Patent Grant
12134773
This statement, made under Rules 77 (b) (5) (ii) and any other applicable rule incorporates into the present specification of an XML file for a “Sequence Listing XML” (see Rule 831 (a)), submitted via the USPTO patent electronic filing system or on one or more read-only optical discs (see Rule 1.52 (e) (8)), identifying the names of each file, the date of creation of each file, and the size of each file in bytes as follows:
The disclosure relates to the field of genetic engineering, and in particular to a mycovirus-induced gene silencing vector and a construction method and application thereof.
Virus-induced gene silencing (VIGS) is a technique to study the function of plant genes by RNA-mediated plant antiviral mechanism. The principle is to insert the target gene fragment into the virus vector and infect the host plant. While the immune system of the plant recognizes the virus and the target gene, it degrades the endogenous target gene mRNA, causing the plant to have a phenotype of loss of function or decreased expression level of the target gene. When VIGS is initiated, a large number of double-stranded RNA (dsRNA) are synthesized by RNA-mediated RNA polymerase. In cells, dsRNA is cleaved into 19-24nt silencing RNA (siRNA) by Dicer analogues of RNaseIII family-specific endonucleases. SiRNA binds to a specific protein in the form of a single strand to form an RNA-induced silencing complex (RISC). The RISC is capable of specifically binding to the mRNA of the target gene in cytoplasm, leading to the degradation of the mRNA of the target gene, so that the infected plant shows the mutation trait of the target gene, thus directly inferring the function of the target gene or providing indirect biological evidence. Under ongoing development and in depth research, the VIGS technology has been widely used in the research and identification of related functional genes such as plant resistance, growth and development, and metabolic regulation, and has a good development and application prospect in plant trait improvement and plant protection.
Fusarium graminearum gemytripvirus 1 (FgGMTV 1) is a multi-component single-stranded circular DNA virus, which contains three single-stranded circular DNA fragments: DNA-A, DNA-B and DNA-C, with molecular sizes of 1316nt, 1320nt and 1309nt respectively and encoded with replication initiation protein (REP), Coat protein, CP) and unknown functional protein respectively. An infectious clone of FgGMTV1 has been successfully constructed in Mycovirus lab in Institute of Plant Protection (IPP), Chinese Academy of Agricultural Sciences (CAAS), and the relationship among the components is made clear: DNA-A and DNA-B are necessary for virus replication and infection, and their co-infection is capable of significantly inhibiting the growth, conidial production and pathogenicity of host fungi, but the virus is unstable and does not spread vertically through conidia; the replication and proliferation of DNA-C depend on DNA-A and DNA-B. The virus containing three components is stable and is capable of spreading through conidia, but it does not affect the phenotype and pathogenicity of host fungi. Therefore, it is possible to construct the vector of VIGS by artificial transfection with the infectious clone of FgGMTV1 as the basic element.
Wheat Fusarium head blight (FHB) is a worldwide wheat disease mainly caused by Fusarium graminearum complex. How to prevent the FHB and its toxin pollution is particularly urgent and important, which is directly related to food safety, food security and people's health. Chemical control is still an important measure for FHB control at present, but excessive dependence on chemical control leads to the resistance of pathogenic fungi to fungicides. In addition, due to the lack of wheat varieties with high resistance to the FHB, wheat FHB remains happening frequently at present and even for a long time to come. It is particularly important to find and explore new control methods of wheat FHB. By successfully constructing the VIGS vector of FgGMTV1 and silencing the genes related to host pathogenicity and toxin production, the obtained hypovirulent strains may be used as biological control factors, providing a new method for controlling the wheat FHB. Meanwhile, the obtained VIGS vector is capable of being widely used to study the gene function of Fusarium graminearum, which solves the problem that a gene knockout method fails to study the lethal genes.
The objective of the present disclosure is to provide a mycovirus-induced gene silencing vector and its construction method and disclosure, so as to solve the problems existing in the prior art. The gene silencing vector induced by Fusarium graminearum single-stranded circular DNA virus FgGMTV1 is capable of effectively reducing the toxin production and pathogenicity of infected Fusarium graminearum, and provide a new direction for preventing and controlling wheat Fusarium head blight (FHB).
In order to achieve the above objectives, the present disclosure provides the following scheme.
The disclosure provides a mycovirus-induced gene silencing vector, and a nucleotide sequence of the mycovirus-induced gene silencing vector is shown in SEQ ID NO: 2.
The disclosure also provides a construction method for the mycovirus-induced gene silencing vector, including following steps:
Optionally, 1.3 copies of the DNA-A, 1.3 copies of the DNA-B and 1.5 copies of the DNA-C are connected in series and then connected to pBluescript II SK(+) to construct the recombinant vector.
Optionally, a nucleotide sequence of the DNA-A is shown in SEQ ID NO: 5, a nucleotide sequence of the DNA-B is shown in SEQ ID NO: 6, and a nucleotide sequence of the DNA-C is shown in SEQ ID NO: 7.
Optionally, the deletion mutation is a deletion of a sequence of 454-603nt of the coding protein p26 of the DNA-C.
The disclosure also provides a recombinant Fusarium graminearum including the mycovirus-induced gene silencing vector.
Optionally, the mycovirus-induced gene silencing vector carries exogenous genes. The exogenous genes include Tri101 gene and FgPP1 gene.
The disclosure also provides an application of the mycovirus-induced gene silencing vector or the recombinant Fusarium graminearum in preventing and controlling wheat FHB.
The disclosure also provides an application of the mycovirus-induced gene silencing vector or the recombinant Fusarium graminearum in preparing a preparation for preventing and controlling wheat FHB.
The disclosure discloses the following technical effects.
On the basis of previous research, the disclosure shortens the repetitive sequences of components DNA-A, DNA-B and DNA-C, and connects the three components in series to the same vector, and then selects the candidate mutant p26-D4 suitable as a VIGS vector by performing serial deletion mutation on the coding protein p26 of the DNA-C component. Through experimental verification, the constructed VIGS silencing vector p26-D4 is capable of accommodating 75-150 bp of exogenous genes, and effectively silence target genes such as GPF, Tri101 and FgPP1 in the wild Fusarium graminearum strain PH-1, which further verifies that this silencing vector p26-D4 carries exogenous genes to infect the Fusarium graminearum strain and is capable of effectively controlling wheat FHB. Therefore, the silencing vector p26-D4 provided by the disclosure offers a new direction for the prevention and control of wheat FHB.
A number of exemplary embodiments of the present disclosure are now described in detail, and this detailed description should not be considered as a limitation of the present disclosure, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present disclosure.
The Fusarium graminearum single-stranded DNA virus FgGMTV1 used in the embodiments of the present disclosure is isolated and identified by Mycovirus lab in Institute of Plant Protection (IPP), Chinese Academy of Agricultural Sciences (CAAS), see Li P. (2020). A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified viruses. Science Advances 6, eaay9634. The wild Fusarium graminearum strain PH-1 and the Fusarium graminearum strain PH-1/GFP with green fluorescent label are preserved in the Mycovirus lab in IPP, CAAS.
1. Construction of the Infectious Clone pSK-ABC with Three Components of DNA Virus FgGMTV1 in Series
Patent CN109810997 A with a name of “Construction method of infectious clone of Fusarium graminearum single-stranded circular DNA virus FgGMTV1/HB58” discloses a construction method of infectious clone of Fusarium graminearum single-stranded DNA virus FgGMTV1/HB58, which includes DNA-A infectious clone construction, DNA-B infectious clone construction and DNA-C infectious clone construction. 2 units of DNA-A and DNA-B molecular sequences and 1.6 units of DNA-C molecular sequences are connected to the cloning vector pBluescript II SK(+) (pSK) respectively, and infectious clones pSK-2A, pSK-2B and pSK-1.6C of the three components of the virus are constructed. Because plasmids of three infectious clones transfect at the same time, and the steps are complicated, the DNA-A and DNA-B molecules herein are reduced to 1.3 units, and DNA-C molecules are reduced to 1.5 units, which only contain coding regions of the three components and two repetitive non-coding regions, respectively, and are connected in series to the vector pBluescript II SK(+). At the same time, suitable restriction enzyme sites are added at both ends of sequences of DNA-A, DNA-B and DNA-C respectively, so as to facilitate further mutant construction (see
2. Protoplast Preparation of Fusarium graminearum
Fusarium graminearum strain PH-1 on PDA plate is inoculated into a 250 mL triangular flask with 100 mL carboxylmethyl cellulose (CMC) liquid medium, and cultured for 3-5 days at 25° C. and 180 rpm (preferably 4 days); the culture medium is filtered with sterilized three-layer lens-wiping paper, centrifuged at 4000 rpm for 5 min at room temperature, and the supernatant is discarded; sterilized water is added, conidia precipitation is resuspended and diluted to the concentration of 107 spores/mL; 3×107 spores are transferred to a 250 mL triangular flask containing 100 mL Yeast Extract Peptone Dextrose (YEPD) liquid medium, and shaken at 25° C. and 180 rpm for 12-14 h; the overnight culture medium is filtered with three-layer sterilized lens-wiping paper, rinsed with sterile water for three times, then rinsed with 1 M sucrose for two times, drained with sterilized filter paper, and mycelium is collected; mycelium is transferred to 20 mL of protoplast hydrolysate with a sterile tweezers, gently shaken at 90 rpm and 28° C. for 2-3 h, and the protoplast granules are examined in terms of quantity by microscope every 0.5 h; the enzymolysis mixture is filtered with two-layer sterilized lens-wiping paper into a sterile 50 mL centrifuge tube, and rinsed twice with 1 M sucrose solution to rinse off as many protoplasts as possible: the protoplasts are centrifuged at 2600 rpm at 4° C. for 1 min, the supernatant is discarded, the protoplast are resuspended with Sucrose-Tris-Calcium (STC) buffer (1 M sorbitol, 50 mM Tris-HCl pH 8.0, and 50 mM CaCl2·2H2O) precooled on ice, and the protoplast are washed for three times according to the above centrifugation conditions: finally, the protoplasts are resuspended with STC buffer, and the final concentration of the protoplasts is adjusted to 2×107 cells/mL, the protoplasts are sub-packaged at 200 μL/tube, added with dimethyl sulfoxide (DMSO) with final concentration of 7%, mixed well and stored at −80° C.
3. Polyethylene Glycol (PEG)-Mediated Transfection of pSK-ABC
The protoplasts are taken out at −80° C. and thawed on ice: 200 μL PH-1 protoplasts and 30 μg plasmid are added into a 2 mL centrifuge tube, mixed gently, and placed on ice for 20 min; 1.25 mL PEG-Tris-Calcium (PTC) buffer (STC buffer containing 40% PEG 4000) is dripped into the 2 mL centrifuge tube, mixed with a gun head, and then allowed to stand at room temperature for 20 min. The protoplasts in the 2 mL centrifuge tube are transferred into the 15 mL centrifuge tube, the 2 mL centrifuge tube is rinsed repeatedly with 5 mL Terrific Broth 3 (TB3) (3 g Yeast Extract, 3 g Casamino Acids, 200 g Sucrose, added with distilled water to constant volume to 1 L) (containing 50 μg/mL ampicillin), then added into the above 15 mL centrifuge tube, shaken lightly at 25° C. and 90 rpm overnight (16 h): the regenerated substance of protoplasts are centrifuged at 4000 rpm for 5 min, the supernatant is discarded, 600 μL STC is added to resuspend and precipitate, 200 μL of the protoplasts are taken respectively in turn and put in the center of a 9 cm culture dish, and added with 20 mL of TB3 solid medium containing 50 μg/mL ampicillin: the plate is placed with face upward, and cultured overnight at 25° C. in the dark. The next day, the plate is tilted to remove STC floating on the surface, and cultured for 5 days. The morphology of mycelium is observed, small pieces of myceliums is cut from different positions on the edge and transferred to PDA plate, cultured, and further experimental observation is conducted after molecular identification.
4. Determination of Colony Morphology and Growth Rate of the Strains
The mycelian blocks of transfectant strain are punched with a 5 mm punch and placed in the center of PDA medium plate to activate the strain. A 5 mm punch is used to punch the blocks on the edge of the colony of the activated strain, and the blocks are transferred to the center of the PDA medium plate, and 5 repetitions are conducted respectively. After 4 days of dark culture at 25° C., the colony morphology and diameter are observed and statistically analyzed.
5. Determination of Pathogenicity of the Strains
Yangmai 158, a wheat variety, is planted and used for wheat spikelet inoculation when the wheat grows to the flowering stage. The conidia of each transfectant strain are prepared according to the above method, and the spore concentration is measured by blood cell counting plate, and then the spore concentration is adjusted to 3×105 spores/mL. Spikelets with basically the same growth are selected, and when the fifth spikelet is found from bottom to top, the inner glume from the outer glume are separated with fingers. 10 μL of the prepared spore solution is sucked and injected into the root between the inner glume and the outer glume, and spikelet is gently closed to keep a natural state. The inoculated spikelet is marked with a marker and a label is put on the wheat stalk, and the inoculation strain, inoculation method, inoculation date, inoculator and other information are recorded. Each strain is inoculated onto at least 15 spikelets, subjected to moisturizing culture with fresh-keeping bag for 48 h: after taking off the fresh-keeping bag, each strain is cultured for 14 days, the symptom of wheat spikelets is observed, and the number of diseased spikelets per wheat head is counted.
6. Results
1. Design and Construction of Gene Silencing Vector Based on FgGMTV1
Based on the infectious clone pSK-ABC, the p26 coding frame of DNA-C component is deleted with the length of 150nt in turn, and five deletion mutants are obtained, named p26-D1, p26-D2, p26-D3 and p26-D4, and p26-D5. The p26-D5 is deleted by 81nt. The method of constructing these mutants is completed by KOD-Plus-Mutagenesis Kit (code NO. SMK-101) of Toyobo Life Science company. The experimental principle of the kit is that by using pSK-ABC as a template through inverse PCR method, two primers are designed in the opposite direction, and restriction enzyme sites NsiI and AgeI are introduced at the same time, and the plasmid is subjected to complete PCR by using high-fidelity KOD-Plus-enzyme, and finally the target mutants are obtained by screening.
The deletion mutants based on DNA-C are constructed by designing specific primers, as shown in Table 1, in which the underlined sequences are introduced restriction enzyme sites NsiI and AgeI.
Protoplast preparation of Fusarium graminearum, transfection of virus deletion mutants mediated by PEG and observation of colony morphology of the strains are all the same as those in Embodiment 1.
The complete sequence (without vector) of the VIGS vector p26-D4 is shown in SEQ ID NO: 2, with the restriction enzyme sites of 1-18nt, 1760-1771nt, 3509-3532nt, 4239-4256nt, 5332-5369nt. 19-1759nt is the sequence of component A, 1772-3508nt is the sequence of component B and 3533-4238nt, 4257-5331nt is the sequence of component C. Hind III (1-6nt) and NotI (5362-5369nt) are used to connect the sequence to pBluescript II SK(+) cloning vector. NsiI (4239-4244nt) and AgeI (4251-4256nt) are used to insert foreign gene fragments. SEQ ID NO: 2 is:
In this embodiment, GFP gene genetically expressed by Fusarium graminearum PH-1 is used as the target gene to illustrate the silencing effect of p26-D4.
1. Construction of Gene Silencing Vector Based on p26-D4
Based on p26-D4 vector, as for the target gene GFP, VIGS vector containing different fragments of GFP gene is constructed to verify the silencing effect and the size range of exogenous fragment insertion. Eight vectors are constructed: p26-D4-GFP75F, p26-D4-GFP75R, p26-D4-GFP150F, p26-D4-GFP150R, p26-D4-GFP300F, p26-D4-GFP300R, p26-D4-GFP450F and p26-D4-GFP450R. The specific construction method is to amplify GFP gene fragments with different lengths and directions by RCR method, connect the amplified fragments to p26-D4 vector by T4 DNA ligase, and finally screen the target silencing vector.
GFP gene fragments with different lengths and directions are amplified by specific primers, and the specific primers are shown in Table 2.
The protoplast preparation of Fusarium graminearum, PEG-mediated transfection of p26-D4-based gene silencing vector and observation of colony morphology of strains are all the same as those in Embodiment 1.
2. Fluorescence Observation
A PDA plate with cellophane is prepared, laid with a sterile cover glass, a 3 mm fresh mycelian block is placed at a distance of 2 cm from the cover glass, and cultured in the dark for 2 days. The fluorescence is observed when the mycelium grows to one third of the cover glass position. The fluorescence intensity is analyzed by ImageJ software.
3. Results
In this embodiment, the endogenous genes Tri101 and FgPP1 of Fusarium graminearum PH-1 are used as target genes to illustrate the silencing effect of p26-D4.
1. Construction of the Silencing Vectors p26-D4-Tri101 and p26-D4-FgPP1
On the basis of p26-D4 vector, VIGS vectors p26-D4-Tri101 and p26-D4-FgPP1 are constructed for target genes Tri101 and FgPP1. The specific construction method is to amplify 150 bp Tri101 and FgPP1 gene fragments by RCR method, connect the amplified fragments to p26-D4 vector by homologous recombination method, and finally obtain the target silencing vectors by screening.
The 150 bp Tri101 and FgPP1 gene fragments are amplified by specific primers, and the specific primers are shown in Table 3.
The protoplast preparation of Fusarium graminearum PH-1, PEG-mediated transfection of p26-D4-based gene silencing vector and observation of colony morphology of strains are all the same as those in Embodiment 1.
The sequence of the target gene Tri101 is shown in SEQ ID NO: 3:
The target gene FgPP1 has a sequence as shown in SEQ ID NO: 4:
2. Results
Based on the results of Embodiment 4, the biological control effect of obtained hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 is further tested. In this embodiment, co-infection method and pre-spray method are selected to verify the biological control effect of VIGS-induced hypovirulent strains.
Co-infection method (Test 1): the mycelian blocks of the hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 are cut into small blocks with the same size and inoculated into the same spikelet together with 10 μL of PH-1 conidia suspension. After 12 days, the incidence of wheat Fusarium head blight (FHB) is observed, the number of diseased spikelets is counted and the content of deoxynivalenol (DON) is determined.
Pre-spray method (Test 2): the hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 are cultured in PDB liquid medium for 3 days, and the mycelium is collected, broken into small mycelium segments, and the OD600≈2.0. First, the mycelium segment suspension is sprayed on wheat spikelets, and then 10 μL of PH-1 conidia suspension is inoculated after 24 h. After 12 days, the incidence of wheat FHB is observed, the number of diseased spikelets is counted and the DON content is determined.
The above-mentioned embodiments only describe the preferred mode of the disclosure, and do not limit the scope of the disclosure. Under the premise of not departing from the design spirit of the disclosure, various modifications and improvements made by ordinary technicians in the field to the technical scheme of the disclosure shall fall within the protection scope determined by the claims of the disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202310441503.1 | Apr 2023 | CN | national |
This application is a continuation of PCT/CN2023/097344, filed on May 31, 2023, and claims priority of Chinese Patent Application No. 202310441503.1, filed on Apr. 23, 2023, the entire contents of which are incorporated herein by reference.
| Number | Date | Country |
|---|---|---|
| 109666655 | Apr 2019 | CN |
| 109666655 | Apr 2019 | CN |
| 109810997 | May 2019 | CN |
| 109810997 | May 2019 | CN |
| 110922457 | Mar 2020 | CN |
| 113136391 | Jul 2021 | CN |
| 114317598 | Apr 2022 | CN |
| Entry |
|---|
| GenBank MK430077.1 (printed May 29, 2024). |
| GenBank MK430077.2 (printed May 29, 2024). |
| Li et al (Science Advances, 2020, eaay9634, 9 pages). |
| CN109666655 English translation (2019). |
| CN109810997 English translation (2019). |
| Li et al Supplemental Material (Science Advances, 2020, eaay9634). |
| Pengfei Li et al., “A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions,” Science Advances, Apr. 2020, vol. 6. |
| Lihang Zhang et al., “Molecular Characterization of a Novel Strain of Fusarium graminearum Virus 1 Infecting Fusarium graminearum,” Viruses, Mar. 2020, 12, 357. |
| Jisuk Yu et al., “Molecular Characterization of Fusarium Graminearum Virus 2 Isolated from Fusarium graminearum Strain 98-8-60,” Plant Pathol. J., Jun. 2011, pp. 285-290, vol. 27, No. 3. |
| Li Ke et al., “Prokaryotic expression of FpgMBV1-P3 and preparation of its polyclonal antibodies,” Acta Phytopathologica Sinica, Jan. 2020, pp. 567-573, vol. 50, No. 5. (abstract translated). |
| Polina Tretiakova et al., “Successful Silencing of the Mycotoxin Synthesis GeneTRI5inFusarium culmorumand Observation of Reduced Virulence inVIGS and SIGS Experiments,” Genes, Feb. 2022, 13, 395. |
| Search Report for China Application No. 202310441503.1, dated Aug. 21, 2023. |
| Notification to Grant Patent for China Application No. 202310441503.1, dated Aug. 23, 2023. |
| International Search Report for PCT/2023/097344, mailed Nov. 13, 2023. |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2023/097344 | May 2023 | WO |
| Child | 18529537 | US |
