Sclerotinia sclerotiorum effector gene and application thereof in plant sclerotiniose resistance breeding

Abstract

The invention discloses a sclerotinia sclerotiorum effector SsV263 gene and application of the sclerotinia sclerotiorum effector SsV263 gene in plant sclerotiniose resistance breeding, and relates to the technical field of gene engineering. The nucleotide sequence of the gene is as shown in SEQ ID NO. 13. A part of coding sequence of the gene is selected as a sense sequence, the sense sequence and an antisense sequence are respectively constructed into a plant expression vector to form a host induced expression silence vector HIGS-SsV263, and after the vector transfects host arabidopsis thaliana, a dsRNA fragment targeting the SsV263 gene can be generated, so that the silence of the SsV263 gene is induced in the sclerotinia sclerotiorum infection process, and the target SsV263 gene is obtained. The resistance of a host to plant diseases caused by sclerotinia sclerotiorum is obviously improved. The gene provided by the invention and a host-induced silent expression vector constructed by adopting the gene have huge application prospects in plant sclerotiniose resistance breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a sclerotinia sclerotiorum effector gene and application thereof in plant sclerotinia disease resistance breeding. Background Art

[0002] Sclerotinia sclerotiorum Sclerotinia sclerotiorum (Lib.) de Bary) is a typical necrotrophic, filamentous plant pathogen with a broad host range. It is distributed in 95 countries and regions and can infect more than 600 plant species, including some important crops such as soybean, rapeseed, sunflower, and Brassica vegetables. Under favorable conditions, Sclerotinia sclerotiorum can rapidly infect host plants and produce sclerotinia disease. The lack of effective antibacterial sclerotinia disease resources in existing Brassica napus and related species has led to a bottleneck in rapeseed disease-resistant breeding. In the past 10 years, with the publication of the S. sclerotiorum genome, researchers have turned their attention to the study of S. sclerotiorum pathogenicity factors, hoping that a deeper understanding of the pathogenic mechanism of S. sclerotiorum will provide new ideas for host disease-resistant breeding.

[0003] With the progress of genomic and proteomic research, nearly 100 S. sclerotinia secreted proteins have been predicted through multi-omics analysis (Guyon et al., 2014; Derbyshire et al., 2017; Ding et al., 2019). However, only a dozen S. sclerotinia secreted proteins have been confirmed to participate in the pathogenic process as effectors, such as SsCP1, SsPINE1, SsERP1, SsITL and SsSSVP1 (Yang et al., 2018; Wei et al., 2022; Fan et al., 2021; Lyu et al., 2016; Tang et al., 2020). Other effector factors, such as SsCaf1, SsRhs1, and SsCVNH, promote the growth of hyphae or sclerotia by participating in the formation of appressorium of S. sclerotiorum (Lyu et al., 2015; Xiao et al., 2014; Yu et al., 2017). Summary of the Invention

[0004] The present invention aims to provide a Sclerotinia effector gene SsV263 that affects the pathogenicity of Sclerotinia sclerotiorum. In order to reduce the damage of Sclerotinia sclerotiorum to plants, the sense sequence and antisense sequence of the partial coding sequence of the gene are respectively connected to the two ends of the amiRNA stemloop of the plant expression vector pLabc by means of the RNAi principle to form a host-induced expression silencing vector HIGS-SsV263. After the vector is transfected into the host Arabidopsis thaliana, a dsRNA fragment targeting the SsV263 gene can be produced, thereby inducing the silencing of the SsV263 gene during the infection process of Sclerotinia sclerotiorum, and significantly increasing the host's resistance to plant diseases caused by Sclerotinia sclerotiorum.

[0005] In order to achieve the above object, the present invention provides a Sclerotinia sclerotiorum effector gene SsV263 , the gene SsV263 The nucleotide sequence is shown in SEQ ID NO.13.

[0006] The Sclerotinia sclerotiorum effector gene provided by the present invention SsV263 The invention can be used to prepare HIGS vectors or Agrobacterium having resistance to plant diseases caused by Sclerotinia sclerotiorum.

[0007] The present invention also provides a method based on the above gene SsV263 The constructed HIGS vector confers resistance to plant diseases caused by Sclerotinia sclerotiorum.

[0008] Preferably, the nucleotide sequences of the sense sequence and antisense sequence selected for the above HIGS vector are shown as SEQ ID NO.15 and SEQ ID NO.16, respectively.

[0009] Preferably, the above-mentioned HIGS vector is selected from plasmid pLabc.

[0010] The present invention also provides a method based on the above gene SsV263 The constructed Agrobacterium has resistance to plant diseases caused by Sclerotinia sclerotiorum.

[0011] Preferably, the above-mentioned Agrobacterium is selected from GV3101.

[0012] The gene provided by the present invention SsV263 Either HIGS vector or Agrobacterium can be used for the prevention and treatment of plant diseases caused by Sclerotinia sclerotiorum, in particular for the prevention and treatment of plant sclerotinia sclerotiorum.

[0013] The gene provided by the present invention SsV263 Either HIGS vectors or Agrobacterium can be used in plant breeding for resistance to Sclerotinia sclerotiorum.

[0014] The present invention has the following advantages: The present invention provides the genome and protein sequence of the SsV263 gene of Sclerotinia sclerotiorum. The gene plays an important role in the pathogenicity of Sclerotinia sclerotiorum and provides a good application prospect for improving the resistance of Sclerotinia sclerotiorum host plants.

[0015] The host-induced silencing expression vector constructed by the construction method provided by the present invention is transformed into cruciferous plants. The plants grown from the seeds harvested from the T0 generation (T1 generation) have resistance to sclerotinia rot and can be inherited by the offspring. It has great application prospects in the breeding of cruciferous plants for sclerotinia rot resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The electrophoresis results of the extracted RNA and cDNA of Sclerotinia sclerotiorum are shown in Figure 1, where A is the RNA electrophoresis diagram; B is the cDNA detection diagram, and lanes 1-7 are technical replicate samples.

[0017] Figure 2 for SsV263 The electrophoresis test results of gene amplification, where A is SsV263 Gene fragment amplification diagram, lanes 1 and 2 are repeated amplification; B is SsV263 PCR detection of gene-linked T-vector bacterial solution, lanes 1-8 are 8 monoclonal bacterial solutions.

[0018] Figure 3 for SsV263 Schematic diagram of the construction of the HIGS vector encoding the gene. The forward fragment of the partial coding nucleotide sequence of the SsV263 gene with a length of 402 bp was connected between the CaMV 35S strong promoter and the amiRNA stem loop using the SpeI and BamHI restriction sites, while the reverse fragment was inserted between the amiRNA stem loop and the T-OCS terminator through the multiple cloning sites HindIII and SalI.

[0019] Figure 4 Figure 3 shows the electrophoresis detection results during the construction of the pLabc-SsV263 forward vector. A is the electrophoresis diagram of the double enzyme digestion (SpeI / BamHI) of the HIGS vector (pLabc), with lanes 1-4 showing repeated enzyme digestion and lane 5 showing the plasmid. B is the amplification diagram of the SsV263 (402 bp) forward gene fragment, with lanes 1-4 showing repeated amplification. C is the PCR detection of the pLabc-SsV263 forward vector clone culture (primer combination: HIGS-SsV263-positive-F + HIGS-introns-R), with lanes 1-8 showing eight single clone culture solutions.

[0020] Figure 5Figure 5. Electrophoresis test results of the reverse fragment ligated based on the successful construction of the pLabc-SsV263 forward vector. Lanes 1-4 show repeated enzyme digestion and lane 5 shows the plasmid. Lane B shows the amplification of the SsV263 (402 bp) reverse gene fragment. Lanes 1-4 show repeated amplification. Lane C shows PCR detection of pLabc-HIGS-SsV263 vector clone culture medium (primer combination: HIGS-introns-F + HIGS-SsV263-reverse-R). Lanes 1-10 show 10 single clone culture mediums.

[0021] Figure 6 These are the electrophoresis results of DNA detection of HIGS transgenic Arabidopsis thaliana. A is the PCR detection of Agrobacterium tumefaciens, i.e., the electrophoresis pattern of PCR amplification using the primer combination HIGS-introns-F + HIGS-SsV263-anti-R; B is the seedling status observed through a red filter under green fluorescence; C is the electrophoresis pattern of transgenic Arabidopsis thaliana detection, i.e., the electrophoresis pattern of PCR amplification using the primer combination HIGS-introns-F + HIGS-SsV263-anti-R; M stands for DNA maker 2000, WT is the wild-type Arabidopsis Col-0, and the remaining lanes are for HIGS transgenic Arabidopsis thaliana.

[0022] Figure 7 The results of identification of sclerotinia resistance of HIGS transgenic Arabidopsis thaliana, where A is SsV263 Phenotypes of two HIGS transgenic Arabidopsis lines, HIGS-SsV263-4 and HIGS-SsV263-5, after inoculation with Sclerotinia sclerotiorum in vitro. B shows the number of plaques in A 24 hours after inoculation. C shows the expression of the SsV263 gene during Sclerotinia sclerotiorum infection in HIGS transgenic Arabidopsis. WT represents wild-type Arabidopsis Col-0. DETAILED DESCRIPTION

[0023] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0024] Note: Unless otherwise noted, the experimental methods in the following examples are conventional methods, performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples, unless otherwise noted, are commercially available.

[0025] The plant material used in the examples is the wild type Col-0 of the Columbia ecotype Arabidopsis thaliana, which is conventionally cultured in a culture chamber.

[0026] The strains used in the examples: wild-type strain 1980 of Sclerotinia sclerotiorum and plant expression vector pLabc were provided by Chongqing Rapeseed Engineering Technology Research Center. DH5α Escherichia coli and GV3101 Agrobacterium tumefaciens were purchased from Qingke Biological Company.

[0027] Reagents and related kits used in the examples: pGEM-T Easy Vector T vector, Nearshore Protein Technology 2x Taq Master Mix, Tiangen TRNzol-A + RNA extraction kit, Tiangen Biochemical Company gel recovery kit, Tiangen Biochemical Company plasmid miniprep kit, BioRAD iScript™ cDNA Synthesis Kit, BioRAD iTaq™ Universal SYBR® Green Supermix kit, ThermoScientific FastDigest Sma I. Hind III. Pst I. EcoR V, Xba I and Xho I, chloroform, anhydrous ethanol, glycerol, mineral oil, sucrose, CTAB, β-mercaptoethanol, isoamyl alcohol, nucleic acid dye, agarose, loading buffer, DNA marker, surfactant SILWET L-77, etc.

[0028] The relevant reagents used in the examples are prepared as follows: LB liquid medium (1 L): Tryptone 10 g; Yeast Extract 5 g; Sodium chloride (NaCl) 10 g, pH 6.8, sterilized at 121°C for 20 min.

[0029] LB solid medium (1 L): Add 10 g of agar powder to LB liquid medium, pH 6.8.

[0030] YEB liquid medium (1 L): 5 g beef extract, 1 g yeast extract, 5 g tryptone, 5 g sucrose, 0.5 g magnesium sulfate monohydrate (MgSO4·H2O), pH 6.8, sterilized at 121°C for 20 min.

[0031] YEB solid medium (1 L): YEB liquid medium plus 10 g agar powder (agar), pH 6.8.

[0032] CTAB Buffer (1 L): CTAB 20 g, Nacl 81.88 g, EDTA (0.5 M, pH 8.0) 40 mL, Tris-HCl (1 M, pH 8.0) 100 mL, sterilized at 121°C for 20 minutes.

[0033] 50 x TAE Buffer (1 L): Tris 242 g, Na2EDTA·2H2O 37.2 g, glacial acetic acid 57.1 mL.

[0034] Antibiotics: kanamycin (kan) 100 mg / mL, streptomycin (str) 25 mg / mL.

[0035] The primer information used in the examples is shown in Table 1 below: Table 1 Primer sequence information

[0036] Experimental Example 1: Total RNA extraction and first-strand cDNA synthesis Tiangen's TRNzol-A + RNA extraction kit was used to extract RNA from Sclerotinia sclerotiorum hyphae according to the kit's procedures.

[0037] Agarose gel electrophoresis: Weigh agarose according to a 1% gel concentration ratio and pour it into a conical flask. Add 1x TAE buffer and heat in a microwave. After the agarose is completely dissolved and clear, remove it and cool to 50℃-60℃. Then add 5 μL of nucleic acid dye to every 100 mL of 1x TAE and mix thoroughly. Pour the mixture into an electrophoresis tank with a small hole comb inserted. After the gel block is completely solidified, carefully remove the comb and place the gel block on ice. Use a pipette to take 2 μL of RNA sample and add it to 2 μL of Loading Buffer, mix thoroughly, and then spot it into the small hole. Use a pipette to take 3 μL of DNA Mark and spot it into the blank small hole as a control. Add 1x TAE to the electrophoresis instrument until the gel block is covered. Start the electrophoresis instrument at 140V, 400A for 25 minutes. Stop the electrophoresis and place the gel block into a gel imaging system for observation and photography.

[0038] Concentration measurement: Use UV-visible spectrophotometer to measure the concentration of 1 μL RNA and store at -80°C.

[0039] The iScript™ cDNA Synthesis Kit from BIO-RAD was used, and the reaction system is shown in Table 2: Table 2 Reverse transcription reaction system

[0040] Note: The total amount of RNA template added is about 1 μg. After adding the sample, mix well and enter the reaction program in the PCR instrument.

[0041] The reaction procedure was as follows: priming at 25°C for 5 min; reverse transcription at 46°C for 20 min; RT inactivation at 95°C for 1 min; and an optional step at 4°C. The obtained cDNA was stored at -20°C.

[0042] Take RNA from Sclerotinia sclerotiorum grown for 1-7 days and test the RNA extraction. The electrophoresis results are shown in Figure 1 In A, it can be seen that the fragment is clear and can be used for subsequent experimental operations.

[0043] The quality of cDNA was tested using the primer combination Sstub1-F + Sstub1-R. The reaction system is shown in Table 3: Table 3 Reaction system

[0044] The reaction procedure is as follows: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 25 s; (3) annealing at 57°C for 25 s; (4) extension at 72°C for 20 s; (5) further extension at 72°C for 5 min; (6) stop at 16°C for 1 min; (2)-(4) are cycled 30 times. The obtained product can be stored in a refrigerator at 4°C for a short period of time. The cDNA amplified fragment is clearly visible, see Figure 1 B in the figure can be used for subsequent experimental operations.

[0045] Experimental Example 2 SsV263 Gene cloning 1. Gene amplification The cDNA mixture sample of Sclerotinia mycelium grown for 1-7 days was used as a template and the primer combination SsV263-F+SsV263-R (sequences shown in Table 1) was used to amplify SsV263 The coding sequence of the gene (468 bp) and the PCR amplification reaction system are shown in Table 4: Table 4 Reaction system

[0046] The PCR reaction procedure was as follows: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 30 s; (5) further extension at 72°C for 5 min; (6) stop at 16°C for 1 min; (2)-(4) were cycled 35 times.

[0047] Agarose gel electrophoresis was performed according to the method described in Experimental Example 1. The gel was placed in a gel cutter and the target gene (468 bp) fragment was excised. The target fragment was recovered using the Tiangen Gel Recovery Kit, following the manufacturer's recommended procedures.

[0048] The amplification was repeated twice, and the electrophoresis results were shown in Figure 2 As shown in A, the two lanes are technical replicates, both with obvious bands at 468 bp, indicating that the amplified SsV263 The nucleotide sequence of the gene was determined as shown in SEQ ID NO.13, and the amino acid sequence encoded by the gene was obtained as shown in SEQ ID NO.14, which are as follows: SsV263 Gene coding sequence (SEQ ID NO.13): ATGCATTTCTCCATGATTGCTTCTATCCCTCCTCTTCCTCTCTGCTCTCACTTCGGCCGCTCCAACTGCCGCTCCAGCAGCCATGGAAGCCAGCGCCTCAAGCTCGACTCAAATCTACTACCTCGTCAACTGCTTCAACAAGAGCTTCGCTGCCTACGCCGAGGCTGACTACTACCCAGAGAAGTCTTTGTCTCTTGCCGGTCAAACACCTACCAAGACAGCTATCTTC AACCCTACCGGTTCTATCGACTTTGAGGATGGAACTTGGACTGTCGACACCCCATTCAAGTTGACTGCTGTCATCGGCGAAGATTCTTACACCGCCAAGGCCGGTACCGTTGTTGGTTCCCGCTACCGTCAGCACATCCAGCTCTAAGTTGTCTTGTGTTCGTCTTGAAAGATTCGTCTTGTATTCGCCAAGTGCTGAAGAGCAATGCTACACCGATTATGCTTGCCAGGCTTAG; SsV263 Amino acid sequence encoded by the gene (SEQ ID NO.14): MHFSMIASIPLLFLSALTSAAPTAAPAAMEASASSSTQIYYLVNCFNNKSFAAYAEADYYPEKSLSLAGQTPTKTAIFNPTGSIDFEDGTWTVDTPFKLTAVIGEDSYTAKAGTVVGSATVSTSSSKLSCVRLERFVLYSPSAEEQCYTDYACQA.

[0049] 2. Construction of cloned bacteria Following the pGEM-T Easy Vector T kit protocol, the target gene fragment gel-recovered product was ligated with the pGEM-T Easy Vector T. The ligation product was then transformed into DH5α E. coli according to the full-strength gold DH5α E. coli competent transformation protocol. After 10 hours of plating, a single colony was picked with a 10 μL pipette tip and transferred to 600 μL of LB liquid medium containing kana resistance. The culture was shaken at 37°C and 250 rpm for 8 hours until turbid. PCR was performed using the primer combination SsV263-F + SsV263-R to detect positive clones. The PCR reaction system is shown in Table 5:

[0050] Table 5 Reaction system

[0051] The PCR reaction procedure was as follows: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 30 s; (5) further extension at 72°C for 5 min; (6) stop at 16°C for 1 min; (2) to (4) were cycled 30 times.

[0052] The results of colony PCR identification are shown in Figure 2 As shown in B, the results showed that among the 8 selected colonies, the primers detected the target bands, and all of them were positive clones except for bacterial solution No. 4. Bacterial solutions No. 1 and No. 2 were selected for sequencing verification.

[0053] Experimental Example 3 Construction of HIGS (host-induced gene silencing) vector 1. Plasmid extraction The plasmid T-SsV263 and the plant expression vector plasmid pLabc containing the target gene in the bacterial solution that was sequenced correctly were extracted respectively, and the plasmid mini-extraction kit of Tiangen Biochemical Company was selected and operated according to the steps provided by the kit.

[0054] 2. Introduce restriction enzyme cutting sites into target fragments The plant overexpression vector is pLabc, which is modified from the binary vector pLH6500 and has a red light selection marker added. The vector contains the CaMV 35S strong promoter and the T-OCS terminator. A partial coding sequence of the SsV263 gene is selected as the basis for constructing the HIGS vector. A conserved sequence of a 402 bp forward fragment in the SsV263 gene is selected as the sense sequence. The specific sequence is shown in SEQ ID NO.15. The forward fragment of the SsV263 gene is inserted between the CaMV 35S strong promoter and the amiRNA stem loop through the multiple cloning site. The multiple cloning site can be specifically selected from SpeI and BamHI. The reverse fragment of the SsV263 gene (antisense sequence, whose nucleotide sequence is shown in SEQ ID NO.16) is inserted between the amiRNA stem loop and the T-OCS terminator through the multiple cloning site. The multiple cloning site is specifically HindIII and SalI. SsV263 Schematic diagram of HIGS vector construction of gene Figure 3 shown.

[0055] Forward fragment of SsV263 gene (SEQ ID NO.15): CCAACTGCCGCTCCAGCAGCCATGGAAGCCAGCGCCTCAAGCTCGACTCAAATCTACTACCTCGTCAACTGCTTCAACAACAAGAGCTTCGCTGCCTACGCCGAGGCTGACTACTACCCAGAGAAGTCTTTGTCTCTTGCCGGTCAAACACCTACCAAGACAGCTATCTTCAACCCTACCGGTTCTATCGACTTTGAGGAT GGAACTTGGACTGTCGACACCCATTCAAGTTGACTGCTGTCATCGGCGAAGATTCTTACACCGCCAAGGCCGGTACCGTTGTTGGTTCCCGCTACCGTCAGCACATCCAGCTCTAAGTTGTCTTGTGTTCGTCTTGAAAGATTCGTCTTGTATTCGCCAAGTGCTGAAGAGCAATGCTACACCGATTATGCTTGCCAGGCT; Reverse fragment of SsV263 gene (SEQ ID NO.16): CATGGCGATGCCTTAAATAAAGATAAACCCAAAATGTTAATTTTACCAGAACTATATATACGAAGGCAGCATATATGTCACTTAGTGGATCAAGCATGTTTTTGTGCAGGAAAGATTAATCAAGAAAATTGGAATACAAAAGAGAGACTGCGGATCATAGTTTGGTAATCAAAGAGAATCAATGATCCAATTTGTCTACCGCA TCATTCATTCATTTAACGAGTCTAGTTTGAATTTTGGCGACTCGGTATTTGGAATGAATGAGTCGGAAGCTAATTGAATCATATCACGACCTGTGAATACCAATCTATGATCCGTAGTCTACATATATATTCCTAAAACATCAATTCAAAACAGCGAGTATTAAGTGTATGAACATGTGTAATATGCGTCCGAGCGTGTGTTTG.

[0056] 3. Double enzyme digestion of vector and forward fragment amplification The double enzyme digestion system of expression vector plasmid is shown in Table 6: Table 6 Enzyme digestion system

[0057] The above enzyme digestion system was digested in a 37℃ incubator for 1 h, and then electrophoresis was performed to detect the vector. Figure 4 A in ).

[0058] Using the cloned plasmid T-SsV263 as a template, the primer combination HIGS-SsV263-positive-F + HIGS-SsV263-positive-R (specific sequences are shown in Table 1) was used to amplify SsV263 Forward fragment of the gene. PCR reaction system is shown in Table 7:

[0059] Table 7 Reaction system

[0060] PCR reaction procedure: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 30 s; (5) further extension at 72°C for 5 min; (6) 16°C for 1 min; (2)-(4) were cycled 35 times.

[0061] Amplified fragment (see Figure 4B) and the digested product were recovered from the gel according to the Tiangen Gum Recovery Kit protocol. The forward fragment was then ligated to the double-digested vector backbone using the ClonExpress® II One Step Cloning Kit. The reaction system was set up on ice. See Table 8 for details:

[0062] Table 8 Reaction system:

[0063] Use a pipette to gently pipette to mix (do not oscillate), briefly centrifuge to collect the reaction solution at the bottom of the tube, and incubate at 37°C for 30 min; then cool to 4°C or immediately place on ice. The ligation product is then transformed into DH5α Escherichia coli. After 10 hours of plating, pick a single colony with a 10 μL pipette tip and place it in 600 μL of LB liquid medium containing Spe resistance. Incubate at 37°C, 250 rpm, and shake in a constant temperature shaker for 8 hours until turbid. Use the primer combination HIGS-SsV263-positive-F + HIGS-introns-R (see Table 1 for specific sequences) to perform bacterial liquid PCR and detect positive clones. The PCR reaction system is shown in Table 9:

[0064] Table 9 Reaction system

[0065] PCR reaction procedure: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 30 s; (5) further extension at 72°C for 5 min; (6) stop at 16°C for 1 min; (2) to (4) were cycled 30 times.

[0066] The results of colony PCR identification showed that (see Figure 4 C in the figure), the primers detected the target bands among the 8 selected colonies, and all 8 bacterial solutions were positive clones. Bacterial solutions No. 4 and No. 5 were selected for sequencing verification.

[0067] 4. Forward fragment amplification and ligation The plasmid pLabc-SsV263 positive containing the positive fragment was extracted and sequenced correctly. The vector was then digested with HindIII and SalI. The double enzyme digestion system is shown in Table 10: Table 10 Reaction system

[0068] The above enzyme digestion system was digested in a 37℃ incubator for 1 h, and then electrophoresis was performed to detect the vector. Figure 5 A in ).

[0069] Using the cloned plasmid T-SsV263 as a template, the primer combination HIGS-SsV263-anti-F + HIGS-SsV263-anti-R (specific sequences are shown in Table 1) was used to amplify SsV263 The reverse fragment of the gene. The PCR reaction system is shown in Table 11:

[0070] Table 11 Reaction system

[0071] The PCR reaction procedure was as follows: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 30 s; (5) further extension at 72°C for 5 min; (6) 16°C for 1 min; (2)-(4) were cycled 35 times.

[0072] Amplified fragments (results see Figure 5 B) and the digested product were recovered using the Tiangen Gum Recovery Kit. The forward fragment was then ligated to the double-digested vector backbone using the ClonExpress® II One Step Cloning Kit. The following reaction system was set up on ice (see Table 12):

[0073] Table 12 Reaction system

[0074] Use a pipette to gently pipette to mix (do not oscillate), briefly centrifuge to collect the reaction solution at the bottom of the tube, and incubate at 37°C for 30 min; then cool to 4°C or immediately place on ice. The ligation product was then transformed into DH5α E. coli. After 10 hours of plating, single colonies were picked with a 10 μL pipette tip and placed in 600 μL of LB liquid medium containing Spe resistance. Incubate at 37°C, 250 rpm, and shaken in a constant temperature shaker for 8 hours until turbid. PCR was performed on the bacterial solution using the primer combination HIGS-introns-F + HIGS-SsV263-anti-R (see Table 1 for specific sequences) to detect positive clones. The PCR reaction system is shown in Table 13:

[0075] Table 13 Reaction system

[0076] PCR reaction procedure: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 30 s; (5) further extension at 72°C for 5 min; (6) stop at 16°C for 1 min; (2) to (4) were cycled 30 times.

[0077] The results of colony PCR identification showed that (see Figure 5 In Figure C), the primers detected the target bands in the 10 selected colonies, and bacterial cultures No. 1-7 were all positive clones. Bacterial cultures No. 1 and No. 7 were selected for sequencing verification.

[0078] The sequencing was correct, indicating that the pLabc-HIGS-SsV263 expression vector was successfully constructed and the plasmid was provided for transformation of Agrobacterium competent cells.

[0079] Thaw GV3101 (pSoup) Agrobacterium competent cells stored at -80°C in an ice-water bath. Aseptically, add 100 ng~1 μg of plasmid DNA to the competent cells, mix gently, and let it stand in an ice-water bath for 5 min. Quick-freeze the centrifuge tube in liquid nitrogen for 5 min. Then quickly place the centrifuge tube in a 37°C water bath and keep it for 5 min. Do not shake the water surface during this process. Return the centrifuge tube to the ice-water bath and let it cool for 5 min. Aseptically add 800 μL of antibiotic-free LB liquid medium and incubate on a shaker at 28°C for 2~3 hours. Centrifuge at 5000 rpm for 5 min to collect the bacteria, retain about 100 μL of supernatant, gently pipette to resuspend the bacteria, take an appropriate amount of bacterial solution, spread it on a Spe+Rif plate, and incubate it upside down in a 28°C incubator overnight. After 36 hours of incubation, single colonies were picked with a 10 μL pipette tip and transferred to 600 μL of LB liquid medium containing Spe+Rif resistance. The culture was shaken at 250 rpm at 28°C for 24 hours until turbidity occurred. PCR was performed using the primer combination HIGS-introns-F + HIGS-SsV263-anti-R (for specific sequences, see Table 1) and the results were positive. The PCR reaction system is shown in Table 14:

[0080] Table 14 Reaction system

[0081] The PCR reaction procedure was as follows: (1) pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 30 s; (5) further extension at 72°C for 5 min; (6) stop at 16°C for 1 min; (2) to (4) were cycled 30 times.

[0082] The results of colony PCR identification showed that (see Figure 6 In Figure A), the primers detected the target bands in the 10 selected colonies, and bacterial solutions 1-10 were all positive.

[0083] Experimental Example 4 Agrobacterium-mediated transformation of Arabidopsis thaliana 1. Cultivation of Arabidopsis thaliana, as follows: (1) Sterilize the nutrient soil at 121°C for 40 minutes. After cooling, transfer it to the flower pot and soak the sterilized soil with the prepared nutrient solution. (2) Arabidopsis seeds that have been vernalized at 4°C for 2 days are sown in sterilized soil and covered with plastic wrap to prevent moisture loss; (3) After sowing, move the seeds to a light incubator (time: 16h / 8h; humidity: 40% / 60%; temperature: 22℃ / 16℃; light: 10000 LX / 0 LX). After the seeds germinate, remove the plastic wrap and water them with nutrient solution and water once a week until the Arabidopsis flowers and bears fruit.

[0084] 2. Transform Arabidopsis thaliana using the floral dip method, as follows: (1) Take 1 mL of Agrobacterium culture containing the recombinant plasmid pRed-HIGS-SsV263 that can express red fluorescent protein and add it to 100 mL of LB liquid medium containing Spe+Rif and culture it at 28°C and 200 rpm for 12 h; (2) Centrifuge at 5000 rpm for 20 min at room temperature, discard the supernatant, resuspend the Agrobacterium pellet in an equal volume of 5% sucrose solution, add 0.2% surfactant, and mix well; (3) Cut off the Arabidopsis pods and flowers, immerse the Arabidopsis inflorescence in the resuspended Agrobacterium solution for 30 seconds, cover with plastic wrap, and keep in the dark for 1 day; (4) Remove the plastic wrap and move the cells to a light incubator for normal culture; (5) One week later, repeat steps (1) to (5) for another transformation, and repeat this process for two weeks.

[0085] (6) When the Arabidopsis pods begin to turn yellow and crack, collect the fallen seeds and dry them in a dry and ventilated place. When the plant is completely dry, cut the entire plant, wrap it, and place it in an oven at 30°C for 2 days. Collect the T0 generation seeds and store them at 4°C.

[0086] 3. Positive seedling detection All the harvested T0 generation seeds were planted to obtain T1 generation transgenic plants. They were irradiated with 540nm excitation light from a handheld LUYOR-3415RG dual fluorescent protein observation lamp. The seedlings that showed red fluorescence under LUV-50A glasses were positive seedlings, and the dark ones were negative seedlings. The results are shown in Figure 6 As shown in B.

[0087] 4. PCR identification DNA was extracted from leaves about 2 weeks old for PCR identification. The primer combination used was HIGS-introns-F + HIGS-SsV263-anti-R (see Table 1 for specific sequences). PCR identification was performed on the sown materials. If there was a clear band at ±500 bp, they were positive seedlings that had successfully undergone genetic modification. The results of agarose gel electrophoresis are shown in Figure 6 As shown in Figure C, the results showed that plants numbered 1, 2, 5, 6, 7, 8, 19, 20 and 22 were positive seedlings.

[0088] Experimental Example 5 Analysis of Sclerotinia sclerotiorum resistance in transgenic Arabidopsis From the T1 generation plants, some red seeds were selected and dark seeds were selected at a ratio of 3:1 for individual plants to be planted in the T2 generation. The T2 generation continued to be screened for red seeds, thereby obtaining pure strains with all single seeds being red. Three strains were randomly selected from them and planted to the T3 generation. The sclerotinia resistance of transgenic Arabidopsis was evaluated by in vitro and in vivo inoculation of leaves with Sclerotinia sclerotiorum. The inoculation method was as follows: after three generations of continuous activation of the wild-type strain 1980 on a PDA plate, a 2 mm punch was used to punch out the mycelial blocks on the edge and inoculate them on Arabidopsis leaves, with the mycelial surface close to the leaf surface. 20 detached leaves were selected for each strain. The temperature was set to 22°C and the humidity was 85%. 24 h after inoculation, the long and short diameters of the plaques were counted, and the plaque area was calculated. The results of the identification of sclerotinia resistance of HIGS transgenic Arabidopsis are shown in Figure 7 The results showed that the leaf plaque area of the transgenic lines HIGS-SsV263-4 and HIGS-SsV263-5 was significantly reduced compared with the positive control empty transgenic line (EV) (for leaf phenotype, see Figure 7 The plaque areas of the two transgenic lines were reduced by 35% and 45%, respectively (see Figure 7 B in ).

[0089] At the same time, the bacterial plaques and surrounding leaves were collected 24 hours after inoculation with Sclerotinia sclerotiorum, and RNA was extracted using the method in Experimental Example 1. cDNA was synthesized using the iScript™ cDNA Synthesis Kit from BIO-RAD.

[0090] RT-PCR was performed using BioRAD's iTaq™ Universal SYBR® Green Supermix Kit. The reaction system is shown in Table 15: Table 15 Reaction system

[0091] The Sstub gene was used as the internal reference gene (primer combination was Sstub1-F + Sstub1-R, the specific sequence is shown in Table 1), and the primers Ssv263-RT-F + Ssv263-RT-R (the specific sequence is shown in Table 1) were used to detect the target gene. SsV263 The expression level of the protein was determined by fluorescence quantitative amplification using a CFX96™ Real-Time PCR instrument. The RT-PCR amplification program was as follows: 95°C for 30s, (95°C for 5s, 55-70°C for 30s) × 39 times; the melting program was 65°C-95°C, increasing by 0.5°C every 5s. The test results are shown in Figure 7 C in the figure shows that RT-PCR analysis showed that Sclerotinia sclerotiorum SsV263 The gene expression level was significantly decreased during infection of transgenic lines compared with that of wild type.

[0092] Arabidopsis thaliana is a model plant for molecular biology research. It belongs to the same family as rapeseed, radish, cabbage and other crops, and has a close relationship with them. SsV263 The HIGS gene was successfully used to obtain transgenic plants with significantly enhanced resistance to sclerotinia sclerotinia. SsV263 The application of this gene in the breeding of Sclerotinia sclerotiorum resistance in cruciferous plants such as rapeseed provides experimental support. SsV263 The genes can be used to increase the host plant's resistance to plant diseases caused by Sclerotinia sclerotiorum.

[0093] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A Sclerotinia sclerotiorum effector gene SsV263 , characterized in that, The gene SsV263 The nucleotide sequence is shown in SEQ ID NO.

13.

2. The Sclerotinia sclerotiorum effector gene according to claim 1 SsV263 Application of the invention in preparing a HIGS vector or Agrobacterium having resistance to plant diseases caused by Sclerotinia sclerotiorum.

3. A method based on the gene according to claim 1 SsV263 The constructed HIGS vector confers resistance to plant diseases caused by Sclerotinia sclerotiorum.

4. The HIGS vector according to claim 3, characterized in that The nucleotide sequences of the sense sequence and antisense sequence selected for the HIGS vector are shown in SEQ ID NO. 15 and SEQ ID NO. 16, respectively.

5. The HIGS vector according to claim 3, characterized in that The HIGS vector is selected from plasmid pLabc.

6. A method based on the gene according to claim 1 SsV263 The constructed Agrobacterium has resistance to plant diseases caused by Sclerotinia sclerotiorum.

7. The Agrobacterium according to claim 6, characterized in that The Agrobacterium was selected from GV3101.

8. The gene according to claim 1 SsV263 Or use of the HIGS vector according to any one of claims 3 to 5 or the Agrobacterium according to any one of claims 6 to 7 in preventing and controlling plant diseases caused by Sclerotinia sclerotiorum.

9. The use according to claim 8, characterized in that The application includes the prevention and treatment of plant sclerotinia disease.

10. The gene according to claim 1 SsV263 Or use of the HIGS vector according to any one of claims 3 to 5 or the Agrobacterium according to any one of claims 6 to 7 in plant breeding for resistance to sclerotinia sclerotinia.

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