Method for screening transgenic plant and bivalent plant expression vector used in the method
A technology of transgenic plants and expression vectors, applied in the field of bivalent plant expression vectors, can solve the problems of single-action safety of screening markers, and achieve the effect of convenient screening
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Embodiment 1
[0022] Example 1. Construction of insect-resistant and glyphosate-resistant plant bivalent expression vectors
[0023] According to the HindIII+SalI double enzyme digestion reaction and electrophoresis system shown in Table 1.1, the plasmid pBtS1m (No. 01129519.8 patent application publication) containing the BtS1m gene fragment was cut with HindIII+SalI, and after agarose gel electrophoresis, the E.Z.N.A GelExtraction kit was used to (Omega, the same below) recovered the band at about 2.7kb in size to obtain the 2E-35sp+BtS1m fragment. Cut the aroAM12PCR2.1 plasmid with HindIII+XhoI (the PCR2.1 plasmid containing the aroAM12 gene, see figure 1 ), reclaim the band at 4100bp to obtain a fragment containing the Nos termination sequence, then use T4DNA ligase to connect the two parts of DNA fragments of 2.7kb and 4100bp recovered (see Table 1.2 for the connection reaction system) to obtain the fragment containing the BtS1m gene pBtPCR2.1 plasmid (see figure 1 ). Cut pCAMBIA130...
Embodiment 2
[0038] Embodiment 2. Obtaining of Escherichia coli transformants
[0039] Preparation of E.coli XL-1-blue competent cells: ①Put a single colony of XL1-blue into 5mL LB liquid medium (see Table 2.1) without antibiotics, shake overnight at 37°C; ②Press 1% The amount of (v / v) was transferred to fresh LB liquid medium and shaken to OD at 37°C 600 =0.3; ③Add 50-100mL LB liquid culture medium (see Table 2.1 for LB preparation) into two sterile centrifuge tubes, place on ice for 30 minutes; ④Centrifuge at 4°C, 4000rpm (rev / min) for 10 minutes , remove the supernatant, and add 10 mL of pre-cooled 0.1% CaCl to each centrifuge tube 2 Resuspend the cells in the solution, and put them in an ice bath for 30 minutes. ⑤ Centrifuge at 4°C and 4000rpm for 10 minutes, remove the supernatant, and suspend the cells in 2 mL of pre-cooled 0.1% CaCl 2 Add 300 μL of sterile glycerol to the solution, mix well, add 100 μL of XL1-blue competent cells to each sterile centrifuge tube, and store at -70°C...
Embodiment 3
[0048] Example 3. Transformation of Agrobacterium tumefaciens
[0049] Preparation of Agrobacterium tumefaciens LBA4404 competent cells: ① Pick LBA4404 colonies from the YEB solid medium (see Table 3.1) plate containing streptomycin (str) and rifampicin (Rif), inoculate into 5ml containing the corresponding antibiotic In the YEB liquid medium (according to Table 3.1, without adding agar), shake overnight at 200 rpm at 28°C. ② Dilute to 50ml with YEB liquid medium, then culture at 28°C and 200rpm for 6-12 hours until 0D 600 The value is around 0.6. ③ Place on ice for 5 minutes, then centrifuge at 4°C, 2000rpm for 7 minutes. ④Take 1ml of ice-cold CaCl with a concentration of 20mmol / L 2 Resuspend the precipitate in the solution and mix well. ⑤ Divide into 10 portions, 100 μl each, and store in a -70°C refrigerator for later use.
[0050] Add 5 μl of the plasmid extracted in Example 2 to 100 μl of Bacillus LBA4404 competent cells stored in a -70°C refrigerator, bathe in water...
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