Method for inducing phytophthora nicotianae to generate pathogenic secretory protein
A technique for secreting protein and Phytophthora nicotiana, which is applied in the field of inducing Phytophthora nicotiana to produce pathogenic secreted protein, which can solve the problem of inability to produce secreted protein
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Embodiment 1
[0013] Take the tobacco black shank sample back to the laboratory, and rinse the stem surface of the diseased plant with tap water. After air-drying, take a 0.4cm×0.4cm marrow tissue piece from the junction of disease and health, place it in oat medium, and culture it in the dark at 28°C for 3-4 days. The oat medium is composed of the following parts: 30g of oatmeal, 20g of agar, and 1000ml of distilled water; the weakly pathogenic strain Y1 of Phytophthora nicotianae isolated and obtained is inoculated into a common solid medium composed of 30g of oatmeal, 20g of agar, and 1000ml of distilled water cultured in the dark at 28°C for 3-4 days; after the formation of larger colonies, transfer the mycelium block to 300mL pathogenic secretory protein liquid induction medium, which consists of the following parts: yeast Extract 0.5 mg / L, glucose 25g / L, NaH 2 PO4 0.5g / L, MgSO4 7H 2 O 0.25g / L, Asparagine 1.5g / L, VB1 1.5mg / L, H 2 O 1L, at 28°C, 120rpm shaking culture for 2 weeks; fi...
Embodiment 2
[0015] Inoculate the isolated strong pathogenic strains Y2 and Y3 of Phytophthora nicotianae into a common solid medium composed of 30 g of oatmeal, 20 g of agar, and 1000 ml of distilled water, and cultivate them under dark conditions at 28°C for 3 to 4 days; After larger colonies, transfer the mycelium block to 300mL liquid induction medium made of pathogenic secretory protein, which consists of the following components: yeast extract 1 mg / L, glucose 30g / L, NaH 2 PO4 0.75g / L, MgSO4 7H 2 O 0.4g / L, Asparagine 2g / L, VB1 2mg / L, H 2 O 1L, at 28°C, 120rpm shaking culture for 2 weeks; filter the mycelium with double-layer sterile filter paper, centrifuge the filtrate at 5000rpm for 20min, add 100% saturated ammonium sulfate solution to the supernatant, centrifuge at 10000rpm for 30min, precipitate through 1KD The dialysis zone was processed, and the Phytophthora nicotianae secreted protein extract was obtained after freeze-drying, and stored at -20°C.
[0016] Inoculate the secre...
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