Detailed Description
The present invention will be described in detail with reference to the following examples, which are to be understood as merely illustrative and explanatory of the invention, and are not in any way limiting to the scope of the invention.
Biological material and carrier
The Tilletia foetida (TILLETIA LAEVIS K. Uhn, TFL) used in the examples below is described in document 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 fordetection of teliospores of Tilletia laevis Kühn.Scientific Reports.2019,9(1):17651. The public can obtain the strain from plant protection research institute of national academy of agricultural science.
Coli Trans2-Blue competent cells were purchased from Beijing full gold Biotechnology Co., ltd.
Agrobacterium GV3101 (pJIC SA-Rep) competent cells were purchased from Shanghai Biotechnology Inc.
The tobacco used in the following examples is burley (Nicotiana benthamiana), provided by the present laboratory.
The pGR vector used in the following examples was modified potato virus X vector pGR, which was presented by the institute of plant protection Chen Jie, national academy of agricultural sciences. Potato X virus vector pGR is a binary expression vector, contains a CaMV 35S promoter and a kanamycin resistance gene, and has a GenBank accession number of AY297842.1 and a full length 10436bp. The sequence between the Cla I and Sal I cloning sites of the modified potato X virus vector pGR was removed. Potato X virus vector pGR107,107 is commercially available.
The pBin-GFP vector is a commercially available vector.
Reagent and consumable
Universal DNA purification recovery kit and rapid plasmid miniprep kit are purchased from Beijing Tiangen Biochemical technology Co. 2 XPro TAQ MASTER Mix (dye plus) was purchased from Hunan Ai Kerui bioengineering Co. High fidelity enzyme 2×Flash Master Mix(Dye Plus)、Ultra One Step Cloning Kit seamless kit was purchased from Nanjinouzan Biotechnology Co., ltd. Green-chain diabodies were purchased from Gibco corporation under the trade designation 15140-122.
Solution formulation
(1) 0.25% NaClO 250. Mu.L NaClO, was fixed to 100mL with deionized water.
(2) 50 XTAE Buffer solution 242g Tris and 37.2g EDTA-Na2·2H2 O were dissolved thoroughly in 600mL deionized water, 57.1mL glacial acetic acid (anhydrous acetic acid, acetic acid) was added to the dissolved solution, the volume was set to 1L, and the pH was adjusted to 8.5 with NaOH.
(3) 2% Of a soil leaching liquid culture medium, wherein the used soil is sterilized in an autoclave at 60-100 ℃ for 30-60 min. Taking 75g of sterilized soil, filtering with 500mL of boiled distilled water under 8 layers of gauze, fixing the volume to 1000mL, and sterilizing at 120 ℃ for 20min. (the solid medium is added with 20g of agar)
(4) LB medium, weighing 3g of sodium chloride, 1.5g of yeast extract powder, 3g of tryptone, fixing the volume to 300mL, and sterilizing at 120 ℃ for 20min. (the solid culture medium is added with 4.5g of agar powder)
(5) Kanamycin stock solution (50 mg/mL) by weighing 2.5g kanamycin, placing into 50mL centrifuge tube, adding 40mL sterilized distilled water, mixing thoroughly, dissolving, fixing volume to 50mL, filtering with 0.22 μm filter membrane for sterilization, packaging small portions, and preserving at-20deg.C.
(6) Rifampicin stock solution (20 mg/mL) 0.2g of Rifampicin was weighed into a 50mL centrifuge tube, 10mL of DMSO (dimethyl sulfoxide) was added, filtered and sterilized, and then sub-packaged and stored at-20 ℃.
(7) Tobacco injection (Buffer) is prepared by taking 5mL of 1M MgCl2 stock solution, 5mL of 1M MES (2-morpholinoethanesulfonic acid) stock solution, and 0.5mL of 0.2M AS (acetosyringone) stock solution, adding sterile water to constant volume to 500mL, and mixing.
(8) The trypan blue staining solution comprises 10mL of lactic acid, 10mL of glycerol, 10mL of phenol, 10mg of trypan blue and 10mL of deionized water, wherein the trypan blue is completely dissolved by the deionized water, added into other solutions, uniformly mixed and stored at 4 ℃.
(9) Trypan blue decoloring solution, 250g of chloral hydrate, deionized water to 100mL, and shaking at a low speed of 37 ℃ or dissolving completely at 65 ℃.
PCR primer
TABLE 1
| Primer name | Primer sequence (5 '-3') |
| g6634F1 | CACCAGCTAGCATCGATTCCCGGGATGGCCCCGAGCCG(SEQ ID NO:3) |
| g6634R1 | AAGCTTATCGGCGGTCGACCCGGGTCACTTGTGCTGGTCGTCAAAG(SEQ ID NO:4) |
| pGR107F | ATAGCAGTCATTAGCACTTCCT(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) |
Main instrument
PCR apparatus, manufactured by Siemens technologies, inc. (Thermo FISHER SCIENTIFIC). Centrifuges, pipetting guns, manufactured by eppendrof life sciences, germany. 50mL large centrifuge, manufactured by sigma, germany. Electrophoresis apparatus, manufactured by bio-rad, U.S.A. Autoclave, manufactured by zealway company, U.S.A. GHB-202 constant temperature metal bath, produced by Hangzhou Bori technology Co., ltd. Thermostatic water bath, shanghai's river Biotechnology Co., ltd. 85-2 digital display constant temperature magnetic stirrer, manufactured by Rong instruments, inc. of Jintan, jiangsu province. High capacity full temperature oscillator, produced by su zhou pekine experiment equipment limited. Ultra clean bench, shanghai Zhi City analytical instruments manufacturing Co., ltd. Confocal laser microscope, zeiss 980.
Unless otherwise indicated, all reagents used in the examples below are conventional in the art and are commercially available or formulated according to conventional methods in the art and are of laboratory grade. Unless otherwise indicated, the experimental procedures and conditions used in the following examples are those conventional in the art and may be referred to the relevant experimental manuals, well-known literature or manufacturer's instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1 cloning and functional verification of Tilletia wheat midwifery Blackia G6634 Gene
1. Culture of Tilletia controversa Kuhn
(1) 5-6 Wheat Tilletia controversa Kuhn (TILLETIA LAEVIS K uhn, TFL) fungus gall is taken and placed into a 2mL centrifuge tube, and 2mL of sterilized distilled water is added and kept stand for 30min at room temperature;
(2) Peeling the seed coat with forceps to obtain small particles, filtering impurities with 150 mesh gauze, and centrifuging;
(3) Centrifuging at 6000rpm for 1min, pouring out supernatant, and adding 2mL of 0.25% NaClO for sterilization for 10min;
(4) Centrifuging at 6000rpm for 1min, removing supernatant, and adding sterilized distilled water;
(5) Repeating the steps (3) and (4) twice;
(6) Observing with an optical microscope through a blood cell counting plate, and adjusting the concentration of the wintergreen spores of the Tilletia graminea to 1X 106/mL after dilution treatment to obtain wintergreen spore suspension;
(7) To a 2% soil extract medium was added cyan-chain diabody (Gibco) in a 1% volume ratio, plates were poured, and each dish was coated with 220. Mu.L of winter spore suspension. The plates were placed in an artificial incubator at 5 ℃, 1Lx and 50% relative humidity for 45d or so under full light, then the germination state of the winter spores was observed under an optical microscope, and after the mycelium grew to the mycelium infection stage, they were collected for extraction of RNA of Tilletia controversa.
2. Extraction of Tilletia controversa Kuhn RNA and synthesis of cDNA
Extracting hypha RNA of Tilletia controversa Kuhn (TILLETIA LAEVIS K uhn, TFL) by using a Trizol method, wherein the method comprises the following specific steps:
(1) Collecting germinated wheat Tilletia controversa mycelia, placing in a 2mL spiral tube without RNase, rapidly placing in liquid nitrogen, and grinding the sample into powder by using a high-flux rapid sample preparation instrument;
(2) Adding 1mL (which can be appropriately increased or decreased according to the amount of the sample) of TRIzol reagent (Invitrogen) into the fully ground mycelium powder, gently shaking and uniformly mixing to fully crack the mycelium powder, and standing at room temperature for 5-10 min;
(3) Adding chloroform into the liquid which is cracked by adding the TRIzol reagent according to the proportion of 0.2mL chloroform into each 1mL TRIzol reagent, shaking vigorously for 15s, extracting, standing at room temperature for 3min, centrifuging at 12000rpm for 15min;
(4) After sufficient centrifugation, the supernatant is sucked into a new 1.5mL RNase-Free centrifuge tube, added with isopropyl alcohol with equal volume for sedimentation, and kept stand on ice for 10min, and centrifuged at 12000rpm for 15min after uniform mixing;
(5) Discarding the supernatant, adding 1mL of 75% ethanol (prepared from absolute ethanol and RNase-Free water), mixing, centrifuging at 12000rpm at 4deg.C for 5min, and removing residual liquid as much as possible;
(6) Repeating the previous operation, and then placing the centrifuge tube in an ultra-clean workbench to be dried for 5-10 min after the centrifuge tube is uncapped until the sediment becomes transparent;
(7) Adding a proper amount of RNase-Free water to dissolve RNA, and preserving in a refrigerator at-80 ℃ for later use.
The obtained total RNA of Tilletia controversa was reverse transcribed using an RNA reverse transcription kit (cat# RR 047A) from TaKaRa company according to the kit instructions to synthesize the first strand of cDNA as a gene cloning template. The obtained wheat light fishy black fungus cDNA is placed in a refrigerator at the temperature of-20 ℃ for preservation.
3. Cloning of the Gene of interest
Based on the wheat Tilletia controversa transcriptome data we found an effector gene designated g 6634. The nucleotide sequence of the Open Reading Frame (ORF) of the g6634 gene is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.
Open reading frame sequence (465 bp) of g6634 gene:
ATGATCGCTATTCCTCGTCTCTTCGCCGCCGTCGCCCTCCTCGCTGCCGTTGTGGTC
AGCGCTGCCCCGAGCCGCGACAAAACCTACCCTGCCTGTGACCCTCCCTCGAAAG
GTCCCGTCCACGGCAAATGCCACCAAATGAACATGAAGACCGACCAAGATCCCTT
CTACATCGCCCCTGGGCTCGGCGCGTGCGGCGTCACCTACAACGACAACGTGATG
GGTGCCTGCCTCAGCCCGGGTTGGATCAACTCCGGCTACTACAGCTCCTGCGGCCG
GAAGACCACCGTCACCAATCCTCGCAACGGCAAGTCGATCCACGTCGTCATCATTG
ACGCCTGTGTATCGGCTTCCTGCAACGACATCATGCTCACGAAGGCGGCCTTCCAG
GCCATCGGCGGTAACATGGCGTCTGGCCACGTCGACAACAAAGTCAACTGGTACTTTGACGACCAGCACAAGTGA(SEQ ID NO:1)。
Amino acid sequence (154 aa) of the protein encoded by the g6634 gene:
MIAIPRLFAAVALLAAVVVSAAPSRDKTYPACDPPSKGPVHGKCHQMNMKTDQDPFY
IAPGLGACGVTYNDNVMGACLSPGWINSGYYSSCGRKTTVTNPRNGKSIHVVIIDAC VSASCNDIMLTKAAFQAIGGNMASGHVDNKVNWYFDDQHK(SEQ ID NO:2).
The N-terminal signal peptide sequence of the protein encoded by the g6634 gene is predicted by using online analysis software SignalP 5.0, the gene sequence encoding the signal peptide is removed, and then primers g6634F1 and g6634R1 (SEQ ID NO:3 and SEQ ID NO: 4) for gene cloning are designed and are entrusted to primer synthesis by Beijing qing department biotechnology Co. The target gene fragment is amplified by PCR with the cDNA of the wheat Tilletia controversa Kuhn synthesized by reverse transcription as a template and the primers g6634F1 and g6634R 1. The PCR reaction system is shown in Table 2.
TABLE 2 PCR reaction System for target Gene
The PCR reaction procedure was 95℃for 5min, 95℃for 1min,57℃for 1min,72℃for 2min,35 cycles, and 72℃for 10min. PCR products were detected by 1.0% agarose gel electrophoresis (150V, 30 min) and the results were shown in FIG. 1, with the correct size. The target band was cut, and recovered using a universal DNA purification recovery kit (Tiangen organism, cat# DP 214-03) according to the kit instructions to obtain the g6634 target gene fragment, which was stored in a-20℃refrigerator.
4. Cleavage of the vector
10. Mu.L of E.coli bacterial liquid containing pGR.sup.107 vector was placed in 10mL of LB liquid medium containing 50. Mu.g/mL kanamycin, cultured at constant temperature of 200rpm for 8-10 hours on a 37℃shaker, and then subjected to plasmid extraction using a rapid plasmid miniprep kit (Tiangen organism, cat. No. DP 105-03) according to the kit instructions. Based on the vector and gene sequence, the pGR plasmid was subjected to double cleavage using Bsu15I enzyme (product number: FD0144, siemens technologies, inc. of U.S.A.) and SalI enzyme (product number: FD0644, siemens technologies, inc.) and the cleavage reaction system is shown in Table 3.
Table 3Bsu15I and Sal I double cleavage reaction System
The prepared cleavage reaction system was placed in a 37℃metal bath for 1h. After the reaction, the digested product was subjected to 1.0% agarose gel electrophoresis. The target band was excised with a sterile razor blade, recovered using a universal DNA purification recovery kit (Tiangen organism, DP 214-03) according to the kit instructions to give pGR linear vectors, which were stored in a-20℃refrigerator.
5. Ligation of the Gene of interest to the vector
(1) Using company VazymeUltra One Step Cloning Kit A seamless ligation kit was used to ligate the purified pGR107 linear vector with the g6634 target gene fragment.
(2) The recombinant ligation system is shown in the following table:
TABLE 4 ligation reaction System of plasmid pGR107,107 and target Gene
(3) The prepared system was gently mixed and placed in a 50 ℃ metal bath for 10min, and then placed on ice for 2min.
(4) And (3) immediately transferring 5-10 mu L of recombinant connection product into 100 mu L of E.coli Trans2-Blue competent cells in a molten state, and standing on ice for 30min.
(5) Heat shock is carried out for 45s in a water bath at the temperature of 42 ℃, and then placing the mixture on ice for 2-3 min.
(6) 800. Mu.L of LB liquid medium without antibiotics was added to the ultra clean bench.
(7) Shaking at 200rpm at 37℃for 1 hour, and uniformly coating the bacterial liquid on an LB solid plate containing 50. Mu.g/mL kanamycin by using a sterile glass rod, and placing the plate in a 37℃incubator for inversion culture overnight.
(8) 6-8 Monoclone are selected for bacterial liquid PCR verification, and the primers are pGR F and pGR R (SEQ ID NO:5 and SEQ ID NO: 6). The PCR reaction system is as follows:
TABLE 5 bacterial liquid PCR reaction system
The PCR reaction procedure was 95℃for 5min, 95℃for 1min,57℃for 1min,72℃for 2min,35 cycles, and 72℃for 10min. After the reaction, the amplified product was sent to Beijing qingke Biotechnology Co.Ltd for sequencing verification.
The positive colonies with correct sequencing were subjected to plasmid extraction using a rapid plasmid miniprep kit (Tiangen organism, cat# DP 105-03) to obtain recombinant plasmids pGR-g 6634.
6. Transformation of Agrobacterium competent cells
The competent cells of Agrobacterium GV3101 (pJIC SA-Rep) were transformed with recombinant plasmid pGR-g 6634 to give recombinant strain GV3101-pGR107-g6634. The agrobacterium transformation method is as follows:
(1) The agro-competent cells stored at-80℃were removed, thawed at room temperature or palm for a period of time, and inserted into ice while in the ice-water mixed state.
(2) And adding the plasmid into the competent cells, adding 0.01-1 mug of plasmid into 100 mug of competent cells, and lightly stirring the bottom of the tube by hand after adding the plasmid and mixing the mixture uniformly.
(3) Sequentially placing on ice for 5min, liquid nitrogen for 5min, and water bath at 37deg.C for 5min and ice bath for 5min.
(4) Adding 700 mu L of LB liquid medium without antibiotics into competent cells, and carrying out shaking culture for 2-3 h at a shaking table of 200rpm at a temperature of 28 ℃;
(5) The cultured bacterial liquid was centrifuged at 6000rpm for 1min to collect bacterial cells, about 100. Mu.L of the supernatant was left to resuspend the bacterial cells, and the bacterial liquid was coated on LB solid plates containing 25. Mu.g/mL of rifampicin and 50. Mu.g/mL of kanamycin with a sterile glass rod.
(6) The plate is inverted and cultured in a28 ℃ incubator for 2-3 days. When single colony grows out on the flat plate, 8 single clones are selected for bacterial liquid PCR verification, and the PCR reaction system and the reaction program are the same as those of the bacterial liquid PCR verification in the step 5.
(7) The colony with correct PCR verification was picked up into 10mL of LB liquid medium containing 25. Mu.g/mL rifampicin and 50. Mu.g/mL kanamycin, cultured at 28℃and 200rpm for 2d, and the bacterial liquid was preserved for later use.
7. Transient expression of genes on tobacco
The BAX gene (GenBank accession number: L22472.1) is one of the members of the Bcl-2 family of apoptosis genes, and the expressed protein has the effect of inducing apoptosis, similar to the allergic necrosis reaction mechanism in pathogenic bacteria-plant interaction. The eGFP gene (GenBank accession number: EU 746493.1) is an enhanced green fluorescent protein gene that autofluoresces under blue excitation. The expression vector pGR-BAX of the BAX gene is prepared in the early stage of the laboratory and is introduced into agrobacterium GV3101 (pJIC SA-Rep) to obtain recombinant bacteria GV3101-pGR107-BAX, the expression vector pGR-eGFP of the eGFP gene is also prepared and is introduced into agrobacterium GV3101 (pJIC SA-Rep) to obtain recombinant bacteria GV3101-pGR107-eGFP. pGR 107A construction method of the BAX comprises the steps of carrying out double digestion on pGR vector and BAX gene fragment by Sal I and Cla I restriction enzymes respectively, and then inserting the BAX gene fragment into pGR vector by ligation reaction. pGR 107A construction method of the eGFP comprises the steps of carrying out double digestion on a pGR vector and an eGFP gene fragment by using Sal I and Cla I restriction enzymes respectively, and then inserting the eGFP gene fragment into a pGR vector through ligation reaction.
The method for transiently expressing genes on tobacco comprises the following steps:
(1) Recombinant bacteria GV 3101-pGR-g 6634, GV 3101-pGR-BAX and GV 3101-pGR-eGFP were inoculated into 10mL of LB liquid medium containing 25. Mu.g/mL rifampicin and 50. Mu.g/mL kanamycin, respectively, and cultured by shaking at 28℃and 200rpm for 2d.
(2) The cultured bacterial cells were collected by centrifugation at 5000rpm for 10min, and the bacterial cells were resuspended in Buffer (tobacco injection) (10 mmol/L MES, 200. Mu. Mol/L AS,10mmol/L MgCl2).
(3) Repeating the steps, repeatedly washing the thalli for 3 times by using a Buffer (tobacco injection), placing the thalli in a dark environment of a 28 ℃ incubator for activation for 3 hours, and adjusting the thalli to OD600nm = 0.3-0.5 by using the Buffer (tobacco injection).
(4) And sucking a proper amount of bacterial liquid by using a l mL sterile injector without a needle, selecting tobacco leaves with good growth state for 4-6 weeks, and injecting the bacterial liquid on the back surfaces of the tobacco leaves by using an infiltration method. GV 3101-pGR-g 6634 bacterial liquid, GV 3101-pGR-eGFP bacterial liquid and Buffer (tobacco injection) are respectively injected from top to bottom on the left side of tobacco leaves by using a 6-zone injection method according to the injection mode shown in FIG. 2A, and the injection range is marked. There were 6 injection areas per tobacco leaf, with Buffer in the lower left corner as a blank. 3-5 leaves are injected into each plant of tobacco. Each sample was repeated 3 times.
(5) After 24h inoculation, the right side of the tobacco leaf (the right side of the last marked area) was injected with equal volumes of GV 3101-pGR-g 6634 bacterial solution and GV 3101-pGR-BAX bacterial solution (g6634+BAX), GV3101-pGR107-eGFP bacterial solution and GV 3101-pGR-BAX bacterial solution (eGFP+BAX), buffer and GV3101-pGR107-BAX bacterial solution (buffer+BAX) respectively from top to bottom according to the injection method shown in FIG. 2A. And placing the tobacco in an incubator at 25 ℃ for continuous culture, observing the necrosis condition of the tobacco leaf injection area after 3-8 d, counting data and photographing.
(6) Mixing trypan blue staining solution and absolute ethyl alcohol according to the volume ratio of 1:1, putting tobacco leaves showing symptoms into the mixed staining solution, boiling for 5min in boiling water, putting the leaves into trypan blue decolorizing solution for decolorizing after dyeing overnight, and photographing after decolorizing is completed.
The results are shown in FIG. 2B (symptomatic diagram of tobacco leaf after gene transient expression) and FIG. 2C (symptomatic diagram of tobacco leaf after trypan blue staining and decoloring after gene transient expression). The left injection area of the tobacco leaf blade is a site (blank control) only injected with Buffer, a site only injected with GV3101-pGR with 107-eGFP bacterial liquid and a site only injected with GV3101-pGR with 107-g6634 bacterial liquid, and no cell necrosis occurs. The right injection area of the tobacco leaf, namely the buffer+BAX injection site and the eGFP+BAX injection site, show the symptoms of dry and necrotic leaf tissue, and the g6634+BAX injection site does not produce the symptoms of necrotic leaf tissue. These phenotypic symptoms indicate that BAX successfully caused allergic necrosis of tobacco cells, that no cell necrosis was generated at the site of GV 3101-pGR-g 6634 bacterial fluid injection alone, indicating that the effector protein expressed by the g6634 gene could not induce allergic necrosis of tobacco, and that no tissue necrosis was generated at the site of g6634+BAX injection, indicating that the g6634 gene could effectively inhibit apoptosis (PCD) of plant cells induced by BAX. Thus, the effector protein expressed by the g6634 gene plays an important role in inhibiting plant defense response.
Example 2 subcellular localization of Tilletia wheat G6634 effector protein
1. Cloning of the Gene of interest
RNA of Tilletia controversa was extracted and reverse transcribed into cDNA according to the method of example 1. PCR amplification was performed using the wheat Tilletia controversa Kuhn cDNA as a template, using primers g6634F2 and g6634R2 (SEQ ID NO:7 and SEQ ID NO: 8), the reaction system being as shown in Table 2, and the reaction procedure being 95℃for 5min, 95℃for 1min,57℃for 1min,72℃for 2min,35 cycles, and 72℃for 10min. After the reaction, the PCR product was detected by 1.0% agarose gel electrophoresis (150V, 30 min), and the g6634 gene fragment was recovered using a universal DNA purification recovery kit (Tiangen, cat# DP 214-03) according to the kit instructions.
2. Cleavage of the vector
The colibacillus liquid containing pBin-GFP carrier is first propagated, and then plasmid extracted with fast plasmid extracting kit (Tiangen organism, product number: DP 105-03) according to the kit instruction. Based on the vector and gene sequence, the pBin-GFP plasmid was digested with Kpn I enzyme (product number: FD0524, siemens technologies, USA) and BamH I enzyme (product number: FD0054, siemens technologies, USA) in the following cleavage system:
TABLE 6Kpn I and BamH I double cleavage reaction System
The prepared enzyme digestion reaction system is placed in a 37 ℃ metal bath for 1h. After the reaction, the digested product was subjected to 1.0% agarose gel electrophoresis, and a pBin-GFP linear vector was recovered using a universal DNA purification recovery kit (Tiangen organism, cat# DP 214-03).
3. Ligation of the Gene of interest to the vector
(1) Using company VazymeUltra One Step Cloning Kit seamless connection kit is used for connecting the recovered pBin-GFP linear vector with the g6634 gene fragment.
(2) The recombinant connection system is as follows:
TABLE 7
(3) The prepared system was gently mixed and placed in a 50 ℃ metal bath for 10min, and then placed on ice for 2min.
(4) And (3) immediately transferring 5-10 mu L of recombinant connection product into 100 mu L of E.coli Trans2-Blue competent cells in a molten state, and standing on ice for 30min.
(5) Heat shock is carried out for 45s in a water bath at the temperature of 42 ℃, and then placing the mixture on ice for 2-3 min.
(6) 800. Mu.L of LB liquid medium without antibiotics was added to the ultra clean bench.
(7) Shaking at 200rpm at 37℃for 1 hour, and uniformly coating the bacterial liquid on an LB solid plate containing 50. Mu.g/mL kanamycin by using a sterile glass rod, and placing the plate in a 37℃incubator for inversion culture overnight.
(8) 6-8 Monoclonals are selected, and bacterial liquid PCR verification is carried out by using primers pBin107F and pBin107R (SEQ ID NO:9 and SEQ ID NO: 10). The PCR reaction system is shown in Table 8.
TABLE 8
The PCR reaction procedure was 95℃for 5min, 95℃for 1min,57℃for 1min,72℃for 2min,35 cycles, and 72℃for 10min. After the reaction, the amplified product was sent to Beijing qingke Biotechnology Co.Ltd for sequencing verification.
The positive colony with correct sequence is extracted by a rapid plasmid small extraction kit (Tiangen organism, product number: DP 105-03) according to the instruction of the kit to obtain recombinant plasmid pBin-g6634.
4. Transformation of Agrobacterium competent cells
The recombinant plasmid pBin-g6634 is used for transforming competent cells of agrobacterium GV3101 (pJIC SA-Rep) to obtain recombinant bacteria GV3101-pBin-g6634. The transformation method of Agrobacterium competent cells was the same as in example 1.
5. Transient expression of genes on tobacco
(1) Recombinant bacteria GV3101-pBin-g6634 are placed in 10mL of LB liquid medium containing 25 mug/mL of rifampicin and 50 mug/mL of kanamycin, and shake culture is carried out at 28 ℃ to OD600 =1.0-2.0.
(2) The bacterial liquid was centrifuged at 5000rpm for 5min to collect the bacterial cells, and the bacterial cells were resuspended with an equal volume of tobacco injection Buffer (10 mmol/L MES, 200. Mu. Mol/L AS,10mmol/L MgCl2), and after 3 replicates, the bacterial liquid OD600nm was adjusted to 0.5 with tobacco injection.
(3) Placing the bacterial liquid at 28℃ activating for 2-3 hours in the dark.
(4) Selecting Nicotiana benthamiana with 4 weeks of seedling culturing age and good growth state, injecting the activated GV3101-pBin-g6634 bacterial liquid from the back of the tobacco by using a 1mL syringe without a needle, and continuously culturing for 2-3 days after injection.
(5) And cutting part of tobacco leaf tissue, making a slice with the leaf back face upwards, and observing the fluorescent expression condition in the leaf by using a confocal laser microscope. The EGFP label is used in the experiment, the excitation wavelength is 488nm, the acquired image wavelength range is 515-540nm, and the EGFP expression condition is observed by using 488nm excitation light and the photo is stored.
As a result, as shown in FIG. 3, green fluorescence representing g6634 effector protein appears on the cell membrane under confocal laser microscopy. It is presumed that the effector protein acts on the cell membrane and acts on the cell.