Method for extracting endophytic fungi genomes of wild roses

An endophytic fungus and genome technology, applied in recombinant DNA technology, DNA preparation, etc., can solve the problems of low purity, poor extraction effect, and low extraction rate of genomic DNA, and achieve improved extraction rate, high yield and high purity. Effect

Inactive Publication Date: 2017-05-31
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Wild rose contains a large amount of polyphenols, esters and other secondary metabolites, pigments, phenolic groups, phenolic hydroxyls and other phenolic compounds, resulting in low extraction rate and low purity of genomic DNA, which hinders subsequent molecular biology analysis
Studies have shown that the improved CTAB method can extract high-quality rose DNA (Yan Tingliang et al., 2014), but there is no research report on the method of extracting high-quality wild rose endophytic fungus genomic DNA, and the extraction effect of ordinary kits and CTAB method is poor , the yield of the extracted endophytic fungal genomic DNA is low and the purity is low

Method used

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  • Method for extracting endophytic fungi genomes of wild roses
  • Method for extracting endophytic fungi genomes of wild roses
  • Method for extracting endophytic fungi genomes of wild roses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Liquid nitrogen grinding combined with CTAB (cetyltrimethylammonium bromide) method to extract wild rose endophytic fungus genome

[0024] (1) Preparation of reagents:

[0025] Sample pretreatment reagents include 0.5M EDTA (pH=8.0), 1M Tris-HCl solution, CTAB solution, β-mercaptoethanol, phenol: chloroform: isoamyl alcohol, isopropanol, ethanol with a concentration of 70% by mass, anhydrous Ethanol, 5% sodium hypochlorite; The preparation method of described reagent is as follows:

[0026] Preparation of 70% alcohol: measure 60mL sterile deionized water and 140mL absolute ethanol respectively, mix and store at room temperature.

[0027] Preparation of 5% sodium hypochlorite: Measure 100 mL of 10% sodium hypochlorite and 100 mL of sterile deionized water respectively, mix them, and store them in the dark at room temperature.

[0028] Preparation of 0.5M EDTA (pH=8.0): Take 186.1g Na 2 EDTA·2H 2 O, adjusted to pH = 8.0 with NaOH (about 20 g), filtered ddH 2 ...

Embodiment 2

[0047] Example 2 Liquid nitrogen grinding combined with soil microbial DNA strong extraction kit to extract wild rose endophytic fungus genome

[0048] (1) Preparation of reagents:

[0049] The sample pretreatment reagent has 70% dehydrated alcohol and 5% sodium hypochlorite. The preparation method is the same as in Example 1, and the DNA extraction kit adopts Reagent test kit, The kit contains Solution C1~Solution C6.

[0050] (2) Surface disinfection of samples

[0051] Weigh 2g of wild rose sample; divide the prepared 70% alcohol and 5% sodium hypochlorite solution into two 250mL sterile beakers; place the plant tissue in 70% alcohol to completely soak all the plant tissue, and the soaking time is 1 to 2 minutes; wash with sterile water three times; place the plant tissue block in 5% sodium hypochlorite solution to completely soak all the plant tissue for 30 to 60 seconds; wash with sterile water for five times; sterilize the surface Plant tissues were collected on st...

Embodiment 3

[0080] Example 3 liquid nitrogen grinding combined with CTAB mixed solution and Kit to extract endophytic fungus genome from wild rose

[0081] (1) Preparation of reagents:

[0082] Sample pretreatment reagents include 0.5M EDTA (pH=8.0), 1M Tris-HCl solution, CTAB extract, 70% absolute ethanol, 5% sodium hypochlorite, CTAB mixed solution, β-mercaptoethanol, PVP (polyvinylpyrrolidone); The preparation method of described reagent is as follows:

[0083] The preparation method of said 0.5M EDTA (pH=8.0), 1M Tris-HCl solution, CTAB extract, 70% dehydrated alcohol, 5% sodium hypochlorite is the same as described in Example 1

[0084] Preparation of the CTAB mixed solution: measure 200 mL of 2% CTAB extract, add 20 g of PVP, sterilize (115° C., 15 min), store at room temperature, add 4 mL of β-mercaptoethanol before use.

[0085] Other reagents can be provided by the kit or purchased through reagent companies.

[0086] (2) Surface disinfection of samples

[0087] Weigh 2g of w...

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Abstract

The invention discloses a method for extracting endophytic fungi genomes of wild roses. The method comprises the following steps: disinfecting the surfaces of wild roses and then performing liquid nitrogen treatment, and extracting endophytic fungi genomes by adopting combination of a CTAB mixed solution and a soil microorganism DNA brutal extracting kit. The method is designed aiming at the specificity of wild rose samples, and the adverse influences of quinones substances, phenol substances, polyphenol oxidase contained in wile rose plants, incomplete dilapidated walls, and the like on the extraction efficiency of genomes DNA can be avoided. The method can be applied to extraction of endophytic fungi genomes of all the wild roses, and has the characteristics of being wide in application range and high in extraction rate. The method can achieve a good extraction effect after being applied to extraction of endophytic fungi genomes in Dali purple flowers of wild roses and the endophytic fungi genomes of seven-sister wild roses, and the extracted genomes DNA can be used for various molecules experiments.

Description

technical field [0001] The invention belongs to the technical field of genome extraction, in particular to a method for extracting the genome of wild rose endophytic fungi. Background technique [0002] Rosa wild species are the main source of rose genetic resources, and more than 25,000 modern rose varieties come from continuous hybridization and backcrossing of 15 original Rosa parents. Due to long-term directional selection and artificial cultivation, some disease-resistant genes of modern roses have been lost and their disease resistance is poor. At present, the excellent genes of wild rose resources are transferred to modern rose cultivars, broadening their genetic basis, and cultivating new disease-resistant varieties are the main directions of disease-resistant breeding. However, due to the problems of hybrid incompatibility and hybrid sterility, the high diversity and variability of physiological races of pathogenic bacteria, and the difficulty in maintaining their ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 李海燕熊帜赵祎李欣亚孙玮宏白维晓吴光丽
Owner KUNMING UNIV OF SCI & TECH
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