Cordyceps sinensis hirsutella sinensis purine anabolism enzyme, gene thereof, and application thereof
A Chinese hairpin, synthetic metabolism technology, applied in the field of purine nucleosidase, can solve the problems of rare gene and protein research, and achieve the effect of major application prospects, expanded production, and high expression
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Embodiment 1
[0027] Example 1: Cultivation of "Bailing" production fungus Cordyceps sinensis
[0028] Source of the strain: Firstly, natural Cordyceps sinensis was collected from Qinghai, and brought back to Hangzhou for isolation and screening, and the L0106 strain was obtained, and the strain was identified as Hirsutella Sinensis, which was preserved in a typical culture in China Objects Collection Center, the deposit number is CCTCC No: M 2011278, which has been disclosed in the previous patent application CN102373190A.
[0029] Inoculate the strain on the slant, and the medium formula (this is the liquid formula before solidification, prepared according to the following proportions before making the slant) is glucose 2.0% (w / v, 1% means that 100mL medium contains 1g , the same below), corn flour 1.0%, potato juice 0.5%, dextrin 0.5%, yeast powder 0.5%, bran 1.0%, silkworm chrysalis powder 2.0%, peptone 1.0%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05% , agar powder 1...
Embodiment 2
[0030] Example 2: Extraction of total RNA of "Bailing" production fungus Cordyceps sinensis
[0031]The total RNA was extracted with TRIzol reagent, and the steps were as follows: 1) Grinding with liquid nitrogen: Take 1 g of fresh bacteria and put it into a mortar, add liquid nitrogen repeatedly to grind until powdery, and divide into pre-cooled 1.5mL centrifuge tubes, Add 1mL TRIzol reagent, mix well, and let stand on ice for 5min to completely separate the nucleic acid-protein complex. 2) RNA isolation: add 0.2mL chloroform, shake vigorously for 15s, let stand on ice for 2~3min, centrifuge at 12000rpm at 4°C for 15min, separate the layers, and take the upper aqueous phase, about 600μL. 3) RNA precipitation: add 500 μL of isopropanol, let stand on ice for 10 minutes, centrifuge at 12000 rpm at 4°C for 10 minutes, and discard the supernatant. 4) RNA washing: add 1 mL of 75% (v / v) ethanol, suspend the pellet, let stand on ice for 10 min, centrifuge at 4°C, 7500 rpm for 15 min...
Embodiment 3
[0032] Example 3: Sequencing of the RNA sample of "Bailing" production fungus Cordyceps sinensis
[0033] After extracting the total RNA from the sample, the mRNA was enriched with Oligo(dT) magnetic beads. Add fragmentation buffer to break mRNA into short fragments (200~700bp), use mRNA as a template, use six base random primers (random hexamers) to synthesize the first cDNA strand, then synthesize the second cDNA strand, and then pass QiaQuickPCR kit After purification and elution with EB buffer, end repair, polyA was added and sequencing adapters were connected, then agarose gel electrophoresis was used for fragment size selection, and finally PCR amplification was performed, and the built sequencing library was sequenced with Illumina GA IIx. The original image data obtained by sequencing is converted into sequence data through base calling, that is, raw data or raw reads. The reads containing only the adapter sequence in the original sequencing reads were removed for sub...
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