CN109912699B - Phytophthora camphora effector protein Avh87 and encoding gene and application thereof - Google Patents

Abstract

The invention discloses an effector protein Avh87 derived from phytophthora cinnamomea, and a coding gene and application thereof. The protein is a protein with an amino acid sequence of SEQ ID No.1 in a sequence table; the nucleotide sequence for expressing the protein is shown as SEQ ID No.2 in the sequence table. Experiments prove that the gene can inhibit the plant cell allergic reaction induced by apoptosis precursor protein Bax or the plant cell death induced by phytophthora infestans INF 1. The invention has important significance for enriching the information of molecular mechanism data of the interaction between the plant pathogenic oomycetes and the host and establishing a comprehensive control technical strategy of the plant pathogenic oomycetes diseases.

Description

Phytophthora camphora effector protein Avh87 and encoding gene and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to an effector protein derived from phytophthora camphora, and a coding gene and application thereof.

Background

Effector proteins (effectors) are a class of exocrine protein molecules secreted by pathogens that alter host plant cell structures and metabolic pathways to facilitate successful infestation of a host plant or to elicit a host defense response. Many plant pathogens secrete effector proteins, such as those found in bacteria, fungi, oomycetes and nematodes. Various classes of effectors are present in plant pathogens, including RxLR, Avr, CRN, PcF, ScR, carbohydrate hydrolase, pectinase, chitinase, and esterase, among others.

During the long-term co-evolution process, plants form two layers of defense mechanisms to resist the attack of pathogenic bacteria. The first layer is immune PTI (protein-triggered immunity, PTI) triggered by Pathogen-associated molecular patterns (PAMPs), namely receptor kinases (PRRs) on the surface of plant cell membranes activate the basic defense system (PTI) of plants by recognizing Pathogen-associated molecular patterns (PAPMs). In most cases, the basal defense response blocks pathogenic bacteria from attacking the plant. Some pathogenic bacteria can secrete Effector proteins to plant cells to break through PTI, and then, the plant bodies recognize the Effector proteins through disease-resistant proteins so as to activate a second layer of defense reaction mechanism, namely Effector-induced immunity (ETI), and activate the expression of defense genes, which are usually expressed as allergic reaction (HR) and infect site apoptosis.

Similar to other types of pathogenic bacteria, the plant pathogenic oomycetes secrete effectors in the process of interacting with hosts to interfere with the immune response of plants and promote the plants to infect the hosts. In the process of infecting host plants by plant pathogenic oomycetes, the host plants are divided into two nutrition stages of living bodies and dead bodies according to the states of the plants, and the life style of the host plants is mostly half living body nutrition parasitism. At different periods, the mode of controlling plant defense reaction by pathogenic oomycetes is different, namely when the HR reaction generated by the plant per se is used for limiting infection and expansion of pathogenic bacteria, the pathogenic bacteria further grow and develop, and the plant is ensured to be in a living state by utilizing a strategy of inhibiting the HR reaction of the plant; on the contrary, when the pathogenic bacteria grow and develop to a certain stage, the pathogenic bacteria can also generate a strategy for promoting plant death, so that the plant death is caused. In the infection process, different effector expressions of plant pathogenic oomycetes are finely regulated and controlled, different functions are born, some pathogenic bacteria can keep the structural integrity in the infection process, some pathogenic bacteria can cause the nutritional defects of hosts, some pathogenic bacteria can help the rapid diffusion of the pathogenic bacteria, and some pathogenic bacteria can inhibit the PTI of the hosts, synergistically regulate the immune response of plants and promote the development of disease courses.

The RxLR effector family (R, arginine; x, any amino acid; L, leucine) is an important family of oomycete effectors that have been discovered from sequence alignments of identified avirulence genes: generally, the protein is short protein without intron, is located at the downstream of a signal peptide at the front end of the sequence and has a conserved RxLR-dEER element at about amino acid 50-70. RxLR effector molecules play an important role in the process that oomycetes escape from plant recognition and evolve. Since RXRR-dEER has a similar N-terminal conserved structure with an oomycete avirulence gene, this type of protein is called an avirulence gene (Avr homologes, Avh). The oomycete genome contains hundreds of highly differentiated RxLR effect genes, and has the characteristic of diversity, namely, the functions of inhibiting apoptosis precursor protein Bax induced cell death, inhibiting PTI and ETI functions, inducing cell death, regulating plant defense response by synergistic action and the like.

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. Besides causing huge economic losses in agriculture, forestry and horticulture, the inadvertent introduction of this pathogen also has disastrous consequences for the natural ecosystem and biodiversity. At home and abroad, the epidemic and harm of the phytophthora cinnamomi are mainly controlled by spraying pesticides, an ideal control effect cannot be achieved, the burden of farmers is increased, and the environmental pollution is caused. Although the growth of the phytophthora cinnamomi can be inhibited by proliferating and secreting cellulase in the mulching film by applying mulching film, compost and other environment-friendly ways, the distribution scale of the phytophthora cinnamomi and the capability of surviving for years in soil or asymptomatic plants make the control of the phytophthora cinnamomi diseases still face huge challenges and difficulties.

In conclusion, according to the molecular mechanism of the interaction between the plant pathogenic oomycetes and the host, effector proteins capable of effectively inhibiting plant cell death can be excavated from a plurality of plant pathogenic oomycete effectors, and the effector proteins are used for inhibiting the plant cell death to keep the plants in a survival state, so that the method has important significance for establishing a comprehensive control technical strategy of the oomycete diseases (especially 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 happens.

Disclosure of Invention

The purpose of the invention is as follows: in view of the above problems of the prior art, the present invention aims to provide a phytophthora cinnamomi effector protein Avh87 having an effect of inhibiting plant cell death, which is effective in inhibiting 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, inhibiting plant cell death caused by various factors and keeping plants in a survival state.

The technical scheme is as follows: in order to solve the problems, the technical scheme adopted by the invention is as follows:

the effector protein provided by the application is specifically derived from a Phytophthora cinnamomi (Phytophthora cinnamomi) strain, is named as Avh87, and has an amino acid sequence shown as SEQ ID No. 1;

the nucleotide sequence of the gene for expressing the protein is shown as SEQ ID No. 2;

wherein, SEQ ID No.2 consists of 390 nucleotides, the 1st to 390 th positions are ORFs, the protein shown by SEQ ID No.1 in the coding sequence table, and the SEQ ID No.1 consists of 129 amino acids in total, wherein, the 1st to 20 th amino acids are signal peptide sequences.

A recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the gene shown in SEQ ID No. 2.

The recombinant vector is a recombinant expression 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 enhanced, constitutive, tissue-specific or inducible promoter can be added in front of the transcription initiation nucleotide, such as a cauliflower mosaic virus 35S promoter, a Ubiquitin gene Ubiquitin promoter, a stress inducible promoter rd29A and the like, and the promoter 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 or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon 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 the present application, the promoter that initiates transcription of the Avh87 gene in the recombinant expression vector is specifically the 35S promoter.

More specifically, the recombinant expression vector is a recombinant plasmid obtained by inserting the Avh87 gene (SEQ ID No.2 in a sequence table) into the enzyme cutting site of the pGR107 vector. The enzyme cutting site is specifically Sma I.

The expression cassette includes a promoter capable of driving expression of the Avh87 gene, the Avh87 gene, and a transcription termination sequence.

The Avh87 protein, the gene for expressing Avh87 protein, or a recombinant expression vector, an expression cassette or a recombinant bacterium containing the gene are applied to any one of the following methods:

1) inhibiting cell death in a plant;

2) preparing a product for inhibiting plant cell death.

The active ingredient of the product for inhibiting the death of plant tissue cells provided by the invention is the Avh87 protein, or the gene for expressing Avh87 protein, or a recombinant expression vector, an expression cassette or a recombinant bacterium containing the gene.

In the use and the product, the plant cell is derived from a dicotyledonous plant or a monocotyledonous plant.

The plant is specifically a dicotyledonous plant, tobacco, more specifically Nicotiana benthamiana (Nicotiana).

Has the advantages that: compared with the prior art, the invention has the advantages that:

the phytophthora camphora effector protein Avh87 capable of effectively inhibiting plant cell death is excavated from a plurality of effectors of phytophthora camphora, and the plant cell survival state can be maintained in the presence of lethal factors by utilizing the function of inhibiting the plant cell death of the phytophthora camphora effector protein Avh87, so that the method has important significance for establishing a comprehensive control technical strategy of oomycete diseases (especially the phytophthora camphora), 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 oomycete epidemic situation is outbreak.

Drawings

FIG. 1 is a graph showing the results of expression levels of Avh87 gene at 24 hours, 48 hours and 96 hours after host inoculation by Phytophthora cinnamomi and at the hypha stage of IF infection;

FIG. 2 is a plasmid map of recombinant expression vector pGR107/Avh 87;

FIG. 3 is a graph showing the results of a lesion in leaf tissue in which Avh87 gene is transiently expressed in tobacco leaves; in the figure, inoculated leaves were free of any symptoms after 5 days; control treatment, no symptoms after 5 days of inoculation of tobacco leaves with empty vector pGR 107/GFP; pGR107/INF1 and pGR107/Bax inoculated leaves produced obvious yellow spots, gradually aggravated until the leaves necrosed;

FIG. 4 is a graph showing the result of inhibiting Bax or INF1 induced cell death in tobacco leaves by Avh87 gene, the left graph is a graph showing that Avh87 gene inhibits INF1 induced cell death in tobacco leaves, and the candidate effector gene Avh87 can significantly inhibit INF1 induced cell death regardless of simultaneous injection with INF1 or 12h and 24h after injection of INF 1. The right picture is a picture of Avh87 gene inhibiting BAX-induced Cell death in tobacco leaves, candidate effector gene Avh87 can inhibit Bax PCD (programmed Cell death) in 12h and 24h treatment, and candidate gene can not inhibit Cell death after agrobacterium containing candidate effector gene Avh87 and agrobacterium containing Bax are injected into tobacco simultaneously.

Detailed Description

The invention is further described with reference to specific examples. The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, 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: set forth in "Sena, kl. variables in fluent photphora cinnamomi distribution with a expressed kentuckyway, expressed Ecology and Management, 2019 (436): 39-44, publicly available from the city delivery college.

Potato Virus X vector (PVX) pGR 107: described in "NasserBeiczadeh. investment on Poto Virus x in North of Khorasan [ A ]. Chinese Plant Protection society of plants Protection the Plant Protection headings the 21st center- - -Proceedings of the International Plant Protection Congress [ C ]. Chinese Plant Protection society: the Chinese plant protection society, 2004: "A article, publicly available from Nanjing university of forestry.

Ben smoke (Nicotiana Benthamiana): the Plant Cell, 1999(11), described in "Louise Jones, Andrew J.Hamilton, Olivier Voinnet, et al.RNA-DNA Interactions and DNA Methylation in Post-transcription: 2291-.

Agrobacterium (Agrobacterium tumefaciens) GV 3101: described in "Gonz-lez-MulaAlmusena, Lang Julien, Grandclertine, Naquin Delphine, Ahmar Mohammed, Soulre Laurent, Queneau Yves, Dessaux Yves, Faure Denis Lifestyle of The biophotobacterium tumefaciens in The ecological environmental constrained on The inostost plant [ J ]. The New phytologist, 2018," a document publicly available from Nanjing university of forestry.

Example 1: acquisition of Effector protein Avh87 encoding gene of Phytophthora camphora

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, primers p1 and p2 are designed according to the obtained gene fragment, and the obtained target gene fragment is amplified and screened.

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

Culturing strains of the Lawsonia inermis on a solid V8 culture medium plate (the formula is that 1.6g of calcium carbonate is added into 170mL of V8 vegetable juice, mixing the mixture evenly, centrifuging the mixture at 2000rpm for 5min to take the supernatant, adding pure water to a constant volume of 1L, then adding 15g of agar powder, and using an autoclave for 20min for later use), culturing the strains in a biochemical incubator at 25 ℃ for 3 days, taking 3 fungus blocks with the diameter of 4mm, transplanting the fungus blocks 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, mixing evenly, centrifuging the mixture at 2000rpm for 5min to take the supernatant, adding the pure water to a constant volume of 1L, subpackaging, sterilizing the mixture in the autoclave for 20min for later use), culturing the strains in the biochemical incubator at 25 ℃ for 5 days, filtering hyphae, adding. Then, extracting the genome DNA of the test strains according to the following steps:

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

(2) The supernatant was added with equal volume of phenol/chloroform/isoamyl alcohol (25: 24: 1), mixed by inversion and centrifuged at 12000rpm for 10 min.

(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 5 min.

(4) The supernatant was transferred to a new tube, 2 volumes of absolute ethanol and 1/10 volumes 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.

PCR amplification of target Gene fragments

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

p1：5′-CTAGCATCGATTCCCGGGATGCTCTCGATGACCACAGC-3′

p2：5′-CTCTAGAGGATCCCGGGGAAATTCTCCTCGCGCGTG-3′

the reaction system is as follows: ddH₂O (22. mu.L), 5 × CE II buffer (2. mu.L), 1. mu.L each of the upstream and downstream primers (p1 and p2), DNA (5. mu.L), Pstar Max (25. mu.L).

The PCR reaction program is: 5min at 98 ℃; circulating for 32 times at 98 deg.C for 30s, 55 deg.C for 30s, and 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 performing homologous sequence alignment on the obtained gene fragments by using a BLAST program in NCBI to determine a target gene Avh87, wherein the nucleotide sequence of the target gene is shown as SEQ ID No.2, the amino acid sequence of the expression protein is shown as SEQ ID No.1, and the nucleotide sequence of the target gene and the amino acid sequence of the expression protein are stored in a database https: do gov cgi bin browserLoad? db ═ Phycil & position ═ scaffold _ 77: 29462 along 29851(scaffold _ 77: 29462 along 29851).

Example 2: avh87 gene expression pattern analysis in process of infecting apple with phytophthora camphora

1. Apple infected by phytophthora camphora strain

Activating the mycelium blocks stored in a refrigerator at 4 deg.C on V8 solid culture medium, and culturing at 25 deg.C for 24-36 h. A sterile filter paper of appropriate size was placed on the bottom of a sterile petri dish (diameter 9cm) 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 sides²And (4) removing the pulp. And picking out colonies, placing the colonies on sterilized filter paper, plugging the filter paper into the cut on the surface of the apple, plugging only three sides of the apple, and using one side of the apple 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 into a tray and culturing in a constant temperature incubator. And after the infection for 24h, 48h, 72h and 96h respectively, collecting hypha and pulp, sucking excess water by using filter paper, and putting the filter paper into liquid nitrogen for later use.

2. RNA extraction and expression verification after apple infection

RNA of an apple infected by the phytophthora camphora 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 ℃ below zero for later use, and the cDNA is used for fluorescent quantitative analysis. The Avh87 gene is detected by using a primer pair consisting of AVH87-QRT-F and AVH87-QRT-R, and the Actin gene (the sequence is stored in https:// genome.jgi.doe.gov/pages/search-for-genes.jsf. The SYBR fluorescent dye method was used, the system was referred to TaKaRa instructions, and 3 replicates were set up for each sample.

The apparatus used was ABI 7500. The Real-Time PCR reagent was SYBR Premix Ex Taq (Peffect Real Time) from TaKaRa. The system is as follows: SYBR Premix Ex Taq (2X) 10. mu.l; PCR Forward Primer (10. mu.M) 0.4. mu.l; PCR reverse Primer (10. mu.M) 0.4. mu.l; ROX Reference Dye II (50X) 0.4. mu.l; DNA template 2.0. mu.l, water addition volume to 20. mu.L, reaction program according to ABI PRISM 7500 and SYBR Premix Ex Taq (Peffect Real Time) by TaKaRa: 30s at 95 ℃; circulating for 40 times at 95 ℃ for 5s and 60 ℃ for 34 s; 95 ℃ for 15s, 60 ℃ for 1min and 95 ℃ for 15 s.

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

table 1: primers for qRT-PCR

Primer name	Sequence of
		AVH87-QRT-F	5′-TTCCCGGGATGCTCTCGATG-3′
AVH87-QRT-R	5′-TACTTCGCATGTTGCTGCAG-3′
		Avh-q F	5′-CGCCAAGATCCCCTCGGCAG-3′
Avh-q R	5′-GCGCGTGATCCTCGGGGACC-3′

The results are shown in FIG. 1, and it can be seen from the figure that Avh87 gene has the highest expression level at 24h, gradually decreases at 48h and 96h after phytophthora camphora infects the host, but still is higher than the hyphal stage. It is shown that Avh87 gene is highly expressed in the early stage of infection.

Example 3: avh87 gene is expressed transiently in tobacco body

Construction of PVX recombinant expression vector

(1) Sma I enzyme digestion PCR amplification target gene fragment Avh87, recovery insert, size is about 327 bp.

Reaction system: ddH₂O (33. mu.L), Sma I (2. mu.L), plasmid (10. mu.L), 10 × cut Buffer (5. mu.L), 37 ℃ for 30 min.

(2) The DNA fragment was ligated with pGR107 vector digested in the same manner to transform E.coli DH 5. alpha.

(3) After the transformation, DH5 alpha was subjected to Kan resistance screening, and the obtained colonies were shaken overnight at 37 ℃ to extract plasmids.

(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 327 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 the restriction sites Sma I of the pGR107 vector as shown by sequencing was designated pGR107/Avh87 (the plasmid map is shown in FIG. 2). In the recombinant expression vector pGR107/Avh87, the promoter for promoting the transcription of the DNA fragment shown in SEQ ID No.2 is a 35S promoter.

In the construction process of the PVX recombinant expression vector, the primer sequences used for PCR amplification of the target gene segment Avh87 are as follows:

table 2: primers for amplification Avh87

Primer name	Sequence of
		AVH87-F	5′-CAATCACAGTGTTGGCTTGC-3′
AVH87-R	5′-GACCCTATGGGCTGTGTTGT-3′

2. Agrobacterium transformation

2.1. Extraction of recombinant plasmid pGR107/Avh87

(1) Escherichia coli containing the recombinant plasmid pGR107/Avh87 was inoculated in LB medium containing an appropriate amount of antibiotic and shake-cultured at 37 ℃ and 220-.

(2) And (3) taking 1-4 mL of bacterial liquid into a 1.5mL centrifuge tube, and centrifuging 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, pH8.0) 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 tubes were inverted and mixed well several times, and centrifuged at 12000rpm for 5 min.

(6) 300. mu.L of precooled solution III (formulation: 3M K) was added⁺，5M A^c-) the mixed solution was inverted, put on an ice layer for 5min, centrifuged at 12000rpm for 5min, and the supernatant was transferred to another centrifuge tube.

(7) Adding equal volume of phenol/chloroform/isoamyl alcohol (volume ratio 25: 24: 1), shaking and mixing evenly, and centrifuging at 12000rpm for 5 min.

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

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

(10) Redissolved in 30. mu.LTE (containing 20. mu.g of RNase), and 5. mu.L of the mixture was electrophoretically detected and 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, shake-culturing in 200ml triangular flask for 8h until OD is about 0.8, and standing on ice for 10 min;

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

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

(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-induced competent cell electric shock transformation

(1) The electric shock cup and the cup holder are placed on ice for cooling. The parameters of the electric converter were set, the capacitance C was 25, the voltage V was 2.5kV (0.2 electric cup), and the pulse control unit was set at 200 Ω.

(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 1 min.

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

(4) Applying a pulse under the above set conditions, the resulting time constant is 4.8-5.1 ms.

(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, 200rpm, 30 ℃ and cultured for 3 h.

(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/Avh87, pGR107/GFP (negative control), pGR107/INF1 and pGR107/Bax of the positive recombinant Agrobacterium transformants selected and identified in step 2 were inoculated with 50mg/ml each of kan and rifampicin, respectively, in LB liquid medium. 30 ℃ for 2 days. 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 10 th day. The results are shown in FIG. 3, from which it can be seen that the Avh87 gene is transiently expressed in tobacco leaves as a result of lesion of leaf tissue, and inoculated leaves after 5 days are free from any symptoms; control treatment, tobacco leaves inoculated with empty vector pGR107/GFP for 5 days, was asymptomatic. pGR107/INF1 and pGR107/Bax inoculated leaves produced marked yellowing spots, which gradually worsened until the leaves necrosed. Indicating that the effector gene Avh87 was unable to cause tobacco cell death.

4. Screening of Effector molecules inhibiting the Induction of HR by Bax or INF1

The tobacco used in the experiment is Bunsen tobacco growing for 4 to 6 weeks, and the tobacco is placed in a greenhouse (22-25 ℃ and high light intensity) in the whole growth and experiment process. A suspension of Agrobacterium carrying the effector gene (pGR107/Avh87) was infiltrated into Nicotiana benthamiana leaves using a 1mL syringe with the needle removed. A small wound was made in the tobacco lower epidermis with a needle, and then 100. mu.L of the cell suspension was injected into the leaf cells around the wound. Three treatments were set for each gene, respectively:

firstly, simultaneously injecting agrobacterium suspension containing candidate genes GV3101/pGR107/Avh87 and agrobacterium suspension containing Bax (PGR107/Bax) genes;

secondly, injecting candidate genes GV3101/pGR107/Avh87, and injecting Bax after 12 h;

thirdly, injecting candidate gene GV3101/pGR107/Avh87, injecting Bax after 24h, and using agrobacterium containing empty vector and GFP (pGR107/GFP) as negative control.

In addition, a small wound was made on the tobacco lower epidermis with a needle, and then 100. mu.L of the cell suspension was injected into between leaf cells around the wound. Three treatments were set for each gene, respectively:

firstly, simultaneously injecting agrobacterium suspension containing candidate genes GV3101/pGR107/Avh87 and agrobacterium suspension containing INF1(PGR107/INF1) genes;

secondly, injecting candidate genes GV3101/pGR107/Avh87, and injecting INF1 after 12 h;

thirdly, injecting candidate gene GV3101/pGR107/Avh87, injecting INF1 after 24h, and using agrobacterium containing empty vector and GFP (pGR107/GFP) as negative control.

As shown in FIG. 4, it can be seen that Avh87 shows a significant inhibitory effect, with a weak inhibitory effect at 0h, and strongly inhibits Bax-induced cell death at 12h and 24 h. The injection of INF1 can obviously inhibit cell death caused by Bax when the injection is simultaneously injected with INF1 or after 12h and 24h of delay.

The foregoing description is intended to be illustrative rather than limiting, and it will be appreciated by those skilled in the art that many modifications, variations or equivalents may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Nanjing university of forestry

<120> phytophthora camphora effector protein Avh87, and coding gene and application thereof

<130>100

<160>10

<170>SIPOSequenceListing 1.0

<210>1

<211>129

<212>PRT

<213>Phytophthora cinnamomi

<400>1

Met Arg Leu Ser Gln Val Leu Val Val Val Thr Val Ser Phe Leu Val

1 5 10 15

Thr Ser Glu Ala Leu Ser Met Thr Thr Ala Ser Asn Gln Ala Lys Ile

20 25 30

Ala Lys Ile Pro Ser Ala Asp Ser Pro Asn Gln Arg Leu Leu Arg Thr

35 40 45

His Arg Glu Ala Val Asp Asp Asp Asp Ser Ser Glu Asp Ser Ser Glu

50 55 60

Glu Arg Thr Ile Ser Ile Ser Gln Met Lys Ala Ile Val Glu Glu Leu

65 70 75 80

Gly Ile Asp Trp Lys Met Val Gln Ala Ser Ser Thr Tyr Leu Gln Gln

85 90 95

His Ala Lys Tyr Glu Gln Tyr Gln Glu Lys Ala Asn Ala Leu Met Lys

100 105 110

Ala Lys Thr Lys Ser His Gly Ser Pro Arg Ile Thr Arg Glu Glu Asn

115 120 125

Phe

<210>2

<211>390

<212>DNA

<213>Phytophthora cinnamomi

<400>2

atgcgtctct ctcaagtctt ggtggtagtc acggtttcct ttctggtcac tagcgaggcc 60

ctctcgatga ccacagcctc caaccaagcc aagatcgcca agatcccctc ggcagatagc 120

cccaaccagc gacttctgag gacacaccgc gaggcagtcg acgatgatga ctcctccgaa 180

gattcctccg aagagaggac gatctcgata tcccaaatga aggcaattgt ggaagaactc 240

ggcatcgact ggaagatggt ccaggcaagc agcacatatc tgcagcaaca tgcgaagtat 300

gagcagtacc aggagaaggc caacgcgctt atgaaggcta aaacgaaaag ccacgggtcc 360

ccgaggatca cgcgcgagga gaatttctag 390

<210>3

<211>38

<212>DNA

<213> p1 primer sequence (Artificial)

<400>3

ctagcatcga ttcccgggat gctctcgatg accacagc 38

<210>4

<211>36

<212>DNA

<213> p2 primer sequence (Artificial)

<400>4

ctctagagga tcccggggaa attctcctcg cgcgtg 36

<210>5

<211>20

<212>DNA

<213> AVH87-QRT-F primer sequence (Artificial)

<400>5

ttcccgggat gctctcgatg 20

<210>6

<211>20

<212>DNA

<213> AVH87-QRT-R primer sequence (Artificial)

<400>6

tacttcgcat gttgctgcag 20

<210>7

<211>20

<212>DNA

<213> Avh-qF primer sequence (Artificial)

<400>7

cgccaagatc ccctcggcag 20

<210>8

<211>20

<212>DNA

<213> Avh-qR primer sequence (Artificial)

<400>8

gcgcgtgatc ctcggggacc 20

<210>9

<211>20

<212>DNA

<213> AVH87-F primer sequence (Artificial)

<400>9

caatcacagt gttggcttgc 20

<210>10

<211>20

<212>DNA

<213> AVH87-R primer sequence (Artificial)

<400>10

gaccctatgg gctgtgttgt 20

Claims (9)

1. An effector protein Avh87 of Phytophthora camphora, the amino acid sequence of which is shown in SEQ ID No. 1.

2. The gene for expressing the protein of claim 1, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 2.

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

4. The recombinant vector according to claim 3, wherein: the recombinant vector is a recombinant expression vector or a recombinant cloning vector.

5. The recombinant vector according to claim 4, wherein: the recombinant expression vector contains a 35S promoter.

6. Use of the protein of claim 1, or the gene 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. Use according to claim 6, characterized in that: the plant cell is derived from tobacco.

8. A product for inhibiting plant cell death, the active ingredient of which is the protein of claim 1, or the gene of claim 2, or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of any one of claims 3, 4 or 5.

9. The product of claim 8, wherein: the plant cell is derived from tobacco.

Images