CN114292785B - Marine streptomycete and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals - Google Patents
Marine streptomycete and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals Download PDFInfo
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Abstract
The invention discloses marine streptomycete and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals, and belongs to the technical field of biological control. The Streptomyces sp.SH5 is named as Streptomyces sp.SH5, accession number: GDMCC No:62057. the SH5 strain is separated from the water environment, and the ecological safety risk is greatly reduced when the strain is applied to the culture water. The SH5 strain is planted in the intestinal tract of the zebra fish, activates a host immune system, inhibits inflammatory reaction, reduces production of aeromonas hydrophila virulence factors and virulence gene expression, and can effectively improve the survival rate of the zebra fish under the stress of aeromonas hydrophila, so that prevention and control of aeromonas hydrophila infection are realized in a friendly mode which is environment-friendly and does not influence the micro-ecological balance of the host.
Description
Technical Field
The invention belongs to the technical field of biological control, relates to marine streptomyces and application thereof in the field of aquatic disease prevention and control, and in particular relates to marine streptomyces SH5 capable of being planted in intestinal tracts of aquatic animals and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals (such as zebra fish).
Background
Aeromonas hydrophila (Aeromonas hydrophila) is a common pathogenic bacterium in water environment, can infect various aquatic animals such as fish, shrimp and shellfish, induces septicemia of the aeromonas hydrophila, and has direct economic loss of billions caused by the outbreak of the disease every year in China. Antibiotic pharmacotherapy is always a traditional means for preventing and treating aquatic diseases commonly used at home and abroad, however, the problems of drug resistance, environmental pollution, ecological balance destruction and the like caused by the large-scale use of broad-spectrum antibiotics force people to develop safer, environment-friendly and reliable novel disease prevention and control drugs and methods as soon as possible so as to meet the national 'green development' strategic requirements. Biological control is considered as a main technical means for replacing antibiotics because of the advantages of environmental protection, safety, health, economy, low cost, convenient use and the like.
Streptomyces receives great attention in the field of biological control due to the advantages of good production of antagonistic active substances, degradative enzymes, good stress resistance and the like. However, the aerobic living characteristics of streptomyces have resulted in no related findings or basis for the biological control of streptomyces in implantable aquatic organisms.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide marine streptomyces. The strain is streptomyces capable of being planted in intestinal tracts of the zebra fish, and fermentation liquor of the strain is applied to a culture water body, so that the survival rate of the zebra fish under the stress of aeromonas hydrophila can be effectively improved, the inflammatory reaction of the zebra fish is inhibited, and the production of aeromonas hydrophila virulence factors and the expression of related virulence genes are reduced.
It is another object of the present invention to provide the use of Streptomyces maritimus as described above.
The aim of the invention is achieved by the following technical scheme:
the invention provides a strain of Streptomyces sp.SH5, which is obtained by separating and purifying the sea sediment of Dalian Starfish Bay.
Preservation information of Streptomyces sp SH 5: preservation unit: the Guangdong province microorganism strain collection center (GDMCC), accession number: GDMCC No:62057, deposit address: the Guangdong province, guangzhou City, first, china, no. 100 institute, no. 59 building, guangdong province microbiological institute, deposit date: 2021, 11, 12.
The 16S rDNA sequence characteristic of Streptomyces sp.) SH5 described: the 16SrDNA sequence has a nucleotide sequence shown as SEQ ID No.1, and the sequence length is 1374bp.
A biological agent is prepared from the marine Streptomyces.
The biological agent is prepared from the marine streptomycete through liquid culture, and preferably comprises the following steps: inoculating the marine streptomyces into ISP1 liquid culture medium for culture to obtain the biological agent.
The culture is carried out at 28-30 ℃ and 180-200 rpm for 48-72 h.
The application of the marine streptomyces SH5 in preventing and controlling aeromonas hydrophila from infecting aquatic animals.
The application of the marine streptomycete SH5 in the analysis of the intestinal tract colonization ability of aquatic animals;
further, the use of Streptomyces maritimus SH5 for enhancing the immune response of aquatic animals.
The application of the marine streptomyces SH5 in regulating the aeromonas hydrophila in the inflammatory reaction process of aquatic animals.
Furthermore, the application of the marine streptomyces SH5 in inhibiting aeromonas hydrophila in intestinal colonization of aquatic animals is provided.
Further, the application of the marine streptomyces SH5 in reducing the inflammatory response of aquatic animals under the stress of pathogenic bacteria; the pathogenic bacteria are aeromonas hydrophila.
Further, the aquatic animal is zebra fish.
The application of the marine streptomyces SH5 in inhibiting the production of aeromonas hydrophila virulence factors and the expression of virulence genes.
The virulence genes are aero, act, ast, hlyA, alt, ahp, ahyR, ompA and fur.
The principle of preventing and controlling the infection of the zebra fish by the aeromonas hydrophila by the SH5 strain is that the SH5 is planted in the intestinal canal of the zebra fish to activate a host immune system, inhibit inflammatory reaction and reduce the virulence gene expression of the aeromonas hydrophila, so that the prevention and control of the infection of the aeromonas hydrophila is realized in a friendly mode without influencing the micro-ecological balance of the host in a green and environment-friendly way.
Compared with the prior art, the invention has the following advantages and effects:
(1) The present invention provides Streptomyces sp.SH5 strains. The SH5 strain is separated from the water environment, and the ecological safety risk is greatly reduced when the strain is applied to the culture water.
(2) The SH5 strain provided by the invention can effectively improve the survival rate of the zebra fish under the stress of aeromonas hydrophila.
(3) The SH5 strain is an aerobic strain, but can be planted in the intestinal tract of zebra fish.
(4) The SH5 strain provided by the invention can effectively reduce the colonization quantity of aeromonas hydrophila in the intestinal tract of zebra fish.
(5) The SH5 strain provided by the invention can effectively inhibit the expression of virulence genes of pathogenic bacteria aeromonas hydrophila.
(6) The SH5 strain provided by the invention can effectively promote the immune response of the zebra fish and obviously reduce the inflammatory response of the zebra fish under the stress of pathogenic bacteria.
Drawings
FIG. 1 is a graph showing the actual effect of the target strain SH5 on improving the survival rate of zebra fish under the stress of aeromonas hydrophila; wherein SH5 (L) represents a low concentration treatment group, i.e., SH5 is at 1: adding the 1000 diluted fermentation liquor into the culture water of the young zebra fish; SH5 (H) represents a high concentration treatment group, i.e., SH5 is at 1: the 100 dilution fermentation liquor is added into the breeding water of the young zebra fish.
FIG. 2 shows the SH5 strain 16S rDNA sequence and the evolutionary tree.
FIG. 3 is a primer SH5F/R specificity assay; wherein M is a marker of DL2000, lane 1 is SH5, lane 2 is Streptomyces TK24, lane 3 is Escherichia coli, lane 4 is Aeromonas hydrophila, lane 5 is Vibrio parahaemolyticus, and lane 6 is zebra fish.
FIG. 4 shows the effect of fluorescent-labeled SH5 strains on colonization of the intestinal tract of zebra fish (A) and quantitative analysis of colonization of SH5 (B).
FIG. 5 is a graph showing the effect of SH5 strain on reducing the colonization of the zebra fish intestinal tract by Aeromonas hydrophila; wherein A is the number of aeromonas hydrophila of each zebra fish at different time points; b is the number of each zebra fish SH5 at different time points.
FIG. 6 is the effect of SH5 strain on the expression of genes associated with the immune response and inflammatory response of zebra fish; wherein A is the result of the attack of aeromonas hydrophila for 6 hours; b is the result of the attack of aeromonas hydrophila for 12 h.
FIG. 7 is the effect of SH5 strain on inhibiting the expression of aeromonas hydrophila virulence gene; wherein A is the result of co-culture of SH5 and aeromonas hydrophila for 6 hours; b is the result of the co-culture of SH5 and aeromonas hydrophila for 12 hours; a is the result of the co-culture of SH5 and Aeromonas hydrophila for 24 hours.
FIG. 8 is an antagonism of SH5 strain against Aeromonas hydrophila.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are those obtained commercially.
The E.coli used in example 2 was E.coli (Escherichia coli) GDMCC 1.215 and the Aeromonas hydrophila was Aeromonas hydrophila (Aeromonas hydrophila) GDMCC 1.2306, all available from the microorganism strain collection in Guangdong province;
vibrio parahaemolyticus is Vibrio parahaemolyticus (Vibrio parahaemolyticus) ATCC 17802 available from Guangdong Kai biotechnology Co., ltd;
streptomyces lividans TK24 to Streptomyces lividans (Streptomyces lividans) TK24 is disclosed in the literature "Xu D, seghezzi N, esnault C, et al, repetition of Antibiotic Production and Sporulation in Streptomyces coelicolor by Overexpression of a TetR Family Transcriptional Regulator [ J ]. Applied and Environmental Microbiology,2010,76 (23): 7741-7753.
Example 1
The screening and identifying method of the Streptomyces sp SH5 comprises the following steps:
(1) 1g of a Dalianxing Bay mud sample is weighed, 10mL of sodium cholate (0.1% w/v) is added, 200r/min is shaken for 30min, and the supernatant is stored after centrifugation (500 g,1 min). 10mL sodium cholate (0.1% w/v) was added to the lower sea mud pellet, the low power sonicated in water was allowed to warm for 1min, and the supernatant was retained after centrifugation (500 g,1 min). Combining the obtained two batches of supernatant, and performing gradient dilution on the supernatant by using sterile seawater to obtain diluted 10 3 The supernatant was multiplied, 0.2mL of the dilution was uniformly spread on HV plates containing 15. Mu.g/mL of nalidixic acid and 50. Mu.g/mL of potassium dichromate, and after 20 days of incubation in a 28℃incubator, streptomyces colonies were observed, single colonies were picked up and streaked on Bennett medium, and after 7 days the growth morphology of Streptomyces strains was observed, confirming no bacterial or fungal contamination. Streptomyces agar blocks are taken from a Bennett culture medium, inoculated into a TSB liquid culture medium, cultured for 7 days at 28 ℃ and 200rpm, and 500 mu L of bacterial suspension is taken, fully and uniformly mixed with 40% glycerol with equal volume, placed into a 1.5mL centrifuge tube and stored in a refrigerator at-70 ℃.
(2) Activation of the strain. Firstly, taking the target streptomycete strain SH5 out of a refrigerator at the temperature of minus 70 ℃, taking 100 mu L of the strain SH5 to be coated in ISP1 solid culture medium, placing the strain SH5 in 28 ℃ for 3 days of culture, and then taking a strain agar block to be inoculated in 50mL of ISP1 liquid culture medium, and culturing the strain agar block at the temperature of 28 ℃ for 3 days of culture at 200rpm for later use. Cleaning TU-type zebra fish embryo (fertilized for 0 days) (purchased from Shanghai Fei Xi Biotechnology Co., ltd.) with sterile water for 3 times, placing into 6-well plate, placing into 28 deg.C incubator, and controlling photoperiod with 14h light and 10h dark to provideFertilization conditions. On day 3 after fertilization (3 dpf), 1:100 (SH 5 (H)), 1:1000 (SH 5 (L)) dilution ratio and the same volume of ISP1 liquid medium were added to the Control group (Control), and the original Streptomyces fermentation broth concentration was maintained after daily water change. After 3 days of fermentation broth treatment, aseptic water is used for culture, and the final concentration is added to be 1 multiplied by 10 8 The survival rate was recorded every 12h for the challenge of the aeromonas hydrophila of CFU/mL. Each set was set up with 3 replicates. The survival results are detailed in figure 1. As can be seen from fig. 1, in the case of no SH5 (Control) addition, the survival rate of the young zebra fish decreases 24 hours after the toxicity attack, and decreases sharply after 36 hours, and the survival rate of the young zebra fish is 0% at 48 hours; under SH5 treatment group, the survival rate of young zebra fish after 24h is 100%, and the survival rate of young zebra fish after 36h is over 80% which is 3 times of that of Control group (Control). The result shows that the survival rate of the zebra fish under the attack of aeromonas hydrophila can be effectively improved under the SH5 treatment.
Wherein, ISP1 liquid culture medium comprises the following formula: 3g of yeast extract powder, 5g of tryptone and sterile water to a volume of 1L, and pH 7.2+/-0.2; ISP1 solid medium also required the addition of 20g of agar.
(3) Identification of the 16S rDNA sequence of the target strain SH5. 200. Mu.L of 24h fermentation broth of SH5 strain was taken, the supernatant was discarded by centrifugation, the collected mycelia were suspended in 100. Mu.L of sterile water, heated to 100℃for 10 minutes, and the resulting solution was directly used as a template for PCR. The universal primer is adopted: 27F:5'-AGAGTTTGATCCTGGCTCAG-3',1492R:5'-TACGGTTACCTTGTTACGACTT-3'.
The PCR reaction system is as follows: ddH 2 O15.00. Mu.L, KOD-Plus buffer 2.5. Mu.L, dNTP mix (200 mmol/L) 2.5. Mu.L, primer 27F 1.0. Mu.L, primer 14992R 1.0. Mu.L, bacterial liquid template 1.0. Mu.L, KOD-Plus enzyme 0.5. Mu.L, mgSO 4 (25 mmol/L) 1.5. Mu.L. The reaction procedure was set as follows: pre-denatured at 95 ℃ for 5min before entering thermal cycle (×30): 94 ℃ for 1min;54 ℃ for 1min; extending at 72 deg.C for 2min and at 72 deg.C for 10min. The resulting PCR amplified product was subjected to 1.2% agarose gel electrophoresis (100V, 30 min), the PCR product (about 1.5kb in size) was recovered by gel cutting, and the purified DNA fragment was cloned into pMD18-T vector, and the resulting T-A ligation product was transformed into E.coli EscThe herichia coli DH5 alpha competent cells were used to detect clones by PCR and to sequence the positive clones.
(4) The evolutionary tree was established based on the SH5 strain 16S rDNA sequence. Firstly, the measured 16S rDNA sequence is imported into an EzTaxon professional database for on-line homologous sequence alignment analysis, then the result is imported into Cluxtalx 1.83 software for multi-sequence matching arrangement, the obtained result is applied to Mega5 software, and a phylogenetic tree is established through a Neighbor-joining method. The results obtained are shown in FIG. 2, in which the SH5 strain is integrated with S.barius NRRL B-2567 (AY 999783). The strain SH5 was identified as Streptomyces sp, designated Streptomyces sp SH5.
Preservation information of Streptomyces sp SH 5: preservation unit: the Guangdong province microorganism strain collection center (GDMCC), accession number: GDMCC No:62057, deposit address: the Guangdong province, guangzhou City, first, china, no. 100 institute, no. 59 building, guangdong province microbiological institute, deposit date: 2021, 11, 12.
The 16S rDNA sequence of the Streptomyces sp SH5 is shown as SEQ ID No.1 (1374 bp).
Example 2
The SH5 strain is planted in the intestinal tract of the zebra fish, and the effect of aeromonas hydrophila on the intestinal tract of the zebra fish is reduced.
(1) And directly observing the colonization condition of SH5 in zebra fish intestinal tracts by a fluorescence microscope. First, SH5 was fluorescently labeled with a green fluorescent tracer dye CFDA-SE. Then, stained SH5 was used as a dye according to 1: 100% strength was added to zebra fish culture water treated with PTU (phenylthiourea) to inhibit melanin production of zebra fish larvae, and fluorescent dye was supplemented to maintain the initial strength after daily water change. After 3 days, the zebra fish is continuously bred by using normal breeding water (no SH5 is added), 5 zebra fish young fish are randomly selected from each group every 24 hours, after the zebra fish young fish are anesthetized by MS-22, the planting condition of SH5 is observed under a fluorescence microscope by utilizing the optical permeability of the zebra fish young fish. The results showed that SH5 can stay in the zebra fish intestine for more than 7 days and the number in the zebra fish foregut and midgut is higher (fig. 4A).
(2) Analysis of the number of SH5 colonization in zebra fish intestinal tracts. The specific number of SH5 colonization in zebra fish intestinal tracts at different time points is identified by adopting an RT-PCR technology, and the specific steps are as follows: firstly, polynucleotide sequence comparison is carried out on 16S rDNA of streptomyces by using ClustalW, a specific primer (SH 5-F:5'-TAACACTCTGTCCCGCATGG-3'; SH5-R: 5'-TTACCCCACCAACAAGCTGA-3') of SH5 is designed according to the conservation and variable regions of the sequences, an amplification region comprises a gamma hypervariable region of the 16S rDNA, and specificity of escherichia coli, aeromonas hydrophila, vibrio parahaemolyticus and other streptomyces (streptomyces plumbum TK 24) and zebra fish verification primer SH5-F/R is adopted, so that only SH5 is amplified to form a band, and the other bands are absent; as can be seen, the primer SH5-F/R has good specificity (FIG. 3). Cloning the specific primer amplified fragment into the pMD18-T vector, identifying the concentration of the constructed plasmid, according to the formula Copies/. Mu.L=6.02X10 23 Concentration of DNA (concentration (ng/. Mu.L). Times.10) -9 DNA length×660, and the copy number of the standard sample was determined. Standard samples were diluted 10-fold as templates and standard curves were drawn. And extracting total DNA of intestinal bacteria of each group of zebra fish by adopting an OMEGA DNA kit, homogenizing the initial concentration, taking the total DNA as a template, taking SH5-F/SH5-R as a specific primer, and identifying the specific number of SH5 colonization in the intestinal tract of the zebra fish by using an RT-PCR experiment. The reaction system of RT-PCR is as follows: SYBR Green Realtime PCR Master Mix 10. Mu.L, primer SH 5-F0.5. Mu.L, primer SH 5-R0.5. Mu.L, template DNA 1. Mu.L, ddH 2 O8. Mu.L. The procedure for the RT-PCR reaction was set as follows: pre-denatured at 95 ℃ for 5min before entering thermal cycle (×40): 94 ℃ for 30s;58 ℃ for 30s;72℃for 20s. The melting signal collection conditions were set as follows: 65 ℃ to 95 ℃ and 0.5 ℃/10s. The results demonstrate that SH5 is capable of staying in the intestinal tract of zebra fish for about 4 days, and that the colonization amount stabilizes at about 1X 10 per fish 2.5 CFU (fig. 4B).
(3) SH5 inhibits the action and effect of aeromonas hydrophila in colonizing zebra fish. The specific effect of SH5 on the intestinal tract of the aeromonas hydrophila field planting zebra fish is identified by adopting an RT-PCR technology, and the related steps are as follows: first, DNA fragments were amplified by PCR technique using Aeromonas hydrophila specific primers (aeromonas 2-F:5'-CGCCAGCTGGTCAAGACTGT-3', aeromonas 2-R: 5'-CCAGTTGGTGGCTGTGTCGT-3'), cloned into pMD18-T vector, and standard sample copy number was determined according to the method described above, and standard curve was drawn. And calculating the quantity of aeromonas hydrophila and SH5 of each zebra fish at different time points according to the standard curve. As a result, SH5 was found to be effective in inhibiting colonization of the zebra fish intestinal tract by aeromonas hydrophila (fig. 5).
Example 3
The SH5 strain affects the expression of zebra fish immune genes.
(1) SH5 pretreatment of zebra fish. After three days of SH5 culture, the fermentation broth was fermented according to SH 5: aquaculture water = 1: 100% SH5 fermentation broth was added to young zebra fish (3 dfp) cultivation water, and water was changed every day and the SH5 fermentation broth was maintained at the original concentration, and treated for 3d. The control group was supplemented with the same amount of ISP1 liquid medium as the experimental group. Each set was set up with 3 parallels.
(2) Aeromonas hydrophila toxicity attack experiment. After the zebra fish is treated by SH5 fermentation liquid for 3d, aeromonas hydrophila 12h TSB culture liquid is added for detoxification, and the final concentration is 1 multiplied by 10 8 CFU/mL. And extracting total RNA from 50 juvenile zebra fish in each group at 0h, 6h and 12h after the toxicity attack.
(3) And (5) extracting total RNA of the zebra fish.
Sample treatment: zebra fish were washed 3 times with PBS, the PBS was drained, and then the zebra fish was triturated with a grind bar. To the mashed sample, 1mL Trizol was added, and the mixture was allowed to stand for 5min.
Chloroform extraction: 400. Mu.L of chloroform was added to the sample, the solution was mixed well by vortexing for 30s with vortexing, and centrifuged (12000 rpm,10min,4 ℃). Carefully aspirate approximately 400 μl of supernatant with a pipette into another clean centrifuge tube.
Isopropanol precipitation: 400. Mu.L of isopropanol was added, mixed upside down, left to stand for 10min, centrifuged (12000 rpm,10min,4 ℃ C.) and the supernatant removed.
Ethanol washing: the pellet was washed twice with 75% ethanol and centrifuged to remove the supernatant. The precipitate was placed in a vent to fully volatilize the ethanol.
An appropriate amount of RNase-free water was added to the pellet for solubilization, and the RNA quality was detected by agarose gel electrophoresis, and the concentration was identified by a nucleic acid quantitative instrument.
(4) qRT-PCR (quantitative reverse transcription-polymerase chain reaction) detection of immune gene expression quantity. cDNA was amplified using reverse transcriptase M-MLV Reverse Transcriptase (Promega) using total RNA as template. First total RNA:2 μg, oligo (dT) 18:0.5 μg was mixed, DEPC treated water was added to 15 μl, and then warmed to 70deg.C for 5min. After cooling on ice, M-MLV 5 Xreaction Buffer 5. Mu.L, dNTPs Mixture (10 mM) 1. Mu.L, M-MLV Reverse Transcriptase. Mu.L, RNase Inhibitor 1. Mu.L, DEPC-treated water to 25. Mu.L were added to the Reaction system. The reaction procedure was as follows: 42 ℃ C:: 1h,72 ℃ C: 10min, termination at 4 ℃. The samples were stored at-20 ℃.
The cDNA synthesized by reverse transcription is used as a template, and specific primers (Table 1) are adopted to detect the expression level of immune genes by RT-PCR experiments. Taking housekeeping gene EF-1 alpha as an internal reference gene, setting 5 biological repeats for each sample, setting an experimental group without adding an amplification template as a blank control group, and detecting whether the sample has genomic DNA pollution or not, and carrying out a relative fluorescence quantitative PCR experimental system and reaction conditions as above. Experimental results show that after the treatment of SH5, after the aeromonas hydrophila attacks the toxin, the expression level of immune related genes such as TLR3, lysozyme, iNOs and the like of the zebra fish is obviously up-regulated, and inflammatory genes such as 1L-1 beta, 1L-6, myD88, TLR4a and the like of the zebra fish are obviously down-regulated, which indicates that the probiotic bacterial strain SH5 has the effects of activating host immunity and reducing inflammatory response (figure 6).
Example 4
The SH5 strain affects the expression of aeromonas hydrophila virulence genes.
(1) Establishment of SH5 and aeromonas hydrophila co-culture system
SH5 and aeromonas hydrophila are fermented and cultured in ISP1 and TSB liquid culture media respectively until the platform phase. Then, 1mL of each bacterial liquid was placed in a baffle flask containing 100mL of TSB, and cultured at 28℃and 180rpm for 48 hours, and the control group replaced SH5 fermentation broth with the same volume of ISP1 medium. Samples were taken at different time points of 0h, 6h, 12h, 24h during the culture for total RNA extraction.
(2) Total RNA extraction
Specific steps refer to the procedure provided by the RNA extraction kit (beijing cool libo biotechnology company):
cell lysis: a1 mL sample of the bacterial suspension was centrifuged (5000 rpm,10 min) in a 1.5mL EP tube and the supernatant was discarded. To the resulting precipitate, 0.5mL of Trizol was added, and after mixing, the mixture was left at room temperature for about 3 minutes until the bacteria were completely lysed.
Chloroform extraction: to the lysate was added 0.1mL chloroform, and the mixture was vortexed and mixed for 30 s. Centrifuge at 12000rpm for 3min at room temperature and carefully aspirate the supernatant into a new centrifuge tube.
Ethanol precipitation: 200 mu L of absolute ethyl alcohol is added into the supernatant, and the mixture is uniformly mixed and loaded into an RNA purification column. Centrifuge at 12000rpm for 1min, discard the penetration. To the purification column, 0.5mL of 75% ethanol was added, followed by centrifugation at 12000rpm at room temperature for 30s, the permeate was discarded, and the washing step was repeated twice.
RNA collection: the RNA-bound purification cartridge was centrifuged at 12000rpm for 1min, the cartridge was transferred to a 1.5mL RNase-free centrifuge tube, 30. Mu.L of the RNA-dissolved solution was added, and the mixture was left at room temperature for 2min, and the mixture was centrifuged at 12000rpm for 1min to elute the RNA. The obtained RNA is subjected to nucleic acid electrophoresis and a nucleic acid quantitative analyzer to identify the concentration and purity, and then is preserved at-80 ℃.
(3) qRT-PCR (quantitative reverse transcription-polymerase chain reaction) detection of virulence gene expression quantity. The cDNA was reverse transcription amplified and qRT-PCR method was as above. The specific primers used are shown in Table 1. The results showed that the expression level of nine virulence genes, luxI/R-type response regulator (ahyR), outer membrane protein A (ompA), ferric uptake regulator (fur), was significantly down-regulated when co-cultured with SH5 strain, aerolysin (aerA), cytotoxic enterotoxin (act), heat-stable cytotonic enterotoxin (ast), hemolysin A (hly), heat labile cytotonic enterotoxin (alt), serine protease (ahp) (FIG. 7).
TABLE 1 primer list
Example 5
Antagonism of the SH5 strain against aeromonas hydrophila.
SH5 agar blocks (diameter: 8 mm) of 7d were obtained by using an agar diffusion method using a punch and a solid medium of A1, and the solid medium was placed on a TSB solid medium coated with 100. Mu.L of Aeromonas hydrophila with the surface on which Streptomyces species had grown facing downward, and the culture was allowed to stand at 28℃for 24 hours, and the diameter of the inhibition zone was measured and recorded, and each experiment was repeated three times. As can be seen from fig. 8, no zone of inhibition appears on the aeromonas hydrophila medium, indicating that SH5 has no antagonism on aeromonas hydrophila.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> and university of south China
<120> a strain of Streptomyces maritimus and its use in preventing and controlling aeromonas hydrophila infection of aquatic animals
<160> 59
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1374
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 16S rDNA sequence of Streptomyces sp.SH5 Strain
<400> 1
cgaacgatga agccgcttcg gtggtggatt agtggcgaac gggtgagtaa cacgtgggca 60
atctgccctt cactctggga caagccctgg aaacggggtc taataccgga taacactctg 120
tcccgcatgg gacggggttg aaagctccgg cggtgaagga tgagcccgcg gcctatcagc 180
ttgttggtgg ggtaatggcc taccaaggcg acgacgggta gccggcctga gagggcgacc 240
ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt ggggaatatt 300
gcacaatggg cgaaagcctg atgcagcgac gccgcgtgag ggatgacggc cttcgggttg 360
taaacctctt tcagcaggga agaagcgaaa gtgacggtac ctgcagaaga agcgccggct 420
aactacgtgc cagcagccgc ggtaatacgt agggcgcaag cgttgtccgg aattattggg 480
cgtaaagagc tcgtaggcgg cttgtcacgt cggatgtgaa agcccggggc ttaaccccgg 540
gtctgcattc gatacgggct agctagagtg tggtagggga gatcggaatt cctggtgtag 600
cggtgaaatg cgcagatatc aggaggaaca ccggtggcga aggcggatct ctgggccatt 660
actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 720
gccgtaaacg ttgggaacta ggtgttggcg acattccacg tcgtcggtgc cgcagctaac 780
gcattaagtt ccccgcctgg ggagtacggc cgcaaggcta aaactcaaag gaattgacgg 840
gggcccgcac aagcagcgga gcatgtggct taattcgacg caacgcgaag aaccttacca 900
aggcttgaca tataccggaa agcatcagag atggtgcccc ccttgtggtc ggtatacagg 960
tggtgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1020
caacccttgt tctgtgttgc cagcatgccc ttcggggtga tggggactca caggagactg 1080
ccggggtcaa ctcggaggaa ggtggggacg acgtcaagtc atcatgcccc ttatgtcttg 1140
ggctgcacac gtgctacaat ggccggtaca atgagctgcg atgccgcgag gcggagcgaa 1200
tctcaaaaag ccggtctcag ttcggattgg ggtctgcaac tcgaccccat gaagtcggag 1260
ttgctagtaa tcgcagatca gcattgctgc ggtgaatacg ttcccgggcc ttgtacacac 1320
cgcccgtcac gtcacgaaag tcggtaacac ccgaagccgg tggcccaacc cctt 1374
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 27F
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 1492R
<400> 3
tacggttacc ttgttacgac tt 22
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> aerA-F
<400> 4
gagaaggtga ccaccaagaa ca 22
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> aerA-R
<400> 5
arctgacatc ggccttgaac tc 22
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ast-F
<400> 6
ctatgagctg agcgatggca 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ast-R
<400> 7
tcccgtcgaa cttgaagtgg 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ahp-F
<400> 8
tctatgcgct ggagtcgttc 20
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ahp-R
<400> 9
aggacatgcc cacgttgtag 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> act-F
<400> 10
tcaaggccga tgtcagctat 20
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> act-R
<400> 11
gtcccactgg taacgaatgc 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> hly-F
<400> 12
tctacctcaa cgtcaaccgc 20
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> hly-R
<400> 13
tccgcactat cttggcatcc 20
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ompA-F
<400> 14
tggatctgca agctcgttac 20
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ompA-R
<400> 15
ctacgtagga agtgcggaac 20
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ompW-F
<400> 16
tacttcggtg atgccaacag 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ompW-R
<400> 17
cattgatcgc catgtccaga 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> fur-F
<400> 18
attggtctcg ctaccgtcta 20
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> fur-R
<400> 19
cggagaactc gatcaccttg 20
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ahyR-F
<400> 20
gcggtgatga acgacagtat 20
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ahyR-R
<400> 21
gcagaccttg cccatttact 20
<210> 22
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> alt-F
<400> 22
tggatgccga gcagaacat 19
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> alt-R
<400> 23
ctctttcacc gaagtcacgc 20
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ela-F
<400> 24
taccgcaact ggtacaacac 20
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> ela-R
<400> 25
cggagttctg ctcggtaaag 20
<210> 26
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> EF1α-F
<400> 26
aacagctgat cgttggagtc aa 22
<210> 27
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> EF1α-R
<400> 27
ttgatgtatg cgctgacttc ct 22
<210> 28
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IL-1β-F
<400> 28
tggacttcgc agcacaaaat g 21
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IL-1β-R
<400> 29
cacttcacgc tcttggatga 20
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IL-6-F
<400> 30
tcaacttctc cagcgtgatg 20
<210> 31
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IL-6-R
<400> 31
tctttccctc ttttcctcct g 21
<210> 32
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Lysozyme-F
<400> 32
cgtggatgtc ctcgtgtgaa g 21
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Lysozyme-R
<400> 33
ccaatggaga atccctcaaa 20
<210> 34
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> TLR1-F
<400> 34
cagagcgaat ggtgccacta t 21
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> TLR1-R
<400> 35
gtggcagagg ctccagaaga 20
<210> 36
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> TLR3-F
<400> 36
tggagcatca cagggataaa ga 22
<210> 37
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> TLR3-R
<400> 37
tgatgcccat gcctgtaaga 20
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> SOD1-F
<400> 38
gtcgtctggc ttgtggagtg 20
<210> 39
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> SOD1-R
<400> 39
tgtcagcggg ctagtgctt 19
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Gpx1a-F
<400> 40
gctttgaggc acaacagtca 20
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Gpx1a-R
<400> 41
tctcccataa gggacacagg 20
<210> 42
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HSP70-F
<400> 42
aagcgacgaa ggatgcagga g 21
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HSP70-R
<400> 43
cacgttgcgc tctgaggatt 20
<210> 44
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> TLR4a-F
<400> 44
tttcacaaga acaagccttt g 21
<210> 45
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> TLR4a-R
<400> 45
tccacaagaa caagcctttg 20
<210> 46
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IAP-F
<400> 46
atgggagtgc cacggtttca g 21
<210> 47
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IAP-R
<400> 47
cgatgccaac agactttcct tg 22
<210> 48
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Bcl-2-F
<400> 48
aggaaaatgg aggttgggat g 21
<210> 49
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Bcl-2-R
<400> 49
tgttaggtat gaaaacgggt gga 23
<210> 50
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> TNFα-F
<400> 50
accaggcctt ttcttcaggt 20
<210> 51
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> TNFα-R
<400> 51
tgcccagtct gtctccttct 20
<210> 52
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> iNOs-F
<400> 52
ggagatgcaa ggtcagcttc 20
<210> 53
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> iNOs-R
<400> 53
ggcaaagctc agtgacttcc 20
<210> 54
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> MyD88-F
<400> 54
aacaacttcg ctggataa 18
<210> 55
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> MyD88-R
<400> 55
gttactggaa tcgcctca 18
<210> 56
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> aerA2-F
<400> 56
cgccagctgg tcaagactgt 20
<210> 57
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> aerA2-R
<400> 57
ccagttggtg gctgtgtcgt 20
<210> 58
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> SH5-F
<400> 58
taacactctg tcccgcatgg 20
<210> 59
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> SH5-R
<400> 59
ttaccccacc aacaagctga 20
Claims (5)
1. A strain of marine streptomyces, characterized in that: the name is streptomyceteStreptomycessp.) SH5, 11 months 12 days 2021, deposited with the Guangdong province microbiological strain collection center, accession number: GDMCC No:62057.
2. a biological agent prepared based on the marine streptomyces species of claim 1.
3. The biologic of claim 2, wherein:
the biological agent is prepared by inoculating the marine streptomycete in the invention 1 into ISP1 liquid culture medium for culture.
4. A biologic according to claim 3, characterized in that:
the culture means 48-72 h under the conditions of 28-30 ℃ and 180-200 rpm.
5. Use of a Streptomyces maritimus according to claim 1 or a biological agent according to any one of claims 2 to 4, characterized in that: the application is one of the following applications:
1) Use of a marine streptomyces sp according to claim 1 or a biological agent according to any one of claims 2 to 4 for the preparation of a medicament for inhibiting aeromonas hydrophila in zebra fish intestinal colonization;
2) Use of a marine streptomyces of claim 1 or a biological agent of any one of claims 2 to 4 in the manufacture of a medicament for treating aeromonas hydrophila mediated inflammation of zebra fish;
3) Use of a marine Streptomyces according to claim 1 or a biological agent according to any one of claims 2 to 4 for the preparation of a reagent for inhibiting the expression of the virulence gene of Aeromonas hydrophilaact，ast，hly，ahp，fur。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202111640257.XA CN114292785B (en) | 2021-12-29 | 2021-12-29 | Marine streptomycete and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111640257.XA CN114292785B (en) | 2021-12-29 | 2021-12-29 | Marine streptomycete and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals |
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| CN114292785B true CN114292785B (en) | 2023-07-28 |
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Citations (6)
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|---|---|---|---|---|
| CN101497867A (en) * | 2009-02-18 | 2009-08-05 | 淮海工学院 | Isolated culture method and use of Streptomycete from sea |
| CN104087523A (en) * | 2014-01-20 | 2014-10-08 | 中国科学院南海海洋研究所 | Marine Streptomyces, method for preparation of Enterocin therefrom and application of the same |
| CN106472932A (en) * | 2016-10-18 | 2017-03-08 | 李德田 | A kind of complex microorganism beverage and preparation method thereof |
| CN111979150A (en) * | 2020-08-21 | 2020-11-24 | 华南农业大学 | Marine streptomyces and isolated culture method and application thereof |
| WO2021209820A1 (en) * | 2020-04-15 | 2021-10-21 | Institut Pasteur | Bacterial strains for use as probiotics, compositions thereof, deposited strains and method to identify probiotic bacterial strains |
| CN113621533A (en) * | 2021-07-09 | 2021-11-09 | 湖南师范大学 | Streptomyces rubiginosus Z1-26, microecological preparation and preparation method thereof |
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2021
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Patent Citations (6)
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| CN101497867A (en) * | 2009-02-18 | 2009-08-05 | 淮海工学院 | Isolated culture method and use of Streptomycete from sea |
| CN104087523A (en) * | 2014-01-20 | 2014-10-08 | 中国科学院南海海洋研究所 | Marine Streptomyces, method for preparation of Enterocin therefrom and application of the same |
| CN106472932A (en) * | 2016-10-18 | 2017-03-08 | 李德田 | A kind of complex microorganism beverage and preparation method thereof |
| WO2021209820A1 (en) * | 2020-04-15 | 2021-10-21 | Institut Pasteur | Bacterial strains for use as probiotics, compositions thereof, deposited strains and method to identify probiotic bacterial strains |
| CN111979150A (en) * | 2020-08-21 | 2020-11-24 | 华南农业大学 | Marine streptomyces and isolated culture method and application thereof |
| CN113621533A (en) * | 2021-07-09 | 2021-11-09 | 湖南师范大学 | Streptomyces rubiginosus Z1-26, microecological preparation and preparation method thereof |
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| Study on peptide hydrolysis by aminopeptidases from Streptomyces griseus, Streptomyces septatus and Aeromonas proteolytica;Jiro Arima等;《Appl Microbiol Biotechnol.》;第70卷(第05期);第541-547页 * |
| 水霉拮抗菌的筛选与拮抗物特点初步分析;雷翠霞等;《生态科学》;第32卷(第02期);第189-193页 * |
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| CN114292785A (en) | 2022-04-08 |
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