CN114891077B - Phytophthora camphora effector protein SCR323321 and coding gene and application thereof - Google Patents

Abstract

The invention discloses an SCR (Smallcysteine-rich) effector protein SCR323321 from phytophthora camphora, a coding gene and application thereof, wherein the protein sequence is an amino acid residue sequence of SEQ ID No.1 in a sequence table. The invention takes agrobacterium GV3101 as a host, utilizes a PVX virus expression vector to carry out transient expression on the gene, and inoculates Nicotiana benthamiana (Nicotiana benthamiana) by an injection inoculation method, thereby proving that the effector protein SCR323321 has the function of inhibiting the death of plant tissue cells and causing obvious necrosis symptoms at the injection part. The invention has important significance for enriching the information of molecular mechanism data of plant pathogenic oomycetes and host interaction and establishing the comprehensive control technical strategy of oomycete diseases.

Description

Phytophthora camphora effector protein SCR323321 and coding gene and application thereof

Technical Field

The invention belongs to the technical field of phytophthora camphora effectors, and particularly relates to phytophthora camphora effector protein SCR323321 and a coding gene and application thereof

Background

Phytophthora camphora (Phytophthora cinnamomi) is one of the most destructive plant pathogenic oomycetes known and is widely distributed worldwide, with a host range approaching 5000 species. Phytophthora camphora is one of soil-borne pathogenic bacteria which seriously harm flowers and forest seedlings worldwide. It is distributed to some extent in the southeast coastal areas of China, including Shanghai, jiangsu, zhejiang, fujian and Taiwan. Not only is widely distributed, but also causes the morbidity and the mortality of certain plants such as camellia, rhododendron, cedar, camphor trees and the like seriously. Foreign research shows that the occurrence of host plant diseases is related to the distribution and growth change of the bacteria in soil. Therefore, after a great deal of research on the density of the bacteria in soil, the bacteria are considered to be seasonally changed in natural soil and related to the change of environmental ecological factors. For example, in the Australian forest region, the density is highest in summer, and the soil temperature and humidity are more suitable during the summer; winter is the least because the soil temperature is below 10 ℃.

As a typical semi-living nutritional parasitic phytopathogen, the oomycete is widely researched for phytophthora in the oomycete at present. Phytophthora has evolved a series of effectors which can be secreted in vitro during long-term interaction with host plants, and these effectors can interact with cell membrane recognition receptor proteins of plant immune response signal pathways or downstream targets of the pathways, interfere with host plant defense functions, promote self-invasion colonization, and cause pathogenicity to plants (Jones and Dangl, 2006). Phytophthora can produce an effector protein, small cysteine-rich (SCR), that induces expression of plant pathogenic genes and programmed cell death. PcF/SCR is considered as another type of toxin capable of causing plant cell death, however, the mechanism of the involvement of the SCR effector protein of Phytophthora camphorae in pathogenesis is not clear at present. SCR is considered as another type of toxin capable of causing plant cell death, however, the mechanism of involvement of SCR effector proteins in pathogenesis is not clear at present. Members of the SCR protein family include proteins encoded by polymorphic genes, usually distinguished by the abbreviation "SCR" from the number of amino acid residues following it, such as SCR70, SCR74, SCR91 from phytophthora infestans and SCR96 in phytophthora infestans (oromando et al, 2011). Phytophthora camphora has the largest family of SCR proteins reported to date-homologues of 1 PCF, 8 SCR96, 15 SCR99, and 1 SCR 121.

In conclusion, according to the molecular mechanism of the interaction between the plant pathogenic oomycetes and the host, the effector protein capable of effectively inhibiting the plant cell death can be excavated from a plurality of plant pathogenic oomycete effectors, and the functions of inhibiting the plant cell death and toxicity are utilized, so that the method has important significance for establishing the comprehensive control technical strategy of the phytophthora cinnamomi, and has important application value for reducing the plant death rate and eliminating or reducing the influence of the epidemic situation when the plant pathogenic oomycetes outbreak is outbreak.

Disclosure of Invention

In view of the above problems in the prior art, the present invention aims to provide a phytophthora cinnamomi effector protein SCR323321 having a function of inhibiting plant cell death, which can effectively inhibit plant cell death. The invention also aims to provide a coding gene of the protein and a recombinant vector thereof, which can be used for transgenic plants or products for inhibiting plant cell death and inhibiting plant cell death caused by various factors.

In a first aspect of the invention, there is provided a Phytophthora cinnamomi effector protein SCR323321, said protein being as described in any one of 1) to 2),

1) The amino acid sequence is shown as SEQ ID NO:1 is shown in the specification;

2) A protein which is obtained by substituting/deleting/adding one or more amino acid residues of the amino acid sequence shown in 1) and has effector functions.

The SCR323321 protein can be artificially synthesized, or can be obtained by synthesizing a coding gene and then performing biological expression. The SCR323321 coding gene can be obtained by deleting one or more codons of amino acid from the nucleotide sequence shown in SEQ ID No.2 in the sequence table, and/or carrying out missense mutation of one or more base pairs, and/or connecting the 5 'end and/or the 3' end of the coding gene with the coding sequence of the tag shown in the table 1.

In a second aspect, the present invention provides a nucleic acid molecule encoding a protein according to the first aspect of the present invention as set forth in any one of a) to b),

a) The nucleotide sequence is shown as SEQ ID NO:2 is shown in the specification;

b) A nucleotide sequence which can be hybridized with the nucleotide sequence in a) under strict conditions.

The stringent conditions may be conditions in which hybridization is carried out at 65 ℃ using a solution obtained by 0.5% SDS in 6 XSSC, and then membrane washing is carried out once using 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS, respectively.

Wherein, the sequence 2 consists of 489 nucleotides, the 1st to 489 th sites are ORFs, the protein shown in SEQ ID No.1 in the coding sequence table, the sequence 1 consists of 163 amino acids in total, and the 1st to 21st amino acids are signal peptide sequences.

In a third aspect, the invention provides a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising a nucleic acid molecule according to the second aspect of the 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 comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or repressible promoters, such as a cauliflower mosaic virus 35S promoter, a Ubiquitin gene Ubiquitin promoter, a stress repressible promoter rd29A and the like, can be added in front of the transcription initiation nucleotide, and can be used alone or combined with other promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or initiation codons of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

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

More specifically, the recombinant expression vector is a recombinant plasmid obtained by inserting the effector SCR323321 (SEQ ID No.2 in a sequence table) into the enzyme cutting site of a pGR107 vector. The restriction enzyme cutting site is specifically SmaI.

The expression cassette consists of a promoter capable of promoting expression of the effector SCR323321, including the effector SCR323321 and a transcription termination sequence.

In a fourth aspect, the present invention provides the use of the protein or the nucleic acid molecule, or the recombinant vector, the expression cassette, the transgenic cell line, or the recombinant bacterium in 1) or 2) as follows:

1) Inhibiting cell death in a plant;

2) Preparing a product for inhibiting plant cell death.

In a fifth aspect, the invention provides a product for inhibiting plant cell death, wherein the active ingredient of the product is the protein, or the nucleic acid molecule, or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium.

In certain embodiments, the plant cell is derived from a dicot or monocot.

In certain embodiments, the plant is specifically the dicotyledonous plant tobacco, more specifically Nicotiana Benthamiana (Nicotiana).

Compared with the prior art, the invention has the advantages that:

according to the application, phytophthora camphora effector protein SCR323321 capable of effectively inhibiting plant cell death is excavated from a plurality of effectors of phytophthora camphora SCR families, and the function of inhibiting the plant cell death can be utilized to maintain the physiological activity of plant cells in the presence of lethal factors, so that practical application value is provided for more comprehensively understanding the gene function of the phytophthora camphora.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1 is a graph showing the results of IF (hyphal stage of infection) expression levels of the effector SCR323321 after phytophthora cinnamomi is inoculated into a host for 24 hours, 48 hours and 96 hours, respectively;

FIG. 2 is a plasmid map of the recombinant expression vector pGR107/SCR 323321;

FIG. 3 is a graph of the results of an injection of the effector SCR323321 transiently expressed in tobacco leaves; the figure shows the results after 5 days of injection, in the control group of leaves, GFP alone was injected, and the leaves were asymptomatic; after GFP and Bax injection, a significantly small area necrosis occurred; the SCR323321 alone showed no obvious symptoms and the degree of necrosis of the leaves was significantly reduced after SCR323321 and Bax injection, indicating that the effector SCR323321 was able to inhibit cellular necrosis caused by Bax.

FIG. 4 is a phylogenetic tree of SCR323321 between species and genus, which was created by Neighbor-Joining (NJ) in the phylogenetic tree MEGA (v 6).

Figure 5 is a homology analysis of SCR effectors with RxLR effectors.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The molecular biological experiments, which are not specifically described in the following examples, were carried out by referring to the specific methods listed in molecular cloning, A laboratory Manual (third edition), or according to the kit and product instructions.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Phytophthora camphora (Phytophthora cinnamomi) strain: in "PHYCI _587572 an RxLR efficiency Gene and New Biomarker in A recombination enzyme Amplification for Rapid Detection of Phytophthora cinmamii, fore sts,2020,11 (3), 306-315", publicly available from Nanjing university of forestry.

Potato Virus X vector (potato Virus X, PVX) pGR107: described in "Nasser Beiczadeh. Investment on post Virus X in North of Khorasan [ A ]. Chinese Plant Protection society of plants Protection the Plant Protection headings the 21st center- - -the International Plant Protection Association [ C ]. Chinese Plant Protection society, china Plant Protection society, 2004 1." A, publicly available from Nanjing university of forestry.

Ben smoke (Nicotiana Benthamiana): described in "Louis Jones, andrew J.Hamilton, olivier Voinnet, et al.RNA-DNA Interactions and DNA methylation in Post-transcription Gene mutation the Plant Cell,1999 (11): 2291-2301," publicly available from Nanjing university of forestry.

Agrobacterium (Agrobacterium tumefaciens) GV3101: described in "Gonz-lez-Mula Almudena, lang Julien, grand executive, naquin Delphine, ahmar Mohammed, soulre Laurent, queneau Yves, dessaux Yves, faure Denis Lifestyle of The biophotobacterium tumefaciens in The ecological environmental constrained on host plant [ J ]. The New phytologist,2018.

Example 1 amplification of the Effector protein SCR 323321-encoding Gene of Phytophthora camphorae

Selecting the phytophthora camphorae strain as a reference material, and analyzing the genome sequence of the phytophthora camphorae according to the reported effector protein gene information to obtain the effector protein gene in the whole genome of the phytophthora camphorae. Then designing primers p1 and p2 according to the obtained gene fragment, and amplifying and screening the obtained target gene fragment.

1. Extraction of high-quality phytophthora camphorata genome DNA by CTAB-SDS method

The strain of a. Camphorata was plated on a solid V8 medium (formulation: adding 1.6g of calcium carbonate into 170mL of V8 vegetable juice, uniformly mixing, centrifuging at 2000rpm for 5min to obtain supernatant, adding pure water to a constant volume of 1L, adding 15g of agar powder, and autoclaving for 20min for later use), culturing in a biochemical incubator at 25 ℃ for 3 days, taking 3 fungus blocks with the diameter of 4mm, transplanting into a triangular flask containing 100mL of liquid V8 culture medium (the formula is that 1.6g of calcium carbonate is added into 70ml of V8 vegetable juice, uniformly mixing, centrifuging at 2000rpm for 5min to obtain supernatant, adding pure water to a constant volume of 1L, subpackaging, sterilizing in an autoclaving pot for 20min for later use), culturing in the biochemical incubator at 25 ℃ for 5 days, filtering hyphae, adding liquid nitrogen into a mortar, and grinding into powder. Then, extracting the genome DNA of the test strains according to the following steps:

(1) Transferring the mycelium powder into 1.5mL centrifuge tube, adding 900 μ L of 2% CTAB extractive solution and 90 μ L of 10% SDS, mixing by vortexing, and turning upside down several times every 10min in 55 deg.C water bath for 1h. Centrifuge at 12000rpm for 10min.

(2) The supernatant was added to an equal volume of phenol/chloroform/isoamyl alcohol (25.

(3) The supernatant was transferred to a new tube, added with an equal volume of chloroform, mixed by gentle inversion and centrifuged at 12000rpm for 5min.

(4) The supernatant was transferred to a new tube, 2 volumes of absolute ethanol and 1/10 volume of 3M NaAc (pH 5.2) were added, precipitated at-20 ℃ (> 1 h); centrifuging at 12000rpm for 10min, decanting the supernatant, washing the precipitate with 70% ethanol twice, and air drying at room temperature.

(5) The precipitate (containing 20. Mu.g/mL RNase) was dissolved in 20. Mu.L of sterilized ultrapure water or TE (pH 8.0) and treated at 37 ℃ for 1 hour.

5 mu L of DNA sample is electrophoresed in 1% agarose gel, the length of the DNA fragment is detected, and then the DNA is stored in a freezer at-20 ℃ for a long time for later use.

2. PCR amplification of target gene fragment

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

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

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

the reaction system is as follows: ddH ₂ O (22. Mu.L), 5 × CE II buffer (2. Mu.L), 1. Mu.L each of the upstream and downstream primers (p 1 and p 2), DNA (5. Mu.L), pstar Max (25. Mu.L).

The PCR reaction program is: 5min at 98 ℃; cycling for 32 times at 98 deg.C 30s,55 deg.C 30s,72 deg.C for 1 min; 10min at 72 ℃.

Taking 10 mu L of reaction product for electrophoresis, determining the target gene containing single clone and sequencing. And (3) carrying out homologous sequence alignment on the obtained gene segments by using a BLAST program in NCBI to determine a target effector SCR323321, wherein the nucleotide sequence of the target effector is shown as SEQ ID No.2, and the amino acid sequence of the expression protein is shown as SEQ ID No. 1.

Example 2 analysis of expression Pattern of Effector SCR323321 during host infection with Phytophthora camphora

1. Phytophthora camphora strain infecting host apple

Activating the mycelia stored in a refrigerator at 4 deg.C on V8 solid culture medium, and culturing at 25 deg.C for 24-36h. A sterile filter paper of appropriate size was placed on the bottom of a sterile petri dish (diameter 9 cm) and wetted with sterile water. Sterilizing the sterile operating platform. Wiping the apple surface with alcohol cotton, and sterilizing. Sterilizing the cutting edge of the cutter with outer flame of alcohol lamp, and cutting apple into 1cm pieces on four sides ² And (4) removing the pulp. The colonies are picked out and put on sterilized filter paper to be plugged into the cut on the surface of the apple, only three sides are plugged, and the other side is used as a reference of a blank solid culture medium. The incision was closed with a cotton wool pad and sterilized tap water was dispensed. Putting the mixture into a constant-temperature incubator by using a tray for culture. Respectively infecting for 24h,48h,72h and 96h, collecting mycelium and pulp, sucking with filter paper to extract excessive water, and adding into liquid nitrogen for use.

2. RNA extraction and expression verification after apple infection

RNA of an apple infected by the phytophthora camphorata strain is extracted by adopting R6834-01 (OMEGA) in the kit, digested for 30min at 37 ℃ by DNaseI (Takara), reverse transcribed by adopting M-MLV (Takara) to synthesize cDNA, diluted by 10 times and stored at-20 ℃ for later use, and the cDNA is used for fluorescent quantitative analysis. By adopting a SYBR fluorescent dye method, the system refers to TaKaRa specification, and each sample is not provided with 3 repeats.

Reaction system: mu.L of the reverse transcription product, 0.4. Mu.L of the primer, 10. Mu.L of 2XSYBR Premix Ex Taq II, and a volume of water supplement of 20. Mu.L. The apparatus used was ABI 7500. The Real-Time PCR reagent was SYBR Premix Ex Taq (Perfect Real Time) from TaKaRa.

The reaction program was set up according to ABI PRISM 7500 and SYBR Premix Ex Taq (Perfect Real Time) from TaKaRa, using a two-step procedure, as follows:

the fluorescence intensity was measured during the extension phase and the signal was collected for 40 cycles. After the amplification is finished, melting curve analysis is carried out to detect the specificity of the amplification product, and the temperature is 60-95 ℃. The melting curve is used for subsequent analysis when it is unimodal. The primer sequences used were as follows: primer sequences

The results are shown in FIG. 1, and it can be seen from the graph that the expression level of the effector SCR323321 is highest at the hyphal stage, is reduced to the lowest at 0 to 24 hours after the host is infected by the phytophthora cinnamomi, gradually increases from the infection stage of 24 hours to the infection stage of 48 hours, and then decreases in small amplitude from the infection stage of 48 hours to the infection stage of 96 hours. The expression level of the effector SCR323321 is reduced in the whole infection stage.

Example 3 transient expression of the Effector SCR323321 in tobacco

1. Construction of PVX recombinant expression vector

(1) SmaI enzyme digestion PCR amplification target gene fragment SCR323321, and recovery insert fragment, size is about 291bp.

Reaction system: ddH ₂ O (33. Mu.L), smaI (2. Mu.L), plasmid (10. Mu.L), 10 Xcut Buffer (5. Mu.L). 30min at 37 ℃.

(2) The resulting plasmid was ligated with the similarly digested pGR107 vector to transform E.coli DH 5. Alpha. Cells.

(3) After the transformation, DH5 alpha is screened by Kan resistance, and the obtained colony is shaken overnight at 37 ℃ to extract plasmid.

(4) And carrying out enzyme digestion identification on the recombinant plasmid by using restriction enzyme Sma I. The recombinant plasmid (with the size of about 10098bp and 142 bp) which is preliminarily identified by enzyme digestion is sent to Jinsry biological Limited company for sequencing. A recombinant plasmid in which the DNA fragment of SEQ ID No.2 was inserted between restriction sites SmaI of the pGR107 vector as shown by sequencing was designated as pGR107/SCR323321 (the plasmid map is shown in FIG. 2). In the recombinant expression vector pGR107/SCR323321, the promoter for promoting transcription of the DNA fragment shown in SEQ ID No.2 is a 35S promoter.

2. Agrobacterium transformation

2.1 extraction of recombinant plasmid pGR107/SCR323321

(1) The Escherichia coli containing the recombinant plasmid pGR107/SCR323321 is inoculated in an LB culture medium containing a proper amount of antibiotics, and is shake-cultured at 37 ℃ and 220-250rpm to the logarithmic phase.

(2) 1-4 mL of the bacterial liquid is taken into a 1.5mL centrifuge tube and centrifuged for 1min at 8000 rpm.

(3) Removing supernatant, and collecting thallus.

(4) 200. Mu.L of the precooled solution I (formulation: 50mM glucose, 25mM Tris-HCl,10mM EDTA, pH 8.0) was added, and the cells were suspended with shaking.

(5) 400 μ L of freshly prepared solution II (formulation: 0.2M NaCl,1% SDS) was added, the mixture was mixed by inverting the centrifuge tube several times, and centrifuged at 12000rpm for 5min.

(6) Adding 300 μ L of pre-cooled solution III (formula: 3M K +,5M Ac-), reversing the mixed solution, placing in an ice layer for 5min, centrifuging at 12000rpm for 5min, and transferring the supernatant into another centrifuge tube.

(7) An equal volume of phenol/chloroform/isoamyl alcohol (volume ratio 25: 24.

(8) Transferring the upper layer water phase into another centrifuge tube, adding equal volume of isopropanol, mixing, standing at room temperature for 10min, centrifuging at 12000rpm for 10min, and removing supernatant.

(9) The precipitate was washed 2 times with 70% (volume fraction) ethanol and dried by inversion.

(10) The mixture was dissolved in 30. Mu.L of TE (containing 20. Mu.g of RNase), 5. Mu.L of the mixture was electrophoretically detected, and the mixture was stored at-20 ℃.

2.2 competent preparation of Agrobacterium

(1) Selecting a single colony of the agrobacterium GV3101 to be cultured in 4ml of LB liquid culture medium for 2 days at 28 ℃ and shaking at 200rpm for 24 hours;

(2) Taking 3ml of culture solution, carrying out shake culture in a 200ml triangular flask for 8h until OD =0.8, and standing on ice for 10min;

(3) Subpackaging in 50ml centrifuge tube, balancing with balance, centrifuging at 5000rpm and 4 deg.C for 10min;

(4) Discarding the supernatant, resuspending in 10ml of 0 ℃ sterilized ultrapure water, balancing with a balance, centrifuging at 5000rpm and 4 ℃ for 10min;

(5) Repeating the above process for 3 times;

(6) Discarding the supernatant, adding 2ml of 5% glycerol, and gently sucking by a pipette to resuspend the cells;

(7) 100 μ L of cells were resuspended in pre-cooled 1.5ml EP tubes on ice;

(8) Quick freezing with liquid nitrogen, and storing in refrigerator at-70 deg.C.

2.3 Agrobacterium-infected competent cell electroporation transformation

(1) The electric shock cup and the cup holder are placed on ice for cooling. The parameters of the electric converter, the capacitance C =25 capacitance, the voltage V =2.5kV (0.2 electric shock cup) are set, and the pulse control unit is set at

(2) To a cooled 1.5ml EP tube, 100. Mu.L of a suspension of competent cells thawed in ice or freshly prepared and plasmid in ice are added, gently mixed and placed on ice for about 1min.

(3) The mixture of cells and plasmid was transferred to a cold cuvette and gently tapped so that the mixture was at the bottom of the cuvette.

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

(5) Immediately add 1ml of LB medium to the cuvette (left at room temperature). The cells were resuspended and transferred to a 17mm X100 ml EP tube and cultured at 200rpm for 3h at 30 ℃.

(6) Cells were harvested by centrifugation at 5000rpm for 3min and plated at the appropriate dilution on LB selective plates containing kanamycin. The colony grown after 2 days of culture at 30 ℃ is the positive clone.

3. Recombination agrobacterium positive transformant injection ben shi tobacco seedling leaf

Nicotiana Benthamiana (Nicotiana Benthamiana) was used as a test plant. Single colonies GV3101/pGR107/SCR323321, pGR107/GFP (negative control), pGR107/Bax, which were screened and identified as positive recombinant Agrobacterium transformants in step 2, were inoculated with LB liquid medium (containing kan and rifampicin each at 50 mg/ml) using toothpicks, respectively, and cultured at 30 ℃ for 48 hours. The positive clones were cultured at 30 ℃ for 48h at 200rpm in 3ml of LB medium supplemented with kanamycin (50. Mu.g/ml), centrifuged at 5000rpm for 3min to collect the cells, resuspended in 10mM MgCl2, and then diluted to OD600=0.5 with 10mM MgCl2 after repeating three times. The experiment used Benzenbach grown in a greenhouse (22-25 ℃, high light intensity) for 4 to 6 weeks, the third to sixth leaves from the top were used for osmotically inoculating Agrobacterium. A small wound was made in the lower epidermis of tobacco using a needle, and 100. Mu.l of Agrobacterium suspension carrying effector molecules (pGR 107:: SCR 323321) was infiltrated into Nicotiana benthamiana leaves using a 1mL needleless syringe. FIG. 3 shows Agrobacterium transformed with GFP (pGR 107:: GFP) as a negative control; GFP and Agrobacterium suspension containing Bax (pGR 107:: bax) gene are injected at the same time to be used as positive control; injecting an effector molecule SCR323321 alone (SCR 323321); simultaneously injecting agrobacterium tumefaciens suspension containing candidate genes and agrobacterium tumefaciens suspension containing Bax genes (SCR 323321+ Bax); transformation of Bax agrobacterium served as a positive control. And (3) culturing the inoculated tobacco leaves in an incubator with the temperature of 22 ℃ and the air humidity of 75% in the dark for 2 days, transferring the tobacco leaves into an artificial climate chamber for culturing, observing and recording the symptom change of an inoculated part every day after inoculation till the 5 th day. Results as shown in fig. 3, the observation of the injection results of the effector SCR323321 transiently expressed in tobacco leaves is 5 days after injection, and in the control group, GFP alone was injected, and the leaves were free of any symptoms; after GFP and Bax injection, a significantly small area necrosis occurred; the SCR323321 alone injected into the leaves has no obvious symptoms, and the necrosis degree of the leaves is obviously reduced after the SCR323321 and Bax are injected, which indicates that the effector SCR323321 can inhibit the cell necrosis caused by Bax.

Example 4 phylogenetic Tree of the Effector SCR323321 between species and genus

Homology search is carried out on various oomycetes in a FungiDB database (http:// fungi db. Org), NCBI (https:// www.ncbi.nlm.nih.gov /) and a genome website by using BlastP (E is less than or equal to 1E-15), 15 species with similar homology are obtained after alignment, and sequence alignment is carried out by using Bio Edit (v7.2.5). The final phylogenetic tree was generated by Neighbor-Joining (NJ) in MEGA (v 6) with parameters default. The reliability of the NJ evolutionary tree is evaluated by bootstrap values obtained by 1000 times of analysis, and finally a phylogenetic tree of the SCR323321 between species and genus is generated and established, as shown in FIG. 4.

Example 5 homology analysis of SCR effectors with RxLR effectors

Previous studies found that phytophthora camphora effector proteins RxLR29, rxLR49, rxLR57, rxLR87, which inhibit apoptosis in plant cells. These effectors all belong to the class of RxLR (R, arginine; x, any amino acid; L, leucine) and are very different from the SCR effectors. The RxLR effector belongs to the oomycete intracellular effector and the SCR effector to the apoplast effector, while SCR323321 has low homology to the effector proteins RxLR29, rxLR49, rxLR87, as shown in fig. 5.

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 otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.

Sequence listing

<110> Nanjing university of forestry

<120> phytophthora camphora effector protein SCR323321 and coding gene and application thereof

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gacgagtact gcgagatcag cactggcatg tgccgcggcc caaactacgt cggcgaatgc 180

ttcaatccag ccacgggggc cttccaagat ggttgtgact cgggcttcga atgcatcgac 240

aacaagtgcg actaccagga gacggaaacg acggcctcaa actcggcatc tgggtgcact 300

gtgatcgcca ccgccactga caattcggtc tgctaccttg tctgctcctc tggcgaatac 360

tgcgaaaacg gcactgatga atgccgtgcc cccaactacg acggcgagtg cttcaaccct 420

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Claims (9)

1. The phytophthora camphora effector protein SCR323321 is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO:1 is shown.

2. Nucleic acid molecule encoding the protein of claim 1, wherein the nucleic acid molecule has the sequence set forth in SEQ ID NO:2, respectively.

3. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising 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 comprises a 35S promoter.

6. Use of the protein of claim 1 or the nucleic acid molecule of claim 2, or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of any one of claims 3, 4 or 5 in 1) or 2) as follows:

1) Inhibiting cell death in a plant;

2) Preparing a product for inhibiting plant cell death.

7. A product for inhibiting cell death in a plant, the active ingredient being the protein of claim 1, or the nucleic acid molecule of claim 2, or the recombinant vector, expression cassette, transgenic cell line, or recombinant bacterium of any one of claims 3-5.

8. Use according to claim 6 or product according to claim 7, characterized in that: the plant cell is derived from a dicot or monocot.

9. Use according to claim 8 or a product according to claim 8, characterized in that: the dicotyledonous plant is tobacco.

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