CN116574161A - Phytophthora capsici growth and development regulation protein PcAGO5, and encoding gene and application thereof - Google Patents
Phytophthora capsici growth and development regulation protein PcAGO5, and encoding gene and application thereof Download PDFInfo
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- CN116574161A CN116574161A CN202310401030.2A CN202310401030A CN116574161A CN 116574161 A CN116574161 A CN 116574161A CN 202310401030 A CN202310401030 A CN 202310401030A CN 116574161 A CN116574161 A CN 116574161A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- C—CHEMISTRY; METALLURGY
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a ArgoN, PAZ, MID and Piwi structural domain-containing growth and development regulatory protein PcAGO5 from phytophthora capsici (Phytophthora capsici) germ, and a coding gene and application thereof. The PcAGO5 protein has an amino acid sequence shown as SEQ ID No.2, and the coding gene thereof has a nucleotide sequence shown as SEQ ID No. 1. The PcAGO5 protein has the main functions of reducing the growth speed of phytophthora capsici, reducing sporangium deformity and quantity, not producing zoospores, reducing the number of oospores and losing pathogenic ability after the protein is deleted. The gene of the invention has high application value in controlling epidemic diseases caused by phytophthora capsici, and the bactericide developed by taking the gene as an action target has important practical significance in controlling the occurrence and epidemic of epidemic diseases caused by phytophthora capsici.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a growth and development regulatory protein PcAGO5 from phytophthora capsici (Phytophthora capsici) containing ArgoN, PAZ, MID and Piwi structural domains, and a coding gene and application thereof.
Background
Phytophthora capsici (Phytophthora capsici) is a typical phytopathogenic oomycete of the genus Phytophthora of the family Pythiaceae and is widely distributed worldwide. As an important soil-borne pathogen, there is a wide host range that infects more than 26 kinds of vegetable crops including capsicum, tobacco in the Solanaceae family and leguminous and cucurbitaceae family, etc. the plant is a plant. 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. Zoospores generated by phytophthora capsici sporangia are an important source for re-infection, can be transmitted through rain, soil, air flow and other ways, become resting spores after being attached to the surfaces of host plants, and then germinate to generate hyphae to infect the hosts. Abnormal cooperation can also occur when two mating types exist in the field, so that the oospores are generated, have the capability of resisting stress, and can germinate to generate hyphae to infect hosts under proper environmental conditions.
One phenomenon of gene silencing initiated by double-stranded RNA is commonly known as RNA interference (RNAi) in organisms and can be divided into transcriptional and posttranscriptional levels. RNA with hairpin structure or double chain can be cut by enzyme to form small RNA of 20-30bp, and the small RNA performs gene silencing function by forming gene silencing complex with protein Argonaute (AGO). RNAi is critical to the growth and development of organisms, and therefore the core protein AGO, which forms the gene silencing complex, plays a very important role. In addition, AGO proteins are also found in plant, pathogen exosomes, revealing key biological functions of the protein in plant immunity and pathogenic bacterial pathogenesis. If the zoospore and oospore of phytophthora capsici are destroyed, the hypha growth rate is reduced, and the pathogenic ability of pathogenic bacteria is further inhibited, the normal disease circulation of the phytophthora capsici is blocked, so that the epidemic of the phytophthora capsici caused by the phytophthora capsici is controlled.
Disclosure of Invention
Based on this, the inventors found that there is a growth and development regulatory protein PcAGO5 containing AGO protein conserved domain ArgoN, PAZ, MID and Piwi in phytophthora capsici. The growth regulating protein is closely related to the hypha growth rate, sporangium yield and morphology, zoospore and oospore quantity of phytophthora capsici and is also related to pathogenicity of phytophthora capsici. Therefore, the normal infection of phytophthora capsici can be blocked by controlling the growth and development regulatory protein PcAGO5, so that the large-scale spread and epidemic of the phytophthora capsici is controlled (inhibited or blocked).
Thus, the invention provides a phytophthora capsici leonian growth and development regulating protein containing ArgoN, PAZ, MID and Piwi structural domains, which is named as PcAGO5 and is A1) or A2) or 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 a similarity of 75% or more, preferably 85% or more, more preferably 95% or more with the amino acid sequence shown in SEQ ID NO.2 and having the same function as the amino acid sequence shown in 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 protein in the A1) -A4) is generally derived from phytophthora capsici in nature, namely generally, the protein is a natural product, can be artificially expressed or synthesized, can be synthesized to code the gene, and can be obtained by biological expression. 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, SEQ ID NO.2 (PcAGO 5) in the sequence table consists of 1269 amino acid residues.
It is a further object of the present invention to provide nucleic acid molecules encoding said PcAGO5 proteins containing ArgoN, PAZ, MID and Piwi domains. 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 the PcAGO5 protein is B1), 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 having 75% or more, 85% or more, or 95% or more identity with the nucleotide sequence shown in B1) and encoding the above PcAGO5 protein;
b3 Under stringent conditions with a nucleotide sequence defined in B1) or B2), and a cDNA molecule or DNA molecule encoding the above PcAGO5 protein. For example, in the present invention, the DNA sequence of the growth regulating protein includes the DNA sequence of the growth regulating protein present in a different strain of Phytophthora capsici, such as Phytophthora capsici LT1534 strain.
In the invention, the DNA sequence (coding gene and cDNA) of the pathogenic regulatory protein can be specifically shown as SEQ ID No.2, wherein SEQ ID No.1 in the sequence table consists of 3807 nucleotides, and nucleotides 1-3807 from the 5' end of the sequence 1 are coding sequences for coding the protein (PcAGO 5) 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 75% or more, more preferably 85% or more, still more preferably 95% 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 comprising the coding gene of claim 2;
d2 A recombinant vector comprising the coding gene of claim 2 or a recombinant vector comprising the expression cassette of D1);
d3 A recombinant microorganism comprising the coding gene of claim 2, or a recombinant microorganism comprising the expression cassette of D1), or a recombinant microorganism comprising the recombinant vector of D2);
d4 A transgenic plant cell line comprising the coding gene of claim 2, or a transgenic plant cell line comprising the expression cassette of D1);
d5 A transgenic plant tissue comprising the coding gene of claim 2, or a transgenic plant tissue comprising the expression cassette of D2);
d6 A transgenic plant organ comprising the coding gene of claim 2, or a transgenic plant organ comprising the expression cassette of D2);
d7 A nucleic acid molecule which inhibits the expression of the coding gene of claim 2; preferably, the nucleic acid molecule is a nucleic acid molecule for knocking out the coding gene of claim 2, or silencing the coding gene of claim 2, and can be a nucleic acid molecule for encoding an sgRNA fragment which targets a target gene to be knocked out, such as a sgRNA sequence (coding sequence) CAAGGGGGAAAGCATGTCCG which targets the PcAGO5 gene;
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 Dodor 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 sgRNA fragment of a target gene to be knocked out and a DNA sequence for expressing Cas9 protein, wherein the target gene to be knocked out is a PcAGO5 gene, and the sgRNA sequence (encoding sequence) of the target PcAGO5 gene is CAAGGGGGAAAGCATGTCCG. Preferably, the sgRNA and Cas9 coexpression plasmid is prepared by taking a pYF515 carrier as a starting carrier, and inserting a double-stranded sgRNA coding sequence obtained by annealing the sgRNA of a PcAGO5 gene between Nhe I and Bsa I enzyme recognition sites of the pYF515 carrier to obtain the sgRNA and Cas9 coexpression plasmid;
d9 A nucleic acid molecule that inhibits translation of the RNA molecule of claim 3;
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 use of the growth regulating protein, its coding gene or the RNA molecule of claim 3 or the biological material of claim 4 for controlling the complete pathogenicity of oomycetes; preferably, the oomycete is phytophthora capsici, most preferably phytophthora capsici, LT1534.
The application is any one or more of the following E1) -E6):
e1 Application in regulating and controlling (reducing) phytophthora capsici leonian growth rate;
e2 Use in modulating (reducing) phytophthora capsici sporangia yield, and/or maintaining or disrupting sporangia wild type morphology;
e3 Use in regulating (inhibiting) zoospore production;
e4 Use in modulating (reducing) phytophthora capsici s pathogenicity to a host;
e5 Use in modulating (reducing) phytophthora capsici i infest host capability;
e6 For inhibiting and/or killing phytophthora capsici leonian.
Preferably, the application comprises the application of E1) -E6) by inhibiting the transcription or inactivating the coding gene of SEQ ID NO.1, or inhibiting the translation of the RNA molecule, or inhibiting and/or inactivating the activity of the PcAGO5 growth and development regulatory protein of SEQ ID NO.2 in a sequence table.
In the application, the phytophthora capsici leonian sporangia and zoospores are regulated (reduced) and/or hypha growth rate is reduced and/or infection capacity and/or pathogenicity (pathogenicity) to a host are reduced by inhibiting transcription of the coding genes, or inhibiting translation of the RNA sequences, or inhibiting and/or inactivating the activity of the PcAGO5 growth regulating protein, so that phytophthora capsici leonian can be inhibited or killed.
Preferably, the application is that the PcAGO5 protein shown in the phytophthora capsici SEQ ID NO.2 is deleted to change the falling form of the phytophthora capsici, slow down the growth of hyphae, and/or malform sporangia and can not produce zoospores, reduce pathogenicity and the like;
the sixth purpose of the invention is to provide the application of the PcAGO5 growth and development regulating protein shown in SEQ ID NO.2 in the sequence table, the coding gene shown in SEQ ID NO.1 in the sequence table, or the RNA molecule, the biological material or the protein or DNA shown in claim 5 in screening phytophthora capsici leonian bacteriostasis and/or bactericide as a bacteriostasis or bactericide target; the antibacterial or bactericide can inhibit or inactivate PcAGO5 growth and development regulating protein shown in the phytophthora capsici SEQ ID No.2 to change the falling form of the phytophthora capsici, slow down the growth of hyphae, reduce the sporangium yield of the phytophthora capsici, inhibit zoospores, reduce the growth rate of the hyphae, and/or reduce the infection capacity and/or pathogenicity (pathogenicity) to a host, thereby being capable of inhibiting or killing phytophthora capsici. 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 diseases caused by phytophthora capsici on production.
The seventh object of the present invention is to provide a method for screening or assisting in screening phytophthora capsici bacteriostasis and/or bactericide, wherein the method comprises applying a substance to be detected to the phytophthora capsici, and when the substance to be detected can inhibit transcription of the above DNA sequence, or inhibit translation of the above RNA sequence, or inhibit and/or inactivate activity of the above PcAGO5 growth and development regulatory protein, the substance to be detected is a candidate phytophthora capsici bacteriostasis and/or bactericide.
An eighth object of the present invention is to provide a method for controlling (inhibiting or blocking) the complete pathogenicity 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 PcAGO5 growth regulating protein as described above;
wherein the control (inhibition or blocking) of the complete pathogenicity of the oomycete is to and/or inhibit (reduce) zoospore production of phytophthora capsici, and/or to mutate (e.g. expand) sporangia, and/or to reduce the hypha growth rate of phytophthora capsici, and/or to reduce the infection capacity of phytophthora capsici to and/or the pathogenicity (pathogenicity) of phytophthora capsici to the host.
The invention also provides a method for reducing the ability of an oomycete to infect a host by controlling full virulence as claimed.
The method for controlling the complete pathogenicity of the phytophthora capsici is a method for controlling the complete pathogenicity of the phytophthora capsici by knocking out any one of the DNA sequences. The phytophthora capsici comprises a phytophthora capsici LT1534 strain.
In one embodiment of the present invention, wherein the above-described gene knockout method employs CRISPR/Cas 9-based gene knockout.
Specifically, the CRISPR/Cas 9-based gene knock-out method is to transfect a target gene Donor vector, sgRNA and Cas9 co-expression plasmid into phytophthora capsici to obtain the target knock-out protein inactivated recombinant strain.
The Donor vector is a recombinant vector containing a sequence of 800-1500bp upstream of a target gene to be knocked out, a Dodor 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 sgRNA fragment of a target gene to be knocked out and a DNA sequence for expressing Cas9 protein, wherein the target gene to be knocked out is a PcAGO5 gene, and the sgRNA sequence (encoding sequence) of the target PcAGO5 gene is CAAGGGGGAAAGCATGTCCG.
Preferably, the sgRNA and Cas9 coexpression plasmid is obtained by taking a pYF515 carrier as a starting carrier, and inserting a double-stranded sgRNA coding sequence obtained by annealing the sgRNA of the PcAGO5 gene between Nhe I and Bsa I enzyme recognition sites of the pYF carrier.
The application of the substance for inhibiting the expression and/or activity of the PcAGO5 growth and development regulation protein in preparing the plant phytophthora capsici leonian antibacterial or bactericide also belongs to the protection scope of the invention.
In the above application, the substance that controls the expression and/or activity of the PcAGO5 growth-regulating protein is a substance that inhibits the expression of the PcAGO5 growth-regulating protein and/or inhibits the transcription of the gene encoding the cellulose synthase protein and/or inhibits the translation of the RNA molecule obtained by the transcription of the gene encoding the PcAGO5 growth-regulating protein.
The invention also provides a primer sequence for amplifying the PcAGO5 growth and development regulatory protein in phytophthora capsici, namely a primer sequence for amplifying a target sequence, wherein the primer sequence is preferably shown as SEQ ID No. 1. The phytophthora capsici comprises a phytophthora capsici LT1534 strain.
A primer and/or primer pair that amplifies the entire length of the DNA sequence encoding the growth regulatory protein described above or any DNA sequence thereon is also within the scope of the present invention.
Experiments prove that the PcAGO5 growth and development regulating protein provided by the invention plays a role in the growth and development process of phytophthora capsici. By PEG-CaCl 2 The knockout mutant obtained by combining the mediated protoplast transformation technology and the CRISPR/Cas9 gene editing technology has obviously changed growth and development compared with the wild type parent strain, and mainly comprises the following components: the PcAGO5 protein is deleted, so that phytophthora capsici falls into a form, hypha growth is slowed down, sporangium is deformed and swelled, zoospores cannot be released, the number of oospores is reduced, and the phytophthora capsici cannot cause diseases to tobacco leaves; therefore, the PcAGO5 growth and development regulation protein in phytophthora capsici can play an important role in the processes of asexual reproduction, sexual reproduction, infection and the like of the phytophthora capsici. The invention provides technical support for further exploring the growth and development process and pathogenic mechanism of phytophthora capsici, and provides technical foundation for preventing and treating plant epidemic diseases caused by phytophthora capsici and developing novel bactericides.
Drawings
FIG. 1 is a schematic representation of the PcAGO5 domain of Phytophthora capsici, comprising ArgoN, PAZ, MID and Piwi domains.
FIG. 2 is a colony morphology of wild type Phytophthora capsici strain LT1534, pcAGO5 gene knockout mutant ΔPcAGO5.
FIG. 3 is a diagram showing the morphology of the wild type Phytophthora capsici strain LT1534, pcAGO5 gene knockout mutant ΔPcAGO5 sporangia (scale 20 μm).
FIG. 4 shows a tobacco leaf pathogenic force profile (4 d shots after inoculation) of wild type Phytophthora capsici strain LT1534, pcAGO5 gene knockout mutant ΔPcAGO5 bacterial cake.
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.
The wild-type strain LT1534 of phytophthora capsici is given away by the Kurt Lammour professor us Tennessee State University, LT1534 strain information is disclosed in the literature "Jason e.s., andrea l.v., howard s.j., gregori m.v., michael a.g., maryn o.c., nicholas d., jennifer j, joann m., kurt h.l., and Christine d.s. (2021), high-Quality Reference Genome Sequence for the Oomycete Vegetable Pathogen Phytophthora capsici Strain LT1534.plant microbiol.10,21. The above strains are merely materials used in the examples of the present invention, and in fact, the phytophthora strains can be obtained using any commercially available route when the screening markers of the present invention are applied.
All of the above strains have been identified by existing morphological and molecular biological methods.
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 pea into 1L deionized water, sterilizing at 121deg.C for 20min, filtering with gauze to obtain pea soup, adding Mannitol 91.1g, caCO 3 2g，CaCl 2 1g, distilled water is fixed to volume of 1L, and 15g of agar powder is added into the solid culture medium, and the culture medium is sterilized for 20min at 121 ℃.
The media used for the knockout transformation experiments were as follows:
NPB medium (Nutrient pea broth): adding 125g 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, caCO 3 2.0g、CaCl 2 0.1g、MgSO 4 0.5g、MgSO 4 0.5g、KNO 3 3.0g、K 2 HPO 4 1.0g、KH 2 PO 4 1.0g, and the volume is fixed to 1L by 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. Sterilizing, adding 0.22 μm filter, and filtering to obtain Vitamin stock2mL(Folic acid 6.7×10 -7 g/mL；Pyridoxine-HCl 6.0×10 -4 g/mL；Biotin 6.7×10 -7 g/mL；Thiamine-HCl 1.3×10 -3 g/mL；L-inositol 4.0×10 -5 g/mL；Nicotinic acid 4.0×10 -5 g/mL；Riboflavin 5.0×10 -5 g/mL) and Vitamin stock2mL (FeC 6 H 5 O 7 ·3H 2 O 5.4×10 -4 g/mL；Na 2 MoO 4 ·H 2 O 3.0×10 -5 g/mL；ZnSO 4 ·7H 2 O 3.8×10 -4 g/mL；MgSO 4 ·H 2 O 3.8×10 -5 g/mL；H 3 BO 3 2.5×10 -5 g/mL；CuSO 4 ·5H 2 O 7.5×10 -4 g/mL)
The knockout conversion test reagents were formulated as follows:
enzymolysis liquid: in the present preparation, lyase (Lysing Enzymes from Trichoderma harzianum, L1412, sigam) 0.12g, cellulase (yakult R10) 0.12g,0.8M Mannitol 10mL, ultrapure water 8mL,0.5M KCl 800. Mu.L, 0.5M MES (pH 5.7) 800. Mu.L, 0.5M CaCl 2 400. Mu.L, 0.22. Mu.M filter membrane.
W5 solution: KCl 0.1g, caCl 2 ·2H 2 O4.6g,NaCl 2.25g,Glucose 7.8g, the ultrapure water is dissolved to 250mL and the volume is fixed, and a 0.22 mu m filter membrane is used for filtration sterilization.
PEG-CaCl 2 Solution (40% w/v): ready-to-use, 6g PEG4000,0.8M Mannitol 3.75mL, 3mL of ultrapure water, 0.5M CaCl 2 3mL,0.22 μm filter sterilized.
MMG solution (250 mL): mannitol 18.22g,0.5M MES (pH 5.7) 2.0mL, mgCl 2 ·6H 2 O0.76 g, ultrapure water to 250mL, and 0.22 μm filter membrane filtration sterilization.
Example 1, acquisition of growth and development regulatory protein PcAGO5 in Phytophthora capsici and its coding Gene
In this example, the phytophthora capsici i growth and development regulatory protein PcAGO5 and its coding gene (or cDNA) can be obtained by amplifying DNA (or cDNA) of phytophthora capsici i strain LT1534 as a template by the primers shown in table 1. Wherein, the DNA or RNA extracted material can be mycelium of phytophthora capsici strain LT1534. Wherein, the DNA of phytophthora capsici leonian strain LT1534 is used as a template, pcAGO5-F and PcAGO5-R are used for amplifying to obtain a coding gene PcAGO5 of PcAGO5, as shown in SEQ ID NO.1 in a sequence table, SEQ ID NO.1 consists of 3807 nucleotides, nucleotides 1-3807 from the 5' end of SEQ ID NO.1 are coding sequences, a protein (PcAGO 5) shown in SEQ ID NO.2 in the sequence table is coded, wherein the sequence of the ArgON domain is the 384-591 amino acid residue sequence from the amino end of SEQ ID NO.2, the sequence of the PAZ domain is the 659-779 amino acid residue sequence from the amino end of SEQ ID NO.2, the sequence of the MID domain is the 842-923 amino acid residue sequence from the amino end of SEQ ID NO.2, and the sequence of the Piwi domain is the 934-1233 amino acid residue sequence from the amino end of SEQ ID NO. 2. The cDNA of phytophthora capsici strain LT1534 is used as a template, and PcAGO5-F and PcAGO5-R are used for amplification to obtain the cDNA of PcAGO5, 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 PcAGO5 full-length coding gene amplification primers
SEQ ID NO.1 shows the following:
SEQ ID NO.2 shows as follows:
example 2 construction of Phytophthora capsici PcAGO5 Gene knockout vector
CRISPR/Cas 9-based gene knockout vector construction method in the embodiment and sequences of related vectors (comprising pBluescript II SK) + And PYF 515) and NPT II gene sequences are disclosed in the literature "Fang, Y, and Tyler, B.M. (2016). Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/cas9.molecular plant geometry, 17 (1), 127-139," and "Fang, Y, cui, L., gu, B., arrendo, F., and Tyler, B.M. (2017). Efficient genome editing in the oomycete Phytophthora sojaeusing CRISPR/cas9.Cur.Protoc.Microbiol.44, 21A.1.1-21A.1.26. pBluescript II SK used in this embodiment + The homology arm vector plasmid (doppler vector), sgRNA and Cas9 co-expression plasmid PYF515 are each given a professor Brett m.tyler, state university of oregon, usa.
The Donor vector pBS-NPTII-AGO5 used in this embodiment; the specific construction method of the sgRNA and Cas9 coexpression plasmid pYF515-AGO5 is as follows:
1) Construction of pBS-NPTII-AGO 5: the DNA of Phytophthora capsici strain LT1534 was used as a template, the primers were designed to amplify the 1000bp upstream sequence (sequence 3 In the sequence list) of the target gene PcAGO5 using TaKaRa-In-fusion_tools on-line site (http:// www.clontech.com/US/Products/cloning_and_component_cells/cloning_resources/online_in-fusion_tools), the NPTII gene sequence (obtained by amplifying the primers shown In Table 2, pBS-NPTII-AGO5-F1 and pBS-NPTII-AGO 5-R1), the NPTII gene sequence (obtained by amplifying the primers shown In Table 2, pBS-NPTII-AGO5-F2 and pBS-NPTII-AGO5-R2 using pYF backbone plasmids as templates), the 812 downstream sequence (sequence 4 In the sequence list, and the primers shown In Table 2, pBS-NPTII-AGO5-F1 and pBS-NPTII-AGO5-R3 were used to amplify the NPTII gene sequence (obtained by amplifying the primers shown In Table 2, pBS-NPTII-AGI-5-F3 and NPTII-3-R3)HD Cloning Kit sequentially fusion-links three amplified fragments into Cloning vector pBluescript II SK + (EcoR V cleavage), the ligation product was transferred into E.coli DH 5. Alpha. Competent cells, cultured overnight at 37℃and then the monoclonal was picked up, the clone was verified by amplification using the universal primer M13F (SEQ ID NO: 5'-TGTAAAACGACGGCCAGT-3')/M13R (SEQ ID NO: 5'-CAGGAAACAGCTATGACC-3'), and the recombinant expression vector containing the 1000bp upstream, NPTII gene and 812bp downstream PcAGO5 sequences, which were sequentially ligated, was designated pBS-NPTII-AGO5.
2) Construction of pYF515-AGO 5: the site EuPaGDT (http:// grna. Ctegd. Uga. Edu /) and RNA structure on-line analysis tool (http:// RNA. Urmc. Rochester. Edu/RNAstructureWeb/Servers/Predict1/predict1. HtmL) were used to design the site EuPaGDT (http:// grna. Ctegd. Uga. Edu /) and to select a sgRNA sequence (sgAGO 5: CAAGGGGGAAAGCATGTCCG) that specifically targets the PcAGO5 gene and forms a weaker secondary structure, the nucleotide sequence at positions 2148-2167 of SEQ ID No.1 that targets the PcAGO5 gene, which was sent to the company to synthesize forward and reverse sgRNA sequence primers with Nhe I and Bsa I cleavage sites and HH ribozyme. The solution was dissolved in sterile water to 100. Mu.M. And (3) synthesizing a double-stranded sgRNA sequence by an annealing reaction, wherein the reaction system is as follows: 3. Mu.l sense strand solution, 3. Mu.l antisense strand solution, 3. Mu.l 10×T4 DNA Ligase Buffer (NEB), 4. Mu.l 0.5M NaCl, 21. Mu.l ultrapure sterile water were mixed by pipetting, reacted at 100℃for 2min, left to cool naturally to room temperature for 4h, and then the reaction solution was diluted 500-fold. Mu.l of 10 XT 4 DNA Ligase Buffer (NEB), 50ng of pYF515 vector (Nhe I/Bsa I double cleavage), 4. Mu.l of diluted double-stranded sgRNA solution, 1. Mu. l T4 DNA Ligase, sterile ultrapure water were then added to 20. Mu.l, reacted at room temperature for 30min, and transferred into E.coli DH 5. Alpha. Competent cells using 5. Mu.l of the ligation product, after overnight incubation at 37℃colony PCR was verified using the primer pair RPL41_Pseq_F (sequence: 5'-CAAGCCTCACTTTCTGCTGACTG-3')/M13F (sequence: 5'-TGTAAAACGACGGCCAGT-3'), and positive clones were verified by sequencing, and the recombinant vector which verified that the above sgRNA could be expressed correctly was named pYF-AGO 5.
TABLE 2 primer sequences for vector construction
Example 3 obtaining of Phytophthora capsici PcAGO5 Gene knockout transformant
PEG-CaCl is adopted 2 Methods of mediated protoplast transformation to prepare PcAGO5 knock-out transformants, methods of oomycete genetic transformation are disclosed in the literature "Wang, Z., tyler, B.M., liu, X.protocol of Phytophthora capsici transformation using the CRISPR-Cas9 system.plant Pathogenic Fungi and Oomycetes Humana Press, new York, N.Y., 2018:265-274".
The obtaining of the PcAGO5 gene knockout transformant specifically comprises transferring the Donor vector, sgRNA and Cas9 coexpression plasmid (pBS-NPTII-AGO 5 and pYF515-AGO 5) of the knockout gene PcAGO5 obtained in example 1 into protoplast of phytophthora capsici LT1534, culturing and screening the grown transformant by G418-resistant V8 solid medium plate at 25 ℃, collecting mycelium of the suspected transformant, extracting DNA for PCR sequencing verification, re-extracting RNA from the positive transformant for qPCR verification. A PcAGO5 knockout transformant (Δpcago 5) was obtained.
Example 4 biological trait analysis of Phytophthora capsici PcAGO5 knockout transformant
1. Hypha growth rate assay
The wild Phytophthora capsici strain LT1534 and the knockout transformant PcAGO5 knockout transformant (. DELTA.PcAGO5) obtained in example 3 were inoculated into 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 the colony diameters of the respective strains were measured by the crisscross method and repeated 3 times per strain.
The results showed that the hyphal growth rate of all the measured PcAGO5 knockout transformant strains was significantly reduced compared to the wild-type phytophthora capsici strain LT1534 (table 3). And the colony morphology of the PcAGO5 knockout transformant was malformed compared to LT1534 (fig. 2). Experimental results show that the phytophthora capsici growth and development regulating protein PcAGO5 participates in regulating and controlling hypha growth and colony normal morphology of the phytophthora capsici.
2. Sporangium morphology observation and quantity detection
The wild phytophthora capsici strain LT1534 and the knockout transformant delta PcAGO5 obtained in example 3 are respectively inoculated to a V8 solid culture medium (15 mL of the culture medium is poured into a 9cm culture dish), cultured for 4d under the dark condition at 25 ℃, cultured for 5d under the illumination condition at 25 ℃ to induce spore production, the morphology of sporangia on the culture dish is observed through a microscope and counted, and each strain is repeated for 3 times.
The results showed that the knockout transformant Δppago 5 sporangia obtained in example 3 swelled and the number significantly decreased compared to the wild phytophthora capsici strain LT1534 (table 3), indicating that the phytophthora capsici growth regulating protein PcAGO5 affects the morphology and number of phytophthora capsici sporangia (fig. 3).
3. Zoospore quantity detection
The wild phytophthora capsici strain LT1534 and the knockout transformant delta PcAGO5 obtained in the example 3 are respectively inoculated to a V8 solid culture medium (15 mL of the culture medium is poured into a 9cm culture dish), cultured for 4d under the dark condition at 25 ℃, cultured for 5d under the illumination condition at 25 ℃ to induce sporulation, 10mL of sterile water is added, the culture medium is placed at 4 ℃ for 30min and then transferred to 25 ℃ for 40min to release zoospores, the zoospore suspension is vortex-oscillated for 1min to stop the zooabout, a blood cell counting plate is used for observing and counting through a microscope, the number of zoospores is counted, and each strain is repeated for 3 times.
The results showed that the sporangia of the knockout transformant Δppago 5 obtained in example 3 did not release zoospore numbers compared to the wild-type phytophthora capsici strain LT1534, indicating that the phytophthora capsici growth regulating protein PcAGO5 affects the production of zoospores of phytophthora capsici (table 3).
4. Oospore number detection
The standard strain of phytophthora capsici A2 mating type was cultivated in a V8 solid medium (15 mL medium was poured into a 9cm dish) in opposition to the wild type phytophthora capsici strain LT1534 and the knockout transformant Δppago 5 obtained in example 3, respectively, and 5d was cultivated in the dark at 25 ℃, then the junction of the two colonies was observed under a microscope, and the number of oospores was counted, and each strain was repeated 3 times.
The results showed that the knockout transformant Δppago 5 obtained in example 3 showed significantly reduced numbers of oospores produced compared to the wild-type phytophthora capsici strain LT1534, indicating that the phytophthora capsici growth and development regulatory protein PcAGO5 was involved in the sexual reproduction phase of phytophthora capsici and affected the production of oospores of phytophthora capsici (table 3).
5. 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 LT1534 and the knockout transformant ΔPcAGO5 obtained in example 3 were inoculated to V8 solid medium (15 mL medium was poured into a 9cm dish), cultured for 4d at 25℃in the dark, and a 5mm bacterial cake was removed from the edge of the colony with 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 indicate that the knockout transformant Δppago 5 obtained in example 3 lost the pathogenicity to tobacco leaves compared to the wild-type phytophthora capsici strain LT1534 (fig. 4 and table 3). This suggests that phytophthora capsici growth and development regulatory protein PcAGO5 is involved in regulating the biological pathway of phytophthora capsici infection to host plants.
Therefore, the phytophthora capsici growth and development regulating protein PcAGO5 can regulate and control the virulence of the phytophthora capsici, and the infection process of the phytophthora capsici can be controlled by inhibiting the function of the protein, so that the diseases are controlled to occur on a large scale.
TABLE 3 biological Property determination results of Phytophthora capsici PcAGO5 Gene knockout transformant
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Claims (9)
1. A growth and development regulatory protein PcAGO5 from phytophthora capsici (Phytophthora capsici) containing ArgoN, PAZ, MID and Piwi domains, which is a protein of the following A1) or A2) or A3) or A4):
a1 A protein consisting of the amino acid sequence shown in SEQ ID No. 2;
a2 Fusion proteins obtained by ligating tags at the N-terminal and/or C-terminal of the protein shown in SEQ ID No. 2;
a3 Protein which is derived from A1) by substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID No.2 and is related to phytophthora capsici growth and development;
a4 Amino acid sequence having the same function as the amino acid sequence shown in SEQ ID No.2, with the amino acid sequence similarity of 93% or more, preferably 95% or more, more preferably 98% or more, with SEQ ID No. 2.
2. A coding gene encoding the growth regulating protein PcAGO5 as set forth in claim 1; preferably, the coding gene is B1) or B2) or B3) as follows:
b1 A DNA molecule represented by the nucleotide sequence shown in SEQ ID No. 1;
b2 A cDNA molecule or a DNA molecule having 75% or more, or 85% or more, or 95% or more identity with the nucleotide sequence shown in B1) and encoding the protein of claim 1;
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 a protein according to claim 1.
3. An RNA molecule transcribed from the coding gene of claim 2;
preferably, the sequence of the RNA molecule is C1) or C2) as follows:
c1 A DNA sequence shown in SEQ ID No. 1) having a similarity of 75% or more, more preferably 85% or more, still more preferably 95% or more, with an RNA sequence having the same function as the RNA sequence transcribed from the DNA sequence shown in SEQ ID No. 1;
c2 RNA sequence transcribed from the DNA sequence shown in SEQ ID No. 1.
4. A biological material related to the growth and development regulatory protein PcAGO5 as set forth in claim 1, the coding gene as set forth in claim 2, or the RNA molecule as set forth in claim 3, which is any one of the following D1) to D10):
d1 An expression cassette comprising the coding gene of claim 2;
d2 A recombinant vector comprising the coding gene of claim 2 or a recombinant vector comprising the expression cassette of D1);
d3 A recombinant microorganism comprising the coding gene of claim 2, or a recombinant microorganism comprising the expression cassette of D1), or a recombinant microorganism comprising the recombinant vector of D2);
d4 A transgenic plant cell line comprising the coding gene of claim 2, or a transgenic plant cell line comprising the expression cassette of D1);
d5 A transgenic plant tissue comprising the coding gene of claim 2, or a transgenic plant tissue comprising the expression cassette of D2);
d6 A transgenic plant organ comprising the coding gene of claim 2, or a transgenic plant organ comprising the expression cassette of D2);
d7 A nucleic acid molecule which inhibits the expression of the coding gene of claim 2; preferably, the nucleic acid molecule is a nucleic acid molecule that knocks out the encoding gene of claim 2, or that silences the encoding gene of claim 2;
d8 An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing or expressing D7) said nucleic acid molecule;
d9 A nucleic acid molecule that interferes with translation of the RNA molecule of claim 3;
d10 An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing or expressing D9) said nucleic acid molecule.
5. Use of the growth and development regulatory protein PcAGO5 of claim 1, the coding gene of claim 2, or the RNA molecule of claim 3, or the biological material of claim 4, for controlling the growth and development of oomycetes or for regulating the pathogenicity of oomycetes;
preferably, the oomycete is phytophthora capsici, most preferably phytophthora capsici, LT1534.
6. Use of the growth and development regulatory protein PcAGO5 as defined in claim 1, the coding gene as defined in claim 2 or the RNA molecule as defined in claim 3 or the biological material as defined in claim 4 as a pesticide formulation for screening for inhibition of oomycete growth and development and/or pathogenicity to a host, characterized by any one or several of the following E1) -E6):
e1 Application in regulating and controlling the growth rate of phytophthora capsici leonian;
e2 Use in regulating phytophthora capsici sporangium yield, and/or maintaining or disrupting sporangia wild type morphology;
e3 Application of phytophthora capsici to control and control zoospore generation;
e4 Application in regulating and controlling pathogenicity of phytophthora capsici on a host;
e5 Application in regulating and controlling phytophthora capsici infection host capacity;
e6 For inhibiting and/or killing phytophthora capsici leonian.
Preferably, comprising effecting E1) to E6) by inhibiting transcription in or inactivating the coding gene according to claim 2, or inhibiting translation of the RNA molecule according to claim 3, or inhibiting and/or inactivating the growth and development regulatory protein PcAGO5 protein according to claim 1.
7. A method of inhibiting and/or killing oomycetes growth comprising inhibiting or inactivating expression of or RNA translation of a gene encoding a growth regulatory protein PcAGO5 comprising ArgoN, PAZ, MID and Piwi domains in oomycetes or inhibiting or inactivating activity of a growth regulatory protein PcAGO5 comprising ArgoN, PAZ, MID and Piwi domains; the amino acid sequence of the growth and development regulating protein PcAGO5 containing ArgoN, PAZ, MID and Piwi structural domains is the amino acid sequence shown in SEQ ID No.2, and the oomycete is phytophthora capsici (Phytophthora capsici);
the method for inhibiting or inactivating the expression of the coding gene of the pathogenic regulatory protein PcAGO5 containing ArgoN, PAZ, MID and Piwi structural domains in oomycetes is preferably to knock out the coding gene, and preferably, the coding gene of the pathogenic regulatory protein PcAGO5 containing ArgoN, PAZ, MID and Piwi structural domains is DNA shown as SEQ ID No. 1.
8. A method of inhibiting or blocking the full virulence of an oomycete, comprising inhibiting or blocking the full virulence of the oomycete by inhibiting or inactivating the expression of the coding gene of claim 2, or inhibiting the translation of the RNA sequence of claim 3, or inhibiting or inactivating the activity of the growth and development regulatory protein PcAGO5 of claim 1; the oomycete is phytophthora capsici leonian (Phytophthora capsici);
wherein the activity of inhibiting or blocking phytophthora capsici is to inhibit the infection capability of phytophthora capsici to a host and/or pathogenicity to the host, and/or inhibit the sporangium yield of phytophthora capsici, and/or inhibit zoospore production, and/or destroy the wild type form of sporangia, and/or reduce the hypha growth rate of phytophthora capsici and/or malform the falling form of phytophthora capsici;
the method of inhibiting or inactivating the expression of the gene of claim 2 is preferably a knockout of the coding gene of claim 2.
9. A method for reducing the capability of an oomycete infected host, which is to inhibit or block complete pathogenicity by the method of claim 8, so as to achieve the aim of reducing the capability of the oomycete infected host; the host is capsicum, tobacco, etc.
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