CN106754897B - A method of extracting Wild Rosa multiflora endogenetic fungus genome - Google Patents
A method of extracting Wild Rosa multiflora endogenetic fungus genome Download PDFInfo
- Publication number
- CN106754897B CN106754897B CN201710153864.0A CN201710153864A CN106754897B CN 106754897 B CN106754897 B CN 106754897B CN 201710153864 A CN201710153864 A CN 201710153864A CN 106754897 B CN106754897 B CN 106754897B
- Authority
- CN
- China
- Prior art keywords
- added
- solution
- supernatant
- rosa multiflora
- wild rosa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The invention discloses a kind of methods for extracting Wild Rosa multiflora endogenetic fungus genome.The method extracts endogenetic fungus genomic DNA using CTAB mixed liquor combination soil microbial DNA strength extracts kit for after Wild Rosa multiflora surface sterilization, liquid nitrogen grinding processing.The present invention is designed for the particularity of Wild Rosa multiflora sample, avoids contained quinones substance in Wild Rosa multiflora plant, phenolic substances, polyphenol oxidase and the adverse effects of the factors to extracting genome DNA efficiency such as broken wall is insufficient.The method can apply to the extraction of all Wild Rosa multiflora endophyte genomes, have the characteristics that have a wide range of application, to propose rate high.Method provided by the invention is applied to the extraction of the pale reddish brown endogenetic fungus genome in Wild Rosa multiflora Dali and Wild Rosa multiflora seven sisters's endogenetic fungus genome, all reaches good extraction effect, and the genomic DNA of extraction is used equally for all kinds of molecules to test.
Description
Technical field
The invention belongs to genome extractive technique field more particularly to a kind of extract Wild Rosa multiflora endogenetic fungus genome
Method.
Background technique
Rosa wild species are the main sources of Chinese rose genetic resources, and modern modern rose cultivars a about more than 25000 are all from 15
The continuous hybridization and backcrossing of a Rosa original parent.Due to long-term M8003 line and artificial cultivation, so that modern distance education system portion
Divide disease-resistant gene loss, disease resistance poor.The favorable genes of Wild Rosa multiflora resource are transferred to the cultivar of modern distance education system at present
In, it widens its hereditary basis, cultivate the Main way that disease-resistant varieties are breeding for disease resistance.But hybridize not close and hybrid due to existing
Infertility, pathogen biological strain have great diversity and variability, and after introducing a fine variety on a large scale, and resistance, which is difficult to maintain, etc. asks
Topic, so still cannot thoroughly solve the problems, such as disease control by crossbreeding.
Plant endogenesis epiphyte is the important component of fungi, refers to and moves in certain or whole stage of its history of life
Inside health plant tissue and organ, without making host plant show the fungi of obvious infection symptoms, in all kinds of flower plants
In, colonizing for endophyte is very universal.Due to living in inside plant tissues for a long time, with host plant coevolution, endophyte
A kind of mutualistic symbiosis relationship: environment needed for plant provides growth for endophyte and all kinds of nutriments is formd between plant,
And endophyte improves it to various then by generating miscellaneous bioactive substance come the growth and development of stimulation of host plant
Biological and abiotic stress resistivity, foreign countries are existing largely to enhance the special of plant disease prevention and control ability about using endophyte
Benefit, so probing into endophyte enhancing host plant has biggish practice significance to the resistance of various pathogens.
It the use of molecular biology method analysis biological community structure is current most important, most rapid Bacterial community and function
One of knowable method.It gives birth to flora inside to fall in structural research, common method is to carry out system hair using specific primer
Educate the amplification of mark molecule, and identified by measuring its sequence microbiologic population species composition and its quantitative abundance.This hair
Bright middle extraction Wild Rosa multiflora endogenetic fungus genomic DNA, using 18S rRNA gene magnification, the method for high-flux sequence, to visit
The peculiar molecular mechanism disease-resistant with the disease-resistant functional group of advantage endogenetic fungus and Wild Rosa multiflora of bright disease-resistant Wild Rosa multiflora lays compacting
Basis.
Contain the phenols chemical combination such as secondary metabolites, pigment, phenolic group, the phenolic hydroxyl groups such as a large amount of polyphenol, esters in Wild Rosa multiflora
Object, causes that extracting genome DNA rate is low, purity is low, causes to hinder to the analysis in terms of subsequent molecular biology.Studies have shown that
Modified CTAB method can extract high quality rose DNA (Yan Ting good etc., 2014), but extract the Wild Rosa multiflora endogenetic fungus base of high quality
Because group DNA method not studies have reported that, and, the Nei Shengzhen that extracts poor with general reagent box and CTAB method extraction effect
It is low that bacterium genomic DNA yield lacks purity.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of method for extracting Wild Rosa multiflora endogenetic fungus genome.This
Invention can be realized the purpose of a large amount of Wild Rosa multiflora endogenetic fungus genomes of high efficiency extraction, obtained endogenetic fungus genomic DNA
Purity is high, yield are high.
Technical scheme is as follows: a method of Wild Rosa multiflora endogenetic fungus genome being extracted, the method will
After Wild Rosa multiflora surface sterilization, liquid nitrogen grinding processing, using CTAB mixed liquor combination soil microbial DNA strength extracts kit
Extract endogenetic fungus genomic DNA.
Further, the mass percent of CTAB is 1.8~2.2%, PVP (polyvinyl pyrrole in the CTAB mixed liquor
Alkanone) concentration be 9~11% (m/v).
Further, the soil microbial DNA strength extracts kit isKit.
Further, the Wild Rosa multiflora surface sterilization operation is as follows: Wild Rosa multiflora plant tissue is placed in 70% alcohol
In, it is completely soaked all plant tissues, soaking time is 1~2min；Sterile water wash for several times after；Plant tissue is set again
In 4~6% liquor natrii hypochloritis, it is completely soaked all plant tissues, soaking time is 30~60s；Sterile water wash
For several times, disinfection is completed.
Preferably, the method operation is as follows: the plant tissue after surface sterilization being shredded, is placed in pre-cooling mortar and adds
Liquid nitrogen covering is firmly fully ground in the CTAB mixed liquor after the powder after weighing grinding to fine powdered is placed in preheating, grinding
To mixture of viscous form, mixture is vortexed after mixing, is addedSolution C1 in kit, turns upside down
It mixes, supernatant is transferred in new collecting pipe for several times after vortex oscillation, centrifugation, Solution C2 is added, be vortexed and mix,
It is incubated for, centrifugation；Supernatant is shifted into new collecting pipe, Solution C3 is added, is vortexed and mixes, is incubated for, centrifugation；In transfer
Clearly into new collecting pipe, be added Solution C4, be vortexed mix after be put into revolving filter, in, centrifugation discard filtrate, after
Continuous load, supernatant, room temperature, centrifugation are repeated up to and have filtered all supernatants；Solution C5 is added into revolving filter, room
Temperature centrifugation after discarding supernatant, shifts revolving filter into collecting pipe, then addition Solution C6 to white filter membrane center,
Centrifugation discards revolving filter；Total DNA sample is obtained, places and is saved in -20 DEG C of refrigerator, it is spare.
Further, the mass percent of CTAB is the dense of 1.8~2.2%, beta -mercaptoethanol in the CTAB mixed liquor
The concentration that degree is 1.8~2.2% (v/v) and PVP is 9~11% (m/v).
Further, the CTAB mixed liquor is using being preceding preheated to 60~70 DEG C.
As further preferred, the method concrete operations are as follows: the plant tissue powder after weighing 0.15g grinding is set
In a clean and sterile grinding；The CTAB mixed liquor that 600 μ L are preheated to 60~65 DEG C is added, is fully ground to thick；Again
The mixture after grinding is collected with aseptic filter paper and is added in a PowerBead Tubes, is gently vortexed and mixes, and is added 60
μ L Solution C1, turns upside down and mixes for several times；PowerBead Tubes is fixed on vortex instrument adapter, 3200r/m
Vortex 10~15min of continuous oscillation, 10000 × g of room temperature are centrifuged 30~60s；Supernatant is shifted to a clean 2mL collecting pipe
In, 250 μ L Solution C2 are added into supernatant, is vortexed and mixes 5~6s, 4 DEG C of incubation 5min, 10000 × g of room temperature centrifugations 1
~2min；Precipitating globule is avoided, is shifted in the new collecting pipe in supernatant≤600 μ L to one, 200 μ L Solution C3 are added and arrive
It in supernatant, is vortexed and mixes, 4 DEG C of incubations 5min, 10000 × g of room temperature are centrifuged 1~2min；Avoid precipitating globule, transfer supernatant≤
In 750 μ L to a new collecting pipes, 1200 μ L Solution C4 are added into supernatant, is vortexed and mixes 5~6s, load is about
For 675 μ L supernatants into revolving filter, 10000 × g of room temperature is centrifuged 1min, discards filtrate, continues to load 675 μ L supernatants, room temperature
10000 × g is centrifuged 1~2min, is repeated up to and has filtered all supernatants；500 μ L Solution C5 are added to revolving filter
In, 10000 × g of room temperature is centrifuged 30~60s, discards supernatant, and 10000 × g of room temperature is centrifuged 1~2min；Careful transfer rotating filter
100 μ L Solution C6 are added to white filter membrane center into 2mL collecting pipe in device, and 10000 × g of room temperature is centrifuged 30~60s,
Discard revolving filter；Total DNA sample is placed and is saved in -20 DEG C of refrigerator, it is spare.
The present invention removes sample surfaces microorganism by surface sterilization, its surface microorganism is avoided internally to give birth to fungal gene group
DNA is polluted, and the processing of liquid nitrogen grinding can effectively crack fungal cell wall；PVP (polyvinyl pyrrole is added in CTAB extracting solution
Alkanone) and mercaptoethanol can play the role of protect DNA, in addition, containing wound in soil microbial DNA strength extracts kit
New patent inhibiting factor removal(IRT), inhibiting factor (such as humic acid, cell fragment, the egg in sample can be effectively removed
White matter etc.) to DNA extract interference.Under the collective effect of mercaptoethanol, PVP and soil microbial DNA strength extracts kit,
Extract genome during DNA from the material damages such as quinones, phenols, polyphenol oxidase and other inhibiting factors interference,
To improve endogenetic fungus extracting genome DNA rate.The DNA yield that the present invention extracts is high, purity is high, obtained high quality
DNA can be directly used for downstream experiment, without being further purified.
Method provided by the invention be applied to pale reddish brown (R.multiflora) the endogenetic fungus genome in Wild Rosa multiflora Dali and
The extraction of Wild Rosa multiflora seven sisters (R.multiflora var.carnea) endogenetic fungus genome, all reaches good extraction
Effect, the genomic DNA of extraction are used equally for all kinds of molecules to test.
Compared with prior art, the invention has the following advantages: the present invention is directed to the particularity of Wild Rosa multiflora sample
It is designed, avoids contained quinones substance in Wild Rosa multiflora plant, phenolic substances, polyphenol oxidase and the factors such as broken wall is insufficient
Adverse effect to extracting genome DNA efficiency.The method can apply to the extraction of all Wild Rosa multiflora endophyte genomes,
Have the characteristics that have a wide range of application, to propose rate high.
Detailed description of the invention
Fig. 1 is that the method that liquid nitrogen grinding is combined with CTAB method extracts Wild Rosa multiflora endogenetic fungus 18S rRNA gene magnification
Agarose gel electrophoretogram；
Fig. 2 is that the method that liquid nitrogen grinding is combined with soil microbial DNA strength extracts kit is extracted in Wild Rosa multiflora
The agarose gel electrophoretogram of raw fungi 18S rRNA gene magnification；
Fig. 3 is the method extraction open country of liquid nitrogen grinding combination CTAB mixed liquor and soil microbial DNA strength extracts kit
The agarose gel electrophoretogram of raw rose endogenetic fungus 18S rRNA gene magnification.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this
The technical solution of invention is described in further details.It should be appreciated that specific embodiment described herein is only used to explain this
Invention, is not intended to restrict the invention.Other various experimental implementations according to the present invention, are the ordinary skill in the art,
The part being not particularly illustrated in text, those skilled in the art be referred to the present patent application day before it is various common
Reference book, scientific and technical literature or relevant specification, handbook etc. are implemented.Material used in embodiment, reagent are such as without spy
Different explanation, can be obtained through commercial channels, U.S. MOBIO strength soil DNA extraction kitKit (DNA Isolation Kit) it buys and pacifies invincible Science and Technology Ltd. in Shenzhen.
When pre-processing Wild Rosa multiflora sample of the present invention, all tools touched have to pass through autoclave sterilization, with
Exempt to bring external microbe contaminated samples into.When grinding plant sample, it is ensured that grinding great efforts, first time ground sample is to thin
It is powdered, when grinding for the second time, it is ensured that milling time is at least 5 minutes, reacts sample sufficiently with CTAB mixed liquor, is milled to
Sample is thick.During extracting DNA using kit, to avoid being drawn to lower sediment thing as far as possible when collecting supernatant
Otherwise matter will affect the purity of DNA, to have filtered all supernatants when loading supernatant liquid filtering, otherwise will affect the acquisition of DNA
Rate.
1 liquid nitrogen grinding combination CTAB (cetyl trimethylammonium bromide) method of embodiment extracts Wild Rosa multiflora endogenetic fungus base
Because of group
(1) preparation of reagent:
Sample pre-treatments reagent has 0.5M EDTA (pH=8.0), 1M Tris-HCl solution, CTAB solution, β-sulfydryl second
Alcohol, phenol: chloroform: isoamyl alcohol, isopropanol, the ethyl alcohol of mass percent concentration 70%, dehydrated alcohol, 5% sodium hypochlorite；The examination
The preparation method of agent is as follows:
The preparation of 70% alcohol: 60mL aseptic deionized water and 140mL dehydrated alcohol, mixing, room temperature preservation are measured respectively.
The preparation of 5% sodium hypochlorite: measuring 10% sodium hypochlorite of 100mL and the aseptic deionized water of 100mL respectively, mixes
It closes, room temperature is kept in dark place.
The preparation of 0.5M EDTA (pH=8.0): 186.1g Na is taken2EDTA·2H2O adjusts pH=with NaOH (about 20g)
8.0, with the ddH of filtering2O is mixed to 1L, is sterilized (121 DEG C, 15min), room temperature preservation.
The preparation of 1MTris-HCl (pH=8.0): weighing Tris-base 121.1g, is dissolved in 800mL deionization
In water, about 42mL concentrated hydrochloric acid is added, its pH is adjusted to 8.0, finally, plus deionized water be settled to 1L, sterilize (121 DEG C, 15min),
Room temperature preservation.
The preparation of 2%CTAB extracting solution: 20g CTAB, 81.9g NaCl are weighed；It measures 1M Tris-HCl (pH=8.0)
Solution 100mL, 0.5M EDTA (pH=8.0) solution 40mL；Add deionized water to be settled to 1L, sterilizes (121 DEG C, 15min), room
Temperature saves, and uses preceding plus 0.2% (V/V) beta -mercaptoethanol.
Other reagents can be bought by Reagent Company.
(2) surface sterilization of sample
Weigh 2g Wild Rosa multiflora sample；To prepare 70% alcohol and 5% liquor natrii hypochloritis dispense to two 250mL without
In bacterium beaker；Plant tissue is placed in 70% alcohol, all plant tissues are completely soaked, soaking time 2min；Nothing
Bacterium water cleans three times；Plant tissue block is placed in again in 5% liquor natrii hypochloritis, all plant tissues are completely soaked, is soaked
The bubble time is 1min；Sterile water wash five times；The plant tissue that surface sterilization is completed is collected in aseptic filter paper.
(3) CTAB method extracts genomic DNA
1) 2g sample is weighed, liquid feeding nitrogen covering in pre-cooling mortar is placed in, is quickly firmly ground to powdered；
2) about 600 μ l are added into mortar, the 2%CTAB extracting solution of 65 DEG C of preheatings mixes it with the powder after grinding；
3) liquid is transferred in the centrifuge tube of 1.5mL, is turned upside down after the concussion that is vortexed in 65 DEG C of water-bath 1h, every 20min
It mixes gently primary；
4) centrifuge tube is taken out after the water bath is over, the phenol after 4 DEG C of isometric pre-coolings are added in each centrifuge tube: chloroform:
Isoamyl alcohol (25:24:1), after mixing of turning upside down, 12000r/min, is centrifuged 15min by 4 DEG C；
5) supernatant is transferred in new 1.5mL centrifuge tube after being centrifuged, the phenol after 4 DEG C of isometric pre-coolings are added again: chlorine
Imitative: isoamyl alcohol (25:24:1), mixing of turning upside down, 12000r/min, are centrifuged 15min by 4 DEG C；
6) supernatant is transferred in new 1.5mL centrifuge tube after being centrifuged, and the isopropanol after -20 DEG C of pre-coolings is added into supernatant
(0.6 times of volume), mixing of turning upside down are placed in -20 DEG C of refrigerator and precipitate at least 2h；
7) centrifuge tube is taken out from refrigerator after sedimentation time, 12000r/min, is centrifuged 15min, takes precipitating by 4 DEG C.To from
1mL, the ethyl alcohol that the volume fraction after -20 DEG C of pre-coolings is 70% are added in heart pipe, piping and druming rinsing repeatedly precipitates, 12000r/min, and 4
DEG C, it is centrifuged 15min, liquid is abandoned, takes precipitating.It is primary to repeat this operation；
8) centrifuge tube equipped with precipitating is inverted on aseptic filter paper, is placed in superclean bench natural air drying, after air-drying,
The distilled water dissolving DNA of 50 μ l is added to every pipe；
9) dissolved DNA is saved in -20 DEG C of refrigerators；
(4) the DNA genome of 18S rRNA gene magnification Detection and Extraction
Using the universal primer of 18S rRNA gene magnification, using the genome of extraction as template, anneals at 53 DEG C, carry out fine jade
Sepharose electrophoresis simultaneously observes result, if it is possible to amplify the 18S rRNA gene of fungi, it was demonstrated that be successfully extracted genome.
Have 4 swimming lanes in Fig. 1 from left to right, by Fig. 1 it can be found that: 2,3,4 swimming lanes do not have apparent DNA purpose band,
Wherein 2,3,4 swimming lanes are the repetition experiment of three samples, show that the DNA extraction of this experiment is failure.
2 liquid nitrogen grinding combination soil microbial DNA strength extracts kit of embodiment extracts Wild Rosa multiflora endogenetic fungus base
Because of group
(1) preparation of reagent:
Sample pre-treatments reagent has 70% dehydrated alcohol and 5% sodium hypochlorite preparation method with embodiment 1, and DNA extracts examination
Agent box usesKit,Contain Solution C1~Solution C6 in kit.
(2) surface sterilization of sample
Weigh 2g Wild Rosa multiflora sample；To prepare 70% alcohol and 5% liquor natrii hypochloritis dispense to two 250mL without
In bacterium beaker；Plant tissue is placed in 70% alcohol, all plant tissues are completely soaked, soaking time is 1~2min；
Sterile water wash is three times；Plant tissue block is placed in again in 5% liquor natrii hypochloritis, all plant tissues are completely soaked,
Soaking time is 30~60s；Sterile water wash five times；The plant tissue that surface sterilization is completed is collected in aseptic filter paper.
(3) method combined with kit is handled using liquid nitrogen grinding extract fungal gene group
The sample of surface sterilization is placed in liquid feeding nitrogen covering in pre-cooling mortar, is firmly fully ground that (liquid feeding nitrogen is ground repeatedly
Mill) to fine powdered.
Total DNA is extracted using soil microbial DNA strength extracts kit, specific steps are as follows:
1) powder after 0.15g grinding is added is into a PowerBead Tubes；
2) it is gently vortexed and mixes；
3) Solution C1, if precipitating, 60 DEG C of water-baths to fully dissolved are detected；
4) 60 μ l Solution C1 are added, turns upside down and mixes for several times；
5) PowerBead Tubes is fixed on vortex instrument adapter, maximum (top) speed (3200rpm) vortex continuous oscillation
10~15min；
6) 10000 × g of room temperature is centrifuged 30~60s；
7) transfer supernatant is into a clean 2mL Collection Tube (kit offer)；
8) 250 μ l Solution C2 are added into supernatant, is vortexed and mixes 5s, 4 DEG C of incubation 5min；
9) 10000 × g of room temperature is centrifuged 1~2min
10) precipitating globule is avoided, is shifted in the new collecting pipe in supernatant≤600 μ l to one
11) 200 μ l Solution C3 are added into supernatant, is vortexed and mixes, 4 DEG C of incubation 5min
12) 10000 × g of room temperature is centrifuged 1~2min；
13) precipitating globule is avoided, is shifted in the new collecting pipe in supernatant≤750 μ l to one；
14) 1200 μ l Solution C4 (Solution C4 is first shaken up using preceding) are added into supernatant, is vortexed and mixes
5s；
15) about 675 μ l supernatants are loaded into Spin Filter, 10000 × g of room temperature is centrifuged 1~2min；Filtrate is discarded, after
675 μ l supernatants of continuous load, 10000 × g of room temperature are centrifuged 1~2min；It is repeated up to and has filtered all supernatants；
16) it is added in 500 μ l Solution C5 to Spin Filter, 10000 × g of room temperature is centrifuged 30~60s；
17) it discards supernatant；
18) 10000 × g of room temperature is centrifuged 1~2min；
19) it is carefully avoided as far as possible in transfer Spin filter to 2mL Collection Tube (kit offer)
Solution C5 pollution；
20) 100 μ l Solution C6 are added to white filter membrane center；
21) 10000 × g of room temperature is centrifuged 30~60s；
22) Spin Filter is discarded.Total DNA sample is placed and is saved in -20 DEG C of refrigerator, it is spare；
(4) the DNA genome of 18S rRNA gene magnification Detection and Extraction；
Using the universal primer of 18S rRNA gene magnification, using the genome of extraction as template, anneals at 53 DEG C, carry out fine jade
Sepharose electrophoresis simultaneously observes result, if it is possible to amplify the 18S rRNA gene of fungi, it was demonstrated that be successfully extracted genome.
By Fig. 2 it can be found that: 2,3,4 swimming lanes have a 18S rRNA gene magnification purpose band, but not obvious enough, and size is about
Show that this experiment DNA yield obtained is lower wherein 2,3,4 swimming lanes are the repetition experiment of three samples for 500bp.
3 liquid nitrogen grinding combination CTAB mixed liquor of embodiment withKit extracts Wild Rosa multiflora endogenetic fungus base
Because of group
(1) preparation of reagent:
Sample pre-treatments reagent has 0.5M EDTA (pH=8.0), 1M Tris-HCl solution, CTAB extracting solution, 70% nothing
Water-ethanol, 5% sodium hypochlorite, CTAB mixed liquor, beta -mercaptoethanol, PVP (polyvinylpyrrolidone)；The preparation side of the reagent
Method is as follows:
The 0.5M EDTA (pH=8.0), 1M Tris-HCl solution, CTAB extracting solution, 70% dehydrated alcohol, 5% time
The preparation method of sodium chlorate is the same as described in embodiment 1
The preparation of the CTAB mixed liquor: measuring 2%CTAB extracting solution 200mL, and 20gPVP is added, sterilize (115 DEG C,
15min), room temperature preservation uses preceding plus 4mL beta -mercaptoethanol.
Other reagents can be provided by kit or be bought by Reagent Company.
(2) surface sterilization of sample
Weigh 2g Wild Rosa multiflora sample；It prepares 70% alcohol and 5% liquor natrii hypochloritis dispenses to two sterile burnings of 250mL
In cup；Plant tissue is placed in 70% alcohol, all plant tissues are completely soaked, soaking time is 1~2min；It is sterile
Water cleans three times；Plant tissue block is placed in again in 5% liquor natrii hypochloritis, all plant tissues are completely soaked, is impregnated
Time is 30~60s；Sterile water wash five times；The plant tissue that surface sterilization is completed is collected in aseptic filter paper.
(3) liquid nitrogen grinding handles sample
The plant tissue of surface sterilization is shredded, liquid feeding nitrogen covering in pre-cooling mortar is placed in, is fully ground to fine powder
Shape.
(4) sample gene group DNA is extracted using CTAB mixed liquor binding reagents box
1) fine powder after weighing the above-mentioned grinding of 0.15g is placed in a clean and sterile grinding.
2) the CTAB mixed liquor that 600 μ l are preheated to 65 DEG C is added, it is mixed with the powder after grinding, is fully ground to viscous
Thick shape, then the sample after grinding is collected with aseptic filter paper.
3) mixture after above-mentioned grinding is added is into a PowerBead Tubes.
4) it is gently vortexed and mixes.
5) Solution C1 is detected, if precipitating, 60 DEG C of water-baths are to being completely dissolved.
6) 60 μ l Solution C1 are added, turns upside down and mixes for several times.
7) PowerBead Tubes is fixed on vortex instrument adapter, maximum (top) speed (3200r/min), which is vortexed, continuously to shake
Swing 10~15min.
8) 10000 × g of room temperature is centrifuged 30~60s.
9) transfer supernatant is into a clean 2mL Collection Tube.
10) 250 μ l Solution C2 are added into supernatant, is vortexed and mixes 5s, 4 DEG C of incubation 5min.
11) 10000 × g of room temperature is centrifuged 1~2min.
12) precipitating globule is avoided, is shifted in the new collecting pipe in supernatant≤600 μ l to one.
13) 200 μ l Solution C3 are added into supernatant, is vortexed and mixes, 4 DEG C of incubation 5min.
14) 10000 × g of room temperature is centrifuged 1~2min.
15) precipitating globule is avoided, is shifted in the new collecting pipe in supernatant≤750 μ l to one.
16) 1200 μ l Solution C4 are added into supernatant, is vortexed and mixes 5s.
17) about 675 μ l supernatants are loaded into Spin Filter, 10000 × g of room temperature is centrifuged 1~2min；Filtrate is discarded, after
675 μ l supernatants of continuous load, 10000 × g of room temperature are centrifuged 1~2min；It is repeated up to and has filtered all supernatants.
18) it is added in 500 μ l Solution C5 to Spin Filter, 10000 × g of room temperature is centrifuged 30~60s.
19) it discards supernatant.
20) 10000 × g of room temperature is centrifuged 1~2min.
21) carefully avoid Solution C5 dirty as far as possible in transfer Spin filter to 2mL Collection Tube
Dye.
22) 100 μ l Solution C6 are added to white filter membrane center.
23) 10000 × g of room temperature is centrifuged 30~60s.
24) Spin Filter is discarded.
(5) the DNA genome of 18S rRNA gene magnification Detection and Extraction
Using the universal primer of 18S rRNA gene magnification, using the genome of extraction as template, anneals at 53 DEG C, carry out fine jade
Sepharose electrophoresis simultaneously observes result, if it is possible to amplify the 18S rRNA gene of fungi, it was demonstrated that be successfully extracted genome.
By Fig. 3 it can be found that: 2,3,4 swimming lanes have an apparent 18S rRNA gene magnification purpose band, and size is about 500bp, 2,3,4 swimming
Road is the repetition experiment of three samples, shows that the DNA extraction of this experiment is successful, and DNA recovery rate is high.
Genomic DNA purity, concentration and the OTU number of 1~embodiment of comparing embodiment 3, the results are shown in Table 1.
Table 1
| Extracting method | Purity (OD260/OD280) | Concentration (ng/ μ l) | OTU number (a) |
| Embodiment 1 | 6.9 | 6.97 | < 25 |
| Embodiment 2 | 1.263 | 28.07 | 75 |
| Embodiment 3 | 1.673 | 37.31 | 1750 |
For DNA purity, 1.6-1.9 is most suitable, shows that protein content is exceeded lower than this range, is higher than this range
Show to contain RNA in sample, therefore uses extracting method DNA purity provided by the invention higher.By can be seen that embodiment 3 in table 1
DNA concentration be higher than embodiment 1 and embodiment 2.For OUT number, OUT number is more, illustrates to extract species gene group DNA
Abundanter, covering monoid is wider, more can really reflect the abundance of endophyte in plant sample.By being also seen that embodiment 3 in table 1
OUT number far more than embodiment 1 and embodiment 2.
Embodiment 4
On the basis of embodiment 3, changes the ingredient of CTAB mixed liquor, be added without PVP.CTAB mixed liquor configuration method is such as
Under: 2%CTAB extracting solution 200mL is measured, preceding addition 4mL mercaptoethanol is used.Other extraction process utilize 18S with embodiment 3
The universal primer of rRNA gene magnification is annealed using the genome of extraction as template at 53 DEG C, is carried out agarose gel electrophoresis and is seen
Examine result, if it is possible to amplify the 18S rRNA gene of fungi, it was demonstrated that be successfully extracted genome.But in gel electrophoresis figure,
The purpose band of 18S rRNA gene magnification is not obvious, and the DNA yield extracted is lower, therefore extracts Wild Rosa multiflora genome
PVP is essential during DNA.
Embodiment 5
On the basis of embodiment 3, changes the ingredient of CTAB mixed liquor, be added without mercaptoethanol.The configuration of CTAB mixed liquor
Method is as follows: 2%CTAB extracting solution 200mL measured, 20gPVP is added, is sterilized (115 DEG C, 15min), room temperature preservation.Other are mentioned
It takes process with embodiment 3, using the universal primer of 18S rRNA gene magnification, using the genome of extraction as template, is moved back at 53 DEG C
Fire carries out agarose gel electrophoresis and observes result, if it is possible to amplify the 18S rRNA gene of fungi, it was demonstrated that successfully extract
Genome.But in gel electrophoresis figure, the purpose band of 18S rRNA gene magnification is more apparent, but band brightness is not as good as embodiment
3, illustrate that the DNA yield extracted is general, measured purity is lower, therefore it is mixed to extract CTAB during Wild Rosa multiflora genomic DNA
It closes in liquid and mercaptoethanol is preferably also added.
Claims (4)
1. a kind of method for extracting Wild Rosa multiflora endogenetic fungus genome, which is characterized in that the method operation is as follows: by surface
Wild Rosa multiflora plant tissue after disinfection shreds, and is placed in liquid feeding nitrogen covering in pre-cooling mortar, is firmly fully ground to fine powdered,
Powder after weighing grinding is placed in the CTAB mixed liquor after preheating, is ground to mixture of viscous form, and mixture is vortexed after mixing,
It is addedSolution C1 in kit, turns upside down and mixes for several times, by supernatant after vortex oscillation, centrifugation
It is transferred in new collecting pipe, Solution C2 is added, be vortexed and mix, be incubated for, centrifugation；Supernatant is shifted to new collecting pipe
In, Solution C3 is added, is vortexed and mixes, is incubated for, centrifugation；Supernatant is shifted into new collecting pipe, Solution C4 is added,
It is vortexed after mixing and is put into revolving filter, centrifugation discards filtrate, continues to load supernatant, room temperature, centrifugation are repeated up to and have filtered
All supernatants；Solution C5 is added into revolving filter, room temperature centrifugation, after discarding supernatant, transfer revolving filter to receipts
In collector, Solution C6 is then added to white filter membrane center, centrifugation discards revolving filter；Total DNA sample is obtained, is put
It sets in -20 DEG C of refrigerator and saves, it is spare；
The mass percent of CTAB is 1.8~2.2% in the CTAB mixed liquor, the concentration of beta -mercaptoethanol is 1.8~2.2%
(v/v) and the concentration of PVP is 9~11% (m/v).
2. extracting the method for Wild Rosa multiflora endogenetic fungus genome as described in claim 1, which is characterized in that the wild rose
Common vetch surface sterilization operation is as follows: Wild Rosa multiflora plant tissue being placed in 70% alcohol, plant tissue is completely soaked, when immersion
Between be 1~2min, sterile water wash for several times after；Plant tissue is placed in again in 4~6% liquor natrii hypochloritis, plant tissue is made
It is completely soaked, soaking time is 30~60s, and sterile water wash for several times, completes disinfection.
3. extracting the method for Wild Rosa multiflora endogenetic fungus genome as described in claim 1, which is characterized in that the CTAB
Mixed liquor is using being preceding preheated to 60~70 DEG C.
4. the method for the extraction Wild Rosa multiflora endogenetic fungus genome as described in claims 1 to 3 is any, which is characterized in that institute
It is as follows to state method concrete operations: the plant tissue powder after weighing 0.15g grinding is placed in a clean and sterile grinding；It is added
600 μ L are preheated to 60~65 DEG C of CTAB mixed liquor, are fully ground to thick；The mixing after grinding is collected with aseptic filter paper again
Object is simultaneously added in a PowerBead Tubes, is gently vortexed and is mixed, and 60 μ L Solution C1 is added, turn upside down number
Secondary mixing；PowerBead Tubes is fixed on vortex instrument adapter, 3200r/m vortex 10~15min of continuous oscillation, room
10000 × g of temperature is centrifuged 30~60s；Supernatant is shifted into a clean 2mL collecting pipe, 250 μ L Solution C2 are added and arrive
It in supernatant, is vortexed and mixes 5~6s, 4 DEG C of incubations 5min, 10000 × g of room temperature are centrifuged 1~2min；Precipitating globule is avoided, in transfer
In μ L to a new collecting pipe clearly≤600,200 μ L Solution C3 are added into supernatant, is vortexed and mixes, 4 DEG C of incubations
5min, 10000 × g of room temperature are centrifuged 1~2min；Precipitating globule is avoided, is shifted in the new collecting pipe in supernatant≤750 μ L to one,
1200 μ L Solution C4 are added into supernatant, is vortexed and mixes 5~6s, load about 675 μ L supernatants into revolving filter, room
10000 × g of temperature is centrifuged 1min, discards filtrate, continues to load 675 μ L supernatants, and 10000 × g of room temperature is centrifuged 1~2min, repeats straight
To having filtered all supernatants；500 μ L Solution C5 are added into revolving filter, 10000 × g of room temperature is centrifuged 30~60s,
It discards supernatant, 10000 × g of room temperature is centrifuged 1~2min；100 μ L are added into 2mL collecting pipe in careful transfer revolving filter
To white filter membrane center, 10000 × g of room temperature is centrifuged 30~60s, discards revolving filter Solution C6；By total DNA sample
It places and is saved backup in -20 DEG C of refrigerator.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710153864.0A CN106754897B (en) | 2017-03-15 | 2017-03-15 | A method of extracting Wild Rosa multiflora endogenetic fungus genome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710153864.0A CN106754897B (en) | 2017-03-15 | 2017-03-15 | A method of extracting Wild Rosa multiflora endogenetic fungus genome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106754897A CN106754897A (en) | 2017-05-31 |
| CN106754897B true CN106754897B (en) | 2019-06-21 |
Family
ID=58962211
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710153864.0A Expired - Fee Related CN106754897B (en) | 2017-03-15 | 2017-03-15 | A method of extracting Wild Rosa multiflora endogenetic fungus genome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106754897B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110468057A (en) * | 2019-09-02 | 2019-11-19 | 昆明理工大学 | One plants endogenetic disk both-end hair spore category fungi M7SB 41 and its application |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109337955B (en) * | 2018-12-07 | 2021-12-03 | 暨南大学 | Third-generation sequencing zooplankton genome DNA extraction method and application |
| CN112094760B (en) * | 2020-10-13 | 2022-01-28 | 昆明理工大学 | Plant endophytic fungus Echinospora terrestris D2G24 and application thereof |
| CN112358974B (en) * | 2020-12-09 | 2022-06-10 | 昆明理工大学 | Plant endophytic fungus epicoccum nigrum FZT214 and application thereof |
| CN113502285A (en) * | 2021-07-02 | 2021-10-15 | 南京派森诺基因科技有限公司 | Method for extracting total DNA of endophyte in plant tissue |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101302555B (en) * | 2008-06-12 | 2012-08-29 | 上海交通大学 | Molecule identification method for taxol-producing endophytic fungi in yew |
| CN102174509B (en) * | 2011-02-18 | 2013-01-23 | 湖南大学 | Extraction and purification method of total plant endophyte genome DNA for colony analysis |
| CN104017804B (en) * | 2014-06-26 | 2017-06-20 | 华南农业大学 | A kind of polyphenol polysaccharide plant sample RNA extraction method of high-quality and high-efficiency |
| CN105505916A (en) * | 2015-12-31 | 2016-04-20 | 安诺优达基因科技(北京)有限公司 | Method for extracting high-quality genome DNA from dry suriana maritime leaf and kit thereof |
| CN106148329A (en) * | 2016-08-31 | 2016-11-23 | 云南中烟工业有限责任公司 | A kind of method extracting ageing tobacco leaf surface fungal gene group |
-
2017
- 2017-03-15 CN CN201710153864.0A patent/CN106754897B/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110468057A (en) * | 2019-09-02 | 2019-11-19 | 昆明理工大学 | One plants endogenetic disk both-end hair spore category fungi M7SB 41 and its application |
| CN110468057B (en) * | 2019-09-02 | 2021-09-24 | 昆明理工大学 | Endophytic pestalotiopsis fungus M7SB41 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106754897A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754897B (en) | A method of extracting Wild Rosa multiflora endogenetic fungus genome | |
| Mahuku | A simple extraction method suitable for PCR-based analysis of plant, fungal, and bacterial DNA | |
| Izumi et al. | Endobacteria in some ectomycorrhiza of Scots pine (Pinus sylvestris) | |
| Vargas et al. | Molecular fingerprinting evidence (ISSR, inter-simple sequence repeats) for a wild status of Olea europaea L.(Oleaceae) in the Eurosiberian North of the Iberian Peninsula | |
| Heck et al. | Axenic in vitro cultivation of 19 peat moss (Sphagnum L.) species as a resource for basic biology, biotechnology, and paludiculture | |
| CN107418952A (en) | A kind of extracting method of edaphon macro genome DNA and corresponding kit | |
| Aydin et al. | Single cotton seed DNA extraction without the use of enzymes and liquid nitrogen | |
| Jahan et al. | Genomic DNA extraction methods: a comparative case study with gram–negative organisms | |
| CN107119048B (en) | Pseudocercospora mori rDNA and application thereof in molecular detection of pseudocercospora mori | |
| CN106754872B (en) | A method of extracting microbe genome DNA from poplar wood | |
| CN109182582A (en) | A kind of withered germ of water-melon qualitative detection primer and detection method | |
| CN109706087B (en) | Penicillium simplicissimum strain and separation method and application thereof | |
| CN107828798A (en) | Application of the Chlamydomonas reinhardtii TRIP12 genes in Chlamydomonas reinhardtii cadmium tolerance is regulated and controled | |
| Zhang et al. | Analysis of endophytic actinobacteria species diversity in the stem of Gynura cusimbua by 16S rRNA gene clone library | |
| Das et al. | Interactions between aquatic bacteria and an aquatic hyphomycete on decomposing maple leaves | |
| Litvintseva et al. | Population structure and ecology of Cryptococcus neoformans and Cryptococcus gattii | |
| CN105505916A (en) | Method for extracting high-quality genome DNA from dry suriana maritime leaf and kit thereof | |
| CN105950611A (en) | Method for extracting surface bacterium genome of aged tobacco leaves | |
| DELBRIDGE et al. | Community profiles of bacterial endosymbionts in four species of Caulerpa | |
| CN116064501A (en) | Crab meat DNA extraction kit and extraction method | |
| Rogers et al. | DNA from ancient cedar wood from King Midas’ tomb, Turkey, and Al-Aksa Mosque, Israel | |
| CN107267498A (en) | DNA kit and method is extracted in a kind of universal material from trace plant | |
| Das et al. | Fungal communities on decaying leaves in streams: a comparison of two leaf species | |
| Reeleder et al. | The extraction of fungal DNA from multiple large soil samples | |
| CN114164183A (en) | Nocardia south Africa bacteriophage P69 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190621 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |