Mutant of nostoc phycophage and application thereof
Through UV mutagenesis screening of YongM in the rosary algae phage, mutants with efficient invasion and lysis ability were obtained, which solved the problems of low efficiency and single host of existing algae, and achieved effective cleavage of multiple algae. It has made wider applications of water growth prevention and environmental governance.
Patent Information
- Application Number
- CN202410864043.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-30
- Publication Date
- 2024-12-20
AI Technical Summary
The existing algae phages have low in infection and cleavage efficiency on cyanobacteria and have a single host, making it difficult to fully solve the problem of cyanobacteria blooms.
By screening the UV mutagenesis of YongM in the rosary algae, amino acid mutants such as tail fibrin and alkaline phosphatase D were obtained to form mutants with efficient invasion and cleavage ability, which can have certain cleavage ability for many algae .
The invasion and lysis efficiency of algae phages on rosary algae is improved, the host range is expanded, and more effective methods for preventing and treating cyanobacteria blooms and environmental governance.
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Figure CN119161420A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a mutant of a Nostoc cyanophage and an application thereof. Background Art
[0002] With the development of industrialization and the increasing frequency of human production activities, eutrophication of water bodies has become more and more serious, leading to frequent blue algae blooms around the world. The toxins produced by blue algae will pollute surface water and drinking water sources, seriously endangering the health of water resources. At present, the main treatment methods for preventing and controlling blue algae blooms include chemical methods, physical methods and biological methods in addition to traditional salvage. Among them, chemical methods include chemical reagents and ferrate oxidation methods, physical methods include aeration methods and ultrasonic treatment methods, and biological methods include blue algae growth inhibitors and algaecides. Although there are many methods to prevent and control blue algae blooms, they all have their inherent limitations. Therefore, it is a daunting challenge to comprehensively solve the dilemma of blue algae blooms.
[0003] Cyanophage, as an algae virus, was first discovered in cyanobacterial cells. It is a virus that can specifically infect and lyse cyanobacteria. As a biological control method, the use of cyanophage to prevent cyanobacterial blooms has significant advantages such as safety, harmlessness, pollution-free, and low cost. The emergence of cyanophages provides a new way to prevent cyanobacterial blooms and reduce environmental pollution. However, most of the isolated cyanophages have the disadvantages of low infection and lysis efficiency and a single host. Therefore, there is an urgent need to obtain a broad-spectrum cyanophage with efficient infection and lysis capabilities. Summary of the invention
[0004] The purpose of the present invention is to provide a mutant of the Nostoc cyanophage in view of the problems existing in the prior art. The mutant has high efficiency of infection and lysis ability on Nostoc and has a large host range, and can be widely used in preventing and controlling algal blooms and managing the environment.
[0005] To this end, the first aspect of the present invention provides an amino acid mutant that affects the activity of the Nostoc cyanophage, which includes a related protein mutant of the original Nostoc cyanophage YongM, wherein the related protein mutant includes a tail fiber protein mutant and / or an alkaline phosphatase D mutant;
[0006] The tail fiber protein mutant includes a mutation in which the amino acid sequence of the tail fiber protein in the proto-Nostoc cyanophage YongM as shown in SEQ ID NO.1 from the N-terminus to the C-terminus is mutated from A to V and / or the amino acid sequence of the tail fiber protein in the proto-Nostoc cyanophage YongM is mutated from G to E;
[0007] And / or, the alkaline phosphatase D mutant includes a frameshift mutation caused by the deletion of base A at position 488 from the N-terminus to the C-terminus of the amino acid sequence of alkaline phosphatase D in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.2.
[0008] The second aspect of the present invention provides a mutant of the Nostoc cyanophage, which comprises related protein mutants of the original Nostoc cyanophage YongM, wherein the related protein mutants include tail fiber protein mutants and / or alkaline phosphatase D mutants; wherein the tail fiber protein mutants include tail fiber protein mutant 1 and tail fiber protein mutant 2.
[0009] In some embodiments of the present invention, the tail fiber protein mutant 1 comprises a mutation in which the amino acid sequence of the tail fiber protein in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.1 from the N-terminus to the C-terminus at position A is mutated to V; preferably, the amino acid sequence of the tail fiber protein mutant 1 is as shown in SEQ ID NO.3.
[0010] In some embodiments of the present invention, the tail fiber protein mutant 2 comprises a mutation in which the amino acid sequence of the tail fiber protein in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.1 from the N-terminus to the C-terminus at position 398 G is mutated to E; preferably, the amino acid sequence of the tail fiber protein mutant 2 is as shown in SEQ ID NO.4.
[0011] In some embodiments of the present invention, the alkaline phosphatase D mutant comprises a frameshift mutation caused by the deletion of base A at position 488 from the N-terminus to the C-terminus of the amino acid sequence of alkaline phosphatase D in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.2; preferably, the amino acid sequence of the alkaline phosphatase D mutant is as shown in SEQ ID NO.5.
[0012] Preferably, the mutant is the cyanophage mutant UV1, which contains a tail fiber protein mutant 1.
[0013] Preferably, the mutant is the cyanophage mutant UV2, which contains tail fiber protein mutant 2.
[0014] Preferably, the mutant is the cyanophage mutant UV3, which contains a tail fiber protein mutant 1 and an alkaline phosphatase D mutant.
[0015] The third aspect of the present invention provides use of the mutant of the Nostoc cyanophage according to the second aspect of the present invention in preventing and controlling Nostoc blooms.
[0016] According to the present invention, the application includes co-culturing the mutant of the Nostoc cyanophage with a cyanobacterial liquid to infect and lyse the cyanobacteria to prevent and control cyanobacterial blooms.
[0017] The invention is based on the cyanophage YongM, and a mutant with high efficiency lysis ability on Nostoc is obtained by UV mutagenesis screening and separation. In addition to high efficiency infection and lysis ability on Nostoc, the mutant also has certain lysis ability on many other algae, which improves the infection and lysis efficiency of the cyanophage YongM on the host algae, and can be better applied to the prevention and control of algal blooms and the management of the environment. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings:
[0019] Figure 1 Shows the yellowing of Nostoc sp. FACHB-596 after being infected and lysed by cyanophage YongM after being induced by UV at different energies
[0020] Figure 2 The test results of UV mutagenesis are shown, wherein the left side shows the phycophagic plaques formed by the cyanophage YongM after a single UV mutagenesis on the double-layer plate of Nostoc sp. FACHB-596, and the right side shows the phycophagic plaques formed by the cyanophage YongM after ten consecutive UV mutagenesis on the double-layer plate of Nostoc sp. FACHB-596.
[0021] Figure 3 The figure shows the cyanophage spots formed when the three highly efficient cyanophages YongM screened and the unmutagenized YongM were diluted in different gradients and dropped onto the double-layer plates of Nostoc sp. FACHB-596.
[0022] Figure 4 The results of gene sequence comparison between the highly efficient mutagenized cyanophage YongM and the non-mutagenized YongM are shown.
[0023] Figure 5 The figure shows the comparison of biological characteristics between the mutated cyanophage YongM and the non-mutated YongM; wherein A is the optimal multiplicity of infection, B is heat sensitivity, C is pH tolerance, and D is metal ion sensitivity.
[0024] Figure 6 The results of lysing Nostoc sp. by mutants of the cyanophage YongM are shown. DETAILED DESCRIPTION
[0025] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.
[0026] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.
[0027] I. Terminology
[0028] The term "amino acid mutant" as used in the present invention refers to the smallest unit in which a mutation may occur in the amino acid sequence of a protein.
[0029] The term "original Nostoc cyanophage YongM" used in the present invention refers to wild Nostoc cyanophage YongM.
[0030] The term "yellowing" as used in the present invention refers to the phenomenon after cyanobacteria are lysed by cyanophages.
[0031] The term "cyanobacteria liquid" used in the present invention refers to a cyanobacteria culture cultured to a logarithmic growth phase, including Nostoc liquid.
[0032] The term "UV mutagenesis" in the present invention refers to ultraviolet light mutagenesis.
[0033] In the present invention, the terms "protein" and "protein" can be used interchangeably.
[0034] II. Implementation Plan
[0035] As mentioned above, most of the isolated cyanophages have the disadvantages of low infection and lysis efficiency and single host, and it is urgent to obtain a broad-spectrum cyanophage with high infection and lysis ability. In view of this, the inventors have conducted a lot of research on cyanophage mutation.
[0036] The inventors have noticed that the Nostoc cyanophage YongM is a new type of cyanophage with a broad host spectrum and a high infection and lysis efficiency for Nostoc sp. FACHB-596. Therefore, the inventors considered mutagenizing the Nostoc cyanophage YongM in order to obtain a broad-spectrum cyanophage mutant with high infection and lysis ability for cyanobacteria (e.g., Nostoc sp.).
[0037] However, the current mutagenesis breeding method for cyanophages is not mature. The inventors use UV mutagenesis to increase the mutation frequency and improve the traits in a relatively short period of time, and further obtain cyanophage mutants with improved infection and lysis efficiency through separation and screening.
[0038] The above-mentioned mutagenesis and screening and separation to obtain the highly efficient Nostoc cyanophage YongM mainly includes the following steps:
[0039] (1) Cultivation of Nostoc sp.FACHB-596
[0040] The algae liquid of Nostoc sp. FACHB-596 was mixed with BG-11 culture medium in a volume ratio of 1:10 and placed in a conical flask. The mixture was cultured in a light incubator at 25°C, with a light intensity of 2000 lx and a light-dark cycle of 12h:12h for one week to obtain the algae liquid of Nostoc sp. FACHB-596 in the logarithmic phase.
[0041] (2) Preparation of cyanophage suspension
[0042] Take the stock solution of Nostoc sp. cyanophage YongM (deposit number CGMCC NO.18383, General Microbiology Center of Microbiological Culture Collection Committee) and the algae solution of Nostoc sp. FACHB-596 (Freshwater Algae Species Bank of Chinese Academy of Sciences) in the logarithmic phase at a volume ratio of 1:10, and place it in a conical flask and culture it in a light incubator at 25°C, light intensity 2000lx, and light and dark cycle of 12h:12h. Until Nostoc sp. FACHB-596 is completely lysed, the algae solution is visibly yellowed. Centrifuge the mixed system at 4700rpm for 10min, take the supernatant and filter it through a 0.22μm nitrocellulose membrane, and remove the lysed algae fragments to obtain the cyanophage YongM suspension.
[0043] (3) Expansion of cyanophage YongM
[0044] The suspension of cyanophage YongM in step (2) was mixed with the algae solution of Nostoc sp. FACHB-596 in the logarithmic phase at a volume ratio of 1:10, and cultured in a light incubator under the same conditions as step (2). When the algae solution turned yellow, the cyanophage-host algae lysate was obtained. The lysate was centrifuged at 4700 rpm for 10 minutes, and the supernatant was taken and the above steps were repeated to expand the culture.
[0045] (4) Isolation of independent algae-eating plaques by double-layer plate method
[0046] The Nostoc sp. FACHB-596 algae solution in the logarithmic phase was centrifuged at 4700 rpm for 10 min, and the supernatant was removed to 1 / 10 of the original volume to obtain a concentrated algae solution. 7 mL / tube of semi-solid BG-11 medium containing 0.7% agar was prepared in advance and placed in a 50°C forced air drying oven for use. The cyanophage suspension was serially diluted (10 -3 -10 -6), take 150μL of the diluted cyanophage YongM suspension and 1.5mL of concentrated algae solution and mix them by inversion. After mixing, incubate at 25℃, 80rpm, for 30min to allow the cyanophage to fully adsorb to the host algae Nostocsp.FACHB-596. After the incubation is completed, add the system to the semi-solid BG11 medium prepared in advance, mix it by inversion several times, and immediately pour it onto the bottom plate of BG-11 medium containing 1.5% agar, buckle the culture dish cover, seal it with sealing film after it solidifies, and invert it in a light incubator at 25℃, with a light intensity of 2000lx and a light-dark cycle of 12h:12h. It takes about 72h to form a clear-border cyanophage spot.
[0047] (5) UV mutagenesis of cyanophage YongM
[0048] The YongM suspension was induced by UV crosslinking instrument with different UV energies (20mJ, 40mJ, 60mJ, 80mJ, 100mJ), and the induced YongM suspension was serially diluted with the uninduced YongM suspension (10 -3 -10 -6 ), take the diluted suspension and use the double-layer plate method to obtain independent algae-phagocytic spots.
[0049] After the mutant cyanophage YongM was obtained, it was expanded and cultured, and its lysis efficiency was verified by drop plate experiment. The genome was extracted and sequenced, and the sequence was analyzed to analyze the function of the mutant gene.
[0050] As can be seen from the above, the present invention obtains five mutant cyanophages YongM by UV mutagenesis of Nostoc cyanophage YongM and screening and separation. The following steps are mainly taken: A) irradiating cyanophage YongM with UV of different energies to mutagenesis it and determining the mutagenesis conditions by double-layer plate method; B) continuously mutagenizing cyanophage YongM with UV of appropriate energy and subculturing it to obtain mutant cyanophage YongM by double-layer plate method; C) comparing the infection and lysis ability of the separated mutant cyanophage YongM with that of the unmutated cyanophage YongM by drop plate experiment. D) extracting the mutant cyanophage YongM genome for sequencing analysis.
[0051] The proto-Nostoc cyanophage YongM is mainly composed of nucleotide molecules located in the head and tail fiber protein and alkaline phosphatase D located in the tail. The inventors have found that the mutations of the cyanophage mutants obtained by mutagenesis of the proto-Nostoc cyanophage YongM mainly occur in the tail fiber protein and / or alkaline phosphatase D in the tail of the Nostoc cyanophage YongM.
[0052] Through further research, the inventors have discovered amino acid mutants that affect the activity of Nostoc cyanophages in infecting and lysing cyanobacteria, including related protein mutants of the original Nostoc cyanophage YongM, and the related protein mutants include tail fiber protein mutants and / or alkaline phosphatase D mutants; wherein the tail fiber protein mutants include a mutation of the amino acid sequence of the tail fiber protein in the original Nostoc cyanophage YongM as shown in SEQ ID NO.1 from the N-terminus to the C-terminus, in which A (alanine) at position 195 is mutated to V (valine) and / or G (glycine) at position 398 is mutated to E (glutamic acid);
[0053] And / or, the alkaline phosphatase D mutant includes a frameshift mutation caused by the deletion of base A at position 488 from the N-terminus to the C-terminus of the amino acid sequence of alkaline phosphatase D in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.2.
[0054] At the same time, the inventors have discovered nucleotide mutants that affect the activity of Nostoc cyanophage in infecting and lysing cyanobacteria, including nucleotide mutants encoding related proteins of the original Nostoc cyanophage YongM, and the nucleotide mutants encoding related proteins include nucleotide mutants encoding tail fiber protein and / or nucleotide mutants encoding alkaline phosphatase D;
[0055] Wherein, the nucleotide mutant encoding the tail fiber protein includes a non-synonymous mutation of G to A at position 59,382 (relative to the entire genome) and / or a non-synonymous mutation of C to T at position 58,773 (relative to the entire genome) of the nucleotide sequence of the gene encoding the tail fiber protein in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.6 from the 5′ end to the 3′ end;
[0056] And / or, the nucleotide mutant encoding alkaline phosphatase D includes a non-synonymous mutation of A deletion from position 6,196 (relative to the entire genome) from 5′ to 3′ of the nucleotide sequence shown in SEQ ID NO.7 of the gene encoding alkaline phosphatase D in the Proto-Nostoc cyanophage YongM.
[0057] The present inventors have obtained mutants of the Nostoc cyanophage capable of effectively preventing and controlling cyanobacterial blooms through the above mutagenesis screening, which include related protein mutants of the original Nostoc cyanophage YongM, wherein the related protein mutants include tail fiber protein mutants and / or alkaline phosphatase D mutants.
[0058] In the present invention, the tail fiber protein mutants include tail fiber protein mutant 1 and tail fiber protein mutant 2.
[0059] Accordingly, the gene encoding the tail fiber protein mutant includes a gene encoding tail fiber protein mutant 1 and a gene encoding tail fiber protein mutant 2.
[0060] In some embodiments of the present invention, the tail fiber protein mutant 1 comprises a mutation in which the amino acid sequence of the tail fiber protein in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.1 from the N-terminus to the C-terminus at position A is mutated to V; preferably, the amino acid sequence of the tail fiber protein mutant 1 is as shown in SEQ ID NO.3.
[0061] Accordingly, the gene encoding tail fiber protein mutant 1 contains a non-synonymous mutation in which the nucleotide sequence of the gene encoding the tail fiber protein in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.6 from the 5′ end to the 3′ end, in which the 59,382nd G is mutated to A; preferably, the nucleotide sequence of the gene encoding tail fiber protein mutant 1 is as shown in SEQ ID NO.8.
[0062] In some embodiments of the present invention, the tail fiber protein mutant 2 comprises a mutation in which the amino acid sequence of the tail fiber protein in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.1 from the N-terminus to the C-terminus at position 398 G is mutated to E; preferably, the amino acid sequence of the tail fiber protein mutant 2 is as shown in SEQ ID NO.4.
[0063] Accordingly, the gene encoding tail fiber protein mutant 2 contains a non-synonymous mutation in which the nucleotide sequence of the gene encoding the tail fiber protein in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.6 from the 5′ end to the 3′ end, in which the 58,773rd C is mutated to T; preferably, the nucleotide sequence of the gene encoding tail fiber protein mutant 2 is as shown in SEQ ID NO.9.
[0064] In some embodiments of the present invention, the alkaline phosphatase D mutant comprises a frameshift mutation caused by the deletion of base A at position 488 from the N-terminus to the C-terminus of the amino acid sequence of alkaline phosphatase D in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.2; preferably, the amino acid sequence of the alkaline phosphatase D mutant is as shown in SEQ ID NO.5.
[0065] Accordingly, the gene encoding the alkaline phosphatase D mutant contains a mutation in which the A at position 6,196 from the 5′ end to the 3′ end of the nucleotide sequence of the gene encoding the alkaline phosphatase D in the Proto-Nostoc cyanophage YongM is deleted as shown in SEQ ID NO.7; preferably, the nucleotide sequence of the gene encoding the alkaline phosphatase D mutant is shown in SEQ ID NO.10.
[0066] The inventors obtained several Nostoc cyanophage mutants with high-efficiency Nostoc infection and lysis activity through further screening.
[0067] For example, in some preferred examples of the present invention, the mutant is a cyanophage mutant UV1, which contains a tail fiber protein mutant 1.
[0068] In some other preferred examples of the present invention, the mutant is the cyanophage mutant UV2, which contains the tail fiber protein mutant 2.
[0069] In some further preferred examples of the present invention, the mutant is the cyanophage mutant UV3, which contains a tail fiber protein mutant 1 and an alkaline phosphatase D mutant.
[0070] The use of the mutant of the present invention in preventing and controlling cyanobacterial blooms can be understood as a method for preventing and controlling cyanobacterial blooms using the mutant of the present invention.
[0071] According to the present invention, the application includes co-culturing the mutant Nostoc sp. cyanophage with cyanobacteria (eg, Nostoc sp. FACHB-596) in an algae solution to infect and lyse the cyanobacteria to prevent and control cyanobacterial blooms.
[0072] In the present invention, the cyanobacteria include Nostoc sp.
[0073] The research results show that the mutant of the efficient cyanophage YongM obtained by UV mutagenesis separation in the present invention has efficient infection and lysis ability on Nostoc, and has high value in preventing and controlling cyanobacterial blooms and managing the environment.
[0074] III. Examples
[0075] The present invention is described in detail below through specific examples. The experimental methods described below are all routine laboratory methods unless otherwise specified. The experimental materials described below are all obtained from commercial channels or conventional methods unless otherwise specified.
[0076] Example 1: Determining the UV Energy Size of the Mutagenic Cyanophage YongM
[0077] (1) Cultivation of Nostoc sp.FACHB-596 (Freshwater Algae Seed Bank, Chinese Academy of Sciences)
[0078] The algae liquid of Nostoc sp. FACHB-596 was mixed with BG-11 culture medium in a volume ratio of 1:10 and placed in a conical flask. The mixture was cultured in a light incubator at 25°C, with a light intensity of 2000 lx and a light-dark cycle of 12h:12h for one week to obtain the algae liquid of Nostoc sp. FACHB-596 in the logarithmic phase.
[0079] (2) Preparation of cyanophage YongM suspension
[0080] Take the cyanophage YongM stock solution and the Nostoc sp.FACHB-596 algae solution in the logarithmic phase at a volume ratio of 1:10, mix them in a conical flask, and culture them in a light incubator at 25°C, a light intensity of 2000lx, and a light-dark cycle of 12h:12h. Until Nostoc sp.FACHB-596 is completely lysed, that is, the algae solution turns yellow visibly to the naked eye. Centrifuge the mixed system at 4700rpm for 10min, take the supernatant and filter it through a 0.22μm nitrocellulose membrane, and after removing the lysed algae fragments, the cyanophage YongM suspension can be obtained.
[0081] (3) Expansion of cyanophage YongM
[0082] The suspension of cyanophage YongM in step (2) was mixed with the algae solution of Nostoc sp. FACHB-596 in the logarithmic phase at a volume ratio of 1:10, and cultured in a light incubator under the same conditions as step (2). When the algae solution turned yellow, the cyanophage-host algae lysate was obtained. The lysate was centrifuged at 4700 rpm for 10 minutes, and the supernatant was taken and the above steps were repeated to expand the culture.
[0083] (4) UV mutagenesis of cyanophage YongM
[0084] The YongM suspension was induced by UV crosslinking instrument with different UV energies (20mJ, 40mJ, 60mJ, 80mJ, 100mJ), and the induced YongM suspension was serially diluted with the uninduced YongM suspension (10 -3 -10 -6 ), take the diluted suspension and use the double-layer plate method to obtain independent algae-phagocytic spots.
[0085] The yellowing of Nostoc sp. FACHB-596 infected with YongM suspension irradiated with different energy UV (20mJ, 40mJ, 60mJ, 80mJ, 100mJ) is shown in the figure. Figure 1 shown.
[0086] (5) Double-layer plate method
[0087] The Nostoc sp. FACHB-596 algae solution in the logarithmic phase was centrifuged at 4700 rpm for 10 min, and the supernatant was removed to 1 / 10 of the original volume to obtain a concentrated algae solution. 7 mL / tube of semi-solid BG-11 medium containing 0.7% agar was prepared in advance and placed in a 50°C forced air drying oven for use. The cyanophage suspension was serially diluted (10 -3 -10 -6), take 150μL of the diluted cyanophage YongM suspension and 1.5mL of concentrated algae solution and mix them by inversion. After mixing, incubate at 25℃, 80rpm, for 30min to allow the cyanophage to fully adsorb to the host algae Nostocsp.FACHB-596. After the incubation is completed, add the system to the semi-solid BG11 medium prepared in advance, mix it by inversion several times, and immediately pour it onto the bottom plate of BG-11 medium containing 1.5% agar, buckle the culture dish cover, seal it with sealing film after it solidifies, and invert it in a light incubator at 25℃, with a light intensity of 2000lx and a light-dark cycle of 12h:12h. It takes about 72h to form a clear-border cyanophage spot.
[0088] (6) Determination of UV mutagenesis conditions
[0089] Suspensions of cyanophage YongM induced and not induced by UV with different energies were cultured using the double-layer plate method to obtain independent cyanophage plaques. The lethality was calculated by calculating the number of cyanophage plaques. The lethality of cyanophage YongM induced by 20mJ UV was about 91%, which confirmed that cyanophage YongM was induced by UV with an energy of 20mJ.
[0090] The number of cyanophage spots and the lethality rate formed by the cyanophage YongM at the same dilution gradient after irradiation with different energies are shown in Table 1:
[0091] Table 1
[0092]
[0093] Example 2: Isolation and screening of mutant cyanophage YongM
[0094] (1) The cyanophage YongM suspension was induced by UV with an energy of 20 mJ. The induced YongM suspension was serially diluted (10-3-10-5) and then independent cyanophage plaques were obtained by double-layer plate method. The results are as follows: Figure 2 Shown on the left.
[0095] (2) The cyanophage spots with larger diameter were dug out and mixed with the algae liquid of Nostoc sp. FACHB-596 in the logarithmic phase for cultivation to obtain the first generation mutant cyanophage YongM. The cyanophage YongM suspension was prepared by lysing the culture liquid of mutant cyanophage-host algae. An appropriate amount of the suspension was taken for mutagenesis using UV with the same energy.
[0096] (3) The UV-mutated YongM suspension was mixed with the logarithmic phase Nostoc sp. FACHB-596 algae liquid at a volume ratio of 1:10 and cultured. After the algae liquid turned yellow, the first generation mutant cyanophage YongM suspension was obtained. The same method was used to induce mutations and expand the culture to the tenth generation.
[0097] (4) The tenth generation mutagenized cyanophages were plated using the double-layer plate method to obtain independent cyanophage plaques. Figure 2 As shown on the right, independent algae-eating spots with larger diameters were dug out for expansion and culture.
[0098] Example 3: Drop plate experiment
[0099] (1) Finally, three mutant cyanophages were isolated and screened, and the cyanophage-host algae culture lysate was obtained by amplification culture. The three mutant cyanophage stock solutions were serially diluted with the non-mutated cyanophage YongM stock solution (10 -1 -10 -5 ), take the cyanophage YongM dilution solution and use the double-layer plate method to obtain independent cyanophage plaques, and calculate the titer of each cyanophage YongM stock solution in PFU / mL.
[0100] (2) Each cyanophage YongM stock solution was mixed with Nostoc sp. FACHB-596 algae solution in the logarithmic phase, and then BG-11 was added to dilute it so that the cyanophage number / algae cell number (MOI) in the cyanophage-host algae solution mixture was 0.01.
[0101] (3) When the algae solution turns yellow, gently shake to evenly distribute the cyanophage YongM in the lysate. Take 1 mL of the cyanophage YongM stock solution and perform a series of gradient dilutions (10 -1 -10 -5 ).
[0102] (4) Take 3.5 μL of each cyanophage YongM stock solution and each diluted lysis solution and drop them on a double-layer plate containing Nostoc sp. FACHB-596. The efficiency of cyanophage YongM in lysing the host algae can be determined by observing the size of the cyanophage plaques. Figure 3 shown.
[0103] Example 4: Sequencing and assembly of the cyanophage YongM genome
[0104] (1) Take 30 mL of cyanophage-host algae culture lysate, centrifuge at 4°C and 6000 g for 10 min, and filter the supernatant through a 0.22 μm nitrocellulose membrane.
[0105] (2) The filtrate in (1) was subjected to density gradient centrifugation using 20% (w / v) and 40% (w / v) sucrose solutions freshly prepared within one week at 4°C and 35,000 g for 1 h.
[0106] (3) Discard the supernatant, resuspend the precipitate in 200 μL 0.01 M PBS, and extract the cyanophage YongM genome using the Roche High Purity Viral Nucleic Acid Extraction Kit.
[0107] (4) After extracting the genome, the cyanophage YongM genome was sequenced and assembled. The mutation site was located in the genome such as Figure 4 As shown, the mutation sites obtained by sequencing are all related to the cyanophage tail fiber protein gene. The research results of the present invention can be concluded that the related gene is related to the cyanophage infection and lysis efficiency.
[0108] Example 5: Biological characteristics test of mutant cyanophage YongM
[0109] (1) Expand the culture of YongM, determine the YongM titer using the double plate method, detect the OD680 of Nostoc sp.FACHB-596, and infect at the optimal multiple of infection (MOI) of 0.01, 0.1, and 1. After incubation in the incubator for 25 minutes, centrifuge at 6000 rpm for 10 minutes at 4°C. Remove the supernatant and resuspend in 5 mL BG11 medium for 24 hours. Use the double-layer plate method to determine the algae phagocytic titer in each group, with 3 parallels in each group.
[0110] (2) Take 1 mL of YongM suspension and incubate it at different temperatures (0℃, 30℃, 60℃) for 1 h. Use the double-layer plate method to determine the algae phagocytic titer in each group. The untreated group served as the control group, and each group had 3 parallels.
[0111] (3) Take 1 mL of YongM suspension and incubate it in buffer solutions of different pH values (pH 3, 7, 10) for 1 h. The algae phagocytic titer in each group was determined using the double-layer plate method. The untreated group was used as the control group, and each group had 3 parallel assays.
[0112] (4) Take 1 mL of YongM suspension and mix it with different concentrations of metal ions (10 mM Ca 2+ 、10mM Mg 2+ ) buffer for 25 min, and the untreated group was used as the control group. The algae phagocytic titer in each group was determined by the double-layer plate method, with 3 parallels in each group.
[0113] (5) Biological characteristics test results such as Figure 5 As shown, the obtained mutant cyanophages all have good biological activity and stability.
[0114] Example 6: Application experiment of Nostoc cyanophage
[0115] (1) The three mutant cyanophage stock solutions were diluted in a gradient manner (10 -1 -10 -5 ), take the mutant cyanophage dilution solution and use the double-layer plate method to obtain independent cyanophage plaques, and calculate the titer of each mutant cyanophage in PFU / mL.
[0116] (2) Each mutant cyanophage stock solution was mixed with Nostoc sp. FACHB-596 algae solution in the logarithmic phase and then diluted with BG-11 so that the cyanophage number / algae cell number (MOI) in the cyanophage-host algae solution mixture was 0.01.
[0117] (3) Place the algae solution in a light incubator with a light intensity of 2000 lx and a light-dark cycle of 12 h:12 h. The algae solution will turn yellow within 24 hours. Figure 6 shown.
[0118] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.
Claims
1. An amino acid mutant affecting the activity of Nostoc cyanophage, comprising a related protein mutant of the original Nostoc cyanophage YongM, wherein the related protein mutant comprises a tail fiber protein mutant and / or an alkaline phosphatase D mutant; in, The tail fiber protein mutant includes a mutation in which the amino acid sequence of the tail fiber protein in the proto-Nostoc cyanophage YongM as shown in SEQ ID NO.1 from the N-terminus to the C-terminus is mutated from A to V and / or the amino acid sequence of the tail fiber protein in the proto-Nostoc cyanophage YongM is mutated from G to E; And / or, the alkaline phosphatase D mutant includes a frameshift mutation caused by the deletion of base A at position 488 from the N-terminus to the C-terminus of the amino acid sequence of alkaline phosphatase D in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.
2.
2. A mutant of Nostoc cyanophage, comprising a related protein mutant of the original Nostoc cyanophage YongM, wherein the related protein mutant comprises a tail fiber protein mutant and / or an alkaline phosphatase D mutant; in, The tail fiber protein mutants include tail fiber protein mutant 1 and tail fiber protein mutant 2; Preferably, the tail fiber protein mutant 1 comprises a mutation in which the amino acid sequence of the tail fiber protein in the proto-Nostoc cyanophage YongM as shown in SEQ ID NO.1 from the N-terminus to the C-terminus at position 195 A is mutated to V; further preferably, the amino acid sequence of the tail fiber protein mutant 1 is as shown in SEQ ID NO.3; And / or, the tail fiber protein mutant 2 comprises a mutation in which the amino acid sequence of the tail fiber protein in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.1 from the N-terminus to the C-terminus, wherein the 398th G is mutated to an E mutation; further preferably, the amino acid sequence of the tail fiber protein mutant 2 is as shown in SEQ ID NO.4; And / or, the alkaline phosphatase D mutant comprises a frameshift mutation caused by the deletion of base A at position 488 from the N-terminus to the C-terminus of the amino acid sequence of alkaline phosphatase D in the Proto-Nostoc cyanophage YongM as shown in SEQ ID NO.2; preferably, the amino acid sequence of the alkaline phosphatase D mutant is as shown in SEQ ID NO.
5.
3. The mutant according to claim 2, It is characterized in that The mutant is a cyanophage mutant UV1, which contains a tail fiber protein mutant 1.
4. The mutant according to claim 2, It is characterized in that The mutant is a cyanophage mutant UV2, which contains a tail fiber protein mutant 2.
5. The mutant according to claim 2, It is characterized in that The mutant is a cyanophage mutant UV3, which contains a tail fiber protein mutant 1 and an alkaline phosphatase D mutant.
6. Use of the mutant according to any one of claims 2 to 5 in preventing and controlling cyanobacterial blooms.
7. The use according to claim 6, It is characterized in that The application comprises co-culturing the mutant cyanophage with a cyanobacteria liquid to infect and lyse the cyanobacteria to prevent and control cyanobacteria blooms.