CN106754897A - A kind of method for extracting Wild Rosa multiflora endogenetic fungus genome - Google Patents
A kind of method for extracting Wild Rosa multiflora endogenetic fungus genome Download PDFInfo
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- CN106754897A CN106754897A CN201710153864.0A CN201710153864A CN106754897A CN 106754897 A CN106754897 A CN 106754897A CN 201710153864 A CN201710153864 A CN 201710153864A CN 106754897 A CN106754897 A CN 106754897A
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- rosa multiflora
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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 method for extracting Wild Rosa multiflora endogenetic fungus genome.After methods described is by Wild Rosa multiflora surface sterilization, liquid nitrogen grinding treatment, endogenetic fungus genomic DNA is extracted using CTAB mixed liquor combination soil microbial DNA strengths extracts kit.The present invention is designed for the particularity of Wild Rosa multiflora sample, it is to avoid contained quinones substance in Wild Rosa multiflora plant, aldehydes matter, polyphenol oxidase and the harmful effect of the factor 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, with the rate that has a wide range of application, carries it is high the characteristics of.The method that the present invention is provided 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 to all kinds of molecules experiments.
Description
Technical field
The invention belongs to genome extractive technique field, more particularly to a kind of Wild Rosa multiflora endogenetic fungus genome that extracts
Method.
Background technology
Rosa wild species are the main sources of Chinese rose genetic resources, and a modern modern rose cultivars about more than 25000 are all from 15
The continuous hybridization and backcrossing of individual 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 current Wild Rosa multiflora resource are transferred to the cultivar of modern distance education system
In, it is the Main way of breeding for disease resistance to widen its hereditary basis, cultivate disease-resistant varieties.But hybridize not close and hybrid due to existing
Infertility, pathogen biological strain have great diversity and a variability, and it is extensive introduce a fine variety after, its resistance is difficult to maintain etc. asks
Topic, so still can not thoroughly solve the problems, such as disease control by crossbreeding.
Plant endogenesis epiphyte is the important component of fungi, refers to be moved 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 relation is formd between plant：Plant environment and all kinds of nutriments for needed for endophyte provides growth,
And endophyte improves it to various then by producing miscellaneous bioactivator growing come stimulation of host plant
The resistivity of biological and abiotic stress, foreign countries are existing a large amount of on using the special of endophyte enhancing plant disease prevention and control ability
Profit, so probe into endophyte enhancing host plant has larger practice significance to the resistance of various pathogens.
It is current most important, most rapid Bacterial community and work(to analyze biological community structure using molecular biology method
One of knowable method.In the research of endophyte structure of community, conventional method is to carry out system hair using specific primer
The amplification of mark molecule is educated, and the species composition of microbiologic population is recognized by determining its sequence and its abundance is quantified.This hair
Bright middle extraction Wild Rosa multiflora endogenetic fungus genomic DNA, using 18S rRNA gene magnifications, the method for high-flux sequence, to visit
Bright disease-resistant Wild Rosa multiflora is peculiar to lay compacting with the disease-resistant functional group of advantage endogenetic fungus, and the disease-resistant molecular mechanism of Wild Rosa multiflora
Basis.
Contain the phenols chemical combination such as secondary metabolite, pigment, phenolic group, the phenolic hydroxyl groups such as a large amount of polyphenol, esters in Wild Rosa multiflora
Thing, causes that extracting genome DNA rate is low, purity is low, to follow-up molecular biology in terms of analysis cause to hinder.Research shows,
The extractable high-quality rose DNA of modified CTAB method (Yan Ting good etc., 2014), but extract high-quality Wild Rosa multiflora endogenetic fungus base
Because group DNA method not studies have reported that, and, the Nei Shengzhen that extracts poor with general reagent box and CTAB method extraction effects
The few purity of bacterium genomic DNA yield is low.
The content of the invention
In view of the shortcomings of the prior art, the present invention provides a kind of method for extracting Wild Rosa multiflora endogenetic fungus genome.This
Invention can realize the purpose of a large amount of Wild Rosa multiflora endogenetic fungus genomes of high efficiency extraction, the endogenetic fungus genomic DNA for obtaining
Purity is high, and yield is high.
Technical scheme is as follows：A kind of method for extracting Wild Rosa multiflora endogenetic fungus genome, methods described will
After Wild Rosa multiflora surface sterilization, liquid nitrogen grinding treatment, using CTAB mixed liquor combination soil microbial DNA strength extracts kits
Extract endogenetic fungus genomic DNA.
Further, the mass percent of CTAB is 1.8~2.2%, PVP (polyvinyl pyrroles in the CTAB mixed liquors
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, all of plant tissue is completely soaked, soak time is 1~2min；Sterile water wash for several times after；Plant tissue is put again
In 4~6% liquor natrii hypochloritises, it is completely soaked all of plant tissue, soak time is 30~60s；Sterile water wash
For several times, sterilization is completed.
Preferably, methods described operation is as follows：Plant tissue after surface sterilization is shredded, is placed in precooling mortar and is added
Liquid nitrogen is covered, and is firmly fully ground to fine powdered, is weighed in the CTAB mixed liquors after the powder after grinding is placed in preheating, is ground
To mixture of viscous form, mixture is vortexed after mixing, and addsSolution C1 in kit, turn upside down
Mix for several times, supernatant is transferred in new collecting pipe after vortex oscillation, centrifugation, add Solution C2, be vortexed and mix,
It is incubated, is centrifuged；Transfer supernatant adds Solution C3 in new collecting pipe, is vortexed and mixes, and is incubated, centrifugation；In transfer
It is clear to add Solution C4 in new collecting pipe, be vortexed after mixing be put into revolving filter, in, centrifugation discards filtrate, after
Continuous loading, supernatant, room temperature, centrifugation are repeated up to filter all supernatants；Solution C5 are added in revolving filter, room
Temperature centrifugation, after supernatant discarded, in transfer revolving filter to collecting pipe, is subsequently adding Solution C6 to white filter membrane center,
Centrifugation discards revolving filter；STb gene sample is obtained, preservation in -20 DEG C of refrigerator is placed, it is standby.
Further, the mass percent of CTAB is the dense of 1.8~2.2%, beta -mercaptoethanol in the CTAB mixed liquors
Spend for the concentration of 1.8~2.2% (v/v) and PVP is 9~11% (m/v).
Further, the CTAB mixed liquors are using being preceding preheated to 60~70 DEG C.
Used as further preferred, methods described concrete operations are as follows：The plant tissue powder after 0.15g grindings is weighed, is put
In a clean aseptic grinding；The CTAB mixed liquors for adding 600 μ L to be preheated to 60~65 DEG C, are fully ground to thick；Again
The mixture after grinding being collected with aseptic filter paper and being added in a PowerBead Tubes, being gently vortexed mixes, and adds 60
μ L Solution C1, turn upside down and mix for several times；PowerBead Tubes are fixed on vortex instrument adapter, 3200r/m
Vortex 10~15min of continuous oscillation, 10000 × g of room temperature are centrifuged 30~60s；Shift supernatant to a clean 2mL collecting pipe
In, in 250 μ L Solution C2 of addition to supernatant, it is vortexed and mixes 5~6s, 4 DEG C of incubation 5min, 10000 × g of room temperature centrifugations 1
~2min；Avoid precipitating globule, in the transfer new collecting pipe in supernatant≤600 μ L to, add 200 μ L Solution C3 to arrive
In supernatant, it is vortexed and mixes, 4 DEG C of incubations 5min, 10000 × g of room temperature is centrifuged 1~2min；Avoid precipitate globule, transfer supernatant≤
In 750 μ L to a new collecting pipes, in 1200 μ L Solution C4 of addition to supernatant, it is vortexed and mixes 5~6s, loading is about
To in revolving filter, 10000 × g of room temperature centrifugation 1min discard filtrate to 675 μ L of supernatant, continue to load 675 μ L of supernatant, room temperature
10000 × g is centrifuged 1~2min, is repeated up to filter all supernatants；Add 500 μ L Solution C5 to revolving filter
In, 10000 × g of room temperature is centrifuged 30~60s, and supernatant discarded, 10000 × g of room temperature is centrifuged 1~2min；Careful transfer rotating filter
Device in 2mL collecting pipes, adding 100 μ L Solution C6 to arrive white filter membrane center, 10000 × g of room temperature, 30~60s of centrifugation,
Discard revolving filter；Preserved in the refrigerator that STb gene sample is placed -20 DEG C, it is standby.
The present invention removes sample surfaces microorganism by surface sterilization, it is to avoid its surface microorganism internally gives birth to fungal gene group
DNA is polluted, and the treatment of liquid nitrogen grinding can effectively crack fungal cell wall；PVP (polyvinyl pyrroles are added in CTAB extract solutions
Alkanone) and mercaptoethanol can play a part 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 effectively be 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,
So as to improve endogenetic fungus extracting genome DNA rate.The DNA yield that the present invention is extracted is high, and purity is high, the high-quality for obtaining
DNA can be directly used for downstream experiment, without being further purified.
The present invention provide method 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 is used equally to all kinds of molecules experiments.
Compared with prior art, the invention has the advantages that：Particularity of the present invention for Wild Rosa multiflora sample
It is designed, it is to avoid contained quinones substance in Wild Rosa multiflora plant, aldehydes matter, polyphenol oxidase and the factor such as broken wall is insufficient
Harmful effect to extracting genome DNA efficiency.The method can apply to the extraction of all Wild Rosa multiflora endophyte genomes,
With the rate that has a wide range of application, carries it is high the characteristics of.
Brief description of the drawings
Fig. 1 is that liquid nitrogen grinding extracts Wild Rosa multiflora endogenetic fungus 18S rRNA gene magnifications with the method that CTAB methods are combined
Agarose gel electrophoretogram；
Fig. 2 is that liquid nitrogen grinding is extracted in Wild Rosa multiflora with the method that soil microbial DNA strength extracts kit is combined
The agarose gel electrophoretogram of raw fungi 18S rRNA gene magnifications；
Fig. 3 is that liquid nitrogen grinding combination CTAB mixed liquors extract wild with the method for soil microbial DNA strength extracts kit
The agarose gel electrophoretogram of raw rose endogenetic fungus 18S rRNA gene magnifications.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with specific embodiment, to this
The technical scheme 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 limit the invention.Other various experimental implementations involved in the present invention, are the ordinary skill in the art,
The part being not particularly illustrated in text, one of ordinary skill in the art is referred to various conventional before the present patent application day
Specification, handbook of reference book, scientific and technical literature or correlation etc. are carried out.Material, reagent used in embodiment is such as without spy
Different explanation, can be obtained, U.S. MOBIO strength soil DNA extraction kits by commercial sourcesKit (DNA Isolation Kit) buy in the invincible Science and Technology Ltd. of Shenzhen's peace.
During early stage treatment Wild Rosa multiflora sample of the present invention, all instruments for touching have to pass through autoclave sterilization, with
Exempt to bring external microbe contaminated samples into.When plant sample is ground, it is ensured that grinding great efforts, first time ground sample is to thin
It is powdered, during second grinding, it is ensured that milling time is at least 5 minutes, make sample abundant with the reaction of CTAB mixed liquors, it is milled to
Sample is thick.During using kit extraction DNA, to avoid being drawn to lower sediment thing as far as possible when collecting supernatant
Matter, can otherwise influence the purity of DNA, and all supernatants have been filtered during loading supernatant liquid filtering, can otherwise influence the acquisition of DNA
Rate.
Embodiment 1 liquid nitrogen grinding combination CTAB (cetyl trimethylammonium bromide) method 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 ethanol of mass percent concentration 70%, absolute ethyl alcohol, 5% sodium hypochlorite；The examination
The compound method of agent is as follows：
The preparation of 70% alcohol：60mL aseptic deionized waters and 140mL absolute ethyl alcohols are measured respectively, are mixed, room temperature preservation.
The preparation of 5% sodium hypochlorite：10% sodium hypochlorite of 100mL and the aseptic deionized water of 100mL are measured respectively, are mixed
Close, room temperature keeps in dark place.
The preparation of 0.5M EDTA (pH=8.0)：Take 186.1g Na2EDTA·2H2O, pH=is adjusted with NaOH (about 20g)
8.0, with the ddH of filtering2O mixes to 1L, sterilizes (121 DEG C, 15min), room temperature preservation.
The preparation of 1MTris-HCl (pH=8.0)：Tris-base 121.1g are weighed, 800mL deionizations are dissolved in
In water, add about 42mL concentrated hydrochloric acids that its pH is adjusted into 8.0, finally, plus deionized water is settled to 1L, sterilizes (121 DEG C, 15min),
Room temperature preservation.
The preparation of 2%CTAB extract solutions：Weigh 20g CTAB, 81.9g NaCl；Measure 1M Tris-HCl (pH=8.0)
Solution 100mL, 0.5M EDTA (pH=8.0) solution 40mL；Plus deionized water is settled to 1L, sterilize (121 DEG C, 15min), room
Temperature is preserved, and uses preceding (V/V) beta -mercaptoethanol of Jia 0.2%.
Other reagents can be bought by Reagent Company.
(2) surface sterilization of sample
Weigh 2g Wild Rosa multiflora samples；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 of plant tissue is completely soaked, soak time is 2min；Nothing
Bacterium water is cleaned three times；Plant tissue block is placed in 5% liquor natrii hypochloritis again, all of plant tissue is completely soaked, soaked
The bubble time is 1min；Sterile water wash five times；The plant tissue that surface sterilization will have been completed is collected in aseptic filter paper.
(3) CTAB methods extract genomic DNA
1) 2g samples are weighed, liquid feeding nitrogen covering in precooling mortar is placed in, is quickly firmly ground to powdered；
2) to adding about 600 μ l, 65 DEG C of 2%CTAB extract solutions of preheating that it is mixed with the powder after grinding in mortar；
3) liquid is transferred in the centrifuge tube of 1.5mL, the concussion that is vortexed is turned upside down after 65 DEG C of water-bath 1h per 20min
Gently mix once；
4) centrifuge tube is taken out after water-bath terminates, the phenol after 4 DEG C of isometric precoolings is added in each centrifuge tube：Chloroform：
Isoamyl alcohol (25:24:1), turn upside down after mixing, 12000r/min, is centrifuged 15min by 4 DEG C；
5) supernatant is transferred in new 1.5mL centrifuge tubes after being centrifuged, the phenol after 4 DEG C of isometric precoolings is added again：Chlorine
It is imitative：Isoamyl alcohol (25:24:1), turn upside down mixing, 12000r/min, is centrifuged 15min by 4 DEG C；
6) supernatant is transferred in new 1.5mL centrifuge tubes after being centrifuged, and to the isopropanol after -20 DEG C of precoolings of addition in supernatant
(0.6 times of volume), mixing of turning upside down is placed in and at least 2h is precipitated in -20 DEG C of refrigerator；
7) sedimentation time terminate after from refrigerator take out centrifuge tube, 12000r/min, 4 DEG C, be centrifuged 15min, take precipitation.To from
1mL is added in heart pipe, the volume fraction after -20 DEG C of precoolings is 70% ethanol, repeatedly piping and druming rinsing precipitation, 12000r/min, 4
DEG C, 15min is centrifuged, liquid is abandoned, take precipitation.Repeat this operation once；
8) centrifuge tube that will be equipped with precipitation 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) DNA after dissolving is preserved in -20 DEG C of refrigerators；
(4) the DNA genomes of 18S rRNA gene magnifications Detection and Extraction
It is template with the genome for extracting using the universal primer of 18S rRNA gene magnifications, in 53 DEG C of annealing, carries out fine jade
Sepharose electrophoresis simultaneously observes result, if it is possible to amplify the 18S rRNA genes 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：2nd, 3,4 swimming lanes do not have obvious DNA purpose bands,
Wherein 2,3, the repetitions experiment that 4 swimming lanes are three samples, the DNA extractions for showing this experiment are failures.
The liquid nitrogen grinding combination soil microbial DNA strength extracts kit of embodiment 2 extracts Wild Rosa multiflora endogenetic fungus base
Because of group
(1) preparation of reagent：
Sample pre-treatments reagent has 70% absolute ethyl alcohol and 5% sodium hypochlorite compound method with embodiment 1, and DNA extracts examination
Agent box is usedKit,Contain Solution C1~Solution C6 in kit.
(2) surface sterilization of sample
Weigh 2g Wild Rosa multiflora samples；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 of plant tissue is completely soaked, soak time is 1~2min；
Sterile water wash three times；Plant tissue block is placed in 5% liquor natrii hypochloritis again, all of plant tissue is completely soaked,
Soak time is 30~60s；Sterile water wash five times；The plant tissue that surface sterilization will have been completed is collected in aseptic filter paper.
(3) method being combined with kit is processed using liquid nitrogen grinding and extracts fungal gene group
The sample of surface sterilization is placed in precooling mortar the covering of liquid feeding nitrogen, is firmly fully ground that (liquid feeding nitrogen grinds repeatedly
Mill) to fine powdered.
STb gene is extracted using soil microbial DNA strength extracts kit, concrete operation step is as follows：
1) powder after 0.15g grindings is added in a PowerBead Tubes；
2) gently it is vortexed and mixes；
3) Solution C1 are detected, if there is precipitation, 60 DEG C of water-baths to CL；
4) 60 μ l Solution C1 are added, is turned upside down and mix for several times；
5) PowerBead Tubes are 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 in a clean 2mL Collection Tube (kit offer)；
8) 250 μ l Solution C2 are added to mix 5s in supernatant, being vortexed, 4 DEG C of incubation 5min；
9) 10000 × g of room temperature is centrifuged 1~2min
10) avoid precipitating globule, in the transfer new collecting pipe in supernatant≤600 μ l to
11) add 200 μ l Solution C3 in supernatant, be vortexed and mix, 4 DEG C of incubation 5min
12) 10000 × g of room temperature is centrifuged 1~2min；
13) avoid precipitating globule, in the transfer new collecting pipe in supernatant≤750 μ l to；
14) add 1200 μ l Solution C4 (Solution C4 are first shaken up using preceding) in supernatant, be vortexed and mix
5s；
15) about 675 μ l supernatants are loaded in Spin Filter, 10000 × g of room temperature is centrifuged 1~2min；Filtrate is discarded, after
675 μ l supernatants of continuous loading, 10000 × g of room temperature is centrifuged 1~2min；It is repeated up to filter all supernatants；
16) add in 500 μ l Solution C5 to Spin Filter, 10000 × g of room temperature is centrifuged 30~60s；
17) supernatant discarded；
18) 10000 × g of room temperature is centrifuged 1~2min；
19) in careful transfer Spin filter to 2mL Collection Tube (kit offer), avoid as far as possible
Solution C5 pollute；
20) 100 μ l Solution C6 to white filter membrane center are added；
21) 10000 × g of room temperature is centrifuged 30~60s；
22) Spin Filter are discarded.Preserved in the refrigerator that STb gene sample is placed -20 DEG C, it is standby；
(4) the DNA genomes of 18S rRNA gene magnifications Detection and Extraction；
It is template with the genome for extracting using the universal primer of 18S rRNA gene magnifications, in 53 DEG C of annealing, carries out fine jade
Sepharose electrophoresis simultaneously observes result, if it is possible to amplify the 18S rRNA genes of fungi, it was demonstrated that be successfully extracted genome.
By Fig. 2 it can be found that：2nd, 3,4 swimming lanes have 18S rRNA gene magnification purpose bands, but not enough substantially, size is about
It is 500bp, wherein 2,3,4 swimming lanes are three repetition experiments of sample, shows that the DNA yield that this experiment is obtained is relatively low.
The liquid nitrogen grinding combination CTAB mixed liquors of embodiment 3 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 extract solutions, 70% nothing
Water-ethanol, 5% sodium hypochlorite, CTAB mixed liquors, 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 extract solutions, 70% absolute ethyl alcohol, 5% time
The compound method of sodium chlorate is with described in embodiment 1
The preparation of the CTAB mixed liquors：2%CTAB extract solution 200mL are measured, 20gPVP is added, sterilize (115 DEG C,
15min), room temperature preservation, uses the beta -mercaptoethanol of preceding plus 4mL.
Other reagents can be provided by kit or bought by Reagent Company.
(2) surface sterilization of sample
Weigh 2g Wild Rosa multiflora samples；Prepare 70% alcohol and 5% liquor natrii hypochloritis is dispensed to two aseptic burnings of 250mL
In cup；Plant tissue is placed in 70% alcohol, all of plant tissue is completely soaked, soak time is 1~2min；It is aseptic
Water is cleaned three times；Plant tissue block is placed in 5% liquor natrii hypochloritis again, all of plant tissue is completely soaked, soaked
Time is 30~60s；Sterile water wash five times；The plant tissue that surface sterilization will have been completed is collected in aseptic filter paper.
(3) liquid nitrogen grinding treatment sample
The plant tissue of surface sterilization is shredded, liquid feeding nitrogen covering in precooling 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 the above-mentioned grindings of 0.15g is weighed, is placed in a clean aseptic grinding.
2) add 600 μ l to be preheated to 65 DEG C of CTAB mixed liquors, it is mixed with the powder after grinding, be fully ground to viscous
Thick shape, then collect the sample after grinding with aseptic filter paper.
3) mixture after above-mentioned grinding is added in a PowerBead Tubes.
4) gently it is vortexed and mixes.
5) Solution C1 are detected, if there is precipitation, 60 DEG C of water-baths are to being completely dissolved.
6) 60 μ l Solution C1 are added, is turned upside down and mix for several times.
7) PowerBead Tubes are fixed on vortex instrument adapter, maximum (top) speed (3200r/min) is vortexed and continuously shakes
Swing 10~15min.
8) 10000 × g of room temperature is centrifuged 30~60s.
9) transfer supernatant is in a clean 2mL Collection Tube.
10) 250 μ l Solution C2 are added to mix 5s in supernatant, being vortexed, 4 DEG C of incubation 5min.
11) 10000 × g of room temperature is centrifuged 1~2min.
12) avoid precipitating globule, in the transfer new collecting pipe in supernatant≤600 μ l to.
13) add 200 μ l Solution C3 in supernatant, be vortexed and mix, 4 DEG C of incubation 5min.
14) 10000 × g of room temperature is centrifuged 1~2min.
15) avoid precipitating globule, in the transfer new collecting pipe in supernatant≤750 μ l to.
16) add 1200 μ l Solution C4 in supernatant, be vortexed and mix 5s.
17) about 675 μ l supernatants are loaded in Spin Filter, 10000 × g of room temperature is centrifuged 1~2min；Filtrate is discarded, after
675 μ l supernatants of continuous loading, 10000 × g of room temperature is centrifuged 1~2min；It is repeated up to filter all supernatants.
18) add in 500 μ l Solution C5 to Spin Filter, 10000 × g of room temperature is centrifuged 30~60s.
19) supernatant discarded.
20) 10000 × g of room temperature is centrifuged 1~2min.
21) in careful transfer Spin filter to 2mL Collection Tube, avoid Solution C5 dirty as far as possible
Dye.
22) 100 μ l Solution C6 to white filter membrane center are added.
23) 10000 × g of room temperature is centrifuged 30~60s.
24) Spin Filter are discarded.
(5) the DNA genomes of 18S rRNA gene magnifications Detection and Extraction
It is template with the genome for extracting using the universal primer of 18S rRNA gene magnifications, in 53 DEG C of annealing, carries out fine jade
Sepharose electrophoresis simultaneously observes result, if it is possible to amplify the 18S rRNA genes of fungi, it was demonstrated that be successfully extracted genome.
By Fig. 3 it can be found that：2nd, 3,4 swimming lanes have obvious 18S rRNA gene magnification purpose bands, and size is about 500bp, 2,3,4 swimming
Road is three repetition experiments of sample, and it is successful to show that the DNA of this experiment is extracted, and DNA recovery rates are high.
The genomic DNA purity of 1~embodiment of comparing embodiment 3, concentration and OTU numbers, as a result as shown in table 1.
Table 1
| Extracting method | Purity (OD260/OD280) | Concentration (ng/ μ l) | OTU numbers (individual) |
| 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 less than this scope, higher than this scope
Show to contain RNA in sample, therefore use the extracting method DNA purity that the present invention is provided higher.By can be seen that embodiment 3 in table 1
DNA concentration be higher than embodiment 1 and embodiment 2.For OUT numbers, OUT numbers are more, illustrate to extract species gene group DNA
Abundanter, covering monoid is wider, more can truly reflect the abundance of endophyte in plant sample.By being also seen that embodiment 3 in table 1
OUT numbers far more than embodiment 1 and embodiment 2.
Embodiment 4
On the basis of embodiment 3, change the composition of CTAB mixed liquors, be added without PVP.CTAB mixed liquors collocation method is such as
Under：2%CTAB extract solution 200mL are measured, preceding addition 4mL mercaptoethanols are used.Other extraction process with embodiment 3, using 18S
The universal primer of rRNA gene magnifications, is template with the genome for extracting, and in 53 DEG C of annealing, enters row agarose gel electrophoresis and sees
Examine result, if it is possible to amplify the 18S rRNA genes of fungi, it was demonstrated that be successfully extracted genome.But in gel electrophoresis figure,
The purpose band of 18S rRNA gene magnifications is not obvious, and the DNA yield for extracting is relatively low, therefore extracts Wild Rosa multiflora genome
PVP is essential during DNA.
Embodiment 5
On the basis of embodiment 3, change the composition of CTAB mixed liquors, be added without mercaptoethanol.CTAB mixed liquors are configured
Method is as follows：2%CTAB extract solution 200mL are measured, 20gPVP is added, sterilized (115 DEG C, 15min), room temperature preservation.Other are carried
Process is taken with embodiment 3, is template with the genome for extracting using the universal primer of 18S rRNA gene magnifications, moved back at 53 DEG C
Fire, enters row agarose gel electrophoresis and observes result, if it is possible to amplify the 18S rRNA genes of fungi, it was demonstrated that successfully extract
Genome.But in gel electrophoresis figure, the purpose band of 18S rRNA gene magnifications is more apparent, but band brightness is not as good as embodiment
3, illustrate that the DNA yield for extracting is general, measured purity is relatively low, therefore extracts CTAB during Wild Rosa multiflora genomic DNA and mix
Mercaptoethanol is preferably also added in conjunction liquid.
Claims (8)
1. a kind of method for extracting Wild Rosa multiflora endogenetic fungus genome, it is characterised in that methods described is by Wild Rosa multiflora surface
After sterilization, liquid nitrogen grinding treatment, endogenetic fungus are extracted using CTAB mixed liquor combination soil microbial DNA strengths extracts kit
Genomic DNA.
2. the method for extracting Wild Rosa multiflora endogenetic fungus genome as claimed in claim 1, it is characterised in that the CTAB is mixed
The mass percent for closing CTAB in liquid is that the concentration of 1.8~2.2%, PVP is 9~11% (m/v).
3. the method for extracting Wild Rosa multiflora endogenetic fungus genome as claimed in claim 1, it is characterised in that the soil is micro-
Biological DNA strengths extracts kit isKit.
4. the method for extracting Wild Rosa multiflora endogenetic fungus genome as claimed in claim 1, it is characterised in that the wild rose
Common vetch surface sterilization operation is as follows：Wild Rosa multiflora plant tissue is placed in 70% alcohol, is completely soaked plant tissue, during immersion
Between be 1~2min, sterile water wash for several times after；Plant tissue is placed in 4~6% liquor natrii hypochloritises again, makes plant tissue
It is completely soaked, soak time is 30~60s, sterile water wash for several times, completes sterilization.
5. the method for extracting Wild Rosa multiflora endogenetic fungus genome as claimed in claim 4, it is characterised in that methods described is grasped
Make as follows：Plant tissue after surface sterilization is shredded, liquid feeding nitrogen covering in precooling mortar is placed in, is firmly fully ground to fine powder
Last shape, weighs in the CTAB mixed liquors after the powder after grinding is placed in preheating, is ground to mixture of viscous form, and mixture is vortexed mixed
After even, addSolution C1 in kit, turn upside down and mix for several times, will be upper after vortex oscillation, centrifugation
Clear liquid is transferred in new collecting pipe, adds Solution C2, is vortexed and is mixed, and is incubated, is centrifuged；Shift supernatant to new receipts
In collector, Solution C3 are added, be vortexed and mix, be incubated, centrifugation；Transfer supernatant adds Solution in new collecting pipe
C4, be vortexed after mixing be put into revolving filter, in, centrifugation discards filtrate, continues to load, supernatant, and room temperature, centrifugation are repeated up to
All supernatants are filtered；Add Solution C5 in revolving filter, room temperature centrifugation after supernatant discarded, shifts rotating filter
Device discards revolving filter to Solution C6 to white filter membrane center, centrifugation in collecting pipe, is subsequently adding；Obtain STb gene sample
Product, place preservation in -20 DEG C of refrigerator, standby.
6. the method for extracting Wild Rosa multiflora endogenetic fungus genome as claimed in claim 5, it is characterised in that the CTAB is mixed
The mass percent for closing CTAB in liquid is that the concentration of 1.8~2.2%, beta -mercaptoethanol is the dense of 1.8~2.2% (v/v) and PVP
It is 9~11% (m/v) to spend.
7. the method for extracting Wild Rosa multiflora endogenetic fungus genome as claimed in claim 5, it is characterised in that the CTA B
Mixed liquor is using being preceding preheated to 60~70 DEG C.
8. as described in claim 1~7 is any extraction Wild Rosa multiflora endogenetic fungus genome method, it is characterised in that institute
State method concrete operations as follows：The plant tissue powder after 0.15g grindings is weighed, is placed in a clean aseptic grinding；Add
600 μ L are preheated to 60~65 DEG C of CTAB mixed liquors, are fully ground to thick；Again the mixing after grinding is collected with aseptic filter paper
Thing is simultaneously added in a PowerBead Tubes, and being gently vortexed mixes, and adds 60 μ L Solution C1, and turn upside down number
Secondary mixing；PowerBead Tubes are fixed on vortex instrument adapter, 3200r/m vortex 10~15min of continuous oscillation, room
10000 × g of temperature is centrifuged 30~60s；In transfer supernatant to a clean 2mL collecting pipe, 250 μ L Solution C2 are added to arrive
In supernatant, it is vortexed and mixes 5~6s, 4 DEG C of incubations 5min, 10000 × g of room temperature is centrifuged 1~2min；Avoid precipitating globule, in transfer
In μ L to clearly≤600 new collecting pipe, in 200 μ L Solution C3 of addition to supernatant, it is vortexed and mixes, 4 DEG C of incubations
5min, 10000 × g of room temperature are centrifuged 1~2min；Avoid precipitating globule, in the transfer new collecting pipe in supernatant≤750 μ L to,
Add 1200 μ L Solution C4 in supernatant, be vortexed and mix 5~6s, load about 675 μ L of supernatant in revolving filter, room
10000 × g of temperature is centrifuged 1min, discards filtrate, continues to load 675 μ L of supernatant, and 10000 × g of room temperature is centrifuged 1~2min, repeats straight
To having filtered all supernatants；Add 500 μ L Solution C5 in revolving filter, 10000 × g of room temperature is centrifuged 30~60s,
Supernatant discarded, 10000 × g of room temperature is centrifuged 1~2min；Careful transfer revolving filter adds 100 μ L in 2mL collecting pipes
To white filter membrane center, 10000 × g of room temperature is centrifuged 30~60s, discards revolving filter Solution C6；By STb gene sample
Saved backup in the refrigerator for placing -20 DEG C.
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