CN110301454B - Application of rape short peptide BnPEP5 - Google Patents
Application of rape short peptide BnPEP5 Download PDFInfo
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- CN110301454B CN110301454B CN201910518850.3A CN201910518850A CN110301454B CN 110301454 B CN110301454 B CN 110301454B CN 201910518850 A CN201910518850 A CN 201910518850A CN 110301454 B CN110301454 B CN 110301454B
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- bnpep5
- short peptide
- rape
- sclerotinia sclerotiorum
- resistance
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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
- 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
- A01N37/44—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 nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- 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
- 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
- A01N47/44—Guanidine; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Pest Control & Pesticides (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a rape seed oil (Brassica napus) The short peptide BnPEP5 stimulates cruciferous plants to generate sclerotinia sclerotiorum (Sclerotinia sclerotiorum)Sclerotinia sclerotiorum) Resistance and application. The rape short peptide BnPEP5 applied by the invention consists of 27 amino acids and is the C-terminal part of the full-length protein. The invention firstly synthesizes the short peptide BnPEP5 through manual synthesis, clarifies the excitation effect of the short peptide on the resistance of sclerotinia sclerotiorum, and provides the short peptide for treating cruciferous plants including rape and mustard (mustard) through infiltration and spraying (Brassica juncea) The application of the compound can stimulate the compound to generate resistance to sclerotinia sclerotiorum so as to prevent and control sclerotinia rot of rape and mustard. The BnPEP5 short peptide provided by the invention is a product synthesized by plants, and has the advantages of natural green, no environmental pollution, no harm to people and livestock, no induction of drug resistance of pathogens and the like by utilizing the BnPEP5 short peptide to stimulate immunity for preventing and treating sclerotinia sclerotiorum. In addition, the BnPEP5 short peptide is easy to artificially synthesize, simple and convenient to obtain, strong in disease resistance excitation effect and low in required concentration, and is a novel ecological and environment-friendly crop disease control preparation.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an immune excitation function of rape short peptide BnPEP5 and application thereof in sclerotinia sclerotiorum prevention and control.
Background
1. Plant immunity excitation technology
Plant immunity is a defense response activated by plants recognizing pathogen molecules through receptors. The plant immune system has multiple levels, wherein the first level is the immunity generated by transmembrane molecular Pattern Recognition Receptors (PRRs) on the surface of plant cells recognizing conserved molecular patterns derived from pathogens and plants themselves, which is called pattern-triggered immunity (PTI). In general, plants can sense the damage to the integrity of plant cells caused by pathogen infestation, etc., and generate injury-associated molecular patterns (DAMPs) which stimulate plants to generate PTI immune responses including active oxygen bursts, activation of kinase cascades, expression of defense-related genes, etc., through PRR receptor recognition, and finally express resistance to pathogens. PTI plays an important role in non-host resistance (nonhost resistance) to prevent non-adaptive microorganisms from infecting plants and basal resistance (basal resistance) to limit infection of adaptive pathogens to host plants that are susceptible. The PTI immunity stimulated by the DAMP has the advantages of broad spectrum, strong, stable and the like. In addition, because the DAMP is a natural product of the plant, the aim of preventing and controlling diseases by stimulating immunity by using the DAMP is a novel environment-friendly green disease prevention and control method.
2. Sclerotinia preventing and controlling technology
Sclerotinia sclerotiorum is caused by infection with Sclerotinia sclerotiorum (sclerotiorum). Sclerotinia sclerotiorum is a necrotizing nutritional (necrotph) pathogenic fungus, has extremely wide host defense and is a main disease of oil crops and vegetable crops. Causing huge economic losses every year. Chemical control remains an important tool due to the lack of high resistance varieties. Because some pesticides have the problems of ecological pollution, human and animal toxicity, easy generation of drug resistance of pathogens and the like, novel green control preparations are urgently needed to be researched and developed.
Disclosure of Invention
The invention aims to provide application of rape (Brassica napus) short peptide BnPEP5 in stimulating cruciferous plants to generate resistance to Sclerotinia sclerotiorum (sclerotiorum). The sequence of the rape short peptide BnPEP5 applied in the invention is shown in SEQ ID NO: 1, which consists of 27 amino acids in total, is the C-terminal part of a full-length protein (the sequence is shown as SEQ ID NO: 2). Prior to the present invention, the function of the short peptide BnPEP5 was not reported in any disclosure. The invention firstly synthesizes the short peptide BnPEP5 through artificial synthesis, clarifies the excitation effect of the short peptide on the resistance of sclerotinia sclerotiorum, and provides the application of the short peptide in preventing and controlling sclerotinia sclerotiorum by processing cruciferous plants to excite the short peptide to generate the resistance of sclerotinia sclerotiorum. The method is realized by the following steps:
(1) artificial synthesis or biological expression acquisition of BnPEP5 short peptide
The peptide can be synthesized by a professional polypeptide synthesis company. The short peptide BnPEP5 can also be obtained through biological expression: the nucleotide sequence corresponding to the BnPEP5 is cloned into an expression vector and is transformed into eukaryotic or prokaryotic microorganisms, and the target short peptide is amplified, extracted and purified by the microorganisms.
(2) Plant treatment of BnPEP5 short peptides
Plant leaves are infiltrated or sprayed by a short peptide BnPEP5 aqueous solution with proper concentration to stimulate plant immunity. The concentration of the short peptide BnPEP5 aqueous solution required by infiltration and spraying and the treatment scale are different. The concentration of the BnPEP5 required by infiltration treatment is lower, generally in the range of 100 nM-200 nM, and is suitable for small-scale plant treatment, while the concentration of the BnPEP5 required by spray treatment is higher, generally in the range of 200 nM-1000 nM, and is suitable for large-scale plant treatment.
(3) BnPEP5 short peptide stimulates plants to generate resistance to sclerotinia sclerotiorum
After the short peptide BnPEP5 is treated, the plant is quickly stimulated to generate an immune response to sclerotinia sclerotiorum. The intensity of the excitation can be detected by pathogen inoculation analysis. The immune response of the plants to sclerotinia sclerotiorum is shown in that the short peptide BnPEP5 treatment makes necrotic lesions smaller after inoculation than when the water control treatment. From the mechanism of action, the stimulation of this immune response by BnPEP5 is associated with its rapid accumulation of reactive oxygen species.
The invention has the advantages that: (1) the BnPEP5 short peptide provided by the invention is a product synthesized by plants, and has the advantages of natural green, no environmental pollution, no harm to people and livestock, no induction of drug resistance of pathogens and the like by utilizing the BnPEP5 short peptide to stimulate immunity for preventing and treating sclerotinia sclerotiorum. (2) The stimulation of plant immunity by DAMP is the result of recent theoretical research. The BnPEP5 has sufficient theoretical basis for applying the rape DAMP to the immunity excitation and the disease control of cruciferous crops, and is also an example for guiding the practice and application of the theory. (3) The BnPEP5 short peptide has only 27 amino acids, is easy to artificially synthesize and is easy and convenient to obtain. (4) The BnPEP5 short peptide has strong disease resistance excitation effect and low required concentration, so the disease control cost is low.
Drawings
FIG. 1 shows that infiltration or spraying of aqueous solution of BnPEP5 short peptide into leaves of oilseed rape (Brassica napus) elicits resistance to Sclerotinia sclerotiorum. Soaking rape plant leaves (b) with a BnPEP5 short peptide aqueous solution with the concentration of 100nM through a sterile disposable syringe, or spraying a BnPEP5 short peptide aqueous solution with the concentration of 200nM to rape leaves (d) through a spray gun, inoculating the leaves with sclerotinia sclerotiorum hypha blocks after 1d, and showing the size of the lesion spots after 2d of inoculation. The results show that the BnPEP5 treated leaves formed significantly smaller lesions compared to the control of the aseptic double-distilled water treatment (a, infiltration of the aseptic double-distilled water syringe; c, spraying by the aseptic double-distilled water spray gun). It was shown that treatment with BnPEP5 elicited resistance to sclerotinia in oilseed rape.
FIG. 2 shows that BnPEP5 short peptide stimulates rape leaves to rapidly accumulate active oxygen. The BnPEP5 short peptide with the concentration of 100nM rapidly induces the rape leaf disc to accumulate a large amount of active oxygen. Sterile water treatment was used as a control. The active oxygen is detected by a chemiluminescence detector by a luminol-based method.
FIG. 3 shows that leaf discs of mustard (Brassica juncea) were challenged by BnPEP5 short peptide aqueous solution infiltration or spraying of leaf discs with mustard. Soaking leaf blades of mustard plants (b) with a BnPEP5 short peptide aqueous solution with the concentration of 100nM through a sterile disposable syringe, or spraying a BnPEP5 short peptide aqueous solution with the concentration of 200nM to leaf blades of mustard (d) through a spray gun, inoculating the leaf blades with sclerotinia sclerotiorum mycelium blocks after 1d, and showing the size of lesion spots after 2d of inoculation. The results show that the BnPEP5 treated leaves formed significantly smaller lesions compared to the control of the aseptic double-distilled water treatment (a, infiltration of the aseptic double-distilled water syringe; c, spraying by the aseptic double-distilled water spray gun). It was shown that treatment with BnPEP5 elicited resistance to sclerotinia in mustard.
Detailed Description
The invention is further explained by the accompanying drawings and examples.
Example 1 application of B nPEP5 short peptide in resisting sclerotinia rot of rape (Brassica napus)
According to the embodiment of the invention, the artificially synthesized BnPEP5 short peptide is used for treating the rape by an infiltration or spraying method to stimulate the rape to generate resistance to sclerotinia sclerotiorum, so that the method is used for controlling sclerotinia rot of the rape. The method mainly comprises the following steps:
1) artificial synthesis of BnPEP5 short peptide
The BnPEP5 short peptide provided by the invention has a sequence shown in SEQ ID NO: 1, which consists of 27 amino acids in total, is the C-terminal part of a full-length protein (the sequence is shown as SEQ ID NO: 2). Prior to the present invention, the function of the short peptide BnPEP5 was not reported in any disclosure.
In the embodiment, professional polypeptide synthesis companies (Qiangyao biotechnology limited) are entrusted to synthesize the BnPEP5 short peptide for the analysis and the application of the functions of rape disease resistance excitation and sclerotinia disease prevention and control.
2) Treatment of rape by BnPEP5 short peptide
And (3) infiltration treatment: infiltrating rape plant leaves with a BnPEP5 short peptide aqueous solution with the concentration of 100nM through a sterile disposable syringe;
spray treatment: spraying 200nM BnPEP5 short peptide water solution to rape leaf via spray gun.
3) The BnPEP5 short peptide stimulates rape to generate resistance to sclerotinia sclerotiorum
The rape leaves can be stimulated to generate resistance to sclerotinia sclerotiorum by soaking or spraying the rape leaves with the BnPEP5 aqueous solution of short peptide. Sclerotinia inoculation analysis results showed that after 1d treatment with BnPEP5 short peptide (100nM) infiltrated or sprayed (200nM), sclerotinia hypha block inoculated leaf lesions were significantly smaller than the sterile double distilled water treated control (figure 1). The BnPEP5 shows that the infiltration or the spraying treatment can quickly stimulate the rape to generate the resistance to the sclerotinia sclerotiorum.
The analysis result of active oxygen quantitative detection shows that BnPEP5 short peptide (100nM) induces rape leaf discs to rapidly and strongly accumulate active oxygen (FIG. 2). The BnPEP5 short peptide is shown to stimulate the generation of resistance of rape to sclerotinia sclerotiorum by inducing the burst of active oxygen.
Example 2 application of BnPEP5 short peptide in mustard (Brassica juncea) for resisting sclerotinia rot
According to the embodiment of the invention, the artificially synthesized BnPEP5 short peptide is used for treating the mustard by an infiltration or spraying method to stimulate the mustard to generate resistance to sclerotinia sclerotiorum, so that the method is used for controlling sclerotinia rot of the mustard. The method mainly comprises the following steps:
1) artificial synthesis of BnPEP5 short peptide
The BnPEP5 short peptide provided by the invention has a sequence shown in SEQ ID NO: 1, which consists of 27 amino acids in total, is the C-terminal part of a full-length protein (the sequence is shown as SEQ ID NO: 2). In the embodiment, professional polypeptide synthesis companies (Qiangyao biotechnology limited) are entrusted to synthesize the BnPEP5 short peptide for the analysis and the application of the mustard disease resistance excitation and sclerotinia disease prevention and control functions.
2) Treatment of mustard with BnPEP5 short peptide
The plant treatment mode of the BnPEP5 short peptide comprises infiltration and spraying. The concentration of the short peptide BnPEP5 aqueous solution and the treatment scale are different. The concentration of BnPEP5 required by infiltration treatment is low, and the method is suitable for small-scale plant treatment, while the concentration of BnPEP5 required by spray treatment is high, and the method is suitable for large-scale plant treatment.
And (3) infiltration treatment: infiltrating leaves of mustard plants with BnPEP5 short peptide aqueous solution with the concentration of 100nM through a sterile disposable syringe;
spray treatment: spraying 200nM BnPEP5 short peptide aqueous solution to leaf of mustard plant via spray gun.
3) BnPEP5 short peptide stimulates mustard to generate resistance to sclerotinia sclerotiorum
The leaf of the mustard can be stimulated to generate resistance to sclerotinia by soaking or spraying BnPEP5 short peptide aqueous solution on the leaf of the mustard. Sclerotinia inoculation analysis results showed that sclerotinia sclerotiorum hypha block inoculated leaf lesions were significantly smaller than the sterile double distilled water treated control (fig. 3) after 1d treatment with BnPEP5 short peptide (100nM) infiltration or spray (200 nM). The BnPEP5 shows that the infiltration or the spraying treatment can quickly stimulate the mustard to generate the resistance to the sclerotinia sclerotiorum.
Through the above examples, those skilled in the art can determine that the short peptide BnPEP5 can effectively stimulate cruciferous plants to generate resistance to Sclerotinia sclerotiorum (sclerotiorum) and is applied to prevention and control of Sclerotinia sclerotiorum.
Sequence listing
<110> Zhejiang university
<120> application of rape short peptide BnPEP5
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>27
<212>PRT
<213> Brassica napus (Brassica napus)
<400>1
Ser Leu Asn Val Ser Ser Lys Leu Thr Arg Lys Leu Pro Val Ser Ser
1 5 10 15
Gly Lys Arg Gly Gly Val Asn Asn His Asp Met
20 25
<210>2
<211>90
<212>PRT
<213> Brassica napus (Brassica napus)
<400>2
Met Gln Lys Glu Ser Asp Lys Lys Arg His Arg Cys Lys Leu Ile Thr
1 5 10 15
Gln Ser Val Met Ala Phe Leu Asp Cys Leu Asn Val Leu Asn Gln Lys
20 25 30
Lys Lys Lys Lys Lys Lys Leu Ser Glu Tyr Met Val Lys Ala Lys Ala
35 40 45
Arg Asn Glu Ala Glu Val Ala Ser Ser Val Glu Thr Ser Asp Arg Ser
50 55 60
Leu Asn Val Ser Ser Lys Leu Thr Arg Lys Leu Pro Val Ser Ser Gly
65 70 75 80
Lys Arg Gly Gly Val Asn Asn His Asp Met
85 90
Claims (1)
1. Rape seedBrassica napusShort peptide BnPEP5 stimulates cruciferous crops to generate sclerotinia sclerotiorumSclerotinia sclerotiorumThe application of resistance is characterized in that the amino acid sequence of the rape short peptide BnPEP5 is shown as SEQ ID NO: 1, the application is to infiltrate or spray the water solution of the short peptide BnPEP5 into the leaves of cruciferous crops to stimulate the plant immunity, wherein the cruciferous crops are rape and mustardBrassica juncea。
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| CN114375963B (en) * | 2021-12-29 | 2023-03-28 | 浙江大学 | Immune excitation function application of rape short peptide BnPEP6 |
| CN114317554B (en) * | 2021-12-29 | 2023-03-21 | 浙江大学 | Application of rape gene BnPEP5 in sclerotinia sclerotiorum prevention and control |
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|---|---|---|---|---|
| CN1251371A (en) * | 1999-08-10 | 2000-04-26 | 南京农业大学 | Exciter for inducing allergic reaction of plant and preparation method and use thereof |
| US20090300802A1 (en) * | 2005-01-26 | 2009-12-03 | Washington State University | Plant defense signal peptides |
| CN104530204A (en) * | 2014-12-19 | 2015-04-22 | 中国农业科学院油料作物研究所 | Rape antibacterial peptide BnPRP1 and application thereof |
| CN108883148A (en) * | 2016-04-06 | 2018-11-23 | 植物保健公司 | The peptide and application thereof in allergic reaction exciton source |
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| US20090300802A1 (en) * | 2005-01-26 | 2009-12-03 | Washington State University | Plant defense signal peptides |
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