[0012] Example 1: VIGS treatment of tomato
[0013] The formula of the LB solid medium used below is: 10g/L Tryptone, 5g/L Yeast extract, 10g/L Sodium Chloride (NaCl), 15-20g/L agar powder, use Adjust the pH to 7.4 with 10M NaOH, with the remainder being water; sterilize at 121°C for 20 minutes, and when the temperature drops to about 55°C (not hot), add the final concentration of 30mg/L Kanamycin (Kanamycin, Kan), or Add the final concentration of 30mg/L Kanamycin (Kanamycin, Kan), 50mg/L Rifampicin (Rifampicin, Rif), 50mg/L Gentamicin (Gentamicin, Gen), pour the plate and store for later use; LB liquid The medium formula is: 10g/L Tryptone, 5g/L Yeast extract, 10g/L Sodium Chloride (NaCl), adjust the pH to 7.4 with 10M NaOH, and the balance is water; Sterilize at 121°C for 20min. After the temperature drops to about 55°C (not hot) or after cooling, add the final concentration of 30mg/L Kanamycin (Kanamycin, Kan), or add the final concentration of 30mg/L Kanamycin (Kanamycin, Kan), 50 mg/L rifampicin (Rifampicin, Rif), 50 mg/L gentamicin (Gentamicin, Gen).
[0014] The specific steps of the embodiment are described below:
[0015] 1) Tomato RNA extraction and cDNA acquisition
[0016] For the tomato RNA extraction method, please refer to the instructions of Huayueyang Biotech Ultrafast Plant RNA Extraction Kit. The specific steps are as follows: Take 0.1g of tomato leaves ground with liquid nitrogen in a 1.5mL centrifuge tube, add 1mL of cell lysate, and mix thoroughly; add 300μL of protein-removing solution and 200μL of chloroform, cover the tube, and shake vigorously to mix Homogenize for 30s; centrifuge at 12,000×g at room temperature for 10 minutes, transfer the supernatant (not more than 700μL) to a 1.5mL RNase-free centrifuge tube; add an equal volume of rinsing solution, invert and mix thoroughly, and mix the mixture (not more than 700μL) Transfer to a centrifugal adsorption column, centrifuge at 12,000×g at room temperature for 1 min, discard the penetrating solution; add 500 μL of wash column, at room temperature 12,000×g, centrifuge for 1 min, discard the penetrating solution, and repeat. Centrifuge at 12,000×g for 1 min at room temperature to remove the remaining liquid; transfer the centrifugal adsorption column to an RNase-free centrifuge tube, add 50 μL of RNA eluate, and leave it at room temperature for 5 min; centrifuge at 12,000×g at room temperature for 1 min, which will contain tomato RNA The penetration liquid is stored in a refrigerator at -80°C.
[0017] Reverse transcription of tomato RNA into cDNA using PrimeScript from Takara TM RT reagent Kitwith gDNA Eraser (Perfect Real Time) kit. Take out -80°C frozen tomato RNA, after thawing, add DNase according to the instructions, digest the DNA at 42°C for 2min, then add reverse transcription reagent, 37°C for 15min, 85°C for 5sec; get reverse transcribed cDNA.
[0018] 2)△ 8 -Cloning of sphingomyelin dehydrogenase gene (SlSLD)
[0019] Using bioinformatics analysis method, search tomato in tomato genome database△ 8 -Sphingomyelin dehydrogenase gene (SlSLD), to get 1 candidate sequence. According to the sequence information, the primer sequence for amplifying the full-length sequence of the SlSLD gene was designed, the upstream primer SlSLDF: 5'-GGATCCCAGGGTAAGGTGTATGATGT-3' and the downstream primer SlSLDR: 5'-TCTAGAGCAATTCCCAGTAAGGAC-3'.
[0020] Refer to the instructions for the reaction system. For PCR amplification, use Takara's PrimeSTAR Max Premix (2×) 10 μL, 10 μM upstream primer SlSLDF and 10 μM downstream primer SlSLDR each 0.5 μL, reverse transcribed cDNA template 0.1 μL, add double distilled water to make up 20 μL. The PCR amplification program is 98°C 30sec, 1 cycle; 98°C 15sec, 60°C 15sec, 72°C 30sec, 35 cycles; 72°C 10min, 1 cycle. From this amplification, △ 8 -The full-length sequence of the sphingomyelin dehydrogenase gene (SlSLD1). The full-length sequence was digested with restriction enzymes BamH I and Xho I (37℃ digestion reaction for 2h), and then connected to the expression vector pTRV2 vector (37℃ Restriction enzymes BamH I and Xho I were digested for 2h) to obtain the recombinant plasmid pTRV2-SlSLD, which was sent to the company for sequencing. △ 8 -The nucleic acid sequence of sphingomyelin dehydrogenase gene (SlSLD) is shown in SEQ ID NO. 1, which encodes △ 8 -The amino acid sequence of the sphingomyelin dehydrogenase gene is shown in SEQ ID NO.2. After confirming that the sequence is correct, the recombinant plasmid pTRV2-SlSLD was transformed into E. coli DH5α by heat shock method (heat shock at 42°C for 90sec), and cultured at 37°C in LB solid containing 30mg/L Kanamycin (Kan) Cultured on the base for 1 day, pick the positive single clone in 1mL of 30mg/L Kanamycin (Kanamycin, Kan) LB liquid medium, incubate at 200 rpm for 1 day at 37℃, to OD 600 The value is about 1.0, stored in 15% glycerin, and placed in the refrigerator at -80°C for later use.
[0021] 3) Agrobacterium-mediated△ 8 -VIGS of sphingomyelin dehydrogenase gene (SlSLD)
[0022] Pick the strains stored in 2), place them in 5mL LB liquid medium (containing 30mg/L Kanamycin (Kanamycin, Kan)), incubate at 200 rpm at 37°C for 1 day, To OD 600 The value is about 1.2, and then the recombinant plasmid pTRV2-SlSLD is extracted with a small plasmid kit. Take 1 μL of pTRV1 plasmid and pTRV2-SlSLD plasmid and add 30 μL of GV3101 competent cells that have been thawed on ice. The competent cells are transferred to a 2mm motor cup and transformed by motor. The specific parameters are as follows: select A. tumefaciens under Bacterial under Pre-Set Protocols, voltage 2400V, capacitance 25μF, resistance 200Ω, electric shock cup Cuvette is 2mm . After the electric shock, add non-resistant LB liquid medium, mix upside down, transfer to a 1.5mL centrifuge tube, incubate at 28°C at 200 rpm for 2h, and then add the bacteria to 30mg/L kanamycin (Kanamycin, Kan), 50mg/L rifampicin (Rifampicin, Rif), 50mg/L gentamicin (Gentamicin, Gen) on LB solid medium for 2-3 days.
[0023] Pick a positive single clone in 1mL of LB liquid medium containing 30mg/L Kanamycin (Kanamycin, Kan), 50mg/L Rifampicin (Rifampicin, Rif), 50mg/L Gentamicin (Gen) Medium, cultured at 200 rpm for 1 day at 28°C, to OD 600 The value is around 1.0. Centrifuge at 4000×g for 15 minutes, collect the precipitate, add 30 mg/L kanamycin (Kanamycin, Kan), 50 mg/L rifampicin (Rifampicin, Rif), 50 mg/L gentamicin (Gentamicin, Gen ) LB liquid medium to make the OD of pTRV1/GV3101 bacterial solution 600 Value is 0.4, the OD of pTRV2-SlSLD/GV3101 bacterial solution 600 The value is 0.4. Then mix the two bacterial liquids at a ratio of 1:1, and let stand at room temperature for 3 to 6 hours. The tomato seedlings that had just grown the first true leaf were infected by the method of vacuum injection, and pTRV2/GV3101 was used as the control, and the infection was carried out at the same time. The infected seedlings were cultured for 30 days under the conditions of 21℃, 16:8 (day: night). Fluorescence quantitative PCR analysis was used to identify the degree of gene suppression. The specific results are shown in Table 1.
[0024] Table 1 Expression of SlSLD gene in tomato plants after VIGS
[0025] Plant SlSLD gene expression *
[0026] Note: *The expression level of the control gene is 1.
