Gene plcamk1 and application thereof in preventing and treating li-chi downy mildew

Abstract

The application discloses a gene PlCaMK1 and application thereof in prevention and treatment of litchi downy mildew, and belongs to the technical field of green prevention and control of crop diseases.The application applies a protoplast transformation technology based on polyethylene glycol and a CRISPR / Cas9 knockout strategy, constructs a PlCaMK1 knockout vector, and uses PEG-mediated protoplast transformation and a homologous recombination method to knockout the gene PlCaMK1 from litchi downy mildew, so as to obtain a knockout mutant.Compared with a wild type, the mutant has basically consistent growth rate, obviously weakened motility of zoospores, and significantly improved germination rate of chlamydospores.The application proves that PlCaMK1 is necessary for growth and development and pathogenic infection of litchi downy mildew.The research is helpful to further clarify the pathogenic molecular mechanism of litchi downy mildew, and provides a target gene for development of an effective fungicide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of green prevention and control of crop diseases, and particularly relates to a gene PlCaMK1 and application thereof in prevention and treatment of litchi downy mildew caused by Peronophythora litchii. BACKGROUND

[0002] The disease caused by the infection of Peronophythora litchii Chen ex Ko et al. is litchi downy mildew, and the host plant is litchi. The pathogen belongs to the subphylum of flagellum, the class of oomycetes, the order of peronosporales, and the family of peronophthoraceae. The litchi downy mildew caused by Peronophythora litchii is most harmful to litchi, and no effective litchi planting variety resistant to the disease has been found. At present, the prevention and treatment of Peronophythora litchii still mainly relies on pesticide control, which has a high risk of drug resistance and pollutes the environment. Therefore, it is of great significance to study the pathogenic mechanism of Peronophythora litchii.

[0003] Studies have shown that the successful infection of Peronophythora litchii on litchi mainly depends on a series of pathogenic factors. Therefore, fully excavating the pathogenic related genes of litchi and carrying out functional research have important significance for effectively controlling the harm of Peronophythora litchii and breeding disease-resistant varieties. CaMKI is a monomer enzyme with wide substrate specificity and can phosphorylate many different proteins. In oomycetes, there are few reports on Ca2+ / calmodulin kinases. Homologous proteins of CaMK1 have been identified in Phytophthora palmivora and P. capsici. After the treatment of P. capsici with a fungicide-cinnamic aldehyde acid, the expression of CAMK / CAMK1 protein kinase is down-regulated, which makes the mycelial morphology of P. capsici change and disturbs the polysaccharide metabolism of the fungus. SUMMARY

[0004] In view of the defects or deficiencies in the prior art, a primary purpose of the present application is to provide a pathogenic related protein PlCaMK1 of Peronophythora litchii.

[0005] Another purpose of the present application is to provide a biological material related to the pathogenic related protein PlCaMK1 of Peronophythora litchii.

[0006] Another purpose of the present application is to provide an application of the pathogenic related protein PlCaMK1 or the biological material of Peronophythora litchii.

[0007] In order to achieve the above-mentioned purposes of the application, the following technical solutions are adopted in the present application:

[0008] The application discloses a previously unknown protein PlCaMK1 of Peronophythora litchii and a coding gene PlCaMK1 thereof. The full length of the gene PlCaMK1 is shown as SEQ ID NO. 1, the CDS sequence thereof is shown as SEQ ID NO. 2, and the coding gene encodes 747 amino acids shown as SEQ ID NO. 3. The application constructs pBSSK::PlCaMK1 and PYF2.3G-ribo-sgRNA1::PlCaMK1 and PYF2.3G-ribo-sgRNA2::PlCaMK1 knockout vectors by using a CRISPR / Cas9 gene editing technology, and knocks out the PlCaMK1 gene by a PEG-mediated protoplast transformation technology. Finally, knockout mutants T4, T22 and T40 (named according to the order of the transformant verification) are obtained, and the three mutants have obvious defects in the pathogenic process of Peronophythora litchii. The pathogenicity determination result shows that the pathogenicity of the knockout mutants T4, T22 and T40 of the gene PlCaMK1 to litchi tender leaves (variety: Guwei) is significantly reduced. The above test proves that the PlCaMK1 gene of Peronophythora litchii is a pathogenicity-related gene of Peronophythora litchii.

[0009] A pathogenicity-related protein PlCaMK1 of Peronophythora litchii, the amino acid sequence of which is shown as SEQ ID NO. 3, or an analogous sequence shown as SEQ ID NO. 3 obtained by one or more amino acid substitutions, insertions or deletions and still having the same or similar functions.

[0010] The biological material related to the pathogenicity-related protein PlCaMK1 of Peronophythora litchii is any one or a combination of the following biological materials:

[0011] 1) a nucleic acid molecule encoding the pathogenicity-related protein PlCaMK1 of Peronophythora litchii;

[0012] 2) an expression cassette containing the nucleic acid molecule in 1);

[0013] 3) a recombinant vector containing the nucleic acid molecule in 1), or a recombinant vector containing the expression cassette in 2);

[0014] 4) a recombinant microorganism containing the nucleic acid molecule in 1), or a recombinant microorganism containing the expression cassette in 2), or a recombinant microorganism containing the recombinant vector in 3);

[0015] 5) a nucleic acid molecule for inhibiting or blocking the gene expression of the pathogenicity-related protein PlCaMK1 of Peronophythora litchii;

[0016] 6) a gene knockout vector prepared by using the nucleic acid molecule in 5) for inhibiting or blocking the gene expression of the pathogenicity-related protein PlCaMK1 of Peronophythora litchii;

[0017] 7) the pathogenicity-related protein PlYPK1 of P. litchi, which is prepared by using the gene knockout vector as described in 6).

[0018] Further, the nucleic acid molecule as described in 1) is the gene sequence of the pathogenicity-related protein PlCaMK1 of P. litchi, as shown in SEQ ID NO: 1, or the CDS sequence of the pathogenicity-related protein PlCaMK1 of P. litchi, as shown in SEQ ID NO: 2, or a similar sequence having the same or similar function as SEQ ID NO. 1 or SEQ ID NO: 2, which is obtained by base insertion, deletion, or substitution.

[0019] Further, the nucleic acid molecule as described in 5) is the antisense RNA, siRNA, shRNA, or sgRNA of PlCaMK1 gene.

[0020] Further, the sgRNA is as shown in any one of the following sequences:

[0021] sgRNA1: 5'-CACTACCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGTAGTGTCTCTGGAGATCTC-3';

[0022] sgRNA2: 5'-TAGAACCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGTTCTAGCGCTTGTGGGTTA-3'.

[0023] The pathogenicity-related protein PlCaMK1 of P. litchi or the biological material described above is used for any one or a combination of the following applications:

[0024] i) application in regulating the pathogenicity of P. litchi;

[0025] ii) application in regulating the infection ability of P. litchi;

[0026] iii) application in regulating the motility of zoospores of P. litchi;

[0027] iv) application in regulating the formation of zoospores of P. litchi;

[0028] v) application in regulating the germination of resting spores of P. litchi;

[0029] vi) application in preventing and treating P. litchi downy mildew;

[0030] vii) application as a target in designing and screening anti-P. litchi drugs.

[0031] A method for preventing and treating litchi downy mildew caused by Peronophythora litchii by inhibiting or blocking the gene expression of a pathogenicity-related protein PlCaMK1 of the Peronophythora litchii.

[0032] A drug screening model against Peronophythora litchii, which is a gene-deficient Peronophythora litchii of a pathogenicity-related protein PlCaMK1 of the Peronophythora litchii.

[0033] A method for constructing the drug screening model against Peronophythora litchii, comprising the following steps:

[0034] (1) According to the sgRNA website, an sgRNA is designed according to the PlCaMK1 gene sequence, the sgRNA is connected with a pYF2.3G-Ribo-sgRNA vector to obtain a PlCaMK1 gene knockout plasmid pYF2.3G-Ribo-sgRNA::PlCaMK1.

[0035] Alternatively, left and right homologous arm amplification primers are designed according to the sequences of about 1 kb upstream and downstream of the PlCaMK1 gene sequence, and the left and right homologous arms are amplified from the Peronophythora litchii genomic DNA as a template and connected with a pBSSK vector to obtain a PlCaMK1 gene knockout plasmid pBSSK::PlCaMK1.

[0036] (2) The PlCaMK1 gene knockout plasmid pYF2.3G-Ribo-sgRNA::PlCaMK1 or pBSSK::PlCaMK1 is introduced into protoplasts of a wild-type strain of Peronophythora litchii, and a PlCaMK1 gene knockout mutant is obtained through screening and verification, which is the drug screening model against Peronophythora litchii.

[0037] Further, the sgRNA in step (1) is as shown in any one of the following sequences:

[0038] sgRNA1: 5'-CACTACCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGTAGTGTCTCTGGAGATCTC-3';

[0039] sgRNA2: 5'-TAGAACCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGTTCTAGCGCTTGTGGGTTA-3'.

[0040] Further, the left and right homologous arm amplification primers in step (1) are as shown in the following respectively:

[0041] Left homologous arm amplification primer:

[0042] PlCaMK1-Left-F: 5'-CTAGAACTAGTGGATCCCCCTGTCGTAACGCAAAGGAAGT-3';

[0043] PlCaMK1-Left-R: 5'-GTATAATACAACAAACAGATTTTAGTGGAAACTTGCTCGA-3';

[0044] Right homologous arm amplification primer:

[0045] PlCaMK1-Right-F: 5'-TCGAGCAAGTTTCCACTAAAATCTGTTTGTTGTATTATAC-3';

[0046] PlCaMK1-Right-R: 5'-ATATCGAATTCCTGCAGCCCTTCGTTAGTTCCTTACTTTC-3'.

[0047] The present application has the following advantages and effects relative to the prior art:

[0048] The present application is proved by experiments, the upstream and downstream sequences of the gene PlCaMK1 are amplified respectively, and are connected with the vector pBSSK (donated by the Oomycetes and Fungal Molecular Biology Laboratory of the Plant Protection College of Nanjing Agricultural University), and after homologous recombination, the protoplast transformation technology mediated by polyethylene glycol (PEG) and the CRISPR / Cas9 knockout strategy are used, the mutant obtained has no obvious difference in growth rate, sporangium and oospore yield compared with the wild type, and the motility test of zoospores shows that the deletion of the gene PlCaMK1 significantly reduces the motility of the zoospores of Peronophythora litchii. The present application proves that the gene PlCaMK1 is necessary for the pathogenicity of Peronophythora litchii. Our research helps to further clarify the pathogenic molecular mechanism of Peronophythora litchii, and provides a theoretical basis for discovering new drug action targets and designing new high-efficiency, low-toxicity and safe fungicides. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is a schematic diagram of homologous recombination of the PlCaMK1 gene of Peronophythora litchii; wherein, A is a schematic diagram of construction of the pYF2.3G-Ribo-sgRNA::PlCaMK1 vector and the pBSSK::PlCaMK1 vector, B is a schematic diagram of design of PlCaMK1 gene knockout, and C is a sequencing result of the knockout mutant.

[0050] Figure 2Fig. 1 is a PCR amplification result diagram of the litchi downy mildew PlCaMK1 gene knockout transformant; wherein, lane WT: litchi downy mildew wild type; lane CK: transformant by PEG but not knocked out; lanes T4, T22, T40 respectively represent three knocked out transformants of the gene PlCaMK1; wherein: a is the PCR amplification result; b is the statistical result.

[0051] Figure 3 Fig. 2 is a growth rate and colony morphology diagram of the wild type WT, non-knocked out transformant CK, knockout mutant T4, T22, T40 on CA medium; wherein: a is the colony morphology diagram; b is the growth rate statistical result.

[0052] Figure 4 Fig. 3 is a zoospore motility analysis result diagram of the PlCaMK1 knockout mutant T4, T22, T40; wherein: a is the number change of wild type WT and Δplcamk1 zoospores, cysts and germination cysts at 10 min, 20 min, 40 min, 1 h, 2 h, and the scale in the diagram is 200 μm; b is the spore number statistics of the litchi downy mildew wild type strain at different times; c is the spore number statistics of the PlCaMK1 knockout mutant at different times.

[0053] Figure 5 Fig. 4 is a pathogenicity analysis result diagram of the PlCaMK1 knockout mutant; wherein: a is the disease spot of the wild type WT, CK and three PlCaMK1 mutants (T4, T22 and T40) zoospore suspension directly dropped on litchi leaves; b is the spot diameter statistics of a; c is the disease spot of the WT, CK and PlCaMK1 mutant infected by zoospore motility; d is the spot diameter statistics of c.

[0054] Figure 6 Fig. 5 is a germination analysis result diagram of the PlCaMK1 knockout mutant cysts; wherein: a is the observation of the cyst germination suspension respectively prepared from the wild type WT, CK and three PlCaMK1 knockout mutants (T4, T22 and T40) under a microscope, and the arrow indicates the cyst germinating into a germ tube, and the scale in the diagram is 100 μm; b is the proportion of germination of every 100 cysts in the cyst germination suspension. DETAILED DESCRIPTION

[0055] The application will be further described in detail below in combination with the embodiments and the drawings, but the embodiments of the application are not limited thereto.

[0056] Unless otherwise indicated, conventional test conditions were used in the following examples or were as recommended by the reagent manufacturer. The materials, reagents, etc. used were commercially available reagents and materials unless otherwise specified.

[0057] Example 1: Construction of PlCaMK1 gene knockout vector

[0058] Test materials

[0059] Test strains, plants and vectors:

[0060] The test strain Peronophythora litchii (Wild Type, referred to as WT) is a conventional Peronophythora litchii and can be obtained through commercial channels or isolated from nature. Eschrichia coli strain JM109 can be obtained through commercial channels. The test inoculated plant material is litchi (Nuo Michi) tender leaves (collected from the horticultural practice orchard of South China Agricultural University). The oomycete knockout and transformation vectors pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA (which have been disclosed in the literature “Yufeng, Fang, Linkai, et al. Efficient Genome Editing in the Oomycete Phytophthora sojae Using CRISPR / Cas9 [J]. Current Protocols in Microbiology, 2017”) and pBSSK (which have been disclosed in the literature “A CRISPR / Cas9-mediated in situ complementation method for Phytophthora sojae mutants [J]. Molecular Plant Pathology, 2021, 22(3)”) are all donated by the Oomycete and Fungal Molecular Biology Laboratory of the College of Plant Protection, Nanjing Agricultural University.

[0061] Main test medium:

[0062] Carrot agar (CA) (1 L): 300 g of carrot juice is filtered with gauze and sterilized at 121°C for 20 min. Add 1.5% (W / V) agar powder to the solid medium.

[0063] LB medium (1 L): 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride (NaCl) are sterilized at 121°C for 20 min. Add 1.5% (W / V) agar powder to the solid medium.

[0064] Nutrition Pea Broth (NPB) (1 L): Fresh peas 120 g, boiled in water for 20 min and filtered. To the filtrate, add 5 g D-Sorbitol, 5 g D-Mannitol, 5 g glucose, 3 g KNO3, 2 g CaCO3, 2 g yeast extract, 1 g K2HPO4, 1 g KH2PO4, 0.5 g MgSO4, 0.1 g CaCl2, 2 mL Vitamin stock and 2 mL Trace elements, ddH2O to 1 L, 121 °C, 20 min sterilization. Solid medium is supplemented with 1.5% (W / V) Difco Bacto Agar. 4) -1

[0065] Pea / Mannitol Broth (PM) (1 L): Fresh peas 120 g, boiled in water for 20 min and filtered. To the filtrate, add 91 g D-Mannitol, 2 g CaCO3 and 1.32 g CaCl2, ddH2O to 1 L, 121 °C, 20 min sterilization. Solid medium is supplemented with 1.5% (W / V) Difco Bacto Agar.

[0066] Pfeffer Broth (1 L): 0.5 g KH2PO4, 0.25 g MgSO4·7H2O, 1 g L-asparagine, 1 mg Vitamin B1, 0.5 g yeast extract, 10 mg β-sitosterol and 5 g glucose, ddH2O to 1 L, 121 °C, 20 min sterilization. Solid medium is supplemented with 1.5% agar powder.

[0067] Construction of CRISPR / Cas9 related vectors:

[0068] (1) Construction of pYF2.3G-Ribo-sgRNA::PlCaMK1

[0069] According to the design sgRNA website (http: / / grna.ctegd.uga.edu / ), the target RNA (sgRNA) on PlCaMK1 gene was selected, and was synthesized by Shenguo Biotechnology. The sgRNA annealing system was prepared according to Table 1, and the metal bath was 37 °C for 30 min.

[0070] ​​The sgRNA sequence is as follows:

[0071] PlCaMK1-sgRNA1-F: 5'-CTAGCCACTACCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGTAGTGTCTCTGGAGATCTC-3'

[0072] PlCaMK1-sgRNA1-R: 5'-AAACGAGATCTCCAGAGACACTACGACGAGCTTACTCGTTTCGTCCTCACGGACTCATCAGGTAGTGG-3'

[0073] PlCaMK1-sgRNA2-F: 5'-CTAGCTAGAACCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGTTCTAGCGCTTGTGGGTTA-3'

[0074] PlCaMK1-sgRNA2-R: 5'-AAACTAACCCACAAGCGCTAGAACGACGAGCTTACTCGTTTCGTCCTCACGGACTCATCAGGTTCTAG-3'.

[0075] Table 1 sgRNA double-stranded synthesis system (30 μL) (Takara)

[0076]

[0077] The reaction conditions are 37°C, 30 min, then 4 μL of 0.5 mol·L -1 NaCl is added to the above system, mixed, boiled in a boiling water bath for 2 min, cooled at room temperature for 3-4 h, and the DNA fragments are annealed to form double-stranded.

[0078] The target RNA (sgRNA) on the PlCaMK1 gene is annealed to form double-stranded, and the pYF2.3G-Ribo-sgRNA vector (double digestion system is shown in Table 2) after enzyme digestion is connected with T4-DNA ligase, the connection system is shown in Table 3, the reaction conditions are 16°C, 12 h, cooled on ice, then E. coli transformation is carried out.

[0079] Table 2 pYF2.3G-Ribo-sgRNA vector double digestion system (50 μL) (NEB)

[0080]

[0081]

[0082] Table 3 pYF2.3G-Ribo-sgRNA vector and double-stranded sgRNA ligation reaction system (10 μL) (NEB)

[0083]

[0084] (2) Construction of pBSSK::PlCaMK1 vector

[0085] The left and right homologous arms and amplification primers (PlCaMK1-Left-F, PlCaMK1-Left-R, PlCaMK1-Right-F, PlCaMK1-Right-R) were designed according to the sequences of about 1 kb upstream and downstream of the PlCaMK1 gene.

[0086] PlCaMK1-Left-F: 5'-CTAGAACTAGTGGATCCCCCCTCAGTAACATATTGGTGGT-3'

[0087] PlCaMK1-Left-R: 5'-GTATAATACAACAAACAGATTTTAGTGGAAACTTGCTCGA-3'

[0088] PlCaMK1-Right-F: 5'-TCGAGCAAGTTTCCACTAAAATCTGTTTGTTGTATTATAC-3'

[0089] PlCaMK1-Right-R: 5'-ATATCGAATTCCTGCAGCCCTTCGTTAGTTCCTTACTTTC-3'.

[0090] The left and right homologous arms were amplified using high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novozyme) with Perkin Elmer PCR Master Mix (2X) (50 μL) as follows:

[0091] Table 4 Phanta Max high-fidelity enzyme PCR amplification system (50 μL)

[0092]

[0093] The amplification procedure is as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 56-72℃ for 15 s, extension at 72℃ for 30 s / kb, 34 cycles, and finally extension for 5 min. After electrophoresis detection, the target band is recovered by using the agarose gel recovery kit of OMEGA Company, and the specific steps are described in the instruction manual. After the product concentration is detected, the directional seamless cloning kit ClonExpress One Step Cloning Kit (Nanjing Novozyme) is connected with the linearized pBSSK carrier (the enzyme cutting system is shown in Table 5), and the connection system is shown in Table 6.

[0094] Table 5 pBSSK enzyme cutting reaction system (10 μL)

[0095]

[0096] Table 6 pBSSK::PlCaMK1 carrier connection reaction system (10 μL)

[0097]

[0098] The constructed pYF2.3G-Ribo-sgRNA1::PlCaMK1, pYF2.3G-Ribo-sgRNA2::PlCaMK1, and pBSSK::PlCaMK1 are transformed into E. coli.

[0099] (3) E. coli transformation and verification

[0100] 100 μL of the divided E. coli competent cells JM109 are freeze-thawed on ice, 10 μL of the connection product is added, the finger is lightly tapped for mixing, and it is placed on ice for 30 min. 42℃ water bath heat shock for 90 s, and then quickly placed on ice for 2 min. 650 μL of LB liquid medium is added to the tube, 37℃, 180 rpm culture for 1 h. The above bacterial solution is centrifuged at 4000 rpm for 4 min, the supernatant is sucked, the remaining 100 μL of LB medium is used to suspend the bacterial body, and it is coated on the LB solid screening plate containing the final concentration of 100 μg·mL -1 Amp, and cultured at 37℃ for 12-16 h.

[0101] The E. coli single colony is used as a template, the verification primers of the pYF2.3G-Ribo-sgRNA carrier are M13F and RPL41_Pseq_F, the verification primers of the pBSSK carrier are M13F and M13R, M13F and M13R are universal primers, and the sequence of the primer RPL41_Pseq_F is as follows: RPL41_Pseq_F: 5'-CAAGCCTCACTTTCTGCTGACTG-3'.

[0102] Colony PCR verification was performed with Green Taq Mix (Nanjing Novozyme), and the system was as shown in Table 7:

[0103] Table 7 Colony PCR reaction system (20 μL)

[0104]

[0105] The PCR amplification program was as follows: pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s / kb, 34 cycles, and then extension for 7 min. The amplification products were detected by gel electrophoresis, and two colonies with amplified bands meeting the size of the target fragments were selected, which were inoculated into 10 mL LB liquid medium containing 100 μg·mL -1 Amp, and the bacteria were shaken for sequencing.

[0106] (4) Large-scale extraction of plasmid DNA

[0107] The single colony of E. coli containing the target plasmid was shaken, and the single colony was inoculated into 200 mL of LB liquid medium containing 100 μg·mL -1 Amp, and the bacteria were cultured at 37℃ and 180 rpm for 12 h, and then inoculated into 200 mL of LB liquid medium containing Amp, and the bacteria were cultured at 37℃ and 180 rpm for 14 h.

[0108] The four plasmids (pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA1::PlCaMK1, pYF2.3G-Ribo-sgRNA2::PlCaMK1, and pBSSK::PlCaMK1) required for PEG-mediated transformation were extracted using the EndoFree Maxi Plasmid Kit of TIANGEN.

[0109] Example 2: Preparation of P. liliies blight protoplasts and PEG-mediated transformation

[0110] Preparation of enzyme solution: 0.15 g of Lysing Enzymes (SIGMA) and 0.06 g of Cellulase (SIGMA) were weighed in a sterilized beaker, 10 mL of 0.8 mol·L -1 Mannitol, 8 mL of sterilized ddH2O, 800 μL of 0.5 mol·L -1 KCl, 800 μL of 0.5 mol·L -1 MES-KOH, and 400 μL of 0.5 mol·L -1 CaCl2, and then transferred to a 50 mL centrifuge tube for use;

[0111] Preparation of W5 solution: 7.8 g Glucose, 4.6 g CaCl2, 2.25 g NaCl, 0.093 g KCl were weighed, ddH2O was added to make up to 250 mL, and it was ready for use;

[0112] Preparation of MMg solution: 18.22 g Mannitol, 0.76 g MgCl2·6H2O, 2 mL MES Buffer were weighed, and water was added to make up to 250 mL, and it was ready for use;

[0113] Peronophythora litchii wild type strain WT was activated on NPB solid medium. The mycelium block was put into a conical flask, 50 mL of NPB liquid medium was added for culture, a total of three bottles were cultured, and the culture was carried out at 25°C in the dark for 3 days, and the shaking was carried out every 12 hours. The mycelium was collected by gauze filtration, and then it was gently squeezed with tweezers and added to a 50 mL centrifuge tube containing the enzyme solution. After gentle mixing, it was incubated at 25°C and 40 rpm for 40-45 min. After the mycelium was enzymatically digested, the mycelium was quickly filtered with a 50 mL beaker wrapped with three layers of Miracloth, and the filtrate was transferred to a 50 mL round-bottom centrifuge tube. It was centrifuged at 4°C and 1500 rpm for 3 min. The supernatant was discarded, 10 mL of W5 solution was added to resuspend the protoplasts, 25 mL of W5 solution was added, and it was gently mixed by inverting up and down. It was centrifuged at 4°C and 1500 rpm for 4 min. The supernatant was discarded, 7 mL of W5 solution was added to resuspend the protoplasts, and it was placed on ice for 30 min. Then it was centrifuged at 4°C and 1500 rpm for 4 min, the supernatant was discarded, and 6 mL of MMg solution was added to resuspend the protoplasts. It was placed in a warm room for 10 min. Six sterilized 50 mL centrifuge tubes were placed on ice, and four plasmids were added to each centrifuge tube: pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA1::PlCaMK 1, pYF2.3G-Ribo-sgRNA2::PlCaMK1, and pBSSK::PlCaMK1, each 30 μg, to obtain MMg solution containing protoplasts.

[0114] 1 mL of MMg solution containing protoplasts was added to each 50 mL centrifuge tube, and it was gently mixed and shaken, and it was placed on ice for 10 min. 580 μL of 40% polyethylene glycol (PEG) solution was added to each centrifuge tube along the wall, and the process was repeated three times. During this process, the centrifuge tube was slowly rotated to mix the PEG and the protoplasts, and it was placed on ice for 20 min. 100 mg·mL -1Amp PM was prepared by ampicillin. In the centrifuge tube, 2 mL Amp PM was added, and gently up and down, placed on ice for 2 min; continue to add 8 mL Amp PM in the centrifuge tube, and gently up and down, placed on ice for 2 min; finally, each centrifuge tube was added with 10 mL Amp PM in turn, and gently up and down, placed obliquely. The protoplasts were regenerated by culturing at 25℃ in the dark for 14-16 h. After overnight culture, the protoplast regeneration was observed under a microscope, and then centrifuged at 2000 rpm for 5 min. The supernatant was discarded to leave 5 mL liquid medium in each centrifuge tube, and then 30 mL of 30 μg·mL -1 The geneticin G418 was prepared into a pea mannitol solid medium, and after being mixed, poured into two 9 cm sterilized culture dishes, and cultured at 25℃ in the dark for 2-3 d. Single colonies were picked and numbered and named for identification.

[0115] Example 3: Verification and determination

[0116] (1) Verification analysis of PlCaMK1 gene knockout transformants

[0117] The wild type WT of Peronophythora litchii and the transformants were subjected to CTAB method for genome DNA extraction, and the genome DNA was used as a template to perform conventional PCR amplification by designing primers PlCaMK1BY-F and PlCaMK1BY-R outside the PlCaMK1 left and right homologous arm fragments in the genome. The band size was detected by gel electrophoresis to verify whether PlCaMK1 was successfully knocked out and sent for sequencing detection.

[0118] PlCaMK1BY-F: 5'-CCATGAAAAAATTATCGGTTGGGTTAGACGA-3'

[0119] PlCaMK1BY-R: 5'-CGGTATTGAGTCATCATCATCAATCTAGTGG-3'.

[0120] The sequencing results proved that the PEG transformation knockout was successful, and three PlCaMK1 gene knockout transformants were obtained, which were numbered according to the verification order of the transformants from T1, and the three knockout successful mutants were named as T4, T22 and T40, respectively.

[0121] (2) Determination of growth rate of knockout mutants

[0122] Wild type (WT) strain of P. litchi, CK, a transformant with unsuccessful knock-out, and T4, T22, T40, mutants with successful knock-out of PlCaMK1 gene were subcultured twice on CA plates without antibiotics, and 9 mm diameter mycelial plugs of WT, CK and T4, T22, T40 with the same mycelial age were punched and inoculated in the center of 15 mL equal amount of carrot medium plates (diameter = 9 cm). Three replicates were set, and the plates were incubated at 25 °C in the dark for 5 days. The colony diameters were measured and the growth rates were calculated. Photographs were taken. The experiment was repeated three times independently, and Duncan’s multiple range test in SPSS software was used for significant difference analysis among strains.

[0123] Growth rate (mm / d) = colony diameter on the 5th day / 5 days.

[0124] (3) Measurement of zoospore motility of knock-out mutants

[0125] The 9 mm diameter mycelial plugs of WT, CK and T4, T22, T40 with the same mycelial age obtained from subculture in step (2) were inoculated, and 5 plugs of each group were placed in 10 mL centrifuge tubes with 2.5 mL ddH2O. The tubes were shaken for 1 min with a vortex shaker to make the sporangia fall off the sporangiophores and obtain the sporangia suspension. The sporangia suspension was placed in a 4 °C refrigerator for 30 min, and then at room temperature for 10 min, 20 min, 40 min, 1 h and 2 h. The number of zoospores in 1 μL liquid surface was counted, and 10 data of each sample were counted and averaged for zoospore motility analysis.

[0126] (4) Pathogenicity test of knock-out mutants

[0127] The 9 mm diameter mycelial plugs of WT, CK and T4, T22, T40 with the same mycelial age obtained from subculture in step (2) were inoculated, and 5 plugs of each group were placed in 2.5 mL ddH2O. The tubes were shaken for 1 min with a vortex shaker to make the sporangia fall off the sporangiophores and obtain the sporangia suspension. The sporangia suspension was placed in a 16 °C incubator for zoospore release, and the number of zoospores was counted under a microscope. The young leaves of litchi (variety Nuomicai) were washed with ddH2O and dried on a clean bench. Each leaf was inoculated with 10 μL of the zoospore suspension, and 6 leaves of similar age were inoculated for each strain. The leaves were incubated at 25 °C with moisture. Photographs were taken and the lesion diameters were measured 48 h later. Duncan’s multiple range test in SPSS software was used for significant difference analysis.

[0128] Litchi (variety is Nuomi' ) tender leaves were soaked in ddH2O and placed on moist filter paper. The WT, CK and T4, T22, T40 mycelial blocks with consistent mycelial age of 9 mm in diameter obtained in step (2) were inoculated on the back of the leaves. Each strain was inoculated on 6 leaves with similar age, and placed in a humid environment at 25°C. After 48 h, the lesion diameters were measured and photographed. Duncan's multiple range test in SPSS software was used for significant difference analysis.

[0129] (5) Determination of oospore production of knockout mutants

[0130] The colonies of WT, CK, T4, T22 and T40 were subcultured twice, and the mycelial discs (d = 9 mm) were taken from the edge of the colonies with a sterile puncher and transferred to Hybond N+ membranes covered with a layer of carrot medium. + After 10 days of dark culture at 25°C, the membranes were removed, and 5 mycelial discs were punched near the inoculation point with a puncher, homogenized in 5 mL ddH2O, and 1 μL was taken onto a glass slide to count the number of oospores of each strain.

[0131] (6) The zoospores obtained in (4) were filtered through a 50 μm pore size nylon membrane to obtain zoospores, and vortexed to release the flagella from the zoospores to form resting spores. The resting spores were incubated at room temperature for 1 h, and then an appropriate amount of copper sulfate solution was added to the suspension. 1 μL of the resting spore suspension was taken under a microscope to count the number of resting spores and germinating resting spores. The data were counted 10 times for each sample, and the average value was taken.

[0132] Example 4: Results and analysis

[0133] (1) Construction of the recombinant fragment of PlCaMK1 gene of Peronophythora litchii

[0134] The Left and Right homologous arm fragments of PlCaMK1 gene were cloned by PCR technology, and the multiple fragments were ligated to linearized pBSSK vector to successfully obtain pBSSK::PlCaMK1 vector. The sgRNA was synthesized as double-stranded, and was ligated to linearized pYF2.3G-Ribo-sgRNA vector to successfully obtain pYF2.3G-Ribo-sgRNA1::PlCaMK1 and pYF2.3G-Ribo-sgRNA2::PlCaMK1 vectors. The knockout schematic diagram is as follows: Figure 1 .

[0135] (2) Screening of PlCaMK1 knockout mutant of Peronophythora litchii

[0136] Design the outer primers of PlCaMK1 left and right arms: PlCaMK1 BY-F / R, extract the DNA of wild type (WT), non-knockout transformants (CK) and PlCaMK1 gene knockout mutants of Peronophythora litchii, and perform PCR amplification with the wild type as a control, the amplification method and system are referred to Example 1, the results show that WT and CK expand about 5000bp fragments, and T4, T22 and T40 three transformants can all amplify about 2000bp fragments, which further indicates that T4, T22 and T40 are PlCaMK1 knockout mutants ( Figure 2 ), and the sequencing results confirm that the knockout is successful. The sequencing results of T4, T22 and T40 are as follows:

[0137] ​

[0138] (3) Analysis of the growth rate of PlCaMKl knockout mutants

[0139] Compared with wild type WT and non-knockout transformant CK, knockout mutants T4, T22 and T40 grow at a rate similar to that of WT, and the mycelium color has no difference compared with that of wild type Figure 3 ).

[0140] (4) Analysis of zoospore motility of PlCaMKl knockout mutants

[0141] After low temperature induction, wild type WT releases more zoospores at the top layer of sporangium suspension at 10 min, and the number of zoospores reaches the highest at 20 min, and the number of zoospores is basically consistent at 40 min to 2 h, a small amount of zoospores become resting spores at 40 min to 1 h, and a small amount of zoospores become resting spores and germinate at 2 h; after low temperature induction, PlCaMKl mutants release more zoospores at the top layer of sporangium suspension at 10 min, and release a large number of zoospores at 20 min, a large number of zoospores become resting spores at 40 min, and basically all become resting spores and partially germinate at 1 h, and the resting spores basically germinate into germ tubes at 2 h. The zoospores of P. litchi Plasmodium WT basically all become resting spores after 5.5 h of zoospore motility at room temperature, i.e. the PlCaMKl knockout mutant shortens the zoospore motility time by nearly 5 h compared with WT. It can be seen that the knockout of PlCaMKl significantly damages the motility of zoospores Figure 4 ).

[0142] (5) Analysis of pathogenicity of PlCaMKl knockout mutants

[0143] Compared with wild type WT and non-knockout transformant CK, the zoospore suspension of knockout mutants T4, T22 and T40 is directly inoculated on litchi leaves, and the pathogenicity of PlCaMKl knockout mutants to litchi leaves has no difference compared with that of WT and CK; the use of zoospore self-motility to infect litchi leaves will lead to a significant decrease in the pathogenicity of PlCaMKl knockout mutants compared with that of WT and CK Figure 5 ).

[0144] (6) Analysis of resting spore germination of PlCaMKl knockout mutants

[0145] Prepare zoospore suspensions of wild type strain WT, CK and three PlCaMKl knockout mutants (T4, T22 and T40), and place them at room temperature at 28°C for 2 h. It is observed that the resting spore germination rate of PlCaMKl knockout mutants is slightly higher than that of WT and CK Figure 6 ).

[0146] The experimental results prove that the gene provided by the application can be used for plant disease prevention and treatment, in particular, litchi blight caused by Peronophythora litchi. In addition, the gene provided by the application can be used as a drug target for plant disease prevention and treatment. A person skilled in the art can develop a drug for preventing and treating plant diseases, in particular, litchi blight, according to the guidance and inspiration of the present specification.

[0147] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent alternative solutions, and are included in the protection scope of the present application.

Claims

1. A pathogenic related protein PlCaMK1 of Peronophythora litchii, characterized in that: The amino acid sequence of which is shown as SEQ ID NO.

3.

2. The biological material related to the pathogenicity-related protein PlCaMKl of P. litchi chikushiian described in claim 1, characterized by: Any one of the following biological materials: 1) a nucleic acid molecule encoding the pathogenicity-related protein PlCaMK1 of Peronophythora litchii ; 2) an expression cassette containing the nucleic acid molecule in 1); 3) a recombinant vector containing the nucleic acid molecule in 1); 4) a recombinant microorganism containing the nucleic acid molecule in 1).

3. The biological material of claim 2, wherein: 1) the nucleic acid molecule is the gene sequence of the pathogenicity-related protein PlCaMK1 of Peronophythora litchii, as shown in SEQ ID NO: 1, or the CDS sequence of the pathogenicity-related protein PlCaMK1 of Peronophythora litchii, as shown in SEQ ID NO:

2.

4. Use of the pathogenic related protein PlCaMKl of P. litchi chiller blight as claimed in claim 1, characterized in that: Any one of the following applications: ⅰ) the application of knocking out the gene of the pathogenicity-related protein PlCaMK1 of Peronophythora litchii in reducing the pathogenicity of Peronophythora litchii ; ⅱ) the application of knocking out the gene of the pathogenicity-related protein PlCaMK1 of Peronophythora litchii in shortening the zoospore motility time of Peronophythora litchii ; ⅲ) the application of knocking out the gene of the pathogenicity-related protein PlCaMK1 of Peronophythora litchii in promoting the germination of Peronophythora litchii quiescent spores; ⅳ) the application of knocking out the gene of the pathogenicity-related protein PlCaMK1 of Peronophythora litchii in preventing and treating Peronophythora litchii.

5. A method of controlling Peronophythora litchii causing litchi downy mildew caused by Peronophythora litchii, characterized by: By knocking out the gene of the pathogenicity-related protein PlCaMK1 of Peronophythora litchii in claim 1.

6. A method for constructing a PlCaMK1 gene knockout mutant of P. liliicola, characterized by: Comprising the following steps: (1) The gene sequence of pathogenic related protein PlCaMK1 of Litchi Phytophthora blight according to claim 1 is used to design sgRNA by sgRNA website, and the sgRNA is connected with pYF2.3G-Ribo-sgRNA vector to obtain PlCaMK1 Gene knockout plasmid pYF2.3G-Ribo-sgRNA: PlCaMK1 ; Or, the gene sequence of the pathogenic related protein PlCaMK1 of P. litchi is used to design left and right homologous arm amplification primers according to claim 1, and the left and right homologous arms are amplified by taking the P. litchi genomic DNA as a template and connected with a pBSSK vector to obtain PlCaMK1 Gene knockout plasmid pBSSK: PlCaMK1 ; (2) The PlCaMK1 Gene knockout plasmid pYF2.3G-Ribo-sgRNA: PlCaMK1 or pBSSK: PlCaMK1 The protoplasts of the wild type strain of Litchi mildew Phytophthora were introduced, screened and verified to obtain PlCaMK1 gene knockout mutants; The left and right homologous arm amplification primers in step (1) are as follows: Left homologous arm amplification primer: PlCaMK1-Left-F: 5'-CTAGAACTAGTGGATCCCCCTGTCGTAACGCAAAGGAAGT-3'; PlCaMK1-Left-R: 5'-GTATAATACAACAAACAGATTTTAGTGGAAACTTGCTCGA-3'; Right homologous arm amplification primer: PlCaMK1-Right-F: 5'-TCGAGCAAGTTTCCACTAAAATCTGTTTGTTGTATTATAC-3'; PlCaMK1-Right-R: 5'-ATATCGAATTCCTGCAGCCCTTCGTTAGTTCCTTACTTTC-3'.

7. The method for constructing the PlCaMK1 gene knockout Peronophythora litchii mutant of claim 6, wherein: The sgRNA in step (1) is any one of the following: sgRNA1: 5'-CACTACCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGTAGTGTCTCTGGAGATCTC-3'; sgRNA2: 5'-TAGAACCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGTTCTAGCGCTTGTGGGTTA-3'. sgRNA2: 5'-TAGAACCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGTTCTAGCGCTTGTGGGTTA-3'. sgRNA

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