Cordyceps sinensis Zjb18002 and its application
A technology of ZJB18002, Trichosporium chinensis, applied in fungi, microorganisms, microorganisms, etc., can solve the problems of long growth cycle and low mannitol content in Trichospora mycelium, and achieve the effect of major application prospects.
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Embodiment 1
[0030] Embodiment 1: Screening culture and identification of wild Cordyceps sinensis Trichosporum sinensis
[0031] 1. Screening and cultivation
[0032]Excavated 45 Cordyceps sinensis complete fungus complexes with thick and plump fruiting bodies and bulky insects from the hillside at an altitude of 4000-4300 meters in Yushu, Qinghai. The host insects are bat moth larvae. Put them in a 15°C incubator for maintenance. Use a cleaning brush to remove impurities on the surface of the fruit body, rinse with sterile purified water, and then disinfect with 0.1% mercuric chloride. In the ultra-clean bench, use a sterile scalpel to cut open the inner sclerotium of the worm body, and pick out the upper, middle, and lower parts of the fruit body. The tissues of the three parts were placed on the sterilized slant medium (50 μg / mL) containing gentamicin, cultured in a constant temperature incubator at 16°C, and observed and recorded every day. The isolated tissue began to germinate afte...
Embodiment 2
[0035] Embodiment 2, the morphological identification of Cordyceps sinensis isolate
[0036] The growth and single colony morphology of the wild-type bacterium L0106 in Example 1 was observed on the slant medium, and the microorganisms on the fixed slide were observed with a scanning electron microscope to understand the morphology of the microorganisms. After identification, the mycelium grows slowly, and the slope needs to be cultivated for about 40 days. The mycelium is dense, difficult to pick, and has a snow-white bulge. No longer round, like vermicompost, many folds, fluffy hyphae outward, such as figure 1 shown. Electron microscope observation of the mycelium morphology showed that the mycelium was well developed, slender, with a transverse septum, and a diameter of 1.5-2.0 μm. The sporophytes that formed spores could be observed, and the results were as follows: figure 2 shown. After morphological identification, it was preliminarily judged that the colony formed b...
Embodiment 3
[0037] Embodiment 3, the molecular biological identification of Cordyceps sinensis isolate
[0038] (1) 18S rDNA primer design
[0039] Primers:
[0040] p1:5'-TCCGTAGGTGAACCTGCCG-3' and
[0041] p2:5'–TCCTCCGCTTATTGATATGC-3'. Primers were synthesized by Shanghai Shenggong Company.
[0042] (2) Genome extraction
[0043] The genomic DNA of the wild-type cell L0106 in Example 1 was extracted by using a rapid nucleic acid extractor and a microbial genome extraction kit (MP Company, USA).
[0044] (3) 18S rDNA sequence amplification
[0045] Using genomic DNA as a template, PCR amplification was performed using universal primers (BioRad, USA, PTC200 amplification instrument). The reaction conditions were: pre-denaturation at 95°C for 5 minutes, cycle parameters were denaturation at 94°C for 45s, annealing at 55°C for 60s, and 72°C. Extended for 90s, repeated 35 cycles, continued to extend for 10min at 72°C, and finally identified the PCR product by 0.9% agarose gel electrop...
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