CN114957419A - Antifungal glycan protein peanut glycan argentamine and preparation and application thereof - Google Patents
Antifungal glycan protein peanut glycan argentamine and preparation and application thereof Download PDFInfo
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- CN114957419A CN114957419A CN202210454062.4A CN202210454062A CN114957419A CN 114957419 A CN114957419 A CN 114957419A CN 202210454062 A CN202210454062 A CN 202210454062A CN 114957419 A CN114957419 A CN 114957419A
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- antifungal
- glycan
- ahgp
- diameter
- protein
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- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
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Abstract
An antifungal glycan protein arachidan argentane and its preparation method and application are provided. The invention discloses liquid nitrogen treated peanut meal, which can be separated to obtain polysaccharide argentaxanthin resisting broad-spectrum fungi. The antifungal anaccharide argentine of the invention can effectively inhibit various plant pathogenic fungi (rust, fusarium, anthrax and rhizoctonia). The arachidan argentamine can effectively improve the antifungal activity of plants.
Description
Technical Field
The invention belongs to the technical field of biological pesticides, and particularly relates to preparation of antifungal arachidonin argentum yellow PG-AHGP and application of the antifungal arachidonin argentum yellow in preparation of antifungal drugs.
Background
Since the discovery of antibiotics, there has been a fundamental improvement in the treatment of fungal infectious diseases, and the use of antibiotics has saved crop economy and increased crop yield. However, with the large-scale use of antibiotics, especially in developing countries, the development and spread of some drug-resistant bacteria are caused, including some very virulent pathogenic bacteria, such as methicillin-resistant staphylococcus and streptococcus pneumoniae. Therefore, the search for safe and effective antibacterial drugs that do not easily develop drug resistance properties is a competitive and endeavor among scientists worldwide.
Higher plants have a wide range of defense mechanisms to cope with physical, chemical and biological stresses such as drought, cold, heavy metals, pollutants and pathogen attack from fungi, bacteria and viruses. In response to infection by various pathogens, plants exhibit an up-regulation of a set of genes associated with systemic acquired resistance [ Stintzi A., Heitz T., Prasad V., Wiedemann-Merdinoglu S., Kauffmann S., Geoffroy P., Legrand M., Fritig B.plant "genes-related" proteins and the control in feedback against genes of pathogenesis pages of biochemics.1993; 75:687-706.]. General resistance is achieved by the release of secondary metabolites such as phytoalexins, tannins and polyphenolic compounds and the production of pathogenesis-related (PR) proteins. PR proteins were first found in tobacco leaves in the early 1970 s to cope with tobacco mosaic virus infection, and were later defined as inducible proteins released during pathogenic outbreaks [ stinzi a., Heitz t., Prasad v., Wiedemann-Merdinoglu s., Kauffmann s., Geoffroy p., Legrand m., friigig b.plant "pathogenesis-related" proteins and the control in a pathogenic agent pathway biochemis.1993; 75: 687-706; sinha M., Singh R.P., Kushwaha G.S., Iqbal N., Singh A., Kaushik S., Kaur P., Sharma S., Singh T.P. Current overview of allogens of plant related proteins J.Sci.J.2014; 2014:543195.]. According to a recent review, at least 17 families have been detected and isolated, which have broad defense-related properties, including antibacterial, antifungal, antiviral, antioxidant activity, chitinase and protease inhibitory activity [ Stintzi A., Heitz T., Prasad V., Wiedemann-Merdinoglu S., Kauffmann S., Geoffree P., Legrand M., Fritig B.plant "pathologenes-related" proteins and the control in a controlled against disease pathway biochemis.1993; 75: 687-706; sinha M., Singh R.P., Kushwaha G.S., Iqbal N., Singh A., Kaushik S., Kaur P., Sharma S., Singh T.P. Current overview of allogens of plant related proteins J.Sci.J. 2014; 2014: 543195; ebrahim s, usaha k, Singh b.pathogenis related (pr) proteins in plant feedback mechanism.sci.against micro b.pathog.2011; 2: 1043-1054; sels J., substances J., De Coninck B.M., Cammmue B.P., De Bolle M.F. plant pathophysiology-related (pr) proteins A focus on pr peptides plant physiol. biochem.2008; 46: 941-950 ], including peptides with antibacterial activity, i.e., thionin (PR-13 family), defensins (PR-12 family), hevein-like peptides, knottins, alpha-hairpin proteins, lipid transfer proteins (PR-14 family) and serpentine.
A novel single-chain antifungal protein with the molecular weight of 13kDa is separated from ginkgo seeds, and the N-terminal sequence of the novel single-chain antifungal protein has obvious similarity with embryo-rich protein from white spruce. The protein is named as Yinhuang, and has effective antifungal activity on various fungi, including Botrytis cinerea, Neurospora arachidicola, Fusarium oxysporum, Rhizoctonia solani and Coprinus comatus. Has moderate antibacterial effect on Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli [ Wang, H.,&Ng,T.B.(2000).Ginkbilobin,a novel antifungal protein from Ginkgo biloba seeds with sequence similarity to embryo-abundant protein.Biochemical and Biophysical Research Communications,279(2),407-411.]. It has also been reported that this argentuxanthin inhibits the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans), but does not exhibit antibacterial effects against E.coli [ Sawano, Y., Miyakawa, T., Yamazaki, H., Tanokura, M.,&Hatano,K.I. (2007).Purification,characterization,and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds.]. The antifungal mechanism is likely to bind to fungal actin, triggering cell death [ Gao, n., Wadhwani, p.,
P.,Liu,Q.,Riemann,M.,Ulrich,A.S.,& Nick,P.(2016).An antifungal protein from Ginkgo biloba binds actin and can trigger cell death.Protoplasma,253(4),1159-1174；Gao,N.(2014).New Weapon from an Ancient Tree-Antifungal protein ginkbilobin binds actin (Doctoral dissertation,Karlsruhe,Karlsruher Institut für Technologie(KIT), Diss.,2014).]. Crystallization and preliminary X-ray analysis of Yinhuang, a novel antifungal protein homologous to the extracellular domain of a plant cysteine-rich receptor kinase [ Miyakawa, T., Sawano, Y., Miyazono, K.I., Hatano, K.I.,&Tanokura,M.(2007). Crystallization and preliminary X-ray analysis of ginkbilobin-2from Ginkgo biloba seeds:a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases.Acta Crystallographica Section F:Structural Biology and Crystallization Communications,63(9), 737-739.]. In addition to this, hydrogen bonding and hydrophobic interactions may also be the antimicrobial effects of such proteins [ Sahu, s.n., Moharana, m., Sahu, r.,&Pattanayak,S.K.(2019,August). Molecular docking approach study of binding performance of antifungal proteins.In AIP Conference Proceedings(Vol.2142,No.1,p.060001).AIP Publishing LLC.]. Glycan modifications of proteins have important effects on the activity and stability of the protein [ Zhou, q.,&Qiu,H.(2019).The mechanistic impact of N-glycosylation on stability,pharmacokinetics,and immunogenicity of therapeutic proteins.Journal of pharmaceutical sciences,108(4),1366-1377.]. However, polysaccharide modification of the argentine is rarely reported, and the polysaccharide-modified argentine is separated from peanut protein, so that a reliable tool is provided for controlling fungi.
Aspergillus niger and Penicillium chrysogenum belong to the biotrophic phytopathogenic fungi and are nutritionally dependent on the metabolism of living cells of plants such as grape. These fungi belong to the group of biotrophic fungi. They penetrate directly into the epidermis of the plant cell, and the infiltrated cells subsequently necrose. After infiltration, the fungus shifts to an obligate biotrophic lifestyle. A sub-population of biotrophic fungal pathogens that primarily follow such infection strategies are semi-dead-body trophic. In contrast to hemibiotrophic pathogens, hemibiotrophic pathogens live in a biotrophic mode for a short period of time and subsequently begin to kill host cells and/or host organisms.
In recent years, fungal diseases such as aspergillus niger have become increasingly important as diseases in agriculture, such as grape production. Wheat stripe rust caused by Puccinia striiformis, among rust fungi, is one of the most important wheat diseases worldwide [ Chen, x.m. epidemic and control of stripe rust striiformis f.sp.tritici on where is Canadian journal of plant disease 2005; 27.3:314-337.]. Among Fusarium fungi, Fusarium oxysporum is well representative in rhizosphere microflora. Almost all strains exist in a saprophytic manner, known to induce wilting or root rot in plants [ franll, d., Chantal Olivain, and Claude alamoutrage. Fusarium oxysporum and its biocontrol. new phytologist 2003; 157.3: 493-502.]. Anthrax species that induce anthrax disease are present in tropical and subtropical regions of the world. Are pathogens of berry diseases, which infect leaves and berries at any stage of their development, most are pathogens of brown rot in crop plants, often infecting mature berries [ Chen Z, Franco CF, Battista RP, Cabral JM, Coelho AV, Rodrigues CJ, Mel EP.purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides, Applied microbiology and biotechnology.2007; 73:1306-13.]. Among the Rhizoctonia fungi, the Rhizoctonia solani complex represents an economically important group of soil-borne basidiomycete pathogens that occur on many plant species worldwide [ Cubeta, m.a., and r.vilgrids., "marketing biology of the Rhizoctonia solani complex." Phytopathology 1997; 87.4: 480-484.].
For a long time, the pest control of crops in China mainly depends on chemical pesticides. Compared with chemical pesticides, biological pesticides have the advantages of safety, environmental protection, long-acting effect, no residue and the like, pay attention to low toxicity, high efficiency and strong selectivity, and become one of the current research hotspots.
Accordingly, there is a need in the art to develop methods of controlling fungal biological agents and providing antifungal plants. The peanut meal is a product obtained by squeezing peanut kernels to extract oil, and is generally divided into primary meal and secondary meal. The primary meal means the peanut residue left after primary pressing, and the secondary meal means the peanut residue pressed twice. The yield of the peanut meal can reach more than 44 percent, and the peanut meal is a rich byproduct resource. Peanut meal is rich in protein and carbohydrate. We obtained a series of proteins from the steep liquor of peanut meal. We isolated these proteins and found that some of them had antifungal activity, and one of them was sequenced to remove glycan modifications to give peanut silver flavin. The protein has glycan modification 10-34kDa higher than theoretical molecular weight, and has resistance to various plant pathogenic fungi including rust, fusarium, anthrax and rhizoctonia.
Disclosure of Invention
The invention aims to provide a novel antifungal peanut polysaccharide, namely, the Yinhuang PG-AHGP for crops and a preparation method thereof.
An antifungal arachidan argentum flavin PG-AHGP, wherein the amino acid sequence of the antifungal arachidan argentum flavin PG-AHGP is MINNASNFAASITISLILCLILLWSSSVCECVPNTNITTILCNSGVYTSGDPF AISLSYVLEELEEVTPTQKNYDYYNISPYPNAFAYGHAACNNNNNNLKK KTNLTSSDCKACLGVAKTTMLSSCEKRIGARSVLHDCTIRYEQYPFDD. The glycan-free sequence contained 150 amino acid residues, a molecular weight of 16.5kDa, and an isoelectric point of 5.22. The degree of polymerization of glycans at each glycosylation site is between 20-60, and the major monosaccharide components are Mannose (Mannose), N-acetylglucosamine (GlcNAc), Galactose (Galactose), Xylose (Xylose), and fructose (Fucose). The molecular weight of the polygalaxanthin PG-AHGP is between 26kDa and 50 kDa.
The preparation method of the antifungal arganian argentaxanthin utilizes an ion exchange method, and the arganian argentaxanthin is separated step by step through ion exchange and gel filtration chromatography, and is desalted and purified through reversed-phase column chromatography.
The application of the antifungal peanut polysaccharide argentaxanthin is characterized in that the antifungal peanut polysaccharide argentaxanthin is used for preparing antifungal anthrax fungus, rust disease fungus, fusarium disease fungus and rhizoctonia disease fungus.
The invention has the beneficial effects that the purified antifungal peanut polysaccharide argentaxanthin has the advantages of moderate molecular weight, stable storage, strong antifungal capability and the like. The antifungal peanut polysaccharide argentine PG-AHGP has strong bacteriostasis to anthrax fungi, rust fungi, fusarium fungi and rhizoctonia fungi, but has weak bacteriostasis to probiotics such as bacillus subtilis and low toxicity, which shows that the antifungal peanut polysaccharide argentine PG-AHGP has good antibacterial selectivity and small toxic and side effects.
Drawings
FIG. 1 shows the elution profile of peanut proteins from Sephacryl S-200 column, resulting in peaks A1 (lane 2), A2 (lane 5) and A4 (lane 10). Elution was carried out with 5ml/min of 20mM PBS buffer (pH 8.0) containing 0.35M NaCl. Starting from the detector that can detect the protein, one tube was collected every 5ml, for a total of 10 tubes. Peaks collectively showing higher protein concentration by measuring absorbance at 280 nm are plots a1 (tube 2), a2 (tube 5), A3 (tube 8) and a4 (tube 10).
FIG. 2. area of bacteriostatic clearness zone for isolated antifungal arachidonin, argentum PG-AHGP (A1 (tube 2), A2 (tube 5), A3 (tube 8) and A4 (tube 10)). The diameter of each inhibition zone was measured 3 times, the average value of the diameters of the inhibition zones was calculated, and the area of the transparent zone was found according to the area formula (S ═ pi × d/2)2, where pi represents the circumference ratio, r represents the radius, d represents the diameter, and S represents the area).
FIG. 3 high pressure liquid chromatography analysis of the purity of antifungal arachidocan, argentine PG-AHGP. And A, removing glycan. And B, not removing glycan modification.
FIG. 4 electrospray mass spectrometry identified antifungal arachidonin Yinhgp. And A, removing glycan. And B, not removing glycan modification. The difference in molecular weight between the three peaks of marker molecular weight (degree of polymerisation for the polysaccharide 93, 126 and 187 respectively) is an integer multiple of 162, representing the corresponding glucose and/or galactose units.
FIG. 5 antifungal peanut glycan argentine PG-AHGP assay. A, amino acid sequence. N glycosylation modifies asparagine sites and the amino acid sequence of the polypeptide molecule in which the modified asparagine sites are located, the sequence of the modified asparagine sites needs to conform to the NXS/T structure (X is non-proline), and the double-dashed letter N represents sites which must be glycosylated. The probability of NIS glycosylation of the single-underlined sequence is low; b, glycan species and binding site. The N4, N36 and N105 positions in the amino acid sequence of the fungal arachidocanon argentine PG-AHGP are the positions where the argentine glycan binds.
FIG. 6. antifungal Echinoglycan, argentinia striata fungus (Puccinia striations). (A) Antifungal activity against fungi (Puccinia striiformis) in solutions of different pH; (B) antifungal activity of heat treatment on fungi (Puccinia striiformis). The diameter of the transparent ring is represented by a heat map, and the brightness represents that the diameter of the transparent ring is large and the diameter of the dark transparent ring is small, corresponding to the diameter value of the transparent ring of the scale.
FIG. 7. antifungal Arginosin PG-AHGP Russian fungus (Fusarium oxysporum). (A) Antifungal activity against fungi (Fusarium oxysporum) in solutions of different pH; (B) antifungal activity of heat treatment on fungi (Fusarium oxysporum). The diameter of the transparent circle is represented by a heat map, and the brightness represents that the diameter of the transparent circle is large and the diameter of the dark transparent circle is small, corresponding to the diameter of the transparent circle of the scale.
FIG. 8 is a diagram of the antifungal arachidocan Yinhuang PG-AHGP Russian fungus (Colletotrichum gloeosporioides). (A) Antifungal activity against fungi (Colletotrichum gloeosporioides) in solutions of different pH; (B) antifungal activity of heat treatment on fungi (Colletotrichum gloeosporioides). The diameter of the transparent ring is represented by a heat map, and the brightness represents that the diameter of the transparent ring is large and the diameter of the dark transparent ring is small, corresponding to the diameter value of the transparent ring of the scale.
FIG. 9. antifungal Arachis hypogaea polysaccharide, Yinhuanil PG-AHGP Russian fungus (Rhizoctonia solani). (a) antifungal activity against fungi (Rhizoctonia solani) in solutions of different pH; (B) antifungal activity of heat treatment against fungi (Rhizoctonia solani). The diameter of the transparent circle is represented by a heat map, and the brightness represents that the diameter of the transparent circle is large and the diameter of the dark transparent circle is small, corresponding to the diameter of the transparent circle of the scale.
Detailed Description
The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1: preparation of antifungal peanut glycan argentaxanthin PG-AHGP
10.0g of peanut meal was weighed, repeatedly ground into a fine powder in liquid nitrogen, added to 100mL of a protein extraction buffer (composition: 20mM Phosphate (PBS), pH7.5,10mM ethylenediaminetetraacetic acid (EDTA), 150mM NaCl, 1% dimethyl sulfoxide (DMSO) by volume, and 1mM Dithiothreitol (DTT) in water) in a 200mL beaker, and mixed well with stirring. Centrifuging at 10000g for 20min, and collecting 50ml supernatant.
767g (NH) 4 ) 2 SO 4 The resulting solution was slowly added to 1 liter of distilled water with stirring, and the pH was adjusted to 7.0 with 25 to 28 mass% aqueous ammonia or 95 mass% sulfuric acid, which was an ammonium sulfate solution having a saturation of 100% (4.1 mol/L,25 ℃).
An equal volume of 100% saturated ammonium sulfate solution was slowly added to the supernatant with stirring. The solution was stirred overnight (4 ℃) on a magnetic stirrer, allowing the protein to precipitate well. The protein solution was centrifuged at 10000g for 30min (4 ℃). The supernatant was discarded and the precipitate was retained. The pellet was dissolved in 20ml of protein extraction buffer (composition: 20mM PBS, pH7.5,10mM EDTA, 150mM NaCl, 1% DMSO by volume, 1mM DTT and 0.2g/L sodium azide in water). The mixture was placed in a 40ml dialysis bag (molecular weight cut-off: 10kDa), and dialyzed against 1 liter of protein extraction buffer for 24 hours (4 ℃ C.), and the dialysis buffer was changed every 6 hours to completely remove ammonium sulfate. A Fast Flow DEAE-Sepharose Fast Flow column (internal diameter and length 2.6X 100cm, merck, type 17-0709-10) was equilibrated with a three-column volume of protein extraction buffer (composition: 20mM PBS pH7.5,10mM EDTA, 150mM NaCl, volume concentration 1% DMSO, and 1mM DTT in water) at a Flow rate of 0.5 ml/min. 20ml of the crude extract solution was loaded onto a DEAE-Sepharose Fast Flow column (2.6X 100cm), and the DEAE-Sepharose Fast Flow column (2.6X 100cm) was equilibrated again with three column volumes of a protein extraction buffer (composition: 20mM PBS, pH7.5,10mM EDTA, 150mM NaCl, volume concentration 1% DMSO and 1mM DTT in water) at a Flow rate of 0.5 ml/min. Finally, the column was eluted with a protein extraction buffer (composition: 20mM PBS, pH7.5,10mM EDTA, 150mM NaCl, volume concentration of 1% DMSO and 1mM DTT in water) and a NaCl gradient (0.35M) at a flow rate of 5.0 mL/min. The protein eluent is collected from the moment that the detector can detect the protein, and 50ml of protein is collected at the moment that the detector can not detect the protein desmin. The collection was applied to a gel filtration column (Sephacryl S-200, internal diameter and length 5X90 cm, GE USA, model 17-0584-01) equilibrated with 3 column volumes of 20mM Tris-HCl buffer (pH 8.0) containing 0.35M NaCl. Elution was carried out with 5ml/min of 20mM PBS buffer (pH 8.0) containing 0.35M NaCl. Starting from the detector that can detect the protein, one tube was collected every 5ml, for a total of 10 tubes. Peaks that collectively showed higher protein concentrations by measuring absorbance at 280 nm are fig. 1.a1 (tube 2), a2 (tube 5), A3 (tube 8) and a4 (tube 10).
Each fraction was analyzed for antifungal activity in tubes 2, 5, 8, and 10 of the peanut proteins eluted with the above buffer. A potato glucose agar plate (diameter 9cm, thickness 0.3-0.4cm) is used (the formula is that 20g of potato is prepared into leachate, 2g of glucose, 1.5g of agar and 100mL of water, pH is not regulated (natural), the preparation method comprises taking 20g of fresh potato, cleaning, peeling, cutting into small pieces, adding 80 mL of water, boiling for 30min, filtering with four layers of gauze, adding 2g of glucose and 1.5g of agar, continuing to heat and stir, slightly cooling, adding water to 100mL, and sterilizing at 121 ℃ for 20 min).
The eluted peanut proteins were evaluated for antifungal activity in tubes 2, 5, 8, 10, respectively. Dripping 10 μ L of frozen bacteria liquid (anthrax fungus, rust fungus, fusarium fungus, rhizoctonia fungus and Bacillus subtilis respectively with density of 1 × 10) into the center of the plate 5 ～2×10 5 cfu/g of lyophilized powder, each gram of sample dissolved in 10 ml of 0.85% normal saline). A cross shape is drawn by taking the center (as the center) of a flat plate as the center of a circular sterile paper disc with the diameter of 1cm, a paper disc is placed at the intersection point of the centers, a paper disc is placed at the position, away from the center, of every 1.5cm along two lines, and 5 paper discs are placed on the flat plate with the diameter of 9 cm. mu.L of protein in 20mM PBS, pH7.5 buffer was added drop wise to the disk, and 10. mu.L of 20mM PBS, pH7.5 was added drop wise to the control. Mycelium growth surrounded the control-containing peripheral disks and produced clear ring inhibition around the disks with antifungal samples incubated at 25 ℃ for 72 hours. The larger the area of the clearing zone represents the higher the inhibition rate of the protein. The bacteriostasis experiment showsThe A3 (eluted 8 th tube) in the eluted peanut polysaccharide argentine has obvious bacteriostatic activity on rust fungi, fusarium fungi, anthrax and rhizoctonia fungi, and the A1 (eluted 2 nd tube), A2 (eluted 5 th tube) and A4 (eluted 10 th tube) have weak bacteriostatic activity on rust fungi, fusarium fungi, anthrax and rhizoctonia fungi (figure 2). All peanut proteins had weak antibacterial activity against Bacillus subtilis (FIG. 2). Therefore, a3 (tube 8 of elution) may be the ideal bacteriostatic arachidan argentum flavin, named antifungal arachidan argentum flavin PG-AHGP, for subsequent experiments.
FIG. 1 shows the elution profile of peanut proteins from Sephacryl S-200 column, resulting in peaks A1 (lane 2), A2 (lane 5) and A4 (lane 10). Elution was carried out with 5ml/min of 20mM PBS buffer (pH 8.0) containing 0.35M NaCl. Starting from the detector that can detect the protein, one tube was collected every 5ml, for a total of 10 tubes. Peaks collectively showing higher protein concentration by measuring absorbance at 280 nm are plots a1 (tube 2), a2 (tube 5), A3 (tube 8) and a4 (tube 10).
FIG. 2 area of bacteriostatic transparent circles of isolated antifungal arachidosans, argentine PG-AHGP (A1 (tube 2), A2 (tube 5), A3 (tube 8) and A4 (tube 10)). The diameter of each inhibition zone was measured 3 times, the average value of the diameters of the inhibition zones was calculated, and the area of the transparent zone was found according to the area formula (S ═ pi × d/2)2, where pi represents the circumference ratio, r represents the radius, d represents the diameter, and S represents the area).
Example 2: molecular weight and sequence identification of antifungal Arachis hypogaea glycan argentums PG-AHGP obtained in example 1
Protein deglycation was performed in order to determine the prosequence. Deglycosylation step 1mL of 100pmol PG-AHGP was prepared as follows using 100mM glycine-HCl buffer, pH 2.5. After 2 minutes, 100. mu.L of the sample was transferred to a tube containing 5. mu. L N-glycosidase F (peptide N-glycosidase F, also known as PNGase F, an amidohydrolase that cleaves the glycosidic bond between glycans and proteins of asparagine-linked high mannose, hybrid and complex oligosaccharide glycoproteins). Enzymatic digestion deglycosylation was performed at 4 ℃ for 30 minutes. The sample is injected into a liquid phase mass spectrum (LC-MS) system to test the deglycosylation degree of the N-glycosidase F, the theoretical molecular weight is 16.5kDa, namely the molecular weight of 150 amino acid residues indicates that all glycosylation groups are completely digested by the N-glycosidase F.
1mL antifungal arachidocan, Yinhuang PG-AHGP or deglycosylated PG-AHGP, was purified by liquid chromatography (HPLC) reverse phase column (WelchXB C18, inner diameter and length 4.6X 150mm) chromatography desalting with a mobile phase of 40% v/v pure water (containing 0.1% trifluoroacetic acid (TFA) by volume) to 60% v/v Acetonitrile (ACN) (containing 0.1% trifluoroacetic acid by volume) at a flow rate of 1mL/min at a wavelength of 220nm, collecting the effluent peak, lyophilizing, and then resuspending in 0.1% formic acid by volume for combined liquid chromatography-mass spectrometry (HPLC-MS) analysis. The molecular weight of the synthesized antifungal arachidonan argentine PG-AHGP was identified using electrospray mass spectrometry. Sample is injected by using a liquid phase system, and the volume concentration of a mobile phase is 50% H 2 O/50% CAN, flow rate of 0.2mL/min, flow rate of protective gas nitrogen of 1.5L/min, collision energy of 4.5kV, using anion mode.
The purity of the purified antifungal peanut polysaccharide, namely the Yinhuang PG-AHGP, is identified by a high performance liquid chromatography (Welch XBC 18, inner diameter and length are 4.6x 250mm) HPLC method, and the structure of electrospray mass spectrometry is adopted for molecular weight determination. The HPLC purity identification result is shown in FIG. 3: the antifungal arachidan argentine PG-AHGP showed a single peak at 20.3min after deglycosylation (FIG. 3A). The antifungal arachidonin, argentuxanthin PG-AHGP, showed a broad peak at 26-40min (FIG. 3B).
FIG. 3 is a high pressure liquid chromatography analysis of the purity of antifungal arachidocan argentum flavin PG-AHGP. And A, removing glycan. And B, not removing glycan modification.
The electrospray mass spectrometry identification result is shown in fig. 4: after removal of glycan modifications, the molecular weight of the protein was close to the theoretical value of 16.5kDa (FIG. 4A). The molecular weight of the non-deglycosylated pre-antifungal arachidan argentine PG-AHGP was 26-50kDa, which is 10-34kDa greater than the theoretical molecular weight (FIG. 4B).
FIG. 4 electrospray mass spectrometry was used to identify antifungal arachidonin Yinhgp. And A, removing glycan. And B, not removing glycan modification. The difference in molecular weight between the three peaks of marker molecular weight (degree of polymerisation for the polysaccharide 93, 126 and 187 respectively) is an integer multiple of 162, representing the corresponding glucose and/or galactose units.
Example 3: example 1 antifungal Arabinoxasan Galanthin sequencing
Reference is made to the previous literature for the sequencing of the antifungal arachidonin, Yinhu PG-AHGP, using the phenyl isothiocyanate method (Edman) degradation [16 ]. Purified deglycosylated peanut luteolin (20 μ g) was dissolved in 50 μ l of 0.2M ammonium bicarbonate (pH 8.0 containing 4M guanidine hydrochloride) in 5ml 45mM dithiothreitol. Automated Edman degradation of S-carboxyamidomethylated peptides and detection of thiophenyl acetal derivatives were performed on an automated protein sequencer (Applied Biosystems, model 476A). Cleavage occurs at the first peptide bond in the presence of 6mol/L HCl, resulting in a peptide fragment minus the first base and a first residue in the form of Anilothiazolinone (ATZ) which is released. The other reactions and released residues were washed out with 20mM PBS (pH7.0) buffer and the shortened peptide fragment was subjected to another round of coupling and cleavage to release the second residue (process and conditions are as above), and so on until the last amino acid residue was released. And (3) determining the peanut antifungal peanut glycan argentine PG-AHGP according to a sequencing result.
SEQ ID No.1 of the sequence table is:
MINNASNFAASITISLILCLILLWSSSVCECVPNTNITTILCNSGVYTSGDPF AISLSYVLEELEEVTPTQKNYDYYNISPYPNAFAYGHAACNNNNNNLKK KTNLTSSDCKACLGVAKTTMLSSCEKRIGARSVLHDCTIRYEQYPFDD
(150aa)
(a) sequence characteristics:
length: 150
Type: amino acid sequence
Chain type: single strand
The topology: linearity
(b) Type of molecule: protein
(c) Suppose that: whether or not
(d) Antisense: whether or not
(e) The initial sources were: peanut
Example 4 antifungal Arachis hypogaea polysaccharide, Yinflavine PG-AHGP sequencing glycosylation fraction analysis obtained in example 1
Digestion with trypsin
1ml of the antifungal arachidan argentamine solution obtained in example 1 (1mg/ml) was added to a final concentration of 10mM Dithiothreitol (DTT) and disulfide bonds were reduced at 55 ℃ for 20 minutes. After cooling to room temperature, Iodoacetamide (IAA) was added to a final concentration of 20mM and protein alkylation was induced in the dark for 30 minutes at room temperature. Digestion was performed with trypsin (enzyme: substrate solution ═ 1:50(w/v, mg/ml)) at 37 ℃ for 16 hours with gentle shaking. The digested peptide was desalted using a C18(Phenomenex Jupiter, Filler diameter 15 μm, Fennom, model 00G-4053-P0) solid phase extraction column, octyl nonpolar column, and dried in a vacuum desiccator for 2 hours.
Enrichment and mass spectrometry of intact glycosylated peptide fragments
The trypsin-digested argannins obtained in example 4 above were desalted using a desalting column with 100. mu.L of binding buffer AT (80% acetonitrile (CAN) by volume, 1% trifluoroacetic acid (TFA) by volume). Specifically, 100. mu.L of the trypsin-digested argosyn-ginoxanthin obtained in example 4 above was loaded on a hydrophilic column ZIC-HILIC for 5 minutes by activating the column hydrophilic column (Merck column ZIC-HILIC, model: 1.50478.0001) with 100. mu.L of ACN and equilibrating with 300. mu.L of a binding buffer AT. Subsequently, the column was washed with 800. mu.L of AT binding buffer. Finally, N-glycopeptide was eluted with 300. mu.L of 0.1% TFA by volume and finally 100. mu.L of 50mM NH4HCO 3. The eluate was dried in a vacuum desiccator and resuspended in 100. mu.L volume concentration of 0.1% TFA.
Reverse phase liquid chromatography electrospray mass spectrometry (RPLC ESI-MS/MS) analysis
The mass of the arachidonoglycan argentumbrin PG-AHGP and unglycosylated arachidoxanthin (100 uL of 1mg/ml protein) was analyzed on a reversed phase liquid chromatography electrospray mass spectrometry system, spectrometer run in positive ion mode. In a C18 column (Waters,
5 μm,4.6mm X250 mm, model 186001265) at a flow rate of 300nL/min, with mobile phases of 99.8% by volume H2O, 0.2% TFA (A) and 95% by volume ACN, 4.8% H2O and 0.2% TFA (B). The LC gradient is as follows: 2% of B, the use time is 10 min; linear 2% -40% B, 190 minutes; linear 40% -95% B, 10 minutes; 95 percentB, taking for 5 minutes; linear 95% -2% B, 5 minutes; 2% B, 20 min. ESI conditions were as follows: spray voltage 2.8kV, capillary temperature 320 ℃, high pressure ring iontophoresis device (S-lens RF) level 75V. Full scan mass spectra were acquired in the 700-2000m/z range, with the main ESI source settings: microscopic scanning 1, mass resolution 70k, Automatic Gain Control (AGC) target 2e5, maximum ion implantation time 50 ms; the MSMS spectra were acquired in Top20 data-dependent acquisition mode, set as follows: micro-scanning 1, mass resolution 17.5k, AGC target 5e5, maximum ion implantation time 250ms, isolation window 3 m/z, higher energy collisional dissociation (HCD), stepwise normalization of collisional energies 20%, 30%, 40%; dynamic exclusion for 20 seconds, charge inclusion was 2-6.
With reference to examples 3 and 4, an antifungal arachidon argentum PG-AHGP has the amino acid sequence shown in FIG. 5A. The glycan-free sequence contained 150 amino acid residues, a molecular weight of 16.5kDa, and an isoelectric point of 5.22. The polymerization degree of the glycan is between 20 and 60, the N4, N36 and N105 positions in the amino acid sequence of the fungal peanut glycan argentuxanthin PG-AHGP are the binding positions of the argentuxanthin glycan, and the N-acetylglucosamine forms a glycosidic bond with the gamma-amide N atom of asparagine. The molecular weight after glycan binding is between 26kDa and 50 kDa. The type of the glycoform of the arachidocane argentine mainly comprises 4 types, and referring to FIG. 5B, 2N-acetylglucosamine is linearly connected in the form of an asparagine-glycosidic bond, the N-acetylglucosamine is further combined with a plurality of branched linearly connected mannose unit glycans (75-90% by total weight of the glycans), the following monosaccharide units are connected in the same way, the N-acetylglucosamine is combined with a plurality of mannose units, the addition of galactose-modified glycans (2-15% by total weight of the glycans), the N-acetylglucosamine is combined with a plurality of mannose units, the addition of galactose-modified glycans (1-10% by total weight of the glycans), the N-acetylglucosamine is combined with a plurality of mannose units, the addition of one or more than two types of galactose, xylose and fructose (0-5%, based on total mass of glycans).
FIG. 5 antifungal peanut glycan argentine PG-AHGP assay. A, amino acid sequence. N glycosylation modifies asparagine sites and the amino acid sequence of the polypeptide molecule in which the modified asparagine sites are located, the sequence of the modified asparagine sites needs to conform to the NXS/T structure (X is non-proline), and the double-dashed letter N represents sites which must be glycosylated. The single underlined sequence NIS is less likely to be glycosylated. B, glycan species and binding site. The N4, N36 and N105 positions in the amino acid sequence of the fungal arachidocanon argentine PG-AHGP are the positions where the argentine glycan binds.
Example 5: experiment of antifungal Agomelanin PG-AHGP Russian fungus Puccinia striiformis (Puccinia striiformis) obtained in example 1
The solid medium used in the antifungal studies included Potato Dextrose Agar (PDA) (potato, 200 g; dextrose, 20 g; agar, 18 g; and distilled water, 1L) for the fungus. To test for antifungal activity, 1mL of a suspension of fungal spores (Puccinia striiformis) (1X 10) 7 spores/mL) was added uniformly to 100mL of PDA medium at 40-50 ℃ to prepare solid plates (plate diameter 90mm, thickness 3-4 mm). After the culture medium solidified, the Oxford cups (stainless steel small tubes, inner diameter 6mm, outer diameter 8mm, height 10mm) were placed at a distance of 2 cm. Then 5 mul of antifungal arachidonin argentum flavin (1, 2, 3, 4, 5, 6, 7, 8, 9, 10mg/ml) with different concentrations is added into the Oxford cup, and the mixture is cultured for 48h at 30 ℃, and the diameter of the antibacterial transparent ring is measured. In the control group, an equal volume of 5. mu.L of physiological saline was added, and the cells were cultured by the same procedure and conditions as described above. Simultaneously detecting the treatment of the antifungal arachidocan argentine at different temperatures (20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃) and different pH values of 0-12 (0, 2, 4, 6, 8, 10, 12 respectively), and then repeatedly testing the antibacterial activity by the above method, wherein the process and the conditions are the same.
The growth inhibition degree of certain fungi and other microorganisms is directly antagonized on an agar plate by adopting an oxford cup method, and the activity of the antifungal wheat stripe rust (Puccinia striiformis) of the antifungal peanut glycan argentuxanthin is determined. In this study, the use of the arachidonin argentine showed a significant resistance to fungal spores (Puccinia striiformis) after 48h (clearing zone diameter <6mm at 2mg/L arachidonin argentine; clearing zone diameter >6mm above 2mg/L arachidonin argentine), the diameter of the clearing zone increasing with increasing concentration in the pH range of 2.0 to 10.0. The antifungal activity (Puccinia striations) is active in the pH range of 2.0 to 10.0, the activity is highest near 8 (FIG. 6A), the activity is higher at 30-60 deg.C, and the activity is sharply reduced at the temperature higher than 70 deg.C (FIG. 6B).
FIG. 6. antifungal arachidonin Agxanthin Rust resisting fungus (Puccinia striiformis). (A) Antifungal activity against fungi (Puccinia striiformis) in solutions of different pH. (B) Antifungal activity of heat treatment against fungi (Puccinia striiformis). The diameter of the transparent ring is represented by a heat map, and the brightness represents that the diameter of the transparent ring is large and the diameter of the dark transparent ring is small, corresponding to the diameter value of the transparent ring of the scale.
Example 6: experiment of antifungal Agaxanthin PG-AHGP obtained in example 1 against Fusarium oxysporum (Fusarium oxysporum)
The solid medium used in the antifungal studies included Potato Dextrose Agar (PDA) (potato, 200 g; dextrose, 20 g; agar, 18 g; and distilled water, 1L) for the fungus. To test for antifungal activity, 1mL of a fungal spore Fusarium oxysporum suspension (1X 10) 7 spores/mL) was added uniformly to 100mL of PDA medium at 40-50 c to make a solid plate (plate diameter 90mm, thickness 3-4 mm). After the medium solidified, the medium was placed on a solid plate of stainless steel small tubes (6 mm in inner diameter, 8mm in outer diameter, and 10mm in height) of an Oxford cup. Then, 5 μ L of antifungal arachidonin argentine (1, 2, 3, 4, 5, 6, 7, 8, 9, 10mg/mL) with different concentrations is added into the Oxford cup, and the mixture is cultured for 48h at 30 ℃ to measure the diameter of the antibacterial transparent ring. In the control group, an equal volume of 5. mu.L of physiological saline was added, and the cells were cultured by the same procedure and conditions as described above. Simultaneously detecting the treatment of antifungal Aggregata with different temperatures (20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C) and different pH values of 0-12 (0, 2, 4, 6, 8, 10, 12 respectively), and repeating the above method for testing antibacterial activityThe properties, procedures and conditions were the same as above.
The growth inhibition degree of certain fungi and other microorganisms is directly antagonized on an agar plate by adopting an oxford cup method, and the antifungal (Fusarium oxysporum) activity of the antifungal peanut polysaccharide argentine PG-AHGP is determined. In this study, the use of the arachidonin argentine showed a significant resistance to fungal spores (Puccinia striiformis) after 48h (clearing zone diameter <5mm at 2mg/L arachidonin argentine; clearing zone diameter >5mm above 2mg/L arachidonin argentine), the diameter of the clearing zone increasing with increasing concentration in the pH range of 2.0 to 10.0. The antifungal (Fusarium oxysporum) activity was found to be active in the pH range of 2.0 to 10.0, with the highest activity around 8 (FIG. 7A), higher at 30-60 deg.C and sharply reduced above 70 deg.C (FIG. 7B).
FIG. 7. antifungal Arginosin PG-AHGP Russian fungus (Fusarium oxysporum). (A) antifungal activity against fungi (Fusarium oxysporum) in solutions of various pH. (B) Antifungal activity of heat treatment on fungi (Fusarium oxysporum). The diameter of the transparent ring is represented by a heat map, and the brightness represents that the diameter of the transparent ring is large and the diameter of the dark transparent ring is small, corresponding to the diameter value of the transparent ring of the scale.
Example 7: experiment of antifungal Agaxanthin PG-AHGP obtained in example 1 against Colletotrichum gloeosporioides
The solid medium used in antifungal studies included Potato Dextrose Agar (PDA) (potato, 200 g; dextrose, 20 g; agar, 18 g; and distilled water, 1L) for fungi. To test for antifungal activity, 1mL of a suspension of fungal spores of Bacillus anthracis (Colletotrichum gloeosporioides) (1X 10) 7 spores/mL) was added uniformly to 100mL of PDA medium at 40-50 ℃ to prepare solid plates (plate diameter 90mm, thickness 3-4 mm). After the medium solidified, the medium was placed on a solid plate of stainless steel small tubes (6 mm in inner diameter, 8mm in outer diameter, and 10mm in height) of an Oxford cup. Adding 5 μ L of antifungal peanut polysaccharide argentine PG-AHGP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10mg/ml) with different concentrations into Oxford cup, culturing at 30 deg.C for 48 hr, and measuring antibacterial round and straightAnd (4) diameter. In the control group, an equal volume of physiological saline was added and cultured under the same conditions. Simultaneously detecting the antifungal arachidonin treated at different temperatures (20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃) and different pH values of 0-12 (0, 2, 4, 6, 8, 10, 12 respectively), and then repeatedly testing the antibacterial activity by using the method, wherein the process and the conditions are the same.
The antifungal (Colletotrichum gloeosporioides) activity of the antifungal arachidan argentine PG-AHGP was determined by directly antagonizing the extent of growth inhibition of certain fungi and other microorganisms on agar plates using the Oxford cup method. In this study, the use of the arachidonin gave a marked resistance to fungal spores (Colletotrichum gloeosporioides) after 48h (arachidonin at 2mg/L, clearing circle diameter <4 mm; arachidonin at more than 2mg/L, clearing circle diameter >4mm), with the diameter of the clearing circle increasing with increasing concentration in the pH range of 2.0 to 10.0. The antifungal (Colletotrichum gloeosporioides) activity was active in the pH range of 2.0 to 10.0, with the highest activity around 8 (FIG. 8A), higher activity at 30-60 deg.C and sharply reduced activity above 70 deg.C (FIG. 8B).
FIG. 8 is a diagram of the antifungal arachidocan Yinhuang PG-AHGP Russian fungus (Colletotrichum gloeosporioides). (A) Antifungal activity against fungi (Colletotrichum gloeosporioides) in solutions of different pH. (B) Antifungal activity of heat treatment on fungi (Colletotrichum gloeosporioides). The diameter of the transparent circle is represented by a heat map, and the brightness represents that the diameter of the transparent circle is large and the diameter of the dark transparent circle is small, corresponding to the diameter of the transparent circle of the scale.
Example 8: antifungal arachidonin PG-AHGP Rhizoctonia solani (Rhizoctonia solani) resisting experiment
The solid medium used in the antifungal studies included Potato Dextrose Agar (PDA) (potato, 200 g; dextrose, 20 g; agar, 18 g; and distilled water, 1L) for the fungus. To test for antifungal activity, 1mL of a suspension of sclerotinia sclerotiorum spores (Rhizoctonia solani) (1X 10) 7 spores/mL) was added uniformly to 100mL of PDA medium at 40-50 c,a solid plate (plate diameter 90mm, thickness 3-4mm) was prepared. After the medium solidified, a stainless steel small tube (6 mm in inner diameter, 8mm in outer diameter, and 10mm in height) of an Oxford cup was placed. Then 5. mu.L of antifungal arachidonin PG-AHGP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10mg/ml) with different concentrations was added in dots in an Oxford cup, cultured for 48h at 30 ℃ and the diameter of the antibacterial circle was measured. In the control group, an equal volume of physiological saline was added and cultured under the same conditions. Simultaneously detecting the treatment of the antifungal arachidocan argentine at different temperatures (20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃) and different pH values of 0-12 (0, 2, 4, 6, 8, 10, 12 respectively), and then repeatedly testing the antibacterial activity by the above method, wherein the process and the conditions are the same.
The antifungal (Rhizoctonia solani) activity of the antifungal arachidon argentine PG-AHGP was determined by directly antagonizing the extent of growth inhibition of certain fungi and other microorganisms on agar plates using the Oxford cup method. In this study, the use of the arachidan argentanin showed a significant resistance to fungal spores (Rhizoctonia solani) after 48h (arachidan argentanin at 2mg/L, clearing circle diameter <4 mm; arachidan argentanin above 2mg/L, clearing circle diameter >4mm), with clearing circle diameter increasing with increasing concentration in the pH range of 2.0 to 10.0. The antifungal (Rhizoctonia solani) activity was active in the pH range of 2.0 to 10.0, with the highest activity around 8 (FIG. 9A), higher activity at 30-60 deg.C and sharply decreased activity at temperatures above 70 deg.C (FIG. 9B).
FIG. 9. antifungal Arachis hypogaea polysaccharide, Yinhuanil PG-AHGP Russian fungus (Rhizoctonia solani). (A) Antifungal activity against fungi (Rhizoctonia solani) in solutions of different pH. (B) Antifungal activity of heat treatment against fungi (Rhizoctonia solani). The diameter of the transparent ring is represented by a heat map, and the brightness represents that the diameter of the transparent ring is large and the diameter of the dark transparent ring is small, corresponding to the diameter value of the transparent ring of the scale.
Appendix
SEQ ID NO.1
Antifungal peanut glycan argentine PG-AHGP amino acid sequence
MINNASNFAASITISLILCLILLWSSSVCECVPNTNITTILCNSGVYTSGDPF AISLSYVLEELEEVTPTQKNYDYYNISPYPNAFAYGHAACNNNNNNLKK KTNLTSSDCKACLGVAKTTMLSSCEKRIGARSVLHDCTIRYEQYPFDD
Sequence listing
<110> Guangzhou Shenjingyuan agriculture science and technology Limited
<120> antifungal glycan protein peanut glycan argentamine and preparation and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 150
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Ile Asn Asn Ala Ser Asn Phe Ala Ala Ser Ile Thr Ile Ser Leu
1 5 10 15
Ile Leu Cys Leu Ile Leu Leu Trp Ser Ser Ser Val Cys Glu Cys Val
20 25 30
Pro Asn Thr Asn Ile Thr Thr Ile Leu Cys Asn Ser Gly Val Tyr Thr
35 40 45
Ser Gly Asp Pro Phe Ala Ile Ser Leu Ser Tyr Val Leu Glu Glu Leu
50 55 60
Glu Glu Val Thr Pro Thr Gln Lys Asn Tyr Asp Tyr Tyr Asn Ile Ser
65 70 75 80
Pro Tyr Pro Asn Ala Phe Ala Tyr Gly His Ala Ala Cys Asn Asn Asn
85 90 95
Asn Asn Asn Leu Lys Lys Lys Thr Asn Leu Thr Ser Ser Asp Cys Lys
100 105 110
Ala Cys Leu Gly Val Ala Lys Thr Thr Met Leu Ser Ser Cys Glu Lys
115 120 125
Arg Ile Gly Ala Arg Ser Val Leu His Asp Cys Thr Ile Arg Tyr Glu
130 135 140
Gln Tyr Pro Phe Asp Asp
145 150
Claims (8)
1. An antifungal glycan protein arachidan argentum flavin is characterized in that the antifungal glycan protein is arachidan argentum flavin (named PG-AHGP), and the amino acid sequence of the antifungal glycan protein is shown as a sequence table SEQ ID NO. 1.
2. The antifungal arachidan argentum flavin PG-AHGP of claim 1, wherein said amino acid sequence is: MINNASNFAASITISLILCLILLWSSSVCECVPNTNITTILCNSGVYTSGDPFAISLSYVLEELEEVTPTQKNYDYYNISPYPNAFAYGHAACNNNNNNLKKKTNLTSSDCKACLGVAKTTMLSSCEKRIGARSVLHDCTIRYEQYPFDD are provided.
3. The antifungal arachidonins as in claim 1, wherein the sequence excluding the glycans comprises 150 amino acid residues, has a molecular weight of 16.5kDa and an isoelectric point of 5.22.
4. The antifungal anaxaglycan argentine according to any one of claims 1 to 3 wherein the amino acid sequence has positions N4, N36 and N105 to which the argentine glycan binds; the degree of polymerization of the saccharides at each position is between 20 and 60, and the main components are one, two, three, four or five of Mannose (Mannose), N-acetylglucosamine (GlcNAc), Galactose (Galactose), Xylose (Xylose) and fructose (Fucose). The molecular weight of the antifungal arachidonin after glycan binding is between 26kDa and 50 kDa.
5. A process for preparing the antifungal arganian luteine as claimed in any one of claims 1 to 4, wherein the arganian luteine is isolated by ion exchange chromatography and gel filtration chromatography, and desalted and purified by reverse phase column chromatography using peanut seeds as raw material.
6. The use of the antifungal arachidocan flavoxanthin of any one of claims 1-4, wherein the antifungal arachidocan flavoxanthin PG-AHGP is used in the inhibition of plant pathogenic fungi (including one or more of the genera Puccinia, Fusarium, Anthrax and Rhizoctonia).
7. The use of the antifungal arachidocan flavoxanthin of any one of claims 1 to 4, wherein the antifungal arachidocan flavoxanthin PG-AHGP is used in the preparation of a medicament or pharmaceutical preparation for inhibiting plant pathogenic fungi (including one or more of the genera Puccinia, Fusarium, Anthrax and Rhizoctonia).
8. A medicament or pharmaceutical preparation against phytopathogenic fungi (including one or more of the genera Puccinia, Fusarium, Anthrax and Rhizoctonia) comprising as active ingredient the antifungal arachidocan Yinfluen PG-AHGP of any one of claims 1 to 4.
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Non-Patent Citations (6)
| Title |
|---|
| SUSANA SERRAZINA等: "Genetic Transformation of Quercus ilex Somatic Embryos with a Gnk2-like Protein That Reveals a Putative Anti-Oomycete Action", PLANTS (BASEL) ., vol. 11, no. 3, pages 304 * |
| YORIKO SAWANO等: "Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds", BIOL CHEM ., vol. 388, no. 3, pages 273 - 280 * |
| 刘缙等: "银杏果仁抗菌蛋白的分离纯化及其基因克隆和原核表达初步研究", 农业生物技术学报, vol. 18, no. 2, pages 246 - 253 * |
| 刘缙等: "黄瓜转新型抗菌蛋白基因GNK2-1及其抗枯萎病的研究", 植物学报, vol. 45, no. 4, pages 411 - 418 * |
| 吴海霞: "银杏种仁抑菌蛋白及其抑菌机制研究", 中国博士学位论文全文数据库, no. 09 * |
| 无: "NCBI Reference Sequence: XP_020972742.1", NCBI, pages 1 * |
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