Agrobacterium tumefaciens-mediated phellinus igniarius genetic transformation method
A genetic transformation method, the technology of Phylophorus spp., applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., can solve the problem of low number of wild fruiting bodies
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Embodiment 1
[0044] Sensitivity detection of Phylonotus to hygromycin: Different strains have different sensitivity to hygromycin. In order to screen transformants, it is necessary to screen the concentration of hygromycin. The screening method is as follows: take modified Martin agar The hyphae of Phylophorus spp. growing vigorously on the slant were inoculated in the modified Martin's medium, cultured at 28°C and 180 rpm. After 10 days, wash the cultured mycelium with sterile water, absorb the surface moisture with sterilized filter paper, add 1 mL of enzyme solution for every 300 mg of wet mycelia, and rotate at 100 rpm at 30°C Enzymolysis was performed by shaking in a water bath for 3 hours. The enzymatic solution was filtered with a sterilized G3 sand core funnel, centrifuged at 3400 rpm for 10 min to remove the supernatant, and washed twice with an osmotic pressure stabilizer to obtain purified white protoplasts. Dilute the protoplasts with an osmotic pressure stabilizer to form a s...
Embodiment 2
[0047] 1. Preparation of Protoplasts from Phylophorus spp.
[0048] Take the mycelia of Phylocarpus hyphae growing vigorously on the slant of modified Martin agar, inoculate it in the modified Martin liquid medium, culture at 28°C for 10 days, then centrifuge at 5000 rpm for 10 minutes, remove the supernatant, and use 0.5mol The mannitol solution of / L washes 3 times, obtains the fresh hyphae of Phylophora spp.;
[0049] Take 1 gram of wet mycelia of Phyllostachys spp., add 3mL of lytic enzyme solution, and enzymatically hydrolyze at 35°C for 2 hours. The enzymatic solution was filtered with a sterilized G3 sand core funnel, centrifuged at 3000 rpm for 10 minutes, the supernatant was removed, and washed 2 times with 0.5mol / L mannitol to obtain the Purified Phylophorus protoplasts.
[0050] 2. Construction of plasmid pCH-hph
[0051] Based on the pCAMBIA1300 vector, the hygromycin resistance gene and the 35S promoter sequence of the cauliflower mosaic virus on the pCAMBIA1300...
Embodiment 3
[0069] Take the mycelia of Phyloporus spp. growing vigorously on the slant of modified Martin agar, inoculate it in the modified Martin liquid medium, cultivate it at 27°C for 12 days, then centrifuge it at 4000 rpm for 10 minutes, remove the supernatant, and use 0.5mol The mannitol solution of / L washes 3 times, obtains the fresh hyphae of Phylophora spp.;
[0070] Take 2 grams of wet mycelia of Phylophorus spp., add 8 mL of lytic enzyme solution, and enzymatically hydrolyze at 30°C for 2.5 hours. The enzymolysis solution is filtered with a sterilized G3 sand core funnel, centrifuged at 4000 rpm for 10 minutes, the supernatant is removed, and washed 2 times with 0.5mol / L mannitol to obtain the Purified Phylophorus protoplasts.
[0071] Take 100 μL of refined and purified Ph. 8 per mL) were mixed with 100 μL of the above-prepared AGL-1. Spread on solid induction medium, culture at 30°C for 2 days, transfer to a culture medium containing 30mg / L hygromycin, 200μM cephalosporin...
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