CN116478258A - Mulberry cup fungus effector protein Cs02526 and application thereof - Google Patents
Mulberry cup fungus effector protein Cs02526 and application thereof Download PDFInfo
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- CN116478258A CN116478258A CN202310434227.6A CN202310434227A CN116478258A CN 116478258 A CN116478258 A CN 116478258A CN 202310434227 A CN202310434227 A CN 202310434227A CN 116478258 A CN116478258 A CN 116478258A
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- C07—ORGANIC CHEMISTRY
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- 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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- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- 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
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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Abstract
The invention discloses a phellinus linteus effector protein Cs02526 and application thereof, wherein the amino acid sequence of the phellinus linteus effector protein is shown as SEQ ID NO.1, and the nucleotide sequence of the phellinus linteus effector protein is shown as SEQ ID NO. 2; according to the invention, through prokaryotic expression of Cs02526 and after the leaf is pretreated by using the recombinant expression of Cs02526 protein, the resistance of the plant to pathogenic bacteria is improved by activating autoimmune reaction, which shows that the cupped fungus effector protein Cs02526 can be used as a target for biological control of the pathogenic bacteria.
Description
Technical Field
The invention relates to the field of biological control, in particular to a phellinus igniarius effector protein Cs02526 and an application of the phellinus igniarius effector protein Cs 02526.
Background
Phytopathogens invade host plants by a variety of means. Wherein, pathogenic bacteria secrete effector proteins into plant cells, and the infection efficiency of the plant is improved by regulating the growth and immune response of the plant. In order to avoid the harm caused by pathogenic bacteria, plants have evolved a series of fine and efficient immune responses. Such as the production of active oxygen, the synthesis of phytohormones, and the expression of defense-related genes. The identification of pathogenic bacteria key effector proteins and plant defense related genes is the key place for development of biological control medicines and plant disease resistance breeding.
The sclerotium rolfsii is a main pathogenic fungus of the mulberry sclerotium disease, and has the characteristics of wide spread, strong pathogenic fungus disease, high epidemic frequency, easy expansion of infection and successive outbreaks, and finally causes large-area yield reduction of the mulberry. At present, the main mode for preventing and treating the diseases is to spray chemical pesticides, and although the transmission of pathogenic bacteria can be reduced to a certain extent, the problems of environmental pollution, extremely easy pesticide resistance, food safety and the like exist. The effector proteins playing a key role in the infection process of the sclerotium rolfsii are selected through screening, the functions of the effector proteins in plants are explored, and molecular targets and theoretical basis can be provided for development of biological control means and disease-resistant breeding of the mulberry sclerotium diseases.
Disclosure of Invention
Accordingly, it is an object of the present invention to provide a sclerotinia cup effector protein Cs02526; the second purpose of the invention is to provide a sclerotinia mulberri effector protein Cs02526 gene; the third object of the present invention is to provide a recombinant expression vector containing the phellinus linteus effector protein Cs02526 gene; the fourth object of the present invention is to provide a host containing the gene of the phellinus linteus effector protein Cs02526; the fifth purpose of the invention is to provide the application of the phellinus linteus effector protein Cs02526 in improving the defensive immunity and/or disease resistance of plants; the sixth object of the present invention is to provide a method for improving the resistance of plants to pathogenic bacteria.
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. the amino acid coded by the phellinus linteus effector protein Cs02526 is shown in SEQ ID No. 4.
2. The nucleotide sequence of the phellinus linteus effector protein Cs02526 gene is shown in SEQ ID NO. 3.
3. Recombinant expression vector containing said Phellinus linteus effector protein Cs02526 gene.
4. A host containing the phellinus linteus effector protein Cs02526 gene.
5. The application of the phellinus linteus effector protein Cs02526 in improving the defensive immunity and/or disease resistance of plants.
Preferably, the plant is tobacco.
Preferably, the disease resistance is that of sclerotium rolfsii.
6. A method of increasing the resistance of a plant to pathogenic bacteria by overexpressing phellinus linteus effector protein Cs02526 in the plant or treating the plant with phellinus linteus effector protein Cs02526; the amino acid coded by the phellinus linteus effector protein Cs02526 is shown in SEQ ID NO. 2.
Preferably, the pathogenic bacteria are sclerotium rolfsii.
Preferably, the plant is tobacco.
The invention has the beneficial effects that: the invention identifies that a phellinus linteus effector protein Cs02526 can cause plant cell death and trigger plant immune responses. After the plant leaves are treated by prokaryotic expression and purification of Cs02526 protein, the disease resistance of the plants can be improved. The invention provides theoretical basis and molecular targets for the development of subsequent biopesticides.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 shows the amplification of Cs02526 gene (1-2: cs02526 gene; M: marker);
FIG. 2 is a phenotype observation diagram of transient expression of the Mulberry cup fungus effector protein Cs02526 in tobacco leaves (A: a phenotype diagram of the tobacco leaves after injection; B: a symptom diagram of the tobacco leaves after injection after staining and decoloring by DAB solution; C: an analysis diagram of plant immune response related gene expression in the tobacco leaves after injection).
FIG. 3 shows analysis of leaf disease resistance after prokaryotic expression of Morganella morganii effector protein Cs02526 and protein treatment (A: SDS-PAGE electrophoresis after purification of prokaryotic expression of Cs02526 protein; B: infection area map of Morganella morganii after protein treatment; C: infection area statistical map).
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
Example 1 cloning of the Gene encoding Cs02526 and construction of expression vector
(1) Cloning of Cs02526 encoding Gene
Extracting mycelium tissue RNA of Phellinus linteus by using Trizol reagent, and usinggDNA Removel and cDNA Synthesis SuperMix (TransGen) kit was reverse transcribed into cDNA and the gene encoding Cs02526 was amplified by PCR using the cDNA as template. Wherein the amplification primers are as follows:
upstream primer F1:5'-CCATCGATATGAAGATTTCCATCAAT-3' (SEQ ID NO. 1);
downstream primer R1:5'-GCGTCGACCTAATTAGCCTCTGCTGC-3' (SEQ ID NO. 2).
PCR amplification reaction System (50. Mu.L): 2X Super Pfx MasterMix. Mu.L, 2. Mu.L of the upstream primer (10. Mu.M), 2. Mu.L of the downstream primer (10. Mu.M), 50-100ng of template cDNA, and water were added to make up to 50. Mu.L.
The reaction procedure was set as follows: pre-denaturation at 98℃for 2min; denaturation at 98 ℃,10s, annealing at 55 ℃,30s (actual annealing temperature depends on primer Tm value), extension at 72 ℃,30s,35 cycles; final extension at 72deg.C for 5min; preserving at 12 ℃.
As shown in FIG. 1, the Cs02526 gene is successfully cloned from the Phellinus linteus cDNA, the nucleic acid sequence obtained by sequencing is shown as SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 4.
Cs02526 gene full-length nucleotide sequence (SEQ ID NO. 3)
ATGAAGATTTCCATCAATCTCATTAGTTTACTTTTCTGCAGCACTTTCGTCGCTGCAGAGGGCTTATCGTTCTTTAGAAATGGTCAAAAAGTCCTCGAAGATAAGGGAGGAGCCGTTCCTGGCAGCAATCCTCTTACATACTGCAAAGCCGACCATTCTTCCGATATTCTAAAGCTCGACCACGTCAATTTGACTCCTAATCCTCCAACTGCGTATGCACAATTTGTGAGATCTTGGGAAATGGCAGCAGAGGCTAATTAG；
Protein amino acid sequence (SEQ ID NO. 4)
MKISINLISLLFCSTFVAAEGLSFFRNGQKVLEDKGGAVPGSNPLTYCKADHSSDILKLDHVNLTPNPPTAYAQFVRSWEMAAEAN；
(2) Construction of expression vectors
The glue recovery was performed according to the Gel Extraction Kit (Omega) instructions. According to the kit, the recovered Cs08297 and PVX plant expression vector were ligated using T4 ligase (cleavage site ClaI, salI), transferred into E.coli, cultured overnight on LB plate (containing 50. Mu.g/ml kanamycin) at 37℃and the monoclonal colonies were picked up for amplification and PCR detection. After the sequencing was correct, the recombinant Plasmid pGR-Cs 02526 was extracted using the Plasmid Mini Kit (Omega) Kit.
Example 2 tobacco transient expression of Cs02526 and detection of the production of plant defenses
(1) Transient expression of tobacco
Recombinant plasmids pGR-Cs 02526 and negative control plasmid pGR-GFP (construction method is the same as in example 1) were transferred into Agrobacterium GV3101 cells, spread on LB plates, cultured at 28℃for 2-3 days, and then monoclonal colonies were picked and PCR detection was performed.
The agrobacteria containing pGR-Cs 02526, pGR-GFP were grown at 28℃and 180rpm overnight, and the cells were collected by centrifugation at 5000rpm for 5min, resuspended in MMA solution, and repeated 3 times to finally adjust the OD=0.4 to 0.6.
The bacterial solutions are respectively injected into leaf blades of Nicotiana benthamiana of 5-8 weeks old by a syringe, repeated for three times, marked and placed in an incubator for culture.
After 5-7 days of injection, the tobacco injection site was observed for symptoms of cell necrosis.
As shown in fig. 2a, transient expression of Cs02526 in tobacco can induce plant cell death compared to negative controls.
(2) Detection of plant immune response
Active oxygen accumulation: soaking tobacco leaves after 48h of injection of the bacterial liquid in 1mg/ml DAB solution, and standing in the dark for 6-8h. Taking out the leaves, decoloring by using absolute ethyl alcohol, and photographing after decoloring is finished.
Detection of defense-related gene expression levels: tobacco leaves after 48 hours of injection of the above bacterial liquid were harvested, total RNA was extracted and cDNA was synthesized (the method was the same as in example 1). The expression levels of the defense-related genes CYP71D20, nbPti5, nbAcre31 and NbWRKY7 were detected using the QIAGEN SYBR Premix Ex TaqTMII (Tli RNaseH Plus) kit. Wherein, the fluorescent quantitative primer is as follows:
CYP71D20-qPCR-F:5’-AAGGTCCACCGCACCATGTCCTTAGAG-3’(SEQ ID NO.5)；
CYP71D20-qPCR-F:5’-AAGAATTCCTTGCCCCTTGAGTACTTGC-3’(SEQ ID NO.6)；
NbPti5-qPCR-F:5’-CCTCCAAGTTTGAGCTCGGATAGT-3’(SEQ ID NO.7)；
NbPti5-qPCR-R:5’-CCAAGAAATTCTCCATGCACTCTGTC-3’(SEQ ID NO.8)；
NbAcre31-qPCR-F:5’-AATTCGGCCATCGTGATCTTGGTC-3’(SEQ ID NO.9)；
NbAcre31-qPCR-R:5’-GAGAAACTGGGATTGCCTGAAGGA-3’(SEQ ID NO.10)；
NbWRKY7-qPCR-F:5’-CACAAGGGTACAAACAACACAG-3’(SEQ ID NO.11)；
NbWRKY7-qPCR-R:5’-GGTTGCATTTGGTTCATGTAAG-3’(SEQ ID NO.12)；
NbActin-qPCR-F:5’-TGGTCGTACCACCGGTATTGTGTT-3’(SEQ ID NO.13)；
NbActin-qPCR-R:5’-TCACTTGCCCATCAGGAAGCTCAT-3’(SEQ ID NO.14)。
fluorescent quantitative PCR reaction procedure: pre-denaturation at 95 °c2min, denaturation 95 ℃,10s, annealing 60 ℃,35s,40 cycles. Three replicates were performed for each reaction, and after the reaction was completed, 2 was used -ΔΔCt The data were calculated to determine the gene expression level.
The results are shown in fig. 2B and C. The results show that the production of active oxygen and the PTI pathway-mediated defense genes in leaves injected with Agrobacterium containing pGR-Cs 02526 have a significant upward trend compared to the control, indicating that expression of Cs02526 in tobacco leaves can activate the tobacco defense response.
Example 3 expression, purification and analysis of plant disease resistance of Cs02526 protein
(1) Construction of prokaryotic expression vectors
The vector construction was identical to that of example 1, and the signal peptide-removed Cs02526 was ligated with the prokaryotic expression vector pET32a (BamHI and EcoRI sites) to obtain the recombinant plasmid pET32a-Cs02526, which was then transferred into the expression strain BL21 (DE 3).
The upstream primer F2:5'-CGGGATCCATGAAGATTTCCATCAAT-3' (SEQ ID NO. 15);
downstream primer R2:5'-CGGAATTCCTAATTAGCCTCTGCTGC-3' (SEQ ID NO. 16);
the above-mentioned correct bacterial liquid was subjected to expansion culture at 37℃and 200rpm for 3-4 hours, and then the inducer IPTG (final concentration: 1.0 mM) was added thereto, followed by culture at 220rpm overnight at 16 ℃. The cells were collected by high-speed centrifugation and resuspended 3 times with PBS buffer. After ultrasonication, the protein was purified using a Ni-NTA 6FF His tag protein purification kit (BBI), 40. Mu.L of protein solution was taken, 10. Mu.L of 5 XSDS loading buffer was added, 10min was added to a boiling water bath, and centrifugation was performed for 5min at 13000 r/min. 10. Mu.L of the supernatant was subjected to SDS-PAGE, and Coomassie brilliant blue R250 was stained to observe the expression.
The results are shown in FIG. 3A. The results showed that Cs02526 recombinant protein was successfully expressed and purified.
(2) Disease resistance analysis
The protein solution was used to measure the protein concentration using BCA protein concentration measurement kit (Beyotime). The inoculation experiment was performed using pathogenic bacteria after injecting the recombinant protein into tobacco leaves for 18 h.
The injection concentration of Cs02526 recombinant protein is preferably 40 μg/ml.
The pathogenic bacteria inoculated in the experiment are at least one kind of sclerotinia sclerotiorum and Botrytis cinerea.
As shown in fig. 3B and C, the areas of necrosis caused by the calix mori on the leaves after treatment of tobacco leaves with Cs02526 protein were reduced compared to the control, as compared to the injection of empty protein. The results show that the Cs02526 protein can be used for improving the resistance of plants to pathogenic bacteria after pretreatment.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. The truffle dish fungus effector protein Cs02526 is characterized in that: the amino acid coded by the phellinus linteus effector protein Cs02526 is shown in SEQ ID NO. 4.
2. The mulberry cup fungus effector protein Cs02526 gene is characterized in that: the nucleotide sequence of the phellinus linteus effector protein Cs02526 gene is shown in SEQ ID NO. 3.
3. A recombinant expression vector comprising the phellinus linteus effector protein Cs02526 gene of claim 2.
4. A host comprising the cupped trazosin Cs02526 gene of claim 2.
5. Application of phellinus linteus effector protein Cs02526 in improving the defensive immunity and/or disease resistance of plants.
6. The use according to claim 5, characterized in that: the plant is tobacco.
7. The use according to claim 5, characterized in that: the disease resistance is against Phellinus linteus.
8. A method for improving the resistance of a plant to pathogenic bacteria, comprising: treating the plant by overexpressing phellinus linteus effector protein Cs02526 in the plant or using phellinus linteus effector protein Cs02526; the amino acid coded by the phellinus linteus effector protein Cs02526 is shown in SEQ ID NO. 4.
9. The method according to claim 8, wherein: the pathogenic bacteria are Phellinus linteus.
10. The method according to claim 8, wherein: the plant is tobacco.
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