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This experiment introduces a method for constructing plant expression vectors.
1. Construction of plant justice expression vectors
(1) Double digestion of pTriplEx2-GhMT3a and plant expression vector pBI121 with SmaI and SacI.
(2) Perform agarose gel electrophoresis to recover the GhMT3a fragment and the pBI121 vector.
(3) Ligation: the ligation system was:
| 10×ligation Buffer | 2.5 μl |
| DNA fragment | 500 ng |
| pBI121 vector | 500 ng |
| T4 DNA ligase | 1 μl |
| ddH2O | 10 μl |
Mix and ligate overnight at 4-16°C.
(4) Transformation: 5ul of ligation product was taken to transform E.coli DH5α receptor cells and the bacterial solution was coated on solid LB plates containing kanamycin.
(5) Recombinant screening: PCR and enzymatic identification of the grown colonies.
2. Preparation and transformation of Agrobacterium tumefaciens receptor cells
Preparation of receptor cells:
(1) Single colonies of Agrobacterium tumefaciens GV3101 were picked and inoculated in 5m1 LB liquid medium at 28°C and incubated overnight.
(2) Take 2 ml of culture into liquid LB and continue incubation until OD600 is about 0.5.
(3) The cultures were centrifuged for 5 min at 4°C, 5000 rpm in an ice bath for 30 min.
(4) Discard the supernatant, 10 ml of 0.1 M cold NaCl to suspend the bacterium, and centrifuge at 4°C, 5000 rpm, for 5 min.
(5) Discard the supernatant, 1 ml of 20 mM cold CaCl2 suspension, dispense into 200 ul/tube, freeze in liquid nitrogen and store at -80°C.
Transformation of Agrobacterium tumefaciens by freeze-thaw method:
(1) Melt the receptor cells on ice, add 1-2 ul of recombinant plasmid DNA, ice bath for 45 min, freeze in liquid nitrogen for 1 min and then water bath at 37°C for 5 min.
(2) Addition of 1 ml of LB without antibiotics, 28°C, 200 rpm, 3 h.
(3) 12000 rpm, centrifugation for 1 min to concentrate the bacterial broth, 100 ul back to lysate the bacteria.
(4) The bacterial solution was applied to solid LB medium containing 50 mg/l kanamycin at 28°C and incubated for 2-3 d.
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