CN114214356B - Construction method of Streptomyces roseoflavus TRM49605 genetic operating system - Google Patents
Construction method of Streptomyces roseoflavus TRM49605 genetic operating system Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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Abstract
The application discloses a construction method of a Streptomyces coronensis TRM49605 genetic operating system, which utilizes Streptomyces coronensis TRM49605 mycelium as a donor cell, and carries out combined transfer with recipient cell escherichia coli to obtain a conjugal transfer transformant, and then purifying and culturing the conjugal. The joint transfer of the escherichia coli and the streptomyces roseofluosus TRM49605 does not depend on the formation and regeneration of protoplasts; can avoid the restriction system barrier of the host bacteria; the carrier can be efficiently transferred from the escherichia coli to the streptomycete by utilizing the piece; the plasmids used for conjugal transfer are shuttle plasmids, and the plasmids can autonomously replicate in escherichia coli, and the construction and operation of the expression vector are further improved.
Description
Technical Field
The application relates to the technical field of genetic operation systems. In particular to a construction method of Streptomyces roseofluosus TRM49605 genetic operating system.
Background
Streptomyces can produce abundant secondary metabolites and has a complex morphological differentiation process, has great value in basic research and industrial application, and can produce various antibiotics with antibacterial, antifungal and antitumor activities, so that Streptomyces has become an important point in microbial research. Streptomyces roseoflavus (Streptmyces luozhongensis) TRM49605 is a novel species of Streptomyces sp which has independent intellectual property rights in China and is found by the laboratory where the technical staff of the application is located, and is also a tunicamycin (tunicamycin) producing strain. The establishment and optimization of the Streptomyces roseoflorius TRM49605 joint transfer system are necessary preconditions for researching the regulation genes and the secondary metabolite synthesis genes of the Streptomyces roseoflorius. Meanwhile, the construction of a genetic operation system also enables the targeted modification of the Streptomyces coronensis TRM49605, the improvement of the expression level of the gene thereof to modify the production capacity of strains, the generation of new compounds by gene recombination and the like to be possible, and lays a foundation for realizing the construction of genetic engineering strains and improving the antibiotic production capacity of Streptomyces.
The establishment of a proper and efficient genetic operating system is an important precondition for genetic modification of strains. The indirect transfer of genus Streptomyces is widely used in the genetic manipulation of Streptomyces with the advantage of simplicity and high efficiency. Traditional Streptomyces conjugal transfer relies mainly on gene transfer when the bacterial pili germinated by E.coli (Escherichia coli) and the hyphae germinated by Streptomyces spores come into contact.
Therefore, the strain spore pre-germination state is required to be searched, and besides, the heat shock temperature and time, the donor-acceptor ratio and the antibiotic coverage time are required to be searched to determine the optimal conjugation transfer condition. For example, streptomyces coelicolor (Streptomyces coelicolor) with a clear genetic background, the conventional method is to scrape fresh spores grown on MS plates for 5-6 days, wash the spores with 2 XYT medium 2 times, heat shock at 50℃for 10min, and rapidly cool the spores to room temperature. Treated donor E.coli ET12567 (pUZ 8002, pSET 152) was mixed with Streptomyces coelicolor spore suspension at a 1:1 ratio, spread evenly on MS medium, incubated at 30℃for 15-16h, covered with 1mL of sterile aqueous solution containing antibiotic and nalidixic acid (working concentration both 50. Mu.g/mL). The constant temperature culture is continued for 5-7 days at 30 ℃ until the zygote grows out.
However, streptomyces roseoflormis TRM49605, and earlier studies have found that Streptomyces roseoflormis TRM49605 has developed hyphae, but no obvious spore production. The conventional Streptomyces conjugal transfer operation is not suitable for actinomycetes which are difficult to produce spores and difficult to culture. It has also been reported that for non-spore-forming bacteria, electroporation or protoplast transformation can be used for treatment, but the method is complicated.
At present, the joint transfer operation of Streptomyces roseoflorius TRM49605 has not been successfully reported, so that the construction of a genetic manipulation system of TRM49605 is necessary for facilitating the subsequent genetic modification of Streptomyces roseoflorius TRM 49605.
Disclosure of Invention
Therefore, the technical problem to be solved by the application is to provide a construction method of Streptomyces roseoflormis TRM49605 genetic operating system, which provides a method for purposefully carrying out directional modification on Streptomyces roseoflormis TRM49605, improving the expression level of the gene so as to modify the production capacity of strains, generating new compounds by gene recombination and the like.
In order to solve the technical problems, the application provides the following technical scheme:
the construction method of Streptomyces roseoflorius TRM49605 genetic operating system uses Streptomyces roseoflorius TRM49605 mycelium obtained directly in liquid culture medium as donor cell, and carries out combination transfer with recipient cell escherichia coli to obtain zygote, and then the zygote is purified and cultured.
The construction method of the Streptomyces roseoflorius TRM49605 genetic manipulation system comprises the steps of culturing Streptomyces roseoflorius TRM49605 mycelium donor cells: streptomyces roseoflorius TRM49605 mycelia stored in glycerol tubes were inoculated directly into TSB mycelia growth medium.
The construction method of the Streptomyces roseoflorius TRM49605 genetic manipulation system comprises the following steps of: culturing at 30deg.C for 30h on a shaking table at 220 rpm/min.
The construction method of the Streptomyces roseoflorius TRM49605 genetic operating system comprises the following steps of culturing recipient cell escherichia coli ET 12567: e.coli ET12567 single colony is selected, inoculated in 5mL LB E.coli liquid culture medium, and cultured by adding chloramphenicol, apramycin and kanamycin mixed antibiotic solution.
The construction method of the Streptomyces roseoflavus TRM49605 genetic operating system comprises the steps of: mu.L of chloramphenicol (25 mg/mL), apramycin (50 mg/mL) in an amount of 2.5. Mu.L, and kanamycin (50 mg/mL) in an amount of 2.5. Mu.L were added, followed by culturing at 37℃for 12 hours.
The construction method of the Streptomyces roseoflorius TRM49605 genetic operating system comprises the steps of combining and transferring donor cells and acceptor cells: washing Streptomyces roseoflorius TRM49605 mycelium and Escherichia coli mycelium with LB Escherichia coli liquid medium for 2 times, re-suspending the mycelium, mixing according to the quantity ratio of Streptomyces roseoflorius TRM49605 mycelium to Escherichia coli serving as a donor cell of 100:1, coating on a first Gao's joint transfer medium plate, covering with nalidixic acid and apramycin solution, spreading the solution on the whole plate uniformly, blow-drying, and culturing in an incubator.
The construction method of the Streptomyces coronensis TRM49605 genetic operating system comprises the steps of mixing the Streptomyces coronensis TRM49605 mycelium of donor cells and escherichia coli of recipient cells, coating on a first-order Gao's joint transfer culture medium plate, placing in a 37 ℃ incubator for culturing for about 16-20 h, adding an antibiotic liquid for covering, and sterilizing ddH with the antibiotic liquid being 1mL 2 10 mu L of apramycin with the mother liquor concentration of 50mg/mL and 20 mu L of nalidixic acid with the mother liquor concentration of 25mg/mL are added into O, so that the solution is uniformly spread on the whole flat plate, and the flat plate is placed in a 30 ℃ incubator for 5-7 days after being dried.
The construction method of the Streptomyces roseoflorius TRM49605 genetic operating system comprises the steps of purifying and culturing the zygote: randomly picking zygotes, streaking on a Gao's first joint transfer medium containing apramycin and nalidixic acid, culturing in an incubator, picking hyphae, inoculating in a TSB mycelium growth medium, culturing, and extracting genome.
The construction method of the Streptomyces roseoflorius TRM49605 genetic operating system comprises the steps of streaking on a Gao's first joint transfer culture medium, culturing for 5 days in a 30 ℃ incubator, picking hyphae, inoculating to a TSB mycelium growth culture medium, culturing for 36 hours, and obtaining purified zygotes for genome extraction and PCR verification; the preparation method of the Gao's first joint transfer medium containing the apramycin and the nalidixic acid comprises the following steps: 100mL of TSB mycelium growth medium is sterilized and cooled, and 200 mu L of nalidixic acid with the concentration of 25mg/mL and 100 mu L of apramycin with the concentration of 50mg/mL are added into the medium, so that the final concentration of the nalidixic acid is 50 mu g/mL and the final concentration of the apramycin is 50 mu g/mL.
The technical scheme of the application has the following beneficial technical effects:
1. for the non-spore-forming actinomycetes such as Streptomyces roseoflorius TRM49605, the conjugation transfer was performed using mycelium. Compared with spore joint transfer, mycelium joint transfer has fewer fumbling conditions and simpler operation. The conjugation transfer of E.coli to Streptomyces roseoflorius TRM49605 as a means of DNA introduction has the following advantages: the method is simple and does not depend on the formation and regeneration of protoplasts; can avoid the restriction system barrier of the host bacteria; the carrier can be efficiently transferred from the escherichia coli to the streptomycete by utilizing the piece; the plasmids used for conjugal transfer are shuttle plasmids, and the plasmids can autonomously replicate in escherichia coli, and the construction and operation of the expression vector are further improved. The conversion efficiency is also significantly improved compared with other methods. The method can be applied to most actinomycetes, and the genetic stability of transformation is high.
2. The ordinary mycelium capable of producing spores can only be cultured by using a solid culture medium, namely, a large amount of fresh spores can be collected after the spores are required to be produced on a flat plate, the spores are not easy to produce directly in a liquid culture medium, and the mycelium can grow in the later growth stage, but the mycelium is old and cannot be used for subsequent study. The Streptomyces roseoflorius TRM49605 has no method for sporulation, and fresh and robust mycelia can be collected in a liquid culture medium, so that the quantity of the mycelia is large and the growth speed is high. Thereby realizing the process of obtaining Streptomyces coronensis TRM49605 mycelium to complete the joint transfer by only adopting a liquid culture medium.
3. Compared with the Streptomyces roseoflorius TRM49605 mycelium joint transfer, the spore joint transfer is complex, the spore state is severe, the spore-producing solid culture medium is required to be screened, and the spore germination state and the heat shock temperature are required to be considered. The direct mycelium liquid culture joint transfer is different, and the mycelium is cultured to the logarithmic phase through the TSB liquid culture medium, so that the gene transfer can be carried out with donor bacteria, and the operation is convenient.
4. Compared with the method for processing the transformation of the non-sporulation electrotransformation and protoplast, the method for transferring the Streptomyces roseoflorius TRM49605 mycelium in a joint way is simple and convenient. Protoplast transformation: the protoplast is prepared, cell walls need to be removed, if the wall thickness is too thick, the protoplast is not removed well, the process is complex, the efficiency is very low, the failure rate of one batch is very high, and the cell wall process is very complex; electric conversion: the efficiency is not high, a few transformants grow out or the false positive proportion is high, and the mycelium results are not good.
Drawings
FIG. 1 is a graph showing the growth of Streptomyces roseoflorius TRM49605 in the examples of the present application;
FIG. 2 is a graph showing the ratio of donor to recipient cells versus the efficiency of conjugation transfer in an embodiment of the present application;
FIG. 3 is a diagram showing PCR verification results of the Streptomyces TRM49605 zygote in the example of the present application.
Detailed Description
1. Experimental materials
1.1 Strain:
streptomyces rosenbergii (Streptmyces luozhongensis) TRM49605, deposited by Tarim basin biological resource protection utilizing weapon focus laboratories and China center for type culture collection, E.coli (Escherichia coli) ET12567 (pUZ 8002, pSET 152) as donor strain; the pSET152 plasmid is an escherichia coli-streptomyces shuttle integrated plasmid and has an integration site attp of an apramycin resistance gene aac (3) IV and a phage phi c 31 integrase gene int.
1.2 Medium:
LB E.coli liquid medium: after 20g of soybean powder, 20g of mannitol and 20g of agar were mixed uniformly, distilled water was added to 1000mL to adjust the pH to 7.2.
TSB mycelium growth medium: TSB (tryptone soy broth) 30g, water 1000mL.
high-No. one conjugal transfer medium: 20.0g of soluble starch and 0.01g of FeSO 4 ·7H 2 O、0.5g K 2 HPO 4 ，1.0g KNO 3 、0.5g MgSO 4 ·7H 2 O, 16.0g of agar and 1000mL of water are mixed evenly, and the pH is regulated to 7.2-7.4.
1.3 reagent:
apramycin, kanamycin, chloramphenicol, thiostrepton, ampicillin, and nalidixic acid.
2. Experimental methods and results
2.1 Streptomyces roseoflavus TRM49605 antibiotic resistance
For streptomycete, there is a great difference between different species, and the tolerance to antibiotics is also different. To determine the resistance marker of Streptomyces roseoflorius TRM49605, six antibiotics were selected as screening markers for genetic manipulation. After activating Streptomyces roseoflorius TRM49605, fresh hyphae are streaked on a plate containing different antibiotics, and are cultured for 3-5 days at 30 ℃ to observe the growth condition of the thalli.
The results are shown in Table 1. As can be seen from Table 1, streptomyces roseofluosus TRM49605 is sensitive to apramycin (Apr), kanamycin (kana), thiostrepton (Thio) and chloramphenicol (Cml), and is insensitive to ampicillin (Amp) and nalidixic acid (Nal).
TABLE 1 sensitivity of Streptomyces roseoflavus TRM49605 to different antibiotics
Note that: "+" means sensitive to antibiotics and "-means insensitive to antibiotics
2.2 determination of Streptomyces roseoflavus TRM49605 growth curve
Compared with mycelium conjunctive transfer, spore conjunctive transfer is complex, the spore is in a severe state, not only the spore culture medium is required to be screened, but also the spore germination state and the heat shock temperature are required to be considered. The mycelium conjugal transfer is different, and depends on that the mycelium is contacted with a donor to carry out gene transfer, so that only the growth state of the mycelium is required to be searched.
And (3) scribing the mycelium preserved in glycerol to a first-order Gaoshan joint transfer medium, culturing for 5 days, picking a loop to a TSB mycelium growth medium, taking samples every 6 hours, and measuring the dry weight of the mycelium. As a result, as shown in FIG. 1, mycelia of 24 to 30 hours in mid-log growth were selected for conjugal transfer.
2.3 Effect of donor-acceptor ratio on binding transfer efficiency
The ratio of Streptomyces roseofluosus in donor cells (S.luozhonensis) to E.coli in recipient cells is also an important factor affecting the efficiency of conjugation transfer, using a number of mycelia of 10 7 Under the Condition of (CFU), the number of the suitable donor bacteria is searched, and the donor-acceptor ratio is 10 9 :10 7 (100:1)，10 8 :10 7 (10:1)，10 7 :10 7 (1:1) ratio A conjugation transfer experiment was performed, and the ratio of donor bacteria to recipient bacteria was determined based on conjugation efficiency. As a result, as shown in FIG. 2, it can be seen from the table that the joint transition frequency is highest when the feed-to-receive ratio is 100:1.
2.4 Effect of antibiotic coverage time on binding transfer efficiency
According to the ratio of Streptomyces roseofluosus to E.coli donor/acceptor of recipient cells of 10 8 :10 7 After being coated on the Gao's first joint transfer medium, after being cultured for 12h,14h,16h,18h,20h,22h,24h, an antibiotic liquid is coated, and the antibiotic liquid is 1mL of sterilized ddH 2 To O, 20. Mu.L of nalidixic acid (50 mg/mL) and 20. Mu.L of apramycin mother liquor (50 mg +.mL), antibiotic coverage time was determined based on conjugation efficiency. As is clear from Table 2, the highest frequency of conjugation transfer was observed when the antibiotic addition time was 20 hours.
TABLE 2 Effect of antibiotic coverage time on binding transfer efficiency
2.5 Streptomyces roseoflavus TRM49605 zygote transfer and zygote verification
Streptomyces is a highly differentiated genus, and there are great differences between different strains, and the transformation method for one Streptomyces is not necessarily applicable to other Streptomyces, so that the optimal transformation method needs to be searched according to the investigated strains.
Earlier studies found that Streptomyces roseoflorius TRM49605 aerial hyphae developed, but no apparent spores were produced. To facilitate subsequent genetic engineering of Streptomyces roseoflorius TRM49605, it is therefore necessary to construct a mycelium-joining transfer system for Streptomyces roseoflorius TRM 49605.
(1) Mycelium culture: streptomyces roseofluosus TRM49605 is directly inoculated into 50mL of TSB mycelium growth medium and cultured on a shaker at 30℃at 220rpm/min for 30h.
(2) Coli ET12567 (puc 8002, pSET 152) culture: a single colony of E.coli ET12567 (pUZ 8002, pSET 152) was picked, inoculated into 5mL of LB E.coli liquid medium, 5. Mu.L of 25mg/mL chloramphenicol, 2.5. Mu.L of 50mg/mL apramycin and 2.5. Mu.L of 50mg/mL kanamycin were added, and cultured at 37℃for 12 hours.
(3) And (3) joint transfer: after collecting Streptomyces roseoflorius TRM49605 mycelium and E.coli, washing 2 times with LB E.coli liquid medium, re-suspending the mycelium with appropriate volume of LB E.coli liquid medium, mixing the treated E.coli with 500 μl of Streptomyces roseoflorius TRM49605 mycelium, wherein the quantity of Streptomyces roseoflorius TRM49605 mycelium and E.coli is 100:1, and coating on the high-I joint transfer medium. After the constant temperature culture at 37 ℃ for 16 to 20 hours, adding antibiotic liquid for covering, wherein the antibiotic liquid is 1mL for killingBacteria ddH 2 10. Mu.L of apramycin with a mother liquor concentration of 50mg/mL and 20. Mu.L of nalidixic acid with a mother liquor concentration of 25mg/mL are added to O, and the culture is continued at 30℃for 5-7 days, and colonies growing on the plates are examined.
2.6 PCR amplification reaction verification of the zygote
(1) Purification and culture of zygotes
Randomly picking a joint transfer rotor, streaking on a Gao's first joint transfer culture medium containing apramycin and nalidixic acid, culturing for 5 days in a 30 ℃ incubator, picking a proper amount of mycelium, inoculating into a mycelium liquid culture medium, and culturing for about 36 hours for genome extraction. The preparation method of the Gao's first joint transfer medium containing the apramycin and the nalidixic acid comprises the following steps: 100mL of TSB mycelium growth medium is sterilized and cooled, and 200 mu L of nalidixic acid with the concentration of 25mg/mL and 100 mu L of apramycin with the concentration of 50mg/mL are added into the medium, so that the final concentration of the nalidixic acid is 50 mu g/mL and the final concentration of the apramycin is 50 mu g/mL.
(2) Genome extraction
Taking a small amount of mycelium, washing 3 times by using a 1 XTE buffer, re-suspending the mycelium by using 500uL of 1 XTE buffer, adding lysozyme to a final concentration of 5mg/mL, incubating for 45min at 37 ℃, adding SDS to a final concentration of 1%, adding RNase to a final concentration of 10 mug/mL after reversing and mixing, incubating for 10min at 37 ℃, adding proteinase K to a final concentration of 0.2mg/mL, incubating for about 30min in a 37 ℃ water bath, adding 200 uL of 5M NaCl, reversing and mixing, cooling to room temperature, adding 500uL of phenol chloroform (phenol: isoamyl alcohol=25:24:1), fully reversing and mixing, centrifuging for 10min at 12000rpm, taking an upper water phase, adding an equal volume of precooled isopropanol, reversing and mixing, picking up DNA by using a gun head, washing twice by using 70% ethanol, and adding a proper amount of water to dissolve DNA after the ethanol volatilizes, thus being applicable to PCR.
(3) PCR verification
1 pair of primers (AprF: 5'-CTTCGCATCCCGCCTCTGG-3' and AprR: 5'-CAATACGAATGGCGAAAAG-3') was designed based on the apramycin resistance gene, and the amplified fragment size was 750bp. As shown in FIG. 3, the PCR detection result shows that the conjugative transformant and the positive control pSET152 plasmid both amplify a band of about 750bp, but the wild-type Streptomyces trM49605 DNA does not amplify the target fragment, thereby confirming that the pSET152 plasmid has been successfully transferred into Streptomyces roseoflorii TRM 49605. In fig. 3: 1-15 is Streptomyces trM49605 zygote; m represents a DNA Marker; "-" means wild type; "+" indicates plasmid pSET152.
2.7 treatment method using electrotransformation and protoplast transformation
(1) Genetic transformation is carried out by adopting an electrotransformation method:
by screening for optimum voltage: adding the same amount of bacterial suspension into an electric conversion cup, and respectively setting electric shock voltages of 1kV,2kV and 3kV, wherein no correct transformant is obtained;
screening optimal electric shock time: adding the same amount of bacterial suspension into an electric transformation cup, and respectively setting three gradients of 4s,5s and 6s for electric shock time, wherein no correct transformant is obtained;
after the electric shock treatment, streptomyces roseofluosus TRM49605 was inoculated into R2YE, RM14, YMS, R3, G+Y, SFM, HI medium, and the most suitable recovery medium was selected, and no correct transformants were obtained.
(2) Genetic transformation is carried out by adopting a protoplast transformation method:
the optimal concentration and the action time of lysozyme are selected, a plurality of lysozyme concentration gradients of 10.0mg/mL,20.0mg/mL,30.0mg/mL and the like are set, suspended mycelia added into 10mL lysozyme solution are subjected to enzymolysis, water bath heat preservation is carried out at 37 ℃ for 15min,30min,45min,60min,75min and 80min, and microscopic examination is carried out, so that the effect of removing cell walls by lysozyme is poor, and protoplast formation is not found.
Streptomyces roseoflorius TRM49605 does not produce spores, but mycelium cannot be obtained by adopting an electrotransformation and protoplast transformation treatment method.
Claims (2)
1. Streptomyces roseofluosus (S. Sp.) Var.Streptmyces luozhongensis) A construction method of a TRM49605 genetic operation system is characterized in that Streptomyces roseoflorius TRM49605 mycelium obtained directly in a liquid culture medium is used as a donor cell and is combined with an acceptor cell escherichia coliEscherichia coli) Performing combination transfer to obtain a zygote, and purifying and culturing the zygote;
culturing Streptomyces roseoflorius TRM49605 mycelium donor cells: streptomyces roseoflorius TRM49605 mycelia stored in a glycerol tube are directly inoculated into a TSB mycelia growth medium;
in TSB mycelium growth medium: culturing at 30deg.C on a shaking table at 220rpm/min for 30 hr;
culturing of recipient cell E.coli ET 12567: e.coli ET12567 single colony is selected, inoculated in LB E.coli liquid medium of 5mL, and added with chloramphenicol, apramycin and kanamycin mixed antibiotic solution for culture; the preparation method of the mixed antibiotic solution of chloramphenicol, apramycin and kanamycin comprises the following steps: mu.L of 25mg/mL chloramphenicol, 2.5. Mu.L of 50mg/mL apramycin, and 2.5. Mu.L of 50mg/mL kanamycin were mixed, followed by culturing at 37℃for 12 hours;
donor cell and recipient cell binding transfer method: washing Streptomyces roseoflorius TRM49605 mycelium and Escherichia coli mycelium with LB Escherichia coli liquid culture medium for 2 times respectively, re-suspending the mycelium, mixing according to the quantity ratio of Streptomyces roseoflorius TRM49605 mycelium of donor cells to Escherichia coli of recipient cells of 100:1, coating the mixture on a first Gao's joint transfer culture medium flat plate, placing the flat plate in a 37 ℃ incubator for culturing for 20 hours, adding antibiotic liquid for covering, uniformly spreading the whole flat plate with the solution, drying, and placing the flat plate in a 30 ℃ incubator for culturing for 5-7 days; wherein the preparation method of the antibiotic liquid is 1mL sterilized ddH 2 10 mu L of apramycin with the mother liquor concentration of 50mg/mL and 20 mu L of nalidixic acid with the mother liquor concentration of 25mg/mL are added into O;
purifying and culturing the zygote: randomly picking zygotes, streaking on a Gao's first joint transfer medium containing apramycin and nalidixic acid, culturing in an incubator, picking hyphae, inoculating in a TSB mycelium growth medium, culturing, and extracting genome.
2. The method for constructing Streptomyces roseoflorius TRM49605 genetic manipulation system according to claim 1, wherein in the purification and culture of the zygote: streaking on a Gao's first joint transfer medium, culturing for 5 days in a 30 ℃ incubator, picking hyphae, inoculating in a TSB mycelium growth medium, culturing for 36h, obtaining purified zygotes, and using for genome extraction and PCR verification; the preparation method of the Gao's first joint transfer medium containing the apramycin and the nalidixic acid comprises the following steps: 100mL of TSB mycelium growth medium is sterilized and cooled, and 200 mu L of nalidixic acid with the concentration of 25mg/mL and 100 mu L of apramycin with the concentration of 50mg/mL are added into the medium, so that the final concentration of the nalidixic acid is 50 mu g/mL and the final concentration of the apramycin is 50 mu g/mL.
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