Wheat Tilletia glabra effector protein gene g6634

Abstract

The present invention relates to wheat smut fungus, and specifically to wheat smut fungus effector protein gene g6634. The present invention provides a wheat smut fungus effector protein gene, and its nucleotide sequence is shown in SEQ ID NO: 1. Also provided is a protein encoded by the wheat smut fungus effector protein gene, and its amino acid sequence is shown in SEQ ID NO: 2. The wheat smut fungus effector protein gene plays an important role in inhibiting plant defense response.

Description

Technical Field

[0001] The invention relates to wheat tinderia, and in particular to a wheat tinderia effector protein gene g6634. Background Art

[0002] Wheat light stinky smut induced by wheat light stinky smut (Tilletia laevis Kühn, TFL) is seriously destructive to wheat production. The diseased wheat ears will be replaced by galls produced by the pathogen, which can reduce wheat yield by more than 80% and even lead to wheat grain failure. At present, most production uses biochemical pesticides to control wheat light stinky smut, and there is a lack of safe and effective prevention and control methods. There are few research results on wheat light stinky smut at home and abroad, and research data on the interaction between wheat light stinky smut and the host are particularly rare. Therefore, analyzing the mechanism of wheat light stinky smut infection of wheat at the genetic level is of great significance for the effective prevention and control of wheat light stinky smut. Summary of the invention

[0003] In order to further study the pathogenicity of wheat smut fungus, we analyzed the transcriptome data of wheat smut fungus and found a gene encoding an effector protein, named g6634. To verify the function of the g6634 gene, we introduced the g6634 gene into an expression vector and transformed Agrobacterium GV3101. Through transient expression in tobacco leaves, we used the BAX protein (a pro-apoptotic member of the Bcl-2 family) to induce apoptosis. We found that the effector protein encoded by the g6634 gene can effectively inhibit the programmed cell death (PCD) of plant cells induced by the BAX protein, indicating that the effector protein plays an important role in inhibiting the plant defense response.

[0004] The present invention provides a wheat smut fungus effector protein gene, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0005] Also provided is an expression cassette containing the wheat smut fungus effector protein gene.

[0006] Also provided is a vector containing the wheat smut fungus effector protein gene.

[0007] The vector may be a cloning vector or an expression vector.

[0008] In some embodiments of the present invention, the vector is pGR107 or pBin-GFP.

[0009] Also provided is a host bacteria containing the wheat smut fungus effector protein gene.

[0010] The host bacteria can be a cloning strain or an expression strain.

[0011] In some embodiments of the present invention, the host bacteria is Escherichia coli or Agrobacterium.

[0012] The present invention also provides a method for isolating the wheat tartaria effector protein gene, comprising: extracting RNA of wheat tartaria and reverse transcribing it into cDNA, performing PCR with the cDNA as a template, and amplifying the gene.

[0013] In some embodiments of the present invention, the PCR reaction system is: 2×PCR Mix 25 μL, 10 μM forward primer 2 μL, 10 μM reverse primer 2 μL, cDNA template 0.5 μg, ddH2O supplemented to 50 μL.

[0014] In some embodiments of the present invention, the primers shown in SEQ ID NO:3 and SEQ ID NO:4 are used to amplify the wheat smut fungus effector protein gene.

[0015] In other embodiments of the present invention, the primers shown in SEQ ID NO:7 and SEQ ID NO:8 are used to amplify the wheat smut fungus effector protein gene.

[0016] In some embodiments of the present invention, the PCR reaction conditions are: 95°C for 5 min; 95°C for 1 min, 57°C for 1 min, 72°C for 2 min, 35 cycles; 72°C for 10 min.

[0017] Also provided is a protein encoded by the wheat smut fungus effector protein gene, whose amino acid sequence is shown in SEQ ID NO: 2.

[0018] The invention provides gene resources for in-depth research on the pathogenicity mechanism of wheat smut and development of wheat varieties resistant to wheat smut. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the agarose gel electrophoresis image of the wheat smut fungus effector protein gene g6634 amplified by PCR.

[0020] Figure 2Figure 1 is the phenotypic observation result of transient expression of wheat smut fungus effector protein gene g6634 in tobacco leaves; A is the injection diagram of tobacco leaves, where g6634: only GV3101-pGR107-g6634 solution was injected, eGFP: only GV3101-pGR107-eGFP solution was injected, Buffer: only tobacco injection solution was injected (blank control), g6634+BAX: GV3101-pGR107-g6634 solution and GV3101 were injected. -pGR107-BAX bacterial solution, eGFP+BAX: injection of an equal volume mixture of GV3101-pGR107-eGFP bacterial solution and GV3101-pGR107-BAX bacterial solution, Buffer+BAX: injection of an equal volume mixture of Buffer and GV3101-pGR107-BAX bacterial solution; B is a symptom image of tobacco leaves after transient gene expression; C is a symptom image of tobacco leaves after transient gene expression after trypan blue staining and decolorization.

[0021] Figure 3 This is the subcellular localization result of the effector protein of wheat smut fungus g6634; the green fluorescence represents the g6634 effector protein. DETAILED DESCRIPTION

[0022] The present invention is described in detail below in conjunction with examples. It should be understood that the following examples are only used as explanations and illustrations of the present invention and do not limit the scope of the present invention in any way.

[0023] Biomaterials and carriers

[0024] The wheat light smut fungus (Tilletia laevis Kühn, TFL) used in the following examples is recorded in the literature Yao ZQ, Qin DD, Chen DL, Liu CZ, Chen WQ*, Liu TG, Liu B, Gao L*. Development of ISSR-derived SCAR marker and SYBR Green I Real-time PCR method for detection of teliospores of Tilletia laevis Kühn. Scientific Reports. 2019, 9 (1): 17651. The public can obtain the strain from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0025] Escherichia coli Trans2-Blue competent cells: purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0026] Agrobacterium GV3101 (pJIC SA-Rep) competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0027] The tobacco used in the following examples was Nicotiana benthamiana, which was provided by our laboratory.

[0028] The pGR107 vector used in the following examples is a modified potato virus X vector pGR107, which was donated by Chen Jieyin, a researcher at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The potato virus X vector pGR107 is a binary expression vector containing the CaMV 35S promoter and the kanamycin resistance gene. The GenBank accession number of the vector sequence is AY297842.1, and the total length is 10436 bp. After modification, the sequence between the Cla I and Sal I cloning sites of the potato virus X vector pGR107 was removed. The potato virus X vector pGR107 is commercially available.

[0029] The pBin-GFP vector is a commercially available vector.

[0030] Reagents and consumables

[0031] Universal DNA purification and recovery kit and rapid plasmid extraction kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd. 2× Pro Taq Master Mix (dye plus) was purchased from Hunan Aikerui Bioengineering Co., Ltd. High-fidelity enzyme 2× Flash Master Mix(Dye Plus), Ultra One Step Cloning Kit was purchased from Nanjing Novozymes Biotech Co., Ltd. Blue-chain bispecific antibody was purchased from Gibco, catalog number: 15140-122.

[0032] Solution formulation

[0033] (1) 0.25% NaClO: 250 μL NaClO, dilute to 100 mL with deionized water.

[0034] (2) 50×TAE Buffer: Dissolve 242 g of Tris and 37.2 g of EDTA-Na2·2H2O in 600 mL of deionized water. Add 57.1 mL of glacial acetic acid (anhydrous acetic acid, acetic acid) to the dissolved liquid to make up to 1 L. Adjust the pH to 8.5 with NaOH.

[0035] (3) 2% soil extract medium: The soil used must be sterilized in an autoclave at 60℃~100℃ for 30~60min. Take 75g of sterilized soil, filter it under 8 layers of gauze with 500mL of boiled distilled water, make up to 1000mL, and sterilize at 120℃ for 20min. (20g of agar must be added to the solid medium)

[0036] (4) LB medium: weigh 3 g sodium chloride, 1.5 g yeast extract powder, and 3 g tryptone, dilute to 300 mL, and sterilize at 120°C for 20 min. (For solid medium, add 4.5 g agar powder)

[0037] (5) Kanamycin stock solution (50 mg / ml): Weigh 2.5 g of kanamycin into a 50-mL centrifuge tube and add 40 mL of sterile distilled water. Mix thoroughly to dissolve and then make up to 50 mL. Filter with a 0.22 μm filter to sterilize. Aliquot and store at -20°C.

[0038] (6) Rifampicin stock solution (20 mg / mL): Weigh 0.2 g of rifampicin into a 50 mL centrifuge tube, add 10 mL of DMSO (dimethyl sulfoxide), filter and sterilize, then aliquot and store at -20°C.

[0039] (7) Tobacco injection buffer: Take 5 mL of 1 M MgCl2 stock solution, 5 mL of 1 M MES (2-morpholineethanesulfonic acid) stock solution, and 0.5 mL of 0.2 M AS (acetosyringone) stock solution, add sterile water to make up to 500 mL, mix well and set aside.

[0040] (8) Trypan blue staining solution: 10 mL of lactic acid, 10 mL of glycerol, 10 mL of phenol, 10 mg of trypan blue, and 10 mL of deionized water. First, completely dissolve the trypan blue in deionized water, then add it to the other solutions, mix well, and store at 4°C.

[0041] (9) Trypan blue destaining solution: 250 g chloral hydrate, dilute to 100 mL with deionized water, and incubate at 37°C with low speed shaking or 65°C until completely dissolved.

[0042] PCR primers

[0043] Table 1

[0044] Primer name Primer sequence (5′-3′) g6634F1 CACCAGCTAGCATCGATTCCCGGGATGGCCCCGAGCCG(SEQ ID NO:3) g6634R1 AAGCTTATCGGCGGTCGACCCGGGTCACTTGTGCTGGTCGTCAAAG(SEQ ID NO:4) pGR107F ATAGCAGTCATAGCACTTCCT(SEQ ID NO:5) pGR107R CACTGGGGCATACTTCATGC(SEQ ID NO:6) g6634F2 ATTTACGAACGATAGGGTAATGGCCCCGAGCCG(SEQ ID NO:7) g6634R2 GCCCTTGCTCACCATGGATCCCTTGTGCTGGTCGTCAAAGTACC(SEQ ID NO:8) pBin107F ACAATCCCACTATCCTTCGC(SEQ ID NO:9) pBin107R AAGTCGTGCTGCTTCATGT(SEQ ID NO:10)

[0045] Main instruments

[0046] PCR instrument, produced by Thermo Fisher Scientific, USA. Centrifuge and pipette, produced by Eppendrof Life Sciences, Germany. 50mL large centrifuge, produced by Sigma, Germany. Electrophoresis instrument, produced by Bio-Rad, USA. Autoclave, produced by Zealway, USA. GHB-202 constant temperature metal bath, produced by Hangzhou Bio-River Technology Co., Ltd. Constant temperature water bath, Shanghai Zhijiang Biotechnology Co., Ltd. 85-2 digital display constant temperature magnetic stirrer, produced by Ronghua Instrument Manufacturing Co., Ltd., Jintan City, Jiangsu Province. Large capacity full temperature oscillator, produced by Suzhou Peiying Experimental Equipment Co., Ltd. Clean bench, produced by Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. Confocal laser microscope, Zeiss 980.

[0047] If not otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure. If not otherwise specified, the experimental methods and conditions used in the following examples are all conventional experimental methods and conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer specifications. Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0048] Example 1. Cloning and functional verification of the g6634 gene of Tilletia tritici

[0049] 1. Cultivation of wheat smut fungus

[0050] (1) Take 5-6 wheat stinky smut fungus (Tilletia laevis Kühn, TFL) galls and place them in a 2 mL centrifuge tube, add 2 mL of sterile distilled water and let stand at room temperature for 30 min;

[0051] (2) Use tweezers to peel off the seed coat and pick out small grains, filter out impurities with 150-mesh gauze, and centrifuge;

[0052] (3) Centrifuge at 6000 rpm for 1 min, discard the supernatant, and add 2 mL of 0.25% NaClO for disinfection for 10 min;

[0053] (4) Centrifuge at 6000 rpm for 1 min, discard the supernatant, and add sterile distilled water;

[0054] (5) Repeat the above steps (3) and (4) twice;

[0055] (6) Using an optical microscope and a hemocytometer, the concentration of winter spores of T. tritici was adjusted to 1 × 10 6 / mL, and a teliospore suspension was obtained;

[0056] (7) Add cyan-chain bispecific antibody (Gibco) to 2% soil extract medium at a volume ratio of 1%, pour the plate, and apply 220 μL of spore suspension to each culture dish. Place the plate in an artificial incubator at 5°C, 1Lx and 50% relative humidity for about 45 days under full light, then observe the germination state of spores under an optical microscope, and collect them after the hyphae grow to the infection hyphae stage for RNA extraction of wheat spores.

[0057] 2. Extraction of RNA from Tilletia sutchuenensis and synthesis of cDNA

[0058] The Trizol method was used to extract RNA from the mycelium of Tilletia laevis Kühn (TFL). The specific steps are as follows:

[0059] (1) The germinated mycelium of wheat smut fungus was collected and placed in a 2 mL spiral tube without RNase, and then quickly placed in liquid nitrogen, and the sample was ground into powder using a high-throughput rapid sample preparation instrument;

[0060] (2) Add 1 mL (the amount can be increased or decreased according to the sample quantity) of TRIzol reagent (Invitrogen) to the fully ground mycelium powder, gently shake to fully lyse it, and let it stand at room temperature for 5 to 10 min;

[0061] (3) Add chloroform to the solution containing TRIzol reagent at a ratio of 0.2 mL chloroform per 1 mL TRIzol reagent, shake vigorously for 15 seconds, extract, let stand at room temperature for 3 minutes, and centrifuge at 4°C, 12,000 rpm for 15 minutes;

[0062] (4) After sufficient centrifugation, the supernatant was aspirated into a new 1.5 mL RNase-Free centrifuge tube, and an equal volume of isopropanol was added for precipitation. After mixing, the tube was placed on ice for 10 min and centrifuged at 4°C, 12,000 rpm for 15 min.

[0063] (5) Discard the supernatant, add 1 mL of 75% ethanol (prepared with anhydrous ethanol and RNase-free water), invert to mix, and centrifuge at 12,000 rpm for 5 min at 4°C to remove as much residual liquid as possible;

[0064] (6) Repeat the previous step, then open the centrifuge tube and place it in a clean bench to dry for 5 to 10 minutes until the precipitate turns transparent;

[0065] (7) Add an appropriate amount of RNase-free water to dissolve the RNA and store it in a -80°C refrigerator for later use.

[0066] The RNA reverse transcription kit (Cat. No.: RR047A) of TaKaRa Company was used to reverse transcribe the obtained total RNA of T. tritici according to the kit instructions to synthesize the first strand of cDNA as a template for gene cloning. The obtained T. tritici cDNA was stored in a -20°C refrigerator.

[0067] 3. Cloning of target gene

[0068] Based on the transcriptome data of wheat smut fungus, we found an effector protein gene named g6634. The nucleotide sequence of the open reading frame (ORF) of the g6634 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO:2.

[0069] Open reading frame sequence of g6634 gene (465 bp):

[0070] ATGATCGCTATTCCTCGTCTCTTCGCCGCCGTCGCCCTCCTCGCTGCCGTTGTGGTC

[0071] AGCGCTGCCCCGAGCCGCGACAAAACCTACCCTGCCTGTGACCCTCCCTCGAAAG

[0072] GTCCCGTCCACCGGCAAATGCCACCAAATGAACATGAAGACCGACCAAGATCCCTT

[0073] CTACATCGCCCCTGGGCTCGGCGCGTGCGGCGTCACCTACAACGACAACGTGATG

[0074] GGTGCCTGCCTCAGCCCGGGTTGGATCAACTCCGGCTACTACAGCTCCTGCGGCCG

[0075] GAAGACCACCGTCACCAATCCTCGCAACGGCAAGTCGATCCACGTCGTCATCATTG

[0076] ACGCCTGTGTATCGGCTTCCTGCAACGACATCATGCTCACGAAGGCGGCCTTCCAG

[0077] GCCATCGGCGGTAACATGGCGTCTGGCCACGTCGACAACAAAGTCAACTGGTACTTTGACGACCAGCACAAGTGA (SEQ ID NO: 1).

[0078] Amino acid sequence of the protein encoded by the g6634 gene (154aa):

[0079] MIAIPRLFAAVALLAAVVVSAAPSRDKTYPACDPPSKGPVHGKCHQMNMKTDQDPFY

[0080] IAPGLGACGVTYNDNVMGACLSPGWINSGYYSSCGRKTTTVTNPRNGKSIHVVIIDAC VSASCNDIMLTKAAFQAIGGNMASGHVDNKVNWYFDDQHK (SEQ ID NO: 2).

[0081] The N-terminal signal peptide sequence of the protein encoded by the g6634 gene was predicted using the online analysis software SignalP 5.0, the gene sequence encoding the signal peptide was removed, and then the primers g6634F1 and g6634R1 (SEQ ID NO: 3 and SEQ ID NO: 4) for gene cloning were designed and commissioned to Beijing Qingke Biotechnology Co., Ltd. for primer synthesis. The target gene fragment was PCR amplified using the reverse transcribed wheat light smut fungus cDNA as a template using primers g6634F1 and g6634R1. The PCR reaction system is shown in Table 2.

[0082] Table 2 PCR reaction system of target gene

[0083]

[0084] The PCR reaction program was: 95°C for 5 min; 95°C for 1 min, 57°C for 1 min, 72°C for 2 min, 35 cycles; 72°C for 10 min. The PCR product was detected by 1.0% agarose gel electrophoresis (150 V, 30 min). Figure 1 The target band was cut from the gel and recovered using a universal DNA purification and recovery kit (Tiangen Biotechnology, catalog number: DP214-03) according to the kit instructions to obtain the g6634 target gene fragment, which was stored in a -20°C refrigerator.

[0085] 4. Enzyme digestion of vector

[0086] 10 μL of E. coli containing the pGR107 vector was placed in 10 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with a shaker at 200 rpm for 8-10 h, and then the plasmid was extracted using a rapid plasmid extraction kit (Tiangen Biotechnology, catalog number: DP105-03) according to the instructions of the kit. According to the vector and gene sequence, the pGR107 plasmid was double-digested using Bsu15I enzyme (Thermo Fisher Scientific, USA, catalog number: FD0144) and Sal I enzyme (Thermo Fisher Scientific, USA, catalog number: FD0644), and the enzyme digestion reaction system is shown in Table 3.

[0087] Table 3 Bsu15I and Sal I double restriction enzyme digestion reaction system

[0088]

[0089] Place the prepared enzyme digestion reaction system in a 37°C metal bath for 1 hour. After the reaction is completed, the enzyme digestion product is detected by 1.0% agarose gel electrophoresis. Cut the target band with a sterile blade and use a universal DNA purification and recovery kit (Tiangen Biotechnology, DP214-03) to recover it according to the kit instructions to obtain the pGR107 linear vector, which is stored in a -20°C refrigerator.

[0090] 5. Ligation of target gene and vector

[0091] (1) Using Vazyme Ultra One Step Cloning Kit seamless ligation kit was used to connect the purified pGR107 linear vector with the g6634 target gene fragment.

[0092] (2) The reorganization connection system is as follows:

[0093] Table 4 Ligation reaction system of plasmid pGR107 and target gene

[0094]

[0095] (3) The prepared system was gently mixed and placed in a 50°C metal bath for 10 min, and then placed on ice for 2 min.

[0096] (4) Immediately transfer 5-10 μL of the recombinant ligation product into 100 μL of molten E. coli Trans2-Blue competent cells and place on ice for 30 min.

[0097] (5) Heat shock the cells in a water bath at 42°C for 45 seconds and then place on ice for 2 to 3 minutes.

[0098] (6) Add 800 μL of antibiotic-free LB liquid culture medium into the clean bench.

[0099] (7) Shake the culture at 200 rpm at 37°C for 1 h. Use a sterile glass rod to evenly spread the bacterial solution on a solid LB plate containing 50 μg / mL kanamycin. Place the plate in an incubator at 37°C and incubate overnight.

[0100] (8) Select 6 to 8 single clones for bacterial liquid PCR verification, and the primers are pGR107F and pGR107R (SEQ ID NO: 5 and SEQ ID NO: 6). The PCR reaction system is as follows:

[0101] Table 5 Bacterial liquid PCR reaction system

[0102]

[0103] The PCR reaction program was: 95°C for 5 min; 95°C for 1 min, 57°C for 1 min, 72°C for 2 min, 35 cycles; 72°C for 10 min. After the reaction, the amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing verification.

[0104] The positive colonies with correct sequencing were used to extract plasmids using a rapid plasmid extraction kit (Tiangen Biotechnology, catalog number: DP105-03) to obtain the recombinant plasmid pGR107-g6634.

[0105] 6. Transformation of Agrobacterium Competent Cells

[0106] The recombinant plasmid pGR107-g6634 was used to transform the competent cells of Agrobacterium GV3101 (pJIC SA-Rep) to obtain the recombinant bacteria GV3101-pGR107-g6634. The Agrobacterium transformation method is as follows:

[0107] (1) Take out the competent Agrobacterium cells stored at -80°C, thaw them at room temperature or in the palm of your hand for a while, and insert them into ice when they are in an ice-water mixture.

[0108] (2) Add the plasmid to the competent cells at a rate of 0.01 to 1 μg of plasmid per 100 μL of competent cells. After addition, gently stir the bottom of the tube to mix.

[0109] (3) Place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0110] (4) Add 700 μL of LB liquid medium without antibiotics to the competent cells and culture at 28°C with shaking at 200 rpm for 2 to 3 h;

[0111] (5) The cultured bacterial liquid was centrifuged at 6000 rpm for 1 min to collect the bacterial cells. About 100 μL of the supernatant was retained to resuspend the bacterial cells. The bacterial liquid was spread on an LB solid plate containing 25 μg / mL rifampicin and 50 μg / mL kanamycin using a sterile glass rod.

[0112] (6) Incubate the plate upside down in a 28°C incubator for 2-3 days. When a single colony grows on the plate, pick 8 single clones for bacterial liquid PCR verification. The PCR reaction system and reaction procedure are the same as the bacterial liquid PCR verification in 5 above.

[0113] (7) The colonies verified by PCR were picked and placed in 10 mL of LB liquid culture medium containing 25 μg / mL rifampicin and 50 μg / mL kanamycin. The culture was cultured at 28°C and 200 rpm for 2 days and the bacterial solution was stored for later use.

[0114] 7. Transient expression of genes in tobacco

[0115] The BAX gene (GenBank accession number: L22472.1) is a member of the apoptosis gene Bcl-2 family. The protein it expresses has the function of inducing cell apoptosis, which is similar to the mechanism of hypersensitive necrosis in pathogen-plant interactions. The eGFP gene (GenBank accession number: EU746493.1) is an enhanced green fluorescent protein gene that can spontaneously fluoresce under blue light excitation. In the early stage of this laboratory, the expression vector pGR107-BAX of the BAX gene was prepared and introduced into Agrobacterium GV3101 (pJICSA-Rep) to obtain the recombinant bacteria GV3101-pGR107-BAX; the expression vector pGR107-eGFP of the eGFP gene was also prepared and introduced into Agrobacterium GV3101 (pJIC SA-Rep) to obtain the recombinant bacteria GV3101-pGR107-eGFP. The construction method of pGR107-BAX is as follows: the pGR107 vector and the BAX gene fragment are double-digested with Sal I and Cla I restriction endonucleases, respectively, and then the BAX gene fragment is inserted into the pGR107 vector by ligation. The construction method of pGR107-eGFP is as follows: the pGR107 vector and the eGFP gene fragment are double-digested with Sal I and Cla I restriction endonucleases, respectively, and then the eGFP gene fragment is inserted into the pGR107 vector by ligation.

[0116] The method for transient gene expression in tobacco is as follows:

[0117] (1) The recombinant bacteria GV3101-pGR107-g6634, GV3101-pGR107-BAX, and GV3101-pGR107-eGFP were inoculated into 10 mL of LB liquid culture medium containing 25 μg / mL rifampicin and 50 μg / mL kanamycin, respectively, and cultured at 28°C and 200 rpm for 2 days.

[0118] (2) The cultured bacterial solution was centrifuged at 5000 rpm for 10 min to collect the bacterial cells, and the cells were resuspended in Buffer (tobacco injection solution) (10 mmol / L MES, 200 μmol / L AS, 10 mmol / L MgCl2).

[0119] (3) Repeat the above steps, wash the cells three times with Buffer (tobacco injection solution), activate the cell suspension in a dark environment at 28°C incubator for 3 h, and adjust the cell suspension to OD 600nm =0.3~0.5.

[0120] (4) Use a 1 mL sterile syringe without a needle to draw an appropriate amount of bacterial solution, select tobacco leaves that are 4 to 6 weeks old and in good growth condition, and use the infiltration method to inject the bacterial solution into the back of the tobacco leaves. Use the 6-zone injection method according to Figure 2 In the injection method shown in A, GV3101-pGR107-g6634 solution, GV3101-pGR107-eGFP solution and Buffer (tobacco injection solution) were injected from top to bottom on the left side of the tobacco leaf, and the injection range was marked. Each tobacco leaf has 6 injection areas, of which the Buffer in the lower left corner is the blank control. Each tobacco plant was injected with 3 to 5 leaves. Each sample was repeated 3 times.

[0121] (5) 24 hours after inoculation, Figure 2 The injection method shown in A is to inject equal volumes of GV3101-pGR107-g6634 and GV3101-pGR107-BAX mixtures (g6634+BAX), equal volumes of GV3101-pGR107-eGFP and GV3101-pGR107-BAX mixtures (eGFP+BAX), and equal volumes of Buffer and GV3101-pGR107-BAX mixtures (Buffer+BAX) on the right side of tobacco leaves (right side of the marked area in the previous step) from top to bottom. The tobacco leaves were placed in an incubator at 25°C for further culture. After 3 to 8 days, the necrosis of the injected area of ​​the tobacco leaves was observed, and the data was counted and photographed.

[0122] (6) Mix trypan blue staining solution and anhydrous ethanol in a volume ratio of 1:1, place tobacco leaves showing symptoms in the mixed staining solution, boil them in boiling water for 5 minutes, and place the leaves in trypan blue decolorizing solution for decolorization after overnight staining. After decolorization, take pictures.

[0123] The results are as follows Figure 2 B (symptoms of tobacco leaves after transient gene expression) and Figure 2 C (symptoms of tobacco leaves after transient gene expression, stained with trypan blue and decolorized). The left injection area of ​​tobacco leaves: the site injected with only Buffer (blank control), the site injected with only GV3101-pGR107-eGFP bacterial solution, and the site injected with only GV3101-pGR107-g6634 bacterial solution did not show cell necrosis. The right injection area of ​​tobacco leaves: the Buffer+BAX injection site and the eGFP+BAX injection site both showed symptoms of leaf tissue dryness and necrosis, while the g6634+BAX injection site did not produce leaf tissue necrosis symptoms. These phenotypic symptoms indicate that BAX can successfully induce hypersensitive necrosis in tobacco cells; no cell necrosis occurred in the site where only GV3101-pGR107-g6634 bacterial solution was injected, indicating that the effector protein expressed by the g6634 gene cannot induce hypersensitive necrosis in tobacco; and no tissue necrosis occurred in the site where g6634+BAX was injected, indicating that the g6634 gene can effectively inhibit the programmed cell death (PCD) induced by BAX. Therefore, the effector protein expressed by the g6634 gene plays an important role in inhibiting the plant defense response.

[0124] Example 2. Subcellular localization of the effector protein of Tilletia tritici g6634

[0125] 1. Cloning of target gene

[0126] RNA of wheat light tartaria was extracted and reverse transcribed into cDNA according to the method in Example 1. Using wheat light tartaria cDNA as a template, PCR amplification was performed using primers g6634F2 and g6634R2 (SEQ ID NO: 7 and SEQ ID NO: 8). The reaction system is shown in Table 2, and the reaction program is: 95°C 5min; 95°C 1min, 57°C 1min, 72°C 2min, 35 cycles; 72°C 10min. After the reaction, 1.0% agarose gel electrophoresis (150V, 30min) was used to detect the PCR product, and a universal DNA purification and recovery kit (Tiangen Bio, catalog number: DP214-03) was used to recover the g6634 gene fragment according to the kit instructions.

[0127] 2. Enzyme digestion of vector

[0128] First, the E. coli containing the pBin-GFP vector was propagated, and then the plasmid was extracted using a rapid plasmid extraction kit (Tiangen Biotechnology, catalog number: DP105-03) according to the kit instructions. According to the vector and gene sequence, the pBin-GFP plasmid was double-digested using Kpn I enzyme (Thermo Fisher Scientific, USA, catalog number: FD0524) and BamH I enzyme (Thermo Fisher Scientific, USA, catalog number: FD0054). The enzyme digestion system is as follows:

[0129] Table 6 Kpn I and BamH I double restriction enzyme digestion reaction system

[0130]

[0131] The prepared enzyme digestion reaction system was placed in a 37°C metal bath for 1 hour. After the reaction, the digestion product was detected by 1.0% agarose gel electrophoresis, and the pBin-GFP linear vector was recovered using a universal DNA purification and recovery kit (Tiangen Biotechnology, catalog number: DP214-03).

[0132] 3. Ligation of target gene and vector

[0133] (1) Using Vazyme Ultra One Step Cloning Kit seamless ligation kit was used to connect the recovered pBin-GFP linear vector and the g6634 gene fragment.

[0134] (2) The recombinant connection system is as follows:

[0135] Table 7

[0136]

[0137] (3) The prepared system was gently mixed and placed in a 50°C metal bath for 10 min, and then placed on ice for 2 min.

[0138] (4) Immediately transfer 5-10 μL of the recombinant ligation product into 100 μL of molten E. coli Trans2-Blue competent cells and place on ice for 30 min.

[0139] (5) Heat shock the cells in a water bath at 42°C for 45 seconds and then place on ice for 2 to 3 minutes.

[0140] (6) Add 800 μL of antibiotic-free LB liquid culture medium into the clean bench.

[0141] (7) Shake the culture at 200 rpm at 37°C for 1 h. Use a sterile glass rod to evenly spread the bacterial solution on a solid LB plate containing 50 μg / mL kanamycin. Place the plate in an incubator at 37°C and incubate overnight.

[0142] (8) Pick 6 to 8 single clones and perform PCR verification using primers pBin107F and pBin107R (SEQ ID NO: 9 and SEQ ID NO: 10). The PCR reaction system is shown in Table 8.

[0143] Table 8

[0144]

[0145] The PCR reaction program was: 95°C for 5 min; 95°C for 1 min, 57°C for 1 min, 72°C for 2 min, 35 cycles; 72°C for 10 min. After the reaction, the amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing verification.

[0146] The positive colonies with correct sequencing were extracted with a rapid plasmid extraction kit (Tiangen Biotechnology, catalog number: DP105-03) according to the kit instructions to obtain the recombinant plasmid pBin-g6634.

[0147] 4. Transformation of Agrobacterium Competent Cells

[0148] The recombinant plasmid pBin-g6634 was used to transform Agrobacterium GV3101 (pJIC SA-Rep) competent cells to obtain the recombinant bacteria GV3101-pBin-g6634. The transformation method of Agrobacterium competent cells was the same as that in Example 1.

[0149] 5. Transient expression of genes in tobacco

[0150] (1) The recombinant bacterium GV3101-pBin-g6634 was placed in 10 mL of LB liquid medium containing 25 μg / mL rifampicin and 50 μg / mL kanamycin, and cultured in a shaking incubator at 28°C until the OD 600 =1.0~2.0.

[0151] (2) The bacterial solution was centrifuged at 5000 rpm for 5 min to collect the cells, and the cells were resuspended in an equal volume of tobacco injection buffer (10 mmol / L MES, 200 μmol / L AS, 10 mmol / L MgCl2); after repeating 3 times, the OD value of the bacterial solution was adjusted with tobacco injection buffer. 600nm To 0.5.

[0152] (3) Activate the bacterial solution in the dark at 28°C for 2 to 3 hours.

[0153] (4) Select Nicotiana benthamiana plants that are 4 weeks old and in good growth condition, and inject the activated GV3101-pBin-g6634 bacterial solution from the back of the tobacco plants using a 1 mL syringe without a needle. Continue culturing for 2 to 3 days after injection.

[0154] (5) Cut some tobacco leaf tissue, make slices with the back of the leaf facing up, and use a confocal laser microscope to observe the fluorescence expression in the leaf. This experiment used the EGFP tag, the excitation wavelength was 488nm, the image acquisition wavelength range was 515-540nm, and the 488nm excitation light was used to observe the EGFP expression and save the photos.

[0155] The results are as follows Figure 3 As shown, under a confocal laser microscope, green fluorescence representing the g6634 effector protein appears on the cell membrane. It is speculated that the effector protein acts on the cell membrane and exerts its effect on the cell.

Claims

1. Use of overexpression of wheat smut fungus effector protein gene in reducing tobacco leaf tissue necrosis caused by BAX protein, characterized in that: The gene inhibits programmed cell death of tobacco plants induced by BAX protein; the nucleotide sequence of the wheat smut fungus effector protein gene is shown in SEQ ID NO:1.

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