address to improve the mutation efficiency of ARTP induced-mutation technique.
Summary of the invention
It is an object of the present invention to provide a kind of compositions, and the composition is as homeo-osmosis agent, to protoplast
With protective effect, it can prevent protoplast from inactivating.When using the composition homeo-osmosis agent, protoplast is carried out
ARTP mutagenesis, can be improved mutation efficiency.It is a further object to provide a kind of application present compositions as infiltration
The method of pressure stabilizer mutagenesis protoplast thoroughly.
In particular it relates to a kind of composition, it includes:
Osmotic pressure maintains 0.5~1.0wt% of agent (such as 0.6wt%, 0.7wt%, 0.8wt% or 0.9wt%),
5~40wt% of moisturizer, preferably 8~35wt%, further preferably 10~30wt% (such as 15wt%,
16wt%, 18wt%, 20wt%, 22wt%, 25wt% or 28wt%),
Surplus is pH buffer,
The pH value of the composition is 6.5~7.5 (such as 6.8,7.0,7.1,7.2 or 7.4).
In one embodiment, osmotic pressure of the present invention maintains agent to be selected from: sodium chloride and potassium chloride.The moisturizer
It is selected from: trehalose, hyaluronic acid, glycerol, polyethylene glycol and glucose.The pH buffer is selected from: PBS buffer solution and Tris-
HCl buffer.
In another embodiment, composition of the present invention, it includes:
Osmotic pressure maintains 0.5~1.0wt% of agent,
15~25wt% of moisturizer,
Surplus is pH buffer,
The pH value of the composition is 6.5~7.5.
In another embodiment, composition of the present invention, it includes:
Osmotic pressure maintains 0.6~0.9wt% of agent,
15~25wt% of moisturizer,
Surplus is pH buffer,
The pH value of the composition is 6.5~7.5.
In another embodiment, composition of the present invention, it includes:
0.5~1.0wt% of sodium chloride or potassium chloride (such as 0.6wt%, 0.7wt%, 0.8wt% or 0.9wt%),
3~8wt% of glycerol (such as 4wt%, 5wt%, 6wt% or 7wt%),
15~22wt% of trehalose (such as 16wt%, 17wt%, 18wt%, 19wt%, 20wt% or 21wt%),
Surplus is Tris-HCl buffer,
The pH value of the composition is 6.8~7.2 (such as 6.9,7.0 or 7.1).
In another embodiment, composition of the present invention, it includes:
0.5~1.0wt% of sodium chloride or potassium chloride (such as 0.6wt%, 0.7wt%, 0.8wt% or 0.9wt%),
3~8wt% of hyaluronic acid (such as 4wt%, 5wt%, 6wt% or 7wt%),
3~8wt% of glycerol (such as 4wt%, 5wt%, 6wt% or 7wt%),
3~8wt% of polyethylene glycol (such as 4wt%, 5wt%, 6wt% or 7wt%),
Surplus is Tris-HCl buffer,
The pH value of the composition is 7.0~7.5 (such as 7.1,7.2,7.3 or 7.4).
In another embodiment, composition of the present invention, it includes:
0.5~1.0wt% of sodium chloride or potassium chloride (such as 0.6wt%, 0.7wt%, 0.8wt% or 0.9wt%),
8~12wt% of glucose (such as 9wt%, 10wt% or 11wt%),
0.5~1.2wt% of hyaluronic acid (such as 0.6wt%, 0.7wt%, 0.8wt%, 1.0wt% or 1.1wt%),
3~8wt% of trehalose (such as 4wt%, 5wt%, 6wt% or 7wt%),
Surplus is PBS buffer solution,
The pH value of the composition is 6.5~7.5 (such as 6.8,6.9,7.0,7.1,7.2,7.3 or 7.4).
The purposes of homeo-osmosis agent the invention further relates to aforementioned described in any item compositions as protoplast.
The invention further relates to application of the aforementioned described in any item compositions in protoplast ARTP mutagenesis.
The invention further relates to a kind of methods of mutagenesis protoplast comprising: it is aforementioned described in any item using the present invention
Homeo-osmosis agent of the composition as protoplast.
In one embodiment, the method for mutagenesis protoplast of the present invention comprising:
(1) Protoplast suspension is provided；
(2) Protoplast suspension is placed in the described in any item compositions of the present invention, obtains protoplast to be mutagenic
Sample；
(3) mutagenesis is carried out to the protoplast sample to be mutagenic that step (2) obtains.
In another embodiment, the method for mutagenesis protoplast of the present invention, this method use atmospheric pressure at room
Plasma carries out mutagenesis to protoplast.
In another embodiment, the method for mutagenesis protoplast of the present invention, comprising the following steps:
A) broken wall treatment is carried out to cell with complex enzyme, obtains Protoplast suspension；
B) Protoplast suspension is placed in the composition, adjustment protoplast concentration is 3 × 106~3 ×
108CFU/mL obtains protoplast sample to be mutagenic；
C) using atmospheric pressure at room plasma breeding instrument treat mutagenesis protoplast sample carry out mutagenesis, volume containing the sample be 10~
30 μ L, operating power are 100~200w, and action time is 15~50 seconds.
In one embodiment, protoplast of the present invention can be bacterium, fungi, actinomyces, mould, algae
Class, macro fungi, plant and zooblast protoplast, preferably fungal protoplasts.
In one embodiment, polyethylene glycol of the present invention refers to polyethylene glycol of the molecular weight less than 2000, is
Conventional commercial reagent, is used as moisturizer in the present compositions.
PBS buffer solution of the present invention is buffer commonly used in the art, can be according to the side recorded in the prior art
Method is prepared.
Tris-HCl buffer of the present invention is buffer commonly used in the art, can be according to recording in the prior art
Method prepare.
Beneficial effects of the present invention:
Composition provided by the invention has the advantages that following one or more as homeo-osmosis agent:
1) it is able to maintain that intraor extracellular osmotic balance, protects protoplast form, prevent protoplast from inactivating；
2) use composition provided by the invention as homeo-osmosis agent, the existing state of protoplast sample is good,
When carrying out ARTP mutagenesis, it is able to ascend Mutagenic Effect, obtains more mutant strains, mutation efficiency is higher, and mutation efficiency improves
50%-200%.
3) method of mutagenesis of the invention under same Screening Platform, can obtain compared with other control method of mutagenesis
More mutant strains, mutation efficiency are higher.
Detailed description of the invention
Fig. 1 establishes the process of ARTP mutagenesis protoplast method.
Fig. 2 aspergillus oryzae protoplast ARTP mutagenesis lethality curve.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unless stated otherwise, implement
Reagent, the method and apparatus used in example is the art conventional reagent, method and apparatus.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Unless stated otherwise, used medium of the embodiment of the present invention and experimental condition are this field conventional medium and test
Condition.
The preparation of example 1 group conjunction object
The preparation of composition 1:
Formula: sodium chloride 0.8wt%,
Glycerol 5wt%,
Trehalose 20wt%,
Surplus is Tris-HCl buffer.
It by sodium chloride, glycerol, the trehalose Tris-HCl buffer solution in above-mentioned formula, is uniformly mixed, adjusts pH
Value obtains composition 1 to 7.10.
The preparation of Tris-HCl buffer used in composition 1: by 50ml 0.1mol/L trishydroxymethylaminomethane
(Tris) after solution and 45.7ml 0.1mol/L hydrochloric acid mix, be diluted with water to 100ml to get.
The preparation of composition 2:
Formula: sodium chloride 0.8wt%,
Hyaluronic acid 5wt%,
Glycerol 5wt%,
Polyethylene glycol 5wt%,
Surplus is Tris-HCl buffer.
By sodium chloride, hyaluronic acid, glycerol, the polyethylene glycol Tris-HCl buffer solution in above-mentioned formula, mixing
Uniformly, pH value is adjusted to 7.50, obtains composition 2.
The preparation of Tris-HCl buffer used in composition 2: by 50ml 0.1mol/L trishydroxymethylaminomethane
(Tris) after solution and 40.3ml 0.1mol/L hydrochloric acid mix, be diluted with water to 100ml to get.
The preparation of composition 3:
Formula: potassium chloride 0.8wt%,
Glucose 10wt%,
Hyaluronic acid 1wt%,
Trehalose 5wt%,
Surplus is PBS buffer solution.
Potassium chloride, glucose, hyaluronic acid, trehalose in above-mentioned formula is dissolved with PBS buffer solution, is uniformly mixed,
PH value is adjusted to 7.40, obtains composition 3.
The preparation of PBS buffer solution used in composition 3: by 0.27g potassium dihydrogen phosphate (KH2PO4), 1.42g disodium hydrogen phosphate
(Na2HPO4), 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl) plus deionized water about 800mL dissolution is sufficiently stirred, be then added
Concentrated hydrochloric acid tune pH value to 7.4, last constant volume to 1L to get.
The foundation of 2 ARTP method of mutagenesis of embodiment
The Establishing process of ARTP method of mutagenesis is as shown in Figure 1.
1) fungi protobiont is conventionally obtained
Plasm is produced with the fungal spore (such as aspergillus oryzae, Trichoderma viride, Pleurotus eryngii, aspergillus niger etc.) just sprouted
Body.Preparing concentration is 5 × 108The monospore suspension of CFU/ml concentration, is inoculated into fluid nutrient medium by 2% inoculum concentration,
Shaking speed is 120rpm/min, and under the conditions of set temperature is 32 DEG C, shaking flask culture 15 hours to most of spores is just sprouted
When, G3 sand core funnel filters out remaining spore, and the spore and mycelium just sprouted is collected by centrifugation.Be added appropriate composite enzyme solution into
Row broken wall treatment 3.5 hours, protoplast then is collected with G2 sand core funnel, prepares Protoplast suspension.
1) preparation of composite enzyme solution described in: cellulase, pectase, lywallzyme 3:3:1 in mass ratio are mixed, and are used
The sodium chloride of 0.8mol/L dissolves, and is configured to the composite enzyme solution that concentration is 10mg/mL, then with 0.22 μm of filtering with microporous membrane
Degerming to get.
1) formula of fluid nutrient medium described in are as follows: by peptone 5g, yeast extract powder 2g, glucose 20g, phosphoric acid hydrogen two
Potassium 1g, magnesium sulfate 0.5g, distilled water 1000mL, pH 6.2~6.6.
2) ARTP action time range is selected
Using ARTP (atmospheric pressure at room plasma) mutation breeding instrument (model: ARTP-IIIS type, purchased from no tin source clear sky
The wooden Biotechnology Co., Ltd) mutagenic treatment is carried out to the suspension of protoplast.
When this phase experiments operates, between equipment power, working gas flow and plasma emission source and load sample platform
The treatment conditions such as distance according to equipment default setting, reach mutagenesis purpose by handling time difference.
Above-mentioned 1) the middle protoplast suspension prepared is placed in (the i.e. chlorination of 0.8mol/L of conventional osmolarity stabilizer first
Sodium solution) in, adjustment protoplast concentration is 3 × 106CFU/mL-3×108CFU/mL obtains protoplast sample to be mutagenic.
The protoplast sample to be mutagenic for drawing 10 μ L volumes is added dropwise on the sample stage of ARTP, and the ARTP for carrying out the different disposal time is lured
Become, it will mixing in treated protoplast suspension and 900 μ L conventional osmolarity stabilizers (sodium chloride solution of 0.8mol/L)
Afterwards, 100 μ L are taken to be coated in protoplast regeneration culture medium respectively, 30 DEG C count after stationary culture 48 hours, after obtaining processing
Regenerate clump count.It, will by 10 μ L protoplast samples to be mutagenic (that is, protoplast sample without ARTP mutagenesis) as compareing
It is mixed with 900 μ L conventional osmolarity stabilizers (sodium chloride solution of 0.8mol/L), and 100 μ L is taken to be coated on protoplast again
In raw culture medium, 30 DEG C are counted after stationary culture 48 hours, obtain regenerating clump count before handling.
The lethality that ARTP mutagenesis protoplast is calculated according to formula, using lethality as ordinate, the processing time is horizontal seat
Mark draws protoplast ARTP mutagenesis lethality curve.Select lethality best as ARTP for 90% or so processing time
Handle the time.
The regeneration culture medium is the basal medium suitable for fungal culture containing 0.8mol/L sodium chloride, such as containing
The PDA or fermented bean drink culture medium of 0.8mol/L sodium chloride solution, preparation method can refer to Pan Li etc., and Aspergillus sojae spore is primary
The preparation of plastid and UV mutation, food and fermentation industries, the 8th phase (p1~4) of volume 32 in 2006.
The calculation formula of lethality are as follows:
Clump count is regenerated before protoplast lethality=(regenerating clump count after regeneration clump count-processing before processing)/processing
By taking aspergillus protoplast as an example, the lethality curve obtained according to the method described above is as shown in Figure 2.It is former to different fungies
Raw matter carries out ARTP mutagenesis, and when lethality is 90% or so, the ARTP optimization process time is about 15-50 seconds, according to different samples
Mutagenesis complexity choose corresponding mutagenesis lethal conditions.
3) ARTP load sample piece is the dedicated load sample piece of ARTP coordinative composition of equipments, and volume containing the sample is 10 μ L, operating power 120w, work
Throughput is 10SLM (Standard Liter per Minute, indicate standard liter per minute), plasma emission source and load
The distance between sample platform is 2mm.
Following embodiment 3-5 carry out mutagenesis to the protoplast of different fungies, investigate composition of the invention as infiltration
Press stabilizer during Protoplast Mutation for the influence of protoplast.
In embodiment 3-5:
The selection of method for preparing protoplast and ARTP processing time are substantially the same manner as Example 2.
Contrast method 1 is Mutagenesis method, after this method first prepares fungal protoplasts, with conventional infiltration
Pressure stabilizer (sodium chloride solution of 0.8mol/L) adjustment protoplast concentration is 3 × 10 thoroughly6CFU/mL-3×108CFU/mL is obtained
To protoplast sample to be mutagenic.5ml protoplast sample to be mutagenic is drawn, is tiled to routine culture ware, is carried out ultraviolet
Mutagenic treatment.Ultraviolet lamp passes through the preheating of 15min, and ultraviolet lamp power is 10W, ultraviolet apart from irradiation position vertical range 15cm
Processing time unification is set as 20 seconds.
Contrast method 2 is ARTP fungi mutagenesis, is that fungal spore is directly used to ATTP mutagenesis, the preparation of sample to be mutagenic
With 1) step is identical in embodiment 2.ARTP action time is uniformly set as 110 seconds.In other mutagenic conditions and embodiment 2
3) condition is identical.
Contrast method 3 is ARTP Protoplast Mutation method, after this method first prepares fungal protoplasts, with conventional infiltration
Pressure stabilizer (sodium chloride solution of 0.8mol/L) adjustment protoplast concentration is 3 × 10 thoroughly6CFU/mL-3×108CFU/mL is obtained
To protoplast sample to be mutagenic.10 μ L protoplast samples to be mutagenic are drawn, it is uniformly applied to load sample on piece, carry out ARTP
Mutagenic treatment, processing time unification are set as 20 seconds.
Method of mutagenesis of the invention is ARTP Protoplast Mutation method, after this method first prepares fungal protoplasts,
Use composition of the invention as homeo-osmosis agent, adjusting protoplast concentration is 3 × 106CFU/mL-3×108CFU/mL,
Obtain protoplast sample to be mutagenic.10 μ L protoplast samples to be mutagenic are drawn, it is uniformly applied to load sample on piece, are carried out
ARTP mutagenic treatment, processing time unification are set as 20 seconds.
Regeneration culture medium used in following embodiment 3-5 is the general fungi conventional medium of the industry, does not do special want
It asks.For example, cellulase selectivity plate can refer to the " screening of a plant height vigor cellulose-decomposing bacterium of University Of Tianjin's Master's thesis
And zymologic property research " (Wan Xiankai, 2004) 1.3.1.1 section described in method prepare；Laccase selectivity plate, which can refer to, " to be produced
Laccase partly knows the separation and producing enzyme research of fungi Myrothecium verrucaria NF-08 bacterial strain " (Gao Dongni etc., forestry section
Learn, 2015,51 (1): 80-87) described in method prepare；Beta-glucosidase enzyme selectivity plate, which can refer to, " produces beta-glucosidase
Screening and identification, purifying and the characterization analysis of enzyme fungi " institute in (old to wait quietly, Food Science, 2013,34 (5): 191-196)
State method preparation；The selective plate of sodium chloride solution containing 0.8mol/L is that 0.8mol/L is added on the above medium
Sodium chloride solution, to play the role of maintaining osmotic pressure.
Embodiment 3 uses aspergillus oryzae breeding cellulase strain
Protoplast, preparation method reference are produced with aspergillus oryzae spore (aspergillus oryzae is that company has the preservation of microorganism resource library by oneself)
1) the method in embodiment 2, preparation concentration is about 3 × 107The Protoplast suspension of CFU/mL.Using mutagenesis of the invention
Method and contrast method 1 and 3 carry out mutagenesis to obtained protoplast respectively, obtain mutagenesis protoplast suspension.Wherein
Using the composition 1 prepared in embodiment 1 as homeo-osmosis agent in method of mutagenesis of the invention.
Mutagenesis is carried out to aspergillus oryzae spore using contrast method 2, obtains mutagenesis spore suspension.
Gained mutagenesis protoplast suspension is used into being diluted for the sodium chloride solution of 0.8mol/L, is diluted to and is suitble to put down
The concentration of plate observation.The present embodiment is placed in gained mutagenesis protoplast suspension in the sodium chloride solution of 900 μ L0.8mol/L
It is diluted, after mixing, the cellulase selection for taking 100 μ L to be coated on the sodium chloride solution containing 0.8mol/L respectively is mild-natured
Gained mutagenesis spore suspension is placed in 900 μ L sterile waters after mixing, 100 μ L is taken to be coated on cellulase selectivity plate by plate,
It is cultivated 48 hours under the conditions of being respectively placed in 28 DEG C, obtains different mutagenesis aspergillus oryzae strains.Different method of mutagenesis are compared respectively to obtain
The Mutagenic Effect obtained, the results are shown in Table 1.
The comparison of the different method of mutagenesis mutagenesis Aspergillus oryzae cells of table 1
As seen from the above table, for the Screening And Fermenting Cultivation cellulase strain of aspergillus oryzae, method of mutagenesis of the invention and its
He compares method of mutagenesis and compares, and under same Screening Platform, can obtain more mutant strains, mutation efficiency is higher.
Embodiment 4 produces laccase bacterial strain using Pleurotus eryngii breeding
Protoplast is produced with Pleurotus eryngii spore (Pleurotus eryngii is the commercially available separation of conventional market), preparation method is referring to embodiment
1) the method in 2, preparation concentration is about 3 × 107The Protoplast suspension of CFU/mL.Use method of mutagenesis of the invention with
And contrast method 1 and 3 carries out mutagenesis to obtained protoplast respectively, obtains mutagenesis protoplast suspension.It is wherein of the invention
Method of mutagenesis in using the composition 2 prepared in embodiment 1 as homeo-osmosis agent.
Mutagenesis is carried out to Pleurotus eryngii spore using contrast method 2, obtains mutagenesis spore suspension.
Gained mutagenesis protoplast suspension is used into being diluted for the sodium chloride solution of 0.8mol/L, is diluted to and is suitble to put down
The concentration of plate observation.The present embodiment is placed in gained mutagenesis protoplast suspension in the sodium chloride solution of 900 μ L 0.8mol/L
It is diluted, after mixing, 100 μ L is taken to be coated on the laccase selectivity plate of the sodium chloride solution containing 0.8mol/L respectively, it will
Gained mutagenesis spore suspension is placed in 900 μ L sterile waters after mixing, is taken 100 μ L to be coated on laccase selectivity plate, is respectively placed in
It is cultivated 48 hours under the conditions of 28 DEG C, obtains different Pleurotus eryngii mutagenic strains.The mutagenesis that different method of mutagenesis obtain is compared respectively
Effect the results are shown in Table 2.
The comparison of the different method of mutagenesis mutagenesis Pleurotus eryngii cells of table 2
As seen from the above table, laccase bacterial strain is produced for the Screening And Fermenting Cultivation of Pleurotus eryngii, the right method of the invention is right with it
It compares according to method of mutagenesis, under same Screening Platform, more mutant strains can be obtained, mutation efficiency is higher.
Embodiment 5 produces beta-glucosidase bacterial strain using the breeding of aspergillus niger body
Protoplast, preparation method reference are produced with aspergillus niger spore (aspergillus niger is that company has the preservation of microorganism resource library by oneself)
1) the method in embodiment 2, preparation concentration is about 3 × 107The Protoplast suspension of CFU/mL.Using mutagenesis of the invention
Method and contrast method 1 and 3 carry out mutagenesis to obtained protoplast respectively, obtain mutagenesis protoplast suspension.Wherein
Using the composition 2 prepared in embodiment 1 as homeo-osmosis agent in method of mutagenesis of the invention.
Mutagenesis is carried out to aspergillus niger spore using contrast method 2, obtains mutagenesis spore suspension.
Gained mutagenesis protoplast suspension is used into being diluted for the sodium chloride solution of 0.8mol/L, is diluted to and is suitble to put down
The concentration of plate observation.The present embodiment is placed in gained mutagenesis protoplast suspension in the sodium chloride solution of 900 μ L0.8mol/L
It is diluted, after mixing, 100 μ L is taken to be coated on the beta-glucosidase enzyme selectivity of the sodium chloride solution containing 0.8mol/L respectively
Gained mutagenesis spore suspension is placed in 900 μ L sterile waters after mixing by plate, and 100 μ L is taken to be coated on beta-glucosidase selection
Mild-natured plate is cultivated 48 hours under the conditions of being respectively placed in 28 DEG C, obtains different aspergillus niger mutagenic strains.Different mutagenesis are compared respectively
The Mutagenic Effect that method obtains, the results are shown in Table 3.
The comparison of the different method of mutagenesis mutagenesis aspergillus niger cells of table 3
As seen from the above table, beta-glucosidase bacterial strain, mutagenesis side of the invention are produced for the Screening And Fermenting Cultivation of aspergillus niger
Method under same Screening Platform, can obtain more mutant strains, mutation efficiency is more compared with other control method of mutagenesis
It is high.
Claims (8)
1. a kind of composition, it includes:
0.5~1.0wt% of sodium chloride or potassium chloride,
3~8wt% of glycerol,
15~22wt% of trehalose,
Surplus is Tris-HCl buffer.
2. a kind of composition, it includes:
0.5~1.0wt% of sodium chloride or potassium chloride,
3~8wt% of hyaluronic acid,
3~8wt% of glycerol,
3~8wt% of polyethylene glycol,
Surplus is Tris-HCl buffer.
3. a kind of method of mutagenesis protoplast comprising: composition described in using as claimed in claim 1 or 22 is as protoplast
Homeo-osmosis agent.
4. the method for mutagenesis protoplast as claimed in claim 3 comprising:
(1) Protoplast suspension is provided；
(2) Protoplast suspension is placed in composition of any of claims 1 or 2, obtains protoplast sample to be mutagenic；
(3) mutagenesis is carried out to the protoplast sample to be mutagenic that step (2) obtains.
5. the method for mutagenesis protoplast as claimed in claim 4, this method is using atmospheric pressure at room plasma to protoplast
Carry out mutagenesis.
6. the method for mutagenesis protoplast described in claim 5, comprising the following steps:
A) broken wall treatment is carried out to cell with complex enzyme, obtains Protoplast suspension；
B) Protoplast suspension is placed in the composition, adjustment protoplast concentration is 3 × 106~3 × 108CFU/
ML obtains protoplast sample to be mutagenic；
C) mutagenesis protoplast sample is treated using atmospheric pressure at room plasma breeding instrument and carries out mutagenesis, volume containing the sample is 10~30 μ
L, operating power are 100~200w, and action time is 15~50 seconds.
7. purposes of the composition of any of claims 1 or 2 as the homeo-osmosis agent of protoplast.
8. application of the composition of any of claims 1 or 2 in protoplast ARTP mutagenesis.
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| CN201710094381.8A CN106754433B (en) | 2017-02-22 | 2017-02-22 | A kind of homeo-osmosis agent and application thereof |
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