CN113354720A

CN113354720A - Plant immune activation protein PsAEP1 and application thereof

Info

Publication number: CN113354720A
Application number: CN202110525967.1A
Authority: CN
Inventors: 王源超; 徐愿鹏; 王燕
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-05-14
Filing date: 2021-05-14
Publication date: 2021-09-07

Abstract

The invention discloses a plant immune activation protein PsAEP1 and application thereof, wherein the protein can induce plant resistance and can be used as a plant immune activation factor to be applied to the field of prevention and control of plant diseases. The protein has an amino acid sequence shown as SEQ ID NO. 2. The high-concentration protein can be obtained by utilizing the expression of the yeast engineering strain, and the disease resistance of the plant to phytophthora can be improved by treating the plant with the protein. The plant immune activation protein not only can obviously improve the disease resistance of plants, but also has low required concentration, quick response and long action time. The PsAEP1 provides a new way for improving the disease resistance of plants and preventing and treating plant diseases.

Description

Plant immune activation protein PsAEP1 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a plant immune activation protein PsAEP1 and application thereof.

Background

Phytophthora is an important plant pathogenic bacterium (Phytophthora), can harm almost all dicotyledonous plants, and causes plant epidemic diseases which seriously threaten global food safety and ecological safety. Phytophthora has the characteristics of complex genome, quick toxic variation and difficult control, and the resistance of a plant to phytophthora disease resistant varieties is easy to lose, so chemical control is always the main mode for controlling phytophthora diseases of plants (Cooke et al, 2012). However, the large amount of bactericide used not only increases the production cost, but also brings a series of problems of crop phytotoxicity, overproof pesticide residue, environmental pollution and the like, seriously affects the sustainable development of agricultural production and harms human health.

In recent years, with the comprehensive and deep research on the plant immune system, the application of the plant autoimmunity mechanism to control crop diseases and insect pests has become an important development direction in the field of plant protection. For example, chitin, which is a conserved Pathogen Pattern molecule (PAMP), can be recognized by plant cell membrane Pattern Recognition Receptors (PRRs), activate plant immune response (PTI), and generate broad-spectrum resistance to pathogenic bacteria (Zipfel,2014, Jones and Dangl, 2006). At present, chitin as a main component of plant resistance inducer has been widely used for preventing and treating fungi, bacteria and viral diseases, and because of its carbohydrate nature, it can induce plant resistance and reduce environmental pollution (Cao et al, 2014, Petutschnig et al, 2014). However, at present, in addition to chitin, elicitors that induce plant resistance and are put into agricultural production are rare, so the discovery of novel plant immune elicitors is important for agricultural sustainability development.

Reference to the literature

CAO Y,LIANG Y,TANAKA K,et al.2014.The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1.Elife[J],3.

COOKE D E,CANO L M,RAFFAELE S,et al.2012.Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen.PLoS Pathog[J],8:e1002940.

JONES J D,DANGL J L 2006.The plant immune system.Nature[J],444:323-329.

JOOSTEN M H A J,VOGELSANG R,COZIJNSEN T J,et al.1997.The biotrophic fungus Cladosporium fulvum circumvents Cf-4-mediated resistance by producing unstable AVR4 elicitors.Plant Cell[J],9:367-379.

KUNZE G,ZIPFEL C,ROBATZEK S,et al.2004.The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants.Plant Cell[J],16:3496-3507.

MA Z,SONG T,ZHU L,et al.2015.A Phytophthora sojae Glycoside Hydrolase 12Protein Is a Major Virulence Factor during Soybean Infection and Is Recognized as a PAMP.Plant Cell[J],27:2057-2072.

PETUTSCHNIG E K,STOLZE M,LIPKA U,et al.2014.A novel Arabidopsis CHITIN ELICITOR RECEPTOR KINASE 1(CERK1)mutant with enhanced pathogen-induced cell death and altered receptor processing.New Phytol[J],204:955-967.

ZIPFEL C 2014.Plant pattern-recognition receptors.Trends Immunol[J],35:345-351.

Disclosure of Invention

One of the purposes of the invention is to provide a plant immune activation protein PsAEP 1.

The second purpose of the invention is to provide a gene sequence for coding the plant immune activation protein PsAEP 1.

The invention also aims to provide a recombinant expression vector containing the plant immune activator protein PsAEP1 gene.

The fourth content of the invention is to provide the plant immune activation protein PsAEP1 and the application of the coding gene thereof.

The content of the invention is detailed as follows:

the invention provides a gene PsAEP1, which is derived from phytophthora sojae, has an amino acid sequence shown as SEQ ID NO.2 or is a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of the SEQ ID NO.2 and is related to phytophthora nicotianae resistance and is derived from the SEQ ID NO. 2.

The invention provides a coding gene of the plant immune activation protein PsAEP 1.

As a preferred technical scheme, the coding gene is as follows (1) or (2):

(1) a nucleotide sequence shown as SEQ ID NO. 1;

(2) a nucleotide sequence which has at least more than 70 percent of homology with SEQ ID NO.1 and encodes a protein PsAEP 1; preferably a nucleotide sequence which has at least 80% or more homology with SEQ ID NO.1 and encodes the protein PsAEP 1; further preferably a nucleotide sequence which has at least 85% or more homology with SEQ ID NO.1 and encodes the protein PsAEP 1; even more preferably a nucleotide sequence which has at least 90% or more homology with SEQ ID NO.1 and encodes the protein PsAEP 1; most preferably a nucleotide sequence which has at least 95% or more homology with SEQ ID NO.1 and encodes the protein PsAEP 1.

The invention also provides a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the coding gene. Preferably, the recombinant vector is a eukaryotic expression vector obtained by inserting a PsAEP1 coding gene into pPIC 9K. The recombinant vector can be used for expressing pichia pastoris KM 71. The obtained fusion expression protein (PsAEP1-his) with the molecular weight of about 34KD can induce tobacco to generate resistance reaction and improve plant immunity. The harm of the tobacco phytophthora nicotianae to tobacco is reduced.

The application of the plant immune activation protein PsAEP1 in inducing plant defense response and improving plant disease resistance. Preferably, the plant is tobacco, potato, pepper, eggplant or tomato. More preferably, the disease resistance is resistance to phytophthora nicotianae.

The invention also provides a method for improving phytophthora resistance of plants, which is to spray the plant immune activation protein PsAEP1 on the surface of the plants or inject the plant immune activation protein PsAEP1 into plant leaves.

In some embodiments, the plant is tobacco, potato, pepper, eggplant or tomato.

By utilizing the PsAEP1 amino acid sequence, a nucleic acid sequence which is codon-optimized and is favorable for expression in pichia pastoris can be designed and artificially synthesized.

The invention has the advantages of

The plant immune activator protein can obviously improve the disease resistance of plants, and has low use concentration, quick response and long action time. PsAEP1 activates the plant immune system, improves the plant resistance and provides a new way and method for preventing and controlling plant diseases in the agricultural production process.

Drawings

FIG. 1 shows that Pichia pastoris strain KM71 expresses PsAEP1 protein, which is detected by SDS-PAGE after protein purification, and a staining agent is Coomassie brilliant blue;

FIG. 2 the plant immune activator protein PsAEP1 induces bursts of tobacco active oxygen;

FIG. 3 the plant immune activator protein PsAEP1 induces burst of active oxygen in potato, pepper, eggplant or tomato;

FIG. 4 shows that the plant immune activation protein PsAEP1 induces the expression of tobacco disease resistance related genes;

FIG. 5 is a graph showing the effect of the plant immune activator protein PsAEP1 in inducing tobacco against Phytophthora nicotianae;

FIG. 6 statistics of biomass of the plant immune activator protein PsAEP1 induced tobacco resistance to Phytophthora nicotianae.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The primer related to the embodiment of the invention is synthesized by Nanjing Kingsrei Biotechnology GmbH.

And (3) microorganism information: the strain P6497 of Phytophthora sojae was given to professor Brett Tyler, university of Oregon, USA.

Example 1 isolation and characterization of the plant immune activator protein PsAEP1

Inducing phytophthora sojae P6497 to produce zoospores, inoculating the zoospores to soybean leaves, infecting for 10 hours, extracting intercellular fluid of the soybean leaves, sending the infected intercellular fluid to Shanghai New Life company for proteome sequencing, comparing the result with the phytophthora sojae genome data, and identifying candidate protein PsAEP1 secreted by the phytophthora sojae in the intercellular fluid, wherein the amino acid sequence of the candidate protein is shown in SEQ ID NO. 2.

Example 2 cloning of Gene encoding plant immune activator protein PsAEP1

(1) Total RNA extraction:

taking hypha of a liquid-cultured phytophthora sojae strain P6497 as a material, extracting total RNA by using an Omega RNA extraction reagent, and detecting the RNA content and quality by using a spectrophotometer according to the operation instruction.

(2) Reverse transcription to generate the first strand:

mu.g of RNA was used as a template, and cDNA synthesis was carried out according to the instructions of the kit for PrimeScript reverse transcriptase of Takara, Inc., and the volume was adjusted to 20. mu.L. Appropriate amounts of the reverse transcription products were taken for subsequent gene cloning PCR.

(3) And (3) performing PCR by using the first strand of the cDNA as an RT-PCR template according to a conventional method, and amplifying the full length of the PsAEP1 coding gene:

PCR primer amplification sequence:

an upstream primer: SEQ ID NO.3

(5’-CAGCACCAGCTAGCATCGATGGTGTTCTCCATC-3’)

A downstream primer: SEQ ID NO.4

(5’-AATCTCTAGAGGATCCCCGGCCGTGGTGACCGA-3’)

A50. mu.L reaction system in which 5 XBuffer 10. mu.L, 2.5mM dNTP 4. mu.L, Takara PrimerSTARTaq enzyme 0.5. mu.L, template cDNA 1. mu.L, water to 50. mu.L; the PCR amplification program comprises pre-denaturation at 98 deg.C for 3min, denaturation at 98 deg.C for 15 s, annealing at 58 deg.C for 15 s, extension at 72 deg.C for 1min, circulating for 35 times, and extension at 72 deg.C for 10 min; the PCR product of the gene encoding PsAEP1 was recovered by electrophoresis on an agarose gel, photographed by Ethidium Bromide (EB) staining, and excised. The electrophoretic band was recovered with an Agarose Gel DNAPuration Kit (TaKaRa). PCR products of PsAEP 1-encoding gene recovered by cutting gel were ligated to SmaI digested pGR107::3HA vector (vector purchased from Biovector plasmid vector bacterial cell Collection) according to the instructions to obtain pGR107:: PsAEP1-3HA plasmid, transformed Escherichia coli competent cell JM109, plated LB (containing 50. mu.g/mL) plate, cultured at 37 ℃ for 16h, colony PCR-verified three positive clones were picked according to the plasmid extraction Kit (Takara) to extract plasmid, and sent to Nanjing Kingsry for sequencing, which sequence should be identical to SEQ ID NO.1 sequence.

Example 3 eukaryotic expression and purification of plant immune activator protein PsAEP 1.

(1) Construction of eukaryotic expression vectors

PsAEP1 was synthesized by Biotech Inc. according to the sequence shown in SEQ ID NO. 1.

The artificially synthesized PsAEP1 encoding gene was ligated to Snab1 digested pPIC9K vector according to the protocol of Cloneexpress II One Step Cloning Kit (Vazyme) to obtain pPIC9K: (PsAEP 1-6his plasmid), Escherichia coli competent cell JM109 was transformed, LB (containing kanamycin 50. mu.g/mL) plate was applied, and after culturing at 37 ℃ for 16h, colony PCR was verified and three positive clones were picked up and plasmid was extracted according to the plasmid extraction Kit (Takara).

(2) Yeast electric shock transformation

Culture medium for yeast protein expression process: YPD 1L: 10g of yeast extract, 20g of peptone and 20g of agar powder were dissolved in 1L of water. BMGY (Buffered Glycerol-complex Medium)/BMMY (Buffered methane-complex Medium)1L 1% yeast extract, 2% peptone, 100mM potassium phosphate pH 6.0, 1.34% YNB, 4X 105% biotin, 1% Glycerol or 0.5% Methanol. 10g of yeast extract, 20g of peptone in 700ml of water were sterilized for 20min, cooled to room temperature and the following mixture was added: 100ml of 1M potassium phosphate buffer pH 6.0; 100ml of 10 XYNB; 2ml of 500 × Biotin; 100ml 10 × GY; when BMMY is prepared, 100ml of 10 XM is added to replace 10 XMG culture medium, and the mixture is stored at 4 ℃ for 2 months after being sterilized. Storage solution: 10 XYNB (13.4% yeast nitrogen source alkaloid YNB, ammonium sulfate containing no amino acids) 134gYNB was dissolved in 1000ml of water, filtering and sterilizing, heating until YNB is completely dissolved, storing at 4 deg.C, or preparing with 34g of YNB (without ammonium sulfate and without amino acid), 100g of ammonium sulfate, and standing for 1 year. 500 × biotin (0.02%): dissolving 20mg biotin in 100ml water, filtering for sterilization, and standing at 4 deg.C for 1 year; 10 × M (5% methanol): mixing 5ml of methanol with 95ml of water, filtering and sterilizing, storing at 4 ℃, and storing for 2 months; 10 × GY (10% glycerol); mixing 100ml glycerol and 900ml water, filtering or autoclaving, standing at room temperature for more than 1 year; 1M potassium phosphate buffer pH 6.0; 132ml 1M K₂HPO₄，868ml 1M KH₂PO₄The pH was adjusted to 6.0. + -. 0.1 (phosphoric acid or KOH if pH adjustment is required). Filtering or autoclaving, and standing at room temperature for more than 1 year.

Yeast electric shock competence preparation and electric shock transformation

Competent preparation of pichia pastoris: a suitable amount of Pichia pastoris strain KM71 (purchased from Biotechnology engineering Co., Ltd., product No. B528425) was inoculated into 10mL of YPD medium and shaken overnight at 30 ℃. 100mL YPD liquid medium was inoculated at 1% inoculum size and shake-cultured at 30 ℃ until OD was 1.3-1.5. Centrifuging at 4 ℃ and 5000rpm for 5min, discarding the supernatant, and resuspending the thalli with ice-cold sterile water. Centrifuging at 4 ℃ and 5000rpm for 5min, discarding the supernatant, and resuspending the thalli with ice-cold sterile water. Centrifuge at 5000rpm for 5min at 4 deg.C, and discard the supernatant. The mixture was washed 3 times with 1M sorbitol and dissolved in 200uL of 1M ice-cold sorbitol to prepare for conversion.

Transformation of pichia pastoris: add plasmid 10 μ l (higher content is better) linearized with suitable enzyme cutting site into 80 μ l yeast competent cell, put on ice for 5min, add into 0.2cm electric shock cup rapidly (ice pre-cooling), shock, add 1mL sorbitol rapidly, spread on His defect plate after 30 deg.C water bath for 2 h.

And (3) yeast colony verification: the recombinant strain phenotype was examined by PCR, and when the colonies on the plate grew to the naked eye (about 2 days), the other components of the PCR reaction solution except the template were prepared and dispensed. The primers preferably use a universal primer on a carrier, a specific primer of a gene, a colony is selected, the colony is firstly spotted on another His defect flat plate, then the rest saccharomycetes are rinsed in a PCR tube, the numbers on the PCR tube and the His flat plate are corresponding, PCR amplification is carried out, 1% agarose electrophoresis is carried out, and the clone of a specific band is shown in the PCR amplification, and the subsequent culture expression protein can be carried out.

(3) Yeast expressed proteins and purification

After yeast BMMY is cultured for 4 days, collecting bacterial liquid, centrifuging at 8000rpm for 30min, collecting supernatant, adding ammonium sulfate powder (475 g per 1L of supernatant) according to the volume of the supernatant, dissolving the ammonium sulfate, and precipitating at 4 ℃ for 24 hours; the precipitate was collected by centrifugation at 8000rpm for 1 hour, and then dissolved in sterilized ultrapure water (50 ml of water per 1L), and the solution was passed through a 0.45 μm filter, and then the filtrate was subjected to protein purification by column chromatography. A purification step: AKTA Using an automated protein purification Instrument^TMavant 25(GE Healthcare), which is passed through a desalting column, a desalted collected solution is passed through a nickel column, and desalted again after imidazole elution, followed by concentration measurement and SDS-PAGE electrophoresis. As a result: the purified plant immune activation protein PsAEP1 eukaryotic expression protein is obtained, and the result is shown in figure 1.

Example 4 defense response of the plant immune activator protein PsAEP1 in tobacco

(1) 1 mu M of the eukaryotic expression plant immune activation protein PsAEP1 purified in example 3 was taken to treat tobacco leaves, and the burst of active oxygen induced by the treatment was detected.

Harvesting leaf discs from 5-week-old tobacco

Then floated overnight in 200ul of sterile water in 96-well plates. Sterile water was replaced with 200ul of reaction buffer containing luminol and peroxidase (35.4. mu.g/ml luminol, 10. mu.g/ml peroxidase) and PsAEP1 protein. Luminescence was measured using a GLOMAX96 microplate luminometer (Promega, Madison, WI, USA).

As a result: the detection result shows that 1 mu M of PsAEP1 protein can induce the burst of active oxygen of tobacco, as shown in figure 2.

(2) 1 mu M of the eukaryotic expression plant immune activation protein PsAEP1 purified in example 3 is taken to treat potato, pepper, eggplant or tomato leaves, and the active oxygen burst induced by the leaves is detected.

Collecting leaf disks from potatoes, hot peppers, eggplants or tomatoes of suitable age

As a result: the detection result shows that 1 mu M of PsAEP1 protein can induce the active oxygen burst of potatoes, hot peppers, eggplants or tomatoes, as shown in figure 3.

(3) The eukaryotic expression plant immune activation protein PsAEP1 can induce the transcription level of disease resistance related gene to be obviously increased when used for treating leaves

The concentration of PsAEP1 protein was adjusted to 200nM after the concentration was determined. Selecting 5-week-old tobacco, injecting PsAEP1 protein into the leaf blade from the back of the leaf blade by using a 1mL syringe without a needle, taking an empty carrier as a control, collecting a sample after 3 hours of injection, and detecting the transcription level of the disease-resistant related gene. The total RNA extraction adopts an Omega RNA extraction kit, and the total RNA extraction kit is operated according to instructions, and the content and the quality of the RNA are detected by a spectrophotometer.

Reverse transcription to generate the first strand: mu.g of RNA was used as a template, and cDNA synthesis was carried out according to the instructions of the kit for PrimeScript reverse transcriptase of Takara, Inc., and the volume was adjusted to 20. mu.L. The reverse transcription product is diluted by 10 times by the user for detecting the gene silencing efficiency by a real-time fluorescent quantitative PCR reaction.

The primers used in the real-time fluorescent quantitative PCR reaction were as follows:

NbCYP71D20 upstream primer: SEQ ID NO.7

(5’-GTTGACGCCATTGTTGAG-3’)

NbCYP71D20 downstream primer: SEQ ID NO.8

(5’-ATCTTCGCCTCCTAATGC-3’)

NbEF1 a upstream primer: SEQ ID NO.9

(5’-GTATGCCTGGGTGCTTGAC-3’)

NbEF1 a downstream primer: SEQ ID NO.10

(5’-ACAGGGACAGTTCCAATACCA-3’)

The PCR reaction system contained 5. mu.L of cDNA, 10. mu.L of SYBR Premix Ex Taq II (Tli RNase H Plus), 0.4. mu.L of each of the front and rear primers, 0.4. mu.L of ROX Reference Dye II, and 13.8. mu.L of water. Reaction procedure: 95 ℃ for 30 seconds, II: 95 degrees 15 seconds. The results of the detection are shown in FIG. 4.

As a result: fluorescent quantitative PCR results show that 3 hours after 200nM PsAEP1 protein is used for treating tobacco leaves, the expression level of the tobacco disease resistance related gene CYP71D20 is induced to be obviously increased (as shown in figure 4).

Example 5 plant immune activator protein PsAEP1 enhances disease resistance in tobacco

(1) The purified PsAEP1 solution was diluted to 100nM, and after the injection of unused tobacco from the back of leaf discs, 20 plants were treated with the empty vector as a control, and the treatment was repeated 3 times. After 24h of induction, the treated leaves were inoculated with phytophthora nicotianae and three days after inoculation, the infestation was observed (fig. 5). The sample was collected.

(2) Extracting a sample genome, and performing phytophthora nicotianae infection biological quantity determination by using fluorescent quantitative PCR (the method is the same as the embodiment 4), wherein Par is selected as an internal reference gene of phytophthora nicotianae, and EF1 alpha is selected as an internal reference gene of the phytophthora nicotianae. The quantitative primer sequences were as follows:

PAR upstream primer: SEQ ID NO.11

(5’-ATGAACTTCCGCGCTCTGTT-3’)

PAR downstream primer: SEQ ID NO.12

(5’-CAGTGACGCGCACGTAGAC-3’)

NbEF1 a upstream primer: SEQ ID NO.13

(5’-GTATGCCTGGGTGCTTGAC-3’)

NbEF1 a downstream primer: SEQ ID NO.14

(5’-ACAGGGACAGTTCCAATACCA-3’)

As a result: compared with the empty vector control, the tobacco leaves treated with PsAEP1 all showed significantly less lesions after inoculation with Phytophthora nicotianae, and the biomass of Phytophthora nicotianae infection was significantly reduced (FIG. 6).

Sequence listing

<110> Nanjing university of agriculture

<120> plant immune activation protein PsAEP1 and application thereof

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 960

<212> DNA

<213> Phytophthora sojae (Phytophthora sojae)

<400> 1

atggtgttct ccatcttcaa gaccgtcgcc ctggtgtcgt cgctcctggc cgccgtcggc 60

gttgccaacg ccgagctcga gaccgtcaag ctcacgcacc cgtacggctc cagcgcggag 120

gtctaccact tcggcgccca cgtcaagtcg ttccaccccg ctatggaccc aaagatggac 180

gtgctgttca tgtcgaagga gtcgttcctc gacggcgtga accccatccg tggcggcatc 240

cctgtcgtgt tccccaactt cggcagcgcc aagggcttcc cgagccacgg cttcgcccgc 300

atcaccaact ggacgctggc cagctacaag aatgctgctg acaagaacag cccgagtgtg 360

gccaagttca ccatggagtc gagcaactct acgcgtaaga tgtggcctgt tgacttcaag 420

cttgagtacg aggtgaagct gtacggcaac gagctcaaga ctgctctgcg cgtgcgcaac 480

acgaacaagg acccgattga gttccacgct ctgctgcaca actacctgtg ggtggacgac 540

gccaccaaca agggcgtgca ggtgttgggc ctgaagggcg tcgactacta cgacaaggtc 600

gccaaggtca acgccacgga gacgcgcgcg tacatcgact tcgctaacca gacggacaac 660

gtgtacagca acgcccccaa cacgctgcac gctgtgatca agggcgtcaa cgccgtggac 720

cgtacggtga cgatcaagaa gtcgggctcc atctccaacg gcagcaaggc cgtcaagacc 780

cagaccgatg ccgtcgtctg gaacccgtgg gccgaccgcg ccaaggccat ggaggacttc 840

ggcgacgagg agtacaagaa catggtcgcc gtcgagcccg gccgcgtgag cgtcaagcag 900

gccctccccg ccggccagac gtacacgctg gcggagagca tttcggtcac cacggcctaa 960

<210> 2

<211> 319

<212> PRT

<213> Phytophthora sojae (Phytophthora sojae)

<400> 2

Met Val Phe Ser Ile Phe Lys Thr Val Ala Leu Val Ser Ser Leu Leu

1 5 10 15

Ala Ala Val Gly Val Ala Asn Ala Glu Leu Glu Thr Val Lys Leu Thr

20 25 30

His Pro Tyr Gly Ser Ser Ala Glu Val Tyr His Phe Gly Ala His Val

35 40 45

Lys Ser Phe His Pro Ala Met Asp Pro Lys Met Asp Val Leu Phe Met

50 55 60

Ser Lys Glu Ser Phe Leu Asp Gly Val Asn Pro Ile Arg Gly Gly Ile

65 70 75 80

Pro Val Val Phe Pro Asn Phe Gly Ser Ala Lys Gly Phe Pro Ser His

85 90 95

Gly Phe Ala Arg Ile Thr Asn Trp Thr Leu Ala Ser Tyr Lys Asn Ala

100 105 110

Ala Asp Lys Asn Ser Pro Ser Val Ala Lys Phe Thr Met Glu Ser Ser

115 120 125

Asn Ser Thr Arg Lys Met Trp Pro Val Asp Phe Lys Leu Glu Tyr Glu

130 135 140

Val Lys Leu Tyr Gly Asn Glu Leu Lys Thr Ala Leu Arg Val Arg Asn

145 150 155 160

Thr Asn Lys Asp Pro Ile Glu Phe His Ala Leu Leu His Asn Tyr Leu

165 170 175

Trp Val Asp Asp Ala Thr Asn Lys Gly Val Gln Val Leu Gly Leu Lys

180 185 190

Gly Val Asp Tyr Tyr Asp Lys Val Ala Lys Val Asn Ala Thr Glu Thr

195 200 205

Arg Ala Tyr Ile Asp Phe Ala Asn Gln Thr Asp Asn Val Tyr Ser Asn

210 215 220

Ala Pro Asn Thr Leu His Ala Val Ile Lys Gly Val Asn Ala Val Asp

225 230 235 240

Arg Thr Val Thr Ile Lys Lys Ser Gly Ser Ile Ser Asn Gly Ser Lys

245 250 255

Ala Val Lys Thr Gln Thr Asp Ala Val Val Trp Asn Pro Trp Ala Asp

260 265 270

Arg Ala Lys Ala Met Glu Asp Phe Gly Asp Glu Glu Tyr Lys Asn Met

275 280 285

Val Ala Val Glu Pro Gly Arg Val Ser Val Lys Gln Ala Leu Pro Ala

290 295 300

Gly Gln Thr Tyr Thr Leu Ala Glu Ser Ile Ser Val Thr Thr Ala

305 310 315

<210> 3

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cagcaccagc tagcatcgat ggtgttctcc atc 33

<210> 4

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aatctctaga ggatccccgg ccgtggtgac cga 33

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaagcttacg tagaagagct cgagaccgtc 30

<210> 6

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gaattaattc gcggctcagt ggtggtggtg gtggtgggcc gtggtgaccg a 51

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gttgacgcca ttgttgag 18

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atcttcgcct cctaatgc 18

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gtatgcctgg gtgcttgac 19

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acagggacag ttccaatacc a 21

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgaacttcc gcgctctgtt 20

<210> 12

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cagtgacgcg cacgtagac 19

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gtatgcctgg gtgcttgac 19

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

acagggacag ttccaatacc a 21

Claims

1. A plant immune activation protein PsAEP1, the amino acid sequence of which is shown in SEQ ID NO.2 or the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of SEQ ID NO.2 and is related to phytophthora resistance and is derived from SEQ ID NO. 2.

2. A gene encoding the plant immune activator protein PsAEP1 of claim 1.

3. The gene according to claim 2, wherein the gene is the following (1) or (2):

(1) a nucleotide sequence shown as SEQ ID NO. 1;

(2) a nucleotide sequence which has at least more than 70 percent of homology with SEQ ID NO.1 and encodes a protein PsAEP 1; preferably a nucleotide sequence which has at least 80% or more homology with SEQ ID NO.1 and encodes the protein PsAEP 1; further preferably a nucleotide sequence which has at least 85% or more homology with SEQ ID NO.1 and encodes the protein PsAEP 1; even more preferably a nucleotide sequence which has at least 90% or more homology with SEQ ID NO.1 and encodes the protein PsAEP 1; most preferred is a nucleotide sequence which has at least 95% homology with SEQ ID NO.1 and encodes the protein PsAEP 1.

4. A recombinant vector, kit, transgenic cell line or recombinant bacterium comprising the gene of claim 2 or 3.

5. The recombinant vector, kit, transgenic cell line, or recombinant bacterium of claim 4, wherein: the recombinant vector is a eukaryotic expression vector obtained by inserting the PsAEP1 gene of claim 2 or 3 into an expression vector pPIC 9K.

6. Use of the plant immune activator protein PsAEP1 of claim 1, the gene of claim 2 or 3, or the recombinant vector, kit, transgenic cell line or recombinant bacterium of claim 4 or 5 for inducing a plant defense response or improving plant disease resistance.

7. Use according to claim 6, wherein the plant is tobacco, potato, pepper, eggplant or tomato.

8. The use of claim 6, wherein the disease resistance is resistance to Phytophthora nicotianae.

9. A method for improving phytophthora resistance in a plant, which comprises spraying the plant immune activating protein PsAEP1 of claim 1 onto the surface of the plant or injecting the plant immune activating protein PsAEP1 into the leaves of the plant.

10. The method of claim 9, wherein the plant is tobacco, potato, pepper, eggplant or tomato.