CN112195111B

CN112195111B - Pythium ultimum PyuLK1 marked by GFP and application thereof

Info

Publication number: CN112195111B
Application number: CN202011074081.1A
Authority: CN
Inventors: 叶文武; 李柯; 郑小波; 王源超; 张正光; 冯慧; 王晓莉
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-10-09
Filing date: 2020-10-09
Publication date: 2022-07-19
Anticipated expiration: 2040-10-09
Also published as: CN112195111A

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 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

Pythium ultimum PyuLK1 marked by GFP and application thereof

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, we find that 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 like, and the original purpose of the strain cannot 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 strain of 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 takes a pythium ultimum wild type strain (Pyu18-6-1) as a transformant, and transfers the green fluorescent protein into the genome of a pythium ultimum strain (Pyu18-6-1) by a PEG-mediated protoplast transformation method to obtain a pythium ultimum strain (PyuLK1) 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, is clear and clear to observe, saves time and labor, 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.

In the figure, the symbols a, b and c represent GFP (green fluorescent marker), BF (Bright field), and BF and GFP coincided graphs, 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 mycelia are gradually digested to form oospores after about 96h of inoculation.

Biological material preservation information

PyuLK1, which is classified and named as Pythium ultimum, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is No. 7/20 in 2020 years, the preservation address is the microorganism research institute of China academy of sciences No. 1 of Beijing, North Chen Xilu No. 3 of the facing-Yang district, 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 strains Pyu18-6-1 and pTOR:: GFP vectors were provided by Oomycetes and fungal molecular biology laboratories, college of plant protection, Nanjing university of agriculture, and pTOR:: GFP vectors are sometimes written as pTOR-GFP. 1L of 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 an ultraviolet lamp of an ultra-clean workbench for 30min, 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 wrapped 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 enzymatic hydrolysate 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 a shaker at 25 ℃ 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 is set at 4 deg.C 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) 580. mu.L of 40% PEG4000 solution was added to each tube in three portions 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 and the regeneration was microscopically monitored, centrifuged at 1700rpm for 5min, and the supernatant was discarded and left for 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. And blowing the plate to dry, performing dark inversion culture in an incubator at 25 ℃, and observing the growth condition of transformants, wherein the normal time range is about 48 hours.

(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. And blowing the plate to dry, performing dark inversion culture in an incubator at 25 ℃, and observing the growth condition of transformants, wherein the normal time range is about 48 hours.

(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 with the size of 2mm multiplied by 2mm are cut from each transformant, added into a 10% V8 liquid medium and cultured for 24 hours, 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

The selected 20 transformants were subcultured on 10% V8 medium without G418 selection pressure for more than 5 generations, several hypha blocks of 2mm × 2mm were cut and added into 10% V8 liquid medium for culturing for 24h, fluorescence was observed under a laser scanning confocal microscope, and 5 transformants with stable heredity were selected, which were PyuLK1, PyuLK12, PyuLK15, PyuLK24 and PyuLK27, respectively, and PyuLK1 was subcultured on 10% V8 solid medium without resistance selection for the 5 th generation of green fluorescence labeling chart shown in FIG. 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 into 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 a lower embryonic axis 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 the soybean hypocotyl tissue at the upper and lower 3cm (total 6cm) positions, 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 between PyuLK1 and WT, 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 above description is of the preferred embodiment of the invention and is not intended to limit the invention. 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

1. Pythium ultimum labeled by GFP (Green fluorescent protein)Pythium ultimum) PyuLK1 preserved in China general microbiological culture Collection center with preservation date of No. 7/20 (2020), and preservation number of CGMCC No. 20225.

2. Use of the GFP-labeled Pythium ultimum PyuLK1 of claim 1 for studying the pathogenesis of Pythium ultimum strains.

3. A method for observing the interaction process of pythium ultimum and host plants, which is characterized in that the GFP-labeled pythium ultimum PyuLK1 hypha blocks are adopted to inoculate hypocotyls of soybean etiolation seedlings, and the cytological morphology of the interaction of pythium ultimum and soybeans at different time points is observed by a laser scanning confocal microscope to obtain a complete infection process.