CN112293415B

CN112293415B - Application of phytophthora capsici effector factor RxLR23 in promoting plant growth

Info

Publication number: CN112293415B
Application number: CN202011058180.0A
Authority: CN
Inventors: 张修国; 朱春原; 艾聪聪; 杨灿灿; 盛慧
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2021-08-24
Anticipated expiration: 2040-09-29
Also published as: CN112293415A

Abstract

The invention relates to the field of molecular biology, and discloses application of a phytophthora capsici effector RxLR23 in promoting plant growth. A new RxLR23 effector factor is cloned and identified in phytophthora capsici strain SD33, an RxLR23 escherichia coli high-efficiency expression strain is prepared by means of a protein expression technology, an RxLR23 protein solution is further obtained, the performance of promoting the growth and vigor of roots of hot pepper, cucumber and tomato seedlings of the RxLR23 protein solution is further proved, and the preparation and function technology of the RxLR23 protein facilitates the development of a vegetable root-strengthening and seedling-strengthening protein preparation technology through later-stage transformation.

Description

Application of phytophthora capsici effector factor RxLR23 in promoting plant growth

Technical Field

The invention relates to the field of molecular biology, in particular to application of a phytophthora capsici effector RxLR23 in promoting plant growth.

Background

During the process of infecting plant hosts by plant pathogenic bacteria, RxLR effector factors are often secreted and combined with host target proteins to interfere the immune system of the plants and promote the infection and the pathogenicity of the pathogenic bacteria, but host plants also correspondingly evolve a defense system to recognize and prevent the infection and the pathogenicity of the pathogenic bacteria, so that the expression of plant resistance proteins, the burst of active oxygen, the deposition of callose and even the generation of allergic necrosis reaction (HR) are stimulated, and the expansion of pathogenic oomycetes is inhibited, therefore, the RxLR effector factors are often called as immune effector factors.

In recent years, researches show that important plant pathogenic oomycetes such as Phytophthora capsici (Phytophthora capsicii), potato Phytophthora infestans (Phytophthora infestans), soybean Phytophthora sojae (sojae) and Phytophthora nicotianae (p.parasitica) contain a large amount of RxLR effector genes, and some RxLR effector genes have immunological properties. Therefore, the immune characteristic of the RxLR effector is correctly identified, and the method has important significance for researching the pathogenic mechanism of the RxLR effector and preventing and treating plant diseases.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of phytophthora capsici effector RxLR23 in promoting the root development and growth performance of crop seedlings.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

the invention provides a phytophthora capsici effector factor RxLR23 and application of a prokaryotic or eukaryotic expression system for expressing the phytophthora capsici effector factor RxLR23 in promoting plant growth.

The phytophthora capsici effector factor RxLR23 is cloned from phytophthora capsici strain SD33, and the amino acid sequence of the phytophthora capsici effector factor RxLR23 is shown in SEQ ID NO 2.

Preferably, the application comprises: preparing the phytophthora capsici effector RxLR23 into a protein solution, and applying the protein solution to a seedling culture substrate; or applying the escherichia coli liquid expressing the phytophthora capsici effector RxLR23 to a seedling culture substrate.

Alternatively, preferably, the application comprises: preparing the phytophthora capsici effector RxLR23 into a protein solution, and soaking the protein solution in seed; or the Escherichia coli liquid expressing the phytophthora capsici effector factor RxLR23 is subjected to seed soaking treatment

Alternatively, preferably, the application comprises: preparing the phytophthora capsici effector RxLR23 into a protein solution, and performing root irrigation treatment on plants by using the protein solution; or the escherichia coli liquid expressing the phytophthora capsici effector RxLR23 is subjected to root irrigation treatment on the plant.

Preferably, the application further comprises diluting the protein solution or the escherichia coli bacterial liquid.

Preferably, the Escherichia coli expressing the phytophthora capsici effector RxLR23 is obtained by introducing the phytophthora capsici effector RxLR23 encoding gene into Escherichia coli competent cells through plasmids.

Preferably, the plasmid is pET28a, and/or the e.coli competent cell is DH5 α.

The second aspect of the invention provides a phytophthora capsici effector RxLR23 and application of a prokaryotic or eukaryotic expression system for expressing the phytophthora capsici effector RxLR23 in preparation of a plant growth promoter.

The third aspect of the invention provides an application of a phytophthora capsici effector RxLR23 and a prokaryotic or eukaryotic expression system for expressing the phytophthora capsici effector RxLR23 in inhibiting host plant infection by phytophthora capsici.

Preferably, the plants include capsicum, cucumber and tomato.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the invention clones and identifies a new RxLR23 effector in phytophthora capsici strain SD33, prepares an RxLR23 escherichia coli high-efficiency expression strain by virtue of a protein expression technology, further obtains an RxLR23 protein suspension, further proves that the RxLR23 protein has the performance of promoting the growth and vigor of roots of hot pepper, cucumber and tomato seedlings, and is expected to be developed into a novel protein preparation for strengthening the roots and seedlings of vegetables.

Drawings

FIG. 1 is a map of pET28a vector.

FIG. 2 shows the PCR amplification result of the RxLR23 gene in example 1 of the present invention, wherein M: DNAmarker, 1-4: DNAPCR amplification of the RxLR23 gene band.

FIG. 3 is the result of SDS-PAGE gel analysis of the expression of RxLR23 in examples of the present invention, wherein M: protein marker; CK: comparison; 1-4: IPTG induces RxLR23 protein expression.

FIG. 4 shows the results of the test that the RxLR23 protein promotes the development of root system of pepper seedlings in the examples.

FIG. 5 shows the results of the test of the RxLR23 protein promoting the growth of pepper seedlings in the examples.

FIG. 6 shows the results of the test that the RxLR23 protein promotes root development of cucumber seedlings in the examples.

FIG. 7 shows the results of the test that the RxLR23 protein promotes the growth of cucumber seedlings in the examples.

FIG. 8 shows the results of the experiments in examples in which RxLR23 protein promoted root development in tomato seedlings at the 5-leaf stage.

FIG. 9 shows the results of the experiments in the examples that RxLR23 protein promotes root development of tomato seedlings at 6-leaf stage.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 cloning and expression of Phytophthora capsici Effector RxLR23 Gene

1. Phytophthora capsici effector RxLR23 gene information prediction

By means of bioinformatics DNAMAN software, the phytophthora capsici whole genome (https:// genome. jgi. doe. gov/portal /) was analyzed and compared, and 1 important RxLR effector factor was screened, wherein the effector factor is named as RxLR23 effector factor, the size of the encoding gene is 1155 bp (containing 54bp of signal peptide and SEQ ID NO:1), 366 amino acid residues (SEQ ID NO:2) are contained after the signal peptide is removed, and the molecular weight is 41kD, and the pI is 9.47.

SignalP 4.0Server (http:// www.cbs.dtu.dk/services/SignalP /) was used to predict signal peptide, which has a signal peptide region of 1-54bp and no transmembrane region.

2. Phytophthora capsici effector RxLR23 gene clone sequencing

2.1 strains of Phytophthora capsici

The highly pathogenic phytophthora capsici strain SD33 is provided by vegetable pest biology focus laboratory of Shandong university of agriculture.

2.2 RNA extraction and reverse transcription of cDNA from the strain of Phytophthora capsici SD33

The strong pathogenic strain SD33 stored in the laboratory was cultured using V8 plates in a constant temperature incubator at 28 ℃.

RNA extraction is carried out on the strong pathogenic strain SD33, and the steps are as follows:

1) grinding a sample: grinding phytophthora capsici SD33 mycelia into powder by using a mortar precooled by liquid nitrogen;

2) homogenizing: taking 1g of hypha grinding powder, adding 10mL of Trizol, homogenizing for 2min by an electric vortex instrument, standing for 3-5min at room temperature to crack fully;

3) centrifuging at 4 deg.C and 12000rpm for 10min, sucking supernatant, and removing precipitate;

4) adding 200 mu L chloroform/mLTrizol into chloroform, shaking and mixing uniformly (the vortex shaking instrument is forbidden), standing for 15min at 25 ℃, and centrifuging for 15min at the temperature of 4 ℃ and the rotation speed of 13000 rpm;

5) absorbing the upper water phase into a new centrifugal tube, discarding the lower phenol phase without absorbing the middle interface, wherein the phenol phase is used for extracting protein;

6) adding 500 mu L of isopropanol into 1ml of the obtained mixture, inverting the mixture, and standing the mixture at room temperature for 5-10 min;

7) centrifuging at 4 deg.C and 12000rpm for 10min, and removing supernatant;

8) adding anhydrous ethanol according to the proportion of 1mL of 75% ethanol/mL of DEPC, wherein the volume ratio of the ethanol to the DEPC is 3:4, gently inverting, centrifuging at the temperature of 4 ℃ and the rotation speed of 8000rpm for 5min, and removing supernatant as much as possible;

9) standing at room temperature, air drying or oven drying for 5-10min, removing ethanol, and preventing RNA sample from drying excessively or dissolving;

10) using 50 μ L H₂O, TE buffer solution or 0.5% SDS, DEPC treatment of the solvent and autoclaving at 121 ℃ for 30 min;

11) OD measurement using ultraviolet spectrophotometry₆₀₀、A₂₆₀/A₂₈₀1.8～2.0，A₂₆₀/A₂₃₀1.8-2.2, which indicates that the pollution is negligible and the RNA purity meets the experimental requirements.

Synthesizing cDNA by reverse transcription of RNA, and the process is as follows:

1) RNA reverse transcription is carried out on the RNA, an RNase removing centrifugal tube is prepared into a PCR system, and the preparation system is shown in table 1.

TABLE 1 RT-PCR reaction System

Composition (I)	Volume (μ L)
		2.5mmol/LdNTP	4μL
10×RTmix	4μL
		RNA(0.5μg/μL)	8μL
QuantReverseTranscriptase	2μL
		RNaseFreeddH₂O	18μL
Primers (Primers)	4μL
		Total of	40μL

Sucking, beating and mixing after mixing, centrifuging and placing on ice.

2) Incubating at 50 deg.C for 15min, incubating at 85 deg.C for 2min, and immediately subjecting the obtained product to PCR reaction, or storing at-20 deg.C for half a year; the long-term preservation is recommended to be carried out after subpackaging and then storing in a freezer at the temperature of-80 ℃, and repeated freeze thawing of cDNA is avoided.

2.3RxLR23 Gene PCR cloning primer design

A pair of specific primers is designed according to the RxLR23 gene sequence (signal peptide sequence) in the phytophthora capsici whole genome (https:// genome. jgi. doe. gov/portal /) and two enzyme cutting sites NcoI and XhoI of a recombinant vector pET28a (Novagen):

the upstream primer RxLR 23F:

5′-CATGCCATGGATGGAAGTCTCAGCTGGCAAG-3′

downstream primer RxLR 23R:

5′-CCGCTCGAGCGCCGCCGCCTTATACTTAT TG-3′。

for later purification of RxLR23 protein, the C-terminal his tag of pET28a vector was ensured to translate normally, and the stop codon of RxLR23 was removed. Wherein the pair of specific primers is synthesized by Qingdao Kangchi Biotechnology Limited. The map of the PET28a vector is shown in FIG. 1.

2.4 purpose RxLR23 gene PCR amplification

The target fragment was amplified from the cDNA by polymerase chain reaction, the PCR reaction system is shown in Table 2.

TABLE 2RxLR23 Gene PCR amplification System

The PCR amplification product is shown by agarose gel electrophoresis, the length of the amplification band is about 1100bp, and the size of the amplification band is basically consistent with that of the target gene band. The PCR amplified target gene fragment is recovered by using a gel recovery kit (DNA recovery kit), and the recovered product can be stored for later use at the temperature of minus 20 ℃ for a short time. The PCR amplification result of the RxLR23 gene of interest is shown in fig. 2, where M: DNAmarker, 1-4: DNAPCR amplification of the RxLR23 gene band.

3. Recombinant vector construction

3.1 double digestion of target Gene and vector

The RxLR23 gene PCR amplification gel recovery product and the vector pET28a are subjected to double enzyme digestion by enzyme 1(NcoI) and enzyme 2(XhoI), and are subjected to water bath at 37 ℃ for 2-3 h. The double digestion reaction system is shown in Table 3. Recovering the RxLR23 and the pET-28a after enzyme digestion by using a glue recovery kit,

TABLE 3 double digestion reaction System for RxLR23 and vector pET28a

RxLR23 double enzyme cutting reagent component	Volume (μ L)	pET-28a double enzyme digestion reagent component	Volume (μ L)
				DNA fragment of target gene	5	pET-28a plasmid	5
10 Xbuffer	4	10 Xbuffer	4
				Enzyme 1(NcoI)	2	Enzyme 1(NcoI)	2
Enzyme 2(XhoI)	2	Enzyme 2(XhoI)	2
				ddH₂O	27	ddH₂O	27
Total of	40	Total of	40

3.2 connection of the Gene of interest RxLR23 to the vector pET28a

The digested RxLR23 gene DNA fragment and the vector pET28a (Novagen) were recovered and ligated with Solution I at 16 ℃ for 3 hours, the ligation system is shown in Table 4.

TABLE 4 reaction System for RxLR23 ligation to vector pET-28a

Composition (I)	Volume (μ L)
		Enzyme-digested fragment of gene	7.5
Vector pET28a	2.5
		SolutionI	10
Total of	20

3.3 transformation of the pET28a recombinant vector into E.coli DH 5. alpha. competent vectors

1) Melting 50 μ L DH5 α competent cells in ice bath, adding ligation product, mixing gently, and ice-cooling for 30 min;

2) thermally shocking for 90s in a water bath at 42 ℃, and then rapidly carrying out ice-bath on the centrifugal tube for 2 min;

3) adding 500 mu L of sterile LB culture medium (without antibiotics) into each centrifuge tube on a super clean bench, uniformly mixing, placing at 37 ℃, performing shaking culture at 200rpm for 45-60 min, and ensuring that host bacteria are completely recovered;

4) centrifuging for 1min at 8000rpm of a normal temperature centrifuge, discarding part of supernatant, resuspending with a pipette thallus, uniformly mixing, coating on an LB agar culture medium (containing corresponding antibiotics), and then performing inverted culture in an incubator at 37 ℃ for 12-16 h.

3.4 identification of the recombinant vector pET28a

After bacterial plaque grows on the plate by the prepared competent cells, adding 1mL of sterile LB liquid medium (containing corresponding antibiotics) into a 1.5mL centrifuge tube, selecting the grown single bacterial colony, placing the single bacterial colony in the LB liquid medium centrifuge tube, performing shake culture at 37 ℃ for 5-6 h to serve as a bacterial liquid PCR template, and performing bacterial liquid PCR identification at proper time, wherein a bacterial liquid PCR reaction system is shown in Table 5.

TABLE 5 PCR reaction system for recombinant vector pET28a bacterial liquid

Composition (I)	Volume (μ L)
		Upstream primer (10. mu.M)	2
Downstream primer (10. mu.M)	2
		Fungus liquid template	3
High fidelity MIX	25
		ddH₂O	18
Total amount of	50

And (3) carrying out agarose gel electrophoresis identification on the bacteria liquid PCR reaction sample, taking the bacteria liquid with the PCR amplification result as a positive sample, taking 500 mu L of the bacteria liquid with the positive sample, sending the bacteria liquid to Qingdao Titanxi biotechnology Limited company for synthesis sequencing, comparing and analyzing the sequencing result with a genome sequence by using DNAman software, taking 500 mu L of the bacteria liquid with the correct sequencing result, adding 500 mu L of 50% sterilized glycerol, storing the bacteria liquid in a freezer at the temperature of-20 ℃, taking another bacteria liquid with the correct sequencing result, extracting plasmids, and storing the extracted plasmids in a freezer at the temperature of-20 ℃ for later use, wherein the plasmid extraction is carried out according to the instruction of the CWBIO high-purity plasmid small-extraction kit.

RxLR23 recombinant protein expression

4.1RxLR23 recombinant protein test expression

1) The pET28a recombinant plasmid with correct sequencing is transformed into an E.coli Rosetta (DE3) strain, centrifuged for 1min at 8000rpm of a normal-temperature centrifuge, a part of supernatant is discarded, a pipettor is used for re-suspending, the mixture is evenly mixed and then coated on an LB agar plate culture medium (containing corresponding antibiotics), and then the inverted culture is carried out for 12 to 16 hours in an incubator at 37 ℃.

2) Selecting a single bacterial plaque, inoculating the bacterial plaque into a centrifugal tube filled with 1.5mL LB liquid medium (added with Kan resistance), and carrying out shaking culture at 37 ℃ and 180rpm for 6 h;

3) inoculating 1mL of shake culture solution into LB liquid culture medium (adding Kan resistance) containing 1.5mL, and culturing to OD₆₀₀Taking 1mL of bacterial liquid as a control before induction, wherein the bacterial liquid is 0.6-0.8;

4) adding Inducer (IPTG) with different concentrations into the induced bacteria liquid and the control bacteria liquid respectively, and inducing for 3h at 37 ℃ and 180 rpm;

5) after induction, 1mL of bacterial liquid is taken respectively, and is centrifuged for 1min at 12000rpm simultaneously with the control; discarding the supernatant, adding 40 μ L of 2 × Binding Buffer respectively, resuspending and mixing, then adding 40 μ L of 2 × adding Buffer (Loading Buffer), and mixing;

6) boiling the sample for 15min, oscillating for 1 time at 5min intervals, oscillating for 2 times, and centrifuging at 12000rpm for 1min before SDS-PAGE electrophoresis;

7) taking 20 mu L of sample to carry out SDS-PAGE electrophoresis, observing the expression condition of the target protein, and determining that the appropriate concentration of an Inducer (IPTG) is 0.5 mM;

8) taking 500 mu L of the expression target protein bacterial liquid, adding 500 mu L of 50% sterilized glycerol, mixing uniformly, filling into a sterilized freezing storage tube, and storing in a freezer at the temperature of-20 ℃.

And then detecting the expression condition of the target protein by using SDS-PAGE electrophoresis technology, wherein SDS-PAGE electrophoresis results show that, compared with a control, after pET-28a-RxLR23 protein is induced by a proper concentration Inducer (IPTG), RxLR23 is induced to express a protein with a molecular weight of 41KDa (signal peptide is removed), the molecular weight of the protein is consistent with the predicted molecular weight of PcRxLR23, so that the pET28a-RxLR23 protein prokaryotic expression system is successfully constructed, and the results are shown in FIG. 3, wherein M: proteinmarker (protein marker); 1-4: IPTG induces RxLR23 protein expression.

4.2 Large-Scale expression of RxLR23 fusion protein

1) Activating the preserved strain with high expression level, inoculating to 1L LB liquid culture medium containing 50mg/mL Kan at a ratio of 1:100, and shake-culturing at 37 deg.C and 180rpm for 3-4 h to make OD₆₀₀＝0.6～0.8；

2) The temperature of the shaker was lowered to 16 ℃ in advance, and an Inducer (IPTG) was added to the shaker to a final concentration of 1mM, 16 ℃Inducing at 120rpm overnight for 16-21 h under the condition to ensure OD₆₀₀The value is 1.8-2.0, and the OD value is monitored by using an ultraviolet spectrophotometer when the induction time reaches 16h (the OD value represents the optical density absorbed by the detected protein, 1OD escherichia coli is approximately equal to 10)⁸-10⁹Cells/ml) were monitored 1 time every 1 h.

Example 2 application of Phytophthora capsici effector RxLR23 in promoting plant growth

Application of RxLR23 protein in promotion of root development and seedling growth of pepper, cucumber and tomato seedlings

1) OD was monitored by induction of large amounts of bacterial suspensions containing RxLR23 protein according to the method described in example 1₆₀₀The value is about 1.8-2.0, and the product is stored overnight at 4 ℃ and is used for seedling pre-treatment of pepper, cucumber and tomato seeds.

2) OD reduction with sterile Water₆₀₀Diluting RxLR23 protein bacteria suspension with a value of 1.8-2.0 by 2-3 times, before seedling raising of pepper, cucumber and tomato seeds, applying the diluent to a seedling raising substrate (wood chips are used as a main substrate, a mixture of dried chicken manure and fermented dried pig manure with a mass ratio of 1:1 is used as an organic fertilizer source, setting 5 treatments according to the volume ratio, and adding N0.3g/kg of nutrients and P₂O₅0.5 g/kg and K₂O1.5 g/kg quick-acting chemical fertilizer), the amount of the diluent of the bacterial suspension is that the seedling substrate is 100ml/100kg, the components of the seedling substrate are used as the Control (CK) after the bacterial liquid of DH5 alpha which is transformed with pET28a empty vector is induced by IPTG.

2RxLR23 protein solution for promoting root development and growth of pepper, cucumber and tomato seedlings

1) Analysis of results of RxLR23 protein on promotion of root development and growth of pepper seedlings

As shown in FIG. 4, in the 5-leaf stage of the pepper seedlings treated with RxLR23 protein, the average length of the young root was 4.6cm (30 seedling root systems were counted), while in the 5-leaf stage of the pepper seedlings treated with the control CK (LB medium), the average length of the young root was 3.1cm (30 seedling root systems were counted), and the average length of the former was increased by 1.5cm compared with that of the latter.

As shown in FIG. 5, the average height of the seedlings at the 5-leaf stage of the pepper seedlings treated with RxLR23 protein was 14.5cm (30 statistical seedlings), while the average height of the seedlings at the 5-leaf stage of the pepper seedlings treated with control CK (LB medium) was 11.5cm (30 statistical seedlings), which was increased by 3cm compared with the average height of the roots of the latter. Therefore, the RxLR23 protein can obviously promote the development and growth of the root system of the pepper seedlings.

2) Analysis of root system development and growth conditions of cucumber seedlings by RxLR23 protein

As shown in FIG. 6, in the 5-leaf stage of cucumber seedlings treated with RxLR23 protein, the average length of the rootlet was 4.5cm (30 seedling roots were counted), while in the 5-leaf stage of cucumber seedlings treated with control CK (LB medium), the average length of the rootlet was 2.5cm (30 seedling roots were counted), and the average length of the rootlet was increased by 2cm in the former compared with the average length of the rootlet in the latter.

As shown in FIG. 7, the average height of seedlings at 6 leaf stage of cucumber seedlings treated with RxLR23 protein was 15.5cm (30 statistical seedlings), while the average height of seedlings at 6 leaf stage of cucumber seedlings treated with control CK (LB medium) was 13.5cm (30 statistical seedlings), which was increased by 2cm compared with the average height of roots. Therefore, the RxLR23 protein can obviously promote the root development and growth of cucumber seedlings.

3) Analysis of results of development and growth of tomato seedling root system by RxLR23 protein

As shown in FIG. 8, in the 5-leaf stage of tomato seedlings treated with RxLR23 protein, the average length of the young root was 3.5cm (30 seedling roots were counted), while in the 5-leaf stage of tomato seedlings treated with control CK (LB medium), the average length of the young root was 2.0cm (30 seedling roots were counted), and the average length of the former was increased by 1.5cm compared with the latter.

As shown in FIG. 9, the average height of seedlings at 6-leaf stage of tomato seedlings treated with RxLR23 protein was 12.5cm (30 statistical seedlings), while the average height of seedlings at 6-leaf stage of tomato seedlings treated with control CK (LB medium) was 9.5cm (30 statistical seedlings), which was increased by 3cm compared to the average height of the latter seedlings. Therefore, the RxLR23 protein can obviously promote the development and growth of tomato seedling root systems.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Shandong university of agriculture

<120> application of phytophthora capsici effector factor RxLR23 in promoting plant growth

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<170> SIPOSequenceListing 1.0

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<213> Phytophthora capsici (Phytophthora capsicii)

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atgcgtcttc attatatcgt tgtgctcgcg gtcattgcct tcgccacgaa cgggaatgaa 60

gtctcagctg gcaagtcccg tgtcgccata actactactg gtgcacttga cacccccaca 120

accagactct tgaggaccca gtacacggac gaagagaggg cgttcggcct caatcttctc 180

cctggaagca agaaaatctc aagtatcata acaaacaaga aactgtccaa gtatctcaag 240

agcaaccaag aattcgacga cgtgttcatc aaactcaagc tcgacaaggc cggagacaag 300

ttgttcgaga acccgaaatt cctcgcttgg gctcaatacg tggacgattt caatcagaaa 360

caccagaccc agaactcgat gctccccacg cttgtgcgac agtttggagg cgatgatctg 420

tcgattatgt tggaaaaggc caagcaggca gacaaaacct acggggtggc gttgagactt 480

cagggcgaac agatgaaact ctggagacgt gaaggtctca ctactgacat gctcttcaaa 540

atctacaaat tagatgatgg ggctacgaat ctgctggaaa acccaggcat caaaatttgg 600

atgaggtacg cagacgaact tttccctgga gactccacac ttctcttcaa gaagctgcaa 660

aagacgtatt cggacgaggc gttatccaaa atcttgatca acgggaaaac agtcgcaagt 720

acggagaagt tggcgtcgga cttgcagaac cagcaacttc gttattggtt gaaggatctt 780

gtgcctccag agaaggcctt ccagctgctg tcactcaaca agggggcgga cgatgtgttt 840

ggtagtcccc aactgcagac gtggattcgg tacaatgcag cttacgccaa gcagaatccg 900

tacgctcaca aggcgacgct gatcgatacg ctcctggaga atttcgacac tgccgctatg 960

gtcaaaatgc tcaaaacgag gccgaataca gcctacggca agcatttggc tggtggggtg 1020

gaacgtgatc tcatcaaaag gtgggttacg gacggaaaac cgctcaaatt tgttgtcgag 1080

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aaggcggcgg cgtag 1155

<210> 2

<211> 366

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<213> Phytophthora capsici (Phytophthora capsicii)

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Met Gly Val Ser Ala Gly Leu Ser Ala Val Ala Ile Thr Thr Thr Gly

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Ala Leu Ala Thr Pro Thr Thr Ala Leu Leu Ala Thr Gly Thr Thr Ala

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Gly Gly Ala Ala Pro Gly Leu Ala Leu Leu Pro Gly Ser Leu Leu Ile

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Ser Ser Ile Ile Thr Ala Leu Leu Leu Ser Leu Thr Leu Leu Ser Ala

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Gly Gly Pro Ala Ala Val Pro Ile Leu Leu Leu Leu Ala Leu Ala Gly

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Ala Leu Leu Pro Gly Ala Pro Leu Pro Leu Ala Thr Ala Gly Thr Val

85 90 95

Ala Ala Pro Ala Gly Leu His Gly Thr Gly Ala Ser Met Leu Pro Thr

100 105 110

Leu Val Ala Gly Pro Gly Gly Ala Ala Leu Ser Ile Met Leu Gly Leu

115 120 125

Ala Leu Gly Ala Ala Leu Thr Thr Gly Val Ala Leu Ala Leu Gly Gly

130 135 140

Gly Gly Met Leu Leu Thr Ala Ala Gly Gly Leu Thr Thr Ala Met Leu

145 150 155 160

Pro Leu Ile Thr Leu Leu Ala Ala Gly Ala Thr Ala Leu Leu Gly Ala

165 170 175

Pro Gly Ile Leu Ile Thr Met Ala Thr Ala Ala Gly Leu Pro Pro Gly

180 185 190

Ala Ser Thr Leu Leu Pro Leu Leu Leu Gly Leu Thr Thr Ser Ala Gly

195 200 205

Ala Leu Ser Leu Ile Leu Ile Ala Gly Leu Thr Val Ala Ser Thr Gly

210 215 220

Leu Leu Ala Ser Ala Leu Gly Ala Gly Gly Leu Ala Thr Thr Leu Leu

225 230 235 240

Ala Leu Val Pro Pro Gly Leu Ala Pro Gly Leu Leu Ser Leu Ala Leu

245 250 255

Gly Ala Ala Ala Val Pro Gly Ser Pro Gly Leu Gly Thr Thr Ile Ala

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Thr Ala Ala Ala Thr Ala Leu Gly Ala Pro Thr Ala His Leu Ala Thr

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Leu Ile Ala Thr Leu Leu Gly Ala Pro Ala Thr Ala Ala Met Val Leu

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Met Leu Leu Thr Ala Pro Ala Thr Ala Thr Gly Leu His Leu Ala Gly

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Gly Val Gly Ala Ala Leu Ile Leu Ala Thr Val Thr Ala Gly Leu Pro

325 330 335

Leu Leu Pro Val Val Gly Ala Leu Gly Ser Ser Ser Pro Ala Leu Leu

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Gly Pro Val Thr Gly Leu Thr Ala Leu Thr Leu Ala Ala Ala

355 360 365

Claims

1. The phytophthora capsici effector factor RxLR23 and the application of a prokaryotic or eukaryotic expression system for expressing the phytophthora capsici effector factor RxLR23 in promoting the growth of plants, wherein the amino acid sequence of the phytophthora capsici effector factor RxLR23 is shown as SEQ ID NO: 2.

2. The application according to claim 1, wherein the application comprises: preparing the phytophthora capsici effector RxLR23 into a protein solution, and applying the protein solution to a seedling culture substrate; or applying the escherichia coli liquid expressing the phytophthora capsici effector RxLR23 to a seedling culture substrate.

3. The application according to claim 1, wherein the application comprises: preparing the phytophthora capsici effector RxLR23 into a protein solution, and soaking the protein solution in seed; or soaking the Escherichia coli liquid expressing the phytophthora capsici effector factor RxLR23 into seed.

4. The application according to claim 1, wherein the application comprises: preparing the phytophthora capsici effector RxLR23 into a protein solution, and performing root irrigation treatment on plants by using the protein solution; or the escherichia coli liquid expressing the phytophthora capsici effector RxLR23 is subjected to root irrigation treatment on the plant.

5. The use according to any one of claims 2 to 4, further comprising diluting the protein solution or the E.coli suspension.

6. The use as claimed in claim 5, wherein the E.coli expressing the Phytophthora capsici effector RxLR23 is obtained by introducing the gene encoding the Phytophthora capsici effector RxLR23 into E.coli competent cells by plasmid.

7. Use according to claim 6, wherein the plasmid is pET28a, and/or

The Escherichia coli competent cell is DH5 alpha.

8. The phytophthora capsici effector factor RxLR23 and the application of a prokaryotic or eukaryotic expression system for expressing the phytophthora capsici effector factor RxLR23 in the preparation of plant growth promoters, wherein the amino acid sequence of the phytophthora capsici effector factor RxLR23 is shown as SEQ ID NO: 2.