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Cordyceps militaris fruiting body development stage internal reference gene, primer, screening method and application

An internal reference gene, developmental stage technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of no internal reference gene research disclosure, etc., and achieve high primer amplification efficiency and stability. Good, low cost effect

Active Publication Date: 2020-02-11
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 the fruiting body development stage of Cordyceps militaris

Method used

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  • Cordyceps militaris fruiting body development stage internal reference gene, primer, screening method and application
  • Cordyceps militaris fruiting body development stage internal reference gene, primer, screening method and application
  • Cordyceps militaris fruiting body development stage internal reference gene, primer, screening method and application

Examples

Experimental program
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Effect test

reference example 1

[0059] The cultivation conditions of Cordyceps militaris are: inoculate the mycelium of Cordyceps militaris in a glass bottle containing rice culture medium on an ultra-clean workbench, and culture at a constant temperature and shade for 25 days at 25°C until the mycelia are covered with rice culture medium, and then it is ready; The formula of described rice culture medium is: 20g rice, 0.5g silkworm chrysalis powder are mixed with 25mL nutrient solution; The formula of described 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

[0061] (1) Select 12 internal reference genes TUB, EF-1α, UBC, GTPB, RPS, ACTIN, FBOX, CYP, GAPDH, PP2A, PGK, and UBQ as candidate internal reference genes, and design their fluorescent quantitative PCR primers;

[0062] 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;

[0063] (2) In each developmental stage, the Cordyceps militaris cultured in Reference Example 1 was continued to be cultured to the hyphae stage (day 30) by light treatment at 22°C for 14 hours every day, and then at 17°C and shading treatment for 10 hours. , primordium stage (40th day), elongation stage (50th day) and maturity stage (65th day), collect samples of Cordyceps militaris mycelium in the hyphae stage, and Cordyceps militaris at the primordium stage, elongation stage and mature stage For fr...

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Abstract

The invention discloses a cordyceps militaris fruiting body development stage internal reference gene, a primer, a screening method and application, wherein the internal reference gene is TUB, and thenucleotide sequence is SEQ ID NO: 1. The nucleotide sequences of the forward and reverse primers of TUB are SEQ ID NO: 13-14. The screening method comprises the following steps: (1) selecting twelveinternal reference genes as candidate internal reference genes, and designing primers; (2) inducing cordyceps militaris fruiting body development, extracting total RNAs at different development stages, measuring RNA concentration, carrying out reverse transcription on the RNA to synthesize cDNA, taking the cDNA as a template, and analyzing the candidate internal reference genes by using real-timefluorescence quantitative PCR; and (3) carrying out Ct value analysis on the real-time fluorescence quantitative PCR data to screen out stable internal reference genes. According to the application, the TUB is used as the internal reference gene to detect the relative expression levels of target genes at different development stages. The stability of the internal reference gene is good; and the method is simple, rapid and low in cost.

Description

technical field [0001] The invention relates to an internal reference gene, primer, screening method and application of Cordyceps militaris, in particular to an internal reference gene, primer, screening method and application of the fruiting body of Cordyceps militaris. 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] The development of Cordyceps militaris f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6811C12N15/11
CPCC12Q1/6895C12Q1/6811C12Q2600/166C12Q2600/158C12Q2531/113C12Q2563/107C12Q2545/101
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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