Bombyx mori actin 6A gene and application in detection of methylation level of bombyx mori actin 6A gene

An actin and methylation technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of difficult control, strong virus infectivity, frequent outbreaks, etc., and achieve the effect of high stability and strong specificity

Inactive Publication Date: 2020-07-31
JIANGSU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus is highly contagious, difficult to control, and has frequent outbreaks, causing huge economic losses to the sericulture industry

Method used

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  • Bombyx mori actin 6A gene and application in detection of methylation level of bombyx mori actin 6A gene
  • Bombyx mori actin 6A gene and application in detection of methylation level of bombyx mori actin 6A gene
  • Bombyx mori actin 6A gene and application in detection of methylation level of bombyx mori actin 6A gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0023] Example 1DNA Extraction

[0024] Use Ezup Column Animal Genomic DNA Purification Kit to extract DNA, the specific steps are as follows:

[0025] (1) The experimental group was BmN cells added with Bombyx mori nuclear polyhedrosis virus, and the control group was normal BmN cells. Add 200 μl Buffer PBS and 20 μl Proteinase K to each well of cells, shake the culture flask gently, collect the cells, and transfer them to a new Add 200μl Buffer CL to the centrifuge tube, shake and mix, and bathe in 56℃ water for 10min;

[0026] (2) Add 200μl Buffer CL and mix thoroughly by inversion;

[0027] (3) Add 200 μl of absolute ethanol, fully invert and mix;

[0028] (4) Put the adsorption column into the collection tube, add all the solution and translucent fibrous suspension into the adsorption column with a pipette, let it stand for 2 minutes, then centrifuge at 10,000 rpm for 1 minute at room temperature, and pour out the waste liquid in the collection tube;

[0029] (5) Put t...

Embodiment 2

[0033] Example 2 DNA bisulfite treatment

[0034] Use the bisulfite treatment reagent provided in the kit to perform bisulfite modification on the DNA extracted in step 1. The specific steps are as follows:

[0035] (1) Add 130μL of CT Conversion Reagent and 20ul of DNA sample to the single PCR tube (if the sample is less than 20ul, fill it up with water);

[0036] (2) Place the above sample in a water bath on a water bath (98°C for 10min; 64°C for 2.5h);

[0037] (3) Add 600μL of M-Binding Buffer to the Zymo-SpinTMIC Column and put it into the collection tube;

[0038] (4) Add the product after PCR in step 2 to the Zymo-SpinTMIC Column containing M-Binding Buffer, close the lid and mix by inverting;

[0039] (5) Centrifuge at 12000rpm for 30s, discard the liquid in the collection tube;

[0040] (6) Add 100μL of M-Wash Buffer and centrifuge at 12000rpm for 30s;

[0041] (7) Add 200 μL of M-Desulphonation Buffer, let stand at room temperature (20°C-30°C) for 15-20min, and c...

Embodiment 3

[0044] Embodiment 3PCR amplification reaction

[0045] The amplification reagent provided in the kit was used to carry out PCR amplification using the bisulfite-modified DNA in step 2 as a template. The amplification system is shown in Table 1.

[0046] Table 1 PCR amplification system

[0047]

[0048] The PCR reaction conditions are shown in Table 2.

[0049] Table 2 PCR reaction conditions

[0050]

[0051] After the PCR reaction was completed, the agarose gel electrophoresis was detected, and the results showed as follows: figure 1 , indicating that the primers of the present invention can amplify specific target fragments.

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Abstract

The invention discloses a bombyx mori actin 6A gene and application in detection of methylation level of the bombyx mori actin 6A gene. The CG methylation level of the gene in a 1884-2071bp nucleotideregion is obviously higher than that of normal cells in silkworm cells infected with nuclear polyhedrosis viruses, and the nucleotide sequence of the region is as shown in SEQ ID NO. 1. The inventionalso provides a primer pair, a kit and a detection method for specifically detecting the CG methylation level in the region. The forward sequence of the primers is shown as SEQ ID NO.2, and the reverse sequence is shown as SEQ ID NO.3. The invention provides a new research direction for researching the interaction mechanism of bombyx mori DNA methylation and the nuclear polyhedrosis virus. The primers and the detection kit for detecting the methylation level of the gene disclosed by the invention have important practical application value for detecting the bombyx mori nuclear polyhedrosis virus.

Description

technical field [0001] The invention relates to a silkworm gene and an application for detecting its methylation level, in particular to a silkworm actin 6A gene and an application for detecting its methylation level, and belongs to the field of biotechnology. Background technique [0002] DNA methylation is an important modification pathway of epigenetics and is widely involved in a series of important life processes including embryonic development, genome imprinting, sex differentiation, aging and virus infection. A large number of studies have found that viral infection, especially viruses that cause tumors, can induce abnormal methylation of host genomic DNA, thereby generating immune response or immune escape. There are significant differences in DNA methylation patterns and methylation levels between cancer patients at different stages and healthy individuals, and in the early stages of cancer, the DNA methylation levels in body fluids will change. Therefore, accurate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12Q1/6888C12Q1/6858C12N15/11
CPCC07K14/43586C12Q1/6888C12Q1/6858C12Q2600/154
Inventor 吴萍黄昊凌张少伦商琪沈曼曼赵卫国印昊彤李涛
Owner JIANGSU UNIV OF SCI & TECH
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