Method for inducing phytophthora infestans to produce pathogenic secretory protein
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A potato late blight, potato late blight technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as inability to produce pathogenic secretory proteins
Inactive Publication Date: 2008-08-27
云南省农业科学院生物技术与种质资源研究所
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[0004] The purpose of the present invention is to overcome the deficiency that P. infestans infestans cannot produce pathogenic secreted proteins under solid culture conditions, and provide a method capable of inducing the pathogenic secreted proteins produced by P. infestans infestans
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Embodiment 1
[0014] Put the potato late blight disease sample on ordinary water agar medium, and cultivate it in the dark and moist at 16-18°C for 2-3 days; the ordinary water agar medium is composed of the following parts: agar powder 10g / L, H 2 O 1L;
[0015] Inoculate the weakly pathogenic strain A1 of P. infestans isolated from 450ml of rye extract, 150ml of tomato juice, 10g / L of agar powder, CaCO 3 1.2g / L, amoxicillin 100mg / L, pentachloronitrobenzene 30mg / L, rifampicin 10mg / L, nystatin 10mg / L, H 2 O On ordinary solid medium consisting of 1L, culture in dark at 16-18°C for 8-10 days; after forming larger colonies, pick 5 pieces of about 0.5cm with an inoculation needle 2 The mycelium block was transferred to 100mL by rye extract 450ml, CaCO 3 1.2g / L, KH 2 PO 4 0.5g / L, MgSO4 7H 2 O 0.25g / L, Asparagine 1g / L, VB1 1mg / L, Amoxicillin 100mg / L, Pentachloronitrobenzene 30mg / L, Rifampicin 10mg / L, Nystatin 10mg / L, H 2 O 1L pathogenic secretory protein liquid induction medium, 18°C, 150...
Embodiment 2
[0017] Inoculate the isolated strong pathogenic strains A2 and A3 of Potato infestans into 450ml of rye extract, 150ml of tomato juice, 10g / L of agar powder, CaCO 3 1.2g / L, amoxicillin 100mg / L, pentachloronitrobenzene 30mg / L, rifampicin 10mg / L, nystatin 10mg / L, H 2 O On ordinary solid medium consisting of 1L, culture in dark at 16-18°C for 8-10 days; after forming larger colonies, pick 5 pieces of about 0.5cm with an inoculation needle 2 The mycelium block was transferred to 100mL by rye extract 450ml, CaCO 3 1.2g / L, KH 2 PO 4 0.5g / L, MgSO4 7H 2 O 0.25g / L, Asparagine 1g / L, VB1 1mg / L, Amoxicillin 100mg / L, Pentachloronitrobenzene 30mg / L, Rifampicin 10mg / L, Nystatin 10mg / L, H 2 O 1L pathogenic secreted protein liquid induction medium, 18°C, 150rpm shaking culture for 14-16 days. Filter the mycelium with double-layer sterile filter paper, centrifuge the filtrate at 4000rpm for 10min, add 100% ammonium sulfate (697g / L) to the supernatant, centrifuge at 10000rpm for 20min, a...
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Abstract
The invention relates to a process which induces potato late blight pathogen to generate pathogenic secretory protein, which comprises placing potato late blight pathogen sample in common water agar culture medium to hydrate and cultivate, selecting a little amount of bacterial filament to graft in the common solid culture medium through a grafting needle when hoary moldy layers grow on the leaves, cultivating in dark under 16-18 DEG C, transferring bacterial filament blocks into 100mL pathogenic protein liquid inducing medium after forming a larger bacterial colony, removing bacterial filament bodies by double-layer sterilizing filtering paper vibrating in 150 rpm for 14-16days under 18 DEG C, centrifuging the filtering liquid in 4000rpm for 10 min , adding ammonium sulfate (697g/L) in supernatant fluid, centrifuging in 10000rpm for 20min conducting dialysis treatment to the sedimentation, obtaining potato late blight pathogen pathogenic secretory protein abstract after freeze drying, and reserving under -20 DEG C. The process can induce potato late blight pathogen to generate a great amount of pathogenic secretory protein, fills the blank that pathogenic secretory protein can not be obtained in potato late blight pathogen pathogenic mechanism research at home and board, and has great significance.
Description
technical field [0001] The invention relates to the technical field of plant protection, in particular to a method for inducing pathogenic secretion protein of potato infestans. Background technique [0002] Potato late blight caused by Phytophthora infestan is the most serious fungal disease in potato production. It occurs in potato production areas all over the world and can cause billions of dollars in losses every year. Physicists and geneticists are widely concerned. Potato infestans is also a good experimental material for classical genetics and molecular genetics. The disease system is a model system for the study of fungi-plant interactions. In recent years, research has obtained many infection-related morphological differentiation and pathogenic factors. The genetic resources and information provided by P. infestans laid a good foundation for the genomics research of P. infestans. [0003] Through the functional research of pathogenicity-related genes, we can gain...
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