CN107540733B

CN107540733B - Rice blast bacterium exciton Mo65 and purification method thereof

Info

Publication number: CN107540733B
Application number: CN201710791374.3A
Authority: CN
Inventors: 李云锋; 聂燕芳; 王振中
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2017-09-05
Filing date: 2017-09-05
Publication date: 2020-10-23
Anticipated expiration: 2037-09-05
Also published as: CN107540733A

Abstract

The invention discloses a rice blast bacterium exciton Mo65, belonging to the protein class, the relative molecular mass of which is 65.5kDa, the isoelectric point is 5.45, the number of amino acid residues is 596, and the amino acid sequence is shown as SEQ ID NO. 1. The method for purifying the exciton comprises the following steps: culturing rice blast hypha, extracting coarse exciton, DEAE-Sepharose FF column chromatography, CM-Cellulose column chromatography and Sephadex G-75 column chromatography. The exciton provided by the invention is stable in acid and alkali, sensitive to proteinase K and heat, and good in stability, can induce the POD and PAL activities and active oxygen levels of interactive rice with different affinities to be obviously increased, is favorable for improving the disease resistance of rice plants, and provides a new way for preventing and treating rice blast.

Description

Rice blast bacterium exciton Mo65 and purification method thereof

Technical Field

The invention relates to the technical field of rice blast prevention and control, and particularly relates to a rice blast bacterium exciton Mo65 and a purification method thereof.

Background

The rice blast is one of the most main rice diseases in China, and the high yield and the stable yield of rice are seriously influenced, so that the prevention and the treatment of the rice blast are always important for people to pay attention. At present, mainly adopts prevention measures of breeding disease-resistant varieties and chemical prevention as main measures for rice blast, but because the genetic background of rice blast germs is complex and easy to change, and the disease-resistant varieties are relatively simple, the disease-resistant breeding is difficult to keep up with the change speed of physiological races of the rice blast germs; chemical control often causes pesticide residues in rice and environmental pollution, so that the search for an efficient, nuisanceless, economical and reasonable method for controlling rice blast becomes inevitable. In recent years, the study of elicitors provides a new way for preventing and treating rice blast.

Elicitors (elicers) are substances that induce plants to produce defense responses, and mainly include proteins, glycoproteins, polypeptides, oligosaccharides, and the like. The elicitor is of two types, a biological elicitor and a non-biological elicitor, wherein the biological elicitor is derived from a microorganism or produced by host-pathogen interaction. The biological source exciton has the characteristics of environmental protection, safety, obvious effect, rich source and the like, so the biological source exciton has wider application prospect in the prevention and treatment of agricultural plant diseases.

Fungi are one of the most important sources of biological elicitors. Pyricularia oryzae (Pyricularia oryzae) reported at presentMagnaporthe oryzae) The elicitors include glycoprotein elicitors with molecular weights of 15.6kDa, 25.0kDa, 49kDa, 66kDa and 102kDa, protein elicitors with molecular weights of 14.3kDa and 16kDa, unsaturated fatty acid elicitors, and the like. The common characteristics of the elicitors are that the elicitors can induce the rise of the activity of plant defense enzymes, cause the increase of the level of active oxygen of plants, and further cause the allergic necrosis reaction of the plants and the like. At present, no report related to 65.5kDa protein elicitors isolated from Magnaporthe grisea exists in the prior art.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides an elicitor Mo65 of rice blast bacterium, wherein the elicitor is derived from Magnaporthe grisea ZC₁₃The mycelia of the small species are obtained by a series of chromatographic purifications, the relative molecular mass of the mycelia of the small species is 65.5kDa, the isoelectric point of the mycelia of the small species is 5.45, and the mycelia of the small species are named as Mo 65. The exciton has good stability, no loss of bioactivity after being placed at-20 ℃ for 4 months, can induce the enhancement of disease-resistant related enzyme activity of interactive rice with different affinities, and is beneficial to improving the disease resistance of rice plants. The rice product line adopted by the invention is a rice blast-resistant near-isogenic line C101LAC (high-resistance line) and a background line CO39 (susceptible control), and is mixed with rice blast fungus ZC₁₃Constitute a high degree of non-affinity interaction and affinity interaction, respectively.

Another object of the present invention is to provide a method for purifying Magnaporthe grisea exciton Mo 65.

In order to achieve the purpose, the invention is realized by the following scheme:

a rice blast bacterium elicitor Mo65 belongs to proteins, has a relative molecular mass of 65.5kDa, an isoelectric point of 5.45, an amino acid residue number of 596, and an amino acid sequence shown as SEQ ID NO. 1.

The invention discovers that the exciton is Uncharactered protein coded by an open reading frame of a rice blast bacterium gene MGG _02297 and is named as Mo65 through mass spectrometry and database search (NCBI _ Magnaporthe _ oryzae). According to the invention, through research on the biological activity of the rice leaf, the Mo65 exciton can induce the activities of Peroxidase (POD) and Phenylalanine Ammonia Lyase (PAL) of the interactive rice with different affinities to be obviously improved after the rice leaf is treated by the exciton, the specific activities of the POD and the PAL are respectively increased by 52.10-76.32% and 58.65-85.36%, and the exciton can also induce the active oxygen level of the interactive rice with different affinities to be obviously improved, thereby being beneficial to improving the disease resistance of rice plants.

The exciton is stable to acid and alkali, sensitive to proteinase K and heat, free of loss of biological activity and good in stability when placed at the temperature of minus 20 ℃ for 4 months, and the effective concentration of the exciton causing resistance reaction of rice (C101 LAC and CO39 strains) is 0.02-0.15 nmol/L.

Preferably, the method for purifying the exciton comprises the following steps:

s1, culturing rice blast fungus hyphae: ZC rice blast bacteria₁₃Placing the microspecies hypha in a liquid culture medium, carrying out constant temperature shaking culture at 25 ℃ for 14d, filtering with a single-layer gauze, and repeatedly washing with double distilled water for 4 times;

s2, extracting a coarse exciton: breaking hypha cells by a freeze thawing method; centrifuging the freeze-thawed mycelia at 2000 Xg for 25min, and removing supernatant; resuspending the precipitate with double distilled water, centrifuging, and repeating for 4 times; homogenizing, and stirring at 4 deg.C overnight; centrifuging at 12000 Xg for 20min, and collecting supernatant; ultrafiltering the supernatant to obtain concentrated solution as coarse exciton;

s3, DEAE-Sepharose FF column chromatography: loading the crude exciton obtained in S2 onto a DEAE-Sepharose FF anion exchange column, sufficiently eluting with a buffer solution, performing linear gradient elution with the buffer solution containing 0-0.75 mol/L NaCl, collecting an active peak sample, and dialyzing and desalting with the buffer solution;

s4, CM-Cellulose column chromatography: further loading the active peak sample obtained in the step S3 to a CM-Cellulose cation exchange column, fully leaching with a buffer solution, eluting with the buffer solution containing 0-0.5 mol/L NaCl, and collecting active components;

s5, Sephadex G-75 column chromatography: and (4) further loading the active component obtained in the step (S4) to a Sephadex G-75 gel column, eluting with a buffer solution, and collecting the active component to obtain the magnaporthe grisea exciton.

Preferably, the formula of the liquid culture medium in S1 is 5 g of yeast extract and 22 g of glucose, and distilled water is added to the mixture to reach a constant volume of 1000 mL.

Preferably, the ultrafiltration membrane described in S2 has a molecular weight cut-off of 10 kDa.

Preferably, the buffer solution in S3-S5 is 0.02 mol/L Tris-HCl buffer solution with pH 7.2.

Compared with the prior art, the invention has the following beneficial effects:

(1) the exciton provided by the invention is stable to acid and alkali, sensitive to proteinase K and heat, free of loss of biological activity and good in stability when placed at-20 ℃ for 4 months, and the effective concentration of the exciton causing resistance reaction of rice (C101 LAC and CO39 strains) is 0.02-0.15 nmol/L.

(2) The exciton provided by the invention can induce the POD and PAL activities and active oxygen levels of the interactive rice with different affinities to be obviously increased, is favorable for improving the disease resistance of rice plants, and provides a new way for preventing and treating rice blast.

(3) The invention takes the rice blast fungus hypha as the raw material to extract the exciton, the raw material is easy to culture, the price is low, the technical route for purifying the exciton is simple, and the invention has good application prospect in the prevention and treatment of rice blast.

Drawings

FIG. 1 is a DEAE-Sepharose FF column chromatography of Magnaporthe grisea exciton Mo 65; note: 3 mL/tube, NaCl gradient elution.

FIG. 2 is a column chromatography diagram of CM-Cellulose of Magnaporthe grisea exciton Mo 65; note: 3 mL/tube, NaCl gradient elution.

FIG. 3 is a Sephadex G-75 column chromatography diagram of Magnaporthe grisea exciton Mo 65; note: 3 mL/tube.

FIG. 4 is an SDS-PAGE electrophoresis test of Magnaporthe grisea exciton Mo 65; m represents a standard molecular weight protein.

FIG. 5 is MS and MS/MS spectra of Magnaporthe grisea exciton Mo65 identified by MALDI-TOF/TOF; a is MS map, B is MS/MS map.

FIG. 6 shows the case where Magnaporthe grisea elicitor Mo65 induces changes in the activities of POD and PAL in rice; a is rice PAL activity and B is rice POD activity.

FIG. 7 shows rice blast fungus elicitor Mo65 inducing rice O₂ ^.-And H₂O₂The case of a change in level; a is rice O₂ ^.-Level, B is rice H₂O₂And (4) horizontal.

FIG. 8 shows the effect of different concentrations of Mo65 exciton on the induction of the change in POD activity in rice.

FIG. 9 shows the effect of different treatments on the induction of rice POD activity by Mo65 elicitors.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

The purification method of the magnaporthe grisea exciton Mo65 specifically comprises the following steps:

first, culture of rice blast fungus hypha

ZC rice blast bacteria₁₃Placing the hyphae of the microspecies in a liquid culture medium (5 g of yeast extract, 22 g of glucose, and adding distilled water to a constant volume of 1000 mL) to perform constant-temperature shaking culture at 25 ℃ for 14d, filtering with a single-layer gauze, and repeatedly washing with double-distilled water for 4 times to obtain a large amount of hyphae.

Second, extraction of crude exciton

Breaking hypha cells by a freeze thawing method; centrifuging the freeze-thawed mycelia at 2000 Xg for 25min, and removing supernatant; resuspending the precipitate with double distilled water, centrifuging, and repeating for 4 times; homogenizing, and stirring at 4 deg.C overnight; centrifuging at 12000 Xg for 20min, and collecting supernatant; and ultrafiltering the supernatant through a filter membrane with the molecular weight cutoff of 10 kDa to obtain a concentrated solution, namely the crude exciton.

Third, DEAE-Sepharose FF column chromatography

And (3) fully eluting the crude exciton obtained in the second step on a DEAE-Sepharose FF anion exchange column by using a buffer solution (0.02 mol/L Tris-HCl buffer solution with the pH value of 7.2), then performing linear gradient elution by using the buffer solution containing 0-0.75 mol/L NaCl, collecting an active peak sample, dialyzing and desalting by using the buffer solution, and performing DEAE-Sepharose FF column chromatography on the exciton as shown in figure 1.

Column chromatography of CM-Cellulose

And (3) further loading the active peak sample obtained in the step three to a CM-Cellulose cation exchange column, fully leaching with a buffer solution, eluting with the buffer solution containing 0-0.5 mol/L NaCl, collecting active components, and carrying out CM-Cellulose column chromatography on the exciton as shown in figure 2.

Fifth, Sephadex G-75 column chromatography

And (3) further loading the active component obtained in the step four onto a Sephadex G-75 gel column, eluting with a buffer solution, collecting the active component, and freeze-drying to obtain the Magnaporthe grisea exciton, wherein a Sephadex G-75 column chromatography diagram of the exciton is shown in figure 3.

The obtained Magnaporthe grisea exciton Mo65 was subjected to SDS-PAGE and stained by a counterstaining method, and the electrophoretic band showed only one band as shown in FIG. 4, indicating that the exciton Mo65 reached electrophoretic purity.

Performing mass spectrum identification on the obtained magnaporthe oryzae exciton Mo 65: carrying out enzymolysis on an exciton Mo65 sample by using Trypsin, and then desalting by using Ziptip; taking 1 μ L of the dissolved sample, and performing mass spectrometry by using a tandem time-of-flight mass spectrometer (5800 MALDI-TOF/TOF, ABCIEX); data are acquired by adopting a positive ion mode and an automatic data acquisition mode, the scanning range of a primary Mass Spectrum (MS) is 800-4000 Da, a parent ion with a signal-to-noise ratio of more than 50 is selected for secondary mass spectrum (MS/MS) analysis, and the obtained MS and MS/MS spectra are shown in figure 5. The mass spectrometry original file is searched by Mascot 2.2 software to a corresponding database (NCBI _ Magnaporthe _ oryzae), and the exciton Mo65 is identified as Uncharactered protein coded by an open reading frame of rice blast germ gene MGG _02297, and the method is characterized in that: the relative molecular mass of the exciton is 65.5kDa, the isoelectric point is 5.45, the number of amino acid residues is 596, and the amino acid sequence is shown as SEQ ID NO. 1.

Example 2

The invention measures the biological activity of the exciton Mo 65:

peroxidase (POD), Phenylalanine Ammonia Lyase (PAL) and active oxygen level are used as activity indexes, and the pressure injury speckle method is adopted to determine the induction activity of elicitor to rice resistance reaction. Wherein, the method of the pressed lesion is a method for rapidly, accurately and quantitatively detecting the activity of an exciton, and the activity change of POD and PAL is an important resistance physiological index for identifying rice blast resistance. The adopted rice product line is a rice blast-resistant near-isogenic line C101LAC (high-resistant line) and a background line CO39 (susceptible control), and is matched with rice blast fungus ZC₁₃Constitute a high degree of non-affinity interaction and affinity interaction, respectively.

After the rice leaves are treated by the Mo65 exciton, the POD and PAL activities of the rice leaves of disease-resistant and disease-susceptible varieties are changed as shown in FIG. 6, and the results show that the Mo65 exciton can induce the POD and PAL activities of the interactive rice with different affinities to be obviously improved, and the specific activity increase amplitudes of the POD and PAL are 52.10-76.32% and 58.65-85.36% respectively.

Active oxygen (O) of rice leaves of disease-resistant and disease-susceptible varieties after Mo65 exciton treatment₂ ^.-And H₂O₂) The level change is shown in FIG. 7, and the results show that the Mo65 exciton can induce the active oxygen level of the rice with different affinity interactions to be obviously increased.

Example 3

The effective concentration of exciton Mo65 induction activity is measured by the invention:

the elicitor was prepared into 5 kinds of solutions with different concentrations, such as 0.5, 1, 10, 50, 100. mu.g/mL, using 0.02 mol/L Tris-HCl (pH 7.2) buffer, and inoculated onto rice leaves with different affinity interactions for biological activity assay.

FIG. 8 shows that the concentration of 10. mu.g/mL (corresponding to an exciton concentration of 0.15 nmol/L) or more induces the activity of POD in rice leaves; the activity of the Mo65 exciton is enhanced along with the increase of the concentration; while 1. mu.g/mL (corresponding to an exciton concentration of 0.02 nmol/L) of Mo65 excitons could not induce an increase in rice POD activity, and as a summary, the effective concentration of Mo65 excitons was between 0.02 to 0.15 nmol/L.

Example 4

The invention researches the stability of the activity of the exciton Mo 65:

first, heat treatment

Heating the exciton solution to boiling for 10 min, cooling, and measuring bioactivity with the activity change of rice POD as monitoring index.

Secondly, protease treatment

Adding proteinase K (0.1 mg/mL) into solution containing 50 μ g/mL exciton, mixing, reacting at 37 deg.C for 30min, and measuring bioactivity with change of rice POD activity as monitoring index. And adding proteinase K into Tris-HCl buffer solution as a control, and treating with an exciton as the rest.

Thirdly, measuring the stability of acid and alkali

Adjusting 0.02 mol/L Tris-HCl buffer solution with HCl and NaOH to prepare 3 solutions with pH values of 2, 8 and 12, respectively, adding excitons into the solutions, treating at 25 ℃ for 22 h, neutralizing with NaOH or HCl until the final concentration of the excitons is 50 mug/mL, and dialyzing the buffer solution overnight. The above solutions of 3 different pH values without exciton were used as controls, and the rest were treated with excitons. The change of the POD activity of the rice is used as a monitoring index to carry out biological activity determination.

The influence of the different treatments on the activity of the rice POD induced by the Mo65 exciton is shown in FIG. 9, and the results show that the activity of the exciton is not lost after the acid-base treatment; after proteinase K and heat treatment, exciton activity is lost. The research of the invention also finds that the Mo65 exciton has no loss of biological activity and better stability when placed for 4 months at the temperature of minus 20 ℃.

Sequence listing

<110> southern China university of agriculture

<120> rice blast bacterium exciton Mo65 and purification method thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>596

<212>PRT

<213> Magnaporthe oryzae ZC13(Magnaporthe oryzae ZC13)

<400>1

Met Arg Gly Leu Lys Tyr Leu Leu Leu Ala Ser Gly Gly Val Gly Leu

1 5 10 15

Cys Ala Ala Arg His Ala Phe Ser Ile His Glu Asp Met Leu Ser Asn

20 25 30

Pro Gln Phe Glu Ile Ile Phe Ser Glu Thr Phe Ile Arg Glu Ser Asp

35 40 45

Ala Leu Ala Leu Met Glu Ser Ala Gly Lys Asn Pro Pro Gln Ala His

50 55 60

Ala Thr Thr Gln Phe Ser Thr Ser Thr Ser Thr Asp Thr Ser Lys Thr

65 70 75 80

Thr Thr Pro Pro Ala Asn Arg Asp Asp Asp Asp Asp Asp Gln Pro Val

85 90 95

Leu Ser Glu Ala Tyr Glu Val Ile Asn Val Pro Pro Ala Arg Tyr Phe

100 105 110

Cys Ala Ile Pro Val Leu Ala Pro Leu Pro Ala Leu Asn Gln Thr Ala

115 120 125

Ile Asp Leu Ala Lys Ala Glu Glu Leu Arg Glu Met Thr Asp Ala Thr

130 135 140

Ser His Gly Trp Glu Leu Leu Asn Lys Leu Asn Gly Gln Cys Leu Tyr

145 150 155 160

Tyr Thr Ala Gly Tyr Trp Ser TyrSer Phe Cys Tyr Asn Arg Asn Val

165 170 175

Ile Gln Tyr His Ala Leu Pro Pro Gly Thr Arg Ser Gly Pro Pro Val

180 185 190

Arg Asp Glu Arg Glu Pro Glu Tyr Val Leu Gly Arg Ala Leu Pro Gln

195 200 205

Thr Pro His Gly Gln Gln Ala Gly Lys Ser Leu Gly Asp Arg Asp His

210 215 220

Gly Lys Glu Gln Ala Val Leu Lys Gln Asp Asp Lys Gly Val Ser Lys

225 230 235 240

Asn Ala Ala Gln Pro Asn Thr Glu Leu Val Ile Lys Gly Asp Gln Arg

245 250 255

Tyr Leu Val Gln Arg Leu Asp Ser Gly Thr Val Cys Asp Leu Thr Gly

260 265 270

Arg Pro Arg Thr Ile Glu Ile Gln Tyr His Cys Ala Leu Gly Thr Thr

275 280 285

Val Asp Arg Ile Gly Trp Ile Lys Glu Val Thr Thr Cys Ser Tyr Leu

290 295 300

Met Met Val Gln Thr Pro Arg Leu Cys Glu Asp Val Ala Phe Leu Pro

305 310 315 320

Pro Lys Pro Thr Arg Ala His Pro Ile SerCys Arg Gln Ile Val Ser

325 330 335

Ser Glu Asp Glu Val Lys Ser Trp Gln Ser Gln Lys Thr Leu Asp Ala

340 345 350

Arg Leu Arg Leu Thr Gly Ala Glu Ser Gln Trp Gln Leu Gly Gly Gln

355 360 365

Gln Gln Gln Gln Gln Gln Gln Asp Ala Asn Gly Gln His Lys Lys His

370 375 380

Gln Lys Gln Gln Gln Arg Ser Phe Thr Gly Val Asn Ile Gly Gly Val

385 390 395 400

Val Ile Gly Ser Arg Asn Val Leu Gly Arg Leu Glu Asp Gly Gln Pro

405 410 415

Val Pro Asn Leu Thr Pro Pro Arg Lys Ala Gly Ser Met Phe Gln Asn

420 425 430

Gly Ala Arg Ala Gly Ala Ala Gln Pro Ile Ile Glu Leu Leu Ala Leu

435 440 445

Ala Lys Ser Lys Ala Glu Gly Gly Gln Ile Glu Val Val Ser Asp Glu

450 455 460

Asp Leu Leu Gln Leu Glu Leu Asp Pro Val Gln Val Glu Glu Leu Arg

465 470 475 480

Glu Ala Leu Ile Lys Leu Ala Gly Asp Lys Gly TrpLys Leu Glu Ile

485 490 495

Leu Glu His Leu Asn Glu Val Ala Glu Phe Arg Gly Val Val Gln Asp

500 505 510

Pro His Asp Pro Asp Asn Asp Val Tyr Tyr Asn Gly Gly Glu Ala Asn

515 520 525

Ala Pro Pro Ile Val Asp Gly Ala Gly Arg Lys Ala Tyr Gly Asn Thr

530 535 540

Ala Glu Asp Asp Arg Arg Ala Val Gly Asn Lys Asp Lys Asp Lys Gly

545 550 555 560

Gly Lys Ala Val Gly Pro Gly Arg Lys Ala Gln Lys Gln Glu Asp Asp

565 570 575

Lys Glu Asp Gly Asp Met Gly Asp Ala Gly Ser Glu Glu Arg Phe Phe

580 585 590

Lys Asp Glu Leu

595

Claims

1. The application of the rice blast pathogen elicitor Mo65 in causing resistance reaction of C101LAC and CO39 rice; the relative molecular mass of the exciton proteins is 65.5kDa, and the amino acid sequence of the exciton proteins is shown as SEQ ID NO 1;

the application is that the rice blast fungus exciton Mo65 can induce the activity of peroxidase and phenylalanine ammonia lyase in C101LAC and CO39 rice and the increase of the active oxygen level;

the effective concentration of the magnaporthe grisea exciton Mo65 is 0.02-0.15 nmol/L.

2. The method for purifying magnaporthe oryzae exciton Mo65 of claim 1, comprising the steps of:

s1, culturing rice blast fungus hyphae: placing the small hyphae of the Magnaporthe grisea ZC13 in a liquid culture medium, carrying out constant temperature shaking culture for 14d at 25 ℃, filtering with a single-layer gauze, and repeatedly washing with double-distilled water for 4 times;

s5, Sephadex G-75 column chromatography: further loading the active component obtained in the step S4 to a Sephadex G-75 gel column, eluting with a buffer solution, and collecting the active component to obtain the magnaporthe grisea exciton;

the amino acid sequence of the magnaporthe grisea exciton Mo65 is shown as SEQ ID NO. 1.