CN110622968B - Phytophthora sojae inhibitor and preparation method thereof - Google Patents
Phytophthora sojae inhibitor and preparation method thereof Download PDFInfo
- Publication number
- CN110622968B CN110622968B CN201910806605.2A CN201910806605A CN110622968B CN 110622968 B CN110622968 B CN 110622968B CN 201910806605 A CN201910806605 A CN 201910806605A CN 110622968 B CN110622968 B CN 110622968B
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- Prior art keywords
- propyl gallate
- phytophthora sojae
- carbon dioxide
- inhibitor
- phytophthora
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Classifications
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- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
- A01N37/38—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system
- A01N37/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system having at least one carboxylic group or a thio analogue, or a derivative thereof, and one oxygen or sulfur atom attached to the same aromatic ring system
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- C—CHEMISTRY; METALLURGY
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Organic Chemistry (AREA)
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Abstract
The invention provides a phytophthora sojae inhibitor and a preparation method thereof, relates to the field of biological control of plant diseases, and comprises propyl gallate which is used as a food additive and is mainly used for the antioxidation of food and has the value EC of phytophthora sojae50％When the concentration is only 0.458mmol/L, namely the inhibition rate is 50%, the concentration of the propyl gallate is only 0.458mmol/L, and the safety is high. The inhibition of the phytophthora sojae is mainly realized by inhibiting the growth of the phytophthora sojae through propyl gallate, an action mechanism and a target site, and the inhibition mechanism of the phytophthora sojae is disclosed by a proteomics technology and is closely related to the great down regulation of protein expression of key pathways such as fat transportation, metabolism, energy metabolism and carbohydrate metabolism; the propyl digallite can stimulate the defense reaction of plants, improve the expression quantity of resistance genes and resist the infection of phytophthora sojae.
Description
Technical Field
The invention relates to the field of biological control of plant diseases, in particular to a phytophthora sojae inhibitor and a preparation method thereof.
Background
The invention relates to the field of biological control of plant diseases, in particular to a phytophthora sojae inhibitor and a preparation method thereof.
Background
The soybean phytophthora caused by phytophthora sojae is one of the devastating diseases in soybean production, and is one kind of plant quarantine dangerous pathogenic fungi published externally in China. The pathogenic fungi are mainly spread in soil, the pathogenic fungi of the phytophthora sojae are typical soil inhabitation fungi, and the oospores with strong stress resistance are mainly used for overwintering and long-term survival in the soil and host residues in the soil. When the field temperature and humidity are proper, the oospores break dormancy to germinate to produce sporangia, when water is accumulated after rain, the sporangia quickly releases a large amount of zoospores, and the zoospores are splashed to the surface of the host soil by rain water or are attracted by host secretions. Move towards the root system and finally infect the host. The existing methods for preventing and treating phytophthora sojae include the following methods: (1) selecting a disease-resistant variety with resistance to local small seeds. (2) Strengthen field management, and realize timely deep ploughing and intertillage ridging. And accumulated water is timely drained after rain to prevent moisture from being retained. (3) Applying metalaxyl granule in furrow during sowing to make soybean root absorb and prevent root infection. (4) The weight of the seeds before sowing is 0.3 percent, and the metalaxyl powder is 35 percent. (5) When necessary, spraying or irrigating 25% metalaxyl wettable powder 800 times liquid, 58% metalaxyl manganese zinc wettable powder 600 times liquid or 64% chalcanthite wettable powder 900 times liquid. It is known that metalaxyl or algicide inhibits the growth of phytophthora sojae as a main control means in the control of phytophthora sojae, and the action mechanism is to inhibit RNA polymerase so as to inhibit RNA biosynthesis.
The pesticide can cause phytotoxicity to plants when being improperly used, cause poisoning to people and livestock, kill beneficial microorganisms, cause pathogen to generate drug resistance, and cause environmental pollution due to high pesticide residue.
Disclosure of Invention
The invention aims to provide a phytophthora sojae inhibitor and a preparation method thereof, and aims to solve the technical problems.
In order to solve the technical problems, the invention adopts the following technical scheme: a soybean phytophthora inhibitor comprises propyl gallate and decanol, wherein the decanol is used as a cosolvent of the propyl gallate, and the dosage ratio is as follows: 0.1mg-0.3mg decanol: 1-2mmol propyl gallate.
The propyl gallate is extracted by adopting a supercritical carbon dioxide extraction method, and the method comprises the following steps:
(1) accurately weighing 1.3-1.8kg of radix Paeoniae Rubra powder with particle size of 2-5mm, adding into 17-22% pure water at 6-8 deg.C, standing at 5 deg.C for 50-70 min, and extracting with supercritical carbon dioxide at supercritical carbon dioxide extraction pressure of 33-37MPa for 54-57 min and carbon dioxide flow rate of 4.5-5.7L/min to obtain propyl gallate crude extract;
(2) and (3) propyl gallate purification: dissolving the propyl gallate crude extract in the step (3) in hot water at 85-90 ℃ according to the mass ratio of 1:5, and filtering macromolecular impurities by using an ultrafiltration membrane with the molecular weight cutoff of 10 kDa; then concentrating in a rotary evaporator until the content of the propyl gallate is more than 20-50 mg/L;
(3) crystallization purification and drying: transferring the propyl gallate in the step (4) into a 50ml centrifugal tube, standing at 4-6 ℃ for 12 hours, allowing a precipitate to appear at the bottom of the centrifugal tube, centrifuging at 10000rpm/min for 5 minutes, removing a supernatant, quickly freezing the precipitate with liquid nitrogen, freeze-drying at-60 ℃ under a vacuum condition of a vacuum degree of 45, and drying to powder to obtain the propyl gallate.
The propyl gallate can also be prepared from gallic acid and propanol under the catalysis of tannase, and comprises the following preparation steps:
(1) the gallic acid extraction process comprises the following steps: weighing 1.3-1.8kg of blueberry leaves or green tea powder with particle size of 2-5mm by using a supercritical carbon dioxide extraction method, adding the blueberry leaves or green tea powder into 17-22% of pure water with temperature of 2-8 ℃, standing for 50-70 minutes at 5 ℃, wherein in the supercritical carbon dioxide extraction, the supercritical carbon dioxide extraction pressure is 23-27MPa, and the extraction time is 32-50 minutes, so as to extract gallic acid;
(2) the preparation process of tannase comprises the following steps: culturing Aspergillus niger in liquid potato culture medium for 3-5 days, filtering, collecting fermentation broth, precipitating with ammonium sulfate precipitation method, re-dissolving to extract crude protein, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 10kDa to remove ammonium sulfate and concentrate protein to obtain tannase;
(3) the synthesis method of propyl gallate comprises the following steps: dissolving 8.9mmol of gallic acid in 8.2% -13.6% propanol solution, adding 0.3-0.6mol of tannase, performing catalytic reaction in a carbon dioxide incubator at a constant temperature of 20 ℃ for 10-15 hours, freeze-drying at-60 ℃ under a vacuum condition of a vacuum degree of 45, and drying to powder to obtain propyl gallate.
The preparation method of the soybean phytophthora inhibitor comprises the following steps of mixing propyl gallate with decanol: mixing 0.1-0.3 mg decanol and 1-2mmol propyl gallate, dissolving completely, and sequentially adding 500mL hot water of 60-70 deg.C and normal temperature water of 20-25 deg.C respectively to obtain soybean phytophthora inhibitor.
The invention has the beneficial effects that:
propyl gallate as a food additive has the following advantages: the safety is high; no pesticide residue is left; can be biosynthesized, has low energy consumption and is environment-friendly; the soybean resistance gene is excited to be obviously up-regulated to express and resist the infection of phytophthora sojae; the control effect on the phytophthora sojae is good, the concentration for inhibiting the growth of 50% of the phytophthora sojae is only 0.458mmol/L, the dosage of the inhibitor for inhibiting the growth of 50% of the phytophthora sojae is smaller, the control effect is better than that of a common pesticide, and the propyl gallate can also stimulate the defense reaction of plants and improve the expression level of a resistance gene to resist the infection of the phytophthora sojae.
Description of the drawings:
FIG. 1 is a histogram of the length distribution of peptide fragments identified by mass spectrometry according to the present invention;
FIG. 2 is a schematic diagram showing the relationship between the molecular weight and the coverage of the protein identified by mass spectrometry according to the present invention;
FIG. 3 is a graph of mass accuracy of mass spectral data according to the present invention;
FIG. 4 is a functional classification distribution diagram of the differentially expressed protein KOG according to the invention;
FIG. 5 is an enrichment analysis diagram of the differentially expressed protein KEGG pathway of the present invention;
FIG. 6 is a schematic illustration of the fatty acid metabolic pathway of the present invention;
FIG. 7 is a schematic of the starch and sugar metabolic pathways of the present invention;
FIG. 8 is a bar graph of the relative gene expression levels of PG treated soybeans of the present invention versus untreated soybeans.
Detailed Description
The present invention will be further described with reference to specific embodiments for the purpose of facilitating an understanding of technical means, characteristics of creation, objectives and functions realized by the present invention, but the following embodiments are only preferred embodiments of the present invention, and are not intended to be exhaustive. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
A phytophthora sojae inhibitor, characterized by: comprises propyl gallate and decanol, wherein the decanol is used as a cosolvent of the propyl gallate, and the dosage ratio is as follows: 0.1mg-0.3mg decanol: 1-2mmol propyl gallate.
As shown in fig. 1-7, the mechanism of propyl gallate inhibition of phytophthora sojae is explored through proteomics: culturing untreated Phytophthora sojae and propyl gallate treated Phytophthora sojae (with gallic acid added to liquid V8 to give a final concentration of 0.5mmol/L) for 3-5 days, collecting mycelia, adding nitrogen, and grinding to powder. The samples of each group were sonicated by adding powdered phenol extraction buffer (containing 1% protease inhibitor) in 4 volumes. Adding equal volume of Tris equilibrium phenol, centrifuging at 4 deg.C and 5500g for 10min, collecting supernatant, adding 5 times volume of 0.1M ammonium acetate/methanol to precipitate overnight, and washing protein precipitate with methanol and acetone respectively to obtain protein solution. Dithiothreitol was added to the protein solution to give a final concentration of 5mM, and the solution was reduced at 56 ℃ for30 min. After that, iodoacetamide was added to give a final concentration of 11mM, and incubated for 15min at room temperature in the absence of light. Finally the urea concentration of the sample was diluted to below 2M. Adding pancreatin in a mass ratio of 1:50 (pancreatin: protein), and carrying out enzymolysis for 12h at 37 ℃. Adding pancreatin in a mass ratio of 1:100 (pancreatin: protein), and continuing enzymolysis for 4 h. The pancreatin peptide fragments were desalted with Strata X C18(Phenomenex) and vacuum freeze-dried. The peptide fragments were solubilized at 0.5M TEAB and labeled according to the protocol of the TMT kit.
The simple operation is as follows: thawing the labeled reagent, dissolving with acetonitrile, mixing with the peptide segment, incubating at room temperature for 2h, mixing the labeled peptide segment, desalting, and vacuum freeze drying. The peptide fragments were fractionated by high pH reverse phase HPLC using an Agilent 300 extended C18 column (5 μm size, 4.6mm inner diameter, 250mm length). The operation is as follows: the peptide fragment gradient is 8-32% acetonitrile, pH is 9, 60 components are separated in 60min, then the peptide fragments are combined into 18 components, and the combined components are subjected to vacuum freeze drying and then are subjected to subsequent operation.
The peptide fragment was dissolved in mobile phase A (0.1% (v/v) formic acid aqueous solution) by liquid chromatography, and then separated by using EASY-nLC 1000 ultra performance liquid system. The mobile phase A is an aqueous solution containing 0.1 percent of formic acid and 2 percent of acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Setting a liquid phase gradient: 0-24 min, 9% -25% of B; 24-32 min, 25% -36% of B; 32-36 min, 36% -80% B; 36-40 min, 80% B, and the flow rate is maintained at 350 nL/min.
The peptide fragments were separated by ultra high performance liquid system, injected into NSI ion source for ionization and then analyzed by QOxective Plus mass spectrometry. The ion source voltage was set at 2.0kV and both the peptide fragment parent ion and its secondary fragment were detected and analyzed using the high resolution Orbitrap. The scanning range of the primary mass spectrum is set to be 350-1800m/z, and the scanning resolution is set to be 70,000; the secondary mass spectral scan range was then fixed with a starting point of 100m/z and the secondary scan resolution was set to 17,500. The data acquisition mode uses a data-dependent scanning (DDA) program, namely, after the primary scanning, the first 20 peptide fragment parent ions with the highest signal intensity are selected to sequentially enter an HCD collision cell for fragmentation by using 31% of fragmentation energy, and secondary mass spectrometry is also sequentially performed. To improve the effective utilization of the mass spectra, the Automatic Gain Control (AGC) was set to 5E4, the signal threshold was set to 10000ions/s, the maximum injection time was set to 200ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 seconds to avoid repeated scans of parent ions.
Database search and protein annotation: secondary mass spectral data were retrieved using Maxquant (v1.5.2.8), retrieving parameter settings: the database is NCBI Phytophthora _ sojae (26489 sequences), a reverse library is added to calculate false positive rate (FDR) caused by random matching, and a common pollution library is added into the database and is used for eliminating the influence of pollution protein in the identification result; the enzyme cutting mode is set as Trypsin/P; the number of missed cutting sites is set to 2; the minimum length of the peptide segment is set to be 7 amino acid residues; the maximum modification number of the peptide fragment is set to be 5; the First-level parent ion mass error tolerance of the First search and the Main search is respectively set to be 20ppm and 5ppm, and the mass error tolerance of the second-level fragment ions is 0.02 Da. Cysteine alkylation is set as fixed modification, and variable modification is oxidation of methionine and acetylation of the N-terminal of the protein. The quantitative method is set as TMT-6plex, and the FDR of protein identification and PSM identification is set as 1%. (1) Gene Ontology analysis Gene Ontology (GO) annotations at the proteomics level were derived from the UniProt-GOA database. If the protein is from the Uniprot database, the Uniprot ID is firstly used for matching the GO ID, and corresponding information is called from the Uniprot-GOA database according to the GO ID. If the UniProt-GOA database does not have the queried protein information, an algorithm software based on protein sequences, InterProScan, is used for predicting the GO function of the protein. This protein is then classified according to cellular composition, molecular function or physiological process. Protein pathways were annotated using the KEGG pathway database: first, the presented proteins were annotated using the KEGG online service tool KAAS, after which the annotated proteins were matched into the corresponding pathways in the database by the KEGG mapper.
As shown in fig. 1, the length distribution result of the peptide fragment of the protein of the sample is shown, after the sample is subjected to pancreatin enzymolysis and TMT labeling, the protein is identified by reverse HPLC (high performance liquid chromatography) and mass spectrometry analysis, and the fragment ions generated by the peptide fragment of less than 5 amino acids are too few, so that the sequence identification is inaccurate; whereas when the peptide fragments are larger than 20 amino acids, they are not suitable for the HCD fragmentation mode due to the high mass and charge number. The experimental result shows that most of the peptide fragments are distributed in the range of 7-20 amino acids, the result shows that the length distribution of the peptide fragments accords with the general rule based on trypsin enzymolysis and HCD fragmentation, and the distribution of the length of the peptide fragments identified by mass spectrometry accords with the quality control requirement.
As shown in fig. 2, the molecular weight and the coverage of the protein component are in inverse relationship, and hydrolysis generates more enzymolysis peptide fragments as the molecular weight of the protein component increases; in other words, the number of peptides with the same coverage rate increases with the increase of the molecular weight of the protein component, and most protein components have the molecular weight of about 50kD and the coverage degree of about 70 percent.
As shown in figure 3, the primary mass error of the protein component spectrograms of the control group and the treated group is less than or equal to 6ppm, and the result shows that the mass spectrometry result conforms to the high-precision characteristic of the orbitrap mass spectrometry, namely, the mass spectrometer operates normally and can meet the qualitative and quantitative analysis requirements of the protein components. The score of the spectrogram matching peptide fragment is high, the protein component identification quality is reflected to be good, the reliability is high, and the requirement of analyzing the protein components of two groups of samples can be met.
As shown in FIG. 4, the collectible of homologous histones (COG) of the difference proteins were classified into 20 types of functions by the alignment analysis and statistics of the eukaryotic KOG database.
The proteins that down-regulate expression are mainly distributed in 16 types of KOG functions such as fat transport and metabolism (Lipid transport and metabolism), Carbohydrate transport and metabolism (Carbohydrate transport and metabolism), Signal transduction mechanisms (Signal transduction mechanisms), Secondary metabolic synthesis, transport and catabolism (Secondary metabolism biosynthesis, transport and metabolism) and Energy production and transformation (Energy production and conversion), and the number of differential proteins involved in these functions is 17, 16, 12, 11 and 10, respectively. Relates to the reasons that the protein expression is greatly reduced and the growth of the soybean phytophthora is inhibited, such as fat transportation and metabolism, energy metabolism, carbohydrate transportation and metabolism and the like.
As shown in fig. 5 is a differential protein KEGG enrichment assay showing that a total of 17 up-regulated expression differential proteins were enriched into "psoj 03010 ribosomes (ribosomes, 6 differentially expressed proteins)", "psoj 00010 Glycolysis/Gluconeogenesis (glycolytic carbohydrate neogenesis, 4 differentially expressed proteins)" and "psoj 01110 biosyntheses of secondary metabolites (production of secondary metabolites, 7 differentially expressed proteins)". From the enrichment pathway, it is known that the proteins are probably related to the translation of proteins by organelle ribosome function, glycolysis of carbohydrate utilization pathway and carbohydrate regeneration, and more different proteins are probably involved in the secondary metabolic process of phytophthora sojae. In addition, the-log 10(p value) of "psoj 03010 Ribosome", "psoj 00010 Glycolysis/Gluconeogenesis" and "psoj 01110 Biosynthesis of second metabolites are 3.01, 1.73 and 1.4, respectively. 159 of the proteins downregulated in expression (75.7% of the total proteins downregulated in expression) were enriched in "psoj 00350Tyrosine metabisulfins" (Tyrosine, 8 differentially expressed proteins), "psoj 00360 phenylalkane metabisulfins" (Phenylalanine metabolism, 36 differentially expressed proteins), "psoj 00130Ubiquinone and other tertiary-quinone biosyntheses (Biosynthesis of ubiquinones and other terpenoid quinones, 6 differentially expressed proteins)," psoj01110 biosyntheses of secondary metabolism metabisulfites "(22 differentially expressed proteins), respectively, the major eight metabolic pathways include "psoj 00071 Fatty acid degradation (6 differentially expressed proteins)", "psoj 00500 Starch and sucrose metabolism (Starch and sugar metabolism, 4 differentially expressed proteins)", "psoj 00010 Glycolysis/Gluconeogenesis (glycolytic carbohydrate neogenesis, 6 differentially expressed proteins)" and "psoj 00460 Cyanoamino acid metabolism (3 differentially expressed proteins)". We silence both succinate dehydrogenase subunit A, B and fumarate reductase of Phytophthora sojae resulted in a decrease in the growth rate of Phytophthora sojae.
As shown in fig. 6, the proteins XP _009534955.1, XP _009522539.1 and XP _009527170.1 are involved in regulating and controlling long-chain acyl-coa synthesis, acetyl-coa oxidation and acetyl-coa dehydrooxidation, and the down regulation of three key proteins leads to phytophthora sojae fat metabolism disorder and the integrity of related cell structures such as cell membranes is impaired, and the protein descent pathway is shaded in the figure.
In addition, as shown in fig. 7, the proteins XP _009526054.1, XP _009526052.1, XP _009526051.1 and XP _009517993.1 are glycoside hydrolase, a glycoside hydrolase family, glycoside hydrolase and glycosyltransferase, respectively, and participate in starch, sugar and energy metabolism processes, the reduction of the translation amount of key proteins in the pathway negatively affects the growth of phytophthora sojae, and the protein reduction pathway is shaded in the figure.
Example 2
The propyl gallate is prepared from gallic acid and propanol under the catalysis of tannase, and comprises the following preparation steps:
(1) the gallic acid extraction process comprises the following steps: weighing 1.3-1.8kg of blueberry leaves or green tea powder with particle size of 2-5mm by using a supercritical carbon dioxide extraction method, adding the blueberry leaves or green tea powder into 17-22% of pure water with temperature of 2-8 ℃, standing for 50-70 minutes at 5 ℃, wherein in the supercritical carbon dioxide extraction, the supercritical carbon dioxide extraction pressure is 23-27MPa, and the extraction time is 32-50 minutes, so as to extract gallic acid;
(2) the preparation process of tannase comprises the following steps: culturing Aspergillus niger in liquid potato culture medium for 3-5 days, filtering, collecting fermentation broth, precipitating with ammonium sulfate precipitation method, re-dissolving to extract crude protein, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 10kDa to remove ammonium sulfate and concentrate protein to obtain tannase;
(3) the synthesis method of propyl gallate comprises the following steps: dissolving 8.9mmol of gallic acid in 8.2% -13.6% propanol solution, adding 0.3-0.6mol of tannase, performing catalytic reaction in a carbon dioxide incubator at a constant temperature of 20 ℃ for 10-15 hours, freeze-drying at-60 ℃ under a vacuum condition of a vacuum degree of 45, and drying to powder to obtain propyl gallate.
The propyl gallate can also be extracted by supercritical carbon dioxide, which comprises the following steps:
(1) accurately weighing 1.3-1.8kg of radix Paeoniae Rubra powder with particle size of 2-5mm, adding into 17-22% pure water at 6-8 deg.C, standing at 5 deg.C for 50-70 min, and extracting with supercritical carbon dioxide at supercritical carbon dioxide extraction pressure of 33-37MPa for 54-57 min and carbon dioxide flow rate of 4.5-5.7L/min to obtain propyl gallate crude extract;
(2) and (3) propyl gallate purification: dissolving the propyl gallate crude extract in the step (3) in hot water at 85-90 ℃ according to the mass ratio of 1:5, and filtering macromolecular impurities by using an ultrafiltration membrane with the molecular weight cutoff of 10 kDa; then concentrating in a rotary evaporator until the content of the propyl gallate is more than 20-50 mg/L;
(3) crystallization purification and drying: transferring the propyl gallate in the step (4) into a 50ml centrifugal tube, standing at 4-6 ℃ for 12 hours, allowing a precipitate to appear at the bottom of the centrifugal tube, centrifuging at 10000rpm/min for 5 minutes, removing a supernatant, quickly freezing the precipitate with liquid nitrogen, freeze-drying at-60 ℃ under a vacuum condition of a vacuum degree of 45, and drying to powder to obtain the propyl gallate. As shown in the table 1 below, the following examples,
TABLE 1 propyl gallate extraction Process optimization Experimental Table
The extraction process is to use a Box-Behnken test model, and obtain a regression equation through experiments and data analysis:
Y＝10.7+0.89A+1.43B+0.94C+0.05AB+0.33AC+0.6BC-0.44A2-1.01B2-0.84C2(ii) a A, B, C, Y in Table 1 represent the extraction pressure, extraction time, CO2 flow rate and gallic acid extraction yield, respectively. According to an equation model, the maximum value of the gallic acid extraction yield is used as an index, and the optimal process for extracting the supercritical carbon dioxide is calculated and verified as follows: the extraction pressure is 33-37MPa, the extraction time is 54-57 minutes, and the flow rate of carbon dioxide is 4.5-5.7L/min.
Example 3
Selection of solvent and preparation of inhibition solution: taking the phytophthora sojae inhibition rate as an index, preferably selecting decanol as a cosolvent of propyl gallate from organic solvents such as ethanol, propanol, butanol, pentanol, hexanol, acetone, dimethyl sulfoxide, ethyl acetate, petroleum ether, decanol and the like, wherein the dosage ratio is as follows: 0.1mg-0.3mg decanol: 1-2mmol of propyl gallate, and after completely dissolving, sequentially adding 500mL of water respectively in 60-70 deg.C hot water and room temperature water, so as to be used for preventing and treating Phytophthora sojae. As shown in table 2
TABLE 2 solvent type and dosage optimization experimental table
The propyl gallate can also stimulate the defense reaction of plants, improve the expression level of resistance genes and resist the infection of phytophthora sojae. Reverse transcription of cDNA to mRNA and the extraction of takara reagents and the qRT-PCR system (25. mu.L) 2. mu.L of c DNA template, 12.5. mu.L of SYBR green, 8.5. mu.L of dd H2O and 1. mu.L of primers PCR program 95 ℃ for30 s; 39 cycles (95 ℃ for 5s,60 ℃ for30 s).
As shown in FIG. 8, the real-time fluorescent quantitative PCR result shows that the soybean resistance gene expression level can be induced to be remarkably increased. The PR3 gene expression level was continuously increased (up to 149 times) within 12 hours after the treatment, and it was found that the gene expression level ratio at the same time as that of the untreated soybean sample was 1 for comparison, and that it was mainly effective against the infection of Phytophthora sojae. "+" and "+" indicate treatment groups, respectively, and the gene expression levels were significantly different at P <0.01 and P <0.05 from those of the control group.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the preferred embodiments of the present invention are described in the above embodiments and the description, and are not intended to limit the present invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (4)
1. A phytophthora sojae inhibitor, characterized by: comprises propyl gallate and decanol, wherein the decanol is used as a cosolvent of the propyl gallate, and the dosage ratio is as follows: 0.1mg-0.3mg decanol: 1-2mmol propyl gallate.
2. The phytophthora sojae inhibitor according to claim 1, wherein: the propyl gallate is extracted by adopting a supercritical carbon dioxide extraction method, and the method comprises the following steps:
(1) accurately weighing 1.3-1.8kg of radix Paeoniae Rubra powder with particle size of 2-5mm, adding into 17-22% pure water at 6-8 deg.C, standing at 5 deg.C for 50-70 min, and extracting with supercritical carbon dioxide at supercritical carbon dioxide extraction pressure of 33-37MPa for 54-57 min and carbon dioxide flow rate of 4.5-5.7L/min to obtain propyl gallate crude extract;
(2) and (3) propyl gallate purification: dissolving the propyl gallate crude extract in the step (3) in hot water at 85-90 ℃ according to the mass ratio of 1:5, and filtering macromolecular impurities by using an ultrafiltration membrane with the molecular weight cutoff of 10 kDa; then concentrating in a rotary evaporator until the content of the propyl gallate is more than 20-50 mg/L;
(3) crystallization purification and drying: transferring the propyl gallate in the step (4) into a 50ml centrifugal tube, standing at 4-6 ℃ for 12 hours, allowing a precipitate to appear at the bottom of the centrifugal tube, centrifuging at 10000rpm/min for 5 minutes, removing a supernatant, quickly freezing the precipitate with liquid nitrogen, freeze-drying at-60 ℃ under a vacuum condition of a vacuum degree of 45, and drying to powder to obtain the propyl gallate.
3. The phytophthora sojae inhibitor according to claim 1, wherein: the propyl gallate can also be prepared from gallic acid and propanol under the catalysis of tannase, and comprises the following preparation steps:
(1) the gallic acid extraction process comprises the following steps: weighing 1.3-1.8kg of blueberry leaves or green tea powder with particle size of 2-5mm by using a supercritical carbon dioxide extraction method, adding the blueberry leaves or green tea powder into 17-22% of pure water with temperature of 2-8 ℃, standing for 50-70 minutes at 5 ℃, wherein in the supercritical carbon dioxide extraction, the supercritical carbon dioxide extraction pressure is 23-27MPa, and the extraction time is 32-50 minutes, so as to extract gallic acid;
(2) the preparation process of tannase comprises the following steps: culturing Aspergillus niger in liquid potato culture medium for 3-5 days, filtering, collecting fermentation broth, precipitating with ammonium sulfate precipitation method, re-dissolving to extract crude protein, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 10kDa to remove ammonium sulfate and concentrate protein to obtain tannase;
(3) the synthesis method of propyl gallate comprises the following steps: dissolving 8.9mmol of gallic acid in 8.2% -13.6% propanol solution, adding 0.3-0.6mol of tannase, performing catalytic reaction in a carbon dioxide incubator at a constant temperature of 20 ℃ for 10-15 hours, freeze-drying at-60 ℃ under a vacuum condition of a vacuum degree of 45, and drying to powder to obtain propyl gallate.
4. A phytophthora sojae inhibitor, characterized by: the preparation method of the soybean phytophthora inhibitor comprises the following steps of mixing propyl gallate with decanol: mixing 0.1-0.3 mg decanol and 1-2mmol propyl gallate, dissolving completely, and sequentially adding 500mL hot water of 60-70 deg.C and normal temperature water of 20-25 deg.C respectively to obtain soybean phytophthora inhibitor.
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| CN101633789A (en) * | 2009-08-20 | 2010-01-27 | 中国科学院广州化学研究所 | Heat oxygen aging resistance high polymer material and preparation method and application thereof |
| CN102325746A (en) * | 2009-02-19 | 2012-01-18 | 赢创德固赛有限公司 | Reactive extraction of free organic acids from ammonium salts thereof |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102325746A (en) * | 2009-02-19 | 2012-01-18 | 赢创德固赛有限公司 | Reactive extraction of free organic acids from ammonium salts thereof |
| CN101633789A (en) * | 2009-08-20 | 2010-01-27 | 中国科学院广州化学研究所 | Heat oxygen aging resistance high polymer material and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
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| "不同浓度的没食子酸丙酯对鲜切生姜保鲜特性的影响";丁君等;《现代食品科技》;20141231;第30卷(第9期);第236-240页 * |
| "没食子酸丙酯处理对库尔勒香梨果实贮藏期品质相关指标变化的影响";许娟等;《新疆农业科学》;20161231;第53卷(第01期);第126-134页 * |
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