CN115806885A - Preparation method of anthrax protoplast and construction method of genetic transformation system - Google Patents

Abstract

The invention belongs to the technical field of microbial genetic transformation, and particularly relates to a preparation method of an anthrax protoplast and a construction method of a genetic transformation system. According to the invention, the phosphate buffer is used for dissolving the collapsing enzyme and the muralytic enzyme to prepare the enzymolysis liquid, the anthrax hyphae are cracked to prepare the anthrax protoplast, the obtained protoplast is large in quantity and good in quality, and the steps are simple and easy to operate. The anthrax protoplast obtained by the method can quickly and effectively construct and obtain a PEG-mediated anthrax genetic transformation system, and the transformation efficiency is high.

Description

A method for preparing protoplasts of anthrax bacteria and a method for constructing a genetic transformation system

technical field

The invention belongs to the technical field of microbial genetic transformation, and in particular relates to a method for preparing protoplasts of anthrax bacteria and a method for constructing a genetic transformation system.

Background technique

Camellia anthracnose is a fungus of the genus Colletotrichum, one of the main dominant pathogenic species of tea anthracnose, and has high pathogenicity and harm to tea trees. In order to reduce the harm of camellia anthracnose to tea trees and prevent and control tea tree anthracnose, it is particularly important to use genetic engineering methods to study the pathogenicity-related genes of camellia anthracnose. However, there are few reports on the functional analysis of camellia anthracnose-related genes The reason is that there is a lack of a stable genetic transformation system of Camellia anthracnose, which cannot meet the basic requirements of the functional analysis of genes related to the pathogenicity of Camellia anthracnose, and it is urgent to establish a highly efficient and stable PEG-mediated genetic transformation system of Camellia anthracnose, which will provide a basis for the follow-up research on Camellia anthracnose The key pathogenic gene of anthrax provides technical basis.

Although there are related studies on the genetic transformation system of Camellia anthracnose in the prior art, most of them use lyase to extract the protoplasts of Camellia anthracnose, and the protoplasts obtained are small in number, poor in quality, and the steps are cumbersome. The genetic transformation efficiency of anthrax bacteria is not high.

Contents of the invention

The object of the present invention is to provide a kind of preparation method of anthrax bacteria protoplast and the construction method of genetic transformation system, the number of protoplasts prepared by the preparation method of described anthrax bacteria protoplast is large, the quality is good and the steps are simple and convenient, and then can improve follow-up Transformation efficiency of genetic transformation systems.

The invention provides a kind of preparation method of protoplast of anthrax bacterium, comprises the steps:

mixing mycelia of the anthracnose bacteria with the enzymatic solution, and cracking to obtain the lysate;

Cultivating the lysate for 3-8 hours to obtain a protoplast body fluid, which includes Bacillus anthracis protoplasts;

The enzymolysis solution includes a solvent and a solute, the solvent includes a phosphate buffer, and the solute includes 1-3 wt.% of a collapse enzyme and 0.5-1 wt.% of a lysozyme.

Preferably, the concentration of the anthrax protoplasts in the protoplast fluid is (1.6-3.6)×10 ⁷ cells/mL.

Preferably, the oscillating frequency of the cracking is 80-120 rpm, the temperature is 28-32° C., and the time is 10-30 min.

Preferably, the anthracnose bacteria include Bacillus anthracis camellia.

Preferably, the preparation method of the mycelium of the anthrax mycelium comprises:

Inoculating the activated anthrax bacterial strain into a spore medium for spore culture to obtain conidia;

The conidia are inoculated into mycelium medium for mycelial culture to obtain anthrax mycelium; the vibration frequency of the mycelium culture is 150-200 rpm, the temperature is 25-28° C., and the time is 12-16 hours.

The present invention also provides the application of the preparation method described in the above technical scheme in constructing the genetic transformation system of Bacillus anthracis.

The present invention also provides a method for constructing an anthrax genetic transformation system, comprising the following steps:

The preparation method described in the above technical scheme is used to prepare the anthrax protoplast;

The anthrax protoplasts are mixed with the STC solution to obtain an anthrax protoplast suspension;

Mix the protoplast suspension of the anthrax bacteria, the plasmid containing the antibiotic marker gene and the target gene, and the sodium heparin solution, and let stand at 0-4°C for 30 minutes to obtain the first protoplast solution;

Add SPTC solution dropwise to the first protoplast solution, and let stand at 0-4°C for 30 minutes to obtain a second protoplast solution;

Suspending the second protoplast solution in the regeneration medium for recovery to obtain a recovered protoplast solution;

The resuscitated protoplast solution is mixed with a selective medium, and cultured in the dark for 3-5 days to obtain an anthracis genetic transformation system.

Preferably, the ratio of the volume of the protoplast suspension of the anthrax bacteria, the mass of the plasmid containing the antibiotic marker gene and the target gene, and the volume of the sodium heparin solution is 0-100 μL: 1-5 μg: 1-2 μL; The concentration of the protoplasts of Camellia anthracis in the protoplast suspension is (1-3)×10 ⁵ /μL; the concentration of the sodium heparin solution is 0.1 g/mL.

Preferably, the regeneration medium uses water as a solvent, including: 1 wt.% of yeast powder, 1 wt.% of casamino acids and 274 wt.% of sucrose; the pH value is 7.0;

The selective medium uses water as a solvent and includes: 20wt.% of potato juice, 2wt.% of glucose, 1.5wt.% of agar and antibiotics corresponding to the antibiotic marker gene.

Preferably, the antibiotic marker gene includes G418 resistance expression gene.

Beneficial effect:

The invention provides a method for preparing protoplasts of Bacillus anthracis, comprising the following steps: mixing mycelia of Bacillus anthracis with an enzymatic hydrolysis solution, shaking and lyzing to obtain a lysate; performing shaking culture on the lysate for 3 to 8 hours to obtain protoplasts Body fluid, the protoplast body fluid includes anthrax protoplasts; the enzymolysis solution uses phosphate buffer as a solvent and includes 1-3 wt.% of collapsing enzyme and 0.5-1 wt.% of lysozyme. The invention uses the enzymolysis solution prepared by dissolving the collapse enzyme and the lysozyme in the phosphate buffer solution to crack the mycelia of the anthrax bacteria to prepare the anthracis bacteria protoplasts, and the obtained protoplasts are large in quantity and good in quality, and the steps are simple and easy to operate. The anthracis protoplasts obtained in this way can quickly and effectively obtain a PEG-mediated anthrax genetic transformation system, and the transformation efficiency is high.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings required in the embodiments.

Fig. 1 is the statistical figure of the anthrax protoplast quantity in the lysate prepared in the embodiment 1～6 and comparative example 1;

Fig. 2 is the anthrax protoplast morphological figure in the lysate prepared in the embodiment 1～6 and comparative example 1;

Fig. 3 is the G418 resistance screening result in embodiment 7;

Fig. 4 is the agarose gel electrophoresis detection figure that transformant genome extracts in test example 2;

Fig. 5 is the agarose gel electrophoresis detection picture of target gene PCR amplification in the transformant in test example 2;

Fig. 6 is the GFP fluorescence picture observed in the transformant under a fluorescence microscope in Test Example 2;

Fig. 7 is a flow chart of the method for constructing the genetic transformation system of Camellia anthracis in Example 8.

Detailed ways

The invention provides a kind of preparation method of protoplast of anthrax bacterium, comprises the steps:

mixing mycelia of the anthracnose bacteria with the enzymatic solution, and cracking to obtain the lysate;

Cultivating the lysate for 3-8 hours to obtain a protoplast body fluid, which includes Bacillus anthracis protoplasts;

The enzymolysis solution includes a solvent and a solute, the solvent includes a phosphate buffer, and the solute includes 1-3 wt.% of a collapse enzyme and 0.5-1 wt.% of a lysozyme.

In the present invention, the anthracnose bacteria preferably include Bacillus anthracis plantarum, more preferably Bacillus anthracis camellia. The present invention has no special limitation on the strain of the camellia anthracnose, and the conventional camellia anthracnose strains in the art can adopt the preparation method in the present invention to prepare protoplasts, such as the camellia anthracnose LS_19 strain used in the embodiment of the present invention.

The present invention preferably prepares anthrax mycelia, and the preparation method of said anthrax mycelia preferably includes:

Inoculating the activated anthrax bacterial strain into a spore medium for spore culture to obtain conidia;

The conidia are inoculated into mycelium medium for mycelial culture to obtain anthrax mycelium; the vibration frequency of the mycelium culture is 150-200 rpm, the temperature is 25-28° C., and the time is 12-16 hours.

In the present invention, preferably, the activated anthracis bacterial strain is inoculated into a spore culture medium for spore culture to obtain a spore culture solution. The volume ratio of the inoculum amount of the anthracis strain of the present invention to the spore culture medium is preferably (1-5):(10-50), more preferably 1:50. In the present invention, there is no special limitation on the activation method of the anthrax strain, and conventional activation methods in the art can be used. The spore culture medium of the present invention preferably comprises PDB liquid culture medium. The spore culture of the present invention is preferably a constant temperature shaking culture, and the spore culture is preferably carried out on a shaker. The shaking frequency of the spore culture in the present invention is preferably 150-200 rpm, more preferably 200 rpm; the temperature is preferably 25-28°C, more preferably 28°C; the time is preferably 60-72h, more preferably 72h. The spore culture of the present invention is preferably dark culture.

After obtaining the spore culture solution, the present invention preferably further includes filtering the spore culture solution, performing first centrifugation on the obtained culture solution, and discarding the supernatant to obtain conidia. The present invention preferably uses a sterilized funnel covered with a filter membrane to carry out the filtration. The filter membrane preferably includes three layers of Miracloth filter membranes, and the pore size of the Miracloth filter membrane is preferably 22-25mm, more preferably 22mm; the material of the Miracloth filter membrane is preferably polyester rayon filter cloth. The present invention preferably further includes autoclaving and drying the filter membrane before performing the filtration, and the temperature of the autoclaving is preferably 121° C. for 20 minutes. The temperature of the first centrifugation in the present invention is preferably 0-4°C, more preferably 4°C, the rotation speed is preferably 3000-5000rpm, more preferably 5000rpm, and the time is preferably 3-10min, more preferably 5min.

After the conidia are obtained, the present invention preferably inoculates the conidia into a mycelial culture medium for mycelial culture to obtain a mycelial culture solution for Mycelium anthracis. The mycelia culture medium of the present invention preferably includes M3S liquid culture medium. The vibration frequency of mycelia culture in the present invention is preferably 150-200 rpm, more preferably 200 rpm; the temperature is preferably 25-28°C, more preferably 28°C; the time is preferably 12-16h, more preferably 14h. The shaking culture for 12-16 hours in the present invention can keep the mycelia of the anthracnose fungus at a suitable maturity, and avoid reducing the quantity and quality of protoplasts obtained by cracking due to over-mature mycelia. The mycelia culture in the present invention is preferably constant temperature culture.

After obtaining the mycelia culture solution of the anthracis bacteria, the present invention preferably filters the culture solution of the bacteria mycelium anthracis. The filter device used for the filtration in the present invention is preferably the same as the filter device used for the above-mentioned filtration of the spore culture solution, and will not be repeated here.

After the filtration, the present invention preferably uses sterile water to rinse the filtered mycelia to obtain the anthracis mycelia. The number of flushing in the present invention is preferably 3 times.

After the mycelia of the anthracis fungus are obtained, the present invention mixes the mycelia of the anthracis fungus with an enzymatic hydrolysis solution, and cracks to obtain a lysate. The mass-to-volume ratio of the mycelium of the anthracnose fungi and the enzymatic hydrolysis solution in the present invention is preferably 0.1-1 g: 5-20 mL, more preferably 0.1-1 g: 10 mL. The enzymolysis solution of the present invention preferably includes a solvent and a solute, the solvent preferably includes a phosphate buffer, the solute preferably includes 1 to 3 wt.% of a collapsing enzyme and 0.5 to 1 wt.% of a lytic enzyme, and more preferably includes 2 wt of a collapsing enzyme .% and lysozyme 1wt.%. Compared with conventional lyases, the enzymatic hydrolysis solution of the present invention has a better lysis effect, and the protoplasts obtained by lysis are more in quantity and better in quality. The oscillation frequency of the cracking in the present invention is preferably 80-120 rpm, more preferably 100 rpm; the temperature is preferably 28-32°C, more preferably 30.5°C; the time is preferably 10-30min, more preferably 20min. The lysis in the present invention is preferably carried out on a shaker.

After obtaining the lysate, the present invention preferably further includes filtering the lysate. In the present invention, a bacterial filter is preferably used for the filtration, and the pore size of the bacterial filter is preferably 0.22 μm.

After the filtration, the present invention preferably cultures the obtained lysate for 2-8 hours to obtain the protoplast body fluid. The shaking frequency of the culture in the present invention is preferably 80-120 rpm, more preferably 100 rpm; the temperature is preferably 28-30°C, more preferably 30.5°C. The time for culturing in the present invention is preferably 5-8 hours, more preferably 6-7 hours, more preferably 7 hours. The protoplast body fluid of the present invention includes Bacillus anthracis protoplasts, and the concentration of the Bacillus anthracis protoplasts in the protoplast body fluid is preferably (1.6-3.6)×10 ⁷ /mL, more preferably 3.6×10 ⁷ /mL mL. After 2 hours of the culture, the present invention preferably further includes observing the body fluid of the cultured protoplast every 1 hour until the above concentration is reached. The cultivation in the present invention is preferably carried out on a shaker.

After the protoplast solution is obtained, the present invention preferably performs second centrifugation on the protoplast body liquid, discards the supernatant, and obtains the anthrax protoplast. The temperature of the second centrifugation in the present invention is preferably 0-4°C, more preferably 4°C, the rotation speed is preferably 2000-4000rpm, more preferably 3000rpm, and the time is preferably 3-10min, more preferably 5min.

The invention prepares an enzymolysis solution by dissolving the collapse enzyme and the wall-lyzing enzyme in a phosphate buffer solution to crack the mycelia of the anthracis fungus to prepare the protoplast of the anthracis fungus, and the protoplasts obtained are large in quantity and good in quality, and the steps are simple and easy to operate.

Based on the above-mentioned advantages, the present invention also provides the application of the preparation method described in the above-mentioned technical solution in constructing an anthracis genetic transformation system. The anthracnose bacteria described in the present invention preferably include plant anthracnose, more preferably include anthracnose camellia.

The present invention also provides a method for constructing an anthrax genetic transformation system, comprising the following steps:

The preparation method described in the above technical scheme is used to prepare the anthrax protoplast;

The anthrax protoplasts are mixed with the STC solution to obtain an anthrax protoplast suspension;

Mix the protoplast suspension of the anthrax bacteria, the plasmid containing the antibiotic marker gene and the target gene, and the sodium heparin solution, and let stand at 0-4°C for 30 minutes to obtain the first protoplast solution;

adding SPTC solution to the first protoplast solution, and standing at 0-4°C for 30 minutes to obtain a second protoplast solution;

Suspending the second protoplast solution in regeneration medium for recovery to obtain a recovered protoplast solution;

The resuscitated protoplast solution is mixed with a selective medium, and cultured in the dark for 3-5 days to obtain an anthracis genetic transformation system.

In the present invention, the anthracis genetic transformation system preferably includes an anthracis genetic transformation system.

The present invention adopts the preparation method described in the above technical scheme to prepare the anthracis protoplast. The steps and process for preparing the protoplasts of B. anthracis in the present invention are preferably the same as the above-mentioned technical solution, and will not be repeated here.

After the anthrax protoplasts are obtained, the present invention mixes the anthrax protoplasts with an STC solution to obtain an anthrax protoplast suspension. The mixing process of the present invention preferably includes: mixing the anthracis protoplasts with the STC solution and centrifuging for the third time, and repeating this process twice. The conditions of the third centrifugation in the present invention are preferably the same as the conditions of the second centrifugation, and will not be repeated here. The concentration of the anthracis protoplasts in the anthracis protoplast suspension in the present invention is preferably (1-3)×10 ⁵ /μL.

After obtaining the anthrax protoplast suspension, the present invention mixes the anthrax protoplast suspension, a plasmid containing an antibiotic marker gene and a target gene, and a sodium heparin solution, and leaves it standing at 0-4°C for 30 minutes to obtain the first protoplast body solution. The ratio of the volume of the anthrax protoplast suspension of the present invention, the quality of the plasmid containing the antibiotic marker gene and the target gene, and the volume of the sodium heparin solution is preferably 20-100 μL: 1-5 μg: 1-2 μL, more preferably 50 μL: 2 μg: 1 μL. The concentration of the heparin sodium solution in the present invention is preferably 0.1 g/mL. The resistance marker gene in the plasmid containing the antibiotic marker gene and the target gene of the present invention preferably includes a G418 resistance expression gene or a hygromycin B resistance expression gene, more preferably a G418 resistance expression gene. Among the plasmids containing antibiotic marker genes and target genes in the present invention, the initial plasmids preferably include pBlueScriptII(+), pBARGPE1-Hygro(+) or pBin(+) plasmids, more preferably pBlueScriptII(+) plasmids. The present invention has no special limitation on the target gene in the plasmid containing the antibiotic marker gene and the target gene, any anthrax-related gene or model marker gene is acceptable, such as the green fluorescent protein GFP gene used in the embodiment of the present invention.

After obtaining the first protoplast solution, the present invention adds SPTC solution to the first protoplast solution, and leaves it standing at 0-4° C. for 30 minutes to obtain the second protoplast solution. The volume ratio of the SPTC solution in the present invention to the anthracis protoplast suspension is preferably 2:1. The addition of the present invention is preferably dropwise addition, and the method of adding the SPTC solution to the first protoplast solution by adding dropwise will interfere with the recognition between cells by causing the disorder of the surface charge of the cell membrane, thereby facilitating Cell-to-cell fusion and entry of foreign DNA molecules into protoplasts.

After the second protoplast solution is obtained, the present invention suspends the second protoplast solution in regeneration medium for recovery to obtain a recovered protoplast solution. The volume ratio of the second protoplast solution in the present invention to the regeneration medium is preferably 1:20. The regeneration medium of the present invention preferably uses water as a solvent, and further includes: 1 wt.% of yeast powder, 1 wt.% of casamino acids and 274 wt.% of sucrose; the pH value of the regeneration medium is preferably 7.0. In the present invention, the resuscitation is preferably carried out in a constant temperature shaking incubator, the temperature of the resuscitation is preferably 25-28°C, more preferably 26°C; the shaking frequency is preferably 80-120rpm, more preferably 100rpm; the time is preferably 12-28°C. 16h, more preferably 16h.

After obtaining the revived protoplast solution, the present invention mixes the revived protoplast solution with a selective medium and cultures in dark for 3-5 days to obtain an anthrax genetic transformation system. The volume ratio of the recovered protoplast solution to the selection medium in the present invention is preferably 1-3:10-30, more preferably 1:20. The present invention preferably further includes pouring the mixture of the resuscitated protoplast solution and the selective medium into a petri dish before performing the dark culture. The dark culture of the present invention is preferably carried out in a constant temperature incubator. The time of the dark cultivation in the present invention is preferably 5 days; the temperature is preferably 25-28°C, more preferably 28°C. The selective medium of the present invention preferably uses water as a solvent, including: 20wt.% of potato juice, 2wt.% of glucose, 1.5wt.% of agar and antibiotics corresponding to the antibiotic marker gene; The pH value is preferably a natural value. The concentration of the antibiotic in the present invention is preferably set according to the antibiotic marker gene; in an embodiment of the present invention, G418 (geneticin) is included in the selection medium, and the concentration of G418 is preferably 150 μg/mL.

In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

Reagents, medium and preparation methods used in the following examples:

PDA solid medium: use water as solvent, potato juice 20wt.%, glucose 2wt.%, agar 2wt.%, pH value is natural value, autoclaved at 121°C for 20min;

PDB liquid medium: use water as solvent, potato juice 20wt.%, glucose 2wt.%, pH value is natural value, autoclave at 121°C for 20min;

M3S liquid medium: use water as solvent, magnesium sulfate heptahydrate 2.5wt.%, potassium dihydrogen phosphate 2.7wt.%, peptone 1wt.%, yeast extract 1wt.%, sucrose 10wt.%, pH natural value, 121°C Autoclave for 20 minutes;

Regeneration medium: use water as solvent, yeast powder 1wt.%, casein amino acid 1wt.%, sucrose 274wt.%, pH value 7.0, autoclave at 121°C for 20 minutes;

Strain selective medium: use water as solvent, potato juice 20wt.%, glucose 2wt.%, agar 1.5wt.%, pH natural value, autoclave at 121°C for 20min, according to the concentration of G418 in the medium to be obtained, add Corresponding concentration of G418;

LB liquid medium: use water as solvent, yeast powder 5wt.%, sodium chloride 10wt.%, tryptone 10wt.%, pH=7.0, autoclave at 121°C for 20 minutes;

Heparin sodium solution: 100mg of heparin sodium powder was dissolved in 1mL sterile distilled water, and sterilized by filtration with a 0.22μm bacterial filter;

G418: purchased from Beijing Kulaibo Technology Co., Ltd.;

Enzymolysis solution: 2wt.% of collapsing enzyme, 1wt.% of lysozyme, 10mL phosphate buffer, 0.22μm bacterial filter for filter sterilization, wherein the phosphate buffer is: water as solvent, sodium chloride 7wt.%, Calcium chloride 1wt.%, disodium hydrogen phosphate 0.5wt.%, pH natural value, use double distilled water to make up to 1L, sterilize at 121°C for 20min;

STC reagents: Sorbitol 145.6g, Tris 6.05g, CaCl ₂ ·H ₂ O 7.35g, dissolved in 1L sterile distilled water, pH value is 7, autoclaved at 121°C for 20min;

SPTC reagent: 40g PEG was dissolved in 100mL STC reagent, and autoclaved at 121°C for 20min.

Example 1

A preparation method of camellia anthracnose protoplast, the steps are as follows:

A1) Inoculate fresh activated Camellia anthracis LS_19 into PDB liquid medium, 28 ℃ constant temperature shaker 200rpm, dark culture 72h, produce a large number of conidia;

A2) Cover three layers of Miracloth filter membranes on the sterilizing funnel, filter the above culture solution, centrifuge at 5000rpm at 4°C for 5min, discard the supernatant, and collect conidia;

A3) Place the conidia in 50 mL of M3S liquid medium, in a constant temperature shaker at 28° C. at 200 rpm, and culture in the dark for 14 hours to produce tender hyphae;

A4) Cover three layers of Miracloth filter membranes on the sterilizing funnel, filter the above solution, collect the young mycelium on the filter membrane, then rinse three times with sterile water, add the 1g young mycelium of collection to the sterile triangular cone in the bottle;

A5) Prepare 10mL enzymatic hydrolysis solution (10mL phosphate buffer contains 2wt.% collapsing enzyme and 1wt.% lysozyme), 30.5°C constant temperature shaker at 100rpm, shaking culture for 10-30min, 0.22μm bacterial filter for filter sterilization After adding to the Erlenmeyer flask;

A6) Place the above solution in a constant temperature shaking incubator at a temperature of 30.5° C. and a rotation speed of 100 rpm, and incubate for 7 hours. After two hours, observe once every hour to obtain protoplast body fluid;

A7) Place the protoplast body fluid obtained in step A6) in a centrifuge, centrifuge at 3000 rpm at 4°C for 5 min, and discard the supernatant for later use.

Example 2

The preparation method in Example 1 was used to prepare protoplasts of Anthracnose camellia, with the difference that the cultivation time in step A6) was 8 hours.

Example 3

The preparation method in Example 1 was used to prepare protoplasts of Anthracnose camellia, with the difference that the cultivation time in step A6) was 6 h.

Example 4

The preparation method in Example 1 was used to prepare protoplasts of Anthracnose camellia, with the difference that the cultivation time in step A6) was 5 h.

Example 5

The preparation method in Example 1 was used to prepare protoplasts of Anthracnose camellia, with the difference that the cultivation time in step A6) was 4 h.

Example 6

The preparation method in Example 1 was used to prepare protoplasts of Anthracnose camellia, with the difference that the cultivation time in step A6) was 3 h.

Comparative example 1

The preparation method in Example 1 was used to prepare protoplasts of Anthracnose camellia, with the difference that the cultivation time in step A6) was 2 h.

test case 1

Absorb appropriate amount of protoplast body fluids prepared in step 6) in Examples 1-6 and Comparative Example 1 respectively, place them on a hemocytometer, observe the quantity and quality of protoplasts under an ordinary optical microscope, and the results are shown in Figures 1-2 , where the left and right figures in Figure 2 are respectively the morphological figures of the anthrax protoplasts prepared in Comparative Example 1 (2h) and Example 1 (7h); the scale bar in Figure 2 is 100 μm.

It can be drawn from Figure 1 that the numbers of protoplasts in the lysed solution prepared by the preparation methods of Examples 1-6 and Comparative Example 1 were 3.6×10 ⁷ , 3.1×10 ⁷ , 3.0×10 ⁷ , and 2.6× 10 ⁷ , 2.2×10 ⁷ , 1.6×10 ⁷ and 0.4×10 ⁷ cells/mL; combined with Figure 2, it can be seen that a certain number of protoplasts can be observed at 2 hours, but the volume of protoplasts is small. The number of protoplasts increased continuously, and at 7 hours, the number of protoplasts was the largest, and the protoplasts were round and plump.

Comparative example 2

The preparation method in Example 1 is used to prepare the protoplasts of Camellia anthracis, the difference is that the enzymolysis solution component in step A5) is 10mL physiological saline containing 2wt.% of collapsing enzyme and 1wt.% of lysozyme, step A6) The cracking time is 2~8h.

The protoplast body fluid prepared in step A6) was placed on a hemocytometer, and the quantity and quality of the protoplasts were observed under an ordinary optical microscope. It was found that the protoplasts obtained in Comparative Example 2 were broken and the quality was not high. The number of protoplasts obtained after 8 hours was between (1-20)×10 ⁵ /mL.

Comparative example 3

The preparation method in Example 1 is used to prepare the protoplasts of Camellia anthracis, the difference is that the enzymolysis solution component in step A5) is 10mL phosphate buffer containing helicase 2wt.% and lysozyme 1wt.%, step A6) The cracking time is 2~8h.

The protoplast body fluid prepared in step A6) was placed on a hemocytometer, and the quantity and quality of the protoplasts were observed under an ordinary optical microscope. As a result, it was found that the protoplasts obtained in Comparative Example 3 were less in number, and the number of lysed 2 to 8 hours was between ( 1～5)×10 ⁵ cells/mL.

Comparative example 4

The preparation method in Example 1 is used to prepare the protoplasts of Camellia anthracis, the difference is that the enzymolysis solution component in step A5) is 10mL phosphate buffer containing lysozyme 2wt.% and collapsing enzyme 1wt.%, step A6 ) cracking time is 2 ~ 8h.

The protoplast body fluid prepared in step A6) was placed on a hemocytometer, and the quantity and quality of the protoplasts were observed under an ordinary optical microscope. As a result, it was found that the protoplasts obtained in Comparative Example 4 were less in number, and the number of lysed 2 to 8 hours was between ( 1～10)×10 ⁵ cells/mL.

Comparative example 5

The preparation method in Example 1 was used to prepare protoplasts of Camellia anthracnose, the difference being that the enzymolysis solution component in step A5) was 10 mL of phosphate buffer containing 2 wt.% of helicase and 1 wt.% of collapsing enzyme, step A6) The cracking time is 2~8h.

The protoplast body fluid prepared in step A6) was placed on a hemocytometer, and the quantity and quality of protoplasts were observed under an ordinary optical microscope. It was found that the number of protoplasts obtained in Comparative Example 5 was small, and the number of lysed 2 to 8 hours was between ( 1～10)×10 ⁵ cells/mL.

Comparative example 6

The preparation method in Example 1 was used to prepare protoplasts of Camellia anthracis, the difference being that the enzymolysis solution component in step A5) contained 2wt.% helicase, 2wt.% cellulase and lysozyme in 10mL phosphate buffer 1wt.%, step A6) cracking time is 2-8h.

The protoplast body fluid prepared in step A6) was placed on a hemocytometer, and the quantity and quality of protoplasts were observed under an ordinary optical microscope. It was found that there were basically no protoplasts after lysis for 2-8 hours in Comparative Example 6.

Example 7

G418 Resistance Screening

Camellia anthracnose LS_19 was cultured in the dark at 28°C for 3-5 days in a constant temperature incubator, and 5mm bacterial cakes were inoculated on PDA solid medium containing G418 at a concentration of 0 (CK), 50, 100 and 150 μg/mL. The colony diameter was counted and photographed and recorded in a constant temperature incubator for 4 days. Three replicates were set for each treatment, and the experiment was repeated three times. The results are shown in Figure 3.

As can be seen from Fig. 3, the PDA solid culture medium of 150 μ g/mL concentration G418 hardly grows, and on the PDA solid medium containing concentration of 0 (CK), 50 and 100 mg/mL, there is Camellia anthracis LS_19 Colony growth, and then the minimum inhibitory concentration of Camellia anthracis LS_19 strain to G418 was 150 μg/mL.

Example 8

A construction method of Camellia anthracnose genetic transformation system, its construction flow chart is as shown in Figure 7, and the steps are as follows:

1. Preparation of plasmid DNA, the steps are as follows:

The plasmid pBlueScriptⅡ(+)-G418-GFP used consists of pBlueScriptⅡ(+) plasmid and G418 resistance expression gene (accession number in NCBI database is AAG34543.1) and green fluorescent protein GFP (accession number in NCBI database is ANA76508 .1) Gene fusion, the specific steps are as follows:

Using the strategy of homologous recombination, the G418 resistance expression gene cloned by PCR was recombined with the XbaⅠ linearized vector pBlueScriptⅡ(+) to obtain the recombinant product; ) method to transfer the recombinant product to DH5α competent cells, insert LB solid medium (containing 100 mg/mL ampicillin), and culture overnight for 12-16 hours, pick monoclonal Escherichia coli colonies and place them in LB liquid medium for overnight culture for 12- 16h, use the plasmid extraction kit (purchased from Beijing Qingke Biotechnology Co., Ltd.) to extract the plasmid pBlueScriptⅡ(+)-G418 for use; further clone the GFP fragment of the green fluorescent protein gene and single-digest KpnⅠ linearization vector pBlueScriptⅡ(+) -G418 was subjected to a recombination reaction to obtain a recombinant product; according to the method of Escherichia coli DH5α (purchased from Beijing Qingke Biotechnology Co., Ltd.), the recombinant product was transferred to DH5α competent cells, and inserted into LB solid medium (containing 100 mg/mL ampicillin) , cultivated overnight for 12-16 hours, picked monoclonal Escherichia coli colonies and placed them in LB liquid medium for overnight cultivation for 12-16 hours, and extracted plasmid pBlueScriptⅡ(+) with the method of plasmid extraction kit (purchased from Beijing Qingke Biotechnology Co., Ltd.) -G418-GFP spare.

2. B1) The protoplasts of Camellia anthracis prepared in Example 1 were washed and precipitated twice with 1 mL of STC solution, placed in a centrifuge each time, and centrifuged at 3000 rpm at 4° C. for 5 minutes to obtain a protoplast solution suspended in the STC solution;

B2) Add 20 μg of the plasmid pBlueScriptⅡ(+)-G418-GFP prepared in step 1 and 10 μL of heparin sodium solution to 500 μL of the protoplast solution suspended in the STC solution obtained in step B1), mix gently and thoroughly, and keep at 0-4°C Stand still for 30min;

B3) Add 1mL SPTC solution dropwise, mix gently and fully, and let stand at 0-4°C for 30min;

B4) Suspend the obtained protoplast solution in 20 mL of regeneration medium, and then place it in a constant temperature shaking incubator at 26° C., with a rotation speed of 100 rpm, and cultivate for 16 hours;

B5) Mix the revived protoplasts with the selective medium, pour them into a plate, put them into a constant temperature incubator and culture them in the dark for 3-5 days, and observe the growth of the transformants.

test case 2

Transformants were screened on G418 (150 μg/mL) selective medium, and after growing for 3-5 days, the insertion of G418 was first detected by PCR, and then whether the transformants had GFP fluorescence was observed by fluorescence microscope.

1. PCR identification of transformants

1.1 Genome extraction:

(1) Cut off the mycelium block from the selective medium plate, place it in a 1.5mL centrifuge tube, add 500μL DEB, and grind it with a tissue disruptor;

(2) Centrifuge at 12,000 rpm for 10 min at 4°C, take 500 μL of supernatant and place in a new 1.5 mL centrifuge tube;

(3) Add an equal volume of isopropanol to the centrifuge tube and invert to mix;

(4) Centrifuge at 12,000 rpm for 5 minutes at 4°C, discard the supernatant;

(5) Add 700 μL of 70% ethanol to the centrifuge tube, mix it upside down, and wash away impurities;

(6) Centrifuge at 12,000 rpm for 5 minutes at 4°C, discard the supernatant;

(7) After the ethanol evaporates, add 50 μL sterile distilled water to the centrifuge tube to dissolve the precipitate;

(8) Centrifuge at 12,000 rpm for 5 minutes at 4°C, take the supernatant into a new 1.5mL centrifuge tube, and store at -20°C for later use.

1% agarose gel electrophoresis to detect the quality of the genome, take pictures with an ultraviolet gel imaging system and save the picture, as shown in Figure 4, where Marker: 5000bp, 1, 2, 3: transformants obtained in Example 8, 4: Camellia anthracnose LS_19;

It can be concluded from Figure 4 that in the figure 1234 have bands, and the total length is greater than 5000bp, no obvious tailing phenomenon, it can be seen that the quality of the genome is good, the extraction method is feasible, and can be used as a template for PCR amplification.

1.2 Target gene PCR amplification

The total genomic DNA of the transformant strain and LS_19, pBlueScriptⅡ(+)-G418-GFP were used as the template control, and the G418 conserved sequence was used to design primers for PCR amplification, and the amplified fragment was about 400bp

G418-F: 5'-CTCTGATGCCGCCGTGTT-3' (SEQ ID NO.1)

G418-R: 5'-CCCTGATGCTCTTCGTCCA-3' (SEQ ID NO.2);

The PCR amplification system and program are shown in Table 1 and Table 2 respectively:

Table 1 PCR amplification system

template 1μL 2×Mix 12.5μL Primer (G418-F) 0.5μL Primer (G418-R) 0.5μL ddH₂O Make up to 25μL

Table 2 PCR amplification program

The PCR products were detected by 1% agarose gel electrophoresis, and were observed and photographed using a UV gel imaging system. The results are shown in Figure 5, where M: 2000bp Maker; 1, 2: mutant transformants; 3: pBlueScriptⅡ(+ )-G418-GFP; 4: Camellia anthracis LS_19.

It can be seen from Fig. 5 that the transformant and pBlueScript II (+)-G418-GFP have a band size of about 400 bp, and the anthracnose camellia LS_19 has no band, and it can be seen that the G418 fragment has been successfully inserted into its genome.

2. Observe the GFP fluorescence of the transformant with a fluorescence microscope. Under sterile conditions, pick different parts of the hyphae and place them on a glass slide, place them under a fluorescence microscope for observation and take pictures for preservation. The results are shown in Figure 6, which is a mycelium sample Local microscopic imaging, in which the left image is bright field, the right image is green fluorescent field, and the scale is 10mm.

It can be concluded from FIG. 6 that both hyphae and spores of the transformant can produce GFP fluorescence, indicating that the GFP gene has been successfully transformed into and expressed in Anthracnose camellia.

From the above examples, it can be concluded that the number of protoplasts prepared by the method for preparing protoplasts of Bacillus anthracis in the present invention is large, the quality is good, and the steps are simple; at the same time, the genetic transformation of Bacillus anthracis mediated by PEG has been successfully constructed successively. system with high conversion efficiency.

Although the foregoing embodiment has described the present invention in detail, it is only a part of the embodiments of the present invention, rather than all embodiments, and people can also obtain other embodiments according to the present embodiment without inventive step, these embodiments All belong to the protection scope of the present invention.

Claims (10)

1. A preparation method of an anthrax protoplast is characterized by comprising the following steps:

mixing the anthrax hypha with the enzymolysis liquid, and cracking to obtain a cracking liquid;

culturing the lysate for 3-8 h to obtain protoplasm liquid, wherein the protoplasm liquid comprises an anthrax protoplast;

the enzymolysis solution comprises a solvent and a solute, wherein the solvent comprises a phosphate buffer solution, and the solute comprises 1-3 wt.% of collapse enzyme and 0.5-1 wt.% of lywallzyme.

2. The method of claim 1, wherein the anthrax protoplast is present in the protoplast fluid at a concentration of (1.6 to 3.6) x 10 ⁷ one/mL.

3. The preparation method of claim 1, wherein the vibration frequency of the pyrolysis is 80-120 rpm, the temperature is 28-32 ℃, and the time is 10-30 min.

4. The method according to any one of claims 1 to 3, wherein said anthrax comprises Camellia anthrax.

5. The method according to claim 1, wherein the anthrax hyphae are prepared by a method comprising:

inoculating the activated anthrax bacterial strain into a spore culture medium for spore culture to obtain conidia;

inoculating the conidia into a hypha culture medium for hypha culture to obtain anthrax hypha; the shaking frequency of the mycelium culture is 150-200 rpm, the temperature is 25-28 ℃, and the time is 12-16 h.

6. Use of the preparation method according to any one of claims 1 to 5 for the construction of a genetic transformation system for anthrax.

7. A method for constructing a anthrax genetic transformation system is characterized by comprising the following steps:

preparing anthrax protoplast by the preparation method of any one of claims 1 to 5;

mixing the anthrax protoplast with an STC solution to obtain an anthrax protoplast suspension;

mixing the anthrax protoplast suspension, plasmid containing antibiotic marker gene and target gene, and heparin sodium solution, and standing at 0-4 deg.C for 30min to obtain first protoplast solution;

adding an SPTC solution into the first protoplast solution, and standing for 30min at 0-4 ℃ to obtain a second protoplast solution;

suspending the second protoplast solution in a regeneration culture medium for resuscitation to obtain a resuscitated protoplast solution;

and mixing the recovered protoplast solution with a selective culture medium, and performing dark culture for 3-5 days to obtain the anthrax genetic transformation system.

8. The construction method according to claim 7, wherein the ratio of the volume of the anthrax protoplast suspension, the mass of the plasmid containing the antibiotic marker gene and the target gene, and the volume of the heparin sodium solution is 0 to 100 μ L: 1-5 μ g: 1-2 mu L; the concentration of the anthrax protoplast in the anthrax protoplast suspension is (1-3) multiplied by 10 ⁵ mu.L/mu.L; the concentration of the heparin sodium solution is 0.1g/mL.

9. The construction method according to claim 7, wherein the regeneration medium uses water as a solvent, and comprises: 1wt.% yeast powder, 1wt.% casamino acid and 274wt.% sucrose; the pH value is 7.0;

the selective culture medium takes water as a solvent and comprises: 20wt.% of potato juice, 2wt.% of glucose, 1.5wt.% of agar and an antibiotic corresponding to the antibiotic marker gene.

10. The method of claim 7 or 9, wherein the antibiotic marker gene comprises a G418 resistance expression gene.

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