Biocontrol Bacillus velezensis SF259 and application thereof
A technology of Bacillus, Veles, applied in the field of microbial technology application
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Embodiment 1
[0039] The acquisition of embodiment 1, Veles bacillus SF259
[0040] 1. Soil source
[0041] Soil of cherry trees in the orchard of the horticultural field in Ledu District, Qinghai Province, China
[0042] 2. Screening of bacterial strains
[0043] (1) Soil sample collection
[0044] Zig-shaped 5-point sampling method for soil sample collection: collect 200g soil sample at each point, mix the soil sample evenly, take 200g by quartering method and put it into a sterilization bag as a soil sample. Three soil samples were collected from each plot as replicates. Record the time, place and type of sampling. The collected soil samples were stored in a refrigerator at 4°C for use in bacterial isolation.
[0045] (2) Isolation of bacteria
[0046] Plate dilution method: Weigh 10g of soil sample into a Erlenmeyer flask, add 90mL of sterile water, then shake at 200rpm, 28°C shaker, take it out after 20min and let it stand at room temperature for 10min to make the stock solution ...
Embodiment 2
[0053] Example 2 Identification of the 16S rRNA gene of Bacillus Velez SF259
[0054] Genomic DNA of strain SF259 was extracted using primers:
[0055] 27F 5'-AGAGTTTGATCCTGGCTCAG-3', and
[0056] 1492R 5'-TACGGCTACCTTGTTACGACTT-3', with the extracted DNA as a template, PCR amplification was performed to obtain the target fragment. The PCR reaction system is:
[0057] Table 1 Taq polymerase chain reaction system
[0058]
[0059] The basic conditions of the PCR reaction were: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 90 s (1 kb / min), pre-extension at 72°C for 8 min, and storage at 4°C for a total of 30 cycles. After the reaction, the PCR products were checked by 1% agarose gel electrophoresis, and the gel imager was used to detect and record the results (see attached Figure 4 ). Send the PCR stock solution to Bosun Biotechnology (Shanghai) Co., Ltd. for sequencing. The sequencing results were ...
Embodiment 3
[0062] Example 3 Identification of the gyrA gene of Bacillus Velez SF259
[0063] The genomic DNA of the strain SF259 was extracted, and the primers: GyrA-F 5'-CAGTCAGGAAATGCGTACGTCCTT-3' and GyrA-R 5'-CAAGGTAATGCTCCAGGCATTGCT-3' were used to perform PCR amplification using the extracted DNA as a template to obtain the target fragment. The PCR reaction system is:
[0064] Table 2 Taq polymerase chain reaction system
[0065]
[0066]
[0067] Reaction conditions: 94°C 10min; 94°C 1min, 55°C 1min, 72°C 1min, 30 cycles; 72°C 10min, 10°C ∞. After the reaction, the PCR products were checked by 1% agarose gel electrophoresis, detected by a gel imager, and the results were recorded. Send the PCR stock solution to Bosun Biotechnology (Shanghai) Co., Ltd. for sequencing. The sequencing results were analyzed by DNAStar and compared with BLAST on the NCBI website to determine the species of closely related bacteria.
[0068]The results showed that the similarity between the gy...
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