Transgenic edible mushroom and its preparation method
An edible fungus and transgenic technology, applied in the field of genetic engineering breeding, can solve the problems of restricting the circulation of high-temperature edible fungus production, fresh fungus, low-temperature freezing and fresh-keeping, and low-temperature storage for export earnings, and achieves the effect of overcoming the difficulty of cultivating new cold-resistant strains.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0032] Example 1 Construction of recombinant plasmid
[0033] Using AFP1 (5'-TGCAGGAATCGGCACGAGGAA-3') and AFP2 (5'-GACTTTCATGGCTTAATTAGC-3') as primers, the antifreeze protein gene (afp) was amplified from the Nordic Budworm living in Sweden by RT-PCR. Connect the afp gene to the T-Vector vector to form the recombinant plasmid pGTHP4;
[0034] The design and synthesis of the linker, according to the experimental requirements of matching the sticky terminal bases left after restriction endonucleases BstZ I and BstE II and adding a restriction site in the middle of the linker, design and synthesize two Oligo nucleotides , that is, 5'-GGCCGCCATGGA-TGGTAACCAAGATCTAG-3'(29bp) and 5'-GTAACCTAGATCTTGGTTACC-ATCCATGGC-3'(30bp), after annealing, it is the linker, which includes Nco, BstE II, Bgl II 3 enzyme cutting sites point. Linkers were synthesized by Shanghai Bioengineering Company.
[0035] Plasmid pCAMBIA1301 (from the Australian Center for the Application of International Ag...
Embodiment 2
[0036] The preparation of embodiment 2 DNA microprojectiles
[0037] Aspirate 50 μL of gold powder suspension (diameter 0.1 μm, concentration 60 mg / mL), add 5.0 μL plasmid pCTH823 (1 μg / μL) and 50 μL 2.5 M CaCl respectively while vortexing 2, 20 μL of spermidine (filter sterilized, ready to use), continue to vortex for 10 minutes, let stand for 1 minute, centrifuge at 12,000 r / min for 5 minutes, discard the supernatant; then add 140 μL of 70% alcohol, and vortex fully , centrifuge at 12,000r / min for 5min, discard the supernatant; then add 140μL absolute alcohol, vortex fully, centrifuge at 12,000r / min for 5min, discard the supernatant; finally add 50μL absolute alcohol, vortex fully, Made DNA microbombs.
Embodiment 3
[0038] Example 3 DNA microprojectile bombardment into the mycelium of straw mushroom
[0039] (1) Select the robust mycelium of straw mushrooms grown on PDSA medium, grow for about 5 days at a growth temperature of 28° C., and divide them into small bacterial blocks of 3 mm square on a clean bench. Get the small bacteria block and spread it in the middle of a petri dish (d=9cm) with two layers of filter paper that absorbs enough PDSB medium to form a circle with a diameter of about 6cm;
[0040] (2) In the ultra-clean workbench of the gene gun, draw 8 μL of DNA microprojectiles and evenly coat them on the macrocarrier, and coat them into small circles with a diameter of about 1 cm;
[0041] (3) Turn on the power, set the bombardment parameters of the gene gun, and scrub the inner wall of the gene gun and the Rupture disk retaining cap with 70% alcohol. After drying, put the split disc into the groove of the installation cap, and then fix it on the top of the gene gun; put the...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More