CN111118039B - Pathogenicity-related botrytis cinerea genes Bcmet3 and Bcmet16 and application - Google Patents
Pathogenicity-related botrytis cinerea genes Bcmet3 and Bcmet16 and application Download PDFInfo
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- CN111118039B CN111118039B CN202010082092.8A CN202010082092A CN111118039B CN 111118039 B CN111118039 B CN 111118039B CN 202010082092 A CN202010082092 A CN 202010082092A CN 111118039 B CN111118039 B CN 111118039B
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Abstract
The invention discloses pathogenicity-related botrytis cinerea genes Bcmet3 and Bcmet16 and application thereof in preventing and treating botrytis cinerea, and belongs to the technical field of agricultural prevention and treatment. The research of the invention firstly shows that Bcmet3 and Bcmet16 are important genes influencing the pathogenicity of the botrytis cinerea, and the infecting capacity of the botrytis cinerea can be obviously reduced by knocking out the two genes. The Bcmet3 and Bcmet16 genes can be used as targets to be applied to designing and screening anti-botrytis cinerea medicaments or other treatments, and provide a new choice for developing novel bactericides.
Description
Technical Field
The invention relates to the technical field of agricultural control, in particular to a method for controlling botrytis cinerea infected fruits by knocking out Bcmet3 gene or Bcmet16 gene.
Background
Botrytis cinerea (Botrytis cinerea) is a typical lethal pathogen that invades the host and causes programmed death of host cells, resulting in significant rot of fruits infected with Botrytis cinerea, which can infect over 200 plants, such as strawberry, tomato, grape, vegetables, etc., with the greatest damage to the mature or senescent tissues of dicotyledonous plant hosts. When the physiology of the host changes and the environment is not conducive to the germination of botrytis cinerea, the fungus can remain stationary for a considerable period of time before the tissue decays, so that infestation of the botrytis cinerea can occur from the seedling stage to the ripening of the product, and after harvesting of a healthy-looking crop, severe spoilage can still occur during the postharvest storage period. The harvested fruits and vegetables have gray mold outbreaks in the retail chain, including storage, transportation to remote markets or during display by retailers, and cause huge economic losses in both pre-harvest and post-harvest stages of agricultural production.
Among the measures for controlling gray mold, the use of fungicides is the most common method for controlling gray mold. The biggest problem caused by the use of fungicides is the increasing resistance of Botrytis cinerea, and a considerable part of the current fungal population is resistant to fungicides, and this multi-drug resistance is mainly caused by the increased gene expression of ABC transporters (Leroch, M., Kretschmer, m.and Hahn, M. (2011) fungal resistance genes of Botrytis cinerea isolates from commercial vireyards in South West germany.j.phytopathothol.159, 63-65.).
In the new approach of preventing and controlling botrytis cinerea, the development of new biological prevention and control agents aiming at specific proteins or genes in botrytis cinerea has been made. For example, Qiuden et al provide a method for inducing disease resistance of fruits and vegetables by using botrytis cinerea secretory protein elicitor BcXyl1 protein, thereby reducing the occurrence of botrytis cinerea (patent application No. 201811041393.5).
The genome of the botrytis cinerea is clarified by sequencing, and the pathogenic mechanism of the botrytis cinerea can be deeply researched by combining a series of current molecular manipulation means (such as gene knockout, gene silencing and the like) so as to find an effective action target for preventing and treating the botrytis cinerea. For example, patent document CN 109467594A discloses that Bcdmt2 protein and coding gene thereof play an important role in regulating and controlling the pathogenicity and conidiospore generation of botrytis cinerea and can be used as a new target site for preventing and controlling botrytis cinerea; patent document CN 106497943a discloses that the protein encoded by the pathogenic gene BcSEP5 of botrytis cinerea can be used as a target in designing and screening anti-botrytis cinerea agents.
Disclosure of Invention
The invention aims to provide a gene related to the pathogenicity of botrytis cinerea, and the gene is used as an action target to realize the prevention and control of the botrytis cinerea.
In order to achieve the purpose, the invention adopts the following technical scheme:
according to the invention, the sulfur assimilation genes Bcmet3 and Bcmet16 of the botrytis cinerea are knocked out by adopting a gene knock-out technology to interfere with the sulfur metabolism pathway of the botrytis cinerea, so that the normal growth of fungi is not influenced, but the infection capacity of the botrytis cinerea is remarkably reduced, and the two genes are related to the pathogenicity of the botrytis cinerea.
The invention provides application of a Bcmet3 gene in reducing botrytis cinerea pathogenicity, wherein the application is to reduce botrytis cinerea infectivity by knocking out a Bcmet3 gene, and the nucleotide sequence of the Bcmet3 gene is shown in SEQ ID No. 1.
The invention provides application of a Bcmet16 gene in reducing botrytis cinerea pathogenicity, wherein the application is to reduce botrytis cinerea infectivity by knocking out a Bcmet16 gene, and the nucleotide sequence of the Bcmet16 gene is shown in SEQ ID No. 2.
The invention proves that the pathogenicity of botrytis cinerea is obviously reduced due to the deletion of the Bcmet3 gene and the Bcmet16 gene, and the Bcmet3 gene and the Bcmet16 gene are important genes for botrytis cinerea of crops caused by botrytis cinerea. Therefore, the screening of the compound capable of preventing the gene expression and the protein expression, modification and positioning can effectively control the occurrence of the botrytis cinerea.
The invention provides application of a Bcmet3 gene or a protein coded by the Bcmet3 gene as a target in preparing a gray mold resistant medicament, wherein the nucleotide sequence of the Bcmet3 gene is shown as SEQ ID No. 1.
The invention provides application of a Bcmet16 gene or a protein coded by the Bcmet16 gene as a target in preparing a gray mold resistant medicament, wherein the nucleotide sequence of the Bcmet16 gene is shown as SEQ ID No. 2.
The invention also provides a method for preventing and treating plant gray mold, which comprises the step of reducing the infection capacity of gray mold by knocking out Bcmet3 gene with a nucleotide sequence shown as SEQ ID No.1 or Bcmet16 gene with a nucleotide sequence shown as SEQ ID No. 2.
The plant is a tomato fruit.
The invention has the following beneficial effects:
the research of the invention firstly shows that Bcmet3 and Bcmet16 are important genes influencing the pathogenicity of the botrytis cinerea, and the infecting capacity of the botrytis cinerea can be obviously reduced by knocking out the two genes. The Bcmet3 and Bcmet16 genes can be used as targets to be applied to designing and screening anti-botrytis cinerea medicaments or other treatments, and provide a new choice for developing novel bactericides.
Drawings
FIG. 1 is a schematic diagram of construction of a knockout strain Δ Bcmet 3.
FIG. 2 shows the PCR-verified electrophoresis of the deletion mutant of Bcmet3, where Δ met3 is the Bcmet3 knock-out strain and B05.10 is the wild type Botrytis cinerea.
FIG. 3 shows the PCR-verified electrophoresis of the deletion mutant of Bcmet16, where Δ met16 is the Bcmet16 knock-out strain and B05.10 is the wild type Botrytis cinerea.
FIG. 4 shows the PCR-verified electrophoretograms of the complementation of Bcmet3 and Bcmet16 genes, wherein pBcmet3 and pBcmet16 are complemented strains.
FIG. 5 is a schematic diagram of the cleavage site of the PNAN-OGG plasmid.
FIG. 6 shows spore germination of Δ Bcmet3 and Δ Bcmet16 Botrytis cinerea.
FIG. 7 shows the mycelial growth of Δ Bcmet3 and Δ Bcmet16 Botrytis cinerea.
FIG. 8 is a comparison of Δ Bcmet3 and Δ Bcmet16 in comparison to wild type strains in the ability to infect tomato fruits.
Detailed Description
The present invention will be further described with reference to the following examples. The methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used in the examples are commercially available unless otherwise specified.
Example 1 construction and Gene complementation of the Thiosynostosis strains Δ Bcmet3 and Δ Bcmet16 of Botrytis cinerea
1.1 knockout vector construction
The Bcmet3 gene (nucleotide sequence is shown as SEQ ID NO: 1) and the Bcmet16 gene (nucleotide sequence is shown as SEQ ID NO: 2) are two key genes on the pathway of the gray mold assimilating external inorganic sulfur (sulfate ions), and the experiment researches the influence of influencing sulfur assimilation on the fruits infected by the fungus by silencing the two genes.
In this case, transformation fragments were constructed by homologous recombination and Hygromycin Phosphotransferase (HPH) was used as a selection marker. As shown in FIG. 1, taking Bcmet3(Bcmet16) gene as an example, first, side fragments located at 1409bp (1214bp) upstream and 1362bp (609bp) downstream of Bcmet3 were amplified from genomic DNA of wild type strain B05.10 using primer pairs P1/P2 and P3/P4, respectively (see Table 1 for primers). Subsequently, 1764bp HPH cassette containing the trpC promoter (hygromycin B resistance) was amplified from the pKKT plasmid using primer pair P5/P6. Two Bcmet3 flanking fragments were then mixed with HPH cassette at a molar ratio of 1:1:3 as templates for fusion PCR, system: mu.l of fusion template, 2. mu.l dNTP (2.5mM), 2.5. mu.l 10 XPCR buffer, 15.25. mu.l ddH2O, 0.25. mu.l Taq polymerase. The procedure was as follows: 92min at 4 ℃, 30s at 94 ℃, 10min at 58 ℃, 5min at 72 ℃, 10 to 15 cycles, and 10min at 72 ℃ for final extension. A3479 bp (3320bp) DNA fragment was amplified from the fusion PCR product using primer pair P7/P8 for transformation of wild type strain B05.10 protoplasts.
TABLE 1
1.2 protoplast transformation
Botrytis cinerea strains PDA was cultured for 2-3 days, and colony-border plates (8 plates/100 mL) were inoculated into YEPD culture medium (10mg/mL peptone, 3mg/mL yeast extract, 20mg/mL glucose). Shaking at 25 deg.C and 175rpm alternately in 16h/8 light and dark for 1 day, collecting fresh mycelia with sterile filter, and adding balance buffer (0.6 MKCL; 50mM CaCl)2) And washing twice. Then, 15ml of 1.5% lyses cell wall lysis buffer (0.6M KCl,50mM CaCl) was placed2) Culturing in shaking table at 30 deg.C and 85rpm for 60-90min, filtering enzyme solution, removing mycelium, washing with balance buffer solution twice, collecting filtrate, and centrifuging at 1500rpm for 5 min. Protoplasts in the filtrate were washed with STC buffer (0.8M sorbitol, 0.05M Tris, pH 8.0,50mm CaCl)2) Resuspend and centrifuge 2 times, STC solution resuspend protoplast, adjusted to 2X108one/mL, and diluted with SPTC (STC: SPTC ═ 4:1) (SPTC: STC solution STC of 40% PEG6000, w/v) on ice for use.
Adding 100 mu L of protoplast suspension, 30 mu g of DNA and 5 mu L of heparin sodium (5mg/mL) solution into a conversion tube, gently mixing uniformly, and carrying out ice incubation for 90 minutes; adding 1ml of PTC solution, mixing, and standing at 25 deg.C for 30 min. Adding the protoplast mixture into 200mL regeneration medium (1g/L yeast extract, 1g/L casein hydrolysate, 274g/L sucrose and 16g/L agar powder) cooled to 43 deg.C, shaking, pouring into flat plates (20 mL each), and culturing at 25 deg.C for 12-16 h; covering 10mL of selection medium (same as regeneration medium, only 10g of agarose replaces 16g of agar powder) containing the screening medicament (100mg/mL of hygromycin B), and culturing for 4-10 days at 25 ℃; transformants were selected on PDA plates of the selection agent (100mg/mL hygromycin B), cultured at 25 ℃ for 1-2 days, and selected for PCR detection. Verification of Bcmet partial fragment (P11/P12), verification of fragment 1(P10/P13) and verification of fragment 2(P9/P14) as shown in FIGS. 2 and 3.
1.3 Bcmet3, Bcmet16 Gene complementation
The target Bcmet3(Bcmet16) gene fragment was obtained by PCR amplification using Botrytis cinerea B05.10 cDNA as a template and primers P15/P16. The PNAN-OGG plasmid (as shown in figure 5) is cut by Not I enzyme, the PNAN-OGG plasmid and a target gene are fused by a seamless cloning kit (EVERBRIGHT, US) to complete directional cloning and are transformed into competent escherichia coli, the competent escherichia coli is evenly coated on an LB solid plate containing Amp antibiotics, the escherichia coli is cultured overnight at 37 ℃, a single colony is selected to be put into an LB liquid culture medium containing the antibiotics for amplification, 1 mu L of the single colony is taken for colony PCR identification, and the colony is identified to be correctly sent for sequencing. And (3) amplifying the plasmid with correct sequencing, extracting the plasmid, cutting the plasmid by using Apa I and Sac II enzymes, separating by gel electrophoresis, and cutting a segment containing a target gene for DNA recovery for transforming mutant strain protoplast.
The protoplast transformation is as shown above, taking Bcmet3 as an example, culturing Bcmet3 mutant strain by PDA to prepare protoplast, taking 100 μ L of protoplast suspension, 30 μ g of the recovered DNA fragment and 5 μ L of heparin sodium, mixing uniformly, transforming, shaking, screening 100mg/mL nourseothricin, selecting transformant for PCR detection, and verifying primer P17/P18 as shown in figure 4.
Example 2 Δ Bcmet3 and Δ Bcmet16 Botrytis cinerea for spore germination and hyphal growth
2.1 materials of the experiment
1) Pathogenic bacteria to be tested: botrytis cinerea (B05.10) strains were used for storage in the laboratory, and Δ Bcmet3 and Δ Bcmet16 were obtained from example 1 and cultured on PDA, in which case spores were used within four generations of PDA medium.
2) Reagent to be tested: PDB Medium (Potato dextrose Broth Medium, American BD Co.)
2.2 Experimental methods
Scraping mature fresh plate spores (cultured in PDA culture medium for 10-12 days) with scraper, washing with 2mL of 0.01% Triton X-100 sterile water solution, pouring the washed spore solution into 15mL centrifuge tube, washing repeatedly for 2 times, pouring all the obtained bacterial solution into the centrifuge tube, vortex shaking at 26 deg.C for 5min, filtering with absorbent cotton to obtain spore suspension, counting Botrytis cinerea spores with blood counting plate, diluting to 1 × 107CFU/mL is ready for use.
And (3) measuring the spore germination rate: after 5h, randomly selecting 9 fields under an optical microscope to take pictures (the magnification is 125), then utilizing a microscope capture software to measure and count all spores in the fields, wherein the number of the spores in each field is about 30, and taking the average spore germination rate of each field as a parallel. And (3) judging spore germination: bacterial elongation to more than twice the average axial length of the spores (about 41 μm) is considered germination (>82 μm); spore germination rate-the number of spores germinated in the visual field/the number of all spores in the visual field.
Measurement of hypha length: after 9h, randomly selecting 9 visual fields under an optical microscope to take pictures (the magnification is 125), and then measuring and counting the total axial length of each thallus in the visual fields by using image measuring software matched with microscope capture software. The number of cells in each visual field was about 30, and the average cell length in each visual field was taken as one parallel.
The entire experiment was independently repeated twice.
2.3 analysis of results
Spore germination of the wild type strain and the knockout strain is shown in FIG. 6. After the PDB culture medium is used for culturing for 5h, the spore germination rates have no significant difference, and the result shows that the Bcmet3 and Bcmet16 genes are knocked out, so that the spore germination of the botrytis cinerea is not significantly different, and the germination of the botrytis cinerea spores is not influenced.
The length of hyphal growth of the wild type strain and the knockout strain is shown in FIG. 7. The growth length of hyphae has no significant difference after the PDB culture medium is used for culturing for 9h, and the result shows that the hyphae has no significant difference on the hyphae growth of botrytis cinerea after the Bcmet3 and Bcmet16 genes are knocked out, and the hyphae growth of the botrytis cinerea is not influenced.
Example 3 Δ Bcmet3 and Δ Bcmet16 Botrytis cinerea infection fruit experiments
3.1 Experimental materials
The tomato fruits and the cherry tomatoes are picked in the field, the ripeness degree of the fruits is 80 percent, the fruits move to a laboratory within 3 hours after picking, the fruits are soaked in 0.5 percent sodium hypochlorite for 4min for surface sterilization, then are soaked in clear water for 2min and cleaned for 2 times, and the fruits are dried in the air for standby application.
Botrytis cinerea (B05.10) strains were kept in the laboratory and used, and were cultured using PDA.
3.2 infection of tomato fruits by Botrytis cinerea
The wild type Botrytis cinerea B05.10(WT) was cultured in PDA medium for 10-12 days to produce spores.
And evaluating the pathogenicity change condition of the mutant strain by adopting a fruit inoculation method. Scraping Botrytis cinerea spore, eluting with Triton X-100 sterile water solution, filtering to obtain spore suspension, counting Botrytis cinerea spore with blood counting plate, and adjusting spore suspension concentration to 4 × 105CFU/mL. Mutant strains Δ Bcmet3 and Δ Bcmet16 and anaplerotic strains were inoculated onto fruits using wild type botrytis cinerea (WT) as a control. Removing fruit stalks of the treated fruits, and carrying out wound treatment on the tomato fruits in a super clean bench under an aseptic condition. Punching a 2X 2mm small hole at the equator of the tomato fruit by using a syringe needle, inoculating 5 mu l of spore suspension, placing at the wound, standing for 30min, and drying in the air. The fruits are placed in a sterilized tray, the sterilized tray is placed in an incubator with the humidity of 23 ℃ and the humidity of 90% for cultivation for 8d, the diameter of the germs on the surfaces of the fruits is measured by a vernier caliper by adopting a cross method, 20 fruits are arranged in each group, and the experiment is repeated for 3 times. The pathogenicity of the mutant strain was evaluated using the plaque diameter size.
The experimental results are shown in fig. 8, and the plaque size of the three bacteria treated on the surface of the tomato is obviously increased in the treatment period. No growth of plaque was observed in day3 and before, and it was observed from day 4 that Δ Bcmet16 inoculated with WT appeared as plaque, whereas Δ Bcmet3 plaque was observed from day 5, however it grew rapidly in plaque diameter from day7 to day 8. Of the three plaques, WT was consistently maximal, Δ Bcmet16 times, Δ Bcmet3 was minimal, and the differences were all statistically significant (P < 0.05). After 8 days of inoculation, Δ Bcmet3 strain caused fruit plaque size 37.5% less than WT strain infected fruit plaque, and Δ Bcmet16 strain group 12.5% less. The results show that: on tomato fruits, the bacterial plaque diameter of the mutant strain is obviously smaller than that of the wild strain, and the pathogenicity of the mutant strain is obviously reduced.
Taken together, Bcmet3 and Bcmet16 affect the ability of Botrytis cinerea to infect tomato fruits.
Sequence listing
<110> Zhejiang university
<120> botrytis cinerea genes Bcmet3 and Bcmet16 related to pathogenicity and application
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Claims (4)
- The application of Bcmet3 gene in reducing the pathogenicity of botrytis cinerea is characterized in that the infectivity of botrytis cinerea is reduced by knocking out Bcmet3 gene, wherein the nucleotide sequence of the Bcmet3 gene is shown in SEQ ID NO. 1.
- The application of the Bcmet3 gene or the protein coded by the gene as a target in preparing a gray mold resistant medicament is characterized in that the nucleotide sequence of the Bcmet3 gene is shown as SEQ ID NO. 1.
- 3. A method of controlling gray mold in plants comprising: the infectivity of the botrytis cinerea is reduced by knocking out Bcmet3 gene with the nucleotide sequence shown in SEQ ID No.1 in the botrytis cinerea.
- 4. A method for controlling plant gray mold as claimed in claim 3, wherein said plant is tomato fruit.
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| CN202010082092.8A CN111118039B (en) | 2020-02-07 | 2020-02-07 | Pathogenicity-related botrytis cinerea genes Bcmet3 and Bcmet16 and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102965382A (en) * | 2012-12-07 | 2013-03-13 | 河北农业大学 | New botrytis cinerea gene related to pathogenicity and application of new botrytis cinerea gene |
| CN105154453A (en) * | 2015-10-19 | 2015-12-16 | 吉林大学 | Pathogenicity related botrytis cinerea gene BcSep4 and application thereof |
| CN106497943A (en) * | 2016-11-04 | 2017-03-15 | 吉林大学 | A kind of ash arrhizus bacteria gene BcSEP5 related to pathogenicity and its application |
| CN109467594A (en) * | 2018-11-28 | 2019-03-15 | 中国科学院植物研究所 | Bcdmt2 protein and its encoding gene are in regulation botrytis cinerea pathogenicity and the aborning application of conidium |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965382A (en) * | 2012-12-07 | 2013-03-13 | 河北农业大学 | New botrytis cinerea gene related to pathogenicity and application of new botrytis cinerea gene |
| CN105154453A (en) * | 2015-10-19 | 2015-12-16 | 吉林大学 | Pathogenicity related botrytis cinerea gene BcSep4 and application thereof |
| CN106497943A (en) * | 2016-11-04 | 2017-03-15 | 吉林大学 | A kind of ash arrhizus bacteria gene BcSEP5 related to pathogenicity and its application |
| CN109467594A (en) * | 2018-11-28 | 2019-03-15 | 中国科学院植物研究所 | Bcdmt2 protein and its encoding gene are in regulation botrytis cinerea pathogenicity and the aborning application of conidium |
Non-Patent Citations (1)
| Title |
|---|
| Botrytis cinerea B05.10 Bcmet3 (Bcmet3),mRNA;Van Kan JA，等;《Genbank登录号XM_001548961.2》;20180417;参见全文 * |
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