A kind of genetic transformation method of poplar alba
A genetic transformation method, the technology of Populus alba, which is applied in the field of genetic transformation of Poplar alba, can solve the problems of shortening the transformation time and the inapplicability of genetic transformation of Poplar alba, and achieve the effect of shortening the transformation time and improving the transformation efficiency
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[0037] In the present invention, the explants are soaked in the infecting bacteria solution, and the soaking time is preferably 10-15 minutes. The preparation method of the infection bacterium liquid described in the present invention preferably comprises the following steps: A. Agrobacterium carrying the recombinant plasmid is streaked on the YEB plate medium to obtain a single colony; the YEB plate medium contains 50 mg / L of kanamycin and 50mg / L of rifampicin;
[0038]B. Inoculate the single colony according to the ratio of 1 single colony: 5mLYEB liquid medium, and culture with shaking until OD 600 0.7~0.9, to get the expanded strain; the YEB liquid culture medium contains the kanamycin of 50mg / L and the rifampicin of 50mg / L;
[0039] C. Inoculate the expanded strains in YEB anti-antibiotic liquid medium and culture with shaking until OD 600 0.7 to 0.9 to obtain an activated bacterial liquid; the volume ratio of the expanded strain to the YEB non-resistant liquid medium ...
Embodiment 1
[0061] Transferring the GUS gene into Populus alba:
[0062] 1. Connect the GUS gene (GenBank: MG687280.1) to the ΔpCAMBIA1302 vector. The ΔpCAMBIA1302 vector is a new binary expression vector transformed from the traditional pCAMBIA1302. It mainly uses the CaMV35S promoter (CaMV35S-P) (835bp) and multiple The cloning site (42bp) replaced the constitutive CaMV35S promoter (CaMV35S-P) (538bp) and the reporter gene GFP on the original T-DNA segment of the pCAMBIA1302 vector. Transfer the recombinant gene into DH10B Escherichia coli competent. After the sequencing was correct, the plasmid was extracted and transformed into EHA105 Agrobacterium competent by electric shock transformation.
[0063] 2. Streak the Agrobacterium (EHA105) carrying the recombinant plasmid on a YEB (50mg / L Kan+50mg / L Rif) plate, culture at 28°C, and a single colony appears after 2 days.
[0064] 3. Pick a single colony for colony PCR, and inoculate it in 1mL YEB (50mg / L Kan+50mg / LRif) liquid medium, and...
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