CN112195111A - Pythium ultimum PyuLK1 marked by GFP and application thereof - Google Patents
Pythium ultimum PyuLK1 marked by GFP and application thereof Download PDFInfo
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- CN112195111A CN112195111A CN202011074081.1A CN202011074081A CN112195111A CN 112195111 A CN112195111 A CN 112195111A CN 202011074081 A CN202011074081 A CN 202011074081A CN 112195111 A CN112195111 A CN 112195111A
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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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The invention discloses a GFP-labeled pythium ultimum PyuLK1 and application thereof. The GFP-labeled Pythium ultimum (Pythium ultimum) PyuLK1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation date of No.20 at 7 months of 2020 and the preservation number of CGMCC No. 20225. The pythium ultimum PyuLK1 has strong fluorescence stability, the strain obtained by the invention is cultured for multiple generations (more than 5 generations) under a G418-free selective medium, and the strain still has fluorescence with stable intensity, thereby greatly facilitating the subsequent research. The strain has no obvious difference with a wild strain in the aspects of growth rate, pathogenicity and number index of oospores, can be used as a genetic engineering strain of pythium ultimum, and provides a theoretical basis for prevention and treatment of pythium ultimum.
Description
Technical Field
The invention relates to the field of plant pathology, and relates to Pythium ultimum PyuLK1 marked by GFP and application thereof.
Background
Pythium is an important plant pathogen in the whole world, can cause diseases of various crops and economic crops, and causes huge economic loss to the nation. Pythium is a type of oomycete belonging to the genera pythium, pythiaceae, order peronosporales, class oomycetes, kingdom algae. Root rot caused by infecting soybean roots by pythium aphanidermatum is mainly caused by seedling stage, seedlings are often caused to die suddenly and directly, diseased plants are short and small, lower leaves become light yellow, root nodules and fibrous roots are reduced and the like, yellow-white mildew is common at the base parts of main roots and stems, soil permeability and poor cultivation conditions are seriously affected by block growth, and the soybean yield is greatly influenced. However, few reports on the pathogenic mechanism of pythium ultimum are reported at home and abroad at present, the interaction process with host plants is not clear, and great difficulty is brought to disease control.
GFP (Green fluorescent protein) was isolated from luminescent jellyfish drifting with ocean currents on the North American West coast by Shimomura et al 1962. In 1994, Chalfie et al applied GFP to the detection of gene expression levels in prokaryotes and eukaryotes and the localization of the expressed product proteins. Since then, the widespread use of fluorescent proteins in various biological research fields has been opened. In the research of plant pathogenic bacteria, GFP plays an important role in the aspects of transcriptional regulation and subcellular localization of pathogenic bacteria functional genes, pathogenic bacteria morphology and subcellular structure markers and the like. At present, GFP fluorescence labeling is one of the main means for researching pathogenic bacteria infection and cell biological processes interacting with hosts in a living mode, so the Pythium strain marked by GFP is very important for researching the pathogenic mechanism of the Pythium strain.
In past researches, the wild pythium ultimum has extremely strong pathogenicity, high growth speed and high genetic variation speed, and a GFP marker gene is randomly inserted into a genomic locus of the strain and has a plurality of uncontrollable factors. This causes problems that the transferred GFP marker gene is easy to lose or the fluorescence intensity is weak, or the biological characteristics of the strain are changed, and the original purpose of the strain can not be achieved. In order to solve the problems existing in the prior art, the applicant researches and obtains the technical scheme of the invention.
Disclosure of Invention
The first object of the present invention is to provide a GFP-labeled Endophytrium ultimum strain (PyuLK 1).
Another object of the present invention is to provide the use of the strain of Pythium ultimum.
The GFP-labeled Pythium ultimum (Pythium ultimum) PyuLK1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation date of No.20 at 7 months of 2020 and the preservation number of CGMCC No. 20225. The invention uses a pythium ultimum wild type strain (Pyu18-6-1) as a transformant, and transfers green fluorescent protein into the genome of a pythium ultimum strain (Pyu18-6-1) by a PEG-mediated protoplast transformation method to obtain the pythium ultimum strain (PyLK 1) marked with the green fluorescent protein.
The GFP-labeled pythium ultimum PyuLK1 is applied to the study of the pathogenesis of pythium strains.
The invention discloses a method for observing the interaction process of pythium and a host plant, which is characterized in that the method for inoculating soybean etiolation seedling hypocotyls by adopting mycelium blocks with GFP marks is adopted, and the cytological morphology of interaction of pythium ultimum and soybeans at different time points is observed by utilizing a laser scanning confocal microscope to obtain a complete infection process. The pythium is an ultimate pythium strain (PyuLK1) with a GFP mark, and the host plant is Hefeng 47 soybean. In another embodiment, the observation is a confocal laser scanning microscope observation, and the observation is a fluorescence observation under blue light with a wavelength of 488 nm.
The invention has the following advantages:
(1) the fluorescence stability is strong. The over-expressed strain obtained by the PEG-mediated protoplast transformation method has random insertion sites due to the fluorescent marker gene and has many uncontrollable factors. In order to overcome the technical problem, the invention obtains a large batch of transformants with fluorescence through repeated tests, subculture and observation are carried out on a non-selection culture medium, and finally a fluorescent strain with continuous stable intensity is obtained by screening, can be cultured for multiple generations (more than 5 generations) on a G418-free selection culture medium, still has stable fluorescence intensity (figure 1), and is greatly convenient for subsequent research.
(2) The Pythium ultimum PyuLK1 marked by the GFP can be used for accurately observing the colonization condition of the Pythium ultimum in soybean hypocotyl tissues at different time points, is simple and convenient to operate, clear in observation, time-saving and labor-saving, and lays a foundation for researching the colonization mechanism of the Pythium ultimum in plants.
(3) The GFP marked strain obtained by the invention has no obvious difference with the wild strain in the aspects of growth rate, pathogenicity and number index of oospores, can be used as a genetic engineering strain of pythium ultimum, and provides a theoretical basis for the prevention and treatment of pythium ultimum.
Drawings
FIG. 1 Green fluorescent marker map of GFP-tagged strains subcultured on 10% V8 solid Medium without resistance selection for passage 5
FIG. 2 colony diameter of wild type, control, GFP-tagged strains after 24h growth on 10% V8 solid Medium
FIG. 3 pathogenicity results (A) and biomass measurements (B) of wild type, control, GFP-tagged strains 24h after soybean hypocotyl inoculation
FIG. 4 determination of the number of ovaries spores after 10 days of growth of the wild-type, control, GFP-tagged strains
FIG. 5A, B, C, D, E is a diagram showing the cell morphology of Pythium ultimum and soybean at 3h, 12h, 24h, 48h and 96h of inoculation respectively, which are the structure diagrams of hypocotyl epidermal cells of soybean etiolation seedlings.
The symbols a, b and c in the figure represent GFP (green fluorescent marker), BF (Bright field), and BF and GFP maps, respectively.
Panel A was inoculated for about 3h and a small amount of hyphae began to invade the host cells.
In FIG. B, C, D, the hyphae extended and extended inside the host cell, and it was clearly seen that the hyphae were significantly constricted while passing through the host cell wall, and the host cell was gradually occupied by a large amount of hyphae.
In the graph E, the inoculation lasts for about 96h, and hyphae gradually digest to form oospores.
Biological material preservation information
PyuLK1, which is classified and named as Pythium ultimum, is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation date is No. 7/20 at 2020, the preservation address is the institute of microbiology of China academy of sciences No. 1, West Lu 3 at the North Chen of the Yangyang area in Beijing, and the preservation number is CGMCC No. 20225.
Detailed Description
The technical scheme of the invention is further described in detail by combining the drawings and the detailed implementation mode:
1. example 1 is PEG-mediated genetic transformation of protoplasts.
Pythium ultimum wild type strain Pyu18-6-1 and pTOR:: GFP vector both supplied by Oomycetes and fungi molecular biology laboratories of the university of agriculture of Nanjing university, pTOR:: GFP vector is sometimes written as pTOR-GFP. 1L KPYG in this embodiment2The medium consisted of 1.5g glucose, 1g yeast extract, 1g peptone, 0.02g cholesterol, 1g corn oil, 109.302g 0.8M Mannitol powder and 0.1g CaCl2.2H2O composition; 20mL of the enzymatic hydrolysate was prepared from 0.15g of lysine Enzymes, 0.06g of Cellulase, 10mL of 0.8M Mannitol reagent, and 8mL of ddH2O, 800. mu.L of 0.5M KCl, 800. mu.L of 0.5M MES (pH5.7) and 400. mu.L of 0.5M CaCl2Composition is carried out; 12mL of 4% PEG was prepared from 6g of PEG4000, 3.75mL of 0.8M Mannitol reagent, 3mL of 0.5M CaCl2Reagent and 3mL ddH2O composition; 250mLW5 solution consisting of 0.093g KCl, 4.6g CaCl2.2H2O, 2.25g NaCl and 7.97g glucose with ddH2O is added to the volume of 250 mL; 250mL of M Mg solution consisting of 18.22g of 0.8M Mannitol powder, 4.6g of MgCl2.2H2O and 2mL0.5M MES (pH5.7) reagent.
(1) KPYG at 1.5% agar2Activating Pythium ultimum strain (Pyu18-6-1) on solid culture medium, culturing at 25 deg.C in dark for 1-2 days, cutting 1mm × 1mm hypha blocks from the edge, and placing into KPYG2In liquid culture medium, liquid culture is carried out for 6-8 dishes, and the mixture is kept standing and cultured for 48 hours in the dark at the temperature of 25 ℃.
(2) Sterilizing for 30min by an ultraviolet lamp on an ultra-clean workbench, putting required reagents and consumables into the ultra-clean workbench (a disposable 50mL centrifuge tube, a 200mL beaker with a gauze bag opening, a 50mL funnel-shaped small beaker packed by mira-cloth, 5mL gun heads and 1mL gun heads which are cut over and not cut over, a culture dish with a larger height, a bacterial filter and the like), and preparing enzymolysis liquid and 4% PEG (prepared at present).
(3) Collecting mycelia with a 200mL beaker wrapped with gauze, rinsing the mycelia in a culture dish containing 0.8M Mannitol for 2min with forceps, filtering with the beaker wrapped with gauze, transferring the rinsed mycelia into a clean sterile centrifuge tube, adding 0.8M Mannitol to about 30mL, and washing with 25 deg.C for 10 min.
(4) Adding the washed mycelium into a centrifuge tube containing the enzymolysis solution, setting the shaker at 25 deg.C and 70rpm, and placing the centrifuge tube on the shaker for enzymolysis for 45 min.
(5) After the end of the enzymatic hydrolysis, the protoplasts were collected by filtering the mycelia through a 50mL beaker with mira-cloth and placed on ice, and then the collected protoplasts were poured into a 50mL round bottom centrifuge tube. The centrifuge was set at 4 ℃ and 1500rpm for 4 min. The supernatant was discarded, 5mL of W5 solution was added to 35mL, and the mixture was centrifuged at 1500rpm for 4min at 4 ℃. Discarding the supernatant, adding 7mL W5 solution to resuspend the protoplast, ice-cooling for more than 30min, and centrifuging at 1500rpm for 4min at 4 ℃.
(6) Discard the supernatant, add the appropriate volume of MMg solution, resuspend the protoplasts, and allow to stand at room temperature for 10 min. Then 40-50. mu.g of plasmid pTOR:GFPto be transformed was added to the bottom of the tube and placed on ice.
(7) Add 1mL of MMg solution resuspended protoplast fluid to each centrifuge tube containing plasmid pTOR:: GFP, tap the tube wall to mix the plasmid and protoplast thoroughly, and incubate on ice for 6 min.
(8) To each580. mu.L of 40% PEG4000 solution was added to each tube three times and incubated on ice for 20 min. At KPYG2Ampicillin (final concentration 50. mu.g/mL) was added to the liquid medium and 20mL of KPYG was added to each centrifuge tube after incubation2Liquid medium. And (4) carrying out dark static culture in an incubator at 25 ℃ and regenerating for 14-16h overnight.
(9) After the overnight regeneration was complete, 5. mu.L of the supernatant was aspirated for microscopic regeneration, the centrifuge was set to 1700rpm for 5min, and the supernatant was discarded and left to hold about 5mL and resuspended.
(10)KPYG2The G418 antibiotic (final concentration: 40. mu.g/mL) and ampicillin (final concentration: 50. mu.g/mL) were added to the solid medium, mixed well by shaking, added to the regenerated protoplast fluid resuspended in the previous step to make the total volume 45mL, mixed well by inversion, and poured into 3 petri dishes, each containing about 15 mL. Drying, sealing, culturing in 25 deg.C incubator in dark place, and observing growth of transformant within 48 h.
(11) After the transformant grew out, the second covering was carried out, and G418 antibiotic (final concentration: 60. mu.g/mL) and ampicillin (final concentration: 50. mu.g/mL) were added to 10% V8 solid medium, and the mixture was poured into a well-grown transformant petri dish and covered. Drying, sealing, culturing in 25 deg.C incubator in dark place, and observing growth of transformant within 48 h.
(12) After the transformants were grown, selection was performed to select single colonies of the transformants which were visible to the naked eye, and to avoid selecting multiple single colonies at the same time, the colonies were transferred to a 10% V8 solid medium supplemented with G418 antibiotic (final concentration of 60. mu.g/mL) and ampicillin (final concentration of 50. mu.g/mL).
To obtain more transformants, this study was performed in triplicate with PEG-mediated protoplast genetic transformation, and 186 transformants were selected. Several hypha blocks of 2mm multiplied by 2mm are cut from each transformant and added into a 10% V8 liquid culture medium for culturing for 24h, fluorescence is observed under a laser scanning confocal microscope, and 20 transformants with strong fluorescence are screened.
Example 2: subculture and stability detection of transformant
Subculturing the selected 20 transformants on 10% V8 culture medium without G418 selection pressure for more than 5 generations, cutting several hypha blocks of 2mm multiplied by 2mm, adding into 10% V8 liquid culture medium for culturing for 24h, observing fluorescence under a laser scanning confocal microscope, and selecting 5 transformants with relatively stable heredity, namely PyuLK1, PyuLK12, PyuLK15, PyuLK24 and PyuLK27, and PyuLK1 subculturing the green fluorescence labeling diagram of the 5 th generation on 10% V8 solid culture medium without resistance selection as shown in figure 1.
Example 3: determination of growth rate, pathogenicity and number of ova and spores of transformant
And (3) measuring the growth rate: inoculating a wild strain (WT), a negative control strain (CK) which is transformed by protoplasts and does not obtain fluorescence, and 5 transformants with stable fluorescence on an equal volume of 10% V8 agar culture medium, repeating the steps for three strains, measuring the diameter by a cross method after 24h, and calculating the significant difference by using T detection. The results showed that one and only PyuLK1 strain showed no significant difference with both WT and CK, while the remaining 4 strains showed a problem of slow growth rate, and significant difference with WT and CK (fig. 2), so that these four transformants were discarded.
And (3) determining pathogenicity: yellow seedlings of a soybean variety Hefeng 47 cultured for 3-4 days at 25 ℃ under the dark condition are used as materials for determining pathogenicity. After a wild type of pythium ultimum (Pyu18-6-1), a negative control strain (CK) which is not transferred with fluorescence and a transformant with stable fluorescence (PyuLK1) are respectively continuously activated for 2 generations on a nonreactive 10% V8 solid plate, the activated strain is transferred to an isovolumetric 10% V8 solid plate, after 24 hours, a perforator is used for punching a hypha block with the edge diameter of 1.5cm and inoculating the hypha block to the hypocotyl of a etiolated seedling, and each strain is inoculated with 5 etiolated seedlings. And (4) carrying out moisture preservation culture for 24 hours at 25 ℃ in the dark, observing the pathogenicity change, recording the pathogenicity result and taking a picture. Taking the inoculation point as the center, cutting 3cm (totally 6cm) soybean hypocotyl tissue at the upper and lower parts, grinding to extract genome, and measuring biomass. The experiment was repeated at least 3 times and significant differences were calculated using T-test. The results showed no significant difference for PyuLK1 from both WT and CK (fig. 3).
And (3) measuring the number of ova spores: inoculating three strains of WT, CK and PyuLK1 on a 90mm plate of an isometric 10% V8 agar culture medium, inoculating 3 plates, picking a hypha block of 1cm multiplied by 1cm at the edge of each plate after growing for 10d, putting the hypha block into a 50mL sterile BD tube, adding 10mL sterile water, crushing the hypha block for one minute by a homogenizer, drawing 10 mu L of homogenate of each strain for three times, streaking the homogenate on a glass slide, observing and recording the number of oospores under a microscope after the strains are dried, and repeating the steps for three times. Significant differences were calculated using T-test. The results showed no significant difference for PyuLK1 from both WT and CK (fig. 4).
Example 4: soybean inoculation and fluorescent observation of colonization process of pythium ultimum in soybeans
The research adopts a method of inoculating hypocotyl of soybean etiolation seedlings, the selected soybean variety is Hefeng 47, the soybean variety is planted in a seedling tray, and vermiculite is selected as a culture medium. Culturing at 25 deg.C in dark for 3-4 days to obtain soybean etiolation seedling.
Inoculating the pythium ultimum strain on a 10% V8 agar culture medium, growing for 24h at 25 ℃, punching a fungus cake by using a puncher with the diameter of 1.5cm multiplied by 1.5cm, inoculating the hypha surface of the fungus cake to the position 2-3cm away from the root of the hypocotyl of the soybean etiolation seedling, and covering the inoculated position with wet cotton for moisturizing. Fluorescence observation was performed at different time points (3h, 12h, 24h, 48h, 96 h). The specific colonization process is shown in FIG. 5.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (3)
1. A GFP-labeled Pythium ultimum PyuLK1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation date of 2020, 7-month and 20-th and the preservation number of CGMCC No. 20225.
2. Use of the GFP-labeled PyuLK1 of claim 1 for studying the pathogenesis of Pythium ultimum strains.
3. A method for observing the interaction process of pythium and host plants is characterized in that a method for inoculating hypocotyls of soybean etiolation seedlings with pythium ultimum silk blocks is adopted, and the cytological morphology of interaction of pythium ultimum and soybeans at different time points is observed by a laser scanning confocal microscope to obtain a complete infection process.
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|---|---|---|---|---|
| US20060029576A1 (en) * | 2004-08-04 | 2006-02-09 | Her Majesty The Queen In Right Of Canada, The Minister Of Agriculture And Agri-Food | Biological control of pythium disease in crops |
| CN105722396A (en) * | 2013-09-24 | 2016-06-29 | 作物营养公司 | Isolated bacterium of the genus streptomyces |
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