Method for producing T-2 and HT-2 toxin by using toxigenic fusarium graminearum
A technology of HT-2 and Fusarium, which is applied in the direction of microbial-based methods, biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of affecting the production of toxins of strains, and the inability to accurately determine the best action conditions, etc. , to achieve the effect of simple method and high yield of toxin
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Embodiment 1
[0067] Example 1.tri5 gene positive (tri5 + ) Fusarium screen
[0068] (1) Put all the tested Fusarium bacteria into 1.5mL Eppendorf tubes containing 1mL GYM liquid medium, and culture them in a constant temperature incubator at 25°C for 3 days, and culture for slow-growing strains for 5 days. Genomic DNA of Fusarium spp. was extracted by micro-drill genomic DNA rapid extraction method, and the genomic DNA of Fusarium spp. For specific PCR, a 25uL PCR reaction system was used to cycle 40 times according to the program of 94°C for 15s, 62°C for 15s, and 72°C for 45s. Before that, a cycle of 94°C for 75s was added, and finally a cycle of 72°C for 4min15s was added for tri5-PCR amplification. The gel imaging system (GBS8000, UVP Company, USA) was used to image and detect the PCR products of 2% agarose gel electrophoresis, as shown in the attached figure 1 As shown, the resulting Fusarium strain with bright bands is tri5 + strains that can be considered to have the potential t...
Embodiment 2
[0070] Example 2. Screening of optimal culture conditions
[0071] Choose a tri5 + The strain (IBE006019) was used as the experimental object, and the orthogonal experiment design method was used to explore the toxin-producing culture conditions for it to determine the best toxin-producing culture conditions for Fusarium toxin-producing culture.
[0072] Orthogonal experimental design method was used to explore the culture conditions of Fusarium toxin production: experimental design with four factors and five levels, using L 25 (5 6 ) orthogonal table, the factors and levels are as follows:
[0073] Temperature: 5°C, 20°C, 35°C, 5-20°C, 5-35°C;
[0074] Lighting: light, darkness, light-dark 24h alternation, early light and late dark, early dark and late light;
[0075] Mode: static, oscillating, static-oscillation 24h alternately, pre-stationary and late-oscillation, early-oscillation and late-stationary;
[0076] Time: 3d, 5d, 7d, 9d, 11d;
[0077] Input the above-menti...
Embodiment 3
[0084] Embodiment 3: Toxic production culture
[0085] With all total 10 strains tri5 obtained through tri5-PCR in embodiment 1 + Fusarium was cultured for toxin production according to the obtained best toxin production culture conditions, namely:
[0086] The toxin-producing medium is Richard’s liquid medium: 25g of sucrose, 3g of yeast extract, 6g of potassium dihydrogen phosphate, 7 water, 2g of magnesium sulfate, 0.02g of magnesium chloride, 1000mL of sterile water, and sterilized by autoclaving at 121°C and 100kPa for 30min;
[0087] The toxin-producing culture conditions are: 5°C and 20°C alternately, and the culture temperature is changed every 24 hours;
[0088] Light culture and dark culture alternate every 24 hours, and the light conditions refer to the instrument parameters;
[0089] Shake culture first and then static culture. Shake culture accounts for 50% of the total culture time, and the shaker speed is 80r / min.
[0090] Culture 9d.
[0091] According to t...
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