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Internal reference gene of cordyceps militaris mycelium under cold stress, and primers, screening method and application of internal reference gene

A technology of Cordyceps militaris mycelium and internal reference genes, which is applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc. It can solve the problems of no internal reference gene research disclosure and achieve high efficiency of primer amplification , low cost and simple screening method

Active Publication Date: 2020-02-14
CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the prior art, there is no related research disclosure of internal reference genes in Cordyceps militaris mycelia under cold stress

Method used

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  • Internal reference gene of cordyceps militaris mycelium under cold stress, and primers, screening method and application of internal reference gene
  • Internal reference gene of cordyceps militaris mycelium under cold stress, and primers, screening method and application of internal reference gene
  • Internal reference gene of cordyceps militaris mycelium under cold stress, and primers, screening method and application of internal reference gene

Examples

Experimental program
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Effect test

reference example 1

[0056] The culture condition of Cordyceps militaris mycelium is: inoculate the mycelium of Cordyceps militaris in the glass bottle containing the rice culture medium on the ultra-clean workbench, at 25 ℃, constant temperature shading culture for 25 days until the mycelium is covered with the rice culture medium, Serve; the formula of the rice culture medium is: 20g rice, 0.5g silkworm chrysalis powder are mixed with 25mL nutrient solution; the formula of the nutrient solution is: every 1000mL distilled water contains glucose 20g, KH 2 PO 4 2g, MgSO 4 1g, ammonium citrate 1g, peptone 5g, vitamin B1 20mg.

Embodiment

[0058] (1) Select 12 internal reference genes UBC, ACTIN, FBOX, TUB, EF-1α, PGK, GTPB, RPS, UBQ, CYP, GAPDH, PP2A as alternative internal reference genes, and design their fluorescent quantitative PCR primers;

[0059] The nucleotide sequences of the 12 internal reference genes are sequentially SEQ ID NO:1-12, and the nucleotide sequences of the forward and reverse primers of the 12 internal reference genes designed using primer5.0 software are sequentially SEQ ID NO :13-36;

[0060] (2) Place the Cordyceps militaris mycelium cultured in Reference Example 1 in a dark incubator, and perform cold stress treatment at 20°C. After treatment, collect and treat them for 0h, 0.5h, 1h, 2h, 4h, 8h (0h is the control sample) Cordyceps militaris mycelium sample, extract the total RNA immediately, measure the RNA concentration, reverse-transcribe the RNA to synthesize cDNA, then use the cDNA as a template, and use the primers in step (1) to use real-time fluorescent quantitative PCR Analy...

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Abstract

The invention discloses an internal reference gene of the cordyceps militaris mycelium under cold stress, and primers, screening method and application of the internal reference gene. The internal reference gene is UBC, a nucleotide sequence is SEQ ID NO: 1, nucleotide sequences of the forward and reverse primers of the UBC are SEQ ID NO: 13-14. The screening method is as follows: (1) selecting 12internal reference genes as candidate internal reference genes, and designing primers; (2) treating cordyceps militaris under cold stress, extracting the total RNA from the mycelium, measuring the concentration of RNA, synthesizing cDNA through reverse transcription of RNA, using cDNA as a template, and analyzing the candidate internal reference genes by using real-time fluorescence quantitativePCR; and (3) performing Ct value analysis on the real-time fluorescence quantitative PCR data to screen the stable internal reference gene. The application is that the UBC is used as the internal reference gene, and the relative expression of the target gene under cold stress is detected by using real-time fluorescence quantitative PCR. The internal reference gene has good stability. The method issimple, fast, and low in cost.

Description

technical field [0001] The present invention relates to an internal reference gene, primers, screening method and application, in particular to an internal reference gene, primer, screening method and application of Cordyceps militaris mycelia under cold stress. Background technique [0002] Cordyceps militaris is a model species of Ascomycota, Hypocreta, Ergotaceae, and Cordyceps. Its scientific name is Cordycepsmilitaris. The fruiting bodies of Cordyceps militaris have anti-inflammatory, antibacterial, anti-tumor, anti-aging and immunomodulatory effects. At present, using insects (such as silkworm chrysalis), rice, etc. as the main raw materials of the medium, a commercial production method for planting fruiting bodies of Cordyceps militaris has been established. However, little research has been done on the molecular mechanisms underlying pupal fruiting body formation, which is a fundamental biological question. [0003] Temperature is one of the key factors affecting t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/166C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 刘勇男刘必扬马酉初刘高强
Owner CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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