CN111172182B

CN111172182B - Phytophthora capsici PcMPK12 gene and vector and application thereof

Info

Publication number: CN111172182B
Application number: CN201911240953.4A
Authority: CN
Inventors: 孙文秀; 朱彤彤; 李伟
Original assignee: Yangtze University
Current assignee: Yangtze University
Priority date: 2019-12-06
Filing date: 2019-12-06
Publication date: 2021-09-24
Anticipated expiration: 2039-12-06
Also published as: CN111172182A

Abstract

The invention discloses a phytophthora capsici PcMPK12 gene and a vector and application thereof, wherein the nucleotide sequence of the phytophthora capsici PcMPK12 gene is shown as SEQ ID NO.1, and the coded amino acid sequence is SEQ ID NO. 2; the mitogen protein kinase PcMPK12 gene of phytophthora capsici is cloned for the first time, and a transient over-expression vector and a silencing vector are constructed; the biological functions of the PcMPK12 gene are preliminarily explored by utilizing an agrobacterium-mediated transient expression system and a silencing vector, and the result shows that the phytophthora capsici PcMPK12 gene promotes pathogen infection and plays a positive regulation role in a pathogenic process; the silent carrier can reduce the growth and development of phytophthora capsici and reduce the pathogenicity of the phytophthora capsici.

Description

Phytophthora capsici PcMPK12 gene and vector and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a phytophthora capsici mitogen protein kinase PcMPK12 gene and a vector and application thereof.

Background

Phytophthora capsici (Phytophthora capsicii) is a plant pathogenic bacterium with a wide existence range and strong destructive power, and can infect various host plants. Phytophthora capsici usually overwinter in soil or disease residues as oospores, spreading the disease by wind, water and other agronomic activities. The phytophthora capsici caused by the phytophthora capsici infecting capsicum, which seriously occurs in countries and regions such as the united states, canada, korea, China and the like, all cause economic losses of different degrees, which forces the effective prevention and control of the phytophthora capsici to become the main task at present. Because the work progress of disease resistance and breeding of pepper phytophthora blight is slow, the prevention and control of diseases at the present stage mainly depend on chemical prevention and control, the drug resistance of phytophthora is continuously improved due to the large amount of chemical agents, and pesticide residue and environmental pollution are caused. Therefore, the search for the key pathogenic gene and pathogenic mechanism of phytophthora capsici becomes a research hotspot in recent years.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a phytophthora capsici mitogen protein kinase PcMPK12 gene and a vector and application thereof.

The invention is realized by the following steps:

one of the purposes of the invention is to provide a phytophthora capsici PcMPK12 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The invention also aims to provide the phytophthora capsici PcMPK12 protein coded by the phytophthora capsici PcMPK12 gene, wherein the amino acid sequence of the protein is shown as SEQ ID NO: 2, respectively.

The invention also aims to provide a recombinant vector containing the phytophthora capsici PcMPK12 gene.

Preferably, the recombinant vector is obtained by inserting the phytophthora capsici PcMPK12 gene into a BamHI enzyme cutting site and a HindIII enzyme cutting site of a pBINGFP2 expression vector by taking a pBINGFP2 expression vector as an initial vector.

More preferably, the recombinant vector takes phytophthora capsici cDNA as a template, and is shown in SEQ ID NO: 3-4, performing PCR amplification; and carrying out double enzyme digestion on the PCR product and the pBINGFP2 expression vector, recovering and purifying, and connecting and transforming escherichia coli to obtain a recombinant expression vector pBIN-PcMPK 12.

The fourth object of the present invention is to provide a transformant comprising the recombinant vector.

Preferably, the transformant is an agrobacterium comprising the recombinant vector. And (3) transforming the obtained agrobacterium tumefaciens strain containing the recombinant vector to obtain a plant over-expression strain, wherein the strain can express phytophthora capsici PcMPK12 protein.

The fifth purpose of the invention is to provide a silencing vector, which takes pBIN-PcMPK12 plasmid as a template, and has the nucleotide sequence shown in SEQ ID NO: 5-6, reversely inserting the PCR product into a pTOReGFP silent vector through EcoRI and EcoRV enzyme cutting sites, recovering and purifying after double enzyme cutting, and connecting and transforming the Escherichia coli to obtain a recombinant silent vector pTOR-PcMPK 12.

The invention also aims to provide the application of the phytophthora capsici PcMPK12 gene in regulation and control of pathogen infection.

The seventh purpose of the invention is to provide the application of the recombinant vector in regulating and controlling the infection of pathogens.

The eighth purpose of the invention is to provide the application of the silencing vector in regulating and controlling the infection of pathogens.

Compared with the prior art, the invention has the following advantages and effects:

the mitogen protein kinase PcMPK12 gene of phytophthora capsici is cloned for the first time, and a transient over-expression vector and a silencing vector are constructed; the biological functions of the PcMPK12 gene are preliminarily explored by utilizing an agrobacterium-mediated transient expression system and a silencing vector, and the result shows that the PcMPK12 gene promotes pathogen infection and plays a positive regulation role in a pathogenic process; the silencing vector (silencing PcMPK12 gene) can reduce the growth and development of phytophthora capsici (hypha side branches of two silencing transformants are increased, sporangium matures later, and spore activity is reduced, so that the spores cannot be completely released from the sporangium), which shows that the PcMPK12 gene silencing transformants can reduce the pepper infecting ability of phytophthora capsici and reduce the pathogenicity of phytophthora capsici.

Drawings

FIG. 1 shows the analysis of the transcription levels of Phytophthora capsici during growth and development and different infection periods;

FIG. 2 shows the subcellular localization and protein expression assays, A, PcMPK12 subcellular localization results and EV subcellular localization results; B. detecting a protein expression result by Western Blot;

FIG. 3 shows pathogen infection detection, and ultraviolet photography results of phytophthora capsici infection A; B. area statistics results;

FIG. 4 shows the growth rate and silencing efficiency of silenced transformants, and photographs of mycelia obtained by culturing A, wild strains, empty control transformants and silenced transformants in NPB medium at 25 ℃ in the dark for 5 days; B. the efficiency of silencing; C. area of hyphae;

FIG. 5 shows the phenotype analysis of silent transformants, and the observation results of hypha morphology of A, wild type hypha and silent transformant T2 after the hypha is cultured in NPB medium at 25 ℃ for 4 days in a climbing culture mode; B. after induction, observing the release state of zoospores of wild type and silent transformants;

FIG. 6 shows the pathogenicity test results of silent transformants;

FIG. 7 is a graph showing the results of measurements of hyphal growth under different stress conditions, wherein A is the hyphal growth of Wild Type (WT), control strain (CK) and PcMPK12 silent transformants on different media; b is the colony diameter of Wild Type (WT), control strain (CK) and PcMPK12 silenced transformants grown on different media for 5 days.

Detailed Description

Example 1 Phytophthora capsici PcMPK12 Gene cloning and sequence analysis

The invention relates to a phytophthora capsici PcMPK12 gene obtained by sequencing a phytophthora capsici LT1534 whole genome and carrying out a large amount of bioinformatics analysis and screening.

Then, taking phytophthora capsici LT1534 whole genome cDNA as a template, and amplifying a PcMPK12 gene sequence according to a corresponding PCR reaction system, wherein the reaction system is as follows: 25 μ L of 2 × Phanta Max Master Mix, 4 μ L DNA, 17 μ L ddH₂O, 2. mu.L Forward Primer (10. mu.M), 2. mu.L Reverse Primer (10. mu.M), 50. mu.L system. The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for several minutes (determined by the length of the cloned gene, about 1kb/min) for 32 cycles; extension at 72 ℃ for 10 min. The same method was used to clone a fragment of the PcMPK12 gene-specific sequence.

After the sequencing of a company, the total length of a PcMPK12 gene sequence is 1476bp, the ORF of an open reading frame is 1476bp, 491 amino acids are coded, and the molecular weight of the protein is about 55 KDa. The sequence was analyzed using a bioinformatics analysis website, and the results showed that the PcMPK12 gene had no signal peptide region and no transmembrane region.

Example 2 Phytophthora capsici PcMPK12 Gene expression Pattern analysis

Respectively collecting samples of phytophthora capsici LT1534 hypha, sporangium stage, zoospore, telosporium and germinal spore stage, and after phytophthora capsici LT1534 zoospore infects pepper leaves, respectively extracting RNA by adopting an OMAGN kit and carrying out reverse transcription by adopting a Norrespect kit to obtain cDNA (complementary deoxyribonucleic acid) at 1.5h, 3h, 6h, 12h, 24h, 48h and 72h, and carrying out qRT-PCR (quantitative reverse transcription-polymerase chain reaction) detection on the expression quantity of the PcMPK12 gene at different stages by adopting the cDNA at a mycelium stage as a control and Phytophthora capsici Actin as an internal reference gene. Taking the mean value of the results of the three qRT-PCR and carrying out variance analysis.

The results are shown in FIG. 1, and bar graphs on the abscissa are the hypha (MY), Sporangium (SP), Zoospore (ZO), resting spore (CY), germinating spore (GC), infected host 1.5h, infected host 3h, infected host 6h, infected host 12h, infected host 24h, infected host 48h and infected host 72h at each stage of the life history; the ordinate is the result of the PcMPK12 transcription level analysis, and the Phytophthora capsici Actin gene is used as an internal reference; expression pattern analysis shows that the phytophthora capsici PcMPK12 gene has the most obvious up-regulation in zoospore and aposporium stages at the early development stage, then the expression level gradually decreases, and slightly increases after 72 hours at the late infection stage.

EXAMPLE 3 construction of the vector

Construction of PcMPK12 Gene overexpression vector: according to the cDNA sequence (SEQ ID NO: MN577461) of phytophthora capsici PcMPK12 gene, a pair of specific primers pBIN-PcMPK 12-F (shown in SEQ ID NO: 3) and pBIN-PcMPK 12-R (shown in SEQ ID NO: 4) are designed, and BamH I and Hind III enzyme cutting sites are respectively added to the upstream primer and the downstream primer. PCR amplification was performed using Phytophthora capsici cDNA as a template and pBIN-PcMPK 12-F and pBIN-PcMPK 12-R as primers. And carrying out double enzyme digestion on the PCR product and the expression vector pBINGFP2, recovering and purifying, and connecting and transforming Escherichia coli DH5 alpha to obtain a recombinant expression vector pBIN-PcMPK 12.

Example 4 Phytophthora capsici PcMPK12 subcellular localization analysis

The transient overexpression vector pBIN-PcMPK2 (prepared in example 3) was prepared by a freeze-thaw method: PcMPK12 is transformed into agrobacterium-infected GV3101 by the following method:

(1) 30 mu L of agrobacterium-infected state is added into a centrifuge tube containing the recombinant plasmid, and ice bath is carried out for 30min after the mixture is gently sucked, beaten and mixed uniformly.

(2) Quick freezing with liquid nitrogen for 1min, and water-bathing in 37 deg.C water bath for 5 min.

(3) Adding 500. mu.L of nonresistant liquid LB, and shake-culturing at 28 ℃ and 200rpm in a shake incubator for 3-5 h.

(4) Centrifuging at low speed for 2min, and uniformly spreading the centrifuged bacteria solution in solid LB culture medium containing Rif and Kana resistance for 2 days in dark.

(5) When the appearance of the monoclonal colony in the solid LB is observed, the monoclonal bacterial plaque is dipped into 1mL of liquid LB culture medium containing Rif and Kana resistance and cultured for 10h-18h with shaking.

(6) And (3) sucking bacterial liquid, carrying out colony PCR (polymerase chain reaction) test by using a carrier primer, and if the test is correct, successfully transforming, and storing the bacterial liquid for subsequent experiments.

Culturing the agrobacterium liquid containing the recombinant plasmid for 16-18 h, centrifugally collecting the liquid, adding a proper amount of 10mM MgCl₂Suspending the thallus in a buffer solution (containing 10mM MES and 200 mu MAs) to make the OD600 value of the suspension reach 0.5-0.6; and (3) inoculating the suspension into Nicotiana benthamiana for 48h by using a syringe, and observing the subcellular localization condition of the Nicotiana benthamiana by using an ultrahigh-resolution confocal microscope ZEISS LSM 800.

Subcellular localization as shown in FIG. 2-A, Phytophthora capsici PcMPK12 localized to the cytoplasmic membrane of tobacco lamina cells; EV subcellular (control) localization to cytoplasm and nucleoplasm.

Example 5 Western blot detection and pathogen infection detection of PcMPK12 in plant overexpression strains

1. Selecting the Nicotiana benthamiana with good growth vigor of 4-6 weeks, and utilizing an agrobacterium transient expression technology (extracting plasmid from the recombinant expression vector pBIN-PcMPK12 obtained in the embodiment 3, and transforming an agrobacterium Gv3101 expression strain) to obtain a plant over-expression strain, so that the PcMPK12 gene is transiently expressed in tobacco leaves.

2. In order to verify that PcMPK12 can be normally expressed in tobacco, after 48 hours, the tobacco is irradiated by a long-wave ultraviolet lamp under dark conditions, leaves with high fluorescence degree are selected, liquid nitrogen is used for grinding, protein is extracted for Western Blot detection, and the detection result is shown in figure 2-B. The results show that the target band is about 82kDa, the GFP control is 28kDa, and the lower part is the staining result of the corresponding reference ginseng Actin ponceau. The fact that the phytophthora capsici PcMPK12 and the GFP protein can be normally expressed in the Nicotiana benthamiana is shown.

The Western Blot detection method is as follows:

(1) picking up the Nicotiana benthamiana leaves after 2 days of agrobacterium transient expression, grinding the Nicotiana benthamiana leaves into powder by using liquid nitrogen, collecting about 100mg of leaf powder into a 2mL centrifuge tube, adding 650 mu L of 2 multiplied sample buffer solution, reversing the upside and mixing the mixture evenly, and placing the mixture on ice.

(2) Boiling the sample in boiling water for 5min, shaking, mixing, and boiling again for 5 min.

(3) Centrifuging at 12000rpm for 5min at room temperature, sucking supernatant into a new centrifuge tube, centrifuging again for 5min, and completely removing precipitate.

(4) The supernatant was aspirated into a new centrifuge tube, ready for loading, and the remaining-20 ℃ was stored.

(5) SDS-PAGE was performed, and 20-40. mu.L of sample and 5-8. mu.L of Marker were added to each lane, and the voltage was adjusted to 150V for about 2 hours.

(6) After electrophoresis, the soaked protein gel and the PVDF membrane are placed in a membrane rotating instrument for 230A constant flow membrane rotation, and the time is about 1.5 h.

(7) After the membrane transfer is finished, the PVDF membrane is taken down to be placed in a square box, TBST is added for washing for 3-5 times (a horizontal shaking table), and each time lasts for about 5 min.

(8) Then 25mL of 5% skimmed milk powder blocking solution was added to the box and blocked at room temperature for 1.5-2h (horizontal shaker).

(9) 25mL of monoclonal antibody to Invitrogen, mouse Anti-GFP (3000: 1) was added and incubated for 90min (horizontal shaker).

(10) Washing with TBST for 5min for 4 times after incubation; 25mL of a rabbit anti-mouse secondary antibody (10000: 1) from Invitrogen was added and reacted at room temperature for 1h (horizontal shaker).

(11) After the reaction was completed, the reaction mixture was washed 3 times with TBST for 5min each.

(12) Adding nunoprazan developing solution (1:1 ratio), and taking pictures by ECL developing.

3. Pathogen infection detection

In order to verify that the PcMPK12 can play a role in the phytophthora capsici infection process, selecting the Benzenbachium which grows for 4-6 weeks and has good growth vigor, utilizing the agrobacterium transient expression technology in example 3, respectively inoculating PcMPK12 and GFP to two sides of a tobacco leaf, inoculating a phytophthora capsici LT1534 block for 24 hours, carrying out dark treatment in an incubator at 25 ℃ for about 2 days, measuring the infection diameter, taking a picture under an ultraviolet lamp, and carrying out ultraviolet photography on the phytophthora capsici infection result as shown in FIG. 3A; area statistics as shown in figure 3B, t-test, the difference was very significant at P <0.05 level. The results thus show that PcMPK12 promotes pathogen infestation, playing a positive regulatory role in the pathogenic process.

Example 6 construction and biological analysis of Phytophthora capsici PcMPK12 silent transformants

1. Construction of recombinant silencing vector pTOR-PcMPK12

Specific fragment primers, pTOR-PcMPK12- -F (shown in SEQ ID NO: 5) and pTOR-PcMPK12- -R (shown in SEQ ID NO: 6), were designed based on the cDNA sequence of the PcMPK12 gene, to which EcoR I and EcoR V cleavage sites were added at the upstream and downstream primers, respectively. PCR amplification was performed using pBIN-PcMPK12 plasmid as a template and pTOR-PcMPK12- -F and pTOR-PcMPK12- -R as primers. And reversely inserting the PCR product into a pTOReGFP silencing vector, carrying out double enzyme digestion, recovering and purifying, and connecting and transforming Escherichia coli DH5 alpha to obtain a recombinant silencing vector pTOR-PcMPK 12.

2. Preparation of silent transformants

Extracting the plasmid of the recombinant silencing vector pTOR-PcMPK12, transforming phytophthora capsici protoplast, and screening a silencing transformant, specifically: PEG-mediated protoplast transformation: comprises three parts of protoplast preparation, protoplast transformation and protoplast regeneration.

A. Preparation of protoplast:

(1) selecting phytophthora capsici LT1534 with better growth vigor on an NPB solid culture medium, punching a circle of small holes along the outer edge of a flat plate by using a puncher with the diameter of 5mm, selecting 10-15 fungus cakes, transplanting the fungus cakes into a sterile culture dish, adding 35mL of liquid NPB culture medium, carrying out dark culture in an incubator at 25 ℃ for 2 days, taking out the fungus cakes for later use, wherein the culture time is too long, and the subsequent plasmolysis can be influenced by over-aged thalli.

(2) The liquid NPB culture in each dish was poured into a waste vat and rinsed once with 25-30mL of sterile deionized water, optionally without removing agar medium.

(3) Rinsed once more with 25-30mL of 0.8M mannitol.

(4) Transferring the mycelium to deep culture dish, adding 30mL of 0.8M mannitol, soaking at room temperature for 10min to allow plasmolysis, and dispersing the mycelium with sterile scalpel to improve plasmolysis efficiency.

(5) During soaking for 10min, preparing enzymolysis solution and PEG4000, standing at room temperature, filtering PEG4000 with 0.45 μm bacterial filter for sterilization, and standing at 4 deg.C.

(6) And after soaking, removing the filtrate, collecting hyphae, adding about 20mL of enzymolysis liquid, and fully dispersing the hyphae to ensure that the enzymolysis effect is optimal.

(7) The petri dish was closed with tinfoil paper and gently shaken at 50rpm for 40-60min at room temperature (horizontal shaker), and the enzymatic effect was determined by observing the amount of released protoplasts under a light mirror.

(8) The enzymatic product was filtered through a 70 μm BD Falcon Cell filter, the media residue and hyphal debris were discarded, and the protoplast filtrate was collected in a 50mLBD tube.

(9) 50mL of BD was placed in a horizontal centrifuge and centrifuged at 1200 Xg for 2min at room temperature.

(10) The filtrate was discarded, and 30mL of W5 solution (5mM KCl 0.093g, 125mM CaCl) was added₂·2H₂O4.6g, 154mM NaCl 2.25g, 177mM Glucose7.8g, and water to 250mL) and washed the protoplasts, and for the sake of resuspension, 15mLW5 solution was added to resuspend the protoplasts, and then 15mL was added to make up to 30 mL.

(11) The solution was centrifuged at 1200 Xg for 2min at room temperature to remove W5.

(12) According to the amount of the vector plasmid sample, an appropriate amount of 10mL MMG solution (0.4mM Mannitol 18.22g, 15mM MgCl) was added₂·6H₂O0.76 g, 4Mm MES 2ml, adding water to reach the volume of 250ml), resuspending the protoplast, and standing at room temperature for 10min to complete the preparation of the protoplast.

B. Protoplast transformation test method:

(1) add 20. mu.L of recombinant silencing vector pTOR-PcMPK12 and 1mL of the protoplast to a 50mL BD tube, mix them by gentle shaking, but not shake vigorously, and then ice-wash for 10-20 min.

(2) PEG4000 stored at 4 ℃ was removed and 1.74mL of PEG4000 was added slowly in three portions (0.58 mL each) to a 50mL BD tube and gently shaken to mix it.

(3) After ice-cooling for 15min, 25mL of liquid PM medium containing Amp (50. mu.g/. mu.L) resistance was added (pre-chilled at 4 ℃ in advance).

(4) After ice-bath for 10min, culturing in a constant temperature incubator at 25 ℃ for 12-18h in the dark (inclined placement).

C. Protoplast regeneration test method:

(1) the protoplasts were removed from the incubator and centrifuged at 2000 Xg for 2min at room temperature.

(2) The supernatant was discarded and approximately 10ml of the solution left to resuspend the protoplasts.

(3) To each 50mL BD tube was added 25mL of solid PM medium containing (G418, Rif 50. mu.g/. mu.L) antibiotics and mixed by gentle inversion.

(4) The PM medium in each BD tube was poured into two sterilized petri dishes on average and incubated at 25 ℃ in the dark in an incubator.

C. Validation of silent suspected transformants:

colonies were grown on the plates after the regeneration, each colony was a suspected transformant, and the colonies were picked up in solid NPB medium containing G418 (50. mu.g/. mu.l) antibiotic and cultured in the dark at 25 ℃ for 5 days. Selecting suspected transformants with good growth vigor on an NPB solid culture medium for transferring and backing up, cutting the rest into small blocks, adding 35mL of liquid NPB culture medium for dark culture for 5 days, taking out, collecting hyphae, extracting total RNA, then designing a primer, testing the silencing efficiency through qRT-PCR, and repeating the experiment for three times by taking LT1534 as a control, namely measuring the relative expression of PcMPK12 in a wild strain, an empty control transformant and a silencing transformant (T2 and T19) through SYBR real-time quantitative RT-PCR to evaluate the silencing efficiency, wherein the silencing efficiency reaches about 80 percent, and the silencing success is determined. Two silent transformants with a silencing efficiency of more than 80% and one empty transformant were obtained by final selection (FIG. 4-B).

3. Phytophthora capsici PcMPK12 silent transformant biological analysis

(1) PcMPK12 silent transformant growth rate analysis

And transferring the phytophthora capsici LT1534 strain, the empty vector transformant and the PcMPK12 silent transformant to a new antibiotic-free solid NPB culture medium at the same time, repeating each strain for 15 times, carrying out dark inversion culture in a constant temperature incubator at 25 ℃ for 5 days, observing the growth condition of colonies, measuring the colony diameter of each plate, photographing and recording, and processing the data of the colony diameter. The experimental results are shown in fig. 4A and 4C. The results show that the growth rate of the silent transformant colonies is significantly reduced compared to phytophthora capsici LT1534 and the empty vector transformants.

(2) PcMPK12 silences transformant hypha morphology and sporangium morphology

The well-cultured phytophthora capsici LT1534 and the insert plate of the transformant thereof are taken out, the slide is taken out of the culture medium by using tweezers, a temporary slide is made, a permanent slide can be made for subsequent observation, and DIC pictures are observed and taken under an OLYMPUS BX-53 fluorescence microscope at 40 x. And then punching the flat plate, washing the flat plate for several hours by using deionized water, placing the flat plate at a low temperature for 6-8 hours to induce the production of sporangia, picking hypha on a glass slide by using a toothpick, making a temporary glass slide, observing the morphology of the sporangia under a microscope at 40 times, taking a DIC picture quickly, continuously placing the flat plate in a low-temperature incubator after the hypha is picked, and preventing the sporangia from breaking at room temperature to release zoospores so as to influence the observation effect on the sporangia. The results of the experiment are shown in FIG. 5. The results show that compared with the LT1534 strain and the empty vector transformant, hyphal side branches of two silent transformants are increased, the sporangium is matured later, the spore activity is reduced, and the sporocysts cannot be completely released from the sporangium, which indicates that the PcMPK12 is important for the growth and development of phytophthora capsici.

(3) Pathogenicity of phytophthora capsici PcMPK12 silent transformant to pepper

And selecting a phytophthora capsici LT1534 strain with consistent growth vigor, an empty vector transformant and a silent transformant, and punching a bacterial cake by using a 5mm puncher for inoculation. Selecting pepper seedlings growing for about 5 weeks, pricking small holes on leaves with the same growth vigor by using a syringe needle, and inoculating a bacterium block for infection. 3 replicates were performed using P.capsorum LT1534 strain as a control and 8 leaves per large dish. After 2 days, symptoms were observed and recorded by photographing, and then ultraviolet observation and trypan blue staining observation were performed. The results of photographs of non-wounded (upper row) and wounded (lower row) pieces of the wild strain, empty control transformant and silent transformant (T2 and T19) inoculated with dark-cultured pepper leaves stained and decolorized with trypan blue are shown in FIG. 6.

As can be seen from FIG. 6, compared with the strain LT1534 and the empty vector transformant, the silent transformant can not infect the leaves of the atraumatic pepper, and water stain-like lesions are left on the leaves of the wounded pepper, which indicates that the PcMPK12 silent transformant has reduced capability of infecting the pepper, and the pathogenicity of Phytophthora capsici is reduced.

Example 7 Phytophthora capsici PcMPK12 silent transformant stress assay

To determine whether PcMPK12 is stress-related, its expression was studied under oxidative, osmotic and salt stress. Transferring the phytophthora capsici LT1534 strain, the empty vector transformant and the PcMPK12 silent transformant to a new stressed solid NPB culture medium at the same time, and respectively adding H₂O₂(2.0, 4.0 and 6.0M M), sorbitol (0.5M, 1.0M, 1.5M) and NaCl (0.2M, 0.4M, 0.6M). Each strain is subjected to 15 repetitions, each experiment is subjected to three repetitions, error bars indicate standard deviation, and after the strains are subjected to dark inversion culture in a constant-temperature incubator at 25 ℃ for 5 days, the growth conditions of observed colonies are shown in FIG. 7A; after 5 days of growth, the colony diameter of each plate is measured and photographed for recording, and the data of the colony diameter is processed as shown in FIG. 7B, and the results are divided into 9 independent experiments, wherein the 9 independent experiments are sequentially H on the abscissa from left to right₂O₂(2.0, 4.0 and 6.0M M), sorbitol (0.5M, 1.0M, 1.5M) and NaCl (0.2M, 0.4M, 0.6M), with each independent experiment including WT, CK, T2, T19.

As can be seen in FIG. 7, the growth rate of the silenced transformants was significantly reduced under each treatment, indicating that Phytophthora capsici PcMPK12 is associated with osmotic stress and various types of stress.

The invention is not to be considered as limited to the particular embodiments shown, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> university of Yangtze river

<120> Phytophthora capsici PcMPK12 gene, and vector and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

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<211> 1476

<212> DNA

<213> Phytophthora capsici (Phytophthora capsicii)

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ccaagagacc tccgcgagat gtacccaggt gcgcctgctg attcgctcga cttgctgaag 1260

caaatgctgt cttttaaccc cgagagccga atttcggtcg acaaagccct tgcgcaccct 1320

ttcttggagt cagtacgaag gacccagtct gagactgtag aaggcaatcc ttttggcatg 1380

gagttcgaaa acgtccccct gaacaaggag gccctgaaag gtttgttaaa ctgcattttt 1440

tgtattgatg gtatctatga tattaacatc aactga 1476

<210> 2

<211> 491

<212> PRT

<213> Phytophthora capsici (Phytophthora capsicii)

<400> 2

Met Pro Pro Thr Pro Ala Phe Val Asn Arg Asn Gly Phe Ser His Thr

1 5 10 15

Pro Asp Gly Phe Ser Ser Thr Pro Ser Asp Gly Phe Pro Ser Asn Ser

20 25 30

Gly Val Ala Gln Gln Ala Ser Thr Pro Ala Asp Asp Asn Gln Thr Ala

35 40 45

Gly Ala Thr Ser Asn Gly His Ser Ser His Ala Pro Thr Pro Ser Pro

50 55 60

Val Pro Arg Ala Ala Pro Leu Asn Leu Arg Asn Phe Ala Asn Trp Asp

65 70 75 80

Val Gly Ser Arg Tyr Thr Leu Val Arg Leu Leu Gly Lys Gly Ser Tyr

85 90 95

Gly Gln Val Ala Glu Ala Phe Asp Asn Glu Arg Gln Lys Lys Val Ala

100 105 110

Ile Lys Lys Ile Ile Asn Val Phe Asp Gln Glu Ile Asp Cys Lys Arg

115 120 125

Leu Tyr Arg Glu Ile Tyr Ile Leu Arg Arg Leu Ser His Pro Gln Val

130 135 140

Ile Asn Leu Ile Asp Val Val Pro Pro Glu Asn Tyr Asp Thr Phe Thr

145 150 155 160

Asp Leu Tyr Leu Val Phe Asp Phe Val Asp Thr Asp Leu His Lys Leu

165 170 175

Ile Met Ser Pro Gln Tyr Leu Thr Ile Arg His Ile Gln Val Phe Leu

180 185 190

Tyr Gln Leu Leu Cys Gly Leu Lys Tyr Ile His Ser Ala Asn Val Ile

195 200 205

His Arg Asp Met Lys Pro Ala Asn Ile Leu Leu Asn Glu Asp Cys Thr

210 215 220

Leu Met Ile Cys Asp Phe Gly Leu Ser Arg Val Met Glu Ser Asp Met

225 230 235 240

Ser Met Glu Glu Leu Ser Lys His Leu Tyr Ser Pro Lys Asp Ser Lys

245 250 255

Ser Pro Thr Thr Ser Asp Gly Gly Thr Thr Ser Ala Pro Ser Pro Gly

260 265 270

Ser Ala Ser Ser Thr Pro Ser Ser Ser Gly Glu Phe Pro Lys Met Arg

275 280 285

Arg Gln Leu Thr Lys His Val Val Thr Arg Trp Tyr Arg Ala Pro Glu

290 295 300

Leu Ile Leu Leu Gln Asp Tyr Asp Phe Ser Val Asp Met Trp Ser Ile

305 310 315 320

Gly Cys Ile Phe Ala Glu Leu Leu Ser Met Gln Val Glu Ser Cys Pro

325 330 335

Arg Tyr Gln Glu Arg Val Pro Leu Phe Pro Gly Arg Ser Cys Phe Pro

340 345 350

Leu Ser Ala Asp Arg Pro Thr Thr Tyr Ser Asp Lys Leu Asp Gln Leu

355 360 365

Asn Val Ile Phe Asn Val Ile Gly Thr Pro Gly Glu Asp Asp Ile Gly

370 375 380

Ser Leu Gly Glu Val Lys Gln Tyr Leu Arg Lys Leu Pro Lys Lys Asp

385 390 395 400

Pro Arg Asp Leu Arg Glu Met Tyr Pro Gly Ala Pro Ala Asp Ser Leu

405 410 415

Asp Leu Leu Lys Gln Met Leu Ser Phe Asn Pro Glu Ser Arg Ile Ser

420 425 430

Val Asp Lys Ala Leu Ala His Pro Phe Leu Glu Ser Val Arg Arg Thr

435 440 445

Gln Ser Glu Thr Val Glu Gly Asn Pro Phe Gly Met Glu Phe Glu Asn

450 455 460

Val Pro Leu Asn Lys Glu Ala Leu Lys Gly Leu Leu Asn Cys Ile Phe

465 470 475 480

Cys Ile Asp Gly Ile Tyr Asp Ile Asn Ile Asn

485 490

<210> 3

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgggatccat gcctcctacg cctgcgttt 29

<210> 4

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cccaagcttt cagttgatgt taatatcata g 31

<210> 5

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cggaattcca ggtgcgcctg ctgattc 27

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcgatatctc agttgatgtt aatatcatag 30

Claims

1. Phytophthora capsiciPcMPK12The gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. Phytophthora capsici according to claim 1PcMPK12The gene coded phytophthora capsici PcMPK12 protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO: 2, respectively.

3. A microorganism comprising the Phytophthora capsici according to claim 1PcMPK12Recombinant vectors of genes.

4. The recombinant vector of claim 3, wherein the recombinant vector uses pBINGFP2 expression vector as starting vector, and the phytophthora capsici of claim 1 is usedPcMPK12The gene is inserted into the expression vector pBINGFP2BamH IAndHind IIIobtained between the cleavage sites.

5. The recombinant vector according to claim 4, wherein the recombinant vector uses Phytophthora capsici cDNA as a template, and has the sequence shown in SEQ ID NO: 3-4, performing PCR amplification; and carrying out double enzyme digestion on the PCR product and the pBINGFP2 expression vector, recovering and purifying, and connecting and transforming escherichia coli to obtain a recombinant expression vector pBIN-PcMPK 12.

6. A non-plant transformant comprising the recombinant vector according to any one of claims 3 to 5.

7. A silencing vector, characterized in that, pBIN-PcMPK12 plasmid is used as a template, and the plasmid is shown as SEQ ID NO: 5-6, and passing the PCR product throughEcoR IAndEcoR Vthe reverse direction of the restriction enzyme cutting site is inserted into pTOReGFP silent vector, after double restriction enzyme cutting, recovery and purification are carried out, and the recombinant silent vector pTOR-PcMPK12 is obtained by connecting and transforming Escherichia coli.

8. Use of the silencing vector of claim 7 for regulating phytophthora capsici infestation on plants.