CN113170792A - Preparation of nano prodigiosin preparation and application of nano prodigiosin preparation in prevention and treatment of vegetable economic crop virus diseases - Google Patents
Preparation of nano prodigiosin preparation and application of nano prodigiosin preparation in prevention and treatment of vegetable economic crop virus diseases Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
- A01N25/10—Macromolecular compounds
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/22—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing ingredients stabilising the active ingredients
Abstract
The invention provides a preparation method of a nano prodigiosin preparation and application of the nano prodigiosin preparation in prevention and treatment of virus diseases of vegetable economic crops, belongs to the technical field of application of secondary metabolites of microorganisms, and can solve the problem that the nano prodigiosin preparation is used for preventing and treating main economic crop virus diseases (PVY, TMV, CMV and TSWV). The preparation is prepared from prodigiosin and chitosan nano materials, the prodigiosin can obviously improve the ubiquitination level of a plant host heat shock protein (HSP 70), promote host HSP70 protein to be ubiquitinated and degraded, degrade or inhibit the expression of the Hsp70 protein, activate the natural immunocompetence of host plants, and realize the inhibition effect on common RNA viruses. The nano prodigiosin preparation provided by the invention is used for preventing and treating virus diseases of main economic crops, can ensure that the prodigiosin has stronger stability and more lasting effect, can play a good effect of preventing and treating the virus diseases, and has good application prospect in the field of multi-virus prevention and treatment of economic crops.
Description
Technical Field
The invention belongs to the technical field of application of microbial secondary metabolites, and particularly relates to a novel nano preparation mode of microbial prodigiosin in medicines for resisting virus diseases of cash crops and a novel application of microbial prodigiosin in prevention and treatment of the virus diseases of the cash crops.
Background
The main vegetable economic crops in China mainly comprise solanaceae, cruciferae, cucurbitaceae, compositae and the like, are main dining-table vegetables of residents in China, occupy main positions in the development of agricultural economy, the occurrence and the harm of virus diseases are main restriction factors for guaranteeing the improvement of yield and quality, the prevention and the treatment are mainly carried out by spraying chemical antiviral agents such as moroxydine hydrochloride and the like in production, and the prevention and the treatment are poor due to long service life, serious drug resistance and poor prevention effect.
At present, the following four virus diseases are mainly existed for the main economic crops of vegetables in China. Potato virus Y (Potato virus YPVY), which is a virus worldwide, can cause necrosis of plant veins, causing significant economic loss, but the virus is still lack of effective control measures so far. From tobacco mosaic virus: (Tobacco mosaic virusTMV) is a common virus of vegetable economic crops, has strong stress resistance, simple transmission path and wide host range. Research reports that TMV can infect more than 350 economic crops such as potatoes, tomatoes, tobaccos and the like, infected leaves are still high in infection activity after being treated at the high temperature of 120 ℃ for 10min, and the infected leaves can survive for 52 years in dry tobacco leaves. If the plants are infected with TMV in the seedling stage, the yield is more influenced andand (4) quality. Cucumber mosaic virus: (Cucumber mosaic virusCMV is Bromus mosaic Virus family (C.)Bromoviridae) Cucumovirus genus (A)Cucumovirus) The typical member of the virus can infect more than 1000 kinds of monocotyledons and dicotyledons, can be spread by 75 kinds of aphids, and some isolates can also be spread by seeds, has the characteristics of more hosts, wide distribution and the like, and is one of plant viruses causing the greatest harm to crops. Tomato spotted wilf virus (B) ((B))Tomato spotted wilt virusTSWV) is a serious disease that compromises quality and yield of commercial crops, with an estimated annual crop loss of over 10 billion dollars, at the forefront of the ten most important plant viruses. Tomato spotted wilt virus causes an increasingly serious problem in crops and ornamental crops worldwide. Tomato spotted wilf virus belongs to the order bunyaviridae: (Bunyaviridae) Tomato spotted wilf virus genus (A)Orthotospovirus) Is the only plant-infecting virus genus of the order bunyaviridae. TSWV is transmitted by thrips feeding, wherein Frankliniella occidentalis (A)Frankliniella occidentalis) Is the main propagation mediator.
Prodigiosin compounds (PGs) are a class of compounds containing Prodigiosin (PG) tripyrrole skeleton, and have a wide range of biological activities, such as antibacterial, immunosuppressive, antitumor, antiviral, etc. In 1978 Fullan N P et al reported the antitumor effect of PG in vivo. Since then, PG and its derivatives have been of continuing interest for use in antitumor research. Representative compounds include UP, GX15-070, mcPG, and the like. Among them, GX15-070 is the only PGs currently entering phase III clinical trials, and is expected to become a novel anticancer drug. But the neurotoxicity caused by high dose limits its clinical independent use to some extent. In addition, PGs have potential application value in the industries of food, medicine and the like. In recent years, researches show that prodigiosin can improve the immune induction resistance of plants to viruses, can induce the alkalization of acidic environment in organelles such as lysosomes and the like, prevent the fusion of the viruses and host cell membranes, and can also inhibit certain pathways which are beneficial to the replication and proliferation of the viruses.
During the infection replication and disease symptom occurrence of the plant virus, host self factors and virus-related pathogenic proteins are required to interact, so that the plant virus can be replicated and proliferated in plant cells, moved between cells and transported for a long distance. Heat shock proteins (Hsp) are important molecular chaperones in plant cells, are key proteins necessary for plant virus infection, and are important functional proteins for maintaining cell life activities. Heat shock proteins are also closely related to various aspects of the viral infection cycle, such as assembly and disassembly of virions, cell entry, genome replication, and viral gene expression, and numerous studies have shown that Hsp70 expression is related to the level of viral replication.
The ubiquitination modification is an important protein posttranslational modification mode, plays an important role in natural immune signal transduction, and can be combined with a plurality of key joint molecules in the natural immune transduction process so as to regulate and control a natural immune signal pathway. The ubiquitin-proteasome pathway was first discovered and is a common endogenous protein degradation mode, and the ubiquitination degradation of Hsp70 is beneficial to inhibit the replication and proliferation of various viruses.
The nano pesticide is developed by applying nano materials and nano technology to the development of pesticides and has good pesticide effect. The nano pesticide is not only simple change of pesticide dosage form, but also obvious effect after pesticide application can be caused by substantial change of pesticide processing process. The quantum size, macroscopic quantum tunnel effect and great specific surface area of the nano material endow the nano material with strong and broad-spectrum antibacterial and antiviral functions, and achieve good prevention and treatment effects on certain plant viruses. As a natural secondary metabolite of microorganisms, prodigiosin is easy to degrade in the natural environment and has short drug effect, so that the plant viruses cannot be effectively controlled by methods such as foliage spraying, root irrigation and the like by using the solution. The selection of a proper nano-carrier can help to improve the targeted selective drug delivery capability and drug effect of prodigiosin, and has considerable prospect.
Disclosure of Invention
The invention aims to provide a preparation method of a nano prodigiosin preparation and application thereof in prevention and treatment of vegetable economic crop virus diseases, the preparation has the advantages of strong stability, difficulty in degradation, promotion of shuttling and transport of prodigiosin in plants to increase drug effect and the like, improves the ubiquitination level of Hsp70 in plants, and can effectively prevent and treat viruses such as PVY, TMV, CMV, TSWV and the like.
In order to achieve the purpose, the invention adopts the following specific technical scheme: the invention provides a preparation method of a nano prodigiosin preparation and application thereof in prevention and treatment of vegetable economic crop virus diseases, which is characterized by comprising the following steps: the nano prodigiosin preparation is prepared from prodigiosin and chitosan-sodium tripolyphosphate nano material, wherein the prodigiosin is serratia marcescensSerratia marcescen-S3.
Preferably, the plants infected with PVY, TMV, CMV and TSWV are treated respectively, and Western blot technology is used for verifying that the whole ubiquitination level is increased after the nano prodigiosin preparation is sprayed on the burley tobacco, and meanwhile the protein level of Hsp70 in the host is reduced. The ubiquitination inhibitor MG-132 is used for inhibiting ubiquitination modification in a host body, the action of ubiquitination in the action mechanism of the prodigiosin antiviral disease is researched, and the result shows that the antiviral effect is achieved by inhibiting the expression of Hsp70 protein through the prodigiosin induced resistance. Inhibition of host ubiquitination modification levels inhibits the antiviral resistance to prodigiosin mediated by Hsp70, suggesting that ubiquitination modification by Hsp70 may play an important role in the mechanism of antiviral resistance to prodigiosin. The nano-grade prodigiosin preparation is proved to be capable of promoting host plant HSP70 protein to be ubiquitinated, degrading or inhibiting the expression of Hsp70 protein, activating the natural immunocompetence of host plants and inducing plant hosts to generate system resistance.
The prodigiosin disclosed by the invention is obtained from a TMV (Tetramethylbenzidine) resistant tricyclic compound, a preparation method and an application thereof, wherein the application number of the prodigiosin is 201310323749.5.
Preferably, the chitosan nano material is prepared by dissolving chitosan in 1% glacial acetic acid, and the final concentration of the chitosan solution is 0.4%, w/v; sodium tripolyphosphate is dissolved in deionized water, and the final concentration of the sodium tripolyphosphate solution is 0.2 percent, w/v.
Preferably, the nano-prodigiosin preparation is prepared by uniformly stirring and mixing a chitosan solution and a sodium tripolyphosphate solution after passing through 125 mm filter paper, and slowly adding prodigiosin into the chitosan nano-material solution for uniform mixing.
Preferably, the mass ratio of the chitosan to the prodigiosin is 2: 1.
Preferably, the application method of the nano-prodigiosin preparation is to uniformly spray the finished solution on plant leaves.
Preferably, the nano-scaled prodigiosin preparation may be applied to tobacco, tomato, pepper or potato leaves.
Compared with the prior art, the invention has the advantages and positive effects that:
(1) the invention provides application of a nanocrystallized prodigiosin preparation in prevention and treatment of virus diseases of economic crops, and the stability of prodigiosin for promoting plant immune induced resistance is improved by combining with a chitosan nano material, so that the prodigiosin can more stably and durably cause immune induced resistance in plants;
(2) the preparation provided by the invention can increase the stability of prodigiosin, and can promote the transportation and shuttling of prodigiosin in plants, thereby achieving the purpose of increasing the drug effect;
(3) the preparation provided by the invention has the advantages that the concentration of prodigiosin is 40mg/ml, the concentration of a chitosan solution is 0.4 percent, w/v, the concentration of a sodium tripolyphosphate solution is 0.2 percent, w/v, and the effect is good after the chitosan nanomaterial solution and the prodigiosin are mixed when the mixing ratio of the chitosan nanomaterial solution to the prodigiosin is 2: 1;
(4) the nano prodigiosin preparation provided by the invention can be applied to prevention and treatment of PVY in a spraying mode;
(5) the nano prodigiosin preparation provided by the invention can be used as a biological growth substance and applied to related production in the aspect of agriculture.
Drawings
Fig. 1 is an electron microscope image of a nano-prodigiosin preparation provided in example 1 of the present invention.
FIG. 2 is the average particle size distribution diagram and Zeta potential distribution diagram of the nano-prodigiosin preparation provided in example 1 of the present invention.
FIG. 3 is a graph showing the comparison of the accumulation of PVY, TMV, CMV and TSWV in host cells after treatment with different solutions in example 2 of the present invention.
Fig. 4 is a phenotypic result of tobacco leaves inoculated with PVY and a nano-prodigiosin preparation provided in example 2 of the present invention, the left of the graph is a PVY tobacco infection condition, and the right of the graph is a nano-prodigiosin preparation plus PVY tobacco infection condition.
Fig. 5 shows the inhibition effect of ubiquitination modification on Hsp70 protein reduction induced by the nano-prodigiosin preparation provided in example 3 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention provides application of a nano-prodigiosin preparation in PVY (physical vapor transport) prevention and treatment, wherein the nano-prodigiosin preparation is prepared from prodigiosin and chitosan nano-materials, and the prodigiosin is serratia marcescensSerratia marcescen-S3.
Prodigiosin isSerratia marcescen-A secondary metabolite of the S3, wherein the metabolite is,Serratia marcescen-the S3 strain is a bacterial strain with antiviral bioactivity, and has obvious effect on preventing and treating PVY, TMV, CMV and TSWV viruses. The Western blot technology is utilized to verify that the whole ubiquitination level of the nano prodigiosin preparation is increased after the nano prodigiosin preparation is sprayed on the Nicotiana benthamiana, and meanwhile, the protein level of Hsp70 in the host is reduced, which is consistent with the previous omics data. The ubiquitination inhibitor MG-132 is used for inhibiting ubiquitination modification in a host body, the action of ubiquitination in the action mechanism of the prodigiosin antiviral disease is researched, and the result shows that the antiviral effect is achieved by inhibiting the expression of Hsp70 protein through the prodigiosin induced resistance. Inhibition of host ubiquitination modification levels inhibits Hsp 70-mediated antiviral resistance to prodigiosin, suggesting ubiquitin of Hsp70Chemical modifications may play an important role in the mechanism of action of the antiviral resistance to prodigiosin. The nano-grade prodigiosin preparation is proved to be capable of promoting host plant HSP70 protein to be ubiquitinated, degrading or inhibiting the expression of Hsp70 protein, activating the natural immunocompetence of host plants and inducing plant hosts to generate system resistance.
In a preferred embodiment, the chitosan nano-material is prepared by dissolving chitosan in 1% glacial acetic acid to obtain chitosan nano-material; the final concentration of the chitosan nano-material solution is 80 mg/ml.
In a preferred embodiment, the concentration of prodigiosin is 40 mg/ml.
The invention also provides a preparation method of the nano-prodigiosin preparation, which is prepared by slowly adding prodigiosin liquid into the chitosan nano-material solution and uniformly mixing.
In a preferred embodiment, the mass ratio of chitosan to prodigiosin is 2: 1.
Wherein, if the mass ratio is too low, a lot of prodigiosin is not combined with the chitosan, and the effect of virus prevention and treatment is finally influenced, and if the ratio is too high, a lot of chitosan nano materials are not attached with prodigiosin, and unnecessary waste is caused.
In a preferred embodiment, the application method of the nano-prodigiosin preparation is to uniformly spray the finished solution on plant leaves.
In a preferred embodiment, the nanoengineered prodigiosin formulation can be applied to tobacco, tomato, pepper or potato.
In order to more clearly and specifically describe the nano-prodigiosin preparation for preventing and treating PVY and the preparation method thereof provided by the embodiments of the present invention, the following description will be made with reference to specific embodiments.
Example 1
The preparation method of the nano prodigiosin preparation for preventing and treating the virus diseases of the economic crops comprises the following steps:
s1: the preparation method of the nano prodigiosin preparation comprises the following specific steps:
s1-1: dissolving chitosan in 1% glacial acetic acid, wherein the final concentration of the chitosan solution is 0.4%, and w/v; dissolving sodium tripolyphosphate in deionized water, wherein the final concentration of the sodium tripolyphosphate solution is 0.2% w/v; the concentration of prodigiosin is 40 mg/ml;
s1-2: filtering the chitosan solution and the sodium tripolyphosphate solution by filter paper with the thickness of 125 mm, and adding the mixture into a beaker;
s1-3: stirring the chitosan solution by a magnetic stirrer at 1500rpm, dropwise adding the sodium tripolyphosphate solution and the prodigiosin solution, and stirring for 1 hour to mix uniformly;
s1-4: centrifuging at 4 deg.C in 11000 Xg low temperature centrifuge for 10 min;
s1-5: the supernatant was removed and washed with deionized water three times.
And finally, storing the prepared nano prodigiosin preparation in the dark, and sending a part of the prepared nano prodigiosin preparation to data such as detection particle size, Zeta potential, PDI and the like.
The detection result shows that the average particle size of the preparation is 200 nm to 400 nm, TEM images also show some anatomical microspheres, and micropores are formed in the microspheres and promote the diffusion of the medicament. Zeta potential +31.5 mV, polydispersity index (PDI) 0.452. These data indicate that the nano-prodigiosin preparation has excellent properties of nano-pharmaceuticals.
Example 2
An application of the nano prodigiosin preparation for preventing and treating various virus diseases of economic crops in the virus treatment of the embodiment 1 comprises the following specific application methods:
s1: planting and transplanting the tobacco in the laboratory:
taking 15 disposable plastic cups, burning a glass rod by an alcohol lamp to poke a hole at the bottom of the plastic cup, and facilitating soil to absorb water. The nutrient soil is mixed with vermiculite 1:1 to ensure sufficient nutrition and strong water absorption capacity. Firstly planting tobacco in a flowerpot, and transplanting the tobacco into a plastic cup after the height of the tobacco is suitable. The root integrity of the tobacco is ensured in the transplanting process, sufficient water supply is ensured, and the constant temperature room illuminates for 18h every day. Waiting for 2-3 weeks.
S2: inoculation of tobacco virus:
200 mg of the source leaves are put into a mortar which is sterilized in advance, 10ml of 1 XPBS buffer solution is added for grinding, the supernatant is taken after centrifugation, and the supernatant is absorbed by a 1ml syringe and then injected into the 3 rd to 4 th true leaves of the Nicotiana benthamiana seedlings (4 weeks). After 24h, the nano-prodigiosin preparation (PG-CS), the Chitosan Solution (CS), the prodigiosin solution (PG), the moroxydine hydrochloride solution (AB 0B) and water are respectively used for being applied to tobacco leaves by a spraying method, 5 viruses of each type are treated for 3 times, and the treatment is respectively as follows: spraying chitosan solution after 3 basins of tobacco are inoculated with PVY only, and spraying nano prodigiosin preparation solution after 3 basins of tobacco are inoculated with PVY to serve as a negative control group.
S3: the spraying of the nano prodigiosin preparation comprises the following specific steps:
the medicament is diluted to 200 mug/ml, 30ml of the medicament is uniformly sprayed on each plant once a day for two days.
S4: obtaining plant cDNA for relative fluorescence quantification, which comprises the following steps:
s4-1: the method comprises the following steps of (1) extracting total RNA of plant leaves by using a total RNA extraction kit:
(1) weighing 50-100 mg of plant leaves, putting the plant leaves into a 1.5ml RNA-Free centrifuge tube, quickly grinding the plant leaves into powder in liquid nitrogen by using a grinding rod, adding 500 mu l of SL (beta-mercaptoethanol) into the powder, and uniformly mixing the powder by vortex. 12000rpm, centrifuging for 2 min;
(2) transferring the supernatant to a filter column CS, placing the filter column CS on a collecting pipe, centrifuging at 12000rpm for 2min, and carefully sucking the supernatant in the collecting pipe to a new 1.5ml RNA-Free centrifuge tube under the condition that a suction head avoids sucking bottom sediment;
(3) the centrifuge tube was charged with 0.4 times the volume of the supernatant of absolute ethanol. Reversing and mixing evenly, transferring all the solution into an adsorption column CR3, centrifuging at 12000rpm for 30s, pouring the waste liquid in the collecting tube, and putting the adsorption column CR3 back into the collecting tube;
(4) adding 350 mu l of deproteinized liquid RW1 into an adsorption column CR3, centrifuging at 12000rpm for 30-60 s, discarding waste liquid, and putting the adsorption column back into a collecting pipe;
(5) preparing DNaseI solution: adding 10 mul DNaseI into 70 mul RDD solution, and slowly mixing;
(6) adding 80 μ l DNaseI solution into adsorption column CR3, and standing at room temperature for 15 min;
(7) adding 350 mu l of deproteinized liquid RW1 into CR3, centrifuging at 12000rpm for 30-60 s, discarding waste liquid, and placing an adsorption column back to the collection tube;
(8) adding 500 mul of rinsing liquid RW (added with absolute ethyl alcohol) into the CR3 in the adsorption column, standing for 2min, centrifuging at 12000rpm for 30-60 s, pouring off waste liquid, putting the adsorption column CR3 back to the collection tube, and removing impurities such as pigment. Placing the adsorption column into a new collection tube, centrifuging at 12000rpm for 2min, standing the adsorption column CR3 at room temperature, and air drying;
(9) placing the adsorption column CR3 into an RNase-Free centrifuge tube, and adding 30-50 μ l of RNase-Free ddH2O, standing at room temperature for 2min, centrifuging at 4 ℃ and 12000rpm for 2min, and storing the extracted RNA in an ultra-low temperature refrigerator at minus 80 ℃.
S4-2: the cDNA synthesis, the reaction utilizes the first strand synthesis Kit of FastKing RT Kit (With gDNase) FastKing cDNA of Tiangen to carry out the reverse transcription process, and the concrete operation is as follows:
(1) template RNA was thawed on ice, 5 Xg DNA Buffer, FQ-RY Primer Mix, 10 XKing RT Buffer, RNase-Free ddH2Unfreezing O at room temperature, then placing on ice, and completing the following steps on the ice;
(2) 2 mul of 5 Xg DNA Buffer and 8 mul of RNA are mixed evenly and centrifuged for 3min at 42 ℃, and then placed on ice after completion;
(3) preparing a mixed solution system of a reverse transcription system, which comprises the following specific steps: 10 XKing RT Buffer 2. mu.l; 1. mu.l of Fasiting RT Enzyme Mix; FQ-RY Primer Mix 2. mu.l; RNase-Free ddH2O 5μl；
(4) Adding the solution generated in the reverse transcription reaction into the mixed solution prepared in the step (3), and uniformly mixing;
(5) incubating at 42 deg.C for 15 min;
(6) incubating for 3min at 95 ℃, and storing the product in an ultra-low temperature refrigerator at-80 ℃.
S4-3: the reaction was amplified on an Applied Biosystems 7500 Real-Time PCR model using the Novozan ChamQ Universal SYBR qPCR Master Mix kit, the expression differences of specific genes in different samples were detected, and actin was set as an internal reference.
The reaction conditions are as follows:
(1) template cDNA was thawed on ice, 2 × ChamQ Universal SYBR qPCR Master Mix, ddH2O, unfreezing the upstream and downstream primers of the PVY CP at room temperature, then placing on ice, and completing the following steps on the ice;
(2) preparing a relative fluorescence quantitative mixed liquid system, which comprises the following specific steps: 2 XChamQ Universal SYBR qPCR Master Mix 10. mu.l; 0.4. mu.l of upstream primer (10. mu.M); 0.4. mu.l of downstream primer (10. mu.M); 2. mu.l of cDNA template; ddH2O 7.2μl。
(3) The reaction conditions are as follows:
at 95 ℃ for 30 s; 95 ℃ for 5 s; 60 ℃, 34 s (40 cycles); 95 ℃ for 5 s; 60 ℃ for 1 min; 95 ℃ for 15 s (dissolution curve).
The relative fluorescence quantitative result shows that compared with a pure prodigiosin solution or a chitosan solution, the nano prodigiosin preparation has better effect of resisting the tobacco viruses and can effectively inhibit the replication and proliferation of various tobacco viruses and the outbreak of virus diseases of economic crops.
Example 3
The influence of the nano-prodigiosin preparation on the host factor Hsp70 protein and the ubiquitination modification thereof:
in order to further confirm the expression level of Hsp70 protein and the change of ubiquitination after the nano-prodigiosin preparation is used for treating plants, the change of Hsp70 protein level and the whole ubiquitination level after the nano-prodigiosin preparation is used for treating Nicotiana benthamiana is detected from the protein level by using a ubiquitination inhibitor MG-132 and Western blot technology.
S1: obtaining plant holoprotein for Western blot, and comprises the following steps:
s1-1: grinding 150mg of plant leaves in liquid nitrogen;
s1-2: adding 1ml of lysis solution, treating at 4 deg.C for 20min, and shaking every 5 min;
s1-3: centrifuging at 14000rpm in a high-speed refrigerated centrifuge at 4 deg.C for 30 min;
s1-4: the supernatant was aspirated into a new centrifuge tube and stored at-80 ℃.
S2: polyacrylamide gel electrophoresis (SDS-PAGE), the concrete steps are as follows:
s2-1: installing an electrophoresis device, pulling out the comb of the rubber plate, and filling electrophoresis buffer solution;
s2-2: protein spotting: loading (10-15 μ L), mixing protein and 2 × loading buffer solution at a ratio of 1:1, boiling in water bath at 100 deg.C for about 5min, and loading sample in predetermined order;
s2-3: electrophoresis: using 1 × Tris glycine running buffer (5 × Tris glycine running buffer diluted), the electrophoresis apparatus and power supply were connected, and 180V voltage was continued for about 40 min, and when blue bromophenol blue was seen to reach the bottom of the separation gel, the power supply was turned off, and the upper and lower ends of the gel were cut off.
S3: immunoblotting (Western blotting) was carried out in the following manner:
s3-1: film transfer: soaking the PVDF membrane in methanol for about 1 min before use to increase the hydrophilicity of the membrane, adding the PVDF membrane and gel according to the direction of an electrophoresis electrode, rotating the membrane for 80-90 min under the current condition of 100V voltage and 350 mA, and transferring a protein band to the PVDF membrane;
s3-2: and (3) sealing: completely soaking the PVDF membrane in a sealing solution, and sealing at room temperature for about 1.5 h;
s3-3: primary antibody reaction. Incubating the primary antibody at 4 ℃ overnight, washing the membrane with 25 ml of LTBST at room temperature for 10min, and repeating twice;
s3-4: and (5) secondary antibody reaction. Incubating for 1 h with HRP-labeled secondary antibody at room temperature, washing the membrane with 25 ml of LTBST for 10min at room temperature, and repeating twice;
s3-5: and (3) detection: and (3) using Gelgap software of a full-automatic chemiluminescence imaging analysis system, precooling the film to-30 ℃ in advance by using a machine, putting the washed film into the machine, enabling the washed film to face upwards, dripping luminous liquid, fully contacting, focusing, shooting and storing.
Western Blot results show that after the nano-prodigiosin preparation is applied, the expression level of host cell Hsp70 is obviously reduced, and after MG-132 and the nano-prodigiosin preparation are treated, the PVY infection histone gray values are 139.33 and 84.59 respectively; after 7dpi is treated by MG-132 and the nano-prodigiosin preparation, the PVY infected histone gray values are 180.33 and 132.04 respectively, which are obviously higher than those of a control group. These results indicate that ubiquitination plays a key role in the ISR process of nano-genized prodigiosin preparations. Inhibition of ubiquitination can lead to reduction of the amount of Hsp70 protein accumulation inhibited by the nano-genized prodigiosin preparation, thereby inhibiting the replication of PVY.
Claims (7)
1. The preparation of the nano prodigiosin preparation is characterized by comprising the following steps: the nano prodigiosin preparation is prepared from prodigiosin, chitosan and sodium tripolyphosphate nano materials.
2. The preparation of a nanosized prodigiosin preparation according to claim 1, characterized in that: the prodigiosin is serratia marcescens (L.)Serratia marcescen-S3) The secondary metabolite of (a); the prodigiosin is a liquid or solid preparation.
3. The preparation of a nanosized prodigiosin preparation according to claim 1, characterized in that: the preparation method of the chitosan nano material is that chitosan is dissolved in 1% glacial acetic acid, and the final concentration of the chitosan solution is 0.4%, w/v; dissolving sodium tripolyphosphate in deionized water, wherein the final concentration of the sodium tripolyphosphate solution is 0.2%, and w/v, so as to obtain the chitosan nano material; the nano-prodigiosin preparation is prepared by filtering a chitosan solution and a sodium tripolyphosphate solution through 125 mm filter paper, stirring the chitosan solution by a magnetic stirrer at 1500rpm, dropwise adding the sodium tripolyphosphate solution and the prodigiosin solution, stirring for 1 hour, and uniformly mixing; centrifuging at 4 deg.C in 11000 Xg low temperature centrifuge for 10 min; removing supernatant, deionized water and washing for three times; and finally, storing the prepared nano prodigiosin preparation in the dark.
4. Preparation of a Nanomethanized prodigiosin preparation according to claim 1 or 3, characterized in that: the mass ratio of the chitosan to the prodigiosin is 2: 1.
5. The application of the nano prodigiosin preparation in preventing and treating the virus diseases of the vegetable economic crops is characterized in that: the application method of the nano prodigiosin preparation comprises the steps of diluting a finished product solution medicament to 200 mu g/ml, uniformly spraying 30ml of the nano prodigiosin preparation on each plant, and spraying the nano prodigiosin preparation once a day for two days.
6. The application of the nano prodigiosin preparation according to claim 5 in the control of viral diseases of vegetable commercial crops, which is characterized in that: the nanometer prodigiosin preparation is applied to vegetable commercial crops of solanaceae, cruciferae, cucurbitaceae, compositae and the like.
7. The application of the nano prodigiosin preparation according to claim 5 in the control of viral diseases of vegetable commercial crops, which is characterized in that: the nanometer prodigiosin preparation can be used for treating PVY, TMV, CMV and TSWV infected plants, promoting host plant HSP70 protein to be ubiquitinated, degrading or inhibiting expression of Hsp70 protein, activating natural immunocompetence of host plants and inducing plant hosts to generate systemic resistance.
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| CN114271279A (en) * | 2022-01-07 | 2022-04-05 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Nano prodigiosin preparation and application thereof in prevention and treatment of virus diseases of economic crops |
| CN114794120A (en) * | 2022-05-25 | 2022-07-29 | 中国农业科学院烟草研究所 | Method for reducing toxic amount of TSWV in thrips adults |
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