A kind of rice blast fungus elicitor mo65 and its purification method

A technology of rice blast fungi and elicitors, applied in chemical instruments and methods, peptide preparation methods, organic chemistry, etc.

Active Publication Date: 2020-10-23
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant report on the 65.5kDa protein elicitor isolated from the rice blast fungus in the prior art

Method used

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  • A kind of rice blast fungus elicitor mo65 and its purification method
  • A kind of rice blast fungus elicitor mo65 and its purification method
  • A kind of rice blast fungus elicitor mo65 and its purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The purification method of rice blast fungus elicitor Mo65 specifically comprises the following steps:

[0036] 1. Cultivation of Mycelia of Magnaporthe grisea

[0037] Magnaporthe oryzae ZC 13 The hyphae of the racemic species were placed in a liquid medium (5 g of yeast extract, 22 g of glucose, and distilled water was added to make the volume to 1000 mL) and cultured at 25 °C for 14 days with constant temperature shaking, filtered with a single layer of gauze, and washed repeatedly with double distilled water. After washing 4 times, a large number of hyphae were obtained.

[0038] 2. Extraction of crude excitons

[0039] Break the mycelial cells by freezing and thawing method; centrifuge the frozen and thawed mycelium at 2000×g for 25 min, discard the supernatant; re-suspend the precipitate with double distilled water and centrifuge, repeat 4 times; homogenize and stir at 4°C Overnight; after centrifugation at 12000×g for 20 min, the supernatant was taken; the sup...

Embodiment 2

[0049] The present invention measures the biological activity of elicitor Mo65:

[0050] Using peroxidase (POD), phenylalanine ammonia lyase (PAL), and reactive oxygen species as activity indicators, the pressure-spot method was used to measure the inducing activity of elicitors to rice resistance. Among them, the pressure wound method is a rapid and accurate quantitative method for detecting elicitor activity, and the changes in POD and PAL activities are important physiological indicators for identifying rice blast resistance. The rice strains used were blast-resistant near-isogenic line C101LAC (highly resistant line) and background line CO39 (susceptible control). 13 Form high non-affinity interaction and affinity interaction respectively.

[0051] After the rice leaves were treated with Mo65 elicitor, the changes of POD and PAL activities in the rice leaves of the disease-resistant and susceptible lines were as follows Figure 6 As shown, the results showed that the Mo6...

Embodiment 3

[0054] The present invention measures the effective concentration of elicitor Mo65 inducing activity:

[0055] Use 0.02 mol / L Tris-HCl (pH7.2) buffer solution to prepare the elicitor into five different concentrations of protein concentration: 0.5, 1, 10, 50, 100 μg / mL, and inoculate to different affinity Bioactivity assays were performed on interacting rice leaves.

[0056] The changes of POD activity in rice leaves induced by different concentrations of elicitor Mo65 are as follows: Figure 8 As shown, the results show that each elicitor concentration of 10 μg / mL (equivalent to 0.15 nmol / L elicitor concentration) and above can induce the increase of POD activity in rice leaves; with the increase of concentration, the activity of Mo65 elicitor enhanced; while Mo65 elicitor at 1 μg / mL (equivalent to elicitor concentration of 0.02 nmol / L) could not induce the increase of rice POD activity. between.

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Abstract

The invention discloses a rice blast bacteria elicitor Mo65 and belongs to protein. The relative molecular weight of the elicitor is 65.5 kDa, the isoelectric point is 5.45, the amino acid residue base number is 596, and the amino acid sequence of the elicitor is as shown in the SEQ ID NO: 1. A purification method of the elicitor comprises the following steps of culture of rice blast bacteria mycelia, extraction of a crude elicitor, DEAE-Sepharose FF column chromatography, CM-Cellulose column chromatography and Sephadex G-75 column chromatography. The elicitor is stable in acid and alkali, sensitive to protease K and heat and good in stability, can induce the POD, the PAL activity and the active oxygen level of rice with different compatible interactions to be obviously improved, improvesthe disease resistance of rice plants, and provides a new way for preventing and treating rice blast.

Description

technical field [0001] The invention relates to the technical field of rice blast control, in particular to a rice blast fungus elicitor Mo65 and a purification method thereof. Background technique [0002] Rice blast is one of the most important rice diseases in my country, which seriously affects the high and stable yield of rice, so its control has always been the focus of attention. At present, the main control measures for rice blast are the selection of disease-resistant varieties and chemical control. However, due to the complex genetic background of the rice blast fungus, which is easy to mutate, and the resistant varieties are relatively single, it is difficult for disease-resistant breeding to keep up with the rice blast fungus. Variation speed of physiological races; chemical control often causes pesticide residues in rice and environmental pollution, so it is inevitable to find efficient, pollution-free, economical and reasonable control methods for rice blast. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/37C07K1/36C07K1/34C07K1/18
Inventor 李云锋聂燕芳王振中
Owner SOUTH CHINA AGRI UNIV
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