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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

Inactive Publication Date: 2015-05-20
HONGYUN HONGHE TOBACCO (GRP) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the deficiency that Phytophthora tabacum can not produce secretory protein under solid culture conditions, and provide a method capable of inducing Phytophthora tabacum to produce pathogenic secretory protein

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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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PUM

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Abstract

The invention relates to a method for inducing phytophthora nicotianae to generate pathogenic secretory protein. The method comprises the following steps: taking a tobacco black shank sample back to a laboratory, flushing the surface of the diseased plant stem with tap water till the surface is clean; after drying, taking a 0.4 cm*0.4 cm pith tissue block at a juncture of the diseased and health tissue, placing the block in an oat medium, performing culture under a dark condition with a temperature of 28 DEG C for 3-4 days to form a large bacterial colony, transferring the mycelium block into 300 mL of secretory protein liquid induction medium, performing shaking culture at 28 DEG C and 120 rpm for 2 weeks, filtering the mycelia by a double-layer sterilization filter paper, centrifuging the filtrate at 5000 rpm for 10 min, adding ammonium sulfate into the supernatant, centrifuging the supernatant at 10000 rpm for 20 min, performing dialysis treatment of the precipitate, performing freeze drying to obtain secretory protein of phytophthora nicotianae, storing the protein at -20 DEG C. The method can induce phytophthora nicotianae to generate a lot of pathogenic secretory protein, fills the gap that secretory protein can not be obtained in phytophthora nicotianae pathogenesis research at home and abroad, 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 Phytophthora nicotiana to produce pathogenic secreted protein. Background technique [0002] Depend on Phytophthora parasiticavar. nicotiana Tobacco black shank caused by tobacco is one of the devastating diseases in tobacco production. It occurs in tobacco growing areas all over the world, and can cause tens of billions of dollars in losses every year. Therefore, it is highly regarded by tobacco breeders, plant pathologists, Widespread concern of geneticists. Phytophthora nicotiana is also a good experimental material for genetics and molecular biology. The disease system is a model system for the study of fungal-plant interaction. In recent years, research has obtained many morphological differentiation and pathogenic factors related to the pathogen infection. Genetic resources and information provided by Phytophthora tobacco laid a good foundat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C12R1/645
Inventor 罗华元常寿荣王绍坤徐洁周晓罡
Owner HONGYUN HONGHE TOBACCO (GRP) CO LTD
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