Bacillus subtilis for enhancing salt stress tolerance of fish and application thereof

Abstract

The application discloses a bacillus subtilis for enhancing salt stress resistance of fish and application thereof, and the bacillus subtilis SCAU-Ohb2 is preserved in the Guangdong Provincial Microbial Culture Collection Center on December 13, 2021, and the preservation number is GDMCC NO:62120. The bacillus subtilis SCAU-Ohb2 is safe and harmless to fish, and can significantly improve the survival rate of fish under acute salt stress and the salt tolerance of gill tissue. The application provides a new choice for enhancing the salt stress resistance of aquatic animals, and provides a new method for cultivating fish germplasm with salt-tolerant excellent traits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic editing technology and crop breeding, in particular, to a Bacillus subtilis strain for enhancing the salt stress tolerance of fish and its application. BACKGROUND

[0002] Salinity is an important ecological factor affecting the living environment of aquatic organisms such as fish, and is also an important abiotic factor affecting various physiological activities (such as respiration, metabolism, growth, etc.) of fish, forcing fish to adjust their physiological activities from the genetic level to the cellular level, from enzyme content to hormone content, to maintain the dynamic balance of body fluid osmotic pressure. Salt stress can cause stress reactions in fish, such as increased energy consumption, accelerated metabolism, increased oxygen consumption, etc., and can lead to many consequences: (1) Changes in fish osmotic pressure, forming osmotic stress and ionic stress; (2) Increase of active oxygen free radicals (ROS) in fish, forming oxidative stress; (3) Fish body quality will deteriorate, immune ability and resistance will decrease, and it will be more susceptible to diseases and even death.

[0003] At present, how fish maintains water and ion balance when facing various osmotic stresses has been studied in depth. For fish, salt tolerance can be improved through salt tolerance training, breeding of salt-tolerant varieties, and induction of salt tolerance by biochemical substances (such as organic osmotic agents, etc.). In plants, many studies have reported that rhizosphere growth-promoting bacteria such as Bacillus subtilis can improve plant salt tolerance, but there is no report on feeding Bacillus subtilis to improve the salt tolerance of aquatic animal hosts. SUMMARY

[0004] The technical problem to be solved by the present application is to screen probiotics that can improve the salt stress tolerance of fish, and to provide a Bacillus subtilis strain for enhancing the salt stress tolerance of fish and its application.

[0005] The first object of the present application is to provide a Bacillus subtilis strain SCAU-Ohb2.

[0006] The second object of the present application is to provide an application of the above-mentioned Bacillus subtilis strain SCAU-Ohb2 in the preparation of a product for enhancing the salt stress tolerance of fish.

[0007] The third object of the present application is to provide a microbial preparation.

[0008] The fourth object of the present application is to provide an application of the above-mentioned microbial preparation in enhancing the salt stress tolerance of fish.

[0009] The fifth object of the present application is to provide a functional feed.

[0010] The sixth object of the present application is to provide a method for enhancing the salt stress tolerance of fish.

[0011] To achieve the above-mentioned purpose, the present application is realized by the following scheme:

[0012] The present application screens a bacillus subtilis strain from the intestinal contents of tilapia, identifies it by 16s rDNA, and performs whole genome sequencing, assembly and annotation, and at the same time, counts the number of compatible solute key genes in the whole genome of the strain. Subsequently, in order to explore the influence of bacillus subtilis on the salt stress tolerance of tilapia, the tilapia is fed with feed added with bacillus subtilis for 21 consecutive days, and after 21 days, the tilapia is subjected to acute salt stress treatment, and the morphological structure of the gill tissue of the tilapia, the salt tolerance survival rate and the detection of the antioxidant capacity of the liver of the tilapia are observed. Finally, in order to further analyze the influence of the time of feeding the tilapia with feed added with bacillus subtilis on the salt stress tolerance of the tilapia, the tilapia is fed with feed added with bacillus subtilis for 3 consecutive days, and after 3 days, the tilapia is subjected to acute salt stress treatment, and the cumulative mortality rate of the tilapia after salt stress is determined.

[0013] Therefore, the present application claims the following content:

[0014] A bacillus subtilis strain (Bacillus subtilis) SCAU-Ohb2, preserved in the Guangdong Microbial Culture Collection Center on December 13, 2021, with the preservation number GDMCC NO:62120.

[0015] Preferably, the genome of the bacillus subtilis SCAU-Ohb2 contains 1 betaine dehydrogenase gene, 3 pyrroline-5-carboxylate reductase genes and 2 proline dehydrogenase genes.

[0016] The application of the above-mentioned bacillus subtilis SCAU-Ohb2 in the preparation of a product for enhancing the salt stress tolerance of fish.

[0017] Preferably, the enhancement of the salt stress tolerance of fish is to improve the survival rate of fish in a salt stress environment and / or to enhance the salt tolerance of the gill tissue of fish.

[0018] A microbial preparation containing the above-mentioned bacillus subtilis SCAU-Ohb2.

[0019] Preferably, the effective viable count of bacillus subtilis SCAU-Ohb2 in the microbial preparation is 0.8x10 7 ~1.2x10 7 CFU / g.

[0020] More preferably, the effective viable count of bacillus subtilis SCAU-Ohb2 in the microbial preparation is 1x10 7 CFU / g.

[0021] The application of the above-mentioned microbial preparation in enhancing the salt stress tolerance of fish.

[0022] Preferably, the enhanced salt stress tolerance of fish is to improve the survival rate of fish in a salt stress environment and / or to enhance the salt tolerance of gill tissue of fish.

[0023] A functional feed containing the above-mentioned Bacillus subtilis SCAU-Ohb2.

[0024] Preferably, the effective viable count of Bacillus subtilis SCAU-Ohb2 in the functional feed is 0.8 x 10 7 ~ 1.2 x 10 7 CFU / g.

[0025] More preferably, the effective viable count of Bacillus subtilis SCAU-Ohb2 in the microbial preparation is 1 x 10 7 CFU / g.

[0026] A method for enhancing the salt stress tolerance of fish, the method being to administer one or more of the above-mentioned Bacillus subtilis SCAU-Ohb2, microbial preparation and functional feed to fish.

[0027] The technical scheme of the present application has the following beneficial effects:

[0028] The present application discloses a Bacillus subtilis for enhancing the salt stress tolerance of fish and its application. The Bacillus subtilis SCAU-Ohb2 was preserved in the Guangdong Microbial Culture Collection Center on December 13, 2021, and the preservation number is GDMCC NO:62120. The Bacillus subtilis SCAU-Ohb2 is not only safe and harmless to fish, but also can significantly improve the survival rate of fish under acute salt stress and the salt tolerance of gill tissue. The present application provides a new choice for enhancing the salt stress tolerance of fish, and also provides a new method for cultivating fish germplasm with excellent salt tolerance. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a genome circle diagram of Bacillus subtilis SCAU-Ohb2.

[0030] Figure 2 It is an evolutionary tree analysis of Bacillus subtilis SCAU-Ohb2.

[0031] Figure 3 It is the effect of feeding fish with feed added with different Bacillus subtilis for 21 consecutive days on the salt stress tolerance of tilapia.

[0032] Figure 4Effects of freshwater treatment and salt stress treatment on gill tissue morphology of tilapia after feeding with feed added with different Bacillus for 21 days consecutively; A): gill filament section of the control group without added bacteria after freshwater treatment, B): gill filament section of the control group without added bacteria after salt stress treatment, C): gill filament section of the Bacillus subtilis SCAU-Ohb2 group after freshwater treatment, D): gill filament section of the Bacillus subtilis SCAU-Ohb2 group after salt stress treatment, E): gill filament section of the Bacillus licheniformis SCAU-090 group after freshwater treatment, F): gill filament section of the Bacillus licheniformis SCAU-090 group after salt stress treatment; the red arrow is a chlorocyte, and the blue arrow is a gill filament bending deformation.

[0033] Figure 5 Effects of freshwater treatment and salt stress treatment on the antioxidant capacity of the liver of tilapia after feeding with feed added with different Bacillus for 21 days consecutively; A): effect on the malondialdehyde (MDA) activity of the liver of tilapia, B): effect on the superoxide dismutase (SOD) activity of the liver of tilapia, C): effect on the total antioxidant (T-AOC) activity of the liver of tilapia.

[0034] Figure 6 Effects of freshwater treatment and salt stress treatment on the mortality rate of tilapia after feeding with feed added with different Bacillus for 3 days consecutively. DETAILED DESCRIPTION

[0035] The present application will be further described in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] Example 1: Screening and identification of Bacillus subtilis SCAU-Ohb2

[0038] I. Experimental methods

[0039] The intestinal contents of tilapia were scraped with tweezers into a sterile 10 ml centrifuge tube, and an appropriate amount of LB liquid medium was mixed. The centrifuge tube was placed in a 75℃ water bath for 15 min, and the sample was gradient diluted, and the appropriate gradient of mixed bacteria was coated on the LB solid plate and incubated in a 30℃ biochemical incubator for 24 h, and the strain was selected.

[0040] The strain obtained by the above screening was inoculated into LB liquid medium for culture, centrifuged at 10,000 rpm for 1 min to discard the supernatant medium, and the bacterial cells were collected in a 1.5 mL centrifuge tube. According to the operation manual of the genomic extraction kit, after extracting the genomic DNA, PCR amplification was performed using 16S rDNA universal primers (341F: CCTAYGGGRBGCASCAG, 806R: GGACTACNNGGGTATCTAAT). After purification of the PCR product, 16S rDNA gene sequence sequencing was performed.

[0041] II. Experimental results

[0042] The sequencing results were subjected to nucleotide sequence homology comparison analysis with the related sequences registered in GenBank, and the results showed that the similarity of the BLAST results of the strain obtained by screening with Bacillus subtilis (CP028217) was 100%, indicating that the strain belongs to Bacillus subtilis.

[0043] The strain was preserved in the Guangdong Microbial Culture Collection Center on December 13, 2021, with the preservation number GDMCC NO:62120, and was classified and named as Bacillus subtilis SCAU-Ohb2.

[0044] A strain of Bacillus was also screened from the intestinal contents of tilapia, which was classified and named as Lysinibacillus sphaericus SCAU-090, and its 16S rDNA sequence was registered in Genbank with the accession number MK281536.

[0045] Example 2 Bacillus subtilis (B. subtilis SCAU-Ohb2) genome sequencing, assembly and annotation and main salt-tolerant gene statistics

[0046] I. Experimental methods

[0047] The extracted genomic DNA of Bacillus subtilis SCAU-Ohb2 was sent to Beijing Baimaikes Technology Co., Ltd. for sequencing, assembly and annotation of the whole genome. The sequencing experimental process was performed according to the standard protocol provided by Oxford Nanopore Technologies (ONT) company, including sample quality detection, library construction, library quality detection and library sequencing processes. Information analysis mainly includes the following steps: raw data quality control, genome assembly, genome component analysis and functional annotation, and genome mapping analysis.

[0048] Circos v0.66 software was used to draw the visual genome circle map to explore the location relationship between the genome components on the genome, and analyze tRNA, rRNA, repeat sequence, GC content and gene function information, etc.

[0049] II. Experimental results

[0050] The genome assembly Scaffold / Contig length of Bacillus subtilis SCAU-Ohb2 is 4168067 bp, and 4143 genes are predicted to be encoded, and the result statistics are shown in Table 1.

[0051] Table 1. Genome assembly and gene prediction results statistics of Bacillus subtilis SCAU-Ohb2

[0052]

[0053] The genome circle map of Bacillus subtilis SCAU-Ohb2 is shown in Figure 1 . The outermost circle of the genome circle map is the size of the genome, and each scale is 5 kb; the second and third circles are genes on the positive and negative strands of the genome, respectively, and different colors represent different COG functional classifications; the fourth circle is the repeat sequence; the fifth circle is tRNA (blue) and rRNA (purple); the sixth circle is the GC content, and the light yellow part indicates that the GC content of the region is higher than the average GC content of the genome, and the higher the peak value, the greater the difference from the average GC content, and the blue part indicates that the GC content of the region is lower than the average GC content of the genome; the innermost circle is GC-skew, and dark gray represents the region with more G content than C, and red represents the region with more C content than G. The phylogenetic tree analysis of 16S rRNA sequence shows that the genome contains 10 16S rRNA sequences, and clusters together with B. subtilis (CP028217) Figure 2

[0054] The results of statistical analysis of compatible solute production related genes in the whole genome of Bacillus subtilis SCAU-Ohb2 showed that there was 1 betaine dehydrogenase gene, 3 pyrroline-5-carboxylate reductase genes, and 2 proline dehydrogenase genes in the genome of Bacillus subtilis SCAU-Ohb2.

[0055] Another strain of bacteria, Lysinibacillus sphaericus SCAU-090, screened, has 1 pyrroline-5-carboxylate reductase gene, 3 proline dehydrogenase genes, and no betaine dehydrogenase gene (Table 2). This indicates that the ability of Bacillus subtilis SCAU-Ohb2 to produce or accumulate organic osmotic agents betaine and proline is greater than that of Lysinibacillus sphaericus SCAU-090. ​

[0056] Table 2 Statistics of compatible solute key genes in the whole genome of Bacillus subtilis SCAU-Ohb2 Example 3 Preparation of Bacillus fermentation broth and preparation of feed

[0057] Bacillus subtilis SCAU-Ohb2 and Bacillus licheniformis SCAU-090 were inoculated into LB liquid medium respectively, and cultured at 30°C on a shaker at a speed of 160 r / min for 24 h. Using a hemocytometer slide to determine the number of bacteria in each group, and the bacteria were prepared into a bacterial solution with a concentration of 1 x 10 8 CFU / mL using sterile LB medium.

[0058] Commercially available fish feed (China Tongwei Co., Ltd., Model 150) was used as the source of the feed. The ingredients of the commercial fish feed included crude protein 38%, crude fiber 3%, crude fat 4%, water 10%, and phosphorus 1%.

[0059] The commercial fish feed was treated as follows:

[0060] The control group without bacteria: 50 mL of sterile LB culture solution was sprayed on each 500 g of feed, and mixed evenly;

[0061] The Bacillus subtilis SCAU-Ohb2 group: 50 mL of bacterial solution containing Bacillus subtilis SCAU-Ohb2 was sprayed on each 500 g of feed, and mixed evenly, to finally prepare feed containing 1 x 10 7 CFU / g of Bacillus subtilis SCAU-Ohb2;

[0062] The Bacillus licheniformis SCAU-090 group: 50 mL of bacterial solution containing Bacillus licheniformis SCAU-090 was sprayed on each 500 g of feed, and mixed evenly, to finally prepare feed containing 1 x 10 7 CFU / g of Bacillus licheniformis SCAU-090;

[0063] The feed was packaged in airtight containers and stored at -20°C.

[0064] Example 4 Effect of feeding feed added with Bacillus subtilis SCAU-Ohb2 on the salt stress tolerance of tilapia

[0065] I. Effect of feeding feed added with Bacillus subtilis SCAU-Ohb2 on the growth of tilapia for 21 consecutive days

[0066] 1. Experimental method

[0067] The feed prepared in Example 3 for the control group without bacterial growth, the Bacillus subtilis SCAU-Ohb2 group, and the Bacillus spheroides lysinophilus SCAU-090 group were fed to tilapia fry in equal amounts (approximately 3g), once in the morning and once in the evening, for 21 consecutive days. After 21 days, the tilapia were weighed to determine their growth performance.

[0068] 2. Experimental Results

[0069] As shown in Table 3, after feeding tilapia fry with diets supplemented with Bacillus subtilis SCAU-Ohb2 and Bacillus spheroidis lysine SCAU-090 for 21 consecutive days, there was no significant difference in body weight gain between the two groups of tilapia and the control group without the bacteria (P>0.05). This indicates that Bacillus subtilis SCAU-Ohb2 has no significant side effects on body weight gain during the growth process of tilapia, and that Bacillus subtilis SCAU-Ohb2 has no toxic effect on the growth of tilapia.

[0070] Table 3. Effects of Bacillus on Tilapia Weight Gain

[0071]

[0072] II. Salt tolerance survival rate of tilapia fed with Bacillus subtilis SCAU-Ohb2 for 21 consecutive days

[0073] 1. Experimental Methods

[0074] The three groups of tilapia were each divided into two parts, and placed in fresh water and 2.5% saline (w / v) respectively. The mortality rates of the tilapia after fresh water and acute salt stress treatment were counted to analyze the differences in their salt stress tolerance. No feed was given during the acute salt stress experiment, and the number of fish deaths was recorded. The main measurement was the cumulative mortality rate after acute salt stress, calculated as: Cumulative Mortality Rate % = (Number of fish that died after stress / Number of fish that died before stress) × 100%. This was used as an indicator of salt stress tolerance.

[0075] 2. Experimental Results

[0076] The results are as follows Figure 3 As shown, at 2 h and 2.5 h of salt stress, the mortality rates in the control group without added bacteria were 16.7% and 40.5%, respectively; the mortality rates in the Bacillus spheroides SCAU-090 group were 12% and 33%, respectively; and the mortality rates in the Bacillus subtilis SCAU-Ohb2 group were both 2%.

[0077] After 3 hours of salt stress, the mortality rate was 95% in the control group without added bacteria, 62% in the Bacillus spheroides SCAU-090 group, and 40% in the Bacillus subtilis SCAU-Ohb2 group.

[0078] The mortality rate of Bacillus licheniformis SCAU-Ohb2 group was 62% while the mortality rate of Bacillus licheniformis SCAU-090 group was 83%.

[0079] The results show that the salt stress tolerance of tilapia is improved after feeding the feed added with Bacillus, and the effect of Bacillus licheniformis SCAU-Ohb2 is better.

[0080] III. Analysis of gill tissue morphology

[0081] 1. Experimental method

[0082] To explore the effect of Bacillus licheniformis SCAU-Ohb2 on the tissue morphology of tilapia after acute salt stress, the gill tissue morphology of tilapia in the non-inoculum control group, Bacillus licheniformis SCAU-Ohb2 group and Bacillus licheniformis SCAU-090 group after freshwater treatment and salt stress treatment was observed.

[0083] Sampling was started at the half-lethal time of high salt stress, and the fish surface was sterilized with 70% ethanol. Gill tissue samples from 5 tilapia in each group were collected under sterile conditions and placed in 4% formalin solution (v / v) for histological section analysis.

[0084] The gill tissue samples were taken out from the 4% formalin solution (v / v), dehydrated with different concentrations of alcohol (50%→70%→80%→95%), and completed in xylene solution. Paraffin embedding was performed, and 5 μm sections were cut and stained with HE. Finally, the images were collected and photographed.

[0085] 2. Experimental results

[0086] The gill tissue morphology is shown in A-F of Figure 4 . The results show that under freshwater conditions, the gill filaments of the three groups are relatively complete, symmetrical, natural and relaxed (A, C and E in Figure 4 ), and the chloride cells are not obvious. After acute salt stress, the gill filaments of the non-inoculum control group and Bacillus licheniformis SCAU-090 group are more deformed and curved (B and F in Figure 4 ), while the gill filaments of Bacillus licheniformis SCAU-Ohb2 group are similar to those under freshwater conditions, basically without bending, relatively uniform and orderly (D in Figure 4 ), and the chloride cells are particularly obvious in the control group after acute salt stress Figure 4B) in FIG. 2. This indicates that the salt tolerance of the gill filament of the fish in the B. subtilis SCAU-Ohb2 group is enhanced after acute salt stress, compared to the no bacteria control group and the B. licheniformis SCAU-090 group.

[0087] IV. Analysis of antioxidant capacity of liver

[0088] 1. Experimental method

[0089] To study the effect of B. subtilis SCAU-Ohb2 on the antioxidant capacity of the liver of the tilapia after acute salt stress, the antioxidant-related indicators of the liver of the tilapia in the no bacteria control group, the B. subtilis SCAU-Ohb2 group and the B. licheniformis SCAU-090 group after freshwater treatment and salt stress treatment were detected, respectively.

[0090] Sampling was started at the time of high salt stress semi-lethality, and the fish surface was disinfected with 70% ethanol, and sampling was performed under sterile conditions. Liver tissue of 5 tilapias in each group was collected for digestive enzyme and antioxidant enzyme activity detection.

[0091] The enzyme activity indicators of the tilapia tissue were determined according to the enzyme activity indicator instructions of the reagent kit of Nanjing Jiancheng Bioengineering Institute. The tissue was homogenized in pre-cooled physiological saline, centrifuged at 2500 rpm for 10 min at 4°C, and the supernatant was collected. The malondialdehyde (MDA) (Nanjing Jiancheng, A003-1-2), superoxide dismutase (SOD) (Nanjing Jiancheng, A001-3-2) and total antioxidant capacity (T-AOC) (Nanjing Jiancheng, A015-2-1) activities of the liver tissue of the tilapia were determined. The above enzyme activity indicator data were expressed as nmol / ml (MDA), U / ml (SOD) and mM (T-AOC), respectively.

[0092] 2. Experimental results

[0093] The results are shown in FIG. 3. Figure 5 For MDA activity, there was no significant difference in MDA activity of the no bacteria control group after freshwater treatment and acute salt stress treatment, while the MDA activity of the B. subtilis SCAU-Ohb2 group significantly increased after acute salt stress treatment.

[0094] For SOD activity, compared to the no bacteria control group, the SOD activity of the B. subtilis SCAU-Ohb2 group did not significantly increase after freshwater treatment and acute salt stress treatment.

[0095] For T-AOC activity, there was no significant change in T-AOC activity of the three groups after freshwater treatment and acute salt stress treatment.

[0096] The above results show that, in general, compared with the non-inoculated control group, feeding the feed added with Bacillus subtilis SCAU-Ohb2 has little effect on the antioxidant capacity of the liver of tilapia.

[0097] Example 5 Effect of feeding the feed added with Bacillus subtilis SCAU-Ohb2 for a short time on the salt stress tolerance of tilapia

[0098] I. Experimental method

[0099] To further improve the application in production, the effect of feeding the feed added with Bacillus subtilis SCAU-Ohb2 for a short time (3 days) on the salt stress tolerance of tilapia was analyzed. The feed prepared in Example 3 was fed to tilapia (about 30 g) in equal amounts, once in the morning and once in the evening every day, for 3 days.

[0100] The above three groups of tilapia were each divided into two parts and placed in fresh water and 2.5% salt water (w / v), respectively. The mortality rate of tilapia after acute salt stress was counted to analyze the difference in salt stress tolerance. No feed was fed during the acute salt stress experiment, and the number of fish deaths during the experiment was recorded. The cumulative mortality rate after acute salt stress was mainly determined, and the formula was: cumulative mortality rate % = number of fish deaths after stress / number of fish tails before stress x 100%, which was used as an index of salt stress tolerance.

[0101] II. Experimental results

[0102] The results are shown in Table 1. Figure 6 As shown in Table 1, at 5h of salt stress, the mortality rate of tilapia in the non-inoculated control group was 24%, and the mortality rate of tilapia in the Bacillus subtilis SCAU-Ohb2 group was 2%.

[0103] At 5.5h of salt stress, the mortality rate of tilapia in the non-inoculated control group was 42%, and the mortality rate of tilapia in the Bacillus subtilis SCAU-Ohb2 group was 18%.

[0104] At 8.5h of salt stress, all tilapia in the non-inoculated control group died; at 9h of salt stress, all tilapia in the Bacillus licheniformis SCAU-090 group died; at 11h of salt stress, all tilapia in the Bacillus subtilis SCAU-Ohb2 group died. This indicates that feeding Bacillus subtilis SCAU-Ohb2 can improve the salt stress tolerance of tilapia and delay the death time.

[0105] After feeding the tilapia with the feed added with Bacillus subtilis SCAU-Ohb2 for 3 days and 21 days continuously, although the time of acute salt stress death is different, compared with the group without adding bacteria, the feed added with Bacillus subtilis SCAU-Ohb2 can improve the salt stress tolerance of the tilapia.

[0106] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis SCAU-Ohb2, characterized in that, The gene of Bacillus subtilis SCAU-Ohb2 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 13, 2021, with accession number GDMCC NO: 62120. The genome of Bacillus subtilis SCAU-Ohb2 contains one betaine dehydrogenase gene, three pyrroline-5-carboxylic acid reductase genes and two proline dehydrogenase genes.

2. The use of Bacillus subtilis SCAU-Ohb2 as described in claim 1 in the preparation of products for enhancing the salt stress tolerance of tilapia.

3. The application according to claim 2, characterized in that, The enhancement of tilapia's salt stress tolerance refers to improving the survival rate of tilapia under salt stress and / or enhancing the salt tolerance of tilapia gill tissue.

4. A microbial preparation, characterized in that, The microbial preparation contains Bacillus subtilis SCAU-Ohb2 as described in claim 1.

5. The microbial preparation according to claim 4, characterized in that, The effective viable count of Bacillus subtilis SCAU-Ohb2 in the microbial preparation was 0.8 × 10⁻⁶. 7 ~1.2×10 7 CFU / g.

6. The use of the microbial preparation according to any one of claims 4 to 5 in enhancing the salt stress tolerance of tilapia.

7. The application according to claim 6, characterized in that, The enhancement of tilapia's salt stress tolerance refers to improving the survival rate of tilapia under salt stress and / or enhancing the salt tolerance of tilapia gill tissue.

8. A functional feed, characterized in that, The functional feed contains Bacillus subtilis SCAU-Ohb2 as described in claim 1.

9. The functional feed according to claim 8, characterized in that, The effective viable count of Bacillus subtilis SCAU-Ohb2 in the functional feed is 0.8 × 10⁻⁶. 7 ~1.2×10 7 CFU / g.

10. A method for enhancing the salt stress tolerance of tilapia, characterized in that, The method involves administering tilapia with one or more of the following: Bacillus subtilis SCAU-Ohb2 as described in claim 1, the microbial preparation as described in any one of claims 4-5, and the functional feed as described in any one of claims 8-9.

Images