Vibrio harveyi flrb gene silencing cell line and its application
A technology of Vibrio harveii and gene silencing, applied in the biological field, can solve the problems of loss of large yellow croaker aquaculture, reduced resistance of large yellow croaker, drug resistance of bacteria and other problems, and achieve the effect of reducing adhesion ability
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Embodiment 1
[0014] Example 1 Construction of Vibrio harveyi flrB gene silencing strain
[0015] The construction method of vibrio harveyi flrB gene silencing strain comprises the following steps:
[0016] 1) Design and synthesis of Vibrio harveyi flrB gene-specific shRNA:
[0017] F: 5-GATCCGCTTGCTACAGTCTCGTATCATTCAAGAGATGATACGAGACTGTAGCAAGCTTTTTTGCATG-3
[0018] R: 5'-CAAAAAAGCTTGCTACAGTCTCGTATCATCTCTTGAATGATAC GAGACTGTAGCAAGCG-3'
[0019] 2) annealing the shRNA to form a double-stranded RNA;
[0020] The annealing reaction system is:
[0021]
[0022] Annealing procedure:
[0023] 95℃ 2min
[0024] Decrease 0.1°C every 8s, down to 25°C for about 90min
[0025] 4℃Forever
[0026] 3) The pACYC184 plasmid was extracted and double digested with BamHI and SpHI enzymes, and the linearized plasmid was purified and recovered after agarose gel electrophoresis.
[0027] 4) The double-stranded RNA in step 2) is ligated with the recovered product in step 3) using T4 ligase, and heat-shoc...
Embodiment 2
[0035] Embodiment 2 biofilm formation experiment
[0036] First adjust the OD550 of the Vibrio harveyi cheA gene silencer strain and the control strain cultivated overnight to about 0.2, and add 100 μl of the bacteria solution to a 96-well microtiter plate. After incubating at 28°C for 24h, wash the wells twice with sterile PBS to remove bacteria that did not adhere to the well plate, dry at 60°C, add 125ul 0.1% crystal violet for room temperature staining for 15min, then wash the wells three times with PBS, add 200uL 33% (v / v) glacial acetic acid, measure OD590 with a microplate reader. The experiment was repeated twice, and each experiment was performed in parallel three times.
[0037] The results of the biofilm formation experiment: the biofilm formation ability of the stably silenced strain flrB-RNAi was significantly reduced, the OD590 value of the wild strain was 0.68, and that of the cheA gene silenced strain was 0.33. like figure 1 shown.
Embodiment 3
[0038] Embodiment 3 Adhesion experiment
[0039] Large yellow croaker mucus (20 μL) was evenly added to a glass slide (22 mm×22 mm) and fixed with methanol for 20 minutes at room temperature. The bacterial suspension was adjusted to a final concentration of OD 600 =0.3 (3.0×10 8 CFU / mL) using sterile PBS. Spread 200 μL of the bacterial suspension evenly on the mucus-coated glass slides, then incubate at 28 °C for 2 h and wash three times with PBS. Bacteria were fixed with 4% methanol for 30 min and bacteria were stained with 1% crystal violet for 3 min. The slides were then examined under a light microscope (× 1000), and 20 microscope fields were selected to count bacteria. Sterile PBS was used as a negative control, and the control strain V. harveyi was used as a positive control. Three trials were performed for each group.
[0040] Adhesion test results: the adhesion amount of the wild strain was 1081.22cells / field of view, and that of the silent strain was 458.67cells / f...
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