Method for extracting fungal genome on surface of aged tobacco

A genome and fungal technology, applied in the field of microbiology, can solve the problems of unstable DNA quality, clogging of DNA adsorption columns by non-microbial substances, genome loss, etc., and achieve the effect of improving DNA extraction efficiency, avoiding adverse effects, and good extraction effects

Pending Publication Date: 2016-11-23
CHINA TOBACCO YUNNAN IND
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although ordinary domestic DNA extraction kits currently on the market can obtain high-quality genomic DNA, they are only aimed at purely cultured microorganisms. For the extraction of microbial genomic DNA directly from environmental samples, the effect of using ordinary kits is poor, and non-microbial Substances can clog the DNA adsorption column, and the quality of the extracted DNA is not stable
The method of manually extracting the genome of the sample is inefficient and easily leads to the loss of the genome
When the genomic DNA of fungi on the surface of tobacco leaves is directly extracted using ordinary kits, small tobacco leaf fragments and dust will block the DNA adsorption column, affecting the efficiency and even causing the interruption of the experiment. After soaking the flue-cured tobacco leaves, some oily substances will overflow. Viscous flocs will be formed, which will also clog the adsorption column

Method used

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  • Method for extracting fungal genome on surface of aged tobacco
  • Method for extracting fungal genome on surface of aged tobacco
  • Method for extracting fungal genome on surface of aged tobacco

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: Utilizing CTAB method to extract the genome of the fungus genome on the surface of tobacco leaves of K326 flue-cured tobacco during aging, including the following steps:

[0055] (1) Preparation of reagents:

[0056] Sample pretreatment reagents include 0.5M EDTA (pH=8.0), 1M Tris-HCl solution, CTAB solution, 0.2% (V / V) β-mercaptoethanol, phenol: chloroform: isoamyl alcohol, isopropanol, mass percent concentration 70% ethanol; The preparation method of described reagent is as follows:

[0057] The preparation of the 0.5M EDTA (pH=8.0): get 186.1g Na 2 EDTA·2H 2 O, adjusted to pH = 8.0 with NaOH (about 20 g), filtered ddH 2 O mixed to 1L, sterilized (121°C, 15min), and stored at room temperature.

[0058] Preparation of the 1MTris-HCl (pH=8.0): Weigh 121.1g of Tris-base, dissolve it in 800ml of deionized water, add about 42ml of concentrated hydrochloric acid to adjust its pH to 8.0, and finally, add deionized water to volume Make up to 1L, sterilize (...

Embodiment 2

[0077] Example 2: Utilizing the portable method urea method to extract the fungal genome on the surface of tobacco leaves in K326 variety aging

[0078] (1) Preparation of sample pretreatment reagents

[0079] Sample pretreatment reagents include pH 7.0 PBS buffer, urea extract (7M Urea, 50mM Tris-HCl (pH=8.0), 62.5mM NaCl, 1% SDS), phenol: chloroform: isoamyl alcohol (25:24:1 ), isopropanol, 3M NaAc, mass percent concentration 70% ethanol.

[0080] Preparation of the 1% SDS (sodium dodecyl sulfate): Weigh 10 g of SDS and dissolve it in about 0.9 L deionized water, stir with a magnetic stirrer until completely dissolved, and finally, dilute to 1 L with deionized water.

[0081]Preparation of the 1M Tris-HCl (pH=8.0, final concentration 1M): Weigh 121.1 g of Tris-base, dissolve it in 800 ml of deionized water, add about 42 ml of concentrated hydrochloric acid to adjust its pH to 8.0, and finally, add Dilute to 1L with deionized water, sterilize (121°C, 15min), and store at ro...

Embodiment 3

[0098] Example 3: The method of combining liquid nitrogen grinding treatment and kits to extract the fungal genome on the surface of tobacco leaves in K326 variety aging

[0099] (1) Preparation of sample pretreatment reagents

[0100] Sample pretreatment reagents include pH 7.0 PBS buffer solution, and its preparation method is shown in Case 1. The DNA extraction kit uses DNA Isolation Kit.

[0101] (2) Pretreatment of tobacco leaf samples

[0102] Weigh 250g-300g of tobacco leaf samples, cut them into pieces, and evenly divide them into 4 portions, add 1000mL of pH 7.0 PBS buffer solution to each portion, and incubate at 28°C and 170rpm shaker for 2-3 hours. Collect the soaking liquid by filtering with double-layer gauze, centrifuge the filtrate (10000×g, 4°C) for 15-20 min, discard the supernatant, add 20 ml of sterile deionized water to dissolve the precipitate, transfer it to a 50 ml centrifuge tube, and then add DNase Ⅰ to a final concentration of 1u / mL, vortex to m...

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Abstract

The invention relates a method for extracting a fungal genome on the surface of aged tobacco. The method includes the two main steps of aged tobacco surface fungus enrichment and genome DNA extraction. Fungi are enriched through buffering solution soaking and gradient centrifugation, grinding and cell disruption are conducted by means of liquid nitrogen, and the DNA of the genome of fungi is extracted by means of a kit shown in the description. By means of liquid nitrogen grinding, fungal cell walls can be fully split, extraction efficiency of the DNA of the fungi is improved, the DNA isolation kit in the description includes the innovation patent inhibitor removal technology, and the yield of the genome can be effectively increased. The method can be used for efficiently and quickly extracting the DNA of the fungal genome on the surface of aged tobacco.

Description

technical field [0001] The invention relates to the field of microbiology, in particular to a method for extracting fungal genome DNA from the surface of aged tobacco leaves. Background technique [0002] The fresh flue-cured tobacco leaves are collected after they are ripe, and are first roasted, and then enter the aging stage after threshing and redrying. The aging stage can adjust and improve the quality of tobacco leaves and maintain the stability of tobacco leaf quality, which is essential in cigarette production. processing stage. The surface and interior of the aged tobacco leaves are rich in microorganisms, and the aged flue-cured tobacco leaves are rich in various compounds, including sugars, proteins, nicotine, etc., which meet the nutritional conditions for the survival of microorganisms. Many substances in the tobacco leaves have affinity Water-based, can absorb water from the aging environment, and under suitable conditions, it will cause a large number of micr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 王毅高锐宋鹏飞张光煦杨威李欣亚李海燕魏云林浦绍占季秀玲
Owner CHINA TOBACCO YUNNAN IND
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