CN110894218B

CN110894218B - Plant immune activator protein SCR50 secreted by phytophthora infestans and application thereof

Info

Publication number: CN110894218B
Application number: CN201911298597.1A
Authority: CN
Inventors: 董莎萌; 王帅帅; 李兰英; 邢荣康; 王源超; 郑小波
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2022-04-29
Anticipated expiration: 2039-12-17
Also published as: CN110894218A

Abstract

The invention discloses a plant immune activator SCR50 secreted by phytophthora infestans and application thereof, wherein the plant immune activator SCR50 has a sequence shown in SEQ ID NO: 2 in the sequence table 2. The research of the invention finds that the plant immune activator protein SCR50 can be recognized by the plant immune system to generate immune response, and provides a new way for improving the plant resistance. Peptide fragment SCR50^M1Can induce plant defense reaction and provide a new material for synthesizing biological pesticide. The invention can be applied to the aspects of directionally transforming novel beneficial symbiotic bacteria, improving the disease resistance of crop breeding, biological pesticides and the like, and is expected to improve the disease resistance of plants to epidemic diseases, thereby achieving the purposes of increasing production and reducing pesticide consumption, and having wide application prospect in agricultural production.

Description

Plant immune activator protein SCR50 secreted by phytophthora infestans and application thereof

Technical Field

The invention belongs to the field of plant molecular biology and plant genetic engineering, and particularly relates to a protein secreted by phytophthora infestans and capable of stimulating plant immune activity, and a coding gene and application thereof.

Background

Phytophthora is an important group of phytopathogens, and the diseases caused by the phytophthora often cause serious harm to crops. Among them, late blight of potato caused by Phytophthora infestans (Phytophthora infestans) is a devastating disease in agricultural production, causing a great loss of crops [1,2 ]. In the middle of the 19 th century, late blight erupts in European planting areas, and potatoes are dead after two years of continuous disaster, so that hundreds of thousands of people die, one hundred and fifty thousand people live in, and the history is known as Ireland hunger \39313. At present, late blight still causes crop losses of nearly billions of dollars worldwide every year, and still is one of the major potential hazards threatening the safety of human food [3 ]. Promoting the domination of potatoes is a national strategy for guaranteeing the food safety in China, however, late blight is seriously harmful to most of potato production areas in China, and is one of the most important factors for restricting the potato yield in China [4 ].

The effector is a kind of secretory protein which is transported to plant cells by phytophthora infestans, has important functions of regulating host physiology and immune response, and is the key for analyzing disease occurrence mechanisms and plant disease resistance traits [5 ]. During the infection of plants with pathogens, plants recognize effectors secreted by pathogens using Pattern Recognition Receptors (PRRs) on the cell surface, triggering the basic immune response of the plant [6 ]. These factors capable of activating a Plant immune response are generally called Plant immune elicitors (PII), of which a very important class is Plant immune activator proteins. At present, the identification of novel plant immunity inducing protein secreted by pathogenic bacteria is the focus of attention of researchers at home and abroad.

The invention identifies a novel phytophthora infestans Small Cysteine Rich (SCR) effector protein SCR50 which can be recognized by plants so as to stimulate plant immune response. Novel plant immune activator protein secreted by phytophthora infestans is identified, and effective protein resources are provided for developing proteome biological pesticides for activating plant immunity.

Disclosure of Invention

The invention aims to provide an SCR effector molecule SCR50 secreted by phytophthora infestans, which has the activity of activating plant immunity.

Another purpose of the invention is to provide a gene sequence of the phytophthora infestans SCR effector molecule SCR 50.

The invention also aims to provide a recombinant expression vector containing the plant immune activator SCR50 encoding gene.

The invention also aims to provide application of the protein SCR50 and the coding gene thereof in activating plant immune response.

The purpose of the invention can be realized by the following technical scheme:

a phytophthora infestans secreted plant immune activator SCR50 is (a) or (b) as follows:

(a) has the sequence shown in SEQ ID NO: 2;

(b) converting SEQ ID NO: 2 and the amino acid residue sequence is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and the amino acid residue sequence is related to the function of activating the plant immune response and is expressed by SEQ ID NO: 2 derived protein.

The gene of the plant immune activator protein SCR50 is derived from phytophthora infestans. The gene is (1) or (2) as follows:

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

(2) a nucleotide sequence having at least 50% homology with SEQ ID NO. 1; preferably a nucleotide sequence having at least 70% or more homology with SEQ ID NO. 1; preferably a nucleotide sequence having at least 80% or more homology with SEQ ID NO. 1; further preferably a nucleotide sequence having at least 85% homology with SEQ ID NO. 1; more preferably a nucleotide sequence having at least 90% or more homology with SEQ ID NO. 1; most preferred is a nucleotide sequence having at least 95% or more homology with SEQ ID NO. 1.

Plant immune activator protein SCR50 single amino acid mutant peptide segment SCR50 capable of inducing plant immune response^M1The peptide segment has the sequence shown in SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof.

The plant expression vector contains a coding gene of plant immune activator protein SCR50 of PR1 signal peptide, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 3.

Plant immune activator protein SCR50 single amino acid mutant SCR50 containing PR1 signal peptide in plant expression vector^M1The nucleotide sequence of the coding gene is shown in SEQ ID NO. 4.

By utilizing the amino acid sequence of the plant immune activation protein SCR50, a PR1 signal peptide is designed and artificially added to replace the signal peptide sequence of SCR50 so as to be beneficial to expression in plants.

SCR50 containing the above gene or the above peptide fragment^M1The recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant strain of the coding gene.

The above recombinant expression vector is obtained by inserting the gene into BsaI restriction site of plant expression vector pICH31160 [7 ]. In the specific implementation process, the recombinant expression vector is a vector pICH31160:: SCR50 obtained by inserting a gene SCR50 into pICH31160:: GFP restriction enzyme cutting site BsaI.

The plant immune activator protein SCR50 and the peptide segment SCR50^M1The gene, the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant strain are applied to inducing plant defense reaction and improving plant disease resistance.

A method for inducing plant resistance reaction comprises transiently expressing the plant immune activator protein SCR50 or peptide fragment SCR50 in plant^M1。

The invention has the beneficial effects that:

the plant immune activator protein SCR50 can be recognized by the plant immune system to generate immunityThe reaction provides a new way for improving the plant resistance. Peptide fragment SCR50^M1Can induce plant defense reaction and provide a new material for synthesizing biological pesticide. The invention can be applied to the aspects of directionally transforming novel beneficial symbiotic bacteria, improving the disease resistance of crop breeding, biological pesticides and the like, and is expected to improve the disease resistance of plants to epidemic diseases, thereby achieving the purposes of increasing production and reducing pesticide consumption, and having wide application prospect in agricultural production.

Reference documents:

1.Haverkort A J,Boonekamp P M,Hutten R,et al.Societal costs of late blight in potato and prospects of durable resistance through cisgenic modification[J].Potato Research,2008,51(1): 47-57.

2.Fry W E,Birch P R,Judelson H S,et al.Five Reasons to Consider Phytophthora infestans a Reemerging Pathogen.[J].Phytopathology,2015,105(7):966.

3.Vleeshouwers,V.G.,et al.,Understanding and exploiting late blight resistance in the age of effectors.Annu Rev Phytopathol,2011.49:p.507-31.

4.Fry,W.2008.Phytophthora infestans:the plant(and R gene)destroyer.Molecular Plant Pathology,9(3):385-402.

5.Kamoun,S.,A catalogue of the effector secretome of plant pathogenic oomycetes.Annu Rev Phytopathol,2006.44:p.41-60.

6.Jones J D G&L,D.J.The plant immune system.Nature,444(7117):323-329(2006).

7.van Demmel E.J.M.,Nausch H.,Mikschofsky H.,et al.High-Level Transient Expression of ER-Targeted Human Interleukin 6in Nicotiana benthamiana.PloS one,2012.7(11):e48938.

drawings

FIG. 1 is a schematic diagram of the plant expression vector pICH31160: SCR 50;

FIG. 2 is a tobacco lamina anaphylactic reaction assay induced after injecting expression SCR50 on tobacco lamina using plant expression vector;

FIG. 3 shows the induction of plant defense-related gene expression after the expression of SCR50 was injected on tobacco leaves using plant expression vectors;

FIG. 4 is an alignment analysis of the protein sequences encoded by SCR50 and its homologous genes;

FIG. 5 shows the tobacco leaf allergy test induced by injecting expression SCR50 and its homologous protein on tobacco leaf using plant expression vector;

FIG. 6 shows that the mutant SCR50 of SCR50 induced strong necrosis reaction obtained by injecting expression screening on tobacco leaves by using plant expression vector^M1；

FIG. 7 shows an artificially synthesized peptide fragment SCR50^M1Inducing the up-regulation expression of tobacco leaf defense related genes.

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.

Example 1: cloning of the coding Gene

(1) Total RNA extraction: the method is characterized in that phytophthora infestans mycelium is used as an experimental material, an RNA extraction kit of Omega company is adopted for extracting total RNA, the operation is carried out according to the instruction, and the RNA content and the quality of the total RNA are detected by a spectrophotometer.

(2) Reverse transcription to generate the first strand: cDNA synthesis was performed using 0.7. mu.g of RNA as a template according to the instructions of the PrimeScript reverse transcription kit of Takara. Appropriate amounts of the reverse transcription products were taken for subsequent gene cloning PCR.

(3) And (3) amplifying a mature gene sequence of the SCR50 gene by taking the cDNA as a PCR template:

PCR primer amplification sequence:

an upstream primer: SEQ ID NO.5

(5’-GCTGTGTCTCCTATCTGCGA-3’)

A downstream primer: SEQ ID NO.6

(5’-TTACTCGCACCAAGTAGAACCCT-3’)

50 μ L reaction: 5 XBuffer 10. mu.L, 2.5mM dNTPs 4. mu.L, TakaraPrimerSTARTaq 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 15s, annealing at 58 deg.C for 15s, extension at 72 deg.C for 1min, circulating for 35 times, and extension at 72 deg.C for 10 min; the PCR products were recovered by agarose gel electrophoresis, Ethidium Bromide (EB) staining, recording, and cutting. The electrophoresis band was recovered with Agarose Gel DNAPURIFICATION kit (TaKaRa) (the PCR product band was the plant immune activator SCR50 gene, and was sequenced by Nanjing Kinshire corporation, the sequence is shown as SEQID NO.1, and the amino acid sequence of the encoded plant immune activator SCR50 is shown as SEQID NO. 2). The PCR product recovered from the gel cut was ligated to BsaI-digested pICH31160:: GFP vector according to the protocol described in Cloneexpress II One Step Cloning Kit (Vazyme) to obtain a ligation product containing pICH31160:: SCR 50. Wherein the pICH31160 vector is a PVX modified vector and contains BsaI enzyme cutting sites. Transforming the ligation product into Escherichia coli competent cells JM109, coating LB (containing 50. mu.g/mL of Carna) plate, culturing at 37 ℃ for 16h, then carrying out colony PCR verification, picking two positive clones, extracting plasmids according to the operation of a plasmid extraction kit (Takara), and sequencing by Nanjing Kinshire company, wherein the sequence is shown as SEQID NO. 3. The plasmid with correct sequencing is transformed into agrobacterium GV3101 by electric shock, coated with LB plate (kanamycin 50 ug/mL, rifampicin 50 ug/mL) containing corresponding antibiotics, cultured at 28 ℃ for 48h, and colony PCR is verified to pick correct clones for subsequent experiments.

As a result: plant expression vector pICH31160 SCR50 positive transformant was obtained, and the pattern diagram is shown in FIG. 1.

Example 2: detection of transient expression SCR50 in tobacco and necrosis reaction induced by homologous protein thereof

(1) Agrobacterium culture

Single colonies of Agrobacterium GV3101 containing the relevant vector were picked from the plates and inoculated into 2mL of LB liquid medium (containing kanamycin 50. mu.g/mL, rifampicin 50. mu.g/mL) on a constant temperature shaker at 28 ℃ and 200rpm overnight to an OD600 of 2.0. The overnight cultured GV3101 Agrobacterium solution was centrifuged at 5000g for 3min to collect the cells. Buffer (composition: 10 mM2- [ N-morpholino)]ethanesulfonic acid，10mM MgCl₂，200μMacetosyringone pH5.6) And (4) suspending the bacterial liquid and then centrifuging to collect the thallus. After washing was repeated 2 times, the bacterial solution was diluted with a buffer solution to a final concentration of 0.5 each.

(2) Tobacco lamina transient expression SCR50

The prepared agrobacterium tumefaciens is injected into tobacco leaves by using a 1mL syringe with a needle removed, and the tobacco after injection is cultured in a greenhouse (21-23 ℃, 16h light/8 h dark).

(3) Detection of necrotic responses

And 4-5 days after the agrobacterium is injected and expressed, observing the necrosis condition of the tobacco leaves, and collecting the leaves for photographing after an obvious phenotype appears. Tobacco leaves were placed in an ultraviolet imager (Tanon 5200 Multi), exposed to UV light at a wavelength of 365nm, photographed and processed using the staining software Tanon image. If the fluorescence color is yellow green, the result proves that the leaf has a necrosis reaction, and the stronger the fluorescence is, the stronger the necrosis degree is; if the fluorescence color is red, the result proves that the leaves have no necrosis phenomenon

As a result: SCR50 was able to induce a necrotic response in tobacco lamina compared to control EV, the results are shown in figure 2; the SCR50 homologous protein has a certain specificity in inducing necrosis, and the results are shown in fig. 5.

Example 3: transient expression of SCR50 in tobacco to induce expression of defense-related genes

(1) Agrobacterium culture

Single colonies of Agrobacterium GV3101 containing the relevant vector were picked from the plates and inoculated into 2mL of LB liquid medium (containing kanamycin 50. mu.g/mL, rifampicin 50. mu.g/mL) on a constant temperature shaker at 28 ℃ and 200rpm overnight to an OD600 of 2.0. The overnight cultured GV3101 Agrobacterium solution was centrifuged at 5000g for 3min to collect the cells. Buffer (composition: 10 mM2- [ N-morpholino)]ethanesulfonic acid，10mM MgCl ₂200. mu. Macetostyringone pH5.6) suspension of the bacterial solution, and then centrifuging and collecting the cells. After washing was repeated 2 times, the bacterial solution was diluted with a buffer solution to a final concentration of 0.5 each.

(2) Tobacco lamina transient expression SCR50

(3) Total RNA extraction

The method is characterized in that collected tobacco samples at different time points after treatment of agrobacterium are taken as experimental materials, total RNA extraction is carried out by adopting an Omega RNA extraction kit according to the instructions, and the RNA content and quality of the total RNA are detected by a spectrophotometer.

(4) Reverse transcription to generate the first strand

cDNA synthesis was performed using 0.7. mu.g of RNA as a template according to the instructions of the PrimeScript reverse transcription kit of Takara. The appropriate amount of reverse transcription product was taken for subsequent real-time quantitative PCR reaction.

(5) Real-time quantitative PCR reaction

NbCYP71D20 quantitative pre-primer: SEQ ID NO.7

(5’-ACCGCACCATGTCCTTAGAG-3’)

NbCYP71D20 quantitative rear primer: SEQ ID NO.8

(5’-CTTGCCCCTTGAGTACTTGC-3’)

NbRbohB quantitative pre-primer: SEQ ID NO.9

(5’-TCACAAGAGCTCAGGCGTTT-3’)

NbRbohB quantitative rear primer: SEQ ID NO.10

(5’-TCATCGAACCGCTTCTCGAC-3’)

The PCR reaction system contained 5uL cDNA, 10uL SYBR Premix Ex Taq II (Tli RNase H Plus), 0.4uL front and back primers, 0.4uL ROX Reference Dye II and 13.8uL water. Reaction procedure: i:95 deg.C for 30 seconds, II:95 deg.C for 5 seconds, 60 deg.C for 34 seconds, and step II should be performed for 40 cycles. The dissolution curve analysis program was: 95 degrees 15 seconds, 60 degrees 1 minute, 95 degrees 15 seconds.

As a result: compared with the control GFP, SCR50 was able to induce the up-regulated expression of genes NbCYP71D20 and NbRbohB, and the results are shown in FIG. 3.

Example 4: evolutionary tree analysis and homologous protein sequence alignment of SCR50 gene coding protein in oomycetes

(1) Extracting homologous protein of SCR gene coding protein in oomycete

The NCBI database and the software SeqHunter are used for extracting the homologous protein sequence of the PC2 encoding protein in the oomycete.

(2) Alignment of the amino acid sequences of PC2 homologous proteins alignment was performed using the biological software BioEdit (version number 7.2.5).

As a result: the sequence alignment of SCR50 homologous proteins is shown in figure 4.

Example 5: SCR50^M1Inducing plant immunity

(1) Transient expression vector pICH31160 SCR50^M1Construction of

SCR50 containing PR1 signal peptide is constructed by taking plasmid pICH31160 (shown in the specification) SCR50 as a template^M1The sequence of the transient expression vector is shown as SEQID NO. 4. The constructed vector is transferred into agrobacterium GV3101 and positive transformant is selected. And (4) carrying out agrobacterium culture, collecting strains and preparing agrobacterium buffer solution.

(2) Tobacco lamina transient expression SCR50^M1

The prepared Agrobacterium buffer (OD ═ 0.5) was injected into tobacco leaf blades using a 1mL syringe with a needle removed, and the tobacco was incubated in a greenhouse (21-23 ℃ C., 16h light/8 h dark) after injection.

(2) Detection of cellular necrosis

And 4-5 days after the agrobacterium is injected and expressed, observing the necrosis condition of the tobacco leaves, and collecting the leaves for photographing after an obvious phenotype appears. Tobacco leaves were placed in an ultraviolet imager (Tanon 5200 Multi), exposed to UV light at a wavelength of 365nm, photographed and processed using the staining software Tanon image. If the fluorescence color is yellow green, the result proves that the leaf has a necrosis reaction, and the stronger the fluorescence is, the stronger the necrosis degree is; if the fluorescence color is red, the leaf is proved to have no necrosis phenomenon.

As a result: SCR50^M1The plant immune response was induced and the necrosis intensity was significantly stronger than SCR50, the results are shown in fig. 6.

Example 6: artificially synthesized SCR50^M1Up-regulated expression of peptide fragment induced tobacco leaf defense related gene

(1) Peptide fragment synthesis

The peptide fragment is synthesized by biological companies, and the amino acid sequence is shown as SEQID NO. 11.

(2) Peptide-treated tobacco lamina

Peptide fragment concentration was configured at 100 nM. Injecting the prepared peptide segment and DMSO contrast into tobacco leaf with syringe with needle removed, and culturing the tobacco in greenhouse (21-23 deg.C, 16h light/8 h dark). Samples were collected at different time points.

(3) Total RNA extraction

Samples of tobacco processed by peptide fragments at different time points are taken as materials, the extraction of total RNA is carried out by adopting an RNA extraction kit of Omega company according to the instructions, and the RNA content and quality are detected by a spectrophotometer.

(4) 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 appropriate amount of reverse transcription product was taken for subsequent real-time quantitative PCR reaction.

(5) Real-time quantitative PCR reaction

NbRbohB quantitative pre-primer: SEQ ID NO.9

(5’-TCACAAGAGCTCAGGCGTTT-3’)

NbRbohB quantitative rear primer: SEQ ID NO.10

(5’-TCATCGAACCGCTTCTCGAC-3’)

As a result: SCR50 compared to control DMSO^M1The peptide fragment can induce the up-regulated expression of the gene NbRbohB, and the result is shown in FIG. 7.

Full-length nucleotide sequence of SCR50 gene: SEQ ID NO.1

ATGTTCAAGTCGTCGATCATCCTCAGTTTCGCCGTCGCTGTCCTCCTGGCTTTCTCCTCTG CCGACGCTGCCGTTAGCCCCATATGCGACCTCGATTGTGACGGGGGTCAAACGTGCCACA TGGGCGATGAAGGATCCACGTGGTGCGAGTAA

The SCR50 gene encodes the full-length protein sequence: SEQ ID NO.2

MFKSSIILSFAVAVLLAFSSADAAVSPICDLDCDGGQTCHMGDEGSTWCE*

SCR50 plant expression nucleotide sequence: SEQ ID NO.3

ATGGGATTTGTTCTCTTTTCACAATTGCCTTCATTTCTTCTTGTCTCTACACTTCTCTTATTC CTAGTAATATCCCACTCTTGCCGTGCCAGGTCAGCTGTGTCTCCTATCTGCGACCTGGACT GCGACGGTGGTCAGACTTGCCACATGGGTGACGAGGGTTCTACTTGGTGCGAGTAA

SCR50^M1Plant expression of the nucleotide sequence: SEQ ID NO.4

ATGGGATTTGTTCTCTTTTCACAATTGCCTTCATTTCTTCTTGTCTCTACACTTCTCTTATTC CTAGTAATATCCCACTCTTGCCGTGCCAGGTCAGCTGTGTCTCCTATCGCTGACCTGGACT GCGACGGTGGTCAGACTTGCCACATGGGTGACGAGGGTTCTACTTGGTGCGAGTAA

Artificially synthesized SCR50^M1Peptide amino acid sequence: SEQ ID NO.11

AVSPIADLDCDGGQTCHMGDEGSTWCE*。

Sequence listing

<110> Nanjing university of agriculture

<120> phytophthora infestans secreted plant immune activation protein SCR50 and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 153

<212> DNA

<213> Phytophthora infestans (Phytophthora infestans)

<400> 1

atgttcaagt cgtcgatcat cctcagtttc gccgtcgctg tcctcctggc tttctcctct 60

gccgacgctg ccgttagccc catatgcgac ctcgattgtg acgggggtca aacgtgccac 120

atgggcgatg aaggatccac gtggtgcgag taa 153

<210> 2

<211> 50

<212> PRT

<213> Phytophthora infestans (Phytophthora infestans)

<400> 2

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

1 5 10 15

Ala Phe Ser Ser Ala Asp Ala Ala Val Ser Pro Ile Cys Asp Leu Asp

20 25 30

Cys Asp Gly Gly Gln Thr Cys His Met Gly Asp Glu Gly Ser Thr Trp

35 40 45

Cys Glu

50

<210> 3

<211> 180

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgggatttg ttctcttttc acaattgcct tcatttcttc ttgtctctac acttctctta 60

ttcctagtaa tatcccactc ttgccgtgcc aggtcagctg tgtctcctat ctgcgacctg 120

gactgcgacg gtggtcagac ttgccacatg ggtgacgagg gttctacttg gtgcgagtaa 180

<210> 4

<211> 180

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgggatttg ttctcttttc acaattgcct tcatttcttc ttgtctctac acttctctta 60

ttcctagtaa tatcccactc ttgccgtgcc aggtcagctg tgtctcctat cgctgacctg 120

gactgcgacg gtggtcagac ttgccacatg ggtgacgagg gttctacttg gtgcgagtaa 180

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gctgtgtctc ctatctgcga 20

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttactcgcac caagtagaac cct 23

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

accgcaccat gtccttagag 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cttgcccctt gagtacttgc 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tcacaagagc tcaggcgttt 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcatcgaacc gcttctcgac 20

<210> 11

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Ala Val Ser Pro Ile Ala Asp Leu Asp Cys Asp Gly Gly Gln Thr Cys

1 5 10 15

His Met Gly Asp Glu Gly Ser Thr Trp Cys Glu

20 25

Claims

1. Plant immune activator protein SCR50 single amino acid mutant peptide segment SCR50 capable of inducing plant immune response^M1The peptide is characterized in that the amino acid sequence of the peptide segment is shown as SEQ ID NO: shown at 11.

2. The coding gene of the plant immune activator protein SCR50 containing PR1 signal peptide has the nucleotide sequence shown in SEQ ID No. 3.

3. Plant immune activator protein SCR50 single amino acid mutant SCR50 containing PR1 signal peptide^M1The nucleotide sequence of the coding gene is shown in SEQ ID NO. 4.

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

5. The recombinant expression vector, expression cassette, transgenic cell line or recombinant strain of claim 4, wherein the recombinant expression vector is obtained by inserting the encoding gene into BsaI cleavage site of plant expression vector pICH 31160.

6. The plant immune activator protein SCR50 secreted by Phytophthora infestans shown in SEQ ID No.2 is used for inducing plant defense reaction and improving plant disease resistance.

7. The gene of the coding plant immune activation protein SCR50 shown in SEQ ID NO.1, or the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium containing the gene of the coding plant immune activation protein SCR50 shown in SEQ ID NO.1 are applied to the induction of plant defense reaction and the improvement of plant disease resistance.

8. The peptide fragment SCR50 of claim 1^M1The coding gene according to claim 2 or 3, or the recombination according to claim 4 or 5The expression vector, the expression cassette, the transgenic cell line or the recombinant bacterium are applied to inducing plant defense reaction and improving plant disease resistance.

9. A method for inducing plant resistance response, characterized in that a plant immune activator protein SCR50 shown in SEQ ID No.2 is transiently expressed in a plant.