Mycovirus-induced gene silencing vector, construction method and application thereof

Abstract

A mycovirus-induced gene silencing vector and a construction method and an application thereof are provided. A nucleotide sequence of the mycovirus-induced gene silencing vector is shown in SEQ ID NO: 2, and construction method for the mycovirus-induced gene silencing vector includes: (1) connecting three single-stranded circular DNA molecules DNA-A, DNA-B and DNA-C of the mycovirus FgGMTV1/HB58 in series and introducing them into a same vector to construct a recombinant vector; and (2) carrying out a deletion mutation on a coding protein p26 of the DNA-C molecule in the recombinant vector to obtain the mycovirus-induced gene silencing vector.

Claims

1. A mycovirus-induced gene silencing vector, wherein its nucleotide sequence is shown in SEQ ID NO: 2.

2. A construction method for the mycovirus-induced gene silencing vector according to claim 1, comprising the following steps: (1) connecting three single-stranded circular DNA molecules DNA-A as shown in SEQ ID NO: 5, DNA-B as shown in SEQ ID NO: 6 and DNA-C as shown in SEQ ID NO: 7 of mycovirus FgGMTV1/HB58 in series and introducing into a same vector to construct a recombinant vector; and (2) carrying out a deletion mutation on a coding protein p26 of the DNA-C molecule in the recombinant vector to obtain the mycovirus-induced gene silencing vector.

3. The construction method according to claim 2, wherein 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.

4. The construction method according to claim 2, wherein the deletion mutation is a deletion of a sequence of 454-603nt of the coding protein p26 of the DNA-C as shown in SEQ ID NO: 7.

5. The mycovirus-induced gene silencing vector according to claim 1, wherein the mycovirus-induced gene silencing vector is contained in Fusarium graminearum.

6. The mycovirus-induced gene silencing vector according to claim 5, wherein the mycovirus-induced gene silencing vector carries exogenous genes, and the exogenous genes comprise Tri101 gene and FgPP1 gene.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic structural diagram of an infectious clone pSK-ABC.

(2) FIG. 2A shows Southern blot detection (results) of a pSK-ABC transfectant.

(3) FIG. 2B shows a comparison of colony morphology of the pSK-ABC transfectant, a strain PH-1 and a strain A+B+C.

(4) FIG. 2C shows a comparison of growth diameters of pSK-ABC transfectant, the strain PH-1 and the strain A+B+C on Potato Dextrose Agar (PDA) medium.

(5) FIG. 2D shows a disease symptom of wheat spikelets when the pSK-ABC transfectant, the strain PH-1 and the strain A+B+C are inoculated onto wheat.

(6) FIG. 2E shows the number of diseased spikelets per wheat head when the pSK-ABC transfectant, the strain PH-1 and the strain A+B+C are inoculated onto wheat.

(7) FIG. 3A is a comparison of colony morphology of deletion mutants p26-D1, p26-D2, p26-D3, p26-D4 and p26-D5, the strain pSK-ABC and a strain PH-1 (VF).

(8) FIG. 3B shows Southern blot detection of the deletion mutants p26-D1, p26-D2, p26-D3, p26-D4 and p26-D5, the strain pSK-ABC and the strain PH-1 (VF).

(9) FIG. 3C shows a comparison of growth diameters of the deletion mutants p26-D1, p26-D2, p26-D3, p26-D4 and p26-D5, the strain pSK-ABC and the strain PH-1 (VF) on PDA medium.

(10) FIG. 4 is a schematic structural diagram of virus-induced gene silencing (VIGS) vector p26-D4.

(11) FIG. 5A is a comparison of colony morphology, fluorescence observation and intensity analysis of a strain PH-1/WT (VF), a strain PH-1/GFP (VF) and strains infected by p26-D4, p26-D4-GFP75F, p26-D4-GFP75R, p26-D4-GFP150F and p26-D4-GFP150R.

(12) FIG. 5B shows Southern blot detection of the stain PH-1/GFP (VF) and the strains infected by p26-D4, 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.

(13) FIG. 5C shows a comparison of GFP gene expression levels of the strains PH-1/WT (VF), PH-1/GFP (VF) and the strains infected by p26-D4, p26-D4-GFP75F, p26-D4-GFP75R, p26-D4-GFP150F and p26-D4-GFP150R.

(14) FIG. 6A shows Southern blot detection of the strain PH-1/WT (VF), and the strains infected by p26-D4, p26-D4-Tri101 and p26-D4-FgPP1.

(15) FIG. 6B shows a comparison of relative expression levels of Tri101 and FgPP1 genes in strains infected by p26-D4, p26-D4-Tri101 and p26-D4-FgPP1.

(16) FIG. 6C shows a comparison of deoxynivaleno (DON) production induced by TBI-induced toxin-producing liquid medium among the strains infected by p26-D4, p26-D4-Tri101 and p26-D4-FgPP1.

(17) FIG. 6D shows a comparison of colony morphology of the strain PH-1/WT (VF), and the strains infected by p26-D4, p26-D4-Tri101 and p26-D4-FgPP1.

(18) FIG. 6E shows disease symptom of wheat spikelets after strains infected by p26-D4, p26-D4-Tri101 and p26-D4-FgPP1 are inoculated onto wheat.

(19) FIG. 6F shows the number of diseased spikelets per wheat head after strains infected by p26-D4, p26-D4-Tri101 and p26-D4-FgPP1 are inoculated onto wheat.

(20) FIG. 7A shows wheat FHB symptom after control of hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 measured by co-infection method (Test 1).

(21) FIG. 7B shows the number of diseased spikelets per wheat head after control of hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 measured by co-infection method (Test 1).

(22) FIG. 7C shows DON concentration in infected spikelets after control of hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 measured by co-infection method (Test 1).

(23) FIG. 7D shows wheat FHB symptom after control of hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 measured by pre-spray method (Test 2).

(24) FIG. 7E shows the number of diseased spikelets per wheat head after control of hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 measured by pre-spray method (Test 2).

(25) FIG. 7F shows DON concentration in infected spikelets after control of hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 measured by pre-spray method (Test 2).

DETAILED DESCRIPTION OF THE EMBODIMENTS

(26) 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.

(27) 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.

Embodiment 1 Construction of an Infectious Clone pSK-ABC with Three Components of Fusarium graminearum DNA Virus FgGMTV1 in Series and Verification of its Infectivity

(28) 1. Construction of the Infectious Clone pSK-ABC with Three Components of DNA Virus FgGMTV1 in Series

(29) 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 FIG. 1). Hind III (1-6nt), SacI (7-12nt), and SphI (13-18nt) restriction enzyme sites are designated at one end of DNA-A. MluI (5464-5469nt), NcoI (5470-5475nt), MfeI (5476-5481nt), SacI (5482-5487nt), PstI (5488-5493nt) and NotI (5494-5501 nt) restriction enzyme sites are designed at one end of DNA-C. The combination of Hind III (1-6nt) and NotI (5494-5501nt) is used to connect the clone to pBluescript II SK(+) cloning vector. Two restriction enzyme sites, SphI (1760-1765nt) and NheI (1766-1771nt), are designed between DNA-A and DNA-B, and four restriction enzyme sites, NheI (3509-3514nt), MluI (3515-3520nt), NcoI (3521-3526nt) and MfeI (3527-3532nt), are designed between DNA-B and DNA-C. Through the rational use of various restriction enzyme sites, the mutation of the three components of DNA-A, DNA-B and DNA-C is realized. The infectious clone sequence is synthesized by Shanghai Generay Biotechnology Co., Ltd. and connected to pBluescript II SK(+) cloning vector to obtain the infectious clone pSK-ABC. The sequence shown in the following SEQ ID NO: 1 is the complete sequence of the infectious clone pSK-ABC (without vector), with restriction enzyme sites of 1-18nt, 1760-1771nt, 3509-3532nt, 5464-5501nt. 19-1759nt is the sequence of component A, 1772-3508nt is the sequence of component B and 3533-5463nt the sequence of component C. Hind III (1-6nt) and NotI (5494-5501nt) double restriction enzyme digestion are used to connect the sequence to pBluescript II SK(+) cloning vector. SEQ ID NO: 1 is:

(30) TABLE-US-00001 5- AAGCTTGAGCTCGCATGCTTCGGCGAGTTTAGTTGCGTCGAACTCATCAGGTGTTTGG GAATAAGTGAAAAGCCATTTTTTACATCGGAGTCGGTATCTGTGATCACTAGTTAGTTA GTTGGGAATAGCTTCACTTTTTGGCAATTGAGTTAAGTAAGGGTCATTGCACCATCAT GGGGGAGGGACCTCCGGGGGGGCCCCATGTGGGGCTGTACCCGCGTCAGAAGCCCA AGCTCTGGAATAAAAAAAATAAAAAATACGTACTGGTTGCGTTGTCGTTTTTGTGGCT GAGGTGTTTCTGAGTCACATGATTCGGAAGCGCGTTTGGACATTGTGGCTGAAGAGT GGCTTTGCGGAGGTCAAGAAATCTAAGAAAGAGAGGATTTAAGATTCTTCTCTTTCTT AGATTTGTGACCGTGACCCAAGTTTGATTTATATACTAGTTATGTGTTGACTAAGGTAC AGAGAGGGGTAGTGATCTCCACAATAATGGCGTTAGCGTCCATCCATTTCCAATCAAT ATCAGTGCAGGTACGGCGTGGATCTTCATTGGCTATGTAGATGCACGGCTTACCCCAC ATGACCCGCTGTTTTCCTTTGTACTTGTCGGTAGCTACGAATTCTCGTTGGCCGAACC ATTGTTTGTAATTTGGAAAAGAGGAGAACCCACCAATCAAGTCATCAAACACCGCGT AGTTTACGTTAGGGTGAAAGTCAGAGAGCATGAACATGCCTGGGAAGTAGGCGTGAT TGGCTAGACTTCGCGCCCATAGAGTTTTACCGAGGCGGGAAGCGCCGAATAAAATGA GACTTATGGGTCGGTATGATGTGTTGTTCGTGACGTAATTGTCAACCCAGTCTTTGAG TTCGGGATAGTCAGAGTAATTGACAGTAATTTGTGGAGATTCGTATGTGCTCTGTGGT GGACCGAATTTCCATTCAGCAAATGACTTGATGTTGTTCCAGCATTTGATGGATTGGTT AGGTAGTTCGGTGGCAGCCAAGTGTAAAAATTCTTTTGCGGAAGTTGATTGGTCGAT GATACGCAACCATTCTTTATCCATGTTCTTTTGGTTCAAGGATGGTTGGTGTGGGGGTG TACCCTCTTCCCATTGGATGTCGTTATCTTTTTTTACGTAATTGACGACAGTGTGTGGG GTTCTGGTGACAACTTCGATGTTGGGGTGAACACCACAGAAATCGAAGTCCCGTGCG TTGTTTGAAGTGTGAATAGTTTCAACTTCCCAATACACGTGATGGTGGAATCCGCCAT CTTTGTGTTGTTCTTTTGAGATGACGAGATAGACAAGAAGTGGACTTTTTTCTTTGAA CATTTCGGCGAGTTTAGTTGCGTCGAACTCATCAGGTGTTTGGGAATAAGTGAAAAG CCATTTTTTACATCGGAGTCGGTATCTGTGATCACTAGTTAGTTAGTTGGGAATAGCTT CACTTTTTGGCAATTGAGTTAAGTAAGGGTCATTGCACCATCATGGGGGAGGGACCTC CGGGGGGGCCCCATGTGGGGCTGTACCCGCGTCAGAAGCCCAAGCTCTGGAATAAA AAAAATAAAAAATACGTACTGGTTGCGTTGTCGTTTTTGTGGCTGAGGTGTTTCTGAG TCACATGATTCGGAAGCGCGTTTGGACATTGTGGCTGAAGAGTGGCTTTGCGGAGGT CAAGAAATCTAAGAAAGAGAGGATTTAAGATTCTTCTCTTTCTTAGATTTGTGACCGT GACCCAAGTTTGATTTATATACTAGGCATGC GCTAGCGGTAATTTTAGTGTAGCAAAATTGAGTTGGTGATAGCTTCATTTTTTTGGATC CACTTTGTGACAATTGAGTTGAAGTAAGGGTCATTGCACCATCATGGGGGAGGGACC TCCGGGGGGGCCCCATGTGGGGCTGTACCCGCGTCAGAAGCCCAAGCTCCGGAATAA AAAAAAATCAAAAGTAGACTGTCGGTTAAAGTTCGGTTGGGTAGGATTAGTCAGCAA ATTTTCAACCAATAGCGGAGGTCAAGAAATCTAAGAAAGAGAGGATTTAAGATTCTT CTCTTTCTTAGATTTGTGACCGTGACCCAGGTTTGATTTTGGGTATATAAGGGAGGGGT AGCCACCATTTTTGCTAGTCTGTTTTTGGACTTAAAAAATTTATTTTTTAACACAAAAC ATTATTACGTCGACAAAAATGGCTTCTACAAAGAAGAAATCATACAACAACAAGAAG GCTTATAAAAAAAAAGAATGGAAGTCGAAGAAGACTTGGGACAAGTCTAGTTATTAC GACAATTACCAGTCGAAGATGAATATTTCGAATATGCAGACGAAGAGGGACAACATG ATGTGTGTGACGTCACATTGTGGTGTTCCGAATGCGGCGTTACTGGAGAATTCTGTTG TGGGTGAAATTCCAGCCAATATGGGAGTTCATTATATTATGTGGTCTCCTACGTATCGA GAGGCGGTACCACCGAATCGAGCGGCACAGTTGGATCGGCAATCCGCAAACACATTT TTTACTGGTTGGAAGGATAATTTGTCCTATCAATTTAAGGGACAGATTACAGGGATTCA CCTGAGGGTTGTGATATCTACCCGAAGAGAAGTGGAGTCCGCGCAGCCTTTTATTGG GCCGGGGAATACGCTGTGCAGAAACTTGGCGGTTCGTGATATGTCGGATGAGACATT GGACCAGTTTTTGTCGGGTACCCGGGATGTTGATTGGACGTTGGTGAATGTGATGGAC ACGATGTTTGATCCGGCGGTGTGCAAGGTGTTGTTTCGGCAGAGGAAGATTTTAGGT GCAGCTGATGCGTTGTTGAAGACGGAGGAGTTTTATCACCGTATCCGTCGGCCTATGG TGTACGGCGATAGGCAGGATGGTTTGGAGTTTGTGTCTAGTGGTTGGGCTGGAAGGG AGTCGGAGAACATATACGTCATTGATATGTACTCTTTGATTTCGGCAGCCCCACCGTTA GGTAATTTGTTGGATGGAGAGGGAAATATTGTTTTGGATGACAAGAAACGGCCTATTC CCGTATATGCGAAGTTAAATATTAGTGGAAATAGTATAGTGTATTGGAGGGAGTAGGGT AATTTTAGTGTAGCAAAATTGAGTTGGTGATAGCTTCATTTTTTTGGATCCACTTTGTG ACAATTGAGTTGAAGTAAGGGTCATTGCACCATCATGGGGGAGGGACCTCCGGGGGG GCCCCATGTGGGGCTGTACCCGCGTCAGAAGCCCAAGCTCCGGAATAAAAAAAAATC AAAAGTAGACTGTCGGTTAAAGTTCGGTTGGGTAGGATTAGTCAGCAAATTTTCAAC CAATAGCGGAGGTCAAGAAATCTAAGAAAGAGAGGATTTAAGATTCTTCTCTTTCTTA GATTTGTGACCGTGACCCAGGTTTGATTTTGGGTATATAAGGGAGGGGTAGCCACCAT TTTTGCTAGTCTGTTTTTGGACTTAAAAAATTTATTTTTTAACACAAAACATTATTACGT CGACAAAAGCTAGCACGCGTCCATGG CAATTGCTTTATATTGTAAAAAATATTTGTAACTGTAAATAATTAGTTGGTGATAGCTTC ATTTTTTTTACTCCACTTTGTGACAATTGAGTTAAGTAAGGGTCATTGCACCATCATGG GGGAGGGACCTCCGGGGGGGCCCCATGTGGGGCTGTACCCGCGTCAGAAGCCCAAG CTCTTTGTTGAGCCGAGCGCAGCGGTAATTTGGAGTCACGTGAGGTAAAATAAAATG TGGACTTACGTTCTTGGAATTGATGATTGAGACATTTTGAAAAAGTGTTGGAGTGGTT GGGGTATTTATGGTCAAGGACATGTTTGGTGGTGTCATTGGTTAATATAGGTACTGTCG GTAGATAGTTGTTGCGGTTGAAGTATAATGCGTGGAGCACCGAGGTCAGAACTTTTAG GAAAGACGATTTAAGATTCTCTTTCCTAAAAGTCTGACCGTGACTCCCTTTGGCCTTG ACGACGTTATTGGTGGAGGATTGGAATGTTACCCGCAATTTCACGTGACATGTGGAAA TGTGGTGACATGAAGAATTGTGGGACGGCACAATTTTAATTGGGTGGAACACAGCAG GGTAGGATTAGGCAGAATGAGGCAGATTTAGGCAGCGGAAATTTATTTTTAAATTGGA GCATTGTCTAAATCTAGAAGTACATGGTACCAGTCATCATCGTTTGGTTGATGATCTGG TATGTCCACCCAATCAGGATCATTTAAATTATGTACAATTGAATTGTTATGATTTGTAAA AAAAAAAGATAGGTAATCGCATAACGTGTTTTTGTTGAATAAAATAATACACGTGTTT GCGTATTGTTGTACCATGTCATGTGGGTCGGGATTGTTGTGGAGATGACACGTGTTAG TGCTGGTATGAACTCGCAGGAGTGTAGTACCAGTCGACAAAGGCTTGGATGTGTTGG CGGTGTCGTTGGAGTAGTGTAGGGTGAGTTTCCTCGAAACGACTAAGAAACGCCCCT TGATTTGGCTCGGAGAAAAGTTGTTGCCAGAAGGGGTCAGATGTGGCTGAGCAAAG ATCTGCATCCCCGAACTGAGCCACAAGCACCTCTTTAGACTGGATGCTCTCGTATACC GGGCCTGCATGTGTGTATATTGGCAGAATAGAAGGCATAACTCCGCTGTAAGCGAGTT GTAGGCTTCGTTGGAGGGGAATGTTTCCAGTATCGATGTAGATATCGAGATCACAGTT CCCATTAGTTCTTGTATTGTAACCGGCGATGAATCTCTTTGGATTGATGTCCATTTGGA GTTGAGAAGTGAAATTATCTGATTGTTTTGGGTCGACATCTTTATATTGTAAAAAATAT TTGTAACTGTAAATAATTAGTTGGTGATAGCTTCATTTTTTTTACTCCACTTTGTGACAA TTGAGTTAAGTAAGGGTCATTGCACCATCATGGGGGAGGGACCTCCGGGGGGGCCCC ATGTGGGGCTGTACCCGCGTCAGAAGCCCAAGCTCTTTGTTGAGCCGAGCGCAGCGG TAATTTGGAGTCACGTGAGGTAAAATAAAATGTGGACTTACGTTCTTGGAATTGATGA TTGAGACATTTTGAAAAAGTGTTGGAGTGGTTGGGGTATTTATGGTCAAGGACATGTT TGGTGGTGTCATTGGTTAATATAGGTACTGTCGGTAGATAGTTGTTGCGGTTGAAGTAT AATGCGTGGAGCACCGAGGTCAGAACTTTTAGGAAAGACGATTTAAGATTCTCTTTC CTAAAAGTCTGACCGTGACTCCCTTTGGCCTTGACGACGTTATTGGTGGAGGATTGGA ATGTTACCCGCAATTTCACGTGACATGTGGAAATGTGGTGACATGAAGAATTGTGGGA CGGCACAATTTTAATTGGGTGGAACACAGCAGGGTAGGATTAGGCAGAATGAGGCAG ATTTAGGCAGCGGAAATTTATTTACGCGTCCATGGCAATTGGAGCTCCTGCAG GCGGCCGC-3.
2. Protoplast Preparation of Fusarium graminearum

(31) 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 10.sup.7 spores/mL; 310.sup.7 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 CaCl.sub.2.Math.2H.sub.2O) 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 210.sup.7 cells/mL, the protoplasts are sub-packaged at 200 L/tube, added with dimethyl sulfoxide (DMSO) with final concentration of 7%, mixed well and stored at 80 C.

(32) 3. Polyethylene Glycol (PEG)-Mediated Transfection of pSK-ABC

(33) The protoplasts are taken out at 80 C. and thawed on ice: 200 L PH-1 protoplasts and 30 g 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 g/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 L STC is added to resuspend and precipitate, 200 L 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 g/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.

(34) 4. Determination of Colony Morphology and Growth Rate of the Strains

(35) 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.

(36) 5. Determination of Pathogenicity of the Strains

(37) 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 310.sup.5 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 L 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.

(38) 6. Results

(39) FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, and FIG. 2E show an infection activity analysis of an infectious clone pSK-ABC of FgGMTV1 with three components connected in series. The results show that the pSK-ABC transfectant strain is obtained through protoplast preparation, transfection and regeneration. By Southern blot analysis (see FIG. 2A), it is confirmed that the virus exists in the infectious clone pSK-ABC transfectant strain, and there is no significant difference in colony morphology, colony growth rate and pathogenicity between the pSK-ABC transfectant strain and the strain A+B+C co-transfected by infectious clones of three components (see FIG. 2B-FIG. 2E). It is confirmed that the infectious clone pSK-ABC with three components of DNA virus FgGMTV1 in series is successfully constructed.

Embodiment 2 Construction of Gene Silencing Vector p26-D4 Based on Virus FgGMTV1

(40) 1. Design and Construction of Gene Silencing Vector Based on FgGMTV1

(41) 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.

(42) 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.

(43) TABLE-US-00002 TABLE1 DNA-Cmutantprimers Primername Primersequence(5-3) p26-D1-F AGGATGCATTACAACTCGCTTACAGCGGAGTTATGCC (SEQIDNO:8) TT p26-D1-R AGGACCGGTCATCTTTATATTGTAAAAAATATTTGTAA (SEQIDNO:9) C p26-D2-F AGGATGCATAACAACTTTTCTCCGAGCCAAATCAAG (SEQIDNO:10) GGG p26-D2-R AGGACCGGTGGCTTCGTTGGAGGGGAATGTTTCCAG (SEQIDNO:11) TAT p26-D3-F AGGATGCATCATCTCCACAACAATCCCGACCCACATG (SEQIDNO:12) AC p26-D3-R AGGACCGGTGCCAGAAGGGGTCAGATGTGGCTGAG (SEQIDNO:13) CAAA p26-D4-F AGGATGCATGATTGGGTGGACATACCAGATCATCAAC (SEQIDNO:14) CA p26-D4-R AGGACCGGTACACGTGTTAGTGCTGGTATGAACTCG (SEQIDNO:15) CAG p26-D5-F AGGATGCATTAAAAATAAATTTCCGCTGCCTAAATCT (SEQIDNO:16) GC p26-D5-R AGGACCGGTAGGATCATTTAAATTATGTACAATTGAA (SEQIDNO:17) TT

(44) 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.

(45) FIG. 3A, FIG. 3B, and FIG. 3C illustrate an analysis of infection activity of deletion mutant based on DNA-C. The results show that five deletion mutants p26-D1, p26-D2, p26-D3, p26-D4 and p26-D5 are constructed based on the infectious clone pSK-ABC. It is confirmed by Southern blot that three components of the mutants p26-D1, p26-D2, p26-D3 and p26-D4 could be effectively replicated, but only p26-D4 has the same phenotype as the wild-type virus, that is, the infection of mutant virus would not cause abnormal phenotype of the host (FIG. 3A, FIG. 3B, FIG. 3C). p26-D4 may be used as a candidate gene silencing vector, named p26-D4, that is, the sequence of 284-434nt of the coding protein p26 of the DNA-C component is deleted, and the restriction enzyme sites NsiI and AgeI are introduced, the structure of the VIGS vector p26-D4 is shown in FIG. 4.

(46) 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:

(47) TABLE-US-00003 5-AAGCTTGAGCTC GCATGCTTCGGCGAGTTTAGTTGCGTCGAACTCATCAGGTGTTTGGGAATAAGTGAA AAGCCATTTTTTACATCGGAGTCGGTATCTGTGATCACTAGTTAGTTAGTTGGGAATAG CTTCACTTTTTGGCAATTGAGTTAAGTAAGGGTCATTGCACCATCATGGGGGAGGGAC CTCCGGGGGGGCCCCATGTGGGGCTGTACCCGCGTCAGAAGCCCAAGCTCTGGAATA AAAAAAATAAAAAATACGTACTGGTTGCGTTGTCGTTTTTGTGGCTGAGGTGTTTCTG AGTCACATGATTCGGAAGCGCGTTTGGACATTGTGGCTGAAGAGTGGCTTTGCGGAG GTCAAGAAATCTAAGAAAGAGAGGATTTAAGATTCTTCTCTTTCTTAGATTTGTGACC GTGACCCAAGTTTGATTTATATACTAGTTATGTGTTGACTAAGGTACAGAGAGGGGTA GTGATCTCCACAATAATGGCGTTAGCGTCCATCCATTTCCAATCAATATCAGTGCAGGT ACGGCGTGGATCTTCATTGGCTATGTAGATGCACGGCTTACCCCACATGACCCGCTGT TTTCCTTTGTACTTGTCGGTAGCTACGAATTCTCGTTGGCCGAACCATTGTTTGTAATT TGGAAAAGAGGAGAACCCACCAATCAAGTCATCAAACACCGCGTAGTTTACGTTAGG GTGAAAGTCAGAGAGCATGAACATGCCTGGGAAGTAGGCGTGATTGGCTAGACTTCG CGCCCATAGAGTTTTACCGAGGCGGGAAGCGCCGAATAAAATGAGACTTATGGGTCG GTATGATGTGTTGTTCGTGACGTAATTGTCAACCCAGTCTTTGAGTTCGGGATAGTCA GAGTAATTGACAGTAATTTGTGGAGATTCGTATGTGCTCTGTGGTGGACCGAATTTCC ATTCAGCAAATGACTTGATGTTGTTCCAGCATTTGATGGATTGGTTAGGTAGTTCGGTG GCAGCCAAGTGTAAAAATTCTTTTGCGGAAGTTGATTGGTCGATGATACGCAACCATT CTTTATCCATGTTCTTTTGGTTCAAGGATGGTTGGTGTGGGGGTGTACCCTCTTCCCAT TGGATGTCGTTATCTTTTTTTACGTAATTGACGACAGTGTGTGGGGTTCTGGTGACAA CTTCGATGTTGGGGTGAACACCACAGAAATCGAAGTCCCGTGCGTTGTTTGAAGTGT GAATAGTTTCAACTTCCCAATACACGTGATGGTGGAATCCGCCATCTTTGTGTTGTTCT TTTGAGATGACGAGATAGACAAGAAGTGGACTTTTTTCTTTGAACATTTCGGCGAGTT TAGTTGCGTCGAACTCATCAGGTGTTTGGGAATAAGTGAAAAGCCATTTTTTACATCG GAGTCGGTATCTGTGATCACTAGTTAGTTAGTTGGGAATAGCTTCACTTTTTGGCAATT GAGTTAAGTAAGGGTCATTGCACCATCATGGGGGAGGGACCTCCGGGGGGGCCCCAT GTGGGGCTGTACCCGCGTCAGAAGCCCAAGCTCTGGAATAAAAAAAATAAAAAATAC GTACTGGTTGCGTTGTCGTTTTTGTGGCTGAGGTGTTTCTGAGTCACATGATTCGGAA GCGCGTTTGGACATTGTGGCTGAAGAGTGGCTTTGCGGAGGTCAAGAAATCTAAGAA AGAGAGGATTTAAGATTCTTCTCTTTCTTAGATTTGTGACCGTGACCCAAGTTTGATTT ATATACTAGGCATGCGCTAGCGGTAATTTTAGTGTAGCAAAATTGAGTTGGTGATAGCT TCATTTTTTTGGATCCACTTTGTGACAATTGAGTTGAAGTAAGGGTCATTGCACCATCA TGGGGGAGGGACCTCCGGGGGGGCCCCATGTGGGGCTGTACCCGCGTCAGAAGCCC AAGCTCCGGAATAAAAAAAAATCAAAAGTAGACTGTCGGTTAAAGTTCGGTTGGGTA GGATTAGTCAGCAAATTTTCAACCAATAGCGGAGGTCAAGAAATCTAAGAAAGAGAG GATTTAAGATTCTTCTCTTTCTTAGATTTGTGACCGTGACCCAGGTTTGATTTTGGGTA TATAAGGGAGGGGTAGCCACCATTTTTGCTAGTCTGTTTTTGGACTTAAAAAATTTATT TTTTAACACAAAACATTATTACGTCGACAAAAATGGCTTCTACAAAGAAGAAATCATA CAACAACAAGAAGGCTTATAAAAAAAAAGAATGGAAGTCGAAGAAGACTTGGGACA AGTCTAGTTATTACGACAATTACCAGTCGAAGATGAATATTTCGAATATGCAGACGAA GAGGGACAACATGATGTGTGTGACGTCACATTGTGGTGTTCCGAATGCGGCGTTACT GGAGAATTCTGTTGTGGGTGAAATTCCAGCCAATATGGGAGTTCATTATATTATGTGGT CTCCTACGTATCGAGAGGCGGTACCACCGAATCGAGCGGCACAGTTGGATCGGCAAT CCGCAAACACATTTTTTACTGGTTGGAAGGATAATTTGTCCTATCAATTTAAGGGACA GATTACAGGGATTCACCTGAGGGTTGTGATATCTACCCGAAGAGAAGTGGAGTCCGC GCAGCCTTTTATTGGGCCGGGGAATACGCTGTGCAGAAACTTGGCGGTTCGTGATATG TCGGATGAGACATTGGACCAGTTTTTGTCGGGTACCCGGGATGTTGATTGGACGTTGG TGAATGTGATGGACACGATGTTTGATCCGGCGGTGTGCAAGGTGTTGTTTCGGCAGA GGAAGATTTTAGGTGCAGCTGATGCGTTGTTGAAGACGGAGGAGTTTTATCACCGTAT CCGTCGGCCTATGGTGTACGGCGATAGGCAGGATGGTTTGGAGTTTGTGTCTAGTGGT TGGGCTGGAAGGGAGTCGGAGAACATATACGTCATTGATATGTACTCTTTGATTTCGG CAGCCCCACCGTTAGGTAATTTGTTGGATGGAGAGGGAAATATTGTTTTGGATGACAA GAAACGGCCTATTCCCGTATATGCGAAGTTAAATATTAGTGGAAATAGTATAGTGTATT GGAGGGAGTAGGGTAATTTTAGTGTAGCAAAATTGAGTTGGTGATAGCTTCATTTTTT TGGATCCACTTTGTGACAATTGAGTTGAAGTAAGGGTCATTGCACCATCATGGGGGAG GGACCTCCGGGGGGGCCCCATGTGGGGCTGTACCCGCGTCAGAAGCCCAAGCTCCG GAATAAAAAAAAATCAAAAGTAGACTGTCGGTTAAAGTTCGGTTGGGTAGGATTAGT CAGCAAATTTTCAACCAATAGCGGAGGTCAAGAAATCTAAGAAAGAGAGGATTTAAG ATTCTTCTCTTTCTTAGATTTGTGACCGTGACCCAGGTTTGATTTTGGGTATATAAGGG AGGGGTAGCCACCATTTTTGCTAGTCTGTTTTTGGACTTAAAAAATTTATTTTTTAACA CAAAACATTATTACGTCGACAAAAGCTAGCACGCGTCCATGGCAATTGCTTTATATTGT AAAAAATATTTGTAACTGTAAATAATTAGTTGGTGATAGCTTCATTTTTTTTACTCCACT TTGTGACAATTGAGTTAAGTAAGGGTCATTGCACCATCATGGGGGAGGGACCTCCGG GGGGGCCCCATGTGGGGCTGTACCCGCGTCAGAAGCCCAAGCTCTTTGTTGAGCCGA GCGCAGCGGTAATTTGGAGTCACGTGAGGTAAAATAAAATGTGGACTTACGTTCTTG GAATTGATGATTGAGACATTTTGAAAAAGTGTTGGAGTGGTTGGGGTATTTATGGTCA AGGACATGTTTGGTGGTGTCATTGGTTAATATAGGTACTGTCGGTAGATAGTTGTTGCG GTTGAAGTATAATGCGTGGAGCACCGAGGTCAGAACTTTTAGGAAAGACGATTTAAG ATTCTCTTTCCTAAAAGTCTGACCGTGACTCCCTTTGGCCTTGACGACGTTATTGGTG GAGGATTGGAATGTTACCCGCAATTTCACGTGACATGTGGAAATGTGGTGACATGAA GAATTGTGGGACGGCACAATTTTAATTGGGTGGAACACAGCAGGGTAGGATTAGGCA GAATGAGGCAGATTTAGGCAGCGGAAATTTATTTTTAAATTGGAGCATTGTCTAAATCT AGAAGTACATGGTACCAGTCATCATCGTTTGGTTGATGATCTGGTATGTCCACCCAATC ATGCATCCTAGGACCGGTACACGTGTTAGTGCTGGTATGAACTCGCAGGAGTGTAGTA CCAGTCGACAAAGGCTTGGATGTGTTGGCGGTGTCGTTGGAGTAGTGTAGGGTGAGT TTCCTCGAAACGACTAAGAAACGCCCCTTGATTTGGCTCGGAGAAAAGTTGTTGCCA GAAGGGGTCAGATGTGGCTGAGCAAAGATCTGCATCCCCGAACTGAGCCACAAGCA CCTCTTTAGACTGGATGCTCTCGTATACCGGGCCTGCATGTGTGTATATTGGCAGAATA GAAGGCATAACTCCGCTGTAAGCGAGTTGTAGGCTTCGTTGGAGGGGAATGTTTCCA GTATCGATGTAGATATCGAGATCACAGTTCCCATTAGTTCTTGTATTGTAACCGGCGAT GAATCTCTTTGGATTGATGTCCATTTGGAGTTGAGAAGTGAAATTATCTGATTGTTTTG GGTCGACATCTTTATATTGTAAAAAATATTTGTAACTGTAAATAATTAGTTGGTGATAGC TTCATTTTTTTTACTCCACTTTGTGACAATTGAGTTAAGTAAGGGTCATTGCACCATCA TGGGGGAGGGACCTCCGGGGGGGCCCCATGTGGGGCTGTACCCGCGTCAGAAGCCC AAGCTCTTTGTTGAGCCGAGCGCAGCGGTAATTTGGAGTCACGTGAGGTAAAATAAA ATGTGGACTTACGTTCTTGGAATTGATGATTGAGACATTTTGAAAAAGTGTTGGAGTG GTTGGGGTATTTATGGTCAAGGACATGTTTGGTGGTGTCATTGGTTAATATAGGTACTG TCGGTAGATAGTTGTTGCGGTTGAAGTATAATGCGTGGAGCACCGAGGTCAGAACTTT TAGGAAAGACGATTTAAGATTCTCTTTCCTAAAAGTCTGACCGTGACTCCCTTTGGCC TTGACGACGTTATTGGTGGAGGATTGGAATGTTACCCGCAATTTCACGTGACATGTGG AAATGTGGTGACATGAAGAATTGTGGGACGGCACAATTTTAATTGGGTGGAACACAG CAGGGTAGGATTAGGCAGAATGAGGCAGATTTAGGCAGCGGAAATTTATTTACGCGTC CATGGCAATTGGAGCTCCTGCAGGCGGCCGC-3.

Embodiment 3 Silencing Effect of p26-D4

(48) 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.

(49) 1. Construction of Gene Silencing Vector Based on p26-D4

(50) 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.

(51) GFP gene fragments with different lengths and directions are amplified by specific primers, and the specific primers are shown in Table 2.

(52) TABLE-US-00004 TABLE2 PrimersforconstructionofGFPgenesilencing vectorbasedonp26-D4 Primername Primersequence(5-3) p26-D4-GFP75F-F CCCACCGGTCAGCACGACTTCTTC (SEQIDNO:18) p26-D4-GFP75F-R CCAATGCATGCCGTCGTCCTTGAAG (SEQIDNO:19) p26-D4-GFP75R-F CCCACCGGTGCCGTCGTCCTTGAAG (SEQIDNO:20) p26-D4-GFP75R-R CCAATGCATCAGCACGACTTCTTC (SEQIDNO:21) p26-D4-GFP150F-F CCCACCGGTATGGTGAGCAAGGGCGAG (SEQIDNO:22) p26-D4-GFP150F-R CCAATGCATGGTGCAGATGAACTTC (SEQIDNO:23) p26-D4-GFP150R-F CCCACCGGTGGTGCAGATGAACTTC (SEQIDNO:24) p26-D4-GFP150R-R CCAATGCATATGGTGAGCAAGGGCGAG (SEQIDNO:25) p26-D4-GFP300F-F CCCACCGGTCAGCACGACTTCTTCAAG (SEQIDNO:26) p26-D4-GFP300F-R CCAATGCATGGCGAGCTGCACGCTG (SEQIDNO:27) p26-D4-GFP300R-F CCCACCGGTGGCGAGCTGCACGCTGC (SEQIDNO:28) p26-D4-GFP300R-R CCAATGCATCAGCACGACTTCTTCAAG (SEQIDNO:29) p26-D4-GFP450F-F CCCACCGGTATGGTGAGCAAGGGCGAG (SEQIDNO:30) p26-D4-GFP450F-R CCAGTTGTGGCTGTTGTAGTTGTAC (SEQIDNO:31) p26-D4-GFP450R-F CCCACCGGTGTTGTGGCTGTTGTAG (SEQIDNO:32) p26-D4-GFP450R-R CCAATGCATATGGTGAGCAAGGGCGAG (SEQIDNO:33)

(53) 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.

(54) 2. Fluorescence Observation

(55) 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.

(56) 3. Results

(57) FIG. 5A, FIG. 5B, and FIG. 5C show the silencing efficiency analysis of GFP gene by VIGS vector p26-D4. The results show that the GFP silencing vectors 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 constructed based on p26-D4 obtain transfectant through transfection by PEG-mediated protoplasts and re-culture (see FIG. 5B). Southern blot and sequencing analysis confirm that in the strains infected by p26-D4-GFP300F, p26-D4-GFP300R, p26-D4-GFP450F and p26-D4-GFP450R, the inserted GFP fragments of 300 bp and 450 bp are lost in the DNA-C component. However, in the strains infected by p26-D4-GFP75F, p26-D4-GFP75R, p26-D4-GFP150F and p26-D4-GFP150R, the inserted GFP fragments of 75 bp and 150 bp are capable of being stably retained in the viral DNA-C component. The strains infected by p26-D4-GFP75F, p26-D4-GFP75R, p26-D4-GFP150F and p26-D4-GFP150R show normal growth phenotype (see FIG. 5A). The fluorescence intensity is observed by fluorescence microscope, and it is found that the fluorescence intensity of GFP in the strains infected by p26-D4-GFP75F, p26-D4-GFP75R, p26-D4-GFP150F and p26-D4-GFP150R is obviously weaker than the fluorescence intensity of GFP in PH-1/GFP strain and PH-1/GFP strain infected by p26-D4 (see FIG. 5A). Quantitative polymerase chain reaction (qPCR) further proves that compared with PH-1/GFP strain and PH-1/GFP strain infected by p26-D4, the mRNA expression level of GFP in strains infected by p26-D4-GFP75F, p26-D4-GFP75R, p26-D4-GFP150F and p26-D4-GFP150R decrease by 55%-75% (as shown in FIG. 5C). Therefore, the present disclosure confirms that the VIGS silencing vector based on p26-D4 may effectively silence GFP, and the inserted fragment is about 75 bp-150 bp.

Embodiment 4 Silencing Effect of p26-D4

(58) 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.

(59) 1. Construction of the Silencing Vectors p26-D4-Tri101 and p26-D4-FgPP1

(60) 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.

(61) The 150 bp Tri101 and FgPP1 gene fragments are amplified by specific primers, and the specific primers are shown in Table 3.

(62) TABLE-US-00005 TABLE3 Primersforconstructionofsilencingvectors p26-D4-Tri101andp26-D4-FgPP1 Primername Primersequence(5-3) p26-D4-Tri101-F CAGCACTAACACGTGTACCGGTGATTTGTACT (SEQIDNO:34) CTGTTCCC p26-D4-Tri101-R GTATGTCCACCCAATCATGCATGGTAGCTGGC (SEQIDNO:35) CGAGGGTGTC p26-D4-FgPP1-F CAGCACTAACACGTGTACCGGTACCATCTGCT (SEQIDNO:36) TGCTCCTCGCC p26-D4-FgPP1-R GTATGTCCACCCAATCATGCATGAAAGTCTTC (SEQIDNO:37) CACAACTTG

(63) 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.

(64) The sequence of the target gene Tri101 is shown in SEQ ID NO: 3:

(65) TABLE-US-00006 GATTTGTACTCTGTTCCCAAGCGTCATCTTTCTCAGCGCAGCACTTCTAT AATTTAGCGGCCTCACCTTCTGTAACACCAACACCAAGTGATTTACAAAC ACCACCAAAATGGCTTTCAAGATACAGCTCGACACCCTCGGCCAGCTAC C.

(66) The target gene FgPP1 has a sequence as shown in SEQ ID NO: 4:

(67) TABLE-US-00007 ACCATCTGCTTGCTCCTCGCCTACAAGATCAAGTACCCCGAAAACTTCTT CATCCTTCGAGGTAACCACGAGTGTGCCTCCATCAACCGTATTTATGGAT TCTACGACGAGTGCAAGCGTCGCTATAACATCAAGTTGTGGAAGACTTT C.
2. Results

(68) FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, and FIG. 6F show an analysis of silencing efficiency of endogenous genes Tri101 and FgPP1 of Fusarium graminearum. The results show that the silencing vectors p26-D4-Tri 101 and p26-D4-FgPP1 constructed based on p26-D4 obtain the transfectant by transfection of PEG-mediated protoplast and re-culture. Southern blot analysis confirms that the virus components could be effectively replicated in strains infected by p26-D4-Tri101 and p26-D4-FgPP1 (see FIG. 6A). Compared with strains infected by p26-D4, the RNA expression of the gene Tri101 in strains infected by p26-D4-Tri101 decreases by 80%, and the RNA expression of FgPP1 in strains infected by p26-D4-FgPP1 decreases by 63% (see FIG. 6B). The phenotypes of the strains infected by p26-D4-Tri101 and p26-D4-FgPP1 are not obviously abnormal, which are consistent with wild-type strain PH-1 and strains infected by p26-D4 (see FIG. 6D). In addition, the experiments of toxin production detection and pathogenicity test show that compared with strains infected by p26-D4, the DON production and pathogenicity of strains infected by p26-D4-Tri101 and p26-D4-FgPP1 are greatly reduced (see FIG. 6C, FIG. 6E and FIG. 6F). It shows that p26-D4 constructed by the disclosure is capable of effectively silencing the endogenous genes of Fusarium graminearum, and at the same time, it shows that the VIGS vector is capable of successfully transforming pathogenic fungal strains into hypovirulent strains.

Embodiment 5 Analysis of Biological Control Effect of VIGS-Induced Hypovirulent Strains

(69) 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.

(70) 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 L 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.

(71) 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 OD.sub.6002.0. First, the mycelium segment suspension is sprayed on wheat spikelets, and then 10 L 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.

(72) FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, and FIG. 7F illustrate an analysis on the control effect of VIGS-induced hypovirulent strains on wheat Fusarium head blight (FHB). The results show that in the co-infection method (Test 1), compared with the control group treated with sterile water, the wheat spikelets treated with hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 show obviously delayed expansion of wheat FHB spot, the protection efficiency reaches 74% and 76% respectively, and the DON content decreases by 21% and 29% respectively (see FIG. 7A, FIG. 7B, FIG. 7C). In the determination test by pre-spray method (Test 2), compared with the control group treated with sterile water, the wheat spikelets treated with hypovirulent strains infected by p26-D4-Tri101 and p26-D4-FgPP1 also show obviously delayed expansion of wheat FHB spot, the protection efficiency reaches 72% and 74% respectively, and the DON content decreases by 25% and 40% respectively (see FIG. 7D, FIG. 7E, FIG. 7F). The results show that the low-virulence strain induced by VIGS vector p26-D4 could effectively control wheat FHB under experimental conditions.

(73) 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.