Construction method of PEG-mediated colletotrichum camelliae protoplast genetic transformation

Abstract

The invention relates to the technical field of microbial genetic transformation, and particularly discloses a PEG (Polyethylene Glycol)-mediated camellia anthracnose protoplast genetic transformation method which comprises the following steps: obtaining young and tender camellia anthracnose mycelia, filtering and collecting protoplast by using double-layer lens wiping paper, obtaining protoplast precipitate, purifying the protoplast by using an STC solution, and resuspending the protoplast by using the STC to obtain the PEG-mediated camellia anthracnose protoplast. The preparation method comprises the following steps: adding plasmids into a camellia anthracnose protoplast, lightly and uniformly mixing, standing at room temperature, adding a PTC solution, uniformly mixing, standing at room temperature, adding a PTC solution, uniformly mixing, standing at room temperature, pouring into a TB3 solid culture medium, uniformly mixing, pouring into a plate, culturing overnight, covering with a potato dextrose agar culture medium containing geneticin, and culturing at room temperature. The transformant is inoculated to a potato dextrose agar culture medium containing heredity mycin, the transformant with stable characters is screened, and the strain obtained through genetic transformation is obtained.By means of the method, construction of genetic transformation of the camellia anthracnose bacteria is achieved, the time needed by genetic transformation is shortened, and the transformant expressing red fluorescence and stably inheriting is obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial genetic transformation, and specifically relates to a construction method for PEG-mediated genetic transformation of camellia anthracnose protoplasts. Background Art

[0002] Camellia anthracnose is one of the main pathogens of tea anthracnose, which harms the growth of tea trees and affects economic benefits. Studying the pathogenic mechanism of camellia anthracnose is crucial to the effective prevention and control of tea anthracnose. Using genetic engineering to verify the function of the pathogenic gene of tea anthracnose is an effective way. At present, there are few reports on the pathogenic gene of camellia anthracnose. Therefore, establishing an efficient and stable genetic transformation system can provide a technical basis for the study of the pathogenic gene of tea anthracnose.

[0003] At present, a PEG-mediated genetic transformation method for protoplasts of Colletotrichum camelliae has been established (patent application number: 202310027554.X). The lysis time of protoplasts is 3-8 hours, and the yield is (1.6-3.6)×10 7 / mL;

[0004] Although there have been reports on the PEG-mediated genetic transformation system of Colletotrichum camelliae protoplasts, the protoplast lysis time is 3-8 hours, and the efficiency is low. In view of this, the inventors propose a method for constructing a PEG-mediated genetic transformation system of Colletotrichum camelliae protoplasts. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing PEG-mediated genetic transformation of camellia anthracnose protoplasts, which can obtain a large number of protoplasts with good vitality in a short period of time and improve the efficiency of genetic transformation. In order to achieve the above purpose, the present invention provides the following basic scheme:

[0006] Step 1: obtain young mycelium of Colletotrichum camelliae, add enzymatic solution to shake the mycelium, collect protoplasts by filtering with double-layer lens paper, centrifuge to obtain protoplast precipitation, suspend the protoplasts with STC solution, centrifuge, discard the supernatant, and resuspend the protoplasts with STC;

[0007] Step 2: Add the plasmid to the protoplasts of Colletotrichum camelliae obtained in step 1, mix gently and let stand at room temperature, add PTC solution, mix and let stand at room temperature, add PTC solution again, mix and let stand at room temperature, then pour into TB3 solid culture medium, mix and pour into plates, cover with geneticin-containing potato dextrose agar medium after overnight culture, culture, and pick out transformants;

[0008] Step 3: inoculating the transformants into a potato dextrose agar medium containing geneticin, culturing, and screening the strain expressing the red fluorescent protein, which is the strain obtained by genetic transformation of Colletotrichum camelliae;

[0009] Step 4: Cultivate the strain expressing red fluorescent protein for multiple generations, prepare slides and observe the fluorescence under a microscope. Transformants that can observe red fluorescence for multiple generations are considered stable transformants.

[0010] Furthermore, the method for obtaining the young mycelium in step 1 is as follows: S01: inoculating the target strain into a potato glucose water culture medium at 28°C-30°C, 200rpm-250rpm, and culturing in a shaking table;

[0011] S02: After centrifugation, conidia are collected, and the conidia are resuspended with sterile water to obtain a conidia suspension, and young mycelium of Colletotrichum japonicum is obtained by culturing the conidia suspension.

[0012] Furthermore, the concentration of the spore suspension was 10 7 / mL, the culture conditions are 28℃-30℃, 200rpm-250rpm, and the culture time is 16h-24h.

[0013] Furthermore, in step 1, the enzymatic hydrolysis solution includes a solvent and a solute, wherein the solvent includes 0.7 M NaCl, and the solute includes 2-4 wt.% of collapsin, 1-1.5 wt.% of lytic enzyme, and 1-1.5 wt.% of snail enzyme.

[0014] Furthermore, in step 1, the oscillating enzymolysis conditions are shaking enzymolysis, the enzymolysis temperature is 28° C., the shaking speed is 80 rpm-90 rpm, and the enzymolysis time is 2 h-3 h.

[0015] Furthermore, in step 1, the centrifugation conditions are 4° C., 3000 rpm-4000 rpm, and 10 min-15 min.

[0016] Further, in step 1, the formula of the STC solution is as follows: each liter of aqueous solution contains 200 g of sucrose, 10 mL of 1M pH 8.0 tris-HCl, and 5.540 g of CaCl2.

[0017] Furthermore, in step 2, the plasmid is pKD7-RED, and the total amount of the plasmid is not less than 2 μg. The eukaryotic screening resistance of the plasmid is geneticin, which can be used to screen transformants. In step 2, the plasmid and the protoplasts are mixed and then left to stand for 25 min-30 min.

[0018] Furthermore, in step 2, the PTC formula contains 0.4 g PEG4000 per milliliter of STC solution, and the TB3 culture medium formula contains 200 g sucrose, 6 g hydrolyzed casein, 6 g yeast extract, and 15 g agar per liter of aqueous solution.

[0019] Furthermore, the standing time at room temperature after adding the PTC solution is 10 min-15 min.

[0020] Beneficial effects:

[0021] The present invention realizes the construction of PEG-mediated genetic transformation of camellia anthracnose protoplasts and establishes an efficient and stable protoplast extraction method. The extraction efficiency of protoplasts can reach 10 within 1-3 hours. 7 / mL. Genetic transformation was achieved, and a transformed strain that stably expressed red fluorescent protein was obtained. The present invention successfully constructed a genetic transformation system of camellia anthracnose fungus and obtained transformants with fluorescence expression by improving transformation conditions including enzymatic conditions, bacterial age, resistance screening concentration and plasmid dosage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 Schematic diagram of the sensitivity test results of Colletotrichum camelliae to Geneticin in the examples;

[0024] Figure 2 This is a graph showing the yield of protoplast enzymatic hydrolysis for 1-3 hours in the example;

[0025] Figure 3 It is a protoplast diagram in the embodiment;

[0026] Figure 4 This is a microscope observation picture of the fluorescent converter in the example. DETAILED DESCRIPTION

[0027] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0028] like Figure 1 , Figure 2 , Figure 3 and Figure 4 :

[0029] The invention discloses an efficient and stable method for preparing protoplasts of camellia anthracnose fungus, comprising the following steps: culturing a conidia suspension to obtain young mycelium, mixing the young mycelium with an enzyme hydrolysate, and cracking the protoplasts, wherein the enzyme hydrolysate comprises a solvent and a solute, wherein the solvent comprises 0.7M NaCl, and the solute comprises 2-4wt.% of collapsin, 1-1.5wt.% of lysozyme, and 1-1.5wt.% of snailase.

[0030] Specific: Detailed steps for obtaining young mycelium by culturing conidia suspension:

[0031] S01: The target strain was inoculated into a potato glucose water medium at 28°C-30°C, 200rpm-250rpm, and cultured in a shaking table; S02: Conidia were collected after centrifugation, and the conidia were resuspended in sterile water to obtain a conidia suspension, and the young mycelium of Colletotrichum camelliae was obtained by culturing the conidia suspension. The concentration of the conidia suspension was 10 7 / mL, the culture conditions are 28℃-30℃, 200rpm-250rpm, and the culture time is 16h-24h, then the young mycelium of Camellia Anthracnose can be obtained.

[0032] A method for constructing PEG-mediated genetic transformation of camellia anthracnose protoplasts, characterized in that it comprises the following steps:

[0033] Step 1: Obtain young mycelium of Colletotrichum camelliae, shake at 80 rpm-90 rpm for 2 h-3 h at 28 ° C, enzymatically hydrolyze the mycelium, filter and collect protoplasts through double-layer lens paper, centrifuge at 3000 rpm-4000 rpm, 4 ° C for 10 min-15 min to obtain protoplast precipitation, suspend the protoplasts in STC solution, centrifuge at 3000 rpm-4000 rpm, 4 ° C for 10 min-15 min, discard the supernatant, and resuspend the protoplasts in STC to a final concentration of 10 8 / mL.

[0034] In step 1: The formula of STC solution: 200 g sucrose, 10 mL 1M pH 8.0 tris-HCl, and 5.540 g CaCl2 per liter of water.

[0035] Step 2: Add plasmid DNA (total amount not less than 2 μg) to the camellia anthracnose protoplasts obtained in step 1, gently mix and let stand at room temperature for 25 min-30 min, add 0.5 mL of PTC solution, mix and let stand at room temperature for 10 min-15 min, add 0.5 mL of PTC solution, mix and let stand at room temperature for 10 min-15 min, then pour 100 mL of TB3 solid culture medium, mix and pour into plates, cover with potato dextrose agar medium containing 80 μg / mL geneticin after overnight culture, culture at 28°C, and pick out transformants;

[0036] In step 2, the plasmid is pKD7-RED, and the plasmid eukaryotic selection resistance is geneticin;

[0037] In step 2, the PTC solution contains 0.4 g per milliliter of STC solution, and the TB3 culture medium formula contains 200 g of sucrose, 6 g of hydrolyzed casein, 6 g of yeast extract, and 15 g of agar per liter of aqueous solution.

[0038] Step 3: Inoculate the transformants into potato dextrose agar medium containing 80 μg / mL Geneticin, culture at 28°C for 5 consecutive generations, prepare slides and observe fluorescence under a microscope. Transformants that can observe red fluorescence for multiple consecutive generations are successful and stable transformants.

[0039] Example 1: Determination of the sensitivity of Colletotrichum camelliae to geneticin:

[0040] The sensitivity of Colletotrichum camelliae to geneticin is required for screening transformants obtained by genetic transformation in step 2;

[0041] The details are as follows:

[0042] Geneticin can be used as a eukaryotic resistance screening marker for the screening of genetic transformation transformants. After the wild-type strain of Colletotrichum camelliae was inoculated on potato dextrose agar medium for 5 days, the edge hyphae were picked up by an inoculation loop and inoculated on potato dextrose agar medium with geneticin concentrations of 0 μg / mL, 40 μg / mL, and 80 μg / mL, respectively. The culture was placed in a 28°C incubator for 7 days and the growth was observed. The growth results are shown in the figure. Figure 1 As shown;

[0043] Example 2: Preparation of protoplasts of Colletotrichum camelliae;

[0044] The young mycelium is mixed with an enzymatic hydrolysis solution and cracked to obtain protoplasts. The enzymatic hydrolysis solution includes a solvent and a solute, wherein the solvent includes 0.7M NaCl, and the solute includes 2-4wt.% of lysozyme, 1-1.5wt.% of lysozyme, and 1-1.5wt.% of snail enzyme. The enzymatic hydrolysis solution is prepared by preparing 50mg / mL of lysozyme, lysozyme, and snail enzyme mother solutions with 0.7M NaCl, filtering the supernatant with a 0.22-micron sterile filter membrane, mixing the lysozyme, lysozyme, and snail enzyme to prepare an enzymatic hydrolysis solution, wherein the final concentrations of lysozyme, lysozyme, and snail enzyme in the enzymatic hydrolysis solution are 20mg, 15mg, and 15mg, respectively, in each milliliter of the enzymatic hydrolysis solution;

[0045] Use double-layer lens paper to collect and filter the protoplasts into a 50 mL centrifuge tube, centrifuge at 3000-4000 rpm at 4°C for 10-15 min to collect the protoplasts, resuspend in STC solution, centrifuge at 3000-4000 rpm at 4°C for 10-15 min to purify the protoplasts, discard the supernatant, and resuspend in STC to a protoplast concentration of 10 8 / mL.

[0046] Example 3: Construction of fluorescently labeled anthrax bacteria;

[0047] Gently mix 10 μL (228 ng / μL) of pKD7-RED plasmid and 150 μL of protoplasts, let stand at room temperature for 25 min, add 0.5 mL of PTC solution, mix gently, let stand for 15 min, add 0.5 mL of PTC solution again, mix gently, let stand for 10 min, add the tea tree camellia anthracnose protoplasts to 100 mL of TB3 solid medium, mix gently and pour into a culture dish, culture at 28°C overnight, add 80 μg / mL geneticin potato dextrose agar medium to the culture dish, select transformants to new 80 μg / mL geneticin potato dextrose agar medium, culture for 5 days, identify fluorescence, and continue to pass on transformants with red fluorescence to multiple generations. Observe fluorescence in each generation to obtain stable inherited fluorescent transformants, as shown in the following example. Figure 4 As shown, Figure 4 Data from the first five generations are shown, all with red fluorescence.

[0048] like Figure 2 As shown, the extraction efficiency of protoplasts can reach 10 within 1h-3h. 7 / mL, genetic transformation was achieved, and a transformed strain stably expressing red fluorescent protein was obtained.

[0049] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for constructing PEG-mediated genetic transformation of camellia anthracnose protoplasts, characterized in that The steps include: Step 1: obtain young mycelium of Colletotrichum camelliae, add enzymatic solution to shake the mycelium, collect protoplasts by filtering with double-layer lens paper, centrifuge to obtain protoplast precipitation, suspend the protoplasts with STC solution, centrifuge, discard the supernatant, and resuspend the protoplasts with STC; Step 2: Add the plasmid to the protoplasts of Colletotrichum camelliae obtained in step 1, mix gently and let stand at room temperature, add PTC solution, mix and let stand at room temperature, add PTC solution again, mix and let stand at room temperature, then pour TB3 solid culture medium, mix and pour into plates, cover with potato dextrose agar medium containing geneticin after overnight culture, culture, and pick out transformants; Step 3: inoculating the transformants into a potato dextrose agar medium containing geneticin, culturing, and screening the strain expressing the red fluorescent protein, which is the strain obtained by genetic transformation of Colletotrichum camelliae; Step 4: Cultivate the strain expressing red fluorescent protein for multiple generations, prepare slides and observe the fluorescence under a microscope. Transformants that can observe red fluorescence for multiple generations are considered stable transformants.

2. The method according to claim 1, characterized in that The method for obtaining the young mycelium of the camellia anthracnose fungus described in step one is as follows: S01: inoculate the target strain into a potato glucose water culture medium, and the culture conditions are 28°C-30°C, 200rpm-250rpm, and shaker culture; S02: collect conidia after centrifugation, resuspend the conidia with sterile water to obtain a conidia suspension, and obtain the young mycelium of the camellia anthracnose fungus by culturing the conidia suspension.

3. The method according to claim 2, characterized in that The concentration of the spore suspension was 10 7 / mL, the culture conditions are 28℃-30℃, 200rpm-250rpm, and the culture time is 16h-24h.

4. The method according to claim 1, characterized in that In step 1, the enzymatic hydrolysis solution includes a solvent and a solute, wherein the solvent includes 0.7M NaCl, and the solute includes 2-4wt.% of collapsin, 1-1.5wt.% of lytic enzyme, and 1-1.5wt.% of snail enzyme.

5. The method according to claim 4, characterized in that In step 1, the oscillating enzymolysis conditions are shaking enzymolysis, the enzymolysis temperature is 28° C., the shaking speed is 80 rpm-90 rpm, and the enzymolysis time is 2 h-3 h.

6. The method according to claim 5, characterized in that In step 1, the centrifugation conditions are 4°C, 3000rpm-4000rpm for 10min-15min.

7. The method according to claim 6, characterized in that In step 1, the formula of the STC solution is as follows: each liter of aqueous solution contains 200 g of sucrose, 10 mL of 1M pH 8.0 tris-HCl, and 5.540 g of CaCl2.

8. The method according to claim 7, characterized in that The plasmid in step 2 is pKD7-RED, and the total amount of the plasmid is not less than 2 μg. The plasmid eukaryotic screening resistance is geneticin, which can be used to screen transformants. In step 2, the plasmid and the camellia anthracnose protoplasts are mixed and then allowed to stand for 25 min-30 min.

9. The method according to claim 8, characterized in that In step 2, the PTC formula contains 0.4 g PEG4000 per milliliter of STC solution, and the TB3 culture medium formula contains 200 g sucrose, 6 g hydrolyzed casein, 6 g yeast extract, and 15 g agar per liter of aqueous solution.

10. The method according to claim 9, characterized in that In step 2, the standing time at room temperature after adding the PTC solution is 10 min-15 min.

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