Method of producing transgenic plant representing phytase
A phytase and plant technology, applied in the field of plant weight, can solve problems such as difficult absorption and utilization of monogastric animals, high phosphorus fecal pollution, and increased feed costs, and achieve rapid expansion of production scale, mitigation of environmental pollution, and low production costs. Effect
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Embodiment 1
[0051] 1. Preparation of materials:
[0052] Phytase gene: the phytase gene and appA-m gene of patent No. ZL 97121731.9 were used as described above.
[0053] Strains: Escherichia coli E.coli DH5α, JM109.
[0054] Tool enzymes and modification enzymes: various restriction enzymes, modification enzymes, pGEM5Zf plasmid and pGEMT-easy vector were purchased from Promega, New England Biolabs and Takara Bio.
[0055] Chemical reagents: Yeast extract and tryptone were purchased from OXOID Company, CHU (N6) bulk salt was purchased from Gibco Company, and other reagents and hormones for tissue culture were purchased from Sigma Company. All other chemical reagents were of domestic analytical grade. Synthesis of primers and fragments: synthesized by Beijing Aoke Bioengineering Co., Ltd. and Shanghai Boya Biotechnology Co., Ltd. Sequencing: completed by Shanghai Shenergy Biotechnology Co., Ltd. Gene gun and consumables used: purchased from Bio-Rad.
[0056] 2. Construction of phytas...
Embodiment 2
[0081] Embodiment 2 transformation recipient crops
[0082] 1. Maize transformation mediated by gene gun
[0083] The preparation of the medium and the transformation method of corn particle gun refer to the method in the book "Plant Cell, Tissue and Organ Culture: fundamental methods / O.L.Gamborg, G.C.Phillips eds., Springer" conduct.
[0084] Selection culture: 3 mg / k BASTA was added to the medium for maintaining the callus as selection pressure, and the transformed materials were selected and cultured, subcultured every two weeks.
[0085] Regeneration: After two months of selective culture, the resistant callus grew rapidly on the selective medium and had a fresh color. Transfer the resistant callus to the regeneration medium, and after two to three weeks, mature embryoid bodies can be obtained.
[0086] Put the embryoid body on the MS medium for rooting, and obtain the regenerated corn seedling. The phytase expression cassette PCR-positive plants were transferred to so...
Embodiment 3
[0087] Example 3 Molecular detection of regenerated gene corn plants
[0088] 1. Extraction of Genomic DNA from Regenerated Maize Plants
[0089] 1) Grind 0.1-0.2 g of plant leaves in liquid nitrogen and transfer to a 1.5 ml Eppendorf tube;
[0090] 2) Add 0.7ml CTAB (Tris 100mM, NaCl 1.4M, 20mM EDTA, CTAB 2%, mercaptoethanol 0.1%), 60°C, 45 minutes, every 10 minutes, invert and mix once.
[0091] 3) Add 0.7ml of phenol:chloroform (1:1), invert several times, centrifuge at 1000rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube. Add an equal volume of chloroform:isoamyl alcohol (24:1), mix well, centrifuge at 1000 rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube.
[0092] 4) Add an equal volume of isopropanol to the centrifuge tube, mix by inverting, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, wash once with 70% ethanol, drain, dissolve in 50 μL sterile water, and use for PCR detection.
[0093] 2. PCR detecti...
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