CN116063419B - Plant immune activating protein PmSCR1 and application thereof - Google Patents
Plant immune activating protein PmSCR1 and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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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
- A01N37/44—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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a plant immune activating protein PmSCR1 and application thereof, belonging to the technical field of biological control of crops. The invention identifies a new plant immune activating protein from the pythium aphanidermatum and names the protein as PmSCR1, the plant immune activating protein has stronger activity, can induce the defense reaction of plants, can be applied to the disease control of crops, and particularly aims at the control of plant diseases caused by the pythium aphanidermatum, the phytophthora sojae and the sclerotinia.
Description
Technical Field
The invention belongs to the technical field of biological control of crops, and particularly relates to a plant immune activating protein PmSCR1 and application thereof.
Background
Pythium group (Pythium myriotylum) is a dead-body nutrient bacteria of Pythum of Oomycetes (Oomycetes), and has a wide host range, and can be infected by dicotyledonous and monocotyledonous plants, and mainly comprises: soybean, peanut, tomato, onion, ginger, and the like, can cause root rot and stem basal rot in a variety of plants (Daly et al 2021). The ginger (Zingiber officinale Roscoe) is one of hosts, has various biological activities such as anti-inflammatory, antibacterial and anticancer, has high economic utilization value, and is widely planted in tropical and subtropical areas (Mao et al, 2019). China is the country with the largest global planting area and the largest total production amount of ginger, however, in recent years, along with the large-scale planting of ginger in China, the occurrence of stem-based rot of the ginger caused by Pythium ultimum is more and more frequent, the hazard is also increased, and in extreme environments, the yield of disease field blocks is reduced by 50% -80%, even the disease field blocks are out of order, so that the ginger production in China is severely restricted (Daly et al, 2021). At present, the prevention and treatment of the diseases mainly depend on chemical pesticides, however, a series of environmental and food safety problems are easily caused by using a large amount of chemical agents, so that development of more efficient and safe prevention and control measures is urgently needed.
In the long-term fight against pathogenic bacteria, plants evolved two broad classes of immune receptors for identifying the invasion of pathogenic bacteria. One of them is pattern recognition receptors (pattern-recognition receptors, PRRs) located on the surface of plant cell membranes, which recognize molecular patterns from invading pathogens (molecular patterns), and thus activate the underlying immune response of the plant itself, thereby protecting itself (Ngou et al, 2021). These molecular patterns capable of activating plant immune responses, also known as plant immune elicitors (plant immunity inducer), are generally a class of substances that are very conserved among microorganisms such as: polypeptides, proteins, oligosaccharides, lipids, etc. (Ngou et al, 2021). In recent years, along with the gradual clarification of plant immunity related mechanisms, a large number of domestic and foreign researchers focus on the identification of plant immunity elicitors so as to provide efficient and environment-friendly biopesticide resources for plant disease and pest control (Zhang Jie and the like, 2019).
Studies have shown that a class of Small molecular weight, cysteine-rich SCR (Small cysteine-rich) proteins secreted by pathogens can play an important role as plant immune elicitors in the process of pathogen infestation of plants (Rocafort et al 2020), also known as plant immune activating proteins. For example: SCR protein-PsPC 2 secreted by the oomycete pathogenic phytophthora infestans (Phytophthora infestans) can be cut by plant protease, and release immune antigen peptide to induce the Solanaceae plant to generate immune response (Wang et al, 2021); in addition, three SCR proteins, vdSCP27 and VdSCP126, secreted by the fungal pathogen verticillium dahliae (Verticillium dahliae), all induce the generation of an immune response in nicotiana benthamiana (Wang et al, 2020); however, the functional and mechanical studies of SCR proteins in saprophytes have not been reported.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a novel plant immune activating protein which is named as PmSCR1, has stronger activity, can induce the defense reaction of plants, can be applied to disease control of crops, and particularly aims at controlling plant diseases caused by pythium aphanidermatum, phytophthora sojae and sclerotinia sclerotiorum.
The invention provides a plant immune activating protein, wherein the amino acid sequence of the plant immune activating protein is shown as SEQ ID NO. 2; or the plant immune activating protein is protein or polypeptide which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO.2, is related to plant resistance and is derived from SEQ ID NO. 2.
The invention also provides a gene for encoding the plant immune activating protein, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID NO.1, and/or the encoding gene has more than 70% of homology with SEQ ID NO.1 and has similar functions.
The invention also provides a primer for amplifying the genes, and the nucleotide sequence of the primer is shown as SEQ ID NO.3 and SEQ ID NO. 4.
The invention also provides application of the plant immune activating protein or the gene for encoding the plant immune activating protein in disease control.
In some embodiments of the invention, the disease is a plant disease caused by a pathogen of the genus pythium, phytophthora sojae or sclerotinia.
In some embodiments of the invention, the plant disease comprises ginger stalk rot or soybean blight.
In yet another aspect, the present invention provides a pesticidal composition comprising the plant immune activating protein described above.
In some embodiments of the invention, the pesticide composition further comprises metalaxyl-m and fludioxonil.
The invention also provides a pesticide preparation, which comprises the plant immune activating protein and pesticide auxiliary materials.
In some embodiments of the invention, the concentration of the plant immune activator protein is in the range of 0.1 to 10mg/L.
The embodiment of the invention has at least the following beneficial effects: the plant immune activating protein PmSCR1 can induce plant immune response, has stronger induced resistance activity, can be applied to disease control of crops, for example, can be used for controlling plant diseases caused by Pythium gracile, phytophthora sojae and sclerotinia sclerotiorum, and particularly can be used for controlling ginger stem basal rot; the plant immune activating protein is SCR plant immune induced resistance protein in the pythium nodosum, lays a foundation for revealing the interaction mechanism of the pythium nodosum and plants, and provides a new protein resource for developing protein biopesticide capable of activating plant immunity.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a photograph of a leaf of Benshi tobacco of example 3 of the present invention, after 7 days of cultivation, after injection of a corresponding Agrobacterium solution;
FIG. 2 is a diagram showing the expression of GFP and PmSCR1 proteins detected by Western blot in example 3 of the present invention;
FIG. 3 is a statistical chart of the disease condition and the lesion area of the inoculated group of the PmSCR124 h after the GFP is overexpressed in the embodiment 4 of the invention;
FIG. 4 is a statistical chart of disease condition and lesion area of sclerotinia inoculated after 124 h of overexpression of GFP and PmSCR in example 4 of the present invention;
FIG. 5 is a SDS-PAGE detection of the purified PmSCR1 protein of example 5 of the present invention;
FIG. 6 is a Western blot detection chart of the PmSCR1 protein purified in the embodiment 5 of the invention;
FIG. 7 is a graph of the PmSCR1 induced by the ROS in Benshi smoke of example 6 of the present invention;
FIG. 8 shows the quantitative PCR detection result of PmSCR1 induced by Benshi smoke resistance related gene expression according to example 7 of the present invention;
FIG. 9 is a statistical chart of the disease condition and the lesion area of the inoculated group of PmSCR1 purified protein for 24 hours in the embodiment 8 of the invention;
FIG. 10 is a statistical chart of the disease occurrence and the lesion area of 36h of phytophthora sojae after spraying PmSCR1 purified protein for 24h in example 8 of the present invention;
FIG. 11 is a statistical chart of the disease states and the lesion areas of the sclerotinia inoculated after 24 hours of injection of the purified protein PmSCR1 in example 8 of the present invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, and the like used, unless otherwise specified, are commercially available (e.g., ordered from conventional biochemical reagent companies). The primers involved in the embodiment of the invention are synthesized by Nanjing division of Beijing qingke biotechnology Co. The specific techniques or conditions not identified in the examples are all carried out according to the specifications of the techniques, conditions or products (kits or reagents) described in the literature in this field.
Example 1: predictive analysis of candidate resistance-inducing proteins in Pythium insidiosum
The saprophytic fungus used in this example is SWQ7 strain, which is isolated from 7 times of diseased ginger tubers in China in 2018-2019 in this laboratory, and is currently stored in the institute of plant protection, academy of agricultural sciences, jiangsu province, and in addition, is also stored in China center for culture collection of microorganisms (China General Microbiological Culture Collection Center, CGMCC), with a collection number of CGMCC No.21459.
The previous study completed whole genome sequencing of SWQ7 and transcriptome sequencing of SWQ7 at different time points of ginger infection, and 47 candidate secreted proteins containing signal peptide sequences, no transmembrane domain, no known functional domain and up-regulated in SWQ7 infection ginger were selected by the present example based on the above-mentioned histologic data through bioinformatics analysis.
In this example, the above 47 candidate secreted protein encoding genes were respectively constructed on pSuper-HA vectors and transiently expressed on Nicotiana benthamiana leaves, and as a result, one of the genes was found to have plant immune activation activity, and was named as PmSCR1. In this name, "Pm" represents Pythium ultimum (Pythium myriotylum), and "SCR" represents the cysteine-rich feature thereof, and "1" is a number.
The nucleotide sequence of the coding plant immune activating protein PmSCR1 is shown as SEQ ID NO. 1; the amino acid sequence of the plant immune activating protein PmSCR1 is shown as SEQ ID NO. 2.
SEQ ID NO.1：
ATGCCTTCGTTCAAGTCGCTCTTCGCCGCCGGCCTCGTGGCCTCGCTCGCCACCGTGGCGAACGCCCACTCGGCGCTGCTCGCCAACGGCAAGCTGCTCGGCAAGCTCAACAGCGGCGAGTGCTTCAACTGCGCCGTGGGTCAGTACCCCGTGCCCAAGTCGATCCCTTCGAGCGCTGTCGACGCGTCGGACCCGGCCGACGGCATCGGCGCCTGGATCAAGTCGTCCGGCACGACCGTCAAGGAGACGTGGAACCTCTTCCACGGCGGCACCTCGTACGGCAGCATCACCGAGCCGGAGGCCATCAAGTCGGCCACGTGTGGTCGCTACGACCCCACGGCCGTCGGCGACGTCCCGTCGGGCGCCAAGATCTCCTACAAGAAGTCGAACCACAACGGTCCGGCCGAGATCTGGGTCGACGGCACCAAGACCTGGTTTGGTAACAACTTCATCGAGGACGGCAAGTCGATCACGGCCGACGCCTTCAAGTGTAACAAGGCGAGCTGCCAGGTCACGTGGTACTGGCTCGGCAAGGTCAAGGTCAAGGACCAGTGGAACTACTACCAGCTCTACGTGCAGTGCTTCGTCGTGAAGGGCTCGGGCAGCGGCGGTTCGGCCTCGGCGCCCGCCCCGGCCTCGGGTAGCAGTGACTCAACTCCGGGCACGGTCGCTCCTGCGAACGGCGCGTCGTCGGGCAACACGTCGGGCAACTCGGCCGACGACAAGGCCGCCCGCAAGGCTGAGAAGGAGAAGAAGAAGGCTGAGAAGGCTGCTGCCAACGAGGCCAAGGAGAAGAACCAGAACCTCCGTTTCTAA。
SEQ ID NO.2：
MPSFKSLFAAGLVASLATVANAHSALLANGKLLGKLNSGECFNCAVGQYPVPKSIPSSAVDASDPADGIGAWIKSSGTTVKETWNLFHGGTSYGSITEPEAIKSATCGRYDPTAVGDVPSGAKISYKKSNHNGPAEIWVDGTKTWFGNNFIEDGKSITADAFKCNKASCQVTWYWLGKVKVKDQWNYYQLYVQCFVVKGSGSGGSASAPAPASGSSDSTPGTVAPANGASSGNTSGNSADDKAARKAEKEKKKAEKAAANEAKEKNQNLRF。
Example 2: cloning of plant immune activating protein PmSCR1 coding gene
Step 1: total RNA extraction and reverse transcription.
The method comprises the steps of taking SWQ 7-infected leaf blades of Benshi cigarettes as materials, extracting total RNA according to the operation of a KK ultrafast plant total RNA extraction kit (Zhuang Meng, ZP 405K) instruction, and detecting the content and quality of the total RNA by using a spectrophotometer. cDNA synthesis was performed using 1. Mu.g of the total RNA as template according to Evo M-MLV RT Kit with gDNA Clean for qPCR (Ai Kerui, AG 11705) protocol.
Step 2: RT-PCR amplifies the full length of the encoding gene of PmSCR1.
According to the coding gene sequence of PmSCR1 shown in SEQ ID NO.1, the following PCR amplification primer sequence is designed:
the upstream primer sequence (shown in SEQ ID NO. 3):
5’-tacaccaaatcgactctagaATGCCTTCGTTCAAGTCGCT-3’；
the downstream primer sequence (shown as SEQ ID NO. 4):
5’-ACGTCGTATGGGTAcccgggGAAACGGAGGTTCTGGTTCT-3’；
and (2) using the cDNA obtained in the step (1) as a template, using the primer sequence as an amplification primer, and using the Northenon high-fidelity DNA polymerase 2X Phanta Max Master Mix (P520) to finish the amplification of the PmSCR1 coding gene according to the specification. Electrophoresis separation was performed on agarose gel, and the target band was recovered using a DNA gel recovery kit (Optimus, praeparata, T-GE 0101).
The recovered PCR product of the PmSCR 1-encoding gene was ligated to the XbaI and SmaI digested pSuper-HA vector using Norpran ClonExpress II One Step Cloning Kit (C112) following the protocol; the recombinant plasmid was transformed into E.coli DH 5. Alpha. Competent cells by the heat shock transformation method, and spread on LB solid medium containing 50. Mu.g/mL kanamycin antibiotic, after 16h of culture at 37 ℃, positive clones were picked up and sent to Beijing division of Biotechnology Co., ltd. In Beijing, and the clones whose sequencing results were identical to the sequence of SEQ ID NO.1 were sequenced, and plasmids were extracted by the high purity plasmid DNA miniprep kit (Optimum, T-PM 0201) according to the instructions for the subsequent experiments, hereinafter referred to as example 2 plasmids.
Example 3: transient expression of plant immune activating protein PmSCR1 coding gene on leaf of Nicotiana benthamiana
The plasmid obtained in example 2 was transformed into competent cells of Agrobacterium GV3101 by cold shock transformation, positive clones verified by PCR were selected, inoculated into 5mL of LB liquid medium containing 50. Mu.g/mL kanamycin and 50. Mu.g/mL rifampicin antibiotic, cultured overnight at 28℃and 220rpm, 1mL of the cultured broth was mixed with 1mL of 50% glycerol, and stored at-80℃for a long period of time for subsequent use, and the resulting mixture was referred to as clone of example 3 below.
In addition, positive control clone-transfected with pSuper-INF1-HA and negative control clone-transfected with pSuper-GFP-HA plasmid were obtained and stored as single colonies of Agrobacterium GV 3101.
The clone of example 3 and the control clone were each incubated overnight at 28℃and 220rpm in LB liquid medium simultaneously containing 50. Mu.g/mL kanamycin and 50. Mu.g/mL rifampicin.5000g,3min, and buffer (10 mM MgCl) 2 After 10mM 2- (N-morpholino) ethanesulfonic acid (MES) and 200. Mu.M acetosyringone, pH=5.7 were resuspended and washed twice, adjusted to OD 600 =0.5. They were injected onto the back of the leaf of Bentonite by using a 1mL syringe with the needle removed, and cultured at 25℃under 12h light/12 h dark conditions. The results were observed after 3 days, photographed after 7 days, and the results were counted.
Referring to FIG. 1, INF1, GFP and PmSCR1 represent leaf discs of Bentonian tobacco 7 days after injection of Agrobacterium strains containing positive control plasmid pSuper-INF1-HA, negative control plasmid pSuper-GFP-HA and example 2 plasmid pSuper-PmSCR1-HA, respectively. As shown in fig. 1, both positive control INF1 and PmSCR1 induced a significant allergic necrosis response compared to the negative control GFP.
And respectively collecting the injected and cultured leaf samples of the Bentonite tobacco for 36 hours, and detecting the protein expression condition. The specific operation is as follows: the collected benthamiana leaf sample is quickly frozen by liquid nitrogen and then ground into powder, quickly transferred to 80 mu L of RIPA lysate (strong) (Biyun Tian, P0013B), vigorously mixed and then added with 20 mu L of 5 XSDS-PAGE protein loading buffer (Biyun Tian, P0015), and uniformly mixed and then subjected to boiling water bath for 5min. 10. Mu.L of the sample was taken and electrophoretically separated in SDS-PAGE gel. After the completion of the reaction, the protein samples were transferred to PVDF membrane using an eBlot L1 rapid wet transfer apparatus (Kirsrui, L00686C), blocked for 1h with a 1 XPBST buffer (Solebao, P1031) containing 5% nonfat milk powder, incubated with Anti-HA-tag mAb-HRP-Direct (Medical & Biological laboratories, M180-7) diluted 1:5000 for 2h, washed with 1 XPBST buffer 3 times for 10min each, and protein bands were detected using a chemiluminescent apparatus by cECL Western Blot Kit (Kangji, CW 0048) following the instructions.
Referring to FIG. 2, western blot detection results show that GFP and PmSCR1 genes can be normally expressed in a transient expression system.
Example 4: resistance of plant immune activating protein PmSCR1 to pythium and sclerotinia
In the embodiment, the transient expression of the plant immune activating protein PmSCR1 coding gene in the Nicotiana benthamiana can induce the Nicotiana benthamiana to verify the resistance of the pythium aphanidermatum and the sclerotinia sclerotiorum.
Pythium group is inoculated to a V8 solid culture medium (1 g CaCO) 3 100mL of V8 juice, 15g of agar powder, constant volume to 1L, autoclaving at 121 ℃ for 15 min) and culturing at 25 ℃ for later use. Inoculating sclerotium rolfsii to PDA solid culture medium (200 g peeled potato, cutting into small pieces, adding water, boiling for 20-30min, filtering with four layers of gauze, adding 20g glucose, 15g agar powder, fixing volume to 1L, sterilizing at 121deg.C under high pressure for 15 min), and culturing at 25deg.C.
GFP is used as a negative control, pmSCR is transiently expressed on the leaf of the Nicotiana benthamiana for 24 hours, then the Mucor group is inoculated with a fungus block (the diameter is about 5 mm), and after 24 hours, the disease condition is observed under an ultraviolet lamp and photographed. Meanwhile, after the PmSCR is overexpressed for 24 hours according to the same method, sclerotinia sclerotiorum bacterial block (with the diameter of about 5 mm) is inoculated, and the disease condition is observed for 24 hours and photographed.
Referring to FIG. 3, pmSCR1 gene is over-expressed and then inoculated with Mucor, and the disease area is obviously smaller than that of GFP, which shows that PmSCR1 can induce resistance of Nicotiana benthamiana to Mucor.
Referring to FIG. 4, after the PmSCR1 gene is overexpressed, sclerotinia sclerotiorum is inoculated, the disease area is obviously smaller than that of the PmSCR1 gene, which indicates that the PmSCR1 can induce the resistance of the cigarette to the Mucor crowd.
Example 5: expression and purification of plant immune activating protein PmSCR1 in yeast cells
Step 1: and (5) constructing a yeast expression vector.
Using the plasmid obtained in example 2 as a template, the inventors designed the following primers based on the sequence of the signal peptide and the pPICK junction sequence removed from the sequence shown in SEQ ID NO. 1:
the upstream primer sequence (shown in SEQ ID NO. 5):
5’-gaattcCATTCTGCTTTGTTGGCTAACGG-3’；
the downstream primer sequence (shown in SEQ ID NO. 6):
5’-gcggccgcTTAGTGGTGATGATGATGATGGAATCTCAAATTTTGGTTCTTT-3’；
the vector construction was completed as described in example 2 to obtain positive clones which were partially identical to SEQ ID NO.1 sequence (nucleotide sequence of PmSCR1 encoding gene) by sequencing, and plasmids were extracted and stored, hereinafter referred to as example 5 plasmids.
Step 2: yeast strain transformation.
The plasmid of example 5 was transformed into competent cells of Pichia pastoris strain GS115 by using an electric shock transformation method, positive clones verified by PCR were selected, inoculated into 5mL of YPD liquid medium, cultured overnight at 30℃and 220rpm, 1mL of bacterial liquid was taken and mixed with 1mL of 50% glycerol, and then stored for a long period of time at-80℃and the mixture was referred to as clone of example 5 hereinafter.
Step 3: optimizing the pPICK9K-PmSCR1 expression system.
The clone of example 5 was inoculated into 4mL BMGY (2% peptone, 1% yeast extract, 100mM potassium phosphate buffer, 1.34% YNB, 4X 10) -5 Biotin, 1% glycerol, ph=6.0) in liquid medium at 30 ℃, 220rpm to OD 600=2-6, 4000rpm, 5min to collect the cells, BMGY liquid medium was used to resuspend and adjust the concentration to OD 600=1, methanol was added to a final concentration of 1% to induce protein expression, SDS-PAGE was examined for different time points (24 h,36h,73 h) for protein expression, and the results showed that the PmSCR1 expression level was relatively good for 72 hours, and this condition was selected for the subsequent protein purification experiments.
Step 4: and (5) purifying the PmSCR1 protein.
The clone of example 3 was grown up to 1L BMGY liquid medium according to the procedure described in step 3, and after 72h of induction expression. And collecting thalli at 10000rpm for 10min, taking supernatant, adding 48% ammonium sulfate to precipitate hybrid protein, and centrifuging at 10000rpm for 10min to obtain supernatant. The target protein was precipitated with 80% ammonium sulfate, and the supernatant was centrifuged at 10000rpm for 10min to retain the precipitate. The precipitated proteins were resuspended in buffer (20 mM Tris-HCl,50mM NaCl,pH =8.0) and dialyzed three times against the buffer to remove part of the pigment. The dialyzed proteins were purified by DEAE column to remove pigments, nucleic acids and some foreign proteins, and the effluent proteins were eluted with buffer and collected. Protein concentration was followed by SDS-PAGE and grey scale analysis to define protein concentration and purity.
Referring to FIGS. 5 and 6, SDS-PAGE was performed to detect the purified target protein, and a single band at 48kDa was seen, which was confirmed as the target band by Western blot using His antibody (FIG. 6).
Example 6: plant immune activating protein PmSCR1 induces active oxygen burst of Benshi smoke
The Benshi tobacco leaf dish was collected by a puncher with a diameter of 5mm and placed in a 96-well plate, 200. Mu.L of sterile water was added, the mixture was left at room temperature in the dark for 12 hours, the sterile water was discarded, 200. Mu. L L-012/horseradish peroxidase reaction solution (20. Mu. M L-012, 20. Mu.g/mL horseradish peroxidase (Sigma-Aldrich), 1. Mu.M PmSCR1 purified protein) was added, and chemiluminescence was detected by an enzyme-labeled instrument.
Referring to FIG. 7, 1. Mu.M PmSCR1 induced rapid bursts of active oxygen compared to the control, and the accumulation of active oxygen reached the peak at 30 min.
Example 7: plant immune activating protein PmSCR1 induces expression of Benshi tobacco resistance related gene
The purified protein 1. Mu.M obtained in example 5 was injected into the back of a well grown leaf of Bentonite by a 1mL syringe without needle, sampled at various time points (0 h,3h,6h,12 h), and RNA was reverse transcribed into cDNA as shown in example 2. Diluted 10 times as template, useGreen Premix Pro Taq HS qPCR Kit (Ai Kerui, AG 11701) the expression level of the above resistance-associated gene was examined according to the protocol.
Selected Benshi smoke resistance genes were: acre31, CYP71D20 and WRKY7.
Referring to fig. 8, qpcr results indicate that: after the plant immune activating protein PmSCR1 is treated, the resistance related genes Acre31, CYP71D20 and WRKY7 of the Nicotiana benthamiana are obviously increased.
Example 8: plant immune activating protein PmSCR1 induces resistance of cigarette to Mucor, phytophthora sojae and Sclerotinia
Injecting the purified protein PmSCR1 obtained in the example 5 on the leaf of Nicotiana benthamiana by taking PBS buffer solution as a control, inoculating the PmSCR1 with the PmSCR1 after 24 hours, and observing the disease condition and counting the area of the disease spots under an ultraviolet lamp and white light after 24 hours; meanwhile, using PBS buffer solution as a control, spraying the purified protein PmSCR1 obtained in the embodiment 5 on soybean seedlings, inoculating 100 phytophthora sojae zoospores after 24 hours, photographing after 36 hours, and counting the lesion length.
Referring to fig. 9, 10 and 11, after injection/spraying of 1 μm PmSCR1 protein, the plaque area/length of pythium nodosum (fig. 9), phytophthora sojae (fig. 10) and sclerotinia sclerotiorum (fig. 11) was significantly smaller than that of PBS control, demonstrating that PmSCR1 can induce resistance of p.
In view of the finding of the plant immune activating protein PmSCR1 and the function thereof in the aspect of crop disease control in the embodiment, the person skilled in the art can reasonably estimate that the plant immune activating protein PmSCR1 can be combined with pesticide auxiliary materials to prepare pesticide preparations on the basis of knowing the application files.
In view of the finding that the plant immune activating protein PmSCR1 and the function thereof in crop disease control are found in the embodiment, on the basis of the knowledge of the application document, a person skilled in the art can reasonably deduce that the plant immune activating protein PmSCR1 can be combined with conventional crop disease control drug components to form a novel pesticide composition, for example, the plant immune activating protein PmSCR1 can be combined with metalaxyl, wherein the plant immune activating protein PmSCR1 can effectively activate a plant immune system to increase crop resistance, and meanwhile, the metalaxyl is used for controlling oomycetes pathogens and the fludioxonil is used for controlling fungal pathogens.
It should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is for clarity only, and that the skilled artisan should recognize that the embodiments may be combined as appropriate to form other embodiments that will be understood by those skilled in the art.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A plant immune activating protein, characterized in that: the amino acid sequence of the plant immune activating protein is shown as SEQ ID NO. 2.
2. A gene encoding the plant immune activator protein according to claim 1, characterized in that: the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.
3. A primer for amplifying the gene of claim 2, wherein: the nucleotide sequences of the primers are shown as SEQ ID NO.3 and SEQ ID NO. 4.
4. Use of the plant immune activating protein according to claim 1 or the gene according to claim 2 for controlling plant diseases.
5. The use according to claim 4, wherein: the disease is plant disease caused by using the pythium aphanidermatum, the phytophthora sojae or the sclerotinia sclerotiorum as pathogenic bacteria.
6. The use according to claim 4, wherein: the plant diseases comprise ginger stem rot or soybean epidemic disease.
7. A pesticidal composition characterized in that: the pesticidal composition comprising the plant immune activating protein of claim 1.
8. The pesticidal composition of claim 7, wherein: the pesticide composition also comprises metalaxyl-M and fludioxonil.
9. A pesticide formulation characterized in that: the pesticide formulation comprises the plant immune activating protein as claimed in claim 1 and a pesticide adjuvant.
10. A pesticide formulation as claimed in claim 9, characterised in that: the concentration range of the plant immune activating protein is 0.1-10 mg/L.
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| CN202210983944.XA CN116063419B (en) | 2022-08-17 | 2022-08-17 | Plant immune activating protein PmSCR1 and application thereof |
| PCT/CN2023/113184 WO2024037548A1 (en) | 2022-08-17 | 2023-08-15 | Plant immunity activation protein pmscr1 and use thereof |
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| CN202210983944.XA CN116063419B (en) | 2022-08-17 | 2022-08-17 | Plant immune activating protein PmSCR1 and application thereof |
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| CN116063419A (en) | 2023-05-05 |
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