CN114989272A - Phytophthora camphora effector protein SCR97226 and application thereof - Google Patents

Abstract

The invention discloses an SCR effector protein SCR97226 derived from Phytophthora cinnamomum, its encoding gene and application. The protein is a protein having one of the following amino acid residue sequences: the amino acid residue sequence of SEQ ID No. 1 in the Sequence Listing. Experiments have shown that the protein controlled by the gene can participate in the process of Phytophthora infestation of Nicotiana benthamiana, and cause the death of plant cells induced by the apoptosis precursor protein Bax. The invention has important significance for enriching the molecular mechanism data information of the interaction between the phytopathogenic oomycete and the host, and for establishing a comprehensive prevention and control technology strategy for the phytopathogenic oomycete disease.

Description

Phytophthora camphora effector protein SCR97226 and its application

technical field

The invention belongs to the technical field of Phytophthora cinnamomum effector, and particularly relates to a Phytophthora cinnamomum effector protein SCR97226 and its application.

Background technique

Effector proteins are a class of exocrine protein molecules secreted by pathogens that can change the cellular structure and metabolic pathways of host plants to promote successful infection of host plants or initiate host defense responses. Many plant pathogens secrete effector proteins, such as those found in bacteria, fungi, oomycetes, and nematodes. There are many types of effectors in plant pathogens, mainly including apoplast effectors and cytoplasmic effectors. Apoplast effectors include elicitors, necrosis and ethylene-inducing peptide 1-like proteins (NLPs), PcF/SCR proteins, Cellulose binding elicitor and lectin- like, CBEL) and enzyme inhibitors. Cytoplasmic effectors include the RxLR and CRN (Crinkler) protein families.

During the long-term co-evolution process, plants have developed a two-layer defense mechanism against pathogenic bacteria. The first layer is PTI (PAMP-triggered immunity, PTI) triggered by pathogen-associated molecular patterns (P AMPs). PAPMs) to activate the basal defense system (PTI) of plants. In most cases, basal defense responses block the infestation of plants by pathogenic bacteria. Some pathogenic bacteria can secrete effector proteins into plant cells to break through PTI. At this time, plants recognize these effector proteins through disease resistance proteins to activate the second layer of defense response mechanism, that is, the defense mechanism stimulated by effector proteins (Effector-triggered immunity, ETI), activates the expression of defense genes, usually manifested as hypersensitive reaction (HR), and cell death at the site of infection.

The phytopathogenic oomycete, similar to other types of pathogens, secretes effectors during the interaction with the host to interfere with the immune response of the plant and promote its infection of the host. In the process of phytopathogenic oomycetes infecting host plants, they are divided into two vegetative stages, living and dead, according to the state of the plants, and their lifestyles mostly belong to semi-living vegetative parasitism. In different periods, pathogenic oomycetes manipulate plant defense responses in different ways. That is, when plants produce HR responses to limit the infection and expansion of pathogens, pathogens use strategies to inhibit plant HR responses for their further growth and development to ensure plant growth. In a living state; on the contrary, when the pathogenic bacteria grow and develop to a certain stage, it will also produce strategies to promote plant death, resulting in plant death. During the infection process, the expression of different effectors of plant pathogenic oomycetes is finely regulated and assumes different functions. Some of them can maintain the integrity of the structure of the pathogenic bacteria during the infection process, some can cause auxotrophy of the host, and some can help the pathogenic bacteria to quickly Proliferation, some can inhibit the host's PTI, synergistically regulate the plant's immune response, and promote the development of the disease course.

Phytophthora cinnamomi is one of the most destructive phytopathogenic oomycetes known, with a worldwide distribution and a host range of nearly 5000 species. In addition to causing huge economic losses in agriculture, forestry and horticulture, the inadvertent introduction of this pathogen has catastrophic consequences for natural ecosystems and biodiversity. At home and abroad, spraying pesticides is mainly used to control the prevalence and harm of Phytophthora cinnamomyces, but they have failed to achieve the desired control effect, which not only increases the burden on farmers, but also causes environmental pollution. Although the growth of Phytophthora cinnamomum can be inhibited by applying mulch, compost and other environmentally friendly methods to make other microorganisms proliferate and secrete cellulase in the mulch, the distribution scale of Phytophthora cinnamomum, the ability to survive for several years in soil or asymptomatic plants , so that the control of Phytophthora cinnamomea still faces great challenges and difficulties.

Phytophthora can produce an effector protein, Small cysteine-rich (SCR), which induces the expression of plant pathogenic genes and programmed cell death. Phytophthora typically includes 3-19 SCR effector proteins. In view of the important role of effectors in the interaction between pathogenic bacteria and host plants, the study of effector biology of P.

In summary, according to the molecular mechanism of the interaction between the phytopathogenic oomycete and the host, the effector proteins that can effectively induce plant cell apoptosis can be excavated from the numerous phytopathogen effectors of Phytophthora cinnamomycetes, and the effector proteins can be used to induce plant cell death. It is of great significance to establish a comprehensive control technology strategy for Phytophthora cinnamomyces disease.

SUMMARY OF THE INVENTION

In view of the above problems existing in the prior art, the purpose of the present invention is to provide a Phytophthora syringae effector protein SCR97226 that induces plant cell death, which can effectively induce plant cell death.

In a first aspect of the present invention, there is provided a Phytophthora effector protein SCR97226, the amino acid sequence of which is as described in any of 1) to 2),

As shown in SEQ ID NO: 1;

The amino acid sequence shown in 1) is substituted/deleted/added by one or several amino acid residues, and has effector function.

The second aspect of the present invention provides a nucleic acid molecule encoding the amino acid of the first aspect of the present invention, the sequence of the nucleic acid molecule is shown in any of the following a) to b),

As shown in SEQ ID NO: 2;

Nucleic acid sequences that hybridize to the nucleotide sequences described in a) under stringent conditions.

The above-mentioned SCR97226 protein can be synthesized artificially, or can be obtained by first synthesizing its encoding gene and then carrying out biological expression. The above-mentioned SCR97226 encoding gene can be obtained by deleting the codon of one or several amino acids from the nucleotide sequence shown in SEQ ID No. 2 in the sequence listing, and/or performing missense mutation of one or several base pairs, and/ Or the coding sequence of the tag shown in Table 1 is attached to its 5' end and/or 3' end.

The above stringent conditions can be hybridized at 65°C with a solution of 6×SSC, 0.5% SDS, and then washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS each.

Among them, SEQ ID NO: 2 consists of 342 nucleotides, and positions 1-342 are ORFs, which encode the protein shown in SEQ ID No. 1 in the sequence listing, and SEQ ID NO: 1 consists of 114 amino acids, of which the first The -20th amino acid is the signal peptide sequence.

In a third aspect, the present invention provides a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the nucleic acid molecule of the second aspect of the present invention.

In certain embodiments, the recombinant vector is a recombinant overexpression vector or a recombinant cloning vector.

The recombinant expression vector can be constructed using existing expression vectors. The expression vector may also contain the 3' untranslated region of the exogenous gene, ie, containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal directs the addition of poly(A) to the 3' end of the mRNA precursor. When using the gene to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or repressive promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus 35S promoter, pan Gene Ubiquitin promoter, stress-inhibiting promoter rd29A, etc., they can be used alone or in combination with other promoters; in addition, when using the gene of the present invention to construct a recombinant expression vector, enhancers can also be used, including translation enhancers Or transcriptional enhancers, these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can be derived from a transcription initiation region or a structural gene.

In certain embodiments, the recombinant expression vector contains a 35S promoter.

More specifically, the recombinant expression vector is a recombinant plasmid obtained by inserting the effector SCR97226 (SEQ ID No. 2 in the sequence listing) into the restriction site of the pGR107 vector. The restriction enzyme cleavage site is specifically SmaI.

The expression cassette consists of a promoter capable of initiating the expression of the effector SCR97226, including the effector SCR97226 and a transcription termination sequence.

In the fourth aspect, the present invention provides the application of the protein or the nucleic acid molecule, or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in the following 1) or 2):

1) induce plant cell death;

2) Preparation of products for inducing plant cell death.

In the fifth aspect, the present invention provides a product for inducing plant cell death, the active component of which is the protein, or the nucleic acid molecule, or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria.

In certain embodiments, the plant cell is derived from a dicotyledonous plant or a monocotyledonous plant.

In certain embodiments, the plant is specifically a dicotyledonous plant Nicotiana, more specifically Nicotiana Benthamiana.

Compared with the prior art, the advantages of the present invention are:

By excavating the effector protein SCR97226 of Phytophthora camphora, which can effectively induce plant cell death from numerous effectors of Phytophthora cinnamomum For field crops, it provides a theoretical basis for increasing plant yield, and provides practical application value for a more comprehensive understanding of the gene function of Phytophthora cinnamomum.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings required for the description of the specific embodiments.

Fig. 1 is the plasmid map of recombinant expression vector pGR107/SCR97226;

Figure 2 shows the results of leaf tissue lesions of the effector SCR97226 transiently expressed in tobacco leaves. In the figure, after 5 days of GFP injection alone, the leaves did not have any symptoms; in the leaves of the control group, after injection of GFP and Bax, obvious small areas appeared Necrosis; After injection of the effectors of interest SCR97226 and Bax, the necrotic area of leaves was significantly larger than that of the control group and leaves injected with Bax alone. It indicates that the effector SCR97226 can induce cell necrosis caused by Bax;

Figure 3 is a phylogenetic tree of SCR97226 among Phytophthora species using the Neighbor-Joining (NJ) method in the phylogenetic tree MEGA (v6). PHYCA 96849 (Phytophthora capsici), PHYCA 96640 (Phytophthora capsici), PHYCI 96480 (Phytophthora cinnamomi), PHYCI 90211 (Phytophthora cinnamomi), PHYCA 96767 (Phytophthora capsici), PHYCA 96549 (Phytophthora capsici), PHYCA4198 (Phytophthora capsici), PHPALM 16185-t46 1 (Phytophthora palmivora), PHPALM36320t46 1 (Phytophthora palmivora), PHPALM 21206-t46 1 (Phytophthora palmivora) mold), PHPALM 29528-t46 1 (Phytophthora palmivora), PHPALM21004t46 1 (Phytophthora palmivora), PHPALM 12174-t46 1 (Phytophthora palmivora), PHPALM 9197t46 1 (Phytophthora palmivora), PHYSODRAFT 336289-t26 1 (Phytophthora sojae), SCR97226 (Phytophthora cinnamomi), PPTG 04328-t26 1 (Phytophthora parasitica), PHYCI237327 (Phytophthora cinnamomi), PHYCI 330581 (Phytophthora cinnamomi), PHYCI 252576 (Phytophthora cinnamomi), PHYCI 234114 (Phytophthora cinnamomi), PHYCI 90122 (Phytophthora cinnamomi).

Figure 4 is an alignment of SCR97226 with five intracellular effector RxLR effectors using DNAMAN (v6.0), and the results show that the gene homology is 25.40%. The comparison results of SCR97226 and 5 intracellular effectors were different, mainly because SCR97226 was an apoplast effector, and the other 5 were intracellular effectors.

Detailed ways

Embodiments of the technical solutions of the present invention will be described in detail below with reference to the accompanying drawings. The molecular biology experimental methods that are not specifically described in the following examples are all carried out with reference to the specific methods listed in the "Molecular Cloning Experiment Guide" (third edition), or according to the kits and product instructions.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Phytophthora cinnamomi strain: described in "Sena, KL. variables influence Phytophthora cinnamomi distribution within a forested Kentuckywatershed. Forest Ecology and Management, 2019(436): 39-44", available to the public from Yuncheng College.

Potato Virus X (PVX) pGR107: described in "Nasser Beiczadeh. Investigation on Potato Virus X in North of Khorasan[A]. Chinese Plant Protection Society. Plant Protection Towards the 21st Century----Proceedings of the International Plant Protection Congress[C]. Chinese Plant Protection Society: Chinese Plant Protection Society, 2004: 1.” article, available to the public from Nanjing Forestry University.

Nicotiana Benthamiana: described in "Louise Jones, Andrew J. Hamilton, Olivier Voinnet, et al. RNA-DNA Interactions and DNA Methylation in Post-Transcriptional Gene Silencing. The Plant Cell, 1999(11):2291- 2301.” article, publicly available from Nanjing Forestry University.

Agrobacterium tumefaciens GV3101: described in "González-Mula Almudena, Lang Julien, Grandclément Catherine, Naquin Delphine, Ahmar M ohammed, Soulère Laurent, Queneau Yves, Dessaux Yves, Faure Denis. Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant.[J].The New phytologist, 2018.”, the article is available to the public from Nanjing Forestry University.

Example 1 Acquisition of the gene encoding the effector protein SCR97226 of Phytophthora cinnamomea

The strain of Phytophthora cinnamomum was selected as the test material, and the genome sequence of Phytophthora cinnamomum was analyzed according to the reported effector protein gene information, and the effector protein gene in the whole genome of Phytophthora cinnamomum was obtained. Then, primers p1 and p2 are designed according to the obtained gene fragments, and the obtained target gene fragments are amplified and screened.

1. Using CTAB-SDS method to extract high-quality Phytophthora cinnamomum genomic DNA

Phytophthora cinnamomum strains were cultured on solid V8 medium plate (recipe: 170ml V8 vegetable juice plus 1.6g calcium carbonate, mixed, centrifuged at 2000rpm for 5min to take the supernatant, add purified water to 1L, add 15g agar powder, autoclave Bacteria pot for 20min standby), after culturing in a biochemical incubator at 25°C for 3 days, take 3 bacterial blocks with a diameter of 4mm and transplant them into a triangular flask containing 100mL of liquid V8 medium (recipe: 70ml V8 vegetable juice plus 1.6g calcium carbonate , Mix well, centrifuge at 2000 rpm for 5 minutes, take the supernatant, add pure water to make up to 1 L, then dispense, sterilize in an autoclave for 20 min for later use), culture in a 25 ℃ biochemical incubator for 5 days, filter the mycelium, put in a mortar Add liquid nitrogen and grind to powder. Then follow the steps below to extract the genomic DNA of the tested strains:

(1) Transfer the mycelial powder to a 1.5 mL centrifuge tube, add 900 μL of 2% CTAB extract and 90 μL of 10% SDS, vortex to mix, and place in a water bath at 55°C for 1 hour, inverting it several times every 10 minutes. Centrifuge at 12000rpm for 10min.

(2) Take the supernatant and add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), invert and mix, and centrifuge at 12000 rpm for 10 min.

(3) Transfer the supernatant to a new tube, add an equal volume of chloroform, gently invert and mix, and centrifuge at 12,000 rpm for 5 min.

(4) Transfer the supernatant to a new tube, add 2 times the volume of absolute ethanol and 1/10 volume of 3M NaAc (pH 5.2), and precipitate at -20°C (>1h); centrifuge at 12000rpm for 10min, and pour off the supernatant , the precipitate was washed twice with 70% ethanol and air-dried at room temperature.

(5) Add 20 μL sterilized ultrapure water or TE (pH 8.0) to dissolve the precipitate (containing 20 μg/mL RNase), and treat at 37°C for 1 h.

5 μL of DNA samples were taken and electrophoresed on a 1% agarose gel to detect the length of DNA fragments, and then the DNA was stored in a -20°C freezer for long-term use.

2. PCR amplification of target gene fragments

The primers are p1 and p2, and the sequences are as follows:

p1: 5'-ATGAAGACCCAGCTCGTTATT-3' (SEQ ID No. 3)

p2: 5'-TTGGTAGCAAGCACGGCGAAA-3' (SEQ ID No. 4)

The reaction system was: ddH ₂ O (22 μL), 5×CEII buffer (2 μL), 1 μL of upstream and downstream primers (p1 and p2), DNA (5 μL), and Pstar Max (25 μL).

The PCR reaction program was: 98°C for 5 min; 98°C for 30 s, 55°C for 30 s, 72°C for 1 min, 32 cycles; 72°C for 10 min.

Take 10 μL of the reaction product for electrophoresis to confirm that the single clone containing the target gene is sequenced. The homologous sequence alignment of the obtained gene fragments was carried out by the BLAST program in NCBI to determine the target effector SCR97226, whose nucleotide sequence is shown in SEQ ID No. 2, and the amino acid sequence of the expressed protein is shown in SEQ ID No. 1.

Example 2 Transient expression of effector SCR97226 in tobacco

1. Construction of PVX recombinant expression vector

(1) The target gene fragment SCR97226 was amplified by SmaI digestion and PCR, and the inserted fragment was recovered, and the size was about 342bp.

Reaction system: ddH ₂ O (33 μL), SmaI (2 μL), plasmid (10 μL), 10×cut Buffer (5 μL). 37℃, 30min.

(2) ligated with pGR107 vector digested by the same enzyme, and transformed into Escherichia coli DH5α.

(3) The transformed DH5α was screened for Kan resistance, and the colonies were obtained and the plasmids were extracted after shaking at 37°C overnight.

(4) The recombinant plasmid was identified by restriction endonuclease SmaI. The correct recombinant plasmid was initially identified by enzyme digestion (two target bands of about 10098 bp and 342 bp were obtained) and sent to GenScript Bio Co., Ltd. for sequencing. The recombinant plasmid with the DNA fragment of SEQ ID No. 2 inserted between the restriction sites SmaI of the pGR107 vector by sequencing was named pGR107/SCR97226 (the plasmid map is shown in Figure 1). In the recombinant expression vector pGR107/SCR97226, the promoter for initiating transcription of the DNA fragment shown in SEQ ID No. 2 is the 35S promoter.

The primer sequences used are as follows: Primer sequence

2. Agrobacterium transformation

2.1 Extraction of recombinant plasmid pGR107/SCR97226

(1) Inoculate Escherichia coli containing the recombinant plasmid pGR107/SCR97226 into LB medium containing appropriate amount of antibiotics, 37° C., 220-250 rpm shaking culture to logarithmic growth phase.

(2) Take 1-4 mL of bacterial solution into a 1.5 mL centrifuge tube, and centrifuge at 8000 rpm for 1 min.

(3) Remove the supernatant and collect the bacterial cells.

(4) Add 200 μL of pre-cooled solution I (recipe: 50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0), and suspend the cells by shaking.

(5) Add 400 μL of freshly prepared solution II (formula: 0.2M NaCl, 1% SDS), invert the centrifuge tube several times to mix, and centrifuge at 12000 rpm for 5 min.

(6) 300 μL of pre-cooled solution III (formula: 3M K ⁺ , 5MAc ^- ) was added, the liquid was mixed by inversion, placed on ice for 5 min, centrifuged at 12000 rpm for 5 min, and the supernatant was transferred to another centrifuge tube.

(7) Add an equal volume of phenol/chloroform/isoamyl alcohol (volume ratio 25:24:1), shake and mix, and centrifuge at 12,000 rpm for 5 min.

(8) The upper aqueous phase was transferred to another centrifuge tube, an equal volume of isopropanol was added, and after mixing, it was placed at room temperature for 10 minutes, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was removed.

(9) The precipitate was washed twice with 70% (volume fraction) ethanol, and dried by inversion.

(10) Redissolve in 30 μL TE (containing 20 μg RNase), take 5 μL for electrophoresis detection, and store the rest at -20°C.

2.2 Agrobacterium competent preparation

(1) Pick a single colony of Agrobacterium GV3101 in 4 ml of LB liquid medium for 2 days, at 28°C, and shake at 200 rpm for 24 hours;

(2) Take 3ml of the culture solution and shake it in a 200ml Erlenmeyer flask for 8h to about OD=0.8, and put it on ice for 10min;

(3) Divide into 50ml centrifuge tubes, balance the balance, centrifuge at 5000rpm and 4°C for 10min;

(4) Discard the supernatant, resuspend in 10ml sterilized ultrapure water at 0°C, balance the balance, and centrifuge at 5000rpm for 10min at 4°C;

(5) Repeat the above process 3 times;

(6) Discard the supernatant, add 2 ml of 5% glycerol, and resuspend the cells by gently pipetting;

(7) Dispense 100 μL of cells on ice and resuspend them in a pre-cooled 1.5 ml EP tube;

(8) Quick-freeze in liquid nitrogen and store in -70°C refrigerator.

2.3 Electroporation transformation of Agrobacterium competent cells

(1) Cool the shock cup and cup holder on ice. Set the parameters of the electrotransformer, capacitance C=25 capacitance, voltage V=2.5kV (0.2 electric shock cup), and the pulse control unit is set at 200∩.

(2) Add 100 μL of thawed or newly prepared competent cell suspension in ice and plasmids in ice into a cooled 1.5 ml EP tube, mix gently and place on ice for about 1 min.

(3) Transfer the mixture of cells and plasmids to a cold electric shock cup, and tap lightly to make the mixture at the bottom of the electric shock cup.

(4) A pulse is applied under the conditions set above, and the resulting time constant is 4.8 to 5.1 ms.

(5) Immediately add 1 ml of LB medium to the electric shock cup (at room temperature). The cells were resuspended and transferred to a 17mm×100ml EP tube, 200rpm, and 30°C for 3h recovery.

(6) Collect cells by centrifugation at 5000 rpm for 3 min, and spread them on LB selective plates containing kanamycin at appropriate dilution. The colonies grown after 2 days of culture at 30°C were positive clones.

3 Injection of recombinant Agrobacterium-positive transformants into leaves of N. benthamiana seedlings

Nicotiana Benthamiana was used as the test plant. Use a toothpick to pick and identify the positive recombinant Agrobacterium transformants in step 2. The single colonies GV3101/pGR107/SCR97226, pGR107/GFP (negative control), and pGR107/Bax were respectively inoculated into LB liquid medium (containing kan and rifampicin). 50mg/ml), cultured at 30°C for 48h. The positive clones were placed in 3 ml of LB medium supplemented with kanamycin (50 μg/ml), cultured at 200 rpm for 48 h at 30°C, centrifuged at 5000 rpm for 3 min to collect the cells, resuspended with 10 mM MgCl ₂ , and after three repetitions, the volume was adjusted to 10 mM MgCl ₂ . OD600=0.4. The experiments used N. benthamiana grown for 4 to 6 weeks in a greenhouse (22-25°C, high light intensity), and the third to sixth leaves from the top were used for infiltration of Agrobacterium. A small wound was made in the lower epidermis of the tobacco with a needle, and 100 μl of the Agrobacterium suspension carrying the effector molecule (pGR107::SCR97226) was infiltrated into N. benthamiana leaves using a 1 mL needleless syringe. In the figure, Agrobacterium transformed with GFP (pGR107::GFP) was used as a negative control; GFP and Agrobacterium suspension containing Bax (pGR107::Bax) gene were injected simultaneously as a positive control; the effector molecule SCR97226 (SCR97226) was injected alone ; Inject the Agrobacterium suspension containing the candidate gene and the Agrobacterium suspension containing the Bax gene at the same time (SCR97226+Bax); Transform Bax Agrobacterium as a positive control. The inoculated tobacco leaves were cultured in the dark for 2 days in a 22°C incubator with an air humidity of 75%, and then transferred to an artificial climate room for cultivation. Figure 2. Observation of the injection results of the transient expression of effector SCR97226 in tobacco leaves; after 5 days, the leaves injected with GFP alone did not have any symptoms; in the leaves of the control group, obvious necrosis appeared in the areas injected with GFP and Bax as a positive control result; After injection of the target effector SCR97226 and Bax, the necrotic area of leaves was significantly larger than that of the control group and leaves injected with Bax alone. It indicated that the effector SCR97226 could promote the cell necrosis induced by Bax.

Example 3 Phylogenetic tree of effector SCR97226 between species and genera

Several oomycete species in FungiDB (http://fungi db.org), NCBI (https://www.ncbi.nlm.nih.gov/) and the Genome website were identified using BlastP (E≤1e-15). Source search. The alignment results obtained 22 species with similar homology, and the sequences were aligned using Bio Edit (v7.2.5). The phylogenetic tree is generated by the Neighbor-Joining (NJ) method in MEGA (v6) with default parameters. The reliability of the NJ phylogenetic tree was evaluated by bootstrap values obtained from 1000 analyses. As shown in Figure 3.

Download the SCR97226 gene and related homologous genes (Phytophthora cinnamomi (Phytophthora cinnamomi), Phytophthora sojae (Phytophthora soybean), Phytophthora palmivora (Palm mildew), Phytophthora capsici (Phytophthora capsici (Phytophthora capsici), etc.) sequences, the MEGA (7.0.20) software was used to construct a phylogenetic tree using the Neighbor-joining (NJ) method, and the reliability of the phylogenetic tree was evaluated by the bootstrap method. The bootstrap value is 1000, and the rest of the parameters are set to default. Finally, the phylogenetic tree of SCR97226 between species and genera is established, as shown in Figure 3.

Example 4 Homology analysis of the apoplast effector SCR97226 and five RxLR family intracellular effectors

SCR97226 and related homologous genes were downloaded from the Phytophthora information resource website (https://fungidb.org/fungidb/app/), five effectors with similar homology were obtained, and the sequences were compared using Bio Edit (v7.2.5). pair clips. Using DNAMAN (v6.0) to align SCR97226 with five intracellular effector RxLR effectors, the results showed that the gene homology was 25.40%. The comparison results of SCR97226 and 5 intracellular effectors were different, mainly because SCR97226 was an apoplast effector, and the other 5 were intracellular effectors. As shown in 4.

Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless stated otherwise, any specific value should be construed as merely exemplary and not as limiting, as other examples of exemplary embodiments may have different values. Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless stated otherwise, any specific value should be construed as merely exemplary and not as limiting, as other examples of exemplary embodiments may have different values.

sequence listing

<110> Nanjing Forestry University

<120> Phytophthora cinnamomum effector protein SCR97226 and its application

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atcatctatg agaactctgg cttcttgacc gcctacttca cccaaaatgc ttgcgccgtg 180

gcaggcggca cgatcgatgc tagcaagaag ggcaaccaga agtgctgcac catccccaaa 240

cgtctcgatg gtcgcagaag tgcctttaac aaagtttgcg aaagtcaaaa ggctggaagt 300

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ctctagagga tccccgggtt ggtagcaagc acggcgaaa 39

Claims (9)

1. Phytophthora effector protein SCR97226, characterized in that the amino acid sequence of the protein is as described in any of 1) to 2), 1) as shown in SEQ ID NO: 1; 2) The amino acid sequence shown in 1) is substituted/deleted/added by one or several amino acid residues, and has effector function. 2. the nucleic acid molecule encoding the amino acid of claim 1, wherein the sequence of the nucleic acid molecule is shown in any of the following a) to b), a) as shown in SEQ ID NO: 2; b) nucleic acid sequences that hybridize to the nucleotide sequences described in a) under stringent conditions. 3. A recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the nucleic acid molecule of claim 2. 4. The recombinant vector according to claim 3, wherein the recombinant vector is a recombinant overexpression vector or a recombinant cloning vector. 5 . The recombinant vector according to claim 4 , wherein the recombinant expression vector contains a 35S promoter. 6 . 6. The protein according to claim 1 or the nucleic acid molecule according to claim 2, or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria according to any one of claims 3-5 in the following 1) or 2) Applications in: 1) induce plant cell death; 2) Preparation of products for inducing plant cell death. 7. a product for inducing plant cell death, its active ingredient is the protein of claim 1, or the nucleic acid molecule of claim 2, or the recombinant vector described in any of claims 3-5, Expression cassettes, transgenic cell lines or recombinant bacteria. 8. The use according to claim 6 or the product according to claim 7, wherein the plant cells are derived from dicotyledonous or monocotyledonous plants. 9. The use according to claim 8 or the product according to claim 8, wherein the dicotyledonous plant is tobacco.

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