CN117088948B - Phytophthora capsici zoospore development regulation related protein, and coding gene and application thereof - Google Patents
Phytophthora capsici zoospore development regulation related protein, and coding gene and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses zoospore development regulatory protein MesA containing Afil and SPA structural domains from Phytophthora (Phytophthora spp.) and a coding gene and application thereof. Wherein the growth and development regulating protein PcMesA of phytophthora capsici leonian (Phytophthora capsici) has an amino acid sequence shown as SEQ ID No.2, and the encoding gene thereof has a nucleotide sequence shown as SEQ ID No. 1. The main function of the MesA protein is that the zoospore of the phytophthora is necessary to form, and after the MesA protein is deleted, the zoosporangium of the phytophthora cannot differentiate to form normal zoospores. The gene of the invention has high application value in controlling epidemic diseases caused by phytophthora, and the novel bactericide developed based on the protein as an action target has important practical significance in controlling the occurrence and epidemic of epidemic diseases caused by phytophthora.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to phytophthora capsici regulating zoospore development related protein, and a coding gene and application thereof; in particular to a zoospore development MesA containing Afil and SPA structural domains from Phytophthora pathogen (Phytophthora spp.) in plant pathogenic oomycetes, and a coding gene and application thereof.
Background
Oomycetes (Oomycetes) are a huge number of eukaryotes, the number of which is known at present to be more than 1800, are widely distributed worldwide, and can cause various animal and plant diseases. Although the oomycetes and fungi are similar in mycelium morphology, nutrient absorption mode and the like, the oomycetes and fungi are far in relation and the diatom is close in relation, and belong to the antler whip biology kingdom. Plant pathogenic oomycetes account for about 60% of the total oomycetes and can be classified into Phytophthora (Phytophthora), pythum (Pythum), peronospora (Peronospora), and the like. Among these Phytophthora pathogens (Phytophthora spp.) are serious hazards in agricultural production, and 6 species of Phytophthora pathogens are listed as ten important plant pathogen oomycetes in the world, including Phytophthora infestans (Phytophthorainfestans), phytophthora sojae (Phytophthora sojea), phytophthora capsici (Phytophthora capsici) and the like. The phytophthora capsici leonian (Phytophthoracapsici) has wide hosts and can infect more than 70 vegetable crops of 26 families such as hot pepper, tobacco, leguminous plants and cucurbitaceae in the Solanaceae. Phytophthora capsici can cause plant diseases from seedling stage to fruit stage, and cause rot of root and stem parts and fruits of crops, and serious plant wilting and even damping-off can be caused when serious, so that the influence on the yield and quality of crops is great, and serious economic loss is caused.
The life history of phytophthora capsici comprises a sexual stage and an asexual stage, wherein the sexual stage generates oospores through heterogeneous cooperation; asexual stage is to release zoospores from the aperture through mature sporangia under low temperature and humidity condition, zoospores reach the surface of the host to form resting spores, and the mycelia germinate under proper condition invade directly or invade plants through wounds or apertures, which is the main infection source. Sporangia and zoospores are spread along with wind and rain and irrigation water remotely, and are the main reasons for causing outbreaks of plant epidemic diseases. Thus, inhibiting zoospore production can further inhibit pathogen infection and pathogenicity to achieve the purposes of cutting off disease circulation and controlling epidemic of plant epidemic diseases caused by phytophthora capsici.
MesA protein is a polar axis stability related protein which is preserved in pathogenic bacteria and plays an important role in maintaining the normal form of hyphae of corn black powder bacteria (Ustilagomaydis), fusarium graminearum (Fusarium graminearum) and aspergillus nidulans (Aspergillus nidulans) and infecting the pathogenic bacteria. MesA homologous proteins are ubiquitous in oomycetes, but their biological functions have not been reported yet.
Disclosure of Invention
The prior studies of the inventor found that, although the MesA protein of phytophthora capsici and the MesA protein of filamentous hyphae both have Afil (Avl 9 superfamily) and SPA (stabilization ofpolarity axis) domains, the protein homology is less than 25%, which indicates that the oomycete MesA protein may have functional characteristics different from that of fungal MesA protein. Further bioinformatics analysis shows that the phytophthora capsici MesA protein and other oomycetes MesA proteins have high homology of more than 60 percent. Phylogenetic relation analysis shows that the phylogenetic relation of the phytophthora MesA proteins is closer, the homology of the phytophthora capsici MesA and the proteins thereof is more than 70%, and the homology of the phytophthora capsici MesA and the proteins of the phytophthora acori (Phytophthora ramorum) MesA is 91.11%. In conclusion, mesA proteins have a high homology in oomycetes, in particular phytophthora.
Based on this, studies by the inventors found that PcMesA containing MesA protein conserved domain Afil and SPA was present in phytophthora capsici. The protein regulates the formation of zoospores, so that the normal infection of oomycetes can be blocked by controlling zoospore development protein PcMesA, and the large-scale spread and epidemic of diseases caused by oomycetes can be controlled (inhibited or blocked).
Thus, the invention provides zoospore development regulatory protein containing Afil and SPA structural domains in phytophthora, which is named MesA and is A1), A2), A3) or A4):
a1 Amino acid sequence is protein shown as SEQ ID NO. 2;
A2 A fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of a protein shown in SEQ ID NO. 2;
a3 A protein derived from a protein shown as SEQ ID NO.2, which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown as SEQ ID NO. 2;
A4 Amino acid sequence having the same function as the amino acid sequence shown in SEQ ID NO.2 with an amino acid sequence similarity of 60% or more, preferably 70% or more, more preferably 90% or more with SEQ ID NO. 2.
In order to facilitate purification of the protein in A1), a fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID No. 2; the tag may be a tag such as Poly-Arg (RRRRR), poly-His (HHHHH), FLAG (DYKDDDDK), strep-tag II (WSHPQFEK), c-myc (EQKLISEEDL), etc.
The growth regulating proteins in A1) -A4) are generally derived from Phytophthora in nature, namely generally, natural products, or can be artificially expressed or synthesized, or can be obtained by synthesizing encoding genes and then biologically expressing the genes. The coding gene of the protein in the A2) -A4) can be obtained by deleting one or more amino acid residues in the DNA sequence shown in SEQ ID No.2 of the sequence list and/or carrying out missense mutation of one or more nucleotide pairs and/or connecting the coding sequences of the tags at the 5 'end and/or the 3' end. Wherein, the sequence of phytophthora capsici leonian (Phytophthora capisici) PcMesA is shown as SEQ ID NO.2, and SEQ ID NO.2 (MesA) in the sequence table consists of 560 amino acid residues.
It is a further object of the present invention to provide nucleic acid molecules encoding the Afil and SPA domain-containing MesA proteins. The nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be an RNA, such as mRNA, hnRNA, tRNA or the like.
Wherein, the coding gene of MesA protein is B1) or B2) or B3) as follows:
b1 A DNA molecule shown in a nucleotide sequence shown in SEQ ID NO.1 of the sequence table;
b2 A cDNA molecule or a DNA molecule which has 60% or more, 70% or more, or 90% or more identity with the nucleotide sequence shown in B1) and encodes the above PcMesA protein;
b3 A cDNA molecule or a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in B1) or B2) and which codes for the above PcMesA protein.
In the invention, the DNA sequence (coding gene and cDNA) of the pathogenic regulatory protein can be specifically shown as SEQ ID No.1, wherein SEQ ID No.1 in the sequence table consists of 2028 nucleotides, and the 1 st to 2028 th nucleotides at the 5' end of the sequence 1 are coding sequences for coding the protein (PcMesA) shown as SEQ ID No.2 in the sequence table.
The third invention provides an RNA sequence transcribed from any DNA sequence as described above, preferably the sequence of the RNA molecule is C1) or C2) as follows:
C1 A RNA sequence having the same function as the RNA sequence transcribed from the DNA sequence shown as SEQ ID NO.1 or SEQ ID NO.1, wherein the similarity of the RNA sequence transcribed from the DNA sequence shown as SEQ ID NO.1 is 60% or more, more preferably 70% or more, still more preferably 90% or more;
c2 Most preferably the RNA sequence is transcribed from the DNA sequence shown in SEQ ID NO. 1.
The fourth aspect of the present invention provides a biological material related to the growth regulating protein, the coding gene, or the RNA molecule, which is any one of the following D1) to D10):
d1 An expression cassette containing the coding gene;
d2 A recombinant vector containing the coding gene or a recombinant vector containing the expression cassette of D1);
D3 A recombinant microorganism containing the coding gene, or a recombinant microorganism containing the expression cassette of D1), or a recombinant microorganism containing the recombinant vector of D2);
D4 A transgenic plant cell line comprising said coding gene, or a transgenic plant cell line comprising D1) said expression cassette;
d5 A transgenic plant tissue comprising said coding gene, or a transgenic plant tissue comprising D2) said expression cassette;
d6 A transgenic plant organ comprising said coding gene, or a transgenic plant organ comprising D2) said expression cassette;
D7 A nucleic acid molecule that inhibits expression of the encoding gene; preferably, the nucleic acid molecule is a nucleic acid molecule for knocking out the coding gene, or silencing the coding gene, and can be a coding sequence for expressing an sgRNA fragment targeting the target gene to be knocked out, such as a sgRNA sequence (coding sequence) AACATCCCACCATTCCCTCT targeting PcMesA genes;
D8 Expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing or expressing D7) the nucleic acid molecule, the recombinant vector may include a CRISPR/Cas 9-based gene knock-out method is to express the gene of interest on the Donor vector and sgRNA and Cas9 protein plasmid; the Donor vector is a recombinant vector containing a sequence of 800-1500bp upstream of a target gene to be knocked out, a Donor DNA sequence (can be a gene sequence such as NPTII, GFP or RFP) and a sequence of 800-1500bp downstream of the target gene to be knocked out, which are connected in sequence. The sgRNA and Cas9 protein coexpression plasmid is a vector for encoding and expressing a target gene to be knocked out sgRNA fragment and a DNA sequence for expressing Cas9 protein, wherein the target gene to be knocked out is PcMesA gene, and the sgRNA sequence (encoding sequence) of the target PcMesA gene is AACATCCCACCATTCCCTCT. Preferably, the sgRNA and Cas9 coexpression plasmid is prepared by taking pYF carrier as a starting carrier, inserting a double-chain sgRNA coding sequence obtained by annealing the sgRNA of PcMesA gene between Nhe I and Bsa I enzyme recognition sites of pYF carrier to obtain the sgRNA and Cas9 coexpression plasmid;
D9 A nucleic acid molecule that inhibits translation of the RNA molecule;
D10 An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing or expressing D9) said nucleic acid molecule.
The fifth object of the present invention is to provide the application of phytophthora capsici PcMesA protein and the nucleic acid molecule encoding PcMesA protein or the biological material containing the nucleic acid molecule encoding PcMesA protein.
The application is any one or more of the following 1) -5):
1) The application in regulating (increasing or decreasing) the yield of oomycete zoospores;
2) Use in modulating (increasing or decreasing) the ability of oomycetes to infect a host;
3) Use in inhibiting and/or killing oomycetes.
Preferably, among said uses, use is made of 1) to 3) by inhibiting transcription or inactivating the coding gene of sequence 1, or inhibiting translation of said RNA molecule, or inhibiting and/or inactivating the activity of the PcMesA protein of sequence 2.
In such applications, zoospore production of oomycetes and the ability to regulate the infecting host is affected by inhibiting transcription of the coding gene described above, or inhibiting translation of the RNA sequence described above, or inhibiting and/or inactivating the activity of the PcMesA protein described above.
The invention provides PcMesA protein shown in SEQ ID NO.2 in the sequence table, a coding gene shown in SEQ ID NO.1 in the sequence table, or application of the RNA molecule or the biological material or the protein or DNA as claimed in claim 5 in screening oomycete bacteriostasis and/or bactericide as a bacteriostasis or bactericide target; the antibacterial or bactericidal agent can inhibit or kill oomycetes by inhibiting or inactivating PcMesA protein shown in SEQ ID NO.2 to inhibit the generation of zoospores. Preferably, the cDNA sequence of the pathogenic regulatory protein is a DNA sequence shown as SEQ ID No. 1. Preferably, the method is used for inhibiting and/or killing oomycete-induced oomycete diseases on production.
The oomycete is preferably phytophthora, more preferably phytophthora capsici leonian (Phytophthora capsici).
The seventh object of the present invention is to provide a method for screening or assisting in screening of an antibacterial and/or bactericidal agent against oomycetes (such as phytophthora capsici, in particular phytophthora capsici), which comprises applying a substance to be tested to said oomycetes, wherein said substance to be tested is a candidate plant oomycete antibacterial and/or bactericidal agent when said substance to be tested is capable of inhibiting transcription of the above DNA sequence, or inhibiting translation of the above RNA sequence, or inhibiting and/or inactivating the activity of the above MesA protein.
The eighth object of the present invention is to provide a method for reducing the activity of oomycetes, comprising the steps of: inhibiting transcription of a coding gene as described above, or inhibiting translation of said RNA molecule, or inhibiting and/or inactivating activity of a MesA protein as described above;
The invention also provides a method for reducing the infection capability of oomycetes, which is to reduce the infection capability of oomycetes by inhibiting the generation of zoospores of oomycetes (such as phytophthora capsici, in particular phytophthora capsici).
In the method, the generation of zoospores of oomycetes is inhibited by knocking out any one of the DNA sequences. The oomycete is preferably phytophthora, more preferably phytophthora capsici (Phytophthora capsici), such as phytophthora capsici BYA strain.
In one embodiment of the present invention, wherein the above described methods of gene knockout and back-filling employ CRISPR/Cas 9-based gene knockout methods.
Specifically, the CRISPR/Cas 9-based gene knockout and anaplerotic method is to select a Donor vector of a target gene, sgRNA and Cas9 co-expression plasmid transfection oomycetes to obtain recombinant bacteria inactivated by the target knockout protein.
The knockout related vector is a recombinant vector containing a sequence of 800-1500bp upstream of a target gene to be knocked out (a gene sequence of NPTII, GFP, RFP or the like) and a sequence of 800-1500bp downstream of the target gene to be knocked out, which are sequentially connected; the repair related vector is a recombinant vector which sequentially contains a sequence of 800-1500bp upstream of the target gene, donorDNA (PcMesA gene sequence) and a sequence of 800-1500bp downstream of the target gene.
The sgRNA and Cas9 protein coexpression plasmid is a vector for encoding and expressing a target gene to be knocked out sgRNA fragment and a DNA sequence for expressing Cas9 protein, wherein the target gene to be knocked out is PCMesA genes and GFP genes, the sgRNA sequence (encoding sequence) of the target PcMesA genes is AACATCCCACCATTCCCTCT, and the sgRNA sequence (encoding sequence) of the target GFP genes is CCACGGAACAGGGAGCTTTC.
Preferably, the sgRNA and Cas9 coexpression plasmid is obtained by taking pYF carrier as starting carrier, respectively annealing the double-chain sgRNA coding sequence obtained by annealing the PcMesA gene and the sgRNA of GFP gene, inserting between Nhe I and Bsa I enzyme recognition sites of pYF carrier, and obtaining the sgRNA and Cas9 coexpression plasmid.
In the above application, the PcMesA expression and/or activity substance is a substance that inhibits PcMesA expression and/or inhibits transcription of its coding gene and/or inhibits translation of an RNA molecule resulting from transcription of the coding gene of PcMesA.
Experiments prove that MesA protein provided by the invention plays a role in the zoospore development process of phytophthora. The knocked-out mutant obtained by combining PEG-CaCl 2 mediated protoplast transformation technology and CRISPR/Cas9 gene editing technology has obviously changed zoospore growth and development compared with wild parent strain, and mainly comprises the following components: the sporangium contents were unable to divide normally after MesA protein deletion, and zoospores of normal morphology were unable to form. Therefore, the zoospore development regulation protein MesA of phytophthora can play an important role in the asexual propagation process of phytophthora, and further cut off disease circulation to influence infection of oomycetes on plants. The invention provides technical support for further exploring zoospore formation process and pathogenic mechanism of phytophthora and oomycetes, and provides technical foundation for prevention and treatment of plant epidemic diseases caused by oomycetes and research and development of novel bactericides.
Drawings
FIG. 1 is a schematic representation of the Phytophthora capsici PcMesA domain, comprising Afil and SPA domains.
FIG. 2 is a cluster analysis of MesA proteins from different species. (note: the numbers on the branch lines represent the evolutionary branch lengths, shorter branch lengths represent smaller differences and closer evolutionary distances).
FIG. 3 shows the growth rate of mycelia, the number of sporangia and the pathogenic symptoms of infection of mycelia into tobacco leaves, sporangia and zoospore morphology of wild phytophthora capsici strain BYA (WT), pcMesA gene knockout mutants delta MesA-T1, delta MesA-T2 and PcMesA gene knockout anaplerotic strain delta MesA-T1 of MesA. Wherein a in fig. 3 is the morphological characteristics of pathogenic bacteria or symptoms of infection; b in fig. 3 is a histogram of data statistics.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Phytophthora capsici wild type strain BYA is stored by seed pathology and bactericide pharmacology laboratory at the university of agriculture, china, and is disclosed in document "Wang,W.,et al.,PcMuORP1,an Oxathiapiprolin-Resistance Gene,Functions as aNovel Selection Marker for Phytophthora Transformation and CRISPR/Cas9 Mediated Genome Editing.Frontiers in Microbiology,2019.10.", and is publicly available from the university of agriculture.
The culture medium and the reagent formula used in the study:
commonly used V8 medium: 340mL V8,4.76g CaCO 3, deionized water is fixed to 3.4L, and 51g of agar is added to prepare a solid culture medium, and the culture medium is sterilized by high-pressure moist heat at 121 ℃ for 20min.
PM medium (Pea Mannitol): adding 125g of pea into 1L of deionized water, sterilizing at 121deg.C for 20min, filtering with gauze to obtain pea soup, adding Mannitol 91.1g, caCO 32 g,CaCl2 1g, adding distilled water to 1L, adding 15g of agar powder into the solid culture medium, and sterilizing at 121deg.C for 20min.
The media used for the knockout transformation experiments were as follows:
NPB medium (Nutrient pea broth): adding 125g of pea into 1L deionized water, sterilizing at 121deg.C for 20min, filtering with gauze to obtain pea soup, adding Yeast Extract 2.0g、Glucose 5.0g、Mannitol 5.0g、Sorbitol 5.0g、CaCO32.0 g、CaCl20.1 g、MgSO40.5g、MgSO40.5 g、KNO33.0 g、K2HPO41.0 g、KH2PO41.0 g, to the above solution, and fixing volume to 1L with deionized water. If the solid culture medium is prepared, 15g of agar powder is added, and the culture medium is sterilized at 121 ℃ for 20min. After sterilization, Vitamin stock 2mL(Folic acid 6.7×10-7g/mL;Pyridoxine-HCl 6.0×10-4g/mL;Biotin 6.7×10-7g/mL;Thiamine-HCl1.3×10-3g/mL;L-inositol 4.0×10-5g/mL;Nicotinic acid 4.0×10-5g/mL;Riboflavin 5.0×10-5g/mL) and filtered through a 0.22 μm filter are added Vitamin stock 2mL(FeC6H5O7·3H2O 5.4×10-4g/mL;Na2MoO4·H2O 3.0×10- 5g/mL;ZnSO4·7H2O 3.8×10-4g/mL;
MgSO4·H2O 3.8×10-5g/mL;H3BO32.5×10-5g/mL;CuSO4·5H2O 7.5×10-4g/mL)
The knockout conversion test reagents were formulated as follows:
Enzymolysis liquid: in the present preparation, 0.12g of lyase (Lysing Enzymes from Trichoderma harzianum, L1412, sigam), 0.12g,0.8M Mannitol 10mL of cellulase (yakult R), 8mL of ultrapure water, 800. Mu.L of 0.5M KCl, 800. Mu.L of 0.5M MES (pH 5.7), 400. Mu.L of 0.5M CaCl 2 and 0.22. Mu.M filter membrane were filtered and sterilized.
W5 solution: KCl 0.1g, caCl 2·2H2 O4.6g,NaCl 2.25g,Glucose 7.8g, ultrapure water dissolved to 250mL,0.22 μm filter membrane filter sterilization.
PEG-CaCl 2 solution (40% w/v): the preparation was prepared immediately, 6g PEG4000,0.8M Mannitol3.75mL, 3mL of ultrapure water, 3mL of 0.5M CaCl 2, and 0.22 μm filter sterilized.
MMG solution (250 mL): mannitol 18.22g,0.5M MES (pH 5.7) 2.0mL, mgCl 2·6H2 O0.76 g, ultra pure water to 250mL, and filter sterilized with 0.22 μm filter.
Example 1, acquisition of growth and development regulatory protein PcMesA in Phytophthora capsici and its coding Gene and homology comparison of PcMesA protein with MesA protein in plant pathogenic bacteria
In this embodiment, the zoospore development regulatory protein PcMesA of phytophthora capsici and its coding gene (or cDNA) can be obtained by amplifying the DNA (or cDNA) of phytophthora capsici strain BYA5 as a template by using the primers shown in Table 1. Wherein the DNA or RNA extracted material can be mycelium of Phytophthora capsici strain BYA. Wherein, DNA of phytophthora capsici strain BYA is used as a template, pcMesA-F and PcMesA-R are used for amplification to obtain PcMesA coding gene PcMesA, as shown in SEQ ID NO.1 in a sequence table, SEQ ID NO.1 is composed of 2028 nucleotides, nucleotides 1-2028 from the 5' end of SEQ ID NO.1 are coding sequences, protein (PcMesA) shown in SEQ ID NO.2 in the sequence table is coded, and the protein comprises an Afi1 domain and an SPA domain, as shown in figure 1, wherein the sequence of the Afi1 domain is the amino acid residue sequence from 13 th to 111 th of the amino end of SEQ ID NO.2, and the sequence of the SPA domain is the amino acid residue sequence from 242 th to 364 of the amino end of SEQ ID NO. 2. cDNA of phytophthora capsici strain BYA is used as a template, and is amplified by PcMesA-F and PcMesA-R to obtain PcMesA cDNA, and the sequence of the cDNA is the same as the sequence of the coding gene, and is shown as SEQ ID NO. 1. The above proteins or genes may also be synthesized artificially.
TABLE 1 PcMESA full-length coding gene amplification primers
Cluster analysis and homology comparison show that, although the MesA protein of phytophthora capsici and the MesA protein of filamentous hypha have Afil (Avl 9 superfamily) and SPA (stabilization ofpolarity axis) domains, the homology of the PcMesA protein and MesA protein in oomycetes is less than 25% as shown in table 2, which indicates that the oomycete MesA protein may have functional characteristics different from that of the fungal MesA protein. Further bioinformatics analysis shows that the phytophthora capsici MesA protein and other oomycetes MesA proteins shown in table 3 have high homology of more than 60 percent. Phylogenetic relationship analysis as shown in FIG. 2, the relatedness of Phytophthora MesA proteins was found to be more recent. The homology of phytophthora capsici MesA and the protein thereof is above 70%, wherein the homology of the phytophthora capsici and the MesA protein of the phytophthora acori (Phytophthora ramorum) is 91.11%. In conclusion, mesA proteins have a high homology in oomycetes, in particular phytophthora.
TABLE 2 homology comparison of MesA proteins in PcMESA protein and fungi
TABLE 3 homology comparison of MesA proteins in PcMESA protein and oomycetes
Example 2 construction of Phytophthora capsici PcMesA Gene knockout and anaplerotic vector
The CRISPR/Cas 9-based gene knockout and back-filling vector construction method and the sequences of the relevant vectors and GFP gene sequences in this example are disclosed in documents "Fang,Y.,andTyler,B.M.(2016).Efficient disruption and replacement ofan effector gene inthe oomycete Phytophthora sojaeusing CRISPR/Cas9.Molecularplantpathology,17(1),127-139." and "Fang,Y.,Cui,L.,Gu,B.,Arredondo,F.,and Tyler,B.M.(2017).Efficient genome editing inthe oomycete Phytophthora sojae using CRISPR/Cas9.Curr.Protoc.Microbiol.44,21A.1.1-21A.1.26.". The pBluescript II SK + homology arm vector plasmid (the Donor vector) used in this example, sgRNA and Cas9 co-expression plasmid PYF515 were both given away by the teachings of BrettM.Tyler, state university of Oregon, U.S.A..
The gene sequence of insert PcMuORP1 used in this example is disclosed in document "Wang,W.,Xue,Z.,Miao,J.,Cai,M.,Zhang,C.,Li,T.,Zhang,B.,Tyler,B.M.and Liu,X.(2019)PcMuORP1,an Oxathiapiprolin-Resistance Gene,Functions as aNovel Selection Marker forPhytophthoraTransformation and CRISPR/Cas9 Mediated Genome Editing.Front.Microbiol.10,2402.".
The knockout related Donor vectors pBS-GFP-MesA, sgRNA and Cas9 co-expression plasmids pYF-MesA used in this embodiment are the co-expression plasmids pYF515-PcMuORP1-MesA with fluorothiazolepezine selection markers; the vector related to the anaplerotic is the Donor vector pBS-MesA, and the co-expression plasmid pYF-PcMuORP 1-GFP with the fluorothiazole piridon screening marker. The specific construction method is as follows:
the Donor vectors pBS-GFP-MesA, pBS-MesA used in this embodiment; the specific construction methods of the sgRNA and Cas9 coexpression plasmids pYF-MesA and pYF-515-GFP are as follows:
1) Construction of pBS-GFP-MesA: the DNA of phytophthora capsici strain BYA5 is used as a template, primers are designed to amplify an upstream 1000bp sequence (shown In sequence 3 In a sequence table and obtained by amplifying primers shown In table 2, namely pBS-GFP-MesA-F1 and pBS-GFP-MesA-R1) of a target gene PcMesA by using TaKaRa-In-fusion_Tools online website (http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Clonin g_Resources/Online_In-Fusion_Tools), a GFP gene sequence (shown In sequence table, wherein the GFP gene is a fragment obtained by amplifying a pTOR backbone plasmid used as a template with primer sequences shown In table 2, namely pBS-GFP-MesA-F2 and pBS-GFP-MesA-R2), a downstream 1000bp sequence (shown In sequence 4 In the sequence table and obtained by amplifying primers shown In table 2, namely pBS-GFP-MesA-F3 and pBS-GFP-MesA-R3) is used HD Cloning Kit three amplified fragments were sequentially fusion-ligated into Cloning vector pBluescript II SK + (EcoRI and BamHI double cleavage), the ligation product was transferred into e.coli DH 5a competent cells, cultured overnight at 37 ℃, and after selection of the monoclonal, the use of universal primer M13F (sequence:
5'-TGTAAAACGACGGCCAGT-3')/M13R (sequence: 5'-CAGGAAACAGCTATGACC-3') amplification, sequencing and cloning, and the correct recombinant expression vector containing the PcMesA upstream 1000bp sequence, GFP gene sequence and PcMesA downstream 1000bp sequence which are connected in sequence is named pBS-GFP-MesA.
2) PYF515 construction of 515-MesA: a specific targeting PcMesA gene is selected and a second-order weak sgRNA sequence (sgMesA: AACATCCCACCATTCCCTCT, target to the 1051-1070 nucleotide sequence of SEQ ID No.1 of PcMesA gene, and forward and reverse sgRNA sequence primers with NheI and BsaI cleavage sites and HH ribozyme are sent to a company to synthesize a double-strand sgRNA sequence by using sterile water, a reaction system of 3 μl sense strand solution, 3 μl antisense strand solution, 3 μl 10×T4 Buser (NEB) DNA LIGASE Buffer (N B), 4 μl 0.5 NaCl M, 21 μl ultra-pure GFP at room temperature, 2 μl ultra-pure water, and 35 to a solution of 35F (F) is added to a solution of 35F, the forward and reverse sgRNA sequence primers of 37A with NheI and BsaI cleavage sites and HH ribozyme are prepared by using sterile water, the reaction system of 3 μl sense strand solution is synthesized by using sterile water, 3 μl 10×Tn 4 Buffer (N B) is prepared by using a Buffer solution, 4 μl 10×4 Buffer (N.N.L) is prepared by using a 3 μl antisense strand solution, 3 μl4 Buffer (N.L) and 3 μl4 Buffe 4 Buffet 3, 3 μl4 Buffet 3, 2F is mixed with sterile water, 2F is mixed at room temperature, and the solution of 35F is diluted to obtain a solution of 35F (F) for 35, and the PCR solution is diluted by using a solution of 35 to dilute the PCR solution (F is diluted to obtain a solution of 35.35.p.5×2.g.g.5) after the gene, and a PCR solution is subjected to verify to be amplified to a positive by using a Buffer solution (F is prepared to be amplified by 35.35.35.35.F after a 35.has been prepared.
3) Construction of pBS-MesA: the DNA of Phytophthora capsici strain BYA is used as a template, and a primer is designed to amplify an upstream 1000bp sequence (shown as a sequence 3 In a sequence table and obtained by amplifying primers shown as pBS-MesA-F1 and pBS-MesA-R1 In a table 2) of a target gene PcMesA, a PcMesA gene sequence (shown as a sequence 1 In the table and obtained by amplifying fragments shown as pBS-MesA-F2 and pBS-MesA-R2 In the table 2) and a downstream 1000bp sequence (shown as a sequence 4 In the sequence table and obtained by amplifying primers shown as pBS-MesA-F3 and pBS-MesA-R3 In the table 2) of PcMesA by using a TaKaRa-In-fusion_Tools online website (http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Clonin g_Resources/Online_In-Fusion_Tools)HD Cloning Kit sequentially fusion-links three amplified fragments to Cloning vector pBluescript II SK +
(EcoRI and BamHI double cleavage), the ligation product was transferred into E.coli DH 5. Alpha. Competent cells, and after overnight incubation at 37℃was picked up and the monoclonal was amplified using the universal primer M13F (sequence:
5'-TGTAAAACGACGGCCAGT-3')/M13R (sequence: 5'-CAGGAAACAGCTATGACC-3') amplification and sequencing to verify cloning, and the correct verified recombinant expression vector containing the PcMesA upstream 1000bp sequence, the PcMesA gene sequence and the PcMesA downstream 1000bp sequence which are connected in sequence is named pBS-MesA.
4) PYF515 construction of 515-GFP: website EuPaGDT was designed with sgRNA (http: v/grna.ctegd.uga.edu /) and RNA structure online analysis tool (http: the// RNA. Urmc. Rochester. Edu/RNAstructureWeb/Servers/Predict 1.Ht mL), the specific targeting GFP gene was selected and a sgRNA sequence with weaker secondary structure was formed (sgGFP: CCACGGAACAGGGAGCTTTC, targeting GFP gene of SEQ ID No.5 of 152-171 nucleotide sequence, send to company synthesis with NheI and BsaI cleavage site and HH ribozyme forward and reverse sgRNA sequence primer, with sterile water to dissolve into 100 u M solution, annealing reaction to synthesize double-stranded sgRNA sequence, reaction system: 3u l sense strand solution, 3u l antisense strand solution, 3u l 10 x T4 DNA LIGASE Buffer (NEB), 4u l 0.5M NaCl,21 u l ultrapure sterile water, pipetting mixing, 100 ℃ reaction 2min, cooling to room temperature naturally 4h, then diluting the reaction solution 500 times, again taking 2u l 10 x T4 DNA LIGASE Buffer (NEB), 50ng pYF515 vector (NheI/BsI double cleavage), 4u l diluted double-stranded sgRNA solution, 1 u l T4 DNA LIGASE, sterile ultrapure water to 20 u l, room temperature reaction 30min, using GFP 5u l connected to the E.mu.5 c cell line to 35F 37-35F 35 cell line to verify the correct expression of the PCR sequence, 35F 35-35 g of the PCR colony was confirmed to be positive (37-35F-35G, 35F-35G PCR sequence.
TABLE 4 primer sequences for vector construction
EXAMPLE 3 obtaining of Phytophthora capsici PcMesA Gene knockout and anaplerotic transformants
The preparation of PcMesA knock-out and PcMesA gene complementation transformants using PEG-CaCl 2 -mediated protoplast transformation methods are disclosed in literature "Wang,Z.,Tyler,B.M.,Liu,X.Protocol ofPhytophthoracapsici transformation using the CRISPR-Cas9 system.Plant Pathogenic Fungi and Oomycetes.Humana Press,NewYork,NY,2018:265-274.".
The PcMesA knockout transformant is obtained by transferring the Donor vector, sgRNA and Cas9 coexpression plasmid (pBS-GFP-MesA and pYF 515-MesA) of the knockout gene PcMesA obtained in example 1 into protoplast of phytophthora capsici BYA5, culturing and screening the grown transformant by using OX-resistant V8 solid medium flat plate at 25 ℃, collecting mycelium of suspected transformant, extracting DNA for PCR sequencing verification, and re-extracting RNA from positive transformant for qPCR verification. PcMesA knockout transformants (. DELTA. MesA-T1,. DELTA. MesA-T2) were obtained.
The PcMesA gene-complemented transformant is obtained by transferring the Donor vector, sgRNA and Cas9 coexpression plasmid (pBS-MesA and pYF 515-GFP) of the GFP to be knocked out obtained in the example 1 into protoplast of a PcMesA knocked out mutant strain delta MesA-T1 of Phytophthora capsici, culturing and screening the grown transformant by using an OX-resistant V8 solid medium flat plate at 25 ℃, collecting mycelium of the suspected transformant, extracting DNA for PCR sequencing verification, and re-extracting RNA from the positive transformant for qPCR verification. A PcMesA make-up knockout transformant (Δ MesA-T1: mesA) was obtained.
Example 4 biological trait analysis of Phytophthora capsici PcMesA knockout and anaplerotic transformants
1. Hypha growth rate assay
The wild Phytophthora capsici strain BYA and the knock-out transformant PcMesA obtained in example 3 (Delta MesA-T1, delta MesA-T2) and the anaplerotic transformant (Delta MesA-T1:: mesA) were respectively inoculated into a V8 solid medium (15 mL of medium was poured into a 9cm dish), cultured for 4 days at 25℃in the dark, the colony diameters of the respective strains were measured by a crisscross method, and each strain was repeated 3 times.
The results showed that all the PcMesA knockout transformant strains tested had no significant differences in hyphal growth rate compared to the wild type phytophthora capsici strain BYA (fig. 3).
2. Sporangium quantity detection
The wild phytophthora capsici strain BYA, the knockout transformants delta MesA-T1, delta MesA-T2 and the reintegration transformant delta MesA-T1 obtained in example 3, wherein MesA are respectively inoculated to a V8 solid culture medium (15 mL of culture medium is poured into a 9cm culture dish), cultured for 4d under the dark condition at 25 ℃, cultured for 5d under the light condition at 25 ℃ to induce sporulation, and the morphology of sporangia on the culture dish is observed by a microscope and counted, and each strain is repeated for 3 times.
The results show that the knockout transformants Δ MesA-T1, Δ MesA-T2 obtained in example 3 do not have significant differences in the number of sporangia produced and the wild type compared to the wild type Phytophthora capsici strain BYA.
3. In vitro leaf pathogenicity detection of transformants
The tobacco plant variety to be tested is Benshi tobacco, which is planted in a seedling tray, the culture medium is turfy soil, and the tobacco plant variety to be tested grows to 4 to 6 weeks for standby. The wild Phytophthora capsici strain BYA and the knockout transformants Delta MesA-T1, delta MesA-T2 and the complementation transformant Delta MesA-T1 obtained in example 3 were inoculated with MesA in a V8 solid medium (15 mL of the medium was poured into a 9cm dish) and cultured for 4 days at 25℃in the dark, and a 5mm cake was removed from the edge of the colony by a puncher. Tobacco leaves with the same leaf position are collected, a bacterial cake is inoculated in the center of the leaf vein, 5 leaves are inoculated in each bacterial strain, and after the tobacco leaves are cultured for 4 days in the dark at 25 ℃ and moisture preservation (RH=60% -80%), the diameter (mm) of the lesions of the phytophthora capsici infected pepper leaves is measured by a crisscross method. The results show that the knock-out transformants obtained in example 3 showed no significant differences in mycelium pathogenicity and wild type as compared to wild type phytophthora capsici strain BYA, Δ MesA-T1, Δ MesA-T2.
4. Zoospore quantity detection
MesA was inoculated with 15ml of V8 solid medium in the center of a sterile dish (diameter 9 cm) and incubated in the dark at 25℃for 3 days, followed by induction of sporulation by incubation at 25℃for 5 days, followed by addition of 10ml of deionized water at 4℃for 30min and then at 25℃for 30min to release zoospores, the zoospore suspension was vortexed for 1min to stop swimming, and the number of zoospores counted by microscopy using a hemocytometer was set 3 replicates per strain.
The results showed that the PcMesA knock-out transformant obtained in example 3 did not release zoospores of normal morphology compared to the wild type Phytophthora capsici strain BYA (WT) and the anaplerotic transformant Delta MesA-T1: mesA, indicating that PcMesA protein affected the number of zoospores of Phytophthora capsici. Therefore, zoospore control eggs PcMesA of the phytophthora capsici are key proteins for zoospore development, and inhibiting the function of the protein can control the infection process of the phytophthora capsici, cut off disease circulation and control the large-scale occurrence of diseases.
Claims (1)
1. The application of inhibiting or inactivating the protein as shown in the sequence 2 or inhibiting the transcription of the gene as shown in the sequence 1 or inactivating the gene in inhibiting the formation of zoospores of phytophthora capsici or reducing the pathogenicity of phytophthora capsici.
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