CN110184199A - A kind of preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria - Google Patents
A kind of preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria Download PDFInfo
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- CN110184199A CN110184199A CN201910433431.XA CN201910433431A CN110184199A CN 110184199 A CN110184199 A CN 110184199A CN 201910433431 A CN201910433431 A CN 201910433431A CN 110184199 A CN110184199 A CN 110184199A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The present invention discloses the preparation and regeneration method of a kind of No. 1 microspecies protoplast of banana blight bacteria.This method includes the preparation of Foc1 conidial suspension, Foc1 mycelial collection, the enzymatic hydrolysis of mycelial cell wall, the collection of Foc1 protoplast, the regeneration of Foc1 protoplast.The present invention digests hyphal cell by the method, and the number for preparing of obtained protoplast is up to 6.0 × 108A/mL, gained protoplast size is uniform, and form is full, approximate circle.The present invention uses the CM solid medium for containing 200g/L glucose as regeneration culture medium, improve the regeneration rate of Foc1 protoplast, up to 28.6%, it is apparently higher than the regeneration method of existing banana blight bacteria protoplast, the pathogenic molecular mechanism of banana germ is studied for subsequent native Plastid transformation technology and provides good basis.The method of the present invention preparation process is simple, efficient, convenient for operation.
Description
Technical field
The present invention relates to filamentous fungal protoplasts preparation and technical field of regeneration in cell engineering, and in particular to one
The preparation and regeneration method of kind No. 1 microspecies (Foc1) protoplast of banana blight bacteria.
Background technique
Banana (Musa spp.) is one of most important fruit of tropical and subtropical zone, deep because its is full of nutrition, delicious flavour
Liked by people.Caused by Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubence, Foc)
Banana blight is the destructive disease in banana production, referred to as " banana cancer ", seriously affects the health hair of banana industry
Exhibition.Banana blight is a kind of typical vascular bundle diseases, can invade plant by the underground bulb of root and injury by Foc,
It is spread along vascular bundle to false stem and blade, and then obstruction conduit.The wilt for endangering China's banana mainly has 2 microspecies, i.e., and 1
Number microspecies (Foc1) and No. 4 microspecies (Foc4).Carry out the functional study of Foc pathogenic related gene, is conducive to the cause for illustrating Foc comprehensively
Anttdisease Mechanism, and the basis of effectively preventing and controlling banana fusarium wilt.
It is the important method for carrying out filamentous fungi gene functional research using the molecule transformation technology that protoplast carries out, and
Efficient protoplast preparation and the basis that regeneration techniques are then that protoplast molecule converts.Due to variety classes filamentous fungi
Eucaryotic cell structure is different, and the structure and composition difference of especially cell wall is obvious, therefore prepares the best of filamentous fungal protoplasts
There is also very big differences for condition.Currently, about banana blight bacteria protoplast preparation method there are reports.For example,
Zhang Leis etc. (2017) are prepared (25g/L driselase, 0.4g/L chitinase to Foc TR4 protoplast using enzymatic isolation method
With 15g/L lyase, 2h is digested, mycelia and enzymatic hydrolysis liquid proportional are 1:15), protoplast yield is about 2 × 107A/mL is (every
The protoplast number that milliliter enzymolysis liquid obtains).Zhang Xin (2007) is prepared Foc4 protoplast using enzymatic isolation method
(lywallzyme of 10g/L digests 3.5h, and mycelia and enzymatic hydrolysis liquid proportional are 1:20), protoplast yield is 3 × 106A/mL, then
Raw rate is 22%.(2012) such as shed howls are prepared (20g/L lywallzyme and 20g/ to Foc4 protoplast using enzymatic isolation method
L driselase digests 2h), protoplast yield is 1 × 106A/mL.On the whole, about banana blight bacteria plasm system
It is standby less with the document report of regeneration method, mostly directly with reference to the preparation and regeneration method of other filamentous fungis；And it is main
Have problems in that protoplast preparation efficiency is lower, regeneration rate is not high.Meanwhile also lack Foc1 protoplast preparation with again
The relevant report of generation method.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a kind of efficient banana blights
The preparation and regeneration method of No. 1 microspecies protoplast of bacterium.Using this method make its protoplast prepare number be up to 6.0 ×
108A/mL, regeneration rate are up to 28.6%.The present invention can provide convenience for banana blight bacteria gene functional research, can also
Efficient preparation and regeneration suitable for other filamentous fungal protoplasts.This method protoplast obtained prepares number and regeneration
No. 4 microspecies protoplast preparations of banana blight bacteria of rate obviously higher than existing report and regenerated correlation technique, and it is first
The protoplast preparation and regeneration method of secondary report No. 1 microspecies of banana blight bacteria.Meanwhile the present invention prepares protoplast and is made
Enzymolysis liquid also has the characteristics that economy.
The purpose of the invention is achieved by the following technical solution:
A kind of preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria, include the following steps:
(1) preparation of Foc1 conidial suspension:
By No. 1 microspecies of Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubence race 1,
Foc1 it) is inoculated in PDA culture medium and cultivates；Mycelia block is taken, is added in czapek's medium, shake culture；By culture solution with 100~
Supernatant is abandoned in the filtering of 200 mesh (preferably 200 mesh) cell sieve, centrifugation；It is resuspended with NCM culture medium and precipitates and be diluted, Foc1 is made
Conidial suspension；
PDA culture medium: potato 200g/L, glucose 20g/L, agar powder 9g/L add ddH2O constant volume, 121 DEG C of sterilizings
20min；
Czapek's medium: NaNO3 3g/L、K2HPO4 1g/L、KCl 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·
7H2O 0.018g/L, sucrose 30g/L adjust pH value to 6.0,121 DEG C of sterilizing 20min with distilled water constant volume；
The condition cultivated in the PDA culture medium is in 25~30 DEG C of 4~7d of culture；Preferably in 28 DEG C of culture 7d；
The condition of the shake culture is in 25~30 DEG C, 130~150rpm shake culture, 3~4d；Preferably in 28
DEG C, 3~4d of 150rpm shake culture；
The condition of the centrifugation is that 9000~12000 × g is centrifuged 10min at 4 DEG C；Preferably 10000 at 4 DEG C ×
G is centrifuged 10min；
The concentration of the Foc1 conidial suspension is 0.5~5 × 108A/mL；Preferably 2.5 × 108A/mL；
(2) the mycelial collection of Foc1:
The conidial suspension prepared is inoculated in NCM culture medium, make conidium final concentration of (1~5) ×
106A/mL；130~150rpm shake culture, 11~12h at 25~30 DEG C, with 100~200 mesh (preferably 200 mesh) cell
It is sieved through filter, and is rinsed 3~5 times with homeo-osmosis agent, fresh mycelia is obtained；
The NCM culture medium contains following components: glucose 10g/L, 4~8g/L of aspartic acid, 20 × nitrate
50mL/L, 200 × molysite 5mL/L, 1000 × vitamin 1mL/L, 1000 × microelement 1mL/L, with deionized water constant volume, pH
Value is 6.2~6.5；
Preferably, the NCM culture medium contains following components: glucose 10g/L, aspartic acid 4g/L, 20 × nitric acid
Salt 50mL/L, 200 × molysite 5mL/L, 1000 × vitamin 1mL/L, 1000 × microelement 1mL/L, with deionized water constant volume,
PH value is 6.5；
20 × the nitrate at being grouped as are as follows: NaNO3 120g/L、KCl 10.4g/L、MgSO4·7H2O 10.4g/
L、KH2PO430.4g/L adds distilled water constant volume；
200 × the molysite at being grouped as are as follows: FeSO4·7H2O 7.5g/L、Na2EDTA·2H2O 5.6g/L adds steaming
Distilled water constant volume；
1000 × the vitamin at being grouped as are as follows: glycine 2g/L, niacin 0.5g/L, biotin 0.5g/L, hydrochloric acid
Pyrrole diindyl alcohol 0.5g/L, thiamine hydrochloride 0.4g/L, riboflavin 0.5g/L, inositol 0.1g/L, add deionized water constant volume；
1000 × the microelement at being grouped as are as follows: ZnSO4·7H2O 22g/L、H3BO3 11g/L、MnCl2·
4H2O 5g/L、KI 8g/L、CoCl2·6H2O 17g/L、CuSO4·5H2O 16g/L、Na2MoO4·2H2O 15g/L adds distillation
Water constant volume；
Preferably, make conidium final concentration of 1 × 106A/mL；
Preferably, the condition of the shake culture is 11~12h of 150rpm shake culture at 28 DEG C；
The homeo-osmosis agent is 0.6~0.8mol/L NaCl solution；Preferably 0.8mol/L NaCl solution.
(3) enzymatic hydrolysis of mycelial cell wall:
The fresh mycelia for taking step (2) to obtain is added according to the ratio of every 0.1g fresh mycelia addition 1mL enzymolysis liquid
Enzymolysis liquid；At 30 ± 0.5 DEG C in the shaking table of 120~150rpm digest 3~4h, protoplast quantity reach (2.0~6.0) ×
108A/mL obtains protoplast enzymolysis liquid；
Preferably, protoplast quantity reaches (4.0~6.0) × 108A/mL；It is furthermore preferred that protoplast quantity reaches
(5.5~6.0) × 108A/mL；
The enzymolysis liquid are as follows: 10~15g/L lywallzyme, 10~15g/L driselase, solvent are 0.6~0.8mol/L
NaCl solution；Alternatively, 15~20g/L driselase, solvent are 0.6~0.8mol/L NaCl solution；It is with diameter after configuration is good
0.22 μm of membrane filtration degerming；
Preferably, the enzymolysis liquid are as follows: 10~15g/L lywallzyme, 10~15g/L driselase, solvent 0.8mol/L
NaCl solution；Alternatively, 15~20g/L driselase, solvent are 0.8mol/L NaCl solution；It is 0.22 μm that diameter is used after configuration is good
Membrane filtration degerming；
It is furthermore preferred that the enzymolysis liquid are as follows: 10g/L lywallzyme, 10g/L driselase, solvent are that 0.8mol/L NaCl is molten
Liquid；Alternatively, 15g/L lywallzyme, 15g/L driselase, solvent are 0.8mol/L NaCl solution；Alternatively, 15g/L driselase, solvent
For 0.8mol/L NaCl solution；Alternatively, 20g/L driselase, solvent are 0.8mol/L NaCl solution；It is with diameter after configuration is good
0.22 μm of membrane filtration degerming；
(4) collection of Foc1 protoplast:
Protoplast enzymolysis liquid is filtered with 3~5 layers of (preferably 4 layers) dust-free paper or lens wiping paper, supernatant is abandoned in centrifugation；It is added pre-
Precipitating is resuspended in cold STC solution；Supernatant is abandoned in centrifugation；Precipitating is resuspended the STC solution for adding pre-cooling, obtains Foc1 plasm
Body suspension；
The STC solution is 10mmol/L Tris-HCl (pH 7.5), 1.2mol/L sorbierite, 50mmol/L
CaCl2；
The condition of the centrifugation is to be centrifuged 10~15min in 4 DEG C, 400~800g；Be preferably all in 4 DEG C, 400g from
Heart 10min；
The concentration of the Foc1 protoplast suspension is 0.5~2 × 107A/mL；Preferably 1 × 107A/mL.
(5) regeneration of Foc1 protoplast:
Foc1 protoplast suspension is diluted to (0.2~1) × 10 with STC solution and sterile water respectively4A/mL is (preferably
It is 1 × 104A/mL)；100~200 μ L (preferably 100 μ L) protoplast dilution and the hypertonic regeneration of 15mL CM is taken to cultivate again
Inverted plate after base mixes is cultivated in 25~30 DEG C (preferably 28 DEG C) inversions, and 2~3d of culture is until grow regeneration bacterium colony (regeneration
The diameter of bacterium colony about 1mm)；Investigation regeneration situation, calculates regeneration rate；Regeneration rate (%)=(grown in STC solution dilution processing
Colony count-sterile water dilution handles the colony count grown) × 100/ total protoplast number；
The hypertonic regeneration culture medium of the CM are as follows: glucose 200g/L, peptone 2g/L, yeast extract 1g/L, junket egg
White hydrolysate 1g/L, 20 × nitrate 50mL/L, 1000 × vitamin 1mL/L, 1000 × microelement 1mL/L, use deionization
Water constant volume, pH value 6.5.
20 × the nitrate, 1000 × vitamin, the ingredient of 1000 × microelement are identical with step 1 (2).
The present invention has the following advantages and effects with respect to the prior art:
1. preparation process is simple, efficient, convenient for operation；
2. protoplast, which prepares number, reaches (2.0~6.0) × 108A/mL, regeneration rate be up to (25.9~
28.6) %.
The NCM culture medium that the present invention uses, can promote the conidial sprouting of banana blight bacteria, and germination rate is up to
92.3%.
The present invention digests hyphal cell by the method, and obtained protoplast prepares high (the every 0.1g mycelia enzymatic hydrolysis of number
Highest can get 6.0 × 108A protoplast), gained protoplast size is uniform, and form is full, approximate circle.Institute of the present invention
The fresh children's tender period (11~12h) for selecting the mycelium of filamentous fungi to sufficiently grow from conidium, what which was obtained
Hyphae length is larger, prepares protoplast with the period mycelia, provides the foundation to obtain the protoplast of high yield.Meanwhile
The enzymolysis liquid that the method uses also has the characteristics that experimentation cost economy.
The present invention uses the CM solid medium for containing 200g/L glucose as regeneration culture medium, and it is withered to improve banana
The regeneration rate of No. 1 microspecies protoplast of germ, up to 28.6%, hence it is evident that higher than existing banana blight bacteria protoplast
Regeneration method studies the pathogenic molecular mechanism of banana germ for subsequent native Plastid transformation technology and provides good basis.
The present invention also optimizes protoplast formation (enzymolysis time, centrifugal force), makes the preparation of Foc1 protoplast more
Easily operated, expense is lower, and significantly improves Foc1 protoplast and prepare number and regeneration rate.
Detailed description of the invention
Fig. 1 is growing state of the Foc1 conidium in NCM culture medium.
Fig. 2 is Foc1 protoplast form.
Fig. 3 is influence of the enzymolysis time to Foc1 protoplast yield.
Fig. 4 is influence of the different enzymolysis liquids to Foc1 protoplast yield.
Fig. 5 is the regeneration rate of Foc1 protoplast in different medium.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The test method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system
Make experiment condition proposed by factory.Used material, reagent etc., unless otherwise specified, for the reagent obtained from commercial channels
And material.
Wherein, No. 1 microspecies of Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubence race
1, Foc1), i.e. No. 1 biological strain of banana blight bacteria, " 201710903818.8, a kind of banana blight bacterium culture medium and
It is disclosed in its application ".
Embodiment 1
1, the preparation and regeneration method of Foc1 protoplast, specific steps are as follows:
(1) the conidial preparation of Foc1
By No. 1 microspecies of Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubence race 1,
Foc1 it) is inoculated in PDA culture medium, in 28 DEG C of culture 7d.It is beaten with the punch that diameter is 5mm and takes mycelia block, added to 250mL and look into
In family name's culture medium；In 28 DEG C, 3~4d of 150rpm shake culture.200 mesh cell sieves of culture solution are filtered, 10000 at 4 DEG C
× g is centrifuged 10min, abandons supernatant.It is resuspended with NCM culture medium after precipitating and being diluted, Foc1 conidial suspension is made, used
Blood counting chamber counts, and makes conidium concentration 2.5 × 108A/mL.
PDA culture medium is prepared: potato 200g, glucose 20g, agar powder 9g add ddH2O is settled to 1L, 121 DEG C of sterilizings
20min。
Czapek's medium is prepared: NaNO3 3g、K2HPO4 1g、KCl 0.5g、MgSO4·7H2O 0.5g、FeSO4·7H2O
0.018g, sucrose 30g are settled to 1L with distilled water, adjust pH value to 6.0,121 DEG C of sterilizing 20min.
(2) the mycelial collection of Foc1
The Foc1 conidial suspension prepared is inoculated in the NCM culture medium of 250mL, conidium final concentration is made
It is 1 × 106A/mL；11~12h of 150rpm shake culture at 28 DEG C is filtered with 200 mesh cell sieves, and is used 0.8mol/L
NaCl solution (homeo-osmosis agent) is rinsed 3~5 times, and fresh mycelia is obtained.
The specific preparation steps of NCM culture medium are as follows: glucose 10g, aspartic acid 4g, 20 × nitrate 50mL, 200 × iron
Salt 5mL, 1000 × vitamin 1mL, 1000 × microelement 1mL are settled to 1L with deionized water, adjust pH value to 6.5.It is described
20 × nitrate at being grouped as are as follows: NaNO3 120g、KCl 10.4g、MgSO4·7H2O 10.4g、KH2PO430.4g adds steaming
Distilled water is to 1L.200 × the molysite at being grouped as are as follows: FeSO4·7H2O 7.5g、Na2EDTA·2H2O 5.6g adds distillation
Water is to 1L.1000 × the vitamin at being grouped as are as follows: glycine 2g, niacin 0.5g, biotin 0.5g, hydrochloric acid pyrrole diindyl alcohol
0.5g, thiamine hydrochloride 0.4g, riboflavin 0.5g, inositol 0.1g, add deionized water to 1L.1000 × the microelement at
It is grouped into: ZnSO4·7H2O 22g、H3BO3 11g、MnCl2·4H2O 5g、KI 8g、CoCl2·6H2O 17g、CuSO4·
5H2O 16g、Na2MoO4·2H2O 15g, adds distilled water to 1L.
(3) enzymatic hydrolysis of mycelial cell wall
0.2g fresh mycelia is taken, 2mL enzymolysis liquid is added.3~4h is digested in the shaking table of 120~150rpm at 30 DEG C,
Protoplast enzymolysis liquid is obtained, and in microscopically observation protoplast production.
The enzymolysis liquid is prepared: driselase 15g/L, solvent are 0.8mol/L NaCl solution, are with diameter after configuration is good
0.22 μm of membrane filtration degerming.
(4) collection of Foc1 protoplast
Protoplast enzymolysis liquid is filtered with 4 layers of dust-free paper (Kimtech), 10min is centrifuged in 4 DEG C, 400 × g, abandons supernatant.
STC solution (10mmol/L Tris-Hcl (pH 7.5), 1.2mol/L sorbierite, the 50mmol/L CaCl of 1mL pre-cooling is added2)
Precipitating is resuspended；Supernatant is abandoned in centrifugation.Precipitating is resuspended the STC for adding 10~20mL pre-cooling, obtains Foc1 protoplast suspension,
Microscopically observation is to calculate protoplast concentration, about 1 × 107A/mL.
(5) regeneration of Foc1 protoplast
By 100 μ L Foc1 protoplast suspension (concentration about 1 × 107A/mL) it is diluted respectively with STC solution and sterile water
To 1 × 104A/mL；Inverted plate after taking 100 μ L protoplast dilutions and the hypertonic regeneration culture medium of 15mL CM to mix again, in 28
DEG C be inverted culture, culture 2~3d until grow diameter about 1mm regeneration bacterium colony；Investigation regeneration situation, calculates regeneration rate.
Regeneration rate (%)=(colony count-sterile water dilution grown in STC solution dilution processing handles the bacterium colony grown
Number) × 100/ total protoplast number.
The hypertonic regeneration culture medium of CM is prepared: glucose 200g, peptone 2g, yeast extract 1g, casein hydrolysate 1g,
20 × nitrate 50mL, 1000 × vitamin 1mL, 1000 × microelement 1mL, agar powder 9g are settled to 1L with deionized water,
PH value is adjusted to 6.5.
20 × the nitrate, 1000 × vitamin, 1000 × microelement ingredient with the present embodiment step 1 (2)
In it is identical.
2, result:
(1) Foc1 spore germination is carried out using NCM culture medium, germination rate may be up to 92.3%；Cultivate 11~12h
Afterwards, it can be obtained the fresh tender mycelium (Fig. 1) for being largely suitable for protoplast preparation.
(2) the mycelium enzymatic hydrolysis of Foc1 is carried out using the isotonic enzymolysis liquid containing driselase (15g/L), protoplast prepares number
Reach as high as 6.0 × 108A/mL, and gained protoplast size is uniform, form is full, and it is approximate circle, conducive to protoplast
It regenerates (Fig. 2).
(3) regeneration of Foc1 protoplast is carried out using the hypertonic regeneration culture medium of CM, regeneration rate is up to 28.6%.
Comparative example 1
By the Foc1 conidial suspension in 1 step 1 of embodiment (2) be inoculated in NCM culture medium be substituted for respectively it is common
PDB culture medium and CM culture medium, its conidia germination rate and mycelial yield are observed.
Interpretation of result: after Foc1 conidial suspension to be inoculated in conventional PDB culture medium and CM culture medium, in 11h
Sampling observation afterwards；The result shows that germination rate of the Foc1 conidium in PDB with CM culture medium is similar, can reach 98% with
On, it is 93% or so in NCM culture medium；Its mycelia weight in wet base is counted, is found in the NCM culture medium of every 250mL,
Foc1 weight is 0.12g, is respectively 0.14g and 0.15g in PDB and CM culture medium.I.e. in different medium, Foc1 is mitogenetic
The germination rate and mycelial yield of spore are not significantly different.
Since NCM culture medium is free of complicated albumen, only using aspartic acid as nitrogen source, preparation method is simple, is suitble to promote
Using；The conidial germination rate of banana blight bacteria is not only improved, and can be reduced (or the macromolecular of the foreign protein in culture medium
Peptide fragment) interference；Therefore, which is conducive to the analysis of Foc1 relevant physiological biochemical indicator, and is conducive to Foc and banana
In the research of cellular level interaction.
PDB culture medium is prepared: potato 200g, glucose 20g add ddH2O is settled to 1L, 121 DEG C of sterilizing 20min.
NCM culture medium prepare: glucose 10g, aspartic acid 4g, 20 × nitrate 50mL, 200 × molysite 5mL, 1000 ×
Vitamin 1mL, 1000 × microelement 1mL are settled to 1L with deionized water, adjust pH value to 6.5.20 × the nitrate,
200 × molysite, 1000 × vitamin, the ingredient of 1000 × microelement are identical with 1 step 1 of embodiment (2).
CM culture medium contains following components: glucose 10g/L, peptone 2g/L, yeast extract 1g/L, casein hydrolysis
Object 1g/L, 20 × nitrate 50mL/L, 1000 × vitamin 1mL/L, 1000 × microelement 1mL/L, with deionized water constant volume,
PH value is 6.5.20 × the nitrate, 1000 × vitamin, 1000 × microelement ingredient with 1 step 1 of embodiment (2)
In it is identical.
Embodiment 2
Enzymolysis time in 1 step 1 of embodiment (3) is substituted for 1.5h, 2h, 2.5h, 3h, 3.5h, 4h and 4.5h respectively,
The production of protoplast is observed.
Interpretation of result: after Foc1 fresh mycelia is digested different time respectively, to the protoplast of acquisition in microscope
Lower observation, the results showed that mycelium is after digesting 1.5h, protoplast yield rapid increase；It can be obtained stabilization when digesting 3h
Protoplast yield, prepare number and reach as high as 6.0 × 108A/mL (Fig. 3).
Embodiment 3
Protoplast centrifugal force in 1 step 1 of embodiment (4) is substituted for 220 × g (1500rpm), 400 × g respectively
(2000rpm), 600 × g (2500rpm), 880 × g (3000rpm) and 1600 × g (4000rpm), then protoplast is carried out
Microexamination.
Interpretation of result: Foc1 protoplast is precipitated under the effect of different centrifugal force respectively, to the plasm of acquisition
Body is observed under the microscope, as a result, it has been found that protoplast is after 400 × g (2000rpm) centrifugation, in supernatant cannot or it is few
It observes conidium, shows to can be obtained good sedimentation effect under the centrifugal force；Since protoplast does not have cell wall
Protection, it is easily broken, therefore with the raising of centrifugal force, the integrality of protoplast may be decreased.
Embodiment 4
Protoplast in 1 step 1 of embodiment (4) is filtered with dust-free paper (Kimtech) and is substituted for miracloth respectively
Filtering, lens wiping paper filtering, then microexamination is carried out to protoplast.
Foc1 protoplast: being filtered by interpretation of result with miracloth, lens wiping paper, dust-free paper respectively, heavy through being centrifuged
It forms sediment, the protoplast of acquisition is observed under the microscope, as a result, it has been found that the Foc1 protoplast obtained after 3 kinds of filtration of material does not have
There is notable difference.It is to collect the most general method of filamentous fungal protoplasts, but its cost is relatively with miracloth filtering
Height, and compare and be difficult to obtain.Dust-free paper and lens wiping paper are cheap, are the common used material in laboratory.Therefore it is recommended to use
Dust-free paper and lens wiping paper obtain Foc1 protoplast to filter.
Embodiment 5
The enzymolysis liquid (15g/L driselase) of mycelial cell wall in 1 step 1 of embodiment (3) is substituted for enzymolysis liquid 1 respectively
(10g/L driselase), enzymolysis liquid 2 (20g/L driselase), enzymolysis liquid 3 (15g/L lywallzyme), enzymolysis liquid 4 (20g/L lywallzyme),
Enzymolysis liquid 5 (10g/L lywallzyme+10g/L driselase), enzymolysis liquid 6 (15g/L lywallzyme+15g/L driselase), enzymolysis liquid 7
(10g/L lywallzyme+20g/L cellulase+30g/L glusulase), to protoplast yield with and protoplast form see
It examines.
Interpretation of result: after respectively digesting Foc1 mycelium with different enzymolysis liquids, protoplast form and yield are carried out
Analysis.As a result, it has been found that enzymolysis liquid, enzymolysis liquid 2, enzymolysis liquid 5,6 effect of enzymolysis liquid are good, protoplast prepare number can stablize 2.5 ×
108A/mL or more (Fig. 4)；And gained protoplast size is uniform, and form is full, and it is approximate circle, again conducive to protoplast
It is raw.Enzymolysis liquid preferred for this invention also has the characteristics that experimentation cost is most economical simultaneously.
Embodiment 6
The hypertonic regeneration culture medium of CM in 1 step 1 of embodiment (5) is substituted for CM sucrose regeneration culture medium, PSA regeneration respectively
Culture medium and YPS regeneration culture medium, investigation regeneration situation, calculate regeneration rate.
Interpretation of result: Foc1 protoplast is regenerated using different regeneration culture mediums, its regeneration rate is counted.
The result shows that protoplast can achieve 28.6% in the regeneration rate highest of the hypertonic regeneration culture medium of CM；PSA regeneration culture medium
Take second place；The regeneration rate of CM sucrose regeneration culture medium and YPS regeneration culture medium is then relatively low (Fig. 5).
The hypertonic regeneration culture medium of CM is prepared: glucose 200g, peptone 2g, yeast extract 1g, casein hydrolysate 1g,
20 × nitrate 50mL, 1000 × vitamin 1mL, 1000 × microelement 1mL, agar powder 9g are settled to 1L with deionized water,
PH value is adjusted to 6.5.
CM sucrose regeneration culture medium: sucrose 200g, peptone 2g, yeast extract 1g, casein hydrolysate 1g, 20 × nitre
Hydrochlorate 50mL, 1000 × vitamin 1mL, 1000 × microelement 1mL, agar powder 9g are settled to 1L with deionized water, adjust pH
It is worth to 6.5.
PSA regeneration culture medium: sucrose 274g (0.8mol/L), potato 200g, agar powder 9g are settled to deionized water
1L。
YPS regeneration culture medium: sucrose 200g, yeast extract 6g, casein hydrolysate 6g, agar powder 9g use deionization
Water is settled to 1L.
Wherein, the 20 × nitrate, 1000 × vitamin, 1000 × microelement ingredient with 1 step 1 of embodiment
(2) identical in.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria, it is characterised in that include the following steps:
(1) preparation of Foc1 conidial suspension:
Foc1 is inoculated in PDA culture medium and is cultivated；Mycelia block is taken, is added in czapek's medium, shake culture；Culture solution is used
Supernatant is abandoned in the filtering of 100~200 mesh cell sieves, centrifugation；It is resuspended with NCM culture medium and precipitates and be diluted, the mitogenetic spore of Foc1 is made
Sub- suspension；
(2) the mycelial collection of Foc1:
The conidial suspension prepared is inoculated in NCM culture medium, final concentration of (1~5) × 10 of conidium are made6A/
mL；130~150rpm shake culture, 11~12h at 25~30 DEG C is filtered with 100~200 mesh cell sieves, and steady with osmotic pressure
Determine agent to rinse 3~5 times, obtains fresh mycelia；
The NCM culture medium contains following components: glucose 10g/L, 4~8g/L of aspartic acid, 20 × nitrate 50mL/L,
200 × molysite 5mL/L, 1000 × vitamin 1mL/L, 1000 × microelement 1mL/L, with deionized water constant volume, pH value 6.2
~6.5；
The homeo-osmosis agent is 0.6~0.8mol/L NaCl solution；
(3) enzymatic hydrolysis of mycelial cell wall:
Enzymatic hydrolysis is added according to the ratio of every 0.1g fresh mycelia addition 1mL enzymolysis liquid in the fresh mycelia for taking step (2) to obtain
Liquid；3~4h is digested in the shaking table of 120~150rpm at 30 ± 0.5 DEG C, protoplast quantity reaches (2.0~6.0) × 108
A/mL obtains protoplast enzymolysis liquid；
The enzymolysis liquid are as follows: 10~15g/L lywallzyme, 10~15g/L driselase, solvent are that 0.6~0.8mol/L NaCl is molten
Liquid；Alternatively, 15~20g/L driselase, solvent are 0.6~0.8mol/L NaCl solution；It is 0.22 μm with diameter after configuration is good to filter
Film filtration sterilization；
(4) collection of Foc1 protoplast:
Protoplast enzymolysis liquid is filtered with 3~5 layers of dust-free paper or lens wiping paper, supernatant is abandoned in centrifugation；The STC solution weight of pre-cooling is added
Outstanding precipitating；Supernatant is abandoned in centrifugation；Precipitating is resuspended the STC solution for adding pre-cooling, obtains Foc1 protoplast suspension；
The condition of the centrifugation is to be centrifuged 10~15min in 4 DEG C, 400~800g；
(5) regeneration of Foc1 protoplast:
Foc1 protoplast suspension is diluted to (0.2~1) × 10 with STC solution and sterile water respectively4A/mL；Take 100 again~
Inverted plate after 200 μ L protoplast dilutions and the hypertonic regeneration culture medium of 15mL CM mix, is cultivated, training in 25~30 DEG C of inversions
2~3d is supported until growing regeneration bacterium colony；Investigation regeneration situation, calculates regeneration rate；Regeneration rate (%)=(STC solution dilution processing
On colony count-sterile water dilution for growing handle the colony count that grows) × 100/ total protoplast number；
The hypertonic regeneration culture medium of the CM are as follows: glucose 200g/L, peptone 2g/L, yeast extract 1g/L, casein water
Object 1g/L, 20 × nitrate 50mL/L, 1000 × vitamin 1mL/L, 1000 × microelement 1mL/L are solved, it is fixed with deionized water
Hold, pH value 6.5.
2. the preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria according to claim 1, feature exist
In:
The condition cultivated in PDA culture medium described in step (1) is in 25~30 DEG C of 4~7d of culture；
The condition of shake culture described in step (1) is in 25~30 DEG C, 130~150rpm shake culture, 3~4d；
The condition of centrifugation described in step (1) is that 9000~12000 × g is centrifuged 10min at 4 DEG C；
The concentration of Foc1 conidial suspension described in step (1) is 0.5~5 × 108A/mL.
3. the preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria according to claim 1 or 2, special
Sign is:
The condition cultivated in PDA culture medium described in step (1) is in 28 DEG C of culture 7d；
The condition of shake culture described in step (1) is in 28 DEG C, 3~4d of 150rpm shake culture；
The condition of centrifugation described in step (1) is that 10000 × g is centrifuged 10min at 4 DEG C；
The concentration of Foc1 conidial suspension described in step (1) is 2.5 × 108A/mL；
The mesh number of cell sieve described in step (1), (2) is 200 mesh.
4. the preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria according to claim 1, feature exist
In:
NCM culture medium described in step (2) contains following components: glucose 10g/L, aspartic acid 4g/L, 20 × nitrate
50mL/L, 200 × molysite 5mL/L, 1000 × vitamin 1mL/L, 1000 × microelement 1mL/L, with deionized water constant volume, pH
Value is 6.5；
In step (2), make conidium final concentration of 1 × 106A/mL；
The condition of shake culture described in step (2) is 11~12h of 150rpm shake culture at 28 DEG C；
Homeo-osmosis agent described in step (2) is 0.8mol/L NaCl solution.
5. the preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria according to claim 1 or 4, special
Sign is:
20 × the nitrate at being grouped as are as follows: NaNO3 120g/L、KCl 10.4g/L、MgSO4·7H2O 10.4g/L、
KH2PO430.4g/L adds distilled water constant volume；
200 × the molysite at being grouped as are as follows: FeSO4·7H2O 7.5g/L、Na2EDTA·2H2O 5.6g/L, adds distilled water
Constant volume；
1000 × the vitamin at being grouped as are as follows: glycine 2g/L, niacin 0.5g/L, biotin 0.5g/L, hydrochloric acid pyrrole diindyl
Alcohol 0.5g/L, thiamine hydrochloride 0.4g/L, riboflavin 0.5g/L, inositol 0.1g/L, add deionized water constant volume；
1000 × the microelement at being grouped as are as follows: ZnSO4·7H2O 22g/L、H3BO3 11g/L、MnCl2·4H2O
5g/L、KI 8g/L、CoCl2·6H2O 17g/L、CuSO4·5H2O 16g/L、Na2MoO4·2H2O 15g/L adds distilled water fixed
Hold.
6. the preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria according to claim 1, feature exist
In:
In step (3), protoplast quantity reaches (4.0~6.0) × 108A/mL；
Enzymolysis liquid described in step (3) are as follows: 10~15g/L lywallzyme, 10~15g/L driselase, solvent 0.8mol/L
NaCl solution；Alternatively, 15~20g/L driselase, solvent are 0.8mol/L NaCl solution；It is 0.22 μm that diameter is used after configuration is good
Membrane filtration degerming.
7. the preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria according to claim 1 or 6, special
Sign is:
In step (3), protoplast quantity reaches (5.5~6.0) × 108A/mL；
Enzymolysis liquid described in step (3) are as follows: 10g/L lywallzyme, 10g/L driselase, solvent are 0.8mol/L NaCl solution；
Alternatively, 15g/L lywallzyme, 15g/L driselase, solvent are 0.8mol/L NaCl solution；Alternatively, 15g/L driselase, solvent are
0.8mol/L NaCl solution；Alternatively, 20g/L driselase, solvent are 0.8mol/L NaCl solution；It is with diameter after configuration is good
0.22 μm of membrane filtration degerming.
8. the preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria according to claim 1, feature exist
In:
STC solution described in step (4), (5) is 10mmol/L Tris-HCl, the 1.2mol/L sorbierite of pH7.5,
50mmol/L CaCl2；
The condition of centrifugation described in step (4) is in 4 DEG C, 400g centrifugation 10min；
The concentration of Foc1 protoplast suspension described in step (4) is 0.5~2 × 107A/mL.
9. the preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria according to claim 1 or 8, special
Sign is:
In step (4), protoplast enzymolysis liquid is filtered with 4 layers of dust-free paper or lens wiping paper；
The concentration of Foc1 protoplast suspension described in step (4) is 1 × 107A/mL.
10. the preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria according to claim 1, feature
It is:
In step (5), Foc1 protoplast suspension is diluted to 1 × 10 with STC solution and sterile water respectively4A/mL；
In step (5), then inverted plate after 100 μ L protoplast dilutions and the hypertonic regeneration culture medium mixing of 15mL CM is taken, in 28
DEG C it is inverted culture, 2~3d is until grow regeneration bacterium colony for culture.
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