Streptomyces bacteriophage St01 and application thereof

Abstract

The invention belongs to the technical field of biological control, and particularly relates to a streptomyces bacteriophage St01 and application thereof. The streptomyces bacteriophage St01 is separated from a soil environment, has the advantages of relatively good propagation performance, short outbreak period, relatively good environmental stability, good genetic stability and good safety, can specifically infect and split pathogenic streptomyces without killing other bacteria, does not cause additional influence on normal flora, and has a good application prospect. As a bacterial virus, the bacteriophage is not influenced by bacterial drug resistance, does not cause the problems of drug residues and the like in application, and has the characteristics of high safety and environmental friendliness; the bacillus subtilis can efficiently kill pathogenic streptomycete in plants and in a soil environment, and has good application prospects in killing the pathogenic streptomycete in the environment, preventing and treating pathogenic streptomycete infection and preventing and treating plant diseases caused by the pathogenic streptomycete; meanwhile, the method can also be used for detecting pathogenic streptomyces and / or identifying plant diseases caused by the pathogenic streptomyces.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and specifically relates to a Streptomyces phage St01 and an application thereof. Background Art

[0002] Plant bacterial diseases have a serious impact on the crop planting industry worldwide. Currently, the prevention and control of such diseases is often limited to the use of antibiotics or chemical pesticides, but this method is prone to the emergence of new pathogens with drug resistance.

[0003] Bacteriophage is a general term for a class of bacterial viruses that can specifically lyse host bacteria, but are non-toxic to non-targeted bacteria, animals and plants, and will not destroy the diversity of microorganisms, meeting the requirements of green and pollution-free agriculture advocated by the contemporary era. Therefore, phage therapy has great potential in the biological control of bacterial diseases. At present, phages have been used in a small number of human diseases, aquatic animal diseases and plant diseases. However, the application of phage preparations to prevent and control plant diseases caused by soil-borne pathogenic Streptomyces is still relatively lacking. The development of this phage not only actively cooperates with my country's strategic goal of reducing the amount of pesticides, but also can effectively promote the green and sustainable development of related planting industries. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide a Streptomyces phage St01.

[0005] The second aspect of the present invention aims to provide a culture.

[0006] The third aspect of the present invention aims to provide a method for preparing the culture of the second aspect of the present invention.

[0007] The fourth aspect of the present invention aims to provide a preparation.

[0008] The fifth aspect of the present invention aims to provide a detection kit.

[0009] The purpose of the sixth aspect of the present invention is to provide the application of the preparation of the fourth aspect of the present invention.

[0010] The purpose of the seventh aspect of the present invention is to provide an application of the detection kit of the fifth aspect of the present invention.

[0011] The eighth aspect of the present invention aims to provide a method for preventing and controlling plant diseases caused by pathogenic Streptomyces infection and / or pathogenic Streptomyces.

[0012] The ninth aspect of the present invention aims to provide a method for killing pathogenic Streptomyces in the environment.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] In a first aspect of the present invention, a Streptomyces phage St01 is provided, wherein the nucleotide sequence contained therein is composed of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 from the 5' end to the 3' end.

[0015] In some embodiments, the Streptomyces phage St01 is a DNA phage; further, a dsDNA phage; thus, it has excellent genetic stability.

[0016] In some embodiments, the classification name of the Streptomyces phage St01 is Streptomyces phage, belonging to Duplodnaviria (double-stranded DNA virus domain), Heunggongvirae (Hong Kong virus kingdom), Uroviricota (tailed bacteriophage), Caudoviricetes (tailed bacteriophage class), Caudovirales (tailed bacteriophage order), and Siphoviridae (long-tailed bacteriophage family).

[0017] In some embodiments, the Streptomyces phage St01 has a typical icosahedral head (about 65 nm in diameter) and a telescopic tail (about 280 nm in length).

[0018] In some embodiments, the optimal infection multiplicity of the Streptomyces phage St01 is 0.001.

[0019] In some embodiments, the Streptomyces phage St01 was cultured for 24 hours at a multiplicity of infection of 0.001, and the titer was as high as 1.11±0.19×10 9 pfu / mL; it has good propagation performance.

[0020] In some embodiments, the incubation period of the Streptomyces phage St01 is about 60 minutes, and the burst period is about 45 minutes; it has the advantage of a short burst period compared with similar phages (for example, Psst1, Psst2, and Psst4 disclosed in the document Shihe Zhang, Cheligeer Zhang, Jian Wu, Simiao Liu, Ruofang Zhang, Utpal Handique, Isolation, characterization and application of noble bacteriophages targeting potato common scab pathogen Streptomyces telliscabiei, Microbiological Research, Volume 283, 2024, 127699, ISSN 0944-5013.).

[0021] In some embodiments, the Streptomyces phage St01 maintains a stable titer under the condition of pH 4-12.

[0022] In some embodiments, the Streptomyces phage St01 maintains a stable titer at a temperature below 40°C (preferably 4-37°C).

[0023] In some embodiments, the Streptomyces phage St01 is exposed to ultraviolet light for a long time (at 20 W, 50 cm, 700 mw / m 2 The titer was maintained at a stable level for 15-60 min.

[0024] The Streptomyces phage St01 has better environmental stability compared with similar phages (for example: SscP1EGY disclosed in the document Abdelrhim, AS, Ahmad, AA, Omar, MOA et al. A new Streptomyces scabies-infecting bacteriophage from Egypt with promising biocontrol traits. Arch Microbiol 203, 4233–4242 (2021).), thereby maintaining higher activity in field environments with complex conditions.

[0025] In some embodiments, the Streptomyces phage St01 has a good lysis effect on pathogenic Streptomyces (including but not limited to: S. scabies (Streptomyces aflumanensis), S. acidiscabies (Streptomyces acidicus), S. europaeiscabiei (Streptomyces europaeiscabiei), S. luridiscabiei (Streptomyces turgidiscabies), S. reticuliscabiei (Streptomyces reticulariscabiei), S. botropensis (Streptomyces bochuensis), S. bobili (Streptomyces bobilis), S. anulatus (Streptomyces anulatus)).

[0026] In some embodiments, the Streptomyces phage St01 has good safety for plants (such as potato chips); it can remain active on plants (such as potato chips) within 96 hours, and the phage residues are completely eliminated after 120 hours.

[0027] In some embodiments, the Streptomyces phage St01 has a good control function on scab of various plants; 10 5 pfu / mL phage St01 can significantly reduce the concentration of pathogens after 5 days, and the therapeutic effect is better than that of similar phages reported before, that is, Streptomyces phage St01 can effectively kill the pathogenic bacteria infected on potato slices.

[0028] In some embodiments, the Streptomyces phage St01 can effectively kill pathogenic Streptomyces contaminated in the soil environment.

[0029] The second aspect of the present invention provides a culture obtained by culturing the Streptomyces phage St01 of the first aspect of the present invention.

[0030] The third aspect of the present invention provides a method for preparing the culture of the second aspect of the present invention, comprising inoculating the Streptomyces phage St01 of the first aspect of the present invention into a host bacterium, culturing, and obtaining.

[0031] In some embodiments, the host bacteria comprises pathogenic Streptomyces; further comprises soil-borne pathogenic Streptomyces; further comprises at least one of S.scabies (Streptomyces aflumanensis), S.acidiscabies (Streptomyces acidicus), S.europaeiscabiei (Streptomyces europaeiscabiei), S.luridiscabiei (Streptomyces luteus), S.turgidiscabies (Streptomyces swollen), S.reticuliscabiei (Streptomyces reticulosus), S.botropensis (Streptomyces bochu), S.bobili (Streptomyces bobili), and S.anulatus (Streptomyces anulatus); further comprises S.turgidiscabies (Streptomyces swollen).

[0032] In some embodiments, the culture system further comprises a culture medium (preferably NB liquid culture medium).

[0033] In some embodiments, the culturing temperature is 24-32°C.

[0034] In some embodiments, the culturing time is 12-48 hours.

[0035] In some embodiments, the culturing is a shaking culture.

[0036] The fourth aspect of the present invention provides a preparation comprising: the Streptomyces phage St01 of the first aspect of the present invention or the culture of the second aspect of the present invention.

[0037] In some embodiments, the preparation further comprises a carrier and / or an excipient.

[0038] In some embodiments, the excipient comprises at least one of a diluent, an adjuvant, a surfactant, an excipient, a vehicle, a dispersant, a stabilizer, a filler and a solvent.

[0039] In some embodiments, the preparation is a solid preparation or a liquid preparation.

[0040] In some embodiments, the solid state formulation comprises a lyophilized formulation.

[0041] In some embodiments, the preparation is in the form of a powder, an aqueous solution, a lyophilized agent, a gel, a cream or an ointment; further in the form of a solution, a suspension, an emulsion, a powder, a tablet, a spray or an injection.

[0042] In some embodiments, the formulation is an agricultural fungicide or an additive thereof, a disinfectant or an additive thereof, or a cleaning agent or an additive thereof.

[0043] In some embodiments, the formulation is used to control pathogenic Streptomyces infection and / or plant diseases caused by pathogenic Streptomyces.

[0044] In some embodiments, the pathogenic Streptomyces comprises soil-borne pathogenic Streptomyces; further comprises at least one of S. scabies (Streptomyces aflumanensis), S. acidiscabies (Streptomyces acidicus), S. europaeiscabiei (Streptomyces europaeiscabiei), S. luridiscabiei (Streptomyces luteus), S. turgidiscabies (Streptomyces swollen), S. reticuliscabiei (Streptomyces reticulus), S. botropensis (Streptomyces bobili), and S. anulatus (Streptomyces anulatus); and further comprises S. turgidiscabies (Streptomyces swollen).

[0045] In some embodiments, the disease comprises scab.

[0046] In some embodiments, the plant comprises at least one of potato, radish, beet, turnip, and peanut; and further comprises potato.

[0047] In some embodiments, the plant disease is potato scab.

[0048] In some embodiments, the formulation further comprises:

[0049] a1) other active ingredients for controlling pathogenic Streptomyces infections and / or plant diseases caused by pathogenic Streptomyces; and / or

[0050] a2) Active ingredients for controlling other pathogens.

[0051] A fifth aspect of the present invention provides a detection kit, comprising: the Streptomyces phage St01 of the first aspect of the present invention or the culture of the second aspect of the present invention.

[0052] In some embodiments, the detection kit is used for any one of b1)-b2):

[0053] b1) Detection of pathogenic Streptomyces;

[0054] b2) Identification of plant diseases caused by pathogenic Streptomyces.

[0055] In some embodiments, the pathogenic Streptomyces comprises soil-borne pathogenic Streptomyces; further comprises at least one of S. scabies (Streptomyces aflumanensis), S. acidiscabies (Streptomyces acidicus), S. europaeiscabiei (Streptomyces europaeiscabiei), S. luridiscabiei (Streptomyces luteus), S. turgidiscabies (Streptomyces swollen), S. reticuliscabiei (Streptomyces reticulus), S. botropensis (Streptomyces bobili), and S. anulatus (Streptomyces anulatus); and further comprises S. turgidiscabies (Streptomyces swollen).

[0056] In some embodiments, the disease comprises scab.

[0057] In some embodiments, the plant comprises at least one of potato, radish, beet, turnip, and peanut; and further comprises potato.

[0058] In some embodiments, the plant disease is potato scab.

[0059] The sixth aspect of the present invention provides the use of the preparation of the fourth aspect of the present invention in any one of c1)-c3);

[0060] c1) Kill pathogenic Streptomyces in the environment;

[0061] c2) Preventing and controlling pathogenic Streptomyces infection;

[0062] c3) Prevent and control plant diseases caused by pathogenic Streptomyces.

[0063] In some embodiments, the pathogenic Streptomyces comprises soil-borne pathogenic Streptomyces; further comprises at least one of S. scabies (Streptomyces aflumanensis), S. acidiscabies (Streptomyces acidicus), S. europaeiscabiei (Streptomyces europaeiscabiei), S. luridiscabiei (Streptomyces luteus), S. turgidiscabies (Streptomyces swollen), S. reticuliscabiei (Streptomyces reticulus), S. botropensis (Streptomyces bobili), and S. anulatus (Streptomyces anulatus); and further comprises S. turgidiscabies (Streptomyces swollen).

[0064] In some embodiments, the disease comprises scab.

[0065] In some embodiments, the plant comprises at least one of potato, radish, beet, turnip, and peanut; and further comprises potato.

[0066] In some embodiments, the plant disease is potato scab.

[0067] The seventh aspect of the present invention provides the use of the detection kit of the fifth aspect of the present invention in any one of b1)-b2):

[0068] b1) Detection of pathogenic Streptomyces;

[0069] b2) Identification of plant diseases caused by pathogenic Streptomyces.

[0070] In some embodiments, the pathogenic Streptomyces comprises soil-borne pathogenic Streptomyces; further comprises at least one of S. scabies (Streptomyces aflumanensis), S. acidiscabies (Streptomyces acidicus), S. europaeiscabiei (Streptomyces europaeiscabiei), S. luridiscabiei (Streptomyces luteus), S. turgidiscabies (Streptomyces swollen), S. reticuliscabiei (Streptomyces reticulus), S. botropensis (Streptomyces bobili), and S. anulatus (Streptomyces anulatus); and further comprises S. turgidiscabies (Streptomyces swollen).

[0071] In some embodiments, the disease comprises scab.

[0072] In some embodiments, the plant comprises at least one of potato, radish, beet, turnip, and peanut; and further comprises potato.

[0073] In some embodiments, the plant disease is potato scab.

[0074] The eighth aspect of the present invention provides a method for preventing and controlling pathogenic Streptomyces infection and / or plant diseases caused by pathogenic Streptomyces, comprising the step of using the preparation of the fourth aspect of the present invention.

[0075] In some embodiments, the method comprises the step of applying the formulation of the fourth aspect of the present invention to the plant or its cultivation medium by seed treatment, foliar application, stem application, or root application.

[0076] In some embodiments, the formulation is applied to the plant or its cultivation medium by drenching, dripping, pouring, spraying, root irrigation, or dusting.

[0077] In some embodiments, the plant comprises at least one of potato, radish, beet, turnip, and peanut; and further comprises potato.

[0078] The ninth aspect of the present invention provides a method for killing pathogenic Streptomyces in an environment, comprising the step of using the preparation of the fourth aspect of the present invention.

[0079] In some embodiments, the method comprises the step of administering the formulation of the fourth aspect of the invention to the environment.

[0080] In some embodiments, the formulation is applied to the environment by drenching, dripping, pouring, spraying, or dusting.

[0081] In some embodiments, the environment is a plantation environment, further comprising plant surfaces, planting soil, farm air, surface water, and agricultural tools within the plantation; and further comprising planting soil.

[0082] The beneficial effects of the present invention are:

[0083] The invention provides a Streptomyces phage St01, which is isolated from a soil environment and has good propagation performance, the advantages of a short outbreak period, good environmental stability, good genetic stability, good safety, and can specifically infect and lyse pathogenic Streptomyces without killing other bacteria and causing no additional impact on normal flora. As a bacterial virus, the phage is not affected by bacterial drug resistance and does not cause problems such as drug residues during application, and has the characteristics of high safety and environmental friendliness. The phage can efficiently kill pathogenic Streptomyces in plants and in soil environments, and has good application prospects in killing pathogenic Streptomyces in the environment, preventing and controlling pathogenic Streptomyces infection, and preventing and controlling plant diseases caused by pathogenic Streptomyces. At the same time, the phage can also be used to detect pathogenic Streptomyces and / or identify plant diseases caused by pathogenic Streptomyces. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Plaques of Streptomyces phage St01 are shown.

[0085] Figure 2 The electrophoresis results of the gene of Streptomyces phage St01 and the gene after enzyme treatment are shown.

[0086] Figure 3 The morphology of Streptomyces phage St01 under transmission electron microscopy is shown (scale bar: 100 nm).

[0087] Figure 4 A one-step growth curve of Streptomyces phage St01 is shown.

[0088] Figure 5The pH stability of Streptomyces phage St01 is shown.

[0089] Figure 6 The temperature stability of Streptomyces phage St01 is shown.

[0090] Figure 7 The UV stability of Streptomyces phage St01 is shown.

[0091] Figure 8 Shown is the in vitro lysis curve of Streptomyces phage St01.

[0092] Fig. 9 The in vivo safety of Streptomyces phage St01 and its preservation effect on potato slices are shown.

[0093] Fig.10 The in vivo therapeutic effect of Streptomyces phage St01 is shown.

[0094] Fig.11 The soil disinfection effect of Streptomyces phage St01 is shown. DETAILED DESCRIPTION

[0095] definition

[0096] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0097] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0098] The present invention is further described in detail below through specific examples.

[0099] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0100] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials and reagents used in the examples are commercially available unless otherwise specified. The manufacturers of the reagents used are indicated, and similar products from other manufacturers are substitutes.

[0101] Example 1: Isolation and purification of Streptomyces phage St01

[0102] 1. Experimental materials:

[0103] Diseased potato and diseased soil samples were collected from potato plantations in Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province. The phage host bacteria was Streptomyces turgidiscabies, ATCC700248 (Tanaka F, et al. A causal pathogen of scab on potato. Ann. Phytopathol. Soc. Jpn. 61: 253-254, 1995. The host bacteria mentioned in the following examples are all ATCC700248).

[0104] 2. Experimental methods:

[0105] Add 10mL of sterile water to the plantation soil sample, mix well, centrifuge at 5000rpm for 20min, and take the supernatant and filter it with 0.45μm and 0.22μm filters respectively for later use. Take 200μL of the above filtrate and mix it with 200μL of host bacterial solution, add 10mL of melted semi-solid NB culture medium, mix well and pour it on the pre-prepared NB solid culture medium plate to make a double-layer plate. The culture plate is inverted and cultured at 28℃ for 36h, and the resulting plaques are the phages that can lyse ATCC700248. Pick the plaques and place them in 20mL NB liquid culture medium and add 100μL of host bacterial solution, culture at 28℃ for 24h, centrifuge and filter the culture solution again, and use the double-layer plate method to pick a single plaque to purify the phage. The purification of the phage needs to be repeated 3 times. After the purification is completed, the titer of the obtained filtrate (i.e., phage stock solution) is calculated, and the phage stock solution is stored at 4℃ for later use. The purified phage St01 stock solution can form clear and regular plaques on solid culture medium, with no halo around them. After 36 hours of culture, the diameter is 2-3 mm ( Figure 1 ).

[0106] Example 2: Massive proliferation of Streptomyces phage St01

[0107] 3 mL of the phage St01 stock solution obtained in Example 1 and 3 mL of the host bacterial solution were added to 200 mL of NB liquid culture medium, cultured at 28°C with shaking for 24 h, centrifuged at 12000 rpm for 30 min, the supernatant was taken, and a large amount of phage proliferation liquid was obtained by suction filtration with a 0.22 μm filter membrane. The phage titer was detected by the double-layer plate method (using the logarithmic phase host bacterial solution).

[0108] Example 3: Genome extraction and sequencing of Streptomyces phage St01

[0109] Use a 30KD ultrafiltration centrifuge tube to concentrate the phage proliferation liquid prepared in Example 2, centrifuge at 8000rpm, 4℃, for 20min, and aspirate the culture liquid on the filter membrane to obtain the concentrated phage pure culture liquid. Use a viral nucleic acid extraction kit to extract the concentrated phage genome. The extracted phage genome was subjected to agarose gel electrophoresis, and it was found that its genome had a band, which is a characteristic of DNA phage. The extracted genome was treated with DNase I enzyme and RNase A enzyme respectively, and a control group without digestive enzyme treatment was set up. 6×DNA Loading buffer was added to the 1% agarose mixed gel for electrophoresis. The results were observed after 20 minutes. If no band was seen in the DNase I treatment group, and there were bands in the RNase A and control groups, the genomic nucleic acid type was DNA, otherwise it was RNA. The results are as follows: Figure 2

[0110] CGTATATTTTCAGCCCCCCTCCAACGTCCAGATGCAGTATCCCTGCATCGTCTACGCCCAGGACA

[0111] ACGCAAAGGTCGAGTTCGCGGGCAACAAGCCATACAGCTACGCCAAGCGATACCAGGTCACGGC

[0112] GATATCCAGGAGTCCGGATTGGGACGTCCCGGACAAGATCGCTCTGCTGCAGTTGTCCAACCTG

[0113] AATCGCATATTCACGGCGGACAACCTCCATCACTACGTCTACAACCTGTACTTCTGAGAGGGAGT

[0114] CTCCAAGCATGACCCAGCTCACCTGGGACAAGAGTGGCGAGCGGTACTACGAGACCGGCGTCG

[0115] ACCACGGTGTCCTCTACATCCCCAACTCGCAGGGCGACTACACCAGCGGCTACGCCTGGAACGG

[0116] TCTCACCACCGTCACCGAGTCGCCGTCGGGTGCCGAGTCCAACCCGCAGTACGCCGACAACATC

[0117] AAGTACCTGAACCTCATCTCGGCCGAGGAGTTCGGCGGCACGATCGAGGCCTTCACCTACCCCG

[0118] ACGAGTTCGGTCAGTGCGACGGCACCGCCGCGCCCACGCCGGGTGTCCTCGTCGGTCAGCAGT

[0119] CCCGCAAGACCTTCGGTCTGTCCTACCGCACCAAGCTCGGCAACGACCTGGACGGCCAGGACCA

[0120] CGGTTACAAGCTGCACCTCGTCTACGGCGCCCTCGCGGCCCCGTCGGAGAAGGCGTACGCCAC

[0121] (SEQ ID NO:1)、CGTCAACGACTCGCCCGAGGCGATCACGTTCTCGTGGGAGTTCACCACCACT

[0122] CCGGTCGAGGTCGGCCAGATCGCCGGTGTCGACTACAAGCCGACGGCCACCCTGACGATCGAC

[0123] TCCACCAAGGTGGACGCCGGTGCGCTCGACACCCTGGAGGAGTTCCTCTACGGCACCGTCGGT

[0124] ACCGACCCGTCCCTTCCCGATCCCGCCACGGTCATCGCCATGTTCACCGGTACCGTGCTCACGG

[0125] CGACCCCGACCGAACCGACCTACGACTCGGCAACCGACACCATCACCATCCCGTCTATCACCGG

[0126] CGTCGAGTACTACATCGACGGAGAGCTCAAGCCCGCCGGTCCCGTAGTGATCTCCTCTGACACC

[0127] ATCGTCACTGCGCGCCCCGCCAAGGGTTACAAGTTCCCGTCCAACGTCGACTCGGACTGGCTCA

[0128] TCACCTACGCGTAACCCAACCATTCGACAGAAAGAGGTTAGAGAGTGCTCGTCATCGAAGTCGTT

[0129] CTCGAAGAGGGCTTCGACGAAGAGAAGCAAAAGTTTGTTGTTGCTAAGTCTTGCGTGCTCGAATT

[0130] GGAGCACTCTCTTGTCTCCCTGTCAAAATGGGAGTCAATCTTCGAGAAGCCGTTCCTGAGCGAAA

[0131] CTGAGAAATCTCCGGAGGAAGTGTTCGAGTACATCCAGATGATGGTTGTAACACCGAATACTCCT

[0132] CCGGAGGTTTTCTACAAACTCACTACGGAAAACGTCGAAGAAATTCGAAAGTACATCGATGCGAA

[0133] GATGACCGCAACGACGTTTAACGATCAAGGGGTCCCTAAGCGAAATCGGGAAGTGATCACGGCG

[0134] GAAATCATTTATCACTGGATGATAGCCGCAGGCGTTCCTTTCGAATGTCAGAACTGGCATCTGAAT

[0135] CGATTGTTGACGCTTATTCGAGTGATCAACCTCAAGAATGCTCCTCCGAAGAAGATGGGCAGGCG

[0136] AGAGATGCTTTCCCAACGTCAAAAGTTGAATGCCCAACGAAGGCAACAATACGGAACCAGTGGTT

[0137] GAGAGGAGGACTATAAATGCCCAGGCTAAACTGGGGTACTTCTGGATCGCGCATCTACGAGACG

[0138] GGTATCGATCGAGGCGTTCTTTATGTTGGTGATCAGGCGGGGGTTGCTTGGACGGGTCTTTCGT

[0139] CCGTCGAGGTTTCACCCACTGGTGGGAACATCAAGTCGTATTACATCGATGGAGTGAAGTATCTC

[0140] CAAGTATCAAGCCTGGAGGAATTTTCGGCCACCATCAATGCCTTTACATATCCAGATGAATTCGGC

[0141] GTATGCGATGGTACCGCTCAAGTGCGTCCCGGATTGTTTTTCACTCAACAACGTCGAAAGACTTT

[0142] CGGATTCTCCTACCGAACTCTAGTCGGTTCCGACCTTACCAGCGACAAAGGCTATAAGATTCACA

[0143] TAGTCTACGGCGCCATGGCCGAACCGAGTCAGACGAGATACAATACGATCGGCGATACAGCGGA

[0144] ACCGGTCGAATTCAGTTGGACCGTTTCGACGAAACCCCCCAGTGTCAGTGGGCACAAGAGAACG

[0145] TCACACATCGTAATAGATTCTCGAACGGTTCATCATACGACGTTGTCCGTACTCGAAGATATTCTC

[0146] TATGGGAACGATGCAAATCTACCCAGGCTTCCGAGTATCGATGAAATCGTGACCATATTCGACAC

[0147] CGTTACGACGCTTGAAATAGTAGCTAATCCTGACGGGACGTACACCATAACCGGTCCGCCTAATG

[0148] CGATACAGGCATTGGATACGGATGCATTCCTGATAGACTGGCCGACTGTTTCGCAAATCAATGCG

[0149] GATACTTATTCCGTGAATTCCGGATAGAAAGGATAGTCCGGTGGCTAGTGTAACCGTAGTAACTG

[0150] CTCAAAAGACGCTCGAGATCGAAGCGGCATCTATCGTTGACGGTTCGGTAAACGAAGCAGGACA

[0151] TCTTATTCTCACGCGACACGACGGATCTGTGATTGATGCTGGTCCCGTCGTGGTATCGCCCTCCG

[0152] ACGCCAGCGCTACAGTCAAGGGTATCGTAGAACTCGCAACGGATATCGAAGCGGCTGATGGAGT

[0153] GGATACCACTAGGGCTCTGACTCCGGCTAACGTCAAGCCTTTGTTTGACGGTAAGCAGAACGCAA

[0154] ATGCGGATCTTTCTGGTATCGCAGGTCTCGCACCGTCGAACGACGATATTCTCCAGAGAAAGTCT

[0155] GGGGTATGGGCGAATCGAACAATTTCGCAATTCAAGAACGATCTCGCTCTGACTAAGTCTGATGT

[0156] TGGTCTTTCCAACGTCGACAACACGAGTGATGTCAACAAGCCGGTGAGTACAGCTACCCAAACTG

[0157] CTCTGAATCTCCTTCTGGCGAAGACCGGTGGAACCATGACCGGTACCATCGATGCAAATCTTTCA

[0158] CTGGCAACTACGCCTTTACTGGCCGCTTTGATCACTGGTGACACGTTCGACCGCATTCGAGTTTA

[0159] TCCTGATGGATCGATCGAATTCGGTAGTGGTGTTGCGTCCCGTGACATGAAGCTTTCGAGAAGCG

[0160] CCGCGAATCAATTGACCCTCACGACCGGTGATTTCCGGATCGGAACCGTCGGTCGAGGTTTGAA

[0161] GGTAGCCGAAGGTTCCAACGCCAAGATGGGACGGGCAACGCTTGTTGGCGGTACCGTTACCGTA

[0162] CCGAACACTTCGGTGACCGCTTCGAGCGAGATATTTGCTTTCCGACAAATCCCCGGTGGTACACC

[0163] AGGTGATCTCGGAGTCAGCTCTCGCATTGCCGGAACTTCTTTCACCATCGCATCGACAAGTGCAT

[0164] CAGACACCAGTACATTCGCATATCTGATCGTGGAACCCGCCTAACAAAGAGGAGGCAGTGTGATT

[0165] TCGTTCGTAACTACTCGTTCCGGCAAGCGAACGGAAGACTCCCTCCGAAAACTCAGCAGGGGCG

[0166] ATATCTACGGTTCCTTGGTCTCAGCCGCCCAAACAGGTGTCTCTGCTTTGGCTTCAGCCACTCCG

[0167] GTTGAGACGGGTTTGACCAAGGACTCATGGGGCTACGATATTCAGCGCTCCGGAAAGTCCGTCA

[0168] CGATCACCTGGACCAACAACGACGTAGAGGACGGTTTCCCGGTTGCGATCATGCTTCAGTACGG

[0169] GCATGGCACCGGCACCGGCGGTTACGTCCAGGGACAGGACTACATAAACCCCGCGATGAAGCC

[0170] GATATTTGACCGTATCGCAGATCAAGTATGGAAGGCGGTGACATCCGCATGAGCACCATCGACGA

[0171] GCGCGTCGTTCAGATGAAGTTCCAGAATGCCGAGTTCCAGCAGGGCGTTCAGCAGACGATCCGC

[0172] TCCCTCGAAGCCCTCAACAAGAGCCTCCAGCTCCAGGGGGCACAGAAGGGTCTCGCGGGGGTC

[0173] GCGCAAGTCAGCCAGGGCTTCAACCAGAGCATGGCGACCAACCGAGACGCCCTGGGTAGGTTC

[0174] ACCAAGGGTGTCTCCGAAGTCACCGAAAATACCCAGCAGTTCGGTCAGAAGATCGAGGCTAACC

[0175] GAGGGTTCCTCGAGCGATTTTCTACCGGGGTAACGACCGTAGCCACTGTGGCTCAGACCTTCGG

[0176] TCACAAGGTCGCCGAGGGCGGTCAGAAGGTCGGCGCCTTCTACGAGAAGATGAAGCAGGGAAC

[0177] TGCTGAAGCCGACAAGCAGAAGAGCTCGCTCAAGAACATCGAAGCCGGTGTTCAGAGCCTGGCC

[0178] GGTAAGTTCACCGCTCTCGGCCAGATAGCCACAGGCGCCTTGCACAACATCGGTGCTCGAGCTG

[0179] CCGAAGCAGGTCAGAGGCTGGTCAACTCGTTCACATTCGGACCGCTCATGGATGGTTTCCGTGA

[0180] GTACGAGACGAACATGAACTCGATCCAGACCATTCTGGCCAACACTCAGGCTGCCGGTACGACT

[0181] CTCAAAGACGTCACCGGAGCCTTGGACGAGCTCAACCACTATTCCGACCAGACCATCTACAACTT

[0182] CTCCGAGATGGCGAAGAATATCGGCACCTTCACGGCTGCTGGTGTTGGACTGAAGGAATCGACG

[0183] GCAGCGATCAAGGGTATCGCCAACCTGGCGGCACTTTCCGGCTCCAACTCGGAGCAGGCGTCC

[0184] GGCGCCATGTACCAGCTTTCGCAGGCCATCTCTTCGGGGACGGTCTCGCTCGAGGACTGGAACT

[0185] CGGTTGTCAACGCTGGTATGGGTGGTACGGTCTTCCAGCGTGCACTTGCCCAGACAGCCGTCAA

[0186] AATGGGAACACTTTCTGAGGGAGCGGTCAAACTCTCCGGGAAGATGAAGAACGTCACGATCGAA

[0187] GGCGAGTCTTTCCGAAACTCCATCTCCGCAGAGAACGGAGAATCGTGGCTCACGTCCAAGGTTC

[0188] TGACGGAAACCCTGGCCCAGTTCACGGGTGACCTTACCGATGCCGAACTCGCTGCGCAGGGGTT

[0189] CAGCAAGGCCCAGATCAAGGCCATCCAGGACCAGGCCAAGATGGCGAAGAGTGCCGCCACCGA

[0190] GGTCAAGACCGCAACACAGCTCTTCGGAACGTTCAAGGAACAGCTCGGTTCCGGTTGGGCTCAA

[0191] ACCTGGCAGATCATTTTCGGTGACTTTGCCGAGGCTAAGGGTCTGTTCACCGGCATCAGCAATTC

[0192] CATCGGCGGGGTACTTCAAAAGTCCTCCGACGCTCGAAACAAGATGCTCAAGGAGTGGGACAAG

[0193] TTCGGCGGGCGTACTGCGCTCATCGATGGTATCACCAACTCCGTAAAGGCCTTGGCGAGTGTCT

[0194] TCACTCCGATCAAGGACGCTTTCCGACAGATATTTCCGCCCACCACCGGCAAGCAACTTGCCGAT

[0195] ATGACCAAGAACTTCCGGGACTTCACTGAGAAGCTCAAGATCGGTAGTGATACAGCAGAGAAGCT

[0196] GAAGAGAACCTTTGCTGGCGTGTTCGCGGTCTTCGGGATCGTGTTTGACGTCGTAAAGGGCGTT

[0197] GCTGGTGTTATATTCGATTTGGTCGGGCAGGTCACCAAGGGCTCCGGCGGTTTCCTAAACTTCAC

[0198] CGCTAAGATCGGTGATTTCCTCGTTGCTTTGCGAAGGGGTATTCAGCAAGGCAAGGGTCTCGAGA

[0199] ATTTCTTCAAGGGTCTAGGAGCAGTGCTCAAGATTCCGATTCAGCTTATTCAGAAGCTGACTGGTT

[0200] TCCTCGGGTCACTGTTCAAGGACACTGATTCCAAGGGCGTCGAGAAGTCTGTCGAAGGCATATCC

[0201] TCGAAGCTTGAACCTCTGGGACGACTTGGTGACGTAGCCTCGAAGGCCTGGGGGAAGACCCTCA

[0202] CGGTCATGAAGAACGTAGGTAATTTCTTCAAGGATCTCGGAAGGCGCATATCTGAGGTCTTCGAG

[0203] AACATCGGCGTTGATGCCGGTACTCTGTTCGATGGTTTCAACTTCGATAACGTGCTGGCAGGACT

[0204] CAACACCGGTTTGCTCGCCGGACTCTTCTTGATCATCAAGAATTTCCTCGGCGGATTCGGCGGAG

[0205] GCGGTATCGGGGGTGTACTCGAAACCATATCTGACGGAATTGAAGACATGACTGGTGTCTTCGGA

[0206] GACATGCAGAACACTCTGAAGGCGGCAACACTGCTTCAGATCGCTTTGGCGATCGGCATCCTCG

[0207] CGGTTTCGATGAACGTCCTTTCTAAGATCGACAAAGAAGGTCTCGTAAAGGCCAGTTCCGCCATG

[0208] ACCGTCATGTTCGGTCAGCTTCTCGGTTCCATGGCCCTGTTCACGAAGTTCATCGGTACGGCTGG

[0209] TTTCGCCAAGATGCCGTTTGTGATGGGATCTTTGATCTTGCTTGCTGCGGCTGTTCTTATTCTGGT

[0210] TCAGGCAGTGAAACAGCTTTCAGAACTGGATTGGAACGAGCTGGCCAAGGGACTTACGGGCCTT

[0211] GCCGTGGTTCTAGGATTGGTAGTCGGTTCGCTCAAACTCATGCCCAATCCAAAGGGACTGATCTC

[0212] TACCGGTCTTGGGATGATTGTTCTTGCTGCGGCAATCAAGATCCTGGCGAGTGCGGTCGAAGATC

[0213] TCTCAGGTCTTGGCTGGAACGAACTTGCCAAGGGTCTAGTCGGGGTCGGAGTCCTTCTGGGGTC

[0214] GCTCACCCTGTTCACCATGTTCGCCAAGGCTAACAAGGGTGGTTTGGCGCAAGGTGCTGGGATC

[0215] ATCCTACTGGCGGCAGGAATCAAGATCCTCGCCAGTGCTGTCGGGGACTTCTCGAAGATGTCTT

[0216] GGAACGAGATCGGTAAGGGCATGGCTGCTCTTGCTGGCGGCCTTGTTCTGATCACGGGAGCTCT

[0217] CAAGCTCATCCCGCCAACGGCGCCTCTAGCAGGCGCTGGCATCCTCCTTGTGGCTTTGTCTATG

[0218] GGGATGGTGGCCGACGCTCTCGCAGACATGGCTAAGATGTCTTGGGGTGAAATCGGCAAGAGTC

[0219] TGACGGTCATGCTGGGTGCTCTAGGTTTGATGGCGGCTGCTTTGTACGTCATTCCTCCGACGGCT

[0220] CCTCTCGGTGCGGCTGCGATGTTGATCACGGCGCTTGCTCTTCAGCAAGTTACCGGCGTTCTCG

[0221] TCAAAATGTCAGAGTTCTCTTGGGAAGAGATCGGCAAGTCCATGACCATGTTGGCAGGTACTCTC

[0222] GGCTTGATCGCCGGGGCCCTGTTCCTCATGACCGGGGCACTTCCCGGAGCAGCAGCTTTGATCA

[0223] TCGTTGCTGCGTCGTTGCGGATACTCCAACCGGTTCTCGTCGCTTTCAGCGAAATGACGTGGGAA

[0224] GAGATCGGCAAGGGACTTACCATGTTGGCCGGGGCTCTAGTGGTCATCG(SEQ ID NO:2)、GTT

[0225] TGGCAGGTCTTATTCTGGCTCCTGCAGTACCGGCCATCATAGGTCTGGGGATTGCCGTCGCTTTG

[0226] CTTGGCGTAGGTATGCTGGCTGCTGGTGCCGGTGTTCTGTTGTTCGCCACTGCTCTAACGGCGC

[0227] TGGCTGCGTCTGGTGGTGCAGCAACAGCGATGATTGTCGGGATTGTTGCCGGTCTTATCGGTCT

[0228] CATTCCGGAAGTCATGAAGCAGATCGGTTTGGGTCTCGTCGCCTTCGCCGAAGTAATCGCCACC

[0229] GCAGGTCCCGCAATTACTGCAGCTTTGGTGACGGTACTCGAGGCACTGATCAAGGCGATCGTCA

[0230] GGGTAACACCGAAGATCGTGGATGCTCTGCTTCGAATGCTGTCTATGATGCTGAGCAAGATGCTG

[0231] CAGTATATTCCTAAGATGGTGGACACTGGTCTGAAGTTGTTGGTGGGTATTCTCACGGGCATAGC

[0232] CAACAACATCGGTAAGGTAATCGATACCGCGACTAAGGTCATCATTAACTTCGTCAGCGGTATATC

[0233] CCGTAACTTGCCGAAGATCATCGATTCCGGCGTGAAGCTGATCATCAGCTTCATCAACGGTGTAT

[0234] CGAAGGCTATCGATTCCCATGCAGAGGAATTGGGCCGTGCCGGTGTTCGACTGGCCACCGCCAT

[0235] CATCAGGGGTATGGCCAAAGGTATCATGGGTGGTCTCAGCGAGATATCTTCGGCAGCGAAGAGC

[0236] GTAGCTAAATCAGCGCTCGACAGTGCCAAGAACTTCCTTGGTATCCACTCACCCTCGAAGGAATT

[0237] CGAGAAGATCGGTAACTACGTCAACGATGGTTTCAGAAAGGGACTGGACGGAAACAAGGACCAG

[0238] ATTTATAAGGCCTTCGACAGTCTGAAGAAGATGCTTTCGGATCTCTCCAAGAGTTCTAAGGCTTCT

[0239] GCTTCGGAACGTAAGAAGGCTGCGACAGCATATTCCACCCTGACGAAGTCACTCAACGACGAGA

[0240] AGTCGACGTTGGGTAAGCTCGCCGACAAGTACGACGTACTCACCGAGAAGATCAAGAAGGCGGA

[0241] CGAAGCATATCAAGCCGCCATCAAGACTCGGGACGATTACCGGAAGCAGATCACCGACAAGTAC

[0242] TCCGATGTCGCAAGCCCCACGGCAGAAACGACATATTCGAGTTACGTGGAGGAACTTAAGAAGC

[0243] AGATCGAGGACACCAAGTTGTTCTCGAACGCCCTTCAGAAGTTGCGAGGGTTCGGACTCAACGA

[0244] CGAACTCTACAAGGATTTGCTGGAGCAGGGGCCGAGTGCTCTTCCGTTCGTCACCGAGCTTCTC

[0245] GACAAGGGTGCTCAGGGCGTCAACGAGGTCAACAAGCTCGGCAAGGATCTCGATGCAGCAGGC

[0246] GCTCATATTGGCAAGATGGGCTCTGACGCTCTCTATCAGGCGGGCGTCGATTCTGCCAAGGGGC

[0247] TGTTGAACGGTCTCAAGTCGCAGCAGAAGTCCCTCGAGAAGATGATGGATATCATCGCTTCGGCC

[0248] ATGATCAGAGCCATCAAGGCCAAGCTCAAGATCAAGTCTCCGTCGCGCGTGTTCATGGAGATGG

[0249] GCGGATATTCCGCGCAGGGACTCATCAAGGGTCTGGACGAAATGTCCGGTTCTGTCGAACGATC

[0250] TGCCGCACGTACTGGAACGGCTGCCGTAGAGTCCCTTCGGAAGTCTCTCGTTGGTTTCTCCGATC

[0251] TGATAACGAACGACGGCGACATGCAGCCTGTCATCACTCCGGTACTGGACTTGTCTAGTGTTCGT

[0252] AGGGACGCAGCCGGAATCAGTGGGATGTTTGATTCGTCTTACATTCCGACTGATTCCGCATACGC

[0253] CAAGGCGAAGTATGTCGCTTCTGGTTTCGCGTCCAACCAGGCTGCAATGGATGATATTCCGGCTG

[0254] GGAAGTCGGTTTCGTATGTCCAGAACAACTACTCGCCCAAGGCATTGTCCTCGGCCGAGATCTAT

[0255] CGCAACACCAAGAACCAGTTGTCTACAGTGAAGGGAGCCCTGGCTACCAGTGCTAACACGGGTG

[0256] GAAGTCCGTAACATTCAGGGCGATCTTTTCAAGCTGGTCCTCGAGGACCCCTCTTCGGGGTTCAT

[0257] TGTTGCGGATATTGACGGGCTGGGTCCAGTGAAGGCCACGTTGGTGTCTTCGAGTTTTGCGGGT

[0258] ATGGACGGAGAGCAGTATCAGTCCAGTCGTCGAGAAGCTCGCAACATCAAACTCAAGCTTGAACT

[0259] GGACCCAGACCCGTCTACCGACACGGTATGGAGTCTTAGAGACAAGCTCTACGATTTCTTCATGC

[0260] CGAAGTTGCAAATCACCCTGCAGTTCTTCCGCGAGGATGGGCTCGTAGTCGAGATTCCGGGCGT

[0261] CGTAGAGACGTGCGAGCCGGATCATTTCGCTCAGGAGCCGACGGTGGATATTTCCATCATGTGC

[0262] TTCAAGCCGGACTTCTACGAGCTCACCTCGCGGACGATCGACACTCTACTCACCACTGACACCGA

[0263] CTTCACAATCTTCGATTACGAGGGTACAGCGGAAACCGGTGTCATATTCCAGTTGACTGTCGACC

[0264] GGGCGATGGACGAGTTTACGATCTATCACCAGATCCCTAGCGGGGAGATCCAAACACTAACCTAC

[0265] GACAACGCACCTTTGGTCGCCGGGGATATTCTGACCATCAGCACGGTTACCGGTGACAAGAGGG

[0266] CGACTTTGAATCGTGGTGGAACTGTCAGTTCGGTTCTATACGGAATCTCGCCGCAATCCAAATGG

[0267] CTCGAGTTGCAGCGAGGGAGTAATGGAATCCGGATGTACGCCACCGGGGCGGCAATACCCGTC

[0268] ACGATGACCTACGTCACTCGATACGGAGGATTGTGATGGAGGCTTACACTCTCGATCCTCTCCTC

[0269] CGCCGTCAGGAAGTCATCGATCAGTTCGAATCGCTAATCTGGACTGAGAGATACCTAGCGTACGG

[0270] CGACTTCGAGATGGACATATTCTCCAACTCAAGGAGTCGAAAACTCTCAAACCGGAACCAAGC

[0271] TCGCCATGAACGATTCACATCGGATCATGACCGTGGAGACGGTGGAGGATTCTCTCGATTCCGA

[0272] GGGTCGGAAGATGTTGAAAGTCAAAGGTCGGTCCATCGAAAGTATCTCCGAAGACAGGGTGGCA

[0273] AAGGAATCCATCTCCGATCTGACCATCTCGCCGAAGTGGACTATCACTGGCACTCCGGCGAACGT

[0274] GGCTAGAAAGATCTTCCACGATATTGCGTCCTCGGATTCTTGATCCTATGGATGTAATCCCGTT

[0275] CGTCGTCGAGGGGACGTTCATGCCCGAAGACACGATCGTGGAACCGATCGACCCCATCACGGTG

[0276] GAGTTGGAACCCACCACAGTCTATAACGCGCTCACGGAAATTTGTGGGATCTGGAATCTCGGTTT

[0277] CAGGATGCTTCGGTACTACGACACTTCTCAACTTTATTTCGATGTGTACACGGGTAGCGACAGAA

[0278] CCACGTCTCAGACGACGTTGGCTCCGGTTGTTTTCACCACTGAACTTGATAATCTACAGAACATCA

[0279] AGAAGCTCACCACCATCGAGAAGGCCAAGAACGTAGCGTACGTATATTCTCCCGCTGGCTTCCAA

[0280] AAGGTGTACCCTGTGGGGGTCGACCCCGACGTCAACGGTTTCGAACGACGAATTCTGGTGGTTA

[0281] ATGCCACAGATATTACGTCCGACAATCCCGACGTCGAAGCAGCTCTACTACAGAGAGGTCGAGAA

[0282] GAACTGGCCAAAAACCAGTCAATCCAGAGCCTCGACGGAGAGATCAACCAGTTCAGTCAGTACAA

[0283] GTATGGTACGCATTACAACCTCGGCGACATTGTCGAGATGCGTAATGACGACGGCGAAACCAACA

[0284] ACATGCGGGTGACGGAACAGATCTTCGTGTCTGACCGAGAGGGTGAACGAACATATCCGACGCT

[0285] CACCGTCAACACCTTCATTACGACGGGTTCTTGGTTGTCCTGGATGAACAACAAGGTGTGGGTGG

[0286] ATCTCGCCGGTGACCCGACGACTTGGTCGGAACAGCCTTGATATTTGTAAGGGAGGTTGGGTGT

[0287] GGCTGAAGGAGATCAGGCAGAAGCCGCTGGTTTTTCGGTAGTTCCGGAAACCGGCGAAGAAGGA

[0288] AGAGTTCGTTGGGGAGCCCGTGAGATAAACCGAACCCGAGATTTCATCGCACAAGTCAAGGCTC

[0289] TGATTCCCGTCGGTAAGGCTGGGTTCAGAACCGCGAGTGGTATATCTTCCGGAACGGCCGACCC

[0290] CACCGGCGGTAACGATGGAGACATCTACTTCAAGATAATCAGCTAGGTGGTTGCGTGACCGATTA

[0291] CACGAAAACCACCGGAGTCAGTGGCAAGATGATGGTCCGGGATACCGGTACCGACGTCGAGTAC

[0292] TGGTTCAAGGCGGGATATTCGAGCGACTGGTGGAACGGTATGCCGTTCAACTGGACCGCCAACG

[0293] GATCAACCACCTATAAGACGATCAATTACCCGACCGGTGCCAATTGGTACAAAGTCGGGGAATCC

[0294] ACGATCACCAAGTCTCAGACCGTCACCTTTCGGTTGCTTGAAGGTTCTAGTTCTTCAGGCATCGG

[0295] TGGTCCGGCTACGTTCAACGTCACGATCGACAGGGCTACGGTTCCGGCCAAGCCCACTACTCCA

[0296] GTCATATCCAACATCACGGCGACGTCTGTTTACGCAACGTTTTCAGATGGTTCGAATGGCGGAGC

[0297] GTCGATCGACTCGAGGCAAATCGGGTACGGCACTAGTTCTACTTCAGTCCAGTCGTACGTAAGTT

[0298] CAGACCGTTCTACGATCATTTCGGGTCTTACTCCGGGAACTACGTACTACTTCTGGGCCAGGACA

[0299] CATAACTCCGAAGGATATTCGGCATGGTCTGGTAGGGCTAGCGCAACAACTCTCAAAGTACCGGA

[0300] TGCCCCAACAGCTCCTTTGCTCGCCGCAGTTCGAATGACCAGTGTAGATGTTGCTTTCAGTGCTA

[0301] ACAACAACGGCGGATCATCCATAACCGGGTATGAAGTCGGTTACAGCACCTCGCCGAGTGGTAA

[0302] CCCCACAACTTCCGTAGCAGCTACGTCACCAATGACTCTCACTGGTCTACAACCAGGAACTACGT

[0303] ACTATATTCGAACCAGGGCGAAAAACTCGGTTGGCTGGAGTTCGTGGTCTGGTTACAGCATTATA

[0304] AAAACCATCGCTGGAGCTTACGTTAAGGACGGTTCGGTTTGGAAGCTAGCCGTACCCTATGTTAA

[0305] CGGCTCGCTCGCAACGTCAACGCAGAGCAACTTGCACAACCGGCCCGAGCCTGGTAAGCTCGG

[0306] AACAGCAACACAGACTTCAGCACGACGCACCGCTCGGCGGTGTAGACGGTGACTCAGGGCAGA

[0307] GGCAAGCAAAGCGCGAGGGTCGGGTATCCGACGCAGTCCGAACGCGGAGCGAACCGATCTCCC

[0308] AAGCGAACGTCAGGGTACAGCGAATGCGAGTGCCCCTCTGAGGCGCAGGCCCGGAGTTGTGGG

[0309] ATGCCACACGATTCCGGGGTGTCGGAGGGGTACGGACAGGACCCAGGGCCCAACTACCCCCCA

[0310] GGGTGGTCCGGAAACGGAACGGACGGCCAAACGGCCGGACGGGAAACGGAAGGCGACTCAAC

[0311] GGGGGGTAGGCACTACCGGCCCTACGGGGCCGGGGGAGCGCCACTACGGGCTCCCGCAAAGC

[0312] CCCCTCGGGACCGCTCGCCAGTACGGGAGCGGGGCCCGTGCATCAAACGCAACAGGGGGTACA

[0313] GGGTCCCGCCCGGAAGGGCGGTTGACCGGCACCGCAAGGTGTGATACTGTTCCTACAGAACGA

[0314] ACGAGGGAGAGCGCGATGCGCTGCACGATCTGCTCCGAGGTTGCGACGCACTACTACCGGACC

[0315] GCCGGTGGCGAGAACGCCGGTCGATGCTGGGAGCACGCTGCACAGTTCGGGTACCCCGAGTAC

[0316] CTGATCGAGCTGGAGCCGCCGAAGGCGGCCCTGCGAGTCCCCAGCTACGTCATCCTCTTCAACG

[0317] AGGGCGACGGGAACGAGGACGAGGTCGCCGTGACCTGGCCCTTCGAGGCCGAGCTCGCCGAG

[0318] TGGTGGGTGCGCAACATCACGGCCTGCGAGTCGGTCGACTCCGACTGGCTGATCGCCGATGCG

[0319] GCGTACGACCCCTACCGCCCCCAGTACATGGAGCGGGGAGGCGTGACCGTCGAGGTGACCCGA

[0320] GTGACCTCGCTGCCACTGCTTCAGTGCAAGGTGTGATACTGTACCAACAGAACGAAGGCGAAAC

[0321] CGCCCGCAAGGGCGGTCGGCCCAGGTGGTTCCTGGGTCCTGACGAGCCAGCCTGCTCACTGAC

[0322] AAGGGAGACCTGTGATGGTGTACCTGGAGAAGCCCGCACTCGATCGCCGTCGGCAAGTTCCA

[0323] GGCCCGAGTGAACCGCGAGCTCCGCCGCAACAACCTCCGGGCCCTGAGTCTGGAGTTCGGCGA

[0324] GATGATCCACCGGTCCGAGGTGCGGGCCTTCGAGGCGCACAAGGCTGAGGTGCTGGGCGCATG

[0325] AGTGACGTGAGGTCTCGGTTCGCCGTCGAGGCGAGCGACGCGAAGGTGGGCCTGACGTACGGG

[0326] GAGCTGAAGAAGCTCCTGGCCACGATCGAGGACGTCTCAGACGTGGACGACGACGCCCCCATC

[0327] AAGGTGAAGGTGGGGTGGAGGTCGCAGATCACGAACATCGAGATCGGATGAAGGCGAAACCCC

[0328] TTCGGGGGTCCGGCCGGCTTGGGTAGCCGACCGCTGATGAGC(SEQ ID NO:3)、CAGCCTGCT

[0329] CAACGTCTGAGGAGACGACTGTGACCACCTACGTACACCCCGCATCCTGGTCCGAGTACACCGC

[0330] CGCCCTGGAGTGGGCGAGGTCGGGCAACGAGCGGATCGCTGGGGCGACCAGCGCACCGAGGG

[0331] AGATGCCTCGCGGTGCGAGGTACTACCTCACCAACGACTTCCAGTCCGGGTTCGGAGTGGCGG

[0332] GCGACGGCACGCTGATCGGCCTGTTCTCCACGGTCAAGGGCCGCGGTGAGGACTTGGTCTGGG

[0333] ACGCGGTGAACCACAAGGGGGCGTCCAAGCTGGACTGCTTCGACGGGTTCCTGCCCGAGTACTA

[0334] CAAGCAGTTCGGGTTCGTCGAGACCGAGCGGGTTGCGAACTGGACGCAGGGTGAGCCGGACGT

[0335] GGTGTTCATGGCGATCCACTGAGCGGTTGTGCAACTGGCACAAGCTGTGATACTGTCACTACATC

[0336] GAGCGAGGGAGACGGCAAGTGGACGGATACAGCATCACCCCGGAGTACACGGTCGAGGAGCTG

[0337] GCTGAGCTCCTGGCGGAGCTGGAGTTCACGGAGCGGCGCCGGGTGATGGAGCTGGCCGACAA

[0338] GATCGCGGCATCGCACTACGAGTGAAGGCGAAACCGGCTGACGCCGGTCTGGCCGGGAGGGGT

[0339] TCCCGGTACTGACGAGACACCCTGTGAAGGTGGTACGGAGATGACTCCGATCGAGAAGATCAAC

[0340] GAGTACAGCCCCGACAAGCTGGCCCGGATCGCTGACTGCACCGTGCCCGACACGAACGTCAAC

[0341] GAGGGCGCCGACTTCCTGGCCCGCACCCGAGACAGCGTGGTCAGCCAGGTCGAGCGGTACTGG

[0342] AGCGGTCGCCCGAGCGAGCTGGCGGATGTGGTGCGAGAGCACCGCGAGCGCATCCAGGACGA

[0343] GGCCAGCCTCGCCGCACCGGGCGCCTTCGCCGACACGATGTGGCGTGAGTACGTCGACCTGCG

[0344] CGCCTACCAGGAAGACCTCACCGAGTTCGGAACCCCGACCGACGACACGATGGAGGGCCGCGC

[0345] CCGGCTGGCGCTGGGCTCGATCGCCTTCCGCCTCGCCTCCGCCCTGCTCCGCCAGATCCAGGA

[0346] GGGCTGACCGATGGGACGCATGAAGGACATCGCCATCGACGTCATGAGCTACGAAGCGGGCGA

[0347] GCTCGACCCGAGGGAGACGCTGGAGCTGTTCAGCCTGCTCGTCAGAAGCGGCATGGCATGGAC

[0348] CCTCCAGGGTTCCTACGGACGCACGGCCAACGAGCTGATCCACGCGGGCTACCTCACCCAAGA

[0349] GGGCAGGGTCACCGAGTTCGCCGAGCTGATGCTGGAGGAGGTGGCGGCGTGAAGATGCCCCG

[0350] CCAGCTCAGCGCCCGAGTGGACGCGAGCATGGCCCGAGACATCCGCTGCCTGCGCCTGGCCGG

[0351] CCTGAGCTGGAGCCAGATGGTCAAGTGGGGGGTTGCCCTCCTCGCAGACGTCTACCGCCAGGC

[0352] GTACATCCACCGCCAGGCCCCCGCCTGCAAGACCCCGATCCTCAAGTCCTACATCTACGCACCG

[0353] TACGACCCCGACCACCAGGGCCCGCCCTGGATCACCGATGAGGAGAACACCGATGAAGACCGC

[0354] GGCGAAGTACGTCCTGACGTTCCTGGCCCTGGCCCTGCTGGGCTCCCTCACCTGGAACTCGCCG

[0355] GCCTCGGCCTCGGACACCAAGCCGGTGACGCTCCCGGCCAAGGTGAAGTACGTCCCCGTGTTC

[0356] CATCTGCCGACGCGGCCGTGCGCTGACGACTTCGACGACACGAACTGCTACTGGGACGGCGCC

[0357] GGACCGGCGTACTACGTCGACCGCGCTGGCAACGTGACGTACCTGAACCCGAAGCTGAACGCC

[0358] CCGGCCAAGCGGAAGGCGTGGGCGAAGCAGAACAAGGCGGCCGGGCGTGAGTACTGGGGCAC

[0359] GGTGTGGGGGCACCGGCTGTGCTACGCGAAGGTCGGCGACACCTCGTACATCTACTGCTTCGAC

[0360] GGGCATCGCGAGACGTCCTGATGTGCAAGTGACCCAAGGCGAAACCCCTTCGGGGGTCGGCGT

[0361] GGGATGGCTGCCCACGCCCTGATGAGCCAAGCCGCGAGACGGGAGACATCCAGTGAAGTGCAG

[0362] CGTGACCAACAGCAAGGGTGTGCAGTGCGACCGCGACGCGAAGGTCAGCGACCTGTGCCCCGG

[0363] CCACAACACCCGGCTGATCCGCAAGGGTGACGTGCTGGCGGACACCCCGCTCCGCTCGTACAC

[0364] CAAGGCGGTCAGCTTCGAGGCTCCGAAGTACGGGCACGGCGACACCGACGAGGAGAAGTTCTT

[0365] CTCCCTGGTGAACCGCACCGCCGACCACTGGCTGTGGGACGGGGGCATCACCAAGAGCACCGG

[0366] CCTCGGGATGTACTCGCTGGAGAACACGCCCAAGACTGCCGGGAGGATCGCCTGGGAGCTGGC

[0367] CTTCGGCCCGCTGCCCGAGGGCGTCAGGATCAAGCACGCCTGCGGCAACAAGCTGTGCGTCCG

[0368] ACTCTCCCACCTGACGGCCGTGCACCACGACGGCACCCCGTACGTGGAGTGGGGAGCCGACGA

[0369] GCTCGCCGAGCTGGAGATCGCCGCGTGAGCCGACAGAGCACCGGCTGGGAGTACATCCGGGGA

[0370] GTCCCGCGGTGGGCCCCCACCGTCGAGGGCGCCATCTCTGAGCTGGTCTACGACAAGTACGGC

[0371] CAGGAGTACGAGGAGTCGGTGACCAAGCTGATGGACATCGCCCGAGCCGCGCAGCGTGACTGC

[0372] GCCGACCGCCTGAGCGAGGCCGGGCACACTGAGGCCGCCGCCCTGATCTTCCCCACCTACCCC

[0373] GAGGAGAACTGAGCAGTGAAGATCGCCATCACCATCACGGTAGACGTCAAGGACCCGGCTGACT

[0374] GGACGCTCGCGTTCGGGGTGGAGGGTGCGGCGAAGATCCGGCAGGACGTCAAGGAGTATGTCG

[0375] GCAACGCCGCGCAGAACCTGCGTGTGTGGGAAGAGGTCGAGGCGGAGGTGAGCTGGAAGTGAC

[0376] CGACCTGATCGTGGGACTGAGCGGCTACGCGAGGTCCGGCAAGAACGAGGCAGCCAACGTGCT

[0377] GGTGACGAGGGGCTGGAGGCAGGCGGCCTTCGCCGACAAGCTCCGAGACTTCCTGTACCAGCT

[0378] CAACCCCCTGATCCCCGGCCACTACGGCGCCGGGAGCCTGCGACTGCGGACCCTGATCGACCG

[0379] GACCGGATGGGACTACGCCAAGGTGACGTACCCCGAGGTGCGTGCCCTGCTTCAGCGGACCGG

[0380] CACGGAGGCCGGCCGCCAGGTGCTCGGCCAGGACGTGTGGGTGGATGCCCTGCTCGACCAGC

[0381] ACCGTGACGCCCCTGCCCTGGTCGTGACGGACGTGCGATTCCCCAACGAGGCTGACGCCATCC

[0382] GCAAGCGGGACGGCATCCTGATCCGAGTGGACCGGCCCGGTGTCGGCCCGGCCAAGGACAAGT

[0383] ACGGCCGAGCCCACATCAGCGAGACCGCGCTCGACGACTACGACTTCGACCACTGGCTGAACAA

[0384] CGACGGCTCGGTGGTCGACCTGCACGAACGGCTGACCGGGGTTGCGAACCTGGTCGAAGCCAA

[0385] CTTGTACGCCTGAGTGTGATGGTGTACCTTGATACTCACGACGCCGAGAGAGCTCGACGAACTG

[0386] CCTGACGGCACGGTGATCGAGCTGCTGGACAAGCGAGGCACGGTCCGCTTCAAGCAGGACGGC

[0387] CACTGGTACGCCGGGGACAAGATCCTCACCCAGAACATCTACGTCTACGTCAACGCCCGACGCT

[0388] ACGGAGCGCGGGTCATCGCAGAGGAGACAGAGAAGTGACCACCGCCACTGACGCCCTGAAGAA

[0389] CATCGAGCTCGCCATCCGCCAGCAGGTCAGCAAGGAGGGGCACGAAGAGGTGAGCGAGGACCG

[0390] CATCACCCTGCTGGTCATCCGCCAGCTTGTCGCCGAGTACCAGCTCGGCGAGACCGAGCGTCAG

[0391] GGCTACCTCGACGAGCTGGTGGAGGAGGGCGAGTACGAGGGCACGTACCGGTACGACTCGCAG

[0392] CTCACCGAGAACGAGACGGCCGCAGCCGGCAGCCTGTCCGGCCTGCTCTCCACCCTCTCCGAG

[0393] CTGCTTCCCTGATCGTGCAAGTGTGCCGAAACCTCGCAGCCAGCGGGGTCCGCGTGGGATGGC

[0394] AGCCCACGCGCTGATGAGGCAGGCCAGACGAGAGGAGAACGGCAGCGTGAGGCGTCAGTGAG

[0395] GATCACCCCGAGGGCCCACGAACTGAAGAAGGTGGTCGACATCCTCGAAGACCCCACCTTCGAC

[0396] TCGCCGGAGCAGCTTGCCAAGGCGGTCATCAAGGAGGTGGGGGACATGCTCCAGATGCGAGAC

[0397] CTGTTCGCCCTCGTCCACACCTGGGACGGCGGTCACAAGGGTTTGAACTACGGGCCCTTCGGCT

[0398] CGACCGCAGAGGCTGAGAGCTGGGCCAAGAAGGTGGCGATCGGCGGCACCGGCAAGGTGGTC

[0399] CCGCTGACGTCATCGGGGATCATCCTCGCCAACACGGAGGGCAAGCCGGACGGCTGGCCTGGC

[0400] TACTGCTGGAACCCTGAGTGCGGGCACAGCCCGAGCAACCACGCCATCGACGGATCGAGTCGA

[0401] GGCAAGTGCCACATCACAACGTGCAACTGTTCCAAGTTCGTCAAGGACGACCCGTCCATCAAGG

[0402] CGAAGAAGAAGACAACCGCCCGCAAGGGCACCGCGAAGGCAGTCAACGAGCTGTAGGAGGCGC

[0403] AAGCAAGTGAGCTGTGACTGGAGGCCCTGCAAGTGCGGGGCGAAGCGAGACTTCATGAGCCAC

[0404] CGCAACGCGGAGAAGGCGCTCGGCAAGGCCCAGGCCAAGCGAGGCCGGCTGGCAACCGCGCG

[0405] AGGCACCATGCGCGGCCAGAAGGTGGAGCACAGGACGTACGTCTGTGAGTTCGGCGGCCACCA

[0406] CCTGACGAGCGAGTCCCGCTCGTCGTACGAGAACCGGATGAGCAAGGAGAGCGCGCAGTGGTG

[0407] AGTCGAGCTGGCGAACCGGAGCGGCCGAAGGGGTACACGGACTGCCTGATGTCGAGGTGCCCG

[0408] AGCCTGTACTACGAGAACGAGATCGGCGGTCAGGCGCGAGGGATCGTGGCGCTGATCGCGAAG

[0409] GCGGTCGGCGTGGAGATGAGCCCGCCCCTCGACGGGAACATCTGGAGCCCCCGCAACATGGCT

[0410] CGCGTGCTCGAAGGGGCTCAGCGAGTGGTCGACGAGAACATCTGGCTGCGACGGGAACTGAAC

[0411] CGCCGAGCCGGCACCCATACCAACACGGTCAAGGCCCTGCATGAGGCCCTGACTCACCTCGGG

[0412] GAGTGAGTGTGCAAGTGAACCAGGCTCCGATCGCCGTCGTCTACGGCTACCACCAGGCCCGACT

[0413] CTTCCCCGAGGTCGAGCGCGAGAACGTCATCCCGTTCCGCCTCCTGCACCTCTTGCGAGGTCGA

[0414] AGGCCCTCCGTCATCTACCGCACCGGGTTCGGCAAGTCCGCGCCGGCCTGGCGCATGCTCGAA

[0415] GCCCTCGAAGGGCTGGCCTGGGACACCGGCGCCGAGATCGTCCACATCAAGCAGCTCACCGAG

[0416] GAGGAACCCACCCCGTGACCGCCATCACCACCCGCCCTGACGTCACCGTCGAGCTCGTCAAGG

[0417] CCAGCGCCACTGACTCCGACGTCGCCACCGCGGCCCGAGTCTCCACCATCGGCGGCTCGCACG

[0418] AACGGGTCGTCGACCTGGAGAAGGACGCCGGCCTCATCAACTTCCTGATGCGGGACCGGCACG

[0419] GCAGCCCCTTCGAGCACACCTCCCTGACGTTCTACGTCGAGGCGCCGATCTTCGTGGCCCGCGA

[0420] ACACTTCCGTCACCGCGCCGGCTGGTCGTACAACGAGGAGAGCGGGCGCTACAAGCAGCTCGC

[0421] CCCCGTCTTCTACCTGCCGGGCAGTCAGCGCAACCTGGTGCAGGTCGGCAAGCCTGGCGCCTA

[0422] CACCTTCCAGCCCGGCACCGACGAGCAGCACGCCCGCATGGTCACCAACACGCTGACCGCGTA

[0423] CGAGGACGCCTTCGGCGCCTACGAGGAGATGCTTCAGGCGGGCATCGCCAAGGAGGTGGCCCG

[0424] CATGGTCCTGCCGGTGGGCACCTTCACCTCCTACTACGCCACCTGCAACGCCCGCAGCCTGATG

[0425] CACTTCCTCGGACTGCGGACCATCAGCAAGGCGGCCACGTTCCCCTCGTTCCCCCAGCGGGAGA

[0426] TCGAGATGGTCGCCGAGATGATGGAGGACCACTTCGCCGAGCAGATGCCGATGACGTACGGCG

[0427] CCTTCAACGCGAACGGGCGGGTGGCGCCGTGACCACGCGGCCCGAGTTCCTGATCCAGCGGTA

[0428] CCCGACGCTCATGAGCTCCGAGCGAGAGGTCCGGCTCCCGAAGTGGGCGCGGGCCAAGCTCGC

[0429] CGACCTCCGCCTCCTCCTGATGCAGGAGGCTGGCCGGTACGAGGACGCACTGAACGAGATCGA

[0430] CCGCCTGGAGAGCGGCCACCACAACTGACGACAGACGAAAGCCCCGGCATCCGAGAGTGGGTG

[0431] ACCGGGGCTTCGTCGTACCTGCTTACTCCTCGACGTCACGCCACTCGACGGTGACGTGGTCGTC

[0432] GACCGACATGCTCTTCATGTGCCGGCCCTTCGCAGGGTTCAGCGTGACCACCAGAGTGAAGTCC

[0433] ACCCACGCCCGCTTCGTCGGGAGCGGACTGTCAGGCCAACCCTTGATC(SEQ ID NO:4)、AGCC

[0434] CGGCGATGCCGGCCACCGGGTCGAACGGAGGGATGCCCGCCGACCCCACCAGCTCCGACTCG

[0435] ACGGCCTCGATCTTCTTCTGGAGTGCCGCCGAGCCTGCCGTCATCTGCGACAGGGTGATCGCAC

[0436] CCTCCGCGTAGGCCGTAGCCAGCCCGTCAAGGCGCTCACGGAGCGTCTGAGCCTCCGCGTGCA

[0437] GGGACGCAGCCGAAGAGCCCTCCTGCTCCTGCTGAGAGCCCAGGATCTGGCTCAGGAAGTCCG

[0438] GGAACATGAGCCGAGCCAGCGTCGCCGAGCTCGCACGGCCATCCGCAACACTCCGAGGCGTCC

[0439] GAGTGTGCGTCGAGCTGCATCCGTACACCAGAACGCCTCGGTAGCCGCGCCCGTTGACCGTCTC

[0440] GCCGCACTCGCCGCACACAGCGATGCCTGCGAGCAGAGTTCCAGGCATGCGACCCGTCCGCTT

[0441] ACCCCCCGAGAACCGGTCAGGGTTGTTCAGGATGGCGACGGCACCGTAGAAGGTCTCCTCGTC

[0442] GAGGATCGCGGGCCACTGACCCTCGCCCATGACCTCTCCCAGGTACGTGGACTGCCCGACGTA

[0443] GCGCGGGGAGGTGAGCACCTTCTTCACGCCTCGCAGGGTCCACGCCTTCCCGCCCGCGGCCAC

[0444] GCCGCGAGGGGACAGCAGTCCGGCCTCCGTCCACTTGCGCGCCACGGCAGACAGAGACTCGCC

[0445] CATCAGGATCATGGACGCGCCCTCACGGATGGCCTCGGCCTCCTTCTCGACCAGCGCGACGTGA

[0446] TCGTCCGCGTACCCGAAGGGCCGGATGCCGGCCGTGTACGGGCGTCCCTGCTGGGCTGCCTGC

[0447] TGGTTGGCCAGCTTCTGGCGCGCGGCCTTCTGCTCACCCTCGTACGTGGCCCACGCCGTCACCG

[0448] TTCGGGCGACAGCTCGACCGGCAGGAGTGGACAGGTCCAGGTGTCCGGCCTCGACCGCGTAGA

[0449] CGTTGATCCCGAGCTCGATCACGCGCTCCAGGTCCCGCGTCACACGGATGAGGCGGTCGGTGT

[0450] GCCAGCAGATGATCGCGCCGGGGCTGCTCTGGAGCAGGGCCTCGAAGTCGGGCCGGATGACGT

[0451] TCTTCTTCGTGGCGCTCAAATCGTTGTCGACCCAGACGTGCCGGACCTTGAGTCCGAGCTGCGC

[0452] GGCGAGTGCCCGGCAGCGCTTCTCCTGCCGCTGGACTCCGTGCTCGTCGCCCGTGAGATCCTG

[0453] GCTGATGCGAACGTAGATGTCGGCCTCTGCTTCAGGCCCTGAGCTGCCGTTCTTCATGCTTCGCA

[0454] TGGTACCCCCAAGGTGTACCGCTCGTTCGAGTGTTACCGCTTGAGGGTATCTTCCCAGCGGACTT

[0455] TCCGCAGCCTCTTTGGGTGACACGGACAACCGCATCTCACCTGGTAGTAGACGACTCCCTGCAC

[0456] CGACACGTCGAGGTTCCCCTCGCAGTACTGATGCAGGTCGACGTCATCCCGGCAGGCGGGCGC

[0457] CTTCTCTACCCCCGCCACGGCTTCAGCCCCCGGTCCTTCACGCAGCCAGCGCAGGCCCGCTGAA

[0458] CCCACTGCGGCCCCGACATGCGCTCGACCACGTTCACGATCGTCAGCTTCTCGGCGACAGCCAG

[0459] CTTCCAGCAGTAGTTACATGCTCGCTCCATGCGATCAGCGTAGCGCGCCGTGGCGGACCGTAGC

[0460] GACCCGTAGTGCACCATTCTCCGATCGCGGACCCATGGTGCACCGTCGTACGGTCCGGGCATGG

[0461] AGATCAGCATCGACCGGCGTCGTTCCGTGTGGCCCCAGGTGGCCACGATCATCCGTGAGCGGG

[0462] TGGAGAGCGGCACCTACGCGGCCGGCGAGGCGATCCCCTCCACCGTCGACCTCGCCGCCGAGT

[0463] TTGAGGTGTCCACCAGCACCATGCGCAAGGCCCTCGTCTCGCTGATCGAGGAGGGCGTGCTCTG

[0464] GACGGAGCCCAGCATGGGCACCTACGCCCGAGACGACAAAAAGCCCCCCACCCAGGACGGGTG

[0465] AGGGGCTTTCGGGTTGCTCGTCAGTGGCCGAGCTGCCGGTTGATCTTCGCCTGTTGCTTCATGA

[0466] GCTTCGTCTGCTTCTTCGCCTGCTTCTTCGTGGCCTTCGTGTAGGCCGCCGTGCGCTCCTTGTCG

[0467] GAGCGGAAGTCGACCAGGCCCAGCGTGGTCACGGACAGGATCTTGCGGGTCATGCCCACGGTT

[0468] GGTACCTCCCAGGTGGAGCGGTGGATGTAGTGCACCGTAGTGCACCGTGGTGCGGGGGCGCAC

[0469] AGGTAATCCTGCACGCCCCCACTGACAACGGTCAGCGCACGGGGCAGGCGCCGTTCGCGCAGT

[0470] TCTCGTCGAAGCTGTCGTCGACCGAGGTGATCTCGTACTCGGCGAACTGCTCGGCGGTCAGGCG

[0471] CTCGTACGGGGCCTGCGGGCGGGTGCCGTCAGGCATGAGGGTCGTACCCTTCAGCTCCGGCAG

[0472] GAAGGAAGCGATCACTCGCTCCGCCTCCTCCTGGGACAGGCTGCCCTCGGGGAAGTTCACCGT

[0473] GTACGAGACGGCGTTGTCGGCGTACTCGGTCTGGTACATGGCCTGGAAGGCGAGCATGTTGGTC

[0474] AGGCTCACCTCGTCGGCCGACTCGACGAGCTTCGGGTCCAGGCCCATGCGCTCGACCTCGTCG

[0475] ACGAGCTTGTCCTTCGTCGGGTAGGCCACGACCATCGTGTTGCCGGACTGGTCGTAGATGCACT

[0476] TCTCGACGAGCAGGCCCTGGGCCATGGCGTCCTCGACCGTCTTCACCTGGGCCGGGTCGACCA

[0477] TGGAGAAGCGGACTCGGCGGTTGAAGTAGCGGGCGTAGATGGTGTGGATGCCCTCGCTCACGC

[0478] CGGGGAGCTTCGCGATCGAGCCGGTCGGGGCGACGGTCGTCACCTTCACGGGCTCCGGCACG

[0479] CGACGCTCGAAGGCGTACTCCCGAGCCTCCTCGCGGACCGTGTCGTACAGGTCGTTGAGCAGG

[0480] TTGCGGAAGTCATACCCGTACGGGGCGTCGGAGTAGCGGATGCCCTGCTTGGCGAGGAAGCCC

[0481] TGCACTCCCAGGTGGCCGACGCCGATGCGTCGGTTCTGGGCGAGCTTGGCGGCCTGCTCGGCG

[0482] TCCGTCACGTCACCGAAGGTGGCTCGGATCAGGAAGCGGGTCATGAGCTGGTGTGCCCGGTGC

[0483] AGGCCCTTGCGGTCCACGCTCCCGCCCTTCACCTTCGGGGCGAAGTGGTCGAGGTTGACGTGC

[0484] CCGAGGTTGCAGTTCTCGGCCGGCTCCAGCGCGATCTCGCCGCACGGGTTGGTCGCGATGACC

[0485] GTGCCGACCTCGCCCTCGTTGGAGTAGCTGGAGTTCCAGTAGCCGGGCTCCCCGTTCAGGAGCA

[0486] TGGCCTCGACTGCCTGGCTGTGGACCGCGGCGGCCTCAGCGTGCCGTCCGTCCGTCACCTCGT

[0487] TGAGCGCCTGGATGAACCTGTTGTCGATCTCGACCGAGATGTTCGTGGTCCAGTGCTTCGAGCC

[0488] GTCCTTCTTGCAGTCGAGGAAGTCCTGGATGAACGGGTCGTCCCACTTGCAGATCGCCATGCGA

[0489] GCCGAGCGGCGGACGCCGCCCGAGACGACGCACTCCGCGATGGCGTGGTCGATCTCCATGGC

[0490] CTCGGTCGGCTTCAGGTGCTTGCCAGTGGCGGACGACAGGGTGCGGCCCACCTCTTCGAGCAT

[0491] CCGCGCGAACGGGCCAGGACCCGAAGCGGTGCCCCCGAACGTCTTCAGTCGGGAGCCCTTGCA

[0492] GCGCACGCGGCTCACGTCGTAGACGCGCTGCTTGTGCTTGACCTCGCCGTCGGTCATGAAGGTG

[0493] TCGATCAGGTCGACGAGGGCGTCGGCCCATCCCTCGCGGGAGTCCTCGACCTCGAAGGCCCCG

[0494] GCCCAGTCGGAGTCGTACTCGGTCGACAGGAGGCCCGCCGCCTTCATCTCCTCGTAGTCCTGGT

[0495] GCATCGGGTCGCACACGACGTGGACTTCGAGCTCGCGCCGCGGGGCGCCGTACGGCTGGAGG

[0496] TAGGTCGAGCTGTAGTTGCCGCCGACACCGCCGCCTTCCATCAGGCGCATGAACGTGAACTCGA

[0497] AGTGCCGGCTGAGCCGGTCTCCCCACGGCGCGACGTGGCAGTTGAACAGGTACTGGCGGCCCT

[0498] TGACTCCGGTCGCCCACAGGTGGCGGCCAGCCGGGATGACGGCGAACACGTCCATGAACTCGA

[0499] CGAGTTCGTCGTGCTCGTCCTTCGCGGCCTGCGGCCAACTCTCCATGTCGGGGCCGTGGACGA

[0500] GGGCGAGGTTGCCGCGAGCGACGCGGCGGACGGTGTCGGGCCAGGTCTCCTTCGAGCCGTCG

[0501] GCCAGCGTGCGGGAGTAGGTGCGGTTGTAGACGAGCTCGCCGGTCGGGCCGAAGGGAACGGT

[0502] GGTCACTGGTGAAGTCCTCCTTGAAGTCGTTGTCTCTCACTGCCTCAAGGCGGCACCCCCGAAG

[0503] AGGTACCGCCTGGGACACTTACACGGGTCGGCCGGTCAGCTCGCCGAGCACCAGGATCATGCG

[0504] CTTCAGGGTGCCGGCGCTGTACGCAGCCAGGTCGCACAGCACCTTGATCTGCGCGGCCGTCTC

[0505] CTCAGCCGTGGGCTCCGGGTTGTTGAGGAAGGCGAGGCTCGGCTTGATCCGCTCACTGAGGTA

[0506] GACGATCGCCGCGTCGGCCTCTCCTCGCAGGCCATCGAGCATCCGTCGCTTCTGAGCGCCAGC

[0507] CCGCTCGGCTTCCGTGAACGGGCGGGAGTAGATCTGCCCGTCACCGTCCGACCGCTCGTCACG

[0508] CCAGAAGTACAGCTCGCGGTCGTCGTCGTAGTAGTCGAAGTCGGTCGGGCTCATCCCTGCCGGG

[0509] GGAGCGATCGGGTCGGGGGCGGTCTCACTCACTCTGCTCATCCTCCAACTGGTCGTATCCGTCG

[0510] ATGTACTTGTCGCCGTTCAGCCAGGCCGTCACCTTGCCGACAGCCCGCTCGGTCGCCTCCTGGG

[0511] CGGACTGCTTGCGCACGCCCCGCGTGTCGGCGATCTCCTCGTAGCCGTAGTCGAAGCCGTACC

[0512] GCAGGACGAGCGACTGCCGCTCGACCAGGGTGAGTTCGGTCCGCTTCCATGCCTGCTTGATGTC

[0513] GGCGATGTGGGCGTACAGCGTGTTCGCCAGCTTCGGGTTCGCCTTCACCTTCGGCATGTCGGCG

[0514] TCCGGGGCGTTCTCCTGCTTGATCCCGTAGACCGCCTCGGCGTCCCAGACGGCCGGGAGGATG

[0515] TGCTCGACGAGAGCTCGGTTGTAGCCGCTCACACACCCTCACCGAGGGCTTCGAGGTTGGCCTC

[0516] GTAGGAGACCTGCTTGCGCCTGTGCGTGGCCTGCGTCTTGAAGAGGTCGCGCAGGATGCGGTA

[0517] GCCGCGGAAGTTCAGGACGCCTGCCGGGTTGTCGGCCTCGGCGATGACCTCGCGGACCATCCT

[0518] GGGCCTGAGCGCGAAGGCGATCAGGAGCTCCTGCTCCATGTCCTCACGCTCGACCACCCCCTC

[0519] GTAGTCCCGAGCGAAGGCGTAGGAGGCGCTACGCGCCCCGTCCGTGACCGGCTTGAGGTCCAG

[0520] GACCGACCAGTCGACCGTCGCCTGCTCAGCGGTCTCGTAGGCGTTCGTGATCTCGATGATGTTC

[0521] ACTGGCTGACTACCTCCTTCACGGGCACGCGGCCCTCTCTCGTTACGGCGACGACCAGGCCAGG

[0522] GGCTCCCGTGGTGCCCTTGGCGTGTCGCCACCAGGTGGACTCGGACTCCATGGCCGGGGGCTG

[0523] GAGGAAGGTCCGCCATCCGTCGGTGTCCAGGAATTCGTGATGCAGGTGGCCGGCCAGGAGCAG

[0524] GTCGACCAGGTGCATGGGCGACGCCTGGTTGAACGCCTGGCCCTTCCACCACTCGAAGTGCTTG

[0525] CCGGGGCGCCACTGGTGGCCGTGCGCGTGGCCCACGACGGTGCCGGAGCACTCGACGACGAC

[0526] GATCAGCTCGTCGGTGTCGGGTACGTAGAACTCGACATGGCCGAACCGGTCGGGGTTCAGGTCC

[0527] GCTGCGTCCTTGACGGCGATCAGGGACTCGGTGTCGTGGCTGTCGTCGTACCGCGTCACGCCCT

[0528] TGCCGTTGATCCGTACCGCCTCGCCGTGGTTGCCCGGCACCGCTGCCATCGTGAGCCGACTACA

[0529] CATCGGCGCGAAGACGAGGAGCGCATGGAGCATCACCCGGCGAGTGAGGCGGATCTGCTCGTT

[0530] CAGCGTGAGCTGTGTCCGCCAGGTGTTGGCCCCGCCCTGCGAGACGAAGCCTTCGATGTGGTC

[0531] ACCCAGCCATGCGATGTGGACGTGGGTGATGTTGAAGCGGAGCCGGTACTCGGCGAGCCGGTC

[0532] GGCTGCCGCGTTGATGCAGGCGATCGTCCGCTCCAGCGTGCCCTCGACGCTGTCGCCGTCGAT

[0533] CTTGCCGAACTGCATGTCGCCGAGCGCGACGATGAAGGTGTACTCCTCGCCGTCCCGGACGAAC

[0534] ATGGTCACCGGGTCGGGCGGGGTGGAGTCGATCGCGGCCAGCAGCTCGTCGATCGGGGGTCG

[0535] CCCACCTGGGACACTTGCACTCTTGCGGGCGAAGGTGAATCGGGTCGAGACCCCGGTGTCTCC

[0536] GTTCGCCATCGTCCACTCCGAGGAGCGGAGCCCGGTCACGGTCCACTCAGCAGGGTCCAGGCC

[0537] CTGGCCGCGGAGGACATCGGCTGCCGAGCCCTCGTTCTGCTCGAAGGTCTCACCCCGGACGGT

[0538] GACGTCGGCCTCGTCGCCCTTGATCTCGATCTGGCGGGTGAAGTCCTTGTCGGGGTCGGTCTGC

[0539] CTGGCCGGTACGGCAGG(SEQ ID NO:5)、GCCCACGGGCTTGGCCAGCAGCTCGTCAAGCAGC

[0540] TCGCTCACTGGAGCCTCCTTCCTGCCGAAGTGCTCGGCGGTACGTACGGATGGTGGTGGCGGA

[0541] CACGTCGTGATCCGCTCTTCGCAGGGCCGAGCTCAGCCAGTCGGCTGACGTCTCGCCCAACAG

[0542] GTGCGGCGCGAAGGCGACCTGCTCCGCCTCGGTCATCGCCGCGTAGATGTCTCGCAGGGTGGG

[0543] GCCCGGTTCACCGGGCAGTTCCGTGTACCTCACTCAGCCGGCCTCGTCACCGCGAGCTCGACG

[0544] ATCTCGACGTCTTCCGGCCAGTCCGTGTGCTCGGTTCCGAAGTAGCTGAAGTACCCAGGGGAGT

[0545] CGGAGTCGTACTGGCAGAACTTCGCCAGGACCACTTCGTGCGTTCCGTGCGGAACAACGTAGGC

[0546] AAGTCGGCCATGCTTCAGTGCGACAGCCACTCAGCCGAACACCCCCAACTCCGAGGCCCAGTAC

[0547] ATCTGGACCAGCTCGAACGTCTCTTCTCTGGTGAAGCCCTCGCCGCGGAGGGCGGACCGCAGG

[0548] TCGCCCACGATCGAGGCGGCCCGCTTCACGTTGCTGAAGTGGTCGACGACCTCGGGCTCGGCG

[0549] TCCTCGGTCCTGTCCCACTCCTCGCTGCTCACTTGCCGGCCTCGTCGATCTGCTCGATGCGACC

[0550] AAGGACGACGGCGGCGAGCTGAGAGAGGGCGTACCGCTCCTCGGCCGGGTTCAGCGCGGCCA

[0551] GCGCCTCGAAGGTGAGCGACAGGAACAGGCTGTCGAGCGCGCCGTCCTTGGCGTACTGCCGGC

[0552] CCCACGCTCGCGCTCGTCCGTCGTGGCTGCGGGCGCGGTGGTTGGGCGTCGGGTCCGTGGCG

[0553] TACTCGCCGAGGTGCTTCGCCGCCTCTCGGACGTCGTTCAGGATGCGGGTCATCGGGTCCGGCT

[0554] TGCGGGCGGTCCGCTTGGGCGCCTGCTTGGCCTCGGTCTTCTCGGTCTCTGCCACTGTCAGTCC

[0555] TCCTTCTTCTGAACCAGGGAGAGCAGGTACTCCGCCCCGTGTTCCATGTATGTGTCGGTCACGTC

[0556] GGCCTTCAGTCGCACCGCCTTCGCGGTACGCAGGGCCCGTGTGATCTTGCCGGTGAGCTCGGC

[0557] TCCCGCGTCGTCCGGATCAGCCCACGTCCACACGCGGTTGAAGCCGGCCAGCATCCTTCGATGC

[0558] CTGCCGAACCACATGTTGGCGCCGGGGATGGCGACCGCGTGAAGGCCGATCTTCCGCAGGATG

[0559] ATGCGGTCCAACTCGCCCTCGGTGACATGGATCTCGCCCTCGGGGCTGCGGGCCGCCTCATGG

[0560] ACGGACTCGATCCCGTACATGCGGGGGATGTCGTCCTTGATCGTGTTGTACTTGCCGTGGAAGT

[0561] AGTCGCGGTGGTTGTGCTCCTGAAGGCAGCGGAACCGGACAGTCAGCGGGTATCCGTCCTTGCT

[0562] CAGGTAGGGGATCGCGAGCATCCCCCGGTACTTCTCATGTCCCGGCGCCGGGTCGGCGACGAT

[0563] CCCGAGCCGGTCGGCCAGGGCCTCGTCCCGCCCGATGCCTCGCTTCATCAGGTAGGCGGCAGT

[0564] CTCGGCGTTCAGGTGCGCCTGGTAGGTGCTCACCGCCTCCTCCAGCATCTCCTTCTGGGATGTC

[0565] GAGAGCGGCGTGAGCGGTTCGTGCTCGGCCAAGCTGGTTCTCCTCCTTACTTCCTCTTCCAGGC

[0566] CGGCACGTAGCCGCTGCCTGCCTTCTTGCCGGGCTTCTTGCTTGCCGCCCGGTGGCCGCCTCC

[0567] GTAGCGGCTGGTGTAGCTGTCGGTCTTGGCGACCGCTCCCTCTTCGAGGCCGTGCTCCTTGGCG

[0568] TACGCCTTGGCCTCCTTGAAGCCGATCTCCTTGGAGAGCTGTTCGGCGTGGTACTCCTGGATCAT

[0569] CGAGAAGCTGTCGCCGCCCTTCCCGCAGGAGTGGCAGTTCCACAGCCCCTCGTCCAGGCGGTA

[0570] GGACATCGACGGGGTGTTGTCCTCATGGAGTGGGCACTTCGCCATGCCGGTGTTGCGCTGGTCG

[0571] TTGAAGTCCACGTCGAAGTGGTGCATCACCGAGTCGAGCAGGGGCTTGCTGTCCGATCCGCCCG

[0572] AGTGGTCGTTGTCGATGCGGTGGAACCTCACGGGTGCGCCACCTCCAGGGCCTGCTCGACGGC

[0573] GAGGATCAGCTCCAGGTCGGAGTCCGGGTCCTCGTTGTAGGCGAAGAACGCCTCTGCCTCGTCG

[0574] TGCAGTTCGTCGAGCTCGGCCTGGGTCATGTCCCAGCCGATGAACCCGATCACTGCGGGTCCAG

[0575] TCCGAGGTAGTCCTCGACGGTCGTGAGCACGAACGCCTTGCGCCAGTTCTTCCCCCGGCGCTTC

[0576] ACGACCACGATCGACTCGACGTCCTCCAGGTCCAGGCCGCGGTGCTTGGCGAAGTTCTCGCGCT

[0577] CGACGATGGCCTGCCCCAGGAAGACGCCCGGCTCGAACTTCGCGTTCTTCGCCTCGATCACCAG

[0578] GTACTTGCCGTCGCCCTCGCGGATGACCATGTCGCCCTCGTCCTCTGCGCCGGCCAGGCGAAG

[0579] GGACTCGACGTCGAAGCCTTCGGTGCGGAGACCGTCACGGAGGTCCGACTCCCAGTCGGCCCC

[0580] CTTGCGCTTGTTGCGCTTGTTCCTCGCCGCGATACTGTTGGACAACTTGCACACCTCCCTCGTTC

[0581] AAAGCCGGGCAGGCGCCCGGCGACTTGTGGAACAGTAGCACACTACGAGCCGAGACTCAACGC

[0582] GACCTTCGTCGGGCTCCACTCCTCGGCCGGCTTCGCCTGCTGGATGATCTCCCGCTTCTCGGCC

[0583] TTCTTGAACCGGGTGTACTCCGGCTGGCAGATCATCGTCGTGTACCGGCCGGCGGTCGGGTCGC

[0584] ACGGGCCCATGCGCTGCTTGATGCACGCCACGTTGTAGGCCAGCGAGGTCGGGTCCAGGGCCA

[0585] CGGAGAGCGAGAGCTCGGGCTTCTCGGACAGGCCGCCCTTGACCTGGTCGCGGGATGGGGGA

[0586] GCCCAAGGGTTCGTCTTGGCTTCCCAGTTCTTGTCGCTCGCGTGGTGCAGGATGACGACCGTCG

[0587] CACCCGTGTGACGGGCCAGCTCGGTGCAGCTCTGCATCACGGCCATCTGCTCGGTGTAGTCCGA

[0588] CTCGGCGCCCTCGAAGTCCATCAGGTTGTCGAAGACGATCACCTGCGGGTAGGCGTCCCACAGC

[0589] TCGACGTACGCCTCCAGCTCCTCGTCGATCCCCCGGTAGGTGATGGGGGAGCCAAAGGAGAAG

[0590] GTGATGTTCGAGGAGGCCAACGCGTCGATGTACGCCTGCCTGTACTTGCCGCCTTCCGCCATGC

[0591] CGGCCTCGACCATCTCGGTCGTGTCCATCGTGGCCATGGAGGCGAGCCGGCTCGACGCCGTGA

[0592] AGGCGCTCATGTCGGCAGAGAAGTACAGCGTCGGCAGGTTCATCTGGGCCACCCAGAACAGGG

[0593] CGAAGCCCGACTTCTGCGTGCCGGAACGGCCGGCGACCATGATGACCTCACCGTGCCGGGGCC

[0594] TCACGCCCATGGCGTACAGGTCGTCGAACGCCTCTACGCGCGGGAGTTCACGGCCCGATTCAGC

[0595] ATGGAGCGCCAGGGACCTTCCGGGGGTGAGCACTGAGTGGACTCCTCTCCTCGGACGACGACG

[0596] CGCCCGATACCGACTGAAGCGATCAGGGTGGTGCAAGCAGGACACGGCTTGCGCGTGACGTAC

[0597] AGCGTCGAGTCCTTCAGCTCGTCAGGGTGGATGCCCTTCACGTCGAGCGCGTCTCGAATCGCGT

[0598] TGCGCTCCGCATGGTCAGCGGCGCAGTTGGAGTAGTCGCTGTCTCGGGCGCACTCCTCGGTGG

[0599] AGAGCTGCCCTCGCGGGCAGTTGCCCTCAGTCCCACAGCCGGGGACTCCTCGCATCAGGCCGT

[0600] TGTATCCGAGCCCGAGTACCGAGTGGCCTCGCTTGGCCACGATGATGGCGCCCACCTGGGCGC

[0601] GCGTACAGTCCGCGATGGTGGACACAGCCTCGGCGATCCCGAGGGCCCACTCGTCGCGGGACG

[0602] GTCGTGTGAACACCGTCCCTCCCTTCCGCCCGCGGGTGCGGGCTCATCGACCCCCTTCCGGTC

[0603] GTAGTGCCACTTACACAGTCAGATCAGTCGAAGTCCGGGACCGCGGCGAGGGCGGCCTCCGCC

[0604] TCCTTCTCCCGCTTGTCGGCGTACGCGATGACGCCGTTACGGACGGCCGCGTCGGTCACCGGA

[0605] CGCCACACCCAGGCCGGGTGCGCGCCGGGCTTCTTCGGGGGGACCTGCTCCAGGCGCACCGAT

[0606] CGTGGCGCCGCCGACGATGGTCTCCAGGTCGCGGGCGAGGATGGTCTGCTCGATCCTCTGGCC

[0607] CTTGGCGACCTGCGGGGTGCCGGCCTGGAGCGCGGAGCCGTCCTGGAAGACGGTCACGTCAG

[0608] CGAGGACGGAGTCCTTCGGGCCGTTCGGGGTGGGGCCTGACGCTCGAACTGGTGCACCTCGA

[0609] TGAGGATGGCCACCGCGCTGATGTTGTCCTTCGGCTTGAACCAGCCGCCGCCCTGGACCGGGAT

[0610] GTCGATGAGGTTGAGAGCCACTGTGTCTGATCTCCTTCGTTCGTTGCCTACGCGGGCAGTTACG

[0611] GTGCGATTCGGGTTGAGGTGTTGCGGTGGGCGCTACGCGCCCTGAAGGGCCTTGCCCTTCGCCTTCCAGGCCGCCATCACGCTGGCGTCCGA (SEQ ID NO: 6). The classification name of Streptomyces phage St01 is Strept omycesphage, belonging to Duplodnaviria (double-stranded DNA virus domain), Heunggongvirae (Hong Kong virus kingdom), Uroviricota (tailed phage), Caudoviricetes (tailed phage class), Caudovirales (tailed phage order), Siphoviridae (long-tailed phage family). No genes related to antibiotic resistance or virulence were found in its genome, indicating that it is suitable for phage biological control.

[0612] Example 4: Transmission electron microscopic morphological observation of Streptomyces phage St01

[0613] Take 10 μL (10 9The bacteriophage St01 proliferation solution prepared in Example 2 (pfu / mL) was dropped on a copper mesh and allowed to stand for 20 minutes. The excess liquid was removed by filter paper. 15 μL of 2% phosphotungstic acid (PTA) was added to the copper mesh for staining for 5 minutes. The residual liquid was removed by filter paper. After drying, the mesh was observed using a transmission electron microscope.

[0614] The results are as follows Figure 3 As shown, the Streptomyces phage St01 has a typical icosahedral head (diameter about 65 nm) and a telescopic tail (length about 280 nm). Phage St01 exhibits a typical head-tail morphology associated with the tailed phages (Caudoviricetes).

[0615] Example 5: Determination of the optimal multiplicity of infection of Streptomyces phage St01

[0616] First, prepare the Streptomyces phage St01 proliferation liquid (method refers to Example 2) and the logarithmic host bacterial liquid, and count and determine the concentration respectively. Mix the phage and host bacteria at a multiplicity of infection of 0.0001, 0.001, 0.01, 0.1, 1 and 10, and place them in a final volume of 10mL NB culture liquid. Culture at 28℃ with shaking for 24h, centrifuge at 12000rpm for 30min, take the supernatant, filter with a 0.22μm filter, and obtain the phage proliferation liquid. Use the double-layer plate method (using the logarithmic host bacterial liquid) to detect the phage titer, the experiment is set up three times, and the optimal multiplicity of infection is determined according to the measurement results.

[0617] The results are shown in Table 1. When the infection multiplicity was 0.001, the titer of Streptomyces phage St01 was the highest, which was 1.11±0.19×10 9 Therefore, the optimal infection multiplicity of this phage is 0.001.

[0618] Table 1 Results of determination of optimal infection multiplicity of bacteriophage St01

[0619]

[0620]

[0621] Example 6: One-step growth curve of Streptomyces phage St01

[0622] 100 μL of Streptomyces phage St01 and host bacteria were mixed at an optimal infection multiplicity of 0.001, incubated at 28°C for 5 min, centrifuged at 12000 rpm for 30 s, washed twice with NB culture medium, and the supernatant was discarded. 10 mL of NB culture medium was added to resuspend the precipitate and cultured at 28°C with shaking. 200 μL of NB culture medium was taken every 15 min, and the phage titer was determined by the double-layer plate method (using logarithmic host bacterial solution). The experiment was repeated three times, and a one-step growth curve was drawn to analyze the incubation period, outbreak period, and lysis amount of the phage.

[0623] The results are as follows Figure 4 As shown, the incubation period of Streptomyces phage St01 is about 60 minutes, and the outbreak period is about 45 minutes, which proves that St01 can lyse pathogens in the field environment, effectively reduce the density of pathogens, and control the disease.

[0624] Example 7: pH stability of Streptomyces phage St01

[0625] Streptomyces phage St01 was placed in 10 mL of NB liquid medium with pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively, and the final concentration of phage was 10 9 After 2 h at 28°C, 200 μL of culture medium was taken and the phage titer was determined by double-layer plate method (using logarithmic phase host bacterial solution). The experiment was repeated three times and a bar graph of pH stability of phage St01 was drawn.

[0626] The results are as follows Figure 5 As shown in the figure, the titer of Streptomyces phage St01 shows stable titer in the pH 4-12 range. When pH <4 or pH>12, the titer of phage St01 decreases significantly with the increase of pH. Therefore, Streptomyces phage St01 is relatively stable in the environment of pH 4-12, and its applicability is significantly wider than other similar phages. Good pH tolerance is the advantage of St01. High concentrations of St01 phage can maintain activity for a longer time in field environments with complex conditions. It will more stably and continuously lyse pathogens and thus control the progression of the disease.

[0627] Example 8: Thermal stability of Streptomyces phage St01

[0628] Use NB culture medium to dilute Streptomyces phage St01 to a final concentration of 10 9 10 mL of phage dilution was taken and incubated in a water bath at 4°C, 25°C, 37°C, and 50°C for 24 h. 200 μL of culture solution was taken every 3 h to determine the titer of phage St01 by the double-layer plate method (using logarithmic host bacterial solution). The experiment was repeated three times to draw the thermal stability curve of phage St01.

[0629] The results are as follows Figure 6 As shown in the figure, the titer of Streptomyces phage St01 did not change significantly in an environment below 40°C. When the temperature exceeded 40°C, the titer of the phage began to decrease, indicating that phage St01 has a certain temperature stability and can maintain a certain activity in the field environment.

[0630] Example 9: UV stability of Streptomyces phage St01

[0631] The concentration is 10 9 pfu / mL of Streptomyces phage St01 was placed in a disposable plate and placed at a distance of 50 cm from a UV lamp (20 W, 700 mw / m 2 ) was irradiated at 40 cm below. 200 μL of phage solution was taken at 15 min, 30 min, 45 min, and 60 min after irradiation, and the titer of phage St01 was determined by double-layer plate method (using logarithmic host bacterial solution) after standing in a dark environment for 30 min. The experiment was repeated three times, and the UV stability curve of phage St01 was drawn.

[0632] The results are as follows Figure 7 As shown in Figure 2, the titer of Streptomyces phage St01 began to decrease after the UV irradiation. After 60 minutes of continuous UV irradiation, the phage titer dropped to about 10 7 pfu / mL. The results show that Streptomyces phage St01 has excellent tolerance to short-term ultraviolet radiation. Phage St01 has excellent ultraviolet tolerance and will continue to lyse pathogens more stably.

[0633] Example 10: Host range detection of Streptomyces phage St01

[0634] Five pathogenic strains of Streptomyces, CGMCC4.1765, CGMCC4.1789, CGMCC4.4739, CGMCC4.3490 and ATCC700248, and 13 pathogenic strains of Streptomyces (A1-4, 6-14) isolated from potato plantations, totaling 18 strains, were selected to determine the host range of Streptomyces phage St01. At the same time, two non-pathogenic Streptomyces strains B1, B2 and two other bacterial strains were selected to detect the specificity of St01. Take 200 μL of the host bacteria to be tested and evenly spread them on the NB solid culture medium plate. After the bacterial solution is completely absorbed, add 5 μL of phage proliferation solution (10 9 pfu / mL) on the plate, and the control group was dripped with NB liquid culture medium. After absorption, it was placed in a 28℃ constant temperature incubator for inverted culture for 24 hours to observe whether clear plaques were produced.

[0635] The results are shown in Table 2. The 18 pathogenic Streptomyces strains to be tested were lysed, and the 2 non-pathogenic Streptomyces strains and the other 2 bacterial strains all grew well on the NB plate. The lysis ability of bacteriophage St01 on each pathogenic bacteria was different. ++++ represents that the bacterial lawn in the phage drop area is completely cleared; +++ represents that the phage drop area is completely cleared but the background is blurred; ++ represents that the cleared area in the phage drop area is turbid; + represents that only individual plaques can be observed in the phage drop area; - represents that the bacterial lawn is not cleared at all. Streptomyces phage St01 can lyse all 18 pathogenic Streptomyces strains, and can achieve +++ and above lysis effects on all 18 pathogenic Streptomyces strains, but has no lysis ability on 2 non-pathogenic Streptomyces strains and the other 2 bacterial strains. This shows that bacteriophage St01 is a broad-spectrum phage that can efficiently and specifically lyse pathogenic Streptomyces without killing common bacteria in other environments. Thus, it can deal with a variety of plant scab diseases caused by a variety of pathogenic Streptomyces.

[0636] Table 2 Results of host range determination of bacteriophage St01

[0637]

[0638]

[0639] Example 11: In vitro lysis test of Streptomyces phage St01

[0640] Streptomyces phage St01 was mixed with 100 μL of pathogenic Streptomyces ATCC700248, CGMCC4.1765, CGMCC4.1789 and CGMCC4.4739 at an optimal infection multiplicity of 0.001, incubated at 28°C for 5 min, centrifuged at 12000 rpm for 30 s, and washed twice with NB culture medium, and the supernatant was discarded. 10 mL of NB culture medium was added to resuspend the precipitate (the final concentration of phage was 10 9 pfu / mL) and cultured with shaking at 28°C. The OD value of the culture solution was measured at 600nm every 2 hours for a total of 24 hours. The experiment was repeated three times to draw the in vitro lysis curve of bacteriophage St01.

[0641] The results are as follows Figure 8 As shown, Streptomyces phage St01 can effectively lyse 4 strains of pathogenic Streptomyces within 24 hours, effectively reduce the concentration of pathogens, alleviate the occurrence of the disease, and has good application prospects.

[0642] Example 12: In vivo safety test of Streptomyces phage St01

[0643] Potato slices with a diameter of 3 cm were made from potatoes and divided into 3 groups, 33 slices in each group. Small holes with a diameter of about 1 cm were punched with a sterile puncher. 10 μL of 10% dapoxetine was added to the small holes in the high-dose group. 8 pfu / mL of bacteriophage St01; in the low-dose group, 10 μL of 10 6 pfu / mL of bacteriophage St01; 10μL of sterile NB culture medium was added to the control group. Three potato slices were taken from each treatment group at 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h and 120h after the addition of phage, 1g of potato flesh was taken from the small hole and the surrounding area, 9mL of physiological saline was added for grinding, centrifuged at 12000rpm for 3min, and the supernatant was filtered through 0.45μm and 0.22μm filters respectively. 200μL of filtrate was taken, and the titer of bacteriophage St01 was determined by double-layer plate method (using logarithmic host bacterial solution), and the experiment was repeated three times.

[0644] like Fig. 9 As shown in the figure, compared with the control group, the potato slices treated with phages had normal potato flesh tissue and no negative impact on the potato slices. Fig. 9 As shown in the figure, both the high and low dose groups of phage St01 can be relatively stable on potato slices for about 96 hours, and the titer begins to decrease significantly after 96 hours, and drops to 0 after 120 hours. The results show that Streptomyces phage St01 has good safety for potato slices, can maintain activity on potato slices within 96 hours, and the phage residues are completely eliminated after 120 hours.

[0645] Example 13: In vivo treatment test of Streptomyces phage St01 against potato scab

[0646] Potato slices with a diameter of 3 cm were made from potatoes. The potato slices were divided into 2 groups, with 3 slices in each group. Small holes with a diameter of about 1 cm were punched with a sterile puncher. 10 μL of 10% 7 CFU / mL pathogen ATCC700248; the treatment group (St01 group) first dripped 10 μL of 10 7 CFU / mL pathogen ATCC700248, 10 μL of 10 5 pfu / mL of bacteriophage St01. After 5 days, the potato slices were observed for pathological changes, and 1g of potato flesh from the small hole and the surrounding area was taken and ground with 9mL of sterile saline. The homogenate was diluted 10 times and the colony count was performed to compare the bacterial concentrations of the two groups of potato slices. The experiment was repeated three times.

[0647] like Fig.10As shown in the figure, the potato slices in the control group had severe lesions after infection, while the phage-treated group was obviously healthy and had no obvious lesions. The results of colony counts after 5 days showed that after phage St01 treatment, the bacterial CFU decreased significantly, and the bacterial concentration was lower than 4lg CFU / g, while the control group was greater than 6lg CFU / g. Therefore, Streptomyces phage St01 has a good therapeutic effect on scab caused by pathogen ATCC700248 in the potato slice experiment.

[0648] Example 14: Soil disinfection test of Streptomyces phage St01

[0649] Add 2.5 mL each of Streptomyces ATCC700248, CGMCC4.1765, CGMCC4.1789 and CGMCC4.4739 to 100 g of sterilized soil, for a total of 10 mL (concentration of 10 7 CFU / mL), and after proper mixing, spray 10mL of bacteriophage St01 (concentration of 10 5 pfu / mL), and the control group was sprayed with an equal amount of sterile NB culture solution. After 24 hours at 28°C, 1g of soil was dissolved in 100mL of sterile water for colony counting. The experiment was repeated three times to detect the bacterial concentration.

[0650] like Fig.11 As shown in the figure, after 24h of treatment with bacteriophage St01, the bacterial concentration of Streptomyces ATCC700248, CGMCC4.1765, CGMCC4.1789 and CGMCC4.4739 in the soil was less than 5lg CFU / g, while that of the control group was greater than 7lg CFU / g. 5 The bacteriophage St01 at a concentration of pfu / mL can effectively kill the contaminated Streptomyces in the soil environment.

[0651] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A Streptomyces phage St01, comprising a nucleotide sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 from the 5' end to the 3' end.

2. A culture obtained by culturing the Streptomyces phage St01 according to claim 1.

3. The method for preparing the culture according to claim 2, comprising inoculating the Streptomyces phage St01 according to claim 1 into a host bacterium, culturing, and obtaining; Preferably, the host bacteria comprises pathogenic Streptomyces.

4. A preparation comprising: the Streptomyces phage St01 according to claim 1 or the culture according to claim 2.

5. The preparation according to claim 4, characterized in that The preparation further comprises a carrier and / or an excipient; Preferably, the excipient comprises at least one of a diluent, an adjuvant, a surfactant, an excipient, a vehicle, a dispersant, a stabilizer, a filler and a solvent; Preferably, the preparation is a solid preparation or a liquid preparation; Preferably, the preparation is in the form of a powder, an aqueous solution, a lyophilized agent, a gel, a cream or an ointment; further in the form of a solution, a suspension, an emulsion, a powder, a tablet, a spray or an injection.

6. The preparation according to any one of claims 4-5, characterized in that The preparation is an agricultural fungicide or an additive thereof, a disinfectant or an additive thereof, or a cleaning agent or an additive thereof; Preferably, the preparation further comprises: a1) other active ingredients for controlling pathogenic Streptomyces infections and / or plant diseases caused by pathogenic Streptomyces; and / or a2) Active ingredients for controlling other pathogens.

7. A detection kit comprising: the Streptomyces phage St01 according to claim 1 or the culture according to claim 2.

8. Any application of A1)-A2): A1) Use of the preparation according to any one of claims 4 to 6 in any one of c1) to c3); c1) Kill pathogenic Streptomyces in the environment; c2) Preventing and controlling pathogenic Streptomyces infection; c3) Control of plant diseases caused by pathogenic Streptomyces; A2) Use of the detection kit according to claim 7 in any one of b1) to b2): b1) Detection of pathogenic Streptomyces; b2) Identification of plant diseases caused by pathogenic Streptomyces.

9. The use according to claim 8, characterized in that: The pathogenic streptomyces include soil-borne pathogenic streptomyces; further include at least one of S. scabies (Streptomyces aflumanensis), S. acidiscabies (Streptomyces acidicus), S. europaeiscabiei (Streptomyces europaeiscabiei), S. luridiscabiei (Streptomyces luteus), S. turgidiscabies (Streptomyces swollenus), S. reticuliscabiei (Streptomyces reticulariscabiei), S. botropensis (Streptomyces botropensis), S. bobili (Streptomyces bobili), and S. anulatus (Streptomyces anulatus); Preferably, the disease comprises scab; Preferably, the plant comprises at least one of potato, radish, beet, turnip and peanut.

10. Any of the methods B1)-B2): B1) A method for preventing and controlling pathogenic Streptomyces infection and / or plant diseases caused by pathogenic Streptomyces, comprising the step of using the preparation according to any one of claims 4 to 6; B2) A method for killing pathogenic Streptomyces in the environment, comprising the step of using the preparation according to any one of claims 4 to 6.

11. The method according to claim 10, characterized in that B1) The method comprises the following steps: applying the formulation according to any one of claims 4 to 6 to the plant or its cultivation medium by seed treatment, foliar application, stem application, or root application; Preferably, B2) the method comprises the following steps: applying the preparation according to any one of claims 4 to 6 to the environment; Preferably, the preparation in B1) is applied to the plant or its cultivation medium by soaking, dripping, pouring, spraying, root irrigation, or dusting; Preferably, the preparation in B2) is applied to the environment by soaking, dripping, pouring, spraying, or dusting.

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