CN110710537A - Alkaline metalloprotease, preparation containing the same and application thereof - Google Patents

Abstract

The invention discloses an alkaline metalloprotease and an application of a preparation containing the alkaline metalloprotease in preventing and treating tobacco mosaic virus, wherein the pH of the alkaline metalloprotease is adjusted to 8.0-9.0, and the alkaline metalloprotease is taken from serratia marcescens. The preparation has stable property, has good antiviral effect by acting on host plants and viruses, and is environment-friendly.

Description

Alkaline metalloprotease, preparation containing the same and application thereof

Technical Field

The invention relates to the technical field of pesticides, in particular to an alkaline metalloprotease and an application of a preparation containing the alkaline metalloprotease in preventing and treating tobacco mosaic virus.

Background

Tobacco Mosaic Virus (TMV) is a pathogen of tobacco mosaic disease and the like, has a wide host range, can infect at least 125 plants of 9 families, such as cruciferae, solanaceae, cucurbitaceae and the like, has high occurrence frequency, is quick in prevalence, is serious in harm and difficult to treat, cannot be effectively controlled once a plant is infected, is very easy to cause the prevalence of the virus disease, and causes serious economic loss. The economic losses due to TMV are as high as billions of dollars per year. At present, modes such as breeding of antiviral varieties, control of virus-carrying media, cross protection and the like are mainly adopted in the treatment of virus diseases, and few effective chemical pesticides aiming at plant virus diseases need to be solved by agricultural scientific researchers.

Serratia marcescens is a gram-negative bacterium widely existing in natural habitat and can live in soil, water and other environments. In soil, some strains are plant growth-promoting rhizobacteria, e.g., Serratia marcescens-90-166 isolated from soil can induce systemic resistance in plants by quorum sensing mechanisms and can enhance plant infestation by several plant pathogens, including cucumber mosaic virus. In addition, Serratia marcescens also produces a variety of extracellular enzymes including sericise, chitinase, metalloprotease, lipase, thiol protease, nuclease, and the like. The serratia marcescens extracellular protein has research and application in the fields of medicine, industry and the like, but has few researches in agriculture, and has no application of related products in the market.

Therefore, it is necessary to provide a further solution to the above problems.

Disclosure of Invention

The invention aims to provide the alkaline metalloprotease and the application of the preparation containing the alkaline metalloprotease in preventing and treating tobacco mosaic virus, so as to overcome the defects in the prior art.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the application of alkaline metalloprotease in preventing and treating tobacco mosaic virus is disclosed.

Preferably, the alkaline metalloprotease is obtained from serratia marcescens.

Preferably, the alkaline metalloprotease has a mass of 45-55 kDa.

Preferably, the alkaline metalloprotease has a mass of 50 kDa.

Preferably, the pH of the alkaline metalloprotease is adjusted to 8.0 to 9.0.

The invention also discloses a preparation containing the alkaline metalloprotease, and the effective components of the preparation are the alkaline metalloprotease and a plant growth regulator and/or a stabilizer.

Preferably, the pH of the alkaline metalloprotease is adjusted to 8.0 to 9.0.

Preferably, the concentration of alkaline metalloprotease in said formulation is 80-120. mu.g/mL.

Preferably, the plant growth regulator is an amino-oligosaccharide,

preferably, the stabilizer is trehalose.

The invention also discloses application of the preparation containing the alkali metal protease in preventing and treating tobacco mosaic virus.

Preferably, the alkaline metalloprotease containing preparation is sprayed after heating to 40-50 ℃.

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

the invention discloses an alkaline metalloprotease and an application of a preparation containing the alkaline metalloprotease in preventing and treating tobacco mosaic virus, which has stable property, good antiviral effect and environmental friendliness by acting on host plants and viruses.

Drawings

FIG. 1 is an agarose gel electrophoresis of a 16SrRNA fragment of a strain resistant to TMV;

FIG. 2 shows the morphological characteristics, anti-TMV effect and phylogenetic tree of Serratia marcescens, wherein a, the semi-leaf method shows the inhibitory effect of S3 on TMV; b, colony morphology; c, an S3 electron microscope picture; and d, analyzing the obtained phylogenetic tree of the 16S sequence of the S3 by using a Clustal W method, wherein a ruler comprises the following steps: 5 percent;

FIG. 3 shows the germination accelerating effect of the fermentation supernatant of the S3 strain;

FIG. 4 shows the growth promoting effect of fermentation supernatant of S3 strain;

FIG. 5 shows the survival of S3 strain in leaves and soil;

FIG. 6 is a standard graph of tyrosine;

FIG. 7 is a graph of the effect of pH on SAMP enzyme activity and enzyme stability;

FIG. 8 is a graph of the effect of temperature on SAMP enzyme activity and enzyme stability;

FIG. 9 is a graph of the inhibitory effect of SAMP at different concentrations on TMV;

FIG. 10 shows the effect of SAMP on TMV inhibition in B.benthamiana leaves.

Detailed Description

The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. The term "prepared from …" as used herein is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or events may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

Approximating language, as used herein throughout the specification and claims, is intended to modify a quantity, such that the invention is not limited to the specific quantity, but includes portions that are literally received for modification without substantial change in the basic function to which the invention is related. Accordingly, the use of "about" to modify a numerical value means that the invention is not limited to the precise value. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. In the present description and claims, range limitations may be combined and/or interchanged, including all sub-ranges contained therein if not otherwise stated.

In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the stated number clearly indicates that the singular form is intended.

The plant material used in this example was TMV, a tobacco-producing host, i.e., nicotiana. tabacum. sunseann, which was grown in the greenhouse by the present applicant. Plant culture conditions: the illumination is 16h, the temperature is 25 +/-1 ℃, the illumination intensity is 2000lux, and the relative humidity is 60 percent; the dark time is 8h, the temperature is 25 +/-1 ℃, and the relative humidity is 60%. The nutrient medium is produced by a Ward nutrient soil processing factory in Shouguang city in Shandong province, and additional fertilization is not needed during the growth of the plants.

TMV virus source is collected from Qingdao city, Shandong province, and propagated and stored on common tobacco NC89 cultivated in greenhouse.

The serratia marcescens is inoculated to a culture medium plate containing rifampicin to be cultured, the concentration of the rifampicin is slowly increased from low to high (50-250 mu mol/mL), and finally the serratia marcescens with the rifampicin resistance to 250 mu mol/mL is obtained through gradient screening.

The rifampicin resistant serratia marcescens was divided into 3 parts on average by overnight shaking culture (28 ℃, 120rpm), two parts of which were mixed with natural soil and sterilized soil, respectively, and the other part was sprayed on leaves of K326 tobacco.

Samples were taken the day after treatment, 1g each, soaked in sterile physiological saline and mixed well, supernatant was taken, diluted in gradient and plated on plates containing 250. mu. mol/mL rifampicin for counting colonies.

3mL of fermentation supernatant of Serratia marcescens is taken, centrifuged (6000g for 10min), the precipitate is discarded, the supernatant is sterilized by a 0.22 mu m filter, and is mixed with deionized water according to the proportion of 1:10, and then the mixture is added into a dish with the diameter of 9cm, and the deionized water without the fermentation supernatant is used as a control. A layer of filter paper is laid in the plate, and then dibbling is carried out. About 100 bungarus seeds were sown on each dish, with 4 replicates per treatment set. Sealing the plate with a sealing film after seeding is finished, carefully transferring the plate into an illumination incubator, culturing for 6-9 days at 25 ℃, counting the germination percentage and weighing, and calculating the fresh weight of the buds.

500mL of Hoagland's nutrient solution and 20mL of fermentation supernatant (centrifuged and sterile filtered) were added to the culture box and mixed well. Taking K326 seedlings with consistent growth vigor in a four-leaf stage, cleaning roots with clear water, washing off root soil, and then inserting the seedlings into culture holes for culture; hoagland culture medium without added supernatant and Hoagland culture medium with added LB medium were used as controls. Each group treated 8 seedlings, 3 replicates. Placing into a light incubator for culture for 14d (at 25 ℃, the light duration is 16h), taking out and weighing the fresh weight of the plant. 10mL of deionized water was added to the culture box daily during the culture.

The inhibition rate of serratia marcescens on TMV reaches 100%.

Serratia marcescens, designated S3, isolated from tobacco rhizosphere soil, (1): the low concentration of the S3 fermentation broth can promote germination. The tobacco seeds used in the experiment are K326 varieties, and the experiment result shows that when the S3 fermentation liquor and water are mixed according to the ratio of 1:10, which is beneficial to the germination of tobacco seeds, and compared with the control, the germination of the seeds treated by the S3 fermentation liquid is faster and the germination vigor is better after the germination. (2): when the fermentation liquor of S3 and the Hoagland hydroponic culture solution are mixed in a ratio of 1: 25, the growth of tobacco seedlings can be obviously promoted. Compared with the control, the leaf growth of the tobacco seedling is good after the S3 fermentation liquid is added into the culture solution.

The S3 strain is used as a growth-promoting microorganism of rhizosphere, and good colonization ability is an important index in application and development. The test result shows that the population number of the living strains in the leaves and the soil is reduced along with the time after the S3 is applied, the survival rate of the S3 is reduced along with the time in the leaves and the soil, but the colonization ability in the soil is obviously higher than that of the leaves. After 3 days of treatment, the colony number of S3 in the leaves and the natural soil is obviously reduced, the reduction range on the leaves is obviously larger than that of the other two treatments, and the colony number of S.marcocens-S3 in the sterile soil is slightly increased; the colony number of S3 in the samples treated by the leaf, the natural soil and the sterilized soil on the 8 th day after the treatment is reduced to about 3 multiplied by 106 CFU/g; on day 30 after treatment, the S3 content in the leaves was only 40CFU/g, and the natural soil was about 4000CFU/g, while the sterile soil was maintained at about 9X 105 CFU/g. Through indoor colonization experiments, the S3 has certain planting capacity on soil and leaves.

Preparing crude extract of the total protein of the serratia marcescens liquid by adopting an ammonium sulfate precipitation method, and separating the total protein by using Q Sephacrylsfast Flow under the following elution conditions:

elution was performed for 1 column volume per concentration of eluate, and each eluate was collected and desalted using a Millipore ultrafiltration tube with a molecular weight cut-off of 3000, concentrated and then assayed for TMV activity by the "half-leaf method".

Separating protein fraction with anti-TMV activity with Sephacryl S100 HR, collecting all the eluted proteins with anti-TMV activity, concentrating, and loading onto Sephacryl S100 HR column. Then washed with eluent (PBS,10mmol/L, pH 7.2).

The chromatographic conditions were as follows:

the elution peaks were collected, concentrated using an ultrafiltration tube with a Millipore molecular weight cut-off of 3000, and the inhibitory effect of each fraction on TMV was determined by the "half-leaf method".

Separation by anion exchange chromatography (Q Sephacryl Fast Flow) and exclusion chromatography (Sephacryl S100 HR) combined with biological activity ("hemiphylly") experiments, a protein with significant inactivation activity against TMV virus was isolated from the secreted protein of Serratia marcescens S3 strain (S. marcocens-S3), with a molecular weight of approximately 50 kDa. The protein is identified as Alkaline Metalloprotease (SAMP) through tandem mass spectrometry, and has high enzyme activity.

The alkaline metalloproteases secreted by Serralysin are a typical representation of serralysin proteins secreted by a number of gram-negative bacteria: it has a zinc ion catalytic domain, belonging to zinc finger protein; the zinc ion binding site amino acid sequence "HEXXHXXGXXH", wherein three histidines (H) are the zinc ion binding site, the middle glutamic acid (G) is the catalytic site, and "X" can be any amino acid; multiple "GGXGXD" sequences exist between 333-374 amino acid residues, which are calcium ion binding sites.

The enzymatic activity of alkaline metalloproteases is determined by the forskolin method.

1 standard curve

Taking 8 test tubes, preparing standard leucine solution according to the following table

Uniformly mixing the diluted leucine solution, respectively taking 1mL, respectively adding 5mL of 0.4mol/L sodium carbonate solution and 1mL of Folin-phenol reagent, placing in a water bath kettle at 40 ℃ for reaction for 20min, taking out, detecting the OD680 nm value by using an enzyme labeling instrument, and drawing a standard curve (figure 6).

2 enzyme Activity assay

Taking a 5mL centrifuge tube, adding 100 μ L enzyme solution, placing in 40 deg.C water bath, preheating for 2-3min, adding 100 μ L casein (2%) preheated at 40 deg.C, and reacting in 40 deg.C water bath for 10min (accurately timing). After the reaction was taken out, 200. mu.L of 0.4mol/L trichloroacetic acid solution was immediately added to terminate the enzyme reaction. Centrifuging after 10min (8000 Xg, 10min), adding 200 μ L of supernatant into 1mL of 0.4mol/L sodium carbonate solution, adding 200 μ L of Folin-phenol reagent, mixing, and reacting in 40 deg.C water bath for 20 min.

Blank control: a5 mL centrifuge tube was charged with 100. mu.L of enzyme solution, 200. mu.L of 0.4mol/L trichloroacetic acid solution, and 100. mu.L of casein (2%) preheated at 40 ℃. Centrifuging after 10min (8000 Xg, 10min), adding 200 μ L of supernatant into 1mL of 0.4mol/L sodium carbonate solution, adding 200 μ L of Folin-phenol reagent, mixing, and reacting in 40 deg.C water bath for 20 min.

3) And (3) calculating: hydrolysis of casein every 1min produced 1. mu.g of tyrosine counted as 1 unit of enzyme activity.

3 influence of pH and temperature on recombinant SAMP enzyme Activity

1) Influence of pH on enzyme Activity and enzyme stability

And (3) adjusting the pH value of the SAMP solution by using buffer solutions with different pH values, and then measuring the enzyme activity according to the method.

SAMP was added to each buffer at different pH values (3.0-11.0) for 10 min. And after the reaction is finished, taking out the reaction product, adjusting the pH value to the optimal reaction pH value, and then measuring the residual enzyme activity of SAMP.

2) Influence of temperature on pH on enzyme activity and enzyme stability

A method for determining SAMP enzyme activity by adopting a forskolin-phenol method and taking casein as a substrate is adopted, different reaction temperatures (30, 40, 50, 60, 70, 80 and 90 ℃) are set under the condition that the pH is 8.0, and the influence of the temperatures on the SAMP enzyme activity is respectively determined.

SAMP was maintained at different temperatures (30, 40, 50, 60, 70, 80 and 90 ℃) for 10min at pH 8.0, and the remaining enzyme activity was measured after the reaction by the Folin-phenol method using casein as a substrate.

Control effect of SAMP on TMV

1 preparation of Virus inoculum

Extracting virus granules and preparing virus inoculation liquid: TMV solutions were prepared using the method of Gooding & Helbert (1967) with minor modifications. Taking new upper leaves of NC89 infected with tobacco mosaic virus, grinding with 10mmol/L PBS buffer solution (pH7.4), and homogenizing; then filtered with double layer gauze and centrifuged (1000 Xg, 20 min); the supernatant was taken and treated twice with polyethylene glycol. Centrifuging again (10000 Xg, 30 min); the precipitate was resuspended in 10mmol/L PBS buffer (pH7.4) to obtain TMV inoculum. The whole operation is carried out at 4 ℃, then a spectrophotometer is used for measuring the absorbance value of 260nm wavelength, and the virus concentration is calculated according to a formula.

Concentration of virus (mg/mL) ═ A260 Xdilution factor/E^0.1％ _1cm260 nm

Note: e is the extinction coefficient, i.e. the value of the optical absorption (optical density) at an optical path length of 1crn for a suspension with a concentration of 0.1% (1mg/mL) at a wavelength of 260 nm. E of TMV^0.1％ _1cmThe concentration of the virus mother liquor is 10 mug/mL, and the concentration of the virus mother liquor is 3.1 at 260 nm.

2-leaf disc method for detecting inhibition effect of SAMP on TMV

The inhibitory effect of SAMP on TMV was examined with TMV 30B (GFP-tagged TMV strain).

1) TMV 30B infectious clones were inoculated by Agrobacterium infiltration method onto 4-week-old Nicotiana benthamiana (N.benthamiana) plants, and the leaves were cut into disks with a diameter of 0.5 cm.

2) The cut leaf disks were placed in 24-well plates and a solution of SAMP was added at a concentration not compatible per well (diluted with MS medium to concentration gradients of 6.5, 12.5, 50 and 100. mu.g/mL), and each concentration was repeated 3 times, adding 3 leaf disks per treatment.

3) The 24-well plate was sealed and incubated at 25 ℃ in the dark for 5 days.

4) The intensity of green fluorescence was detected with a multispectral fluorescence imaging system (FluorCam 7.0), from which the degree of TMV 30B development was indicated.

6.1.3.2 SAMP interferes TMV proliferation and diffusion in plants

100 μ L of TMV 30 infectious clone Agrobacterium suspension was infiltrated into 4 weeks of leaf of Nicotiana benthamiana (N.benthamiana) plants to form a circular infiltration area with a diameter of 1 cm.

After 48h, a loop of SAMP was infiltrated around the TMV 30B zone. The fluorescent condition on the leaves can be observed by an ultraviolet searchlight every 24h, and the intensity and diffusion of the green fluorescent light can indicate the occurrence and diffusion condition of the TMV virus. The control group was treated with PBS buffer (10mmol/L, pH 7.0).

Protective and therapeutic effects of alkaline metalloproteases on TMV

The prophylactic and therapeutic effects of purified SAMP on TMV were tested by the "semi-lobe method". Ningnanmycin (NNM) and BSA were used as positive and negative controls, respectively.

1) First, TMV inoculum was diluted to 1. mu.g/mL and SAMP was diluted to different concentrations (6.5, 12.5, 50 and 100. mu.g/mL).

2) Prevention effect experiment: applying 50 μ L of SAMP diluent to the right half of the leaf of the tobacco leaf, and applying PBS buffer (10mmol/L, pH 7.0) to the left half; after 1h, 20. mu.L of tobacco mosaic virus inoculum (TMV concentration 1. mu.g/mL) was inoculated onto the experimental tobacco leaf discs.

3) Therapeutic effect of SAMP on TMV experiment: mu.L (1. mu.g/mL) of the tobacco mosaic virus inoculum was frictionally inoculated onto the experimental leaves using quartz sand, washed with water, and air-dried. After 6 hours, 50. mu.L of SAMP dilutions of different concentrations were inoculated onto half of the TMV inoculated leaves, and the left half was treated with the same volume of PBS buffer (10mmol/L, pH 7.0).

Enzyme activity tests show that the single protein component obtained by chromatographic separation has strong metalloprotease activity; when the pH value of the reaction buffer solution is 8.0-9.0, the enzyme activity value is highest; when the reaction temperature reaches 40-50 ℃, the enzyme activity is highest. The stability experiment of the enzyme on the pH value proves that when the pH value of the reaction solution is lower than 5.0, the enzyme activity of alkaline metal protease (SAMP) is reduced to about 40 percent of the original enzyme activity; under alkaline conditions (pH is more than 7.0), the enzyme activity of SAMP is stable and is kept above 70%. The thermal stability experiment shows that: when the experimental temperature is 30-50 ℃, the SAMP activity is relatively stable and is kept above 80%; when the temperature is higher than 50 ℃ (60-70 ℃), the enzyme activity of SAMP is rapidly reduced to about 20%, and when the temperature is continuously increased to 80 ℃ or above, the enzyme activity is restored to about 40%, which indicates that SAMP has certain thermal stability.

The results of detecting the inhibition effect of SAMP on TMV by the hemiphyll method show that SAMP has good inhibition effect on TMV, the inhibition effect on TMV can reach 100% when the SAMP concentration is 50 mug/mL, and the inhibition effect on TMV is still maintained to be more than 75% when the SAMP concentration is reduced to 6.5 mug/mL.

In the experiment, active protein with good inhibition effect on TMV virus is separated from extracellular protein of S.marcocensstrain-S3 by chromatographic analysis methods such as anion exchange chromatography (Q Sephacryl FF) and exclusion chromatography (Sephacryl S100 HR) and combined with a biological activity (a half-leaf method) experiment, and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detects that the active protein presents an obvious and single protein band with the size of about 50 kDa. The analysis and identification are carried out by a tandem mass spectrometry (MS/MS) method (Katayama et al, 2010; Su et al, 2015), and the result shows that the anti-TMV Protein of S.marcocens-S3 has 7 peptide fragments matched with the secretory metalloprotease (Protein View No. wP _015377659.1), wherein the peptide fragment 'DTFVYFAAEESTAAAPDWIR' is a unique sequence of the metalloprotease, and the similarity of the two reaches 84%. The anti-TMV active protein isolated in s.marcescens-S3 was preliminarily judged to be alkaline metalloprotease (SAMP) from the results of tandem mass spectrometry, and its structure was analyzed by bioinformatics, demonstrating that SAMP has a hydrolysis domain.

The enzyme activity experiment shows that SAMP has high activity of metalloprotease, highest enzyme activity at pH 8.0-9.0 and temperature 40-50 deg.C, and certain thermal stability. The anti-TMV activity of SAMP is verified by a half-leaf method, and the result shows that SAMP obtained by Q Sephacryl FF and Sephacryl S100 HR chromatographic separation has good anti-TMV activity. The leaf disc method proves that SAMP can be absorbed by leaves and inhibits the proliferation of TMV in cells; the method for infiltrating SAMP proves that SAMP can effectively inhibit TMV virus from diffusing and remotely transporting from infected parts to other healthy parts in plant leaves; the three-generation tobacco is taken as a material, and the counting of the scorched spots proves that the SAMP protein has good prevention effect on TMV virus diseases and also has a certain treatment effect. In conclusion, SAMP has the potential for developing and applying anti-TMV medicaments in agricultural production.

The applicant has further developed a preparation containing an alkaline metalloprotease, the active ingredient of which is the alkaline metalloprotease, and further comprising a plant growth regulator and/or a stabilizer, the plant growth regulator being an amino-oligosaccharide and the plant growth regulator being an amino-oligosaccharide. The preparation is heated to 40-50 deg.C and sprayed on tobacco mosaic virus infected leaf.

In conclusion, the invention discloses the application of alkaline metalloprotease and the preparation containing the alkaline metalloprotease in preventing and treating tobacco mosaic virus, which has stable property, good antiviral effect by acting on host plants and viruses, and environmental protection.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (10)

1. The application of the alkaline metalloprotease in preventing and treating the tobacco mosaic virus is characterized in that the mass of the alkaline metalloprotease is 45-55kDa, and the pH of the alkaline metalloprotease is adjusted to 8.0-9.0.

2. The use according to claim 1, wherein the alkaline metalloprotease is derived from Serratia marcescens.

3. Use according to claim 1, characterized in that the alkaline metalloprotease has a mass of 50 kDa.

4. A preparation containing alkaline metalloprotease, characterized in that the active ingredients of the preparation are alkaline metalloprotease, and plant growth regulator and/or stabilizer.

5. The alkaline metalloprotease-containing preparation according to claim 4, wherein said alkaline metalloprotease is adjusted to a pH of 8.0 to 9.0.

6. The alkaline metalloprotease-containing preparation according to claim 4, wherein said concentration of alkaline metalloprotease in said preparation is 80 to 120. mu.g/mL.

7. The alkaline metalloprotease containing preparation according to claim 4, wherein said plant growth regulator is an amino-oligosaccharide.

8. The alkaline metalloprotease-containing preparation according to claim 4, wherein said stabilizer is trehalose.

9. Use of the alkaline metalloprotease-containing preparation according to any one of claims 4 to 8 for controlling tobacco mosaic virus.

10. Use according to claim 9, wherein the alkali metalloprotease containing preparation is sprayed after heating to 40-50 ℃.

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