CN113736745B - Nostoc high-efficiency lytic phaeophaga YonggM and application thereof - Google Patents
Nostoc high-efficiency lytic phaeophaga YonggM and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10151—Methods of production or purification of viral material
Abstract
The invention discloses a nostoc high-efficiency lytic phycophage and a separation method and application thereof, and is characterized in that the phagosome is a novel myocaudal Myoviridae phagosome named YongM which is preserved in the common microorganism center of the microorganism strain preservation management committee at 1 month 13 of 2020 and has the number of CGMCC NO.18383.YongM has the characteristics of high efficiency and broad spectrum, and can infect and crack various microcystis, chlorella, anabaena, planktonic alga and africaria alboldii besides high-efficiency cracking of nostoc. YongM can crack and clarify nostoc FACHB-596, anabaena flos-aquae FACHB-245, chlorella FACHB-193 and planktonic alga Oscillatoria planctonica FACHB-708 alga liquid within 24 hours.
Description
Technical Field
The invention relates to a blue algae phagosome, in particular to a nostoc high-efficiency virulent phagosome YonggM and application thereof.
Background
With the development of economy in China in recent years, the degree of eutrophication of water bodies and the increasing severity of cyanobacteria bloom in human beings are advanced in the world, the prevention and control of bloom is urgent, and the current technology for controlling bloom can be divided into a mechanical cleaning method, an aeration mixing method, a nutrition control method, a chemical method, a hydrodynamic control method, a biological control method and the like, wherein the biological control method for removing algae by adopting microorganisms has wide application prospect.
Phagosomes are a group of viruses that specifically infect prokaryotes. The algae phagosome is a composition factor of the ecological environment, is closely related to the life activities of a host, and can regulate the density and the community structure of microalgae in a water body. The research shows that the daily lethality of the marine phaeophaeophaeophaeophaeophaea to the Synechococcus is 5-14%. Invasion of the phagosome can also change the photosynthesis center of a host, so that the photosynthesis incidence rate is reduced, and the primary productivity of the water body is indirectly influenced. In addition, the phagosome is also helpful for the nutrition circulation and regulation of the microbial ecosystem, and thus can be used as a harmless environmental factor for controlling blue algae.
Disclosure of Invention
The invention aims to provide an algae-phagosome capable of efficiently and rapidly cracking blue algae, in particular to a candida cracking algae and application thereof.
The technical scheme adopted for solving the technical problems is as follows: a high-efficiency lytic phagosome obtained by taking Nostoc sp.FACHB-596 as a target is classified and named as cyanomoyovirinuse SP., and the strain name YongM (YongM is the abbreviation of Yong and Myovirinae of Myovirinae) belongs to Myovirinae of Myovirinae. The strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.18383 in the year 1 and 13 of 2020, and the addresses of the preservation institutions are: the institute of microbiology, national institute of sciences, no. 3, north chen west way 1, region of korea, beijing city, postal code: 100101.
the biology characteristics of the phagosome are as follows: the phagosome YongM has a head that presents an icosahedral approximately spherical shape, a diameter of 62nm (+ -5 nm), and a collapsible tail of 93nm (+ -10 nm) length; the phagosome YongM can form transparent plaque on the algae plate of Nostoc sp. FACHB-596; the algae phagosome YongM can rapidly crack Nostoc sp.FACHB-596 to clarify algae liquid; in addition to the lysis of nostoc, microcystis, chroococcus and tremella can also be infected; BLASTN alignment with the DNA polymerase II gene of YongM did not find a sequence identical thereto, indicating that YongM is a novel phagosome.
The separation and purification method of the phage YongM specifically comprises the following steps:
(1) Culture of Nostoc sp.FACHB-596
5mL of Nostoc sp.FACHB-596 algae liquid is diluted into a conical flask filled with 500mL of BG11 liquid culture medium according to the volume ratio of 1:100, and is placed into an illumination incubator with the temperature of 25 ℃ and the illumination intensity of 2000lx and the light-dark period of 12h to 12h, and the logarithmic phase FACHB-596 algae liquid is obtained after about 10 days.
(2) Enrichment and screening of phagostimulants
And (3) water sample treatment: and collecting surface water samples of the Yunnan pond all over, and carrying the water samples back to a laboratory for treatment as soon as possible. The water sample was centrifuged at 12000g for 20min at 4℃to remove the precipitate. The supernatant was taken for the experiment.
Taking a number of sterile conical flasks corresponding to the number of samples, and adding into each conical flask: 80mL of the supernatant of the water sample treated in the above way is added with 20mL of 5 XBG 11 culture medium and 20mL of log-phase FACHB-596 algae liquid. Culturing in an illumination incubator after mixing, and manually shaking the algae for 2-3 times per day. Meanwhile, water taken by the water purifier is used for replacing water sample supernatant as a control. The yellow culture broth was centrifuged at 12000g for 10 minutes, and the supernatant was filtered through a 0.45 μm nitrocellulose filter and a 0.22 μm nitrocellulose filter in this order. Adding the filtrate into FACHB-596 algae solution in logarithmic phase of 10 times volume, mixing, and culturing until obvious yellowing phenomenon appears to obtain the primary screening infection solution.
(3) Preliminary purification of phagosome
Preparing concentrated algae liquid (1:20-1:40): taking FACHB-596 algae liquid in logarithmic phase, centrifuging 10000g for 10min, and retaining supernatant equivalent to 1/20-1/40 of original volume for suspension precipitation;
the melted BG11 medium containing 0.7% (W/V) agar is split into 8 mL/tube in an ultra clean bench, and placed in a constant temperature water bath at 45 ℃ for 30min until the temperature is constant.
Taking the primary screening virus-containing infection solution obtained in the step (2), and carrying out serial gradient dilution on the primary screening virus-containing infection solution by using BG11 liquid culture medium (10) -1 - 10 -6 ) The method comprises the steps of carrying out a first treatment on the surface of the Adding the diluted solution of each dilution into 0.9mL of concentrated FACHB-596 algae solution according to the volume ratio of 1:9, uniformly mixing, placing into a shaking table with the rotation speed of about 70rpm, and incubating for 20-30 minutes at 25 ℃ to enable viruses to be fully adsorbed to algae cells;
taking 8 mL/tube of BG11 culture medium containing 0.7% agar from 45 ℃ constant temperature water bath, immediately wiping water outside the tube with paper, pouring the virus-algae mixed solution into the tube, rapidly shaking the tube with a vortex oscillator for 3-5s to fully mix the virus-algae mixed solution, immediately pouring the virus-algae mixed solution onto a BG11 culture substrate layer flat plate (about 110mm in diameter) containing 1.5% (W/V) agar, sealing a sealing film for sealing a sealing hole (at a sealing film at the junction of a dish and a cover) after the virus-algae mixed solution is solidified, and inverting the sealing film at 2000lx,25 ℃ for illumination period: culturing in a 12h/12h incubator and observing plaque formation (7 days were required). The independent phycophage formed under the maximum dilution is dug and suspended in 500 mu L of BG11 culture medium, and is placed in a refrigerator at 4 ℃ for overnight or at 25 ℃ for light-proof incubation for 1 hour, so that the phycophage is thoroughly released, 12000g is centrifuged for 10 minutes, and the supernatant is taken to obtain the first generation of phycophage.
(4) Further purification of phagosome
Taking the first-generation phagosome obtained in the step (3), and serially and gradiently diluting the first-generation phagosome into 10 by using BG11 culture medium -1 To 10 -6 Repeating the plaque experiment in the step (3) for at least 3 times to obtain the plaque with uniform shape and size. The single plaque is dug and suspended in 1mL BG-11 culture medium, and is placed in a refrigerator at 4 ℃ for overnight or at 25 ℃ for light-proof incubation for 1 hour, 10000g is centrifuged for 10-20 minutes, and the supernatant is taken to be the suspension of the phagosome YongM.
(5) Enlarged culture of phaga
Adding 1mL of purified algae phagosome YongM suspension obtained in the step (4) into 9mL of fresh cultivated FACHB-596 algae liquid in logarithmic phase (or platform phase), uniformly mixing, culturing in an illumination incubator for 24 hours, and yellowing the algae liquid to obtain algae phagosome YongM-algae culture lysate, centrifuging 12000g of lysate for 10 minutes, and collecting supernatant. The supernatant was added to fresh cultured FACHB-596 algae liquid in the logarithmic phase at a ratio of 1:10 (V/V), mixed well, and cultured in an illumination incubator for 24 hours to yellow the algae liquid. The expansion culture was repeated in this manner.
(6) Preparation of algae-phagostimulant suspension
Centrifuging the phycophage-algae culture lysate prepared in the step (5) (4 ℃,10000g and 10 min), taking supernatant, and filtering sequentially through nitrocellulose filter membranes with the pore diameters of 0.45 μm and 0.22 μm to obtain the phycophage YongM suspension.
Compared with the prior art, the invention has the advantages that: the invention discloses a novel nostoc lytic phaeophaga Yonggm, a separation method and application thereof, and the lytic virus Yonggm has the characteristics of high replication rate and high infection rate, can efficiently lyse nostoc, and can obviously yellow a host algae liquid within 24 hours; besides the candidiasis, the virus can infect microcystis, chlorella and tremella, and has application value in preventing and treating water bloom caused by blue algae. Culturing a large amount of YongM is easier; the operation is simple, and the development prospect is good.
In summary, the invention provides a novel nostoc lytic phaeophaga Yonggm, a separation method and application thereof, and the virus can infect microcystis, chlorella and tremella besides high-efficiency nostoc lytic phaeophaeophaea, and has application value in preventing and treating water bloom caused by blue algae.
Drawings
FIG. 1 shows the formation of a plaque by the phaeophagosome YongM on a double-layered plate of the nostoc FACHB-596
The test tube 7 on the right side of FIG. 2 is nostoc FACHB-596 control algae liquid, and the nostoc FACHB-596 algae liquid in the test tube 1 on the left side is yellow due to algae cell lysis caused by adding phagosome YongM.
FIG. 3 is a transmission electron microscope image of a negatively stained phagosome YonggM, and the right image is a complete phagosome particle
FIG. 4 is a right image of microscopic lesions of the nostoc FACHB-596 cells infected with phaga-phaga YonggM, and a left image of the control nostoc FACHB-596 cells
FIG. 5 shows the results of Blastn alignment of the DNA polymerase gene of the YongM phycophage in Genbank.
Detailed Description
The invention will be described in further detail with reference to the following examples of the drawings
Example 1
Preparation of algae phagosome YongM
(1) Culture of Nostoc sp.FACHB-596
5mL of Nostoc sp.FACHB-596 algae liquid is diluted into a conical flask filled with 500mL of BG11 liquid culture medium according to the volume ratio of 1:100, and is placed into an illumination incubator with the temperature of 25 ℃ and the illumination intensity of 2000lx and the light-dark period of 12h to 12h, and the logarithmic phase FACHB-596 algae liquid is obtained after about 10 days.
(2) Enrichment and screening of phagostimulants
And (3) water sample treatment: and collecting surface water samples of the Yunnan pond all over, and carrying the water samples back to a laboratory for treatment as soon as possible. The water sample was centrifuged at 12000g for 20min at 4℃to remove the precipitate. The supernatant was taken for the experiment.
Taking a number of sterile conical flasks corresponding to the number of samples, and adding into each conical flask: 80mL of the supernatant of the water sample treated in the above way is added with 20mL of 5 XBG 11 culture medium and 20mL of log-phase FACHB-596 algae liquid. Culturing in an illumination incubator after mixing, and manually shaking the algae for 2-3 times per day. Meanwhile, water taken by the water purifier is used for replacing water sample supernatant as a control. The yellow culture broth was centrifuged at 12000g for 10 minutes, and the supernatant was filtered through a 0.45 μm nitrocellulose filter and a 0.22 μm nitrocellulose filter in this order. Adding the filtrate into FACHB-596 algae solution in logarithmic phase of 10 times volume, mixing, and culturing until obvious yellowing phenomenon appears to obtain the primary screening infection solution.
(3) Preliminary purification of phagosome
Preparing concentrated algae liquid (1:20-1:40): taking FACHB-596 algae liquid in logarithmic phase, centrifuging 10000g for 10min, and retaining supernatant equivalent to 1/20-1/40 of original volume for suspension precipitation;
the melted BG11 medium containing 0.7% (W/V) agar is split into 8 mL/tube in an ultra clean bench, and placed in a constant temperature water bath at 45 ℃ for 30min until the temperature is constant.
Taking the primary screening virus-containing infection solution obtained in the step (2), and carrying out serial gradient dilution on the primary screening virus-containing infection solution by using BG11 liquid culture medium (10) -1 - 10 -6 ) The method comprises the steps of carrying out a first treatment on the surface of the Adding the diluted solution of each dilution into 0.9mL of concentrated FACHB-596 algae solution according to the volume ratio of 1:9, uniformly mixing, placing into a shaking table with the rotation speed of about 70rpm, and incubating for 20-30 minutes at 25 ℃ to enable viruses to be fully adsorbed to algae cells;
taking 8 mL/tube of BG11 culture medium containing 0.7% agar from 45 ℃ constant temperature water bath, immediately wiping water outside the tube with paper, pouring the virus-algae mixed solution into the tube, rapidly shaking the tube with a vortex oscillator for 3-5s to fully mix the virus-algae mixed solution, immediately pouring the virus-algae mixed solution onto a BG11 culture substrate layer flat plate (about 110mm in diameter) containing 1.5% (W/V) agar, sealing a sealing film for sealing a sealing hole (at a sealing film at the junction of a dish and a cover) after the virus-algae mixed solution is solidified, and inverting the sealing film at 2000lx,25 ℃ for illumination period: culturing in a 12h/12h incubator and observing plaque formation (7 days were required). The independent phycophage formed under the maximum dilution is dug and suspended in 500 mu L of BG11 culture medium, and is placed in a refrigerator at 4 ℃ for overnight or at 25 ℃ for light-proof incubation for 1 hour, so that the phycophage is thoroughly released, 12000g is centrifuged for 10 minutes, and the supernatant is taken to obtain the first generation of phycophage.
(4) Further purification of phagosome
Taking the first-generation phagosome obtained in the step (3), and serially and gradiently diluting the first-generation phagosome into 10 by using BG11 culture medium -1 To 10 -6 Repeating the plaque experiment in the step (3) for at least 3 times to obtain the plaque with uniform shape and size. The single plaque is dug and suspended in 1mLBG-11 culture medium, and incubated at 4 deg.C overnight or 25 deg.C in dark place for 1 hr, 10000g is centrifugated for 10-20 min, and the supernatant is the suspension of the phaeophagosome YongM.
(5) Enlarged culture of phaga
Adding 1mL of purified algae phagosome YongM suspension obtained in the step (4) into 9mL of fresh cultivated FACHB-596 algae liquid in logarithmic phase (or platform phase), uniformly mixing, culturing in an illumination incubator for 24 hours, and yellowing the algae liquid to obtain algae phagosome YongM-algae culture lysate, centrifuging 12000g of lysate for 10 minutes, and collecting supernatant. The supernatant was added to fresh cultured FACHB-596 algae liquid in the logarithmic phase at a ratio of 1:10 (V/V), mixed well, and cultured in an illumination incubator for 24 hours to yellow the algae liquid. The expansion culture was repeated in this manner.
(6) Preparation of algae-phagostimulant suspension
Centrifuging the phycophage-algae culture lysate prepared in the step (5) (4 ℃,10000g and 10 min), taking supernatant, and filtering sequentially through nitrocellulose filter membranes with the pore diameters of 0.45 μm and 0.22 μm to obtain the phycophage YongM suspension.
Wherein the formula of the BG-11 culture medium is as follows:
the 5 XBG 11 medium is 5 times mother liquor of the BG-11 medium.
The purified phagosome is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms, and has the preservation number: CGMCC No.18383, the preservation date is 2020, 1 month and 13 days, the preservation unit address: beijing, chaoyang area, north Chenxi Lu No.1, 3, postal code 100101.
Example 2
Morphological observations of algae phagosome YongM
The separation and purification of the phycophage-algae culture lysate of example 1 was collected by low-speed centrifugation (4 ℃,5000g,5 min) to discard the pellet, the supernatant was equally distributed to two tubes, 5-10mL of 20% (w/v) sucrose solution was slowly and gently injected from the bottom by syringe, and the supernatant was discarded by centrifugation at 35000g at 4℃for 1h. The solution was gently added with 0.01M PBS, the PBS was discarded, and the solution was again added with 0.01M PBS and left at 4℃overnight, after which the precipitate was suspended. A drop of the phaeophaga suspension is taken out by a pipetting gun and put on a copper net, and after standing for 10min, the excessive water is sucked from the side by neutral filter paper. A drop of 3% uranyl acetate was dropped on the copper mesh, and after 20s of staining, the stain was sucked off from the side with neutral filter paper. After standing for 10min and air-drying, the morphology of the phagosome was observed by using a transmission electron microscope (Hitachi H-7650).
As a result, as shown in FIG. 2, the phycophage YongM has a head portion exhibiting an icosahedral approximately spherical structure, a diameter of 62nm (+ -5 nm), and a contractile tail portion, a length of 93nm (+ -10 nm).
Example 3
Sequence alignment of DNA polymerase genes of algae phagosome YongM
Genome extraction: DNase I and RNase A were added to the suspension of the phage YongM to a final concentration of 1. Mu.g/mL, digested overnight at 37℃and inactivated at 80℃for 15min. Lysates (0.5% SDS, 50. Mu.g/mL proteinase K,20nM EDTA, all final concentrations) were added to the system and incubated for 1h at 56 ℃. Equal volume of balance phenol was added and 10000g was centrifuged at 4℃for 5min after gentle shaking. The upper liquid was collected, added with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), gently shaken and centrifuged at 10000g for 5min at 4 ℃. Collecting upper liquid, adding equal volume of chloroform, mixing thoroughly, centrifuging for 5min at 10000g, collecting upper liquid, and repeating for 2 times. Adding equal volume of isopropanol, standing at-20deg.C for at least 30min, centrifuging at 4deg.C for 20min, and washing the precipitate with 75% ethanol for 2 times. The nucleic acid pellet was resuspended in deionized water and stored at-20 ℃.
Genome library construction: usingKit NEBNext Ultra II DNA Library Prep Kit for Illumina (#E7645) constructs a genomic library. The method comprises the steps of 1, genome fragmentation: the extracted phagosome nucleic acid was randomly disrupted by a covarias ultrasonic disruptor (30 s,90s,11min, L) and the resulting DNA fragment length was concentrated mainly at 500bp. 2. And (3) terminal repair: 50. Mu.L of the excised DNA fragment was placed in a 1.5ml EP tube without nuclease, and 3. Mu. L NEBNext Ultra II End Prep Enzyme Mix and 7. Mu. L NEBNext Ultra II End Prep Reaction B uffer were added, respectively, and mixed. Incubation at 20℃for 30min,65℃for 30min, and preservation at 4 ℃. 3. Adding a sequencing linker: 30 mu L NEBNext Ultra II Ligation Master Mix, 1 mu L NEBNext Ligation Enhancer and 2.5 mu L NEBNext Adaptor are added into the system in the last step and are mixed uniformly. Incubate at 20℃for 15min. Add 3. Mu.LEnzyme was thoroughly mixed and incubated at 37℃for 15min. 4. Magnetic bead fragment screening: 20. Mu.L of resuspended AMPure XP magnetic beads were added to the above system, mixed well and incubated for 5min at room temperature. The EP tube was placed on a magnetic rack and the solution was gently swirled to separate the beads, and after clarification, the supernatant was transferred to a new EP tube. 10. Mu.L of resuspended AMPure XP beads were added to the supernatant, mixed well and incubated for 5min at room temperature. The EP tube was placed on a magnetic rack and gently rotated to separate the solution from the beads, after which the solution was clarified, the supernatant was discarded and the beads containing the desired DNA were retained. 200 μl of freshly prepared 80% ethanol was added, incubated at room temperature for 30s, the supernatant discarded, and repeated 2 times. The EP tube was placed on a magnetic rack and the magnetic beads were dried for 5min by opening the lid. The EP tube was removed, 17. Mu.L of nuclease-free water was added to elute the DNA, and the mixture was thoroughly mixed on a vortex shaker and incubated at room temperature for 2min. The EP tube was placed on a magnetic rack and left to stand for 3min, after which 15. Mu.L of supernatant was aspirated into the PCR tube for further use. 5. Index addition and PCR amplification: to 15. Mu.L of the nucleic acid obtained in the above step, 25. Mu. L NEBNext Ultra II Q5.5 Master Mix, 5. Mu.L of Index Primer/i7 Primer, 5. Mu. L Universal PCR Primer/i5 Primer were added and mixed well. PCR amplification was performed under the following reaction conditions: 98 ℃ for 30 seconds; 98℃for 10 seconds, 65℃for 70 seconds, 10 cycles; finally, the extension is carried out for 5min at 65 ℃. 6. And (3) purifying a PCR product: PCR productionTransfer the material to a 1.5mL EP tube without nuclease, add 45. Mu.L of resuspended AMPure XP magnetic beads, mix well and incubate for 5min at room temperature. The EP tube was placed on a magnetic rack and after clarification of the solution, the supernatant was discarded and the magnetic beads containing the desired DNA were retained. 200 μl of freshly prepared 80% ethanol was added, incubated at room temperature for 30s, the supernatant discarded, and repeated 2 times. The EP tube was placed on a magnetic rack and the magnetic beads were dried for 5min by opening the lid. The EP tube was removed, 33. Mu.L nuclease-free water was added to elute the DNA, and the mixture was thoroughly mixed and incubated at room temperature for 2min. The EP tube was placed on a magnetic rack and 30. Mu.L of the solution was removed to a new EP tube. The DNA content was determined using Qubit.
Sequencing on a machine: fragment distribution of the purified PCR product was identified according to Agilent High sensitivity DNA kit 2100 instructions. And quantifying by real-time fluorescent quantitative PCR, mixing DNA samples according to the requirement of an on-machine, and finally adding NaOH to denature the DNA into a single chain. Illumina MiSeq sequencing was performed using Illumina PE300 kit.
Sequence splicing and annotation: the quality of the sequenced data was assessed by FastQC (http:// www.bioinformatics.babraham.ac.uk/subjects/FastQC /), then the low quality value of the sequenced data was removed with trimmic v0.36 software, finally the filtered data was spliced using SPADes v3.13.0 software, and the YongM genome was functionally annotated with RAST (Rapid Annotation using Subsystem Technology, http:// RAST. Nmddr. Org).
Sequence alignment: the DNA polymerase gene (specific nucleotide sequence is shown in sequence table SEQ ID NO. 1) in the YongM genome was aligned with all sequences using BLASTN tool provided by NCBI. BLAST alignment shows that YongM sequences are not present in the existing database (FIG. 5), and are a new unreported phagosome. BLAST shows that the highest homology to YongM is Nostoc phase A1, with a DNA polymerase gene homology of 84.38%.
Example 4
Algicidal range test of YongM of phaga
Twenty-five cyanobacteria contained in Table 1 were cultured to logarithmic phase (OD) 680 0.8). The logarithmic phase algae liquid and the phaeophaga YongM suspension are mixed according to the volume ratio of 6:1 to be used as experimental groupsThe control group replaced the phagosome with BG11 medium, and each group was placed in an illumination incubator at 25℃under an illumination intensity of 2000lx and a light-dark period of 12h to 12 h. OD of each group was measured daily with an enzyme-labeled instrument 680 . Average values of the parallel groups are taken, and the ratio of the average values of the control group to the average values of the experimental group is calculated. If the ratio is greater than 1.2, the phagosome can be considered to infect the algae, and the result is positive; otherwise, the phagosome can not infect the algae, and the result is negative; and observing infection and cleavage by naked eyes and a microscope, and confirming the result.
TABLE 1 algicidal extent test results for YongM phagosome
"+" represents infection and "-" represents no infection
Results YongM infected and lysed microcystis Microcystis aeruginosa FACHB-905, FACHB-915, M.wesenbergii FACHB-908, FACHB-929, M viridis FACHB-979, M.Flos-aquae FACHB-1028, chlorella Chroococcus FACHB-193, anabaena flos-aquae FACHB-245, phaescilla floating Oscillatoria planctonica FACHB-708, phaescilla albo Planktothrix agardhii FACHB-920 (Table 1), wherein YongM allowed the Phaescilla FACHB-596, anabaena flos-aquae FACHB-245, chlorella FACHB-193, and Phaescilla floating Oscillatoria planctonica FACHB-708 to lyse within 24 hours.
The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions that would be within the spirit and scope of the invention are also within the scope of the invention, which is defined by the following claims.
Claims (2)
1. A highly efficient lytic phagosome, characterized in that: the algae phagosome is named YongM and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.18383 in the year of 2020, 1 and 13.
2. Use of a highly efficient lytic phagosome according to claim 1, characterized in that said phagosome YongM is used for lysing candida fach b-596, microcystis Microcystis aeruginosa FACHB-905, fach b-915, m.wesenbergii fach b-908, fach b-929, m.viridis fach b-979, m.flos-aquae fach b-1028, chroococcus fach b-193, anabaena flos-aquae fach b-245, plankton alga Oscillatoria planctonica FACHB-708 or africaria albopictus Planktothrix agardhii FACHB-920.
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