CN107312847A - Endophytic bacterium diversity 16SrDNA amplicon preparation methods - Google Patents
Endophytic bacterium diversity 16SrDNA amplicon preparation methods Download PDFInfo
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Abstract
The invention provides a set of primer for high-flux sequence, the primer is made up of the Barcode sequences at the primer pair 799F/1193R for endophytic bacterium 16S rDNA V5~V7 variable region fragments and the 5' ends for being connected to the primer pair 799F/1193R, wherein, the sequence of the 799F is SEQ ID NO：1, the 1193R sequence are SEQ ID NO：4, the Barcode sequences being connected with the 5' ends of the 799F are selected from SEQ ID NO：Sequence in 5 28, the Barcode sequences being connected with the 5' ends of the 1193R are selected from SEQ ID NO：Sequence in 29 52.The present invention specifically expands endophytic bacterium 16S rDNA V5~V7 variable regions by the nest-type PRC of two-wheeled, obtains the amplicon suitable for current two generations high-flux sequence platform that 5' ends carry Barcode sequences.The method provided using the present invention carries out endophytic bacterium Study on Diversity, is remarkably improved endophytic bacterium 16S rDNA amplification efficiency, greatly reduces the accounting of host data in sequencing data, reduces research cost.
Description
Technical field
The present invention relates to microbial diversity high throughput sequencing technologies field, more particularly to endophytic bacterium are various
The research of property.
Background technology
Endophytic bacterium (Endophytic bacteria) can be colonized in health plant space between cells or cell
It is interior, and set up the harmonious quasi-microorganism for combining relation with host plant.They can provide mineral matter nutritional for plant,
It can not encroached on indirect protection plant by pathogen.But in the research process for carrying out endophytic bacterium, endogenetic bacteria
Separation identification it is general relatively difficult.Set 16S rDNA specific fragments PCR is expanded to be carried out with second generation high throughput sequencing technologies
The identification of endophytic bacterium is a kind of method popular in recent years, but contains substantial amounts of host in the STb gene extracted
DNA, including substantial amounts of mitochondrial DNA and chloroplast DNA etc., the preparation to bacterial 16 S rDNA amplicon is caused greatly
Interference.Using traditional primer such as 338F/806R, 515F/907R etc. can not by bacterial 16 S rDNA and host's mitochondria or
Chloroplast DNA is efficiently separated before high-flux sequence, often leads to the presence of substantial amounts of plant host sequence in sequencing result
Row.Inventor is being carried out finding when endophytic bacterium 16S rDNA are sequenced using primer 338F/806R, 515F/907R, surveyed
Ordinal number accounting of host in is up to 59.4%~92.8%.High host data accounting will serious waste sequencing data amount,
Influence is to the multifarious analysis of endophytic bacterium.In order to obtain micro- life of certain data volume in the related research of endogenetic bacteria
Thing data, it usually needs the flux of increase sequencing, this also implies that research cost will be greatly increased.
Disclosed in document [1] using primer 799F/1492R amplification endophytic bacterium 16S rDNA V5~V8 areas, can
Effectively to reduce the amplification of host's chloroplast DNA, while the product of primer amplification host's mitochondrial DNA and bacterial 16 S rDNA
There is obvious difference in size.Document [2] is disclosed can also be realized to the expansion of host's chloroplast DNA using primer 799F/1392R
The effective reduction and the notable differentiation of mitochondrial DNA and bacterial 16 S rDNA amplicons increased.Based on the method in document [3],
The present inventor in SILVA SSU 128RefNR databases using TestPrimer instruments test primer 799F/1492R and
As a result 799F/1392R has found to the coverage of bacterium, when sequence matches (0mismatch) completely, primer 799F/1492R
It is respectively 29.2% and 69.8% to the coverage of bacterium with 799F/1392R.The sequence of the 799F is SEQ ID NO：1, institute
The sequence for stating 1492R is SEQ ID NO：3, the 1392R sequence are SEQ ID NO：2.But either primer 799F/
1492R or primer 799F/1392R, its bacterial 16 S rDNA amplicon expanded is all higher than 600bp, in conventional research
454 microarray datasets are based primarily upon to be sequenced.However, due to Roche (Roche) company, reproduction is not used for 454 microarray datasets
Reagent, causes the platform to be withdrawn from the market in 2016.At present in two generations sequencing market, the MiSeq of Illumina companies is surveyed
The sequencing reading length of sequence platform is most long, up to 300bp.But, even if being surveyed using 2 × 300 sequencing patterns of MiSeq microarray datasets
Sequence, can not also assemble the DNA fragmentation of so long (>=600bp) in the research of 16S rDNA amplicon sequencing projects.
Many research displays can also effectively reduce the amplification of host's chloroplast DNA using primer 799F/1193R, simultaneously
The separation of mitochondrial DNA and bacterial 16 S rDNA amplicons is realized, wherein the clip size of amplification mitochondrial DNA is about 600bp,
The size of bacterial 16 S rDNA amplicons is about 400bp, and it is flat that its purpose expanding fragment length is applicable to current two generations sequencing
Platform (bibliography [4] and document [5] and document [6]).But the present inventor is more using primer progress endophytic bacterium
Find that host's chloroplast number is still higher according to accounting during sample 16S rDNA sequencing researchs, reference table 1.
Bibliography:
[1].Chelius M K,Triplett E W.The Diversity of Archaea and Bacteria in
Association with the Roots of Zea mays L[J].Microbial ecology,2001,41(3):252-
263.
[2].Bai Y,Müller D B,Srinivas G,et al.Functional overlap of the
Arabidopsis leaf and root microbiota[J].Nature,2015,528(7582):364-9.
[3].Quast C,Pruesse E,Yilmaz P,et al.The SILVA ribosomal RNA gene
database project:improved data processing and web-based tools[J].Nucleic
acids research,2013,41:590-596.
[4].Bulgarelli D,Rott M,Schlaeppi K,et al.Revealing structure and
assembly cues for Arabidopsis root-inhabiting bacterial microbiota[J].Nature,
2012,488(7409):91-95.
[5].Bodenhausen N,Horton M W,Bergelson J.Bacterial communities
associated with the leaves and the roots of Arabidopsis thaliana[J].PloS one,
2013,8(2):e56329.
[6].Bulgarelli D,Garrido-Oter R,Münch P C,et al.Structure and
function of the bacterial root microbiota in wild and domesticated barley[J]
.Cell hostµbe,2015,17(3):392-403.
The content of the invention
It is an object of the present invention to which the problem of existing for more than, is used for high-flux sequence the invention provides one group
5' ends carry the primer of Barcode sequences.
Term " Barcode sequences " distinguishes the sequence label of different samples when referring to sequencing.
Further aim of the present invention is primer pair 799F/1392R and 799F/1193R is used in combination there is provided one kind,
The side of the endophytic bacterium diversity 16S rDNA amplicons suitable for current two generations microarray dataset is prepared by nest-type PRC
Method, this method can greatly reduce the accounting of host data in sequencing data.
The purpose of the present invention is realized by following technical scheme：It is described to draw for the primer of high-flux sequence
Thing is by the primer pair 799F/1193R for endophytic bacterium 16S rDNA V5~V7 variable region fragments and is connected to described draw
Thing is constituted to the Barcode sequences at 799F/1193R 5' ends, wherein, the sequence of the 799F is SEQ ID NO：1, it is described
1193R sequence is SEQ ID NO：4, the Barcode sequences being connected with the 5' ends of the 799F are selected from SEQ ID NO：5-28
In sequence, the Barcode sequences being connected with the 5' ends of the 1193R be selected from SEQ ID NO：Sequence in 29-52.
Further, present invention offer is a kind of is applied to using above-mentioned primer and primer pair 799F/1392R progress preparations
The method of the endophytic bacterium diversity 16S rDNA amplicons of two generation microarray datasets, including：
Step one：Sample DNA is obtained, endophytic bacterium 16Ss of the primer pair 799F/1392R to the sample DNA is used
RDNA V5~V8 variable region fragments carry out the amplification of first round PCR, obtain first round pcr amplification product；The sequence of the 799F is
SEQ ID NO：1, the 1392R sequence are SEQ ID NO：2；
Step 2：Screening described first round PCR primer acquisition endophytic bacterium 16S rDNA V5~V8 using electrophoresis can
Become area's fragment；
Step 3：Using the primer, and to screen endophytic bacterium 16S rDNA V5~V8 of acquisition in step 2
Variable region fragment is template, and second is carried out to endophytic bacterium 16S rDNA V5~V7 and takes turns PCR amplifications, second is obtained and takes turns PCR
Amplified production；Wherein, the sequence of the 1193R is SEQ ID No：4, the Barcode sequences being connected with the 5' ends of the 799F
Selected from SEQ ID NO：Sequence in 5-28, the Barcode sequences being connected with the 5' ends of the 1193R are selected from SEQ ID NO：
Sequence in 29-52；
Step 4：The second wheel PCR primer, which is screened, using electrophoresis obtains the plant endogenesis that 5' ends carry Barcode sequences
Bacterial 16 S rDNA V5~V7 variable region fragments.
Further, the process of the first round PCR amplifications includes：
1) by the PCR reaction solutions expanded for first round PCR in 90-100 DEG C of pre-degeneration 2-5 minutes；
2) will be from the step 1) obtain product 90-100 DEG C be denatured 20-40 seconds；
3) will be from the step 2) obtain product 50-60 DEG C anneal 20-40 seconds；
4) will be from the step 3) obtain product 70-75 DEG C extend 1-3 minutes；
5) by the step 2) arrive step 4) process repeat 20-35 times；
6) will be from the step 5) obtain product 70-75 DEG C extend 7-15 minute, complete first round PCR expand.
Further, the component for the first round PCR PCR reaction solutions expanded includes：
Volume parts are 3-10 buffer solution；
Concentration is 2.5mM, and volume parts are 2-5 deoxynucleotide；
Concentration is 3-8 μM, and volume parts are 0.5-3 799F；
Concentration is 3-8 μM, and volume parts are 0.5-3 1392R；
Volume parts are 0.5-2 archaeal dna polymerase；
Concentration is 1-400ng/ μ l, and volume parts are 5-15 sample DNA；
The sterile deionized water of 10-20 volume parts.
Further, the electrophoresis screening process in the step 2 includes：2% fine jade of first round pcr amplification product
Sepharose electrophoresis is detected, using DNA fragmentation of the glue reclaim kit gel extraction size between 600-750bp, is made
Endophytic bacterium 16S rDNA V5~V8 variable region fragments are obtained after being reclaimed with sterile deionized water elution.
Further, the process of the second wheel PCR amplifications includes：
1) by for the PCR reaction solutions of the second wheel PCR amplifications in 90-100 DEG C of pre-degeneration 2-5 minutes；
2) will be from the step 1) obtain product 90-100 DEG C be denatured 20-40 seconds；
3) will be from the step 2) obtain product 50-60 DEG C anneal 20-40 seconds；
4) will be from the step 3) obtain product 70-75 DEG C extend 1-3 minutes；
5) by the step 2) arrive step 4) process repeat 10-20 times；
6) will be from the step 5) obtain product 70-75 DEG C extend 7-15 minute, complete second take turns PCR expand.
Further, the reaction solution that the second wheel PCR amplifications are used includes：
Volume parts are 5-15 buffer solution；
Concentration is 2.5mM, and volume parts are 3-6 deoxynucleotide；
Concentration is 3-8 μM, and volume parts are 0.5-3 799F；
Concentration is 3-8 μM, and volume parts are 0.5-3 1193R；
Volume parts are 0.5-2 archaeal dna polymerase；
16S rDNA V5~V8 variable region fragments of acquisition are screened in the step 2；
With the sterile deionized water of 10-25 volume parts.
Further, the electrophoresis screening process in the step 4 includes：Second wheel, 2% agarose of pcr amplification product
Gel electrophoresis is detected, using DNA fragmentation of the glue reclaim kit gel extraction size between 400-500bp, is gone using sterilizing
Ion water elution obtains endophytic bacterium 16S rDNA V5~V7 variable region fragments that 5' ends are connected with Barcode sequences after reclaiming.
Further, the sample DNA is the mixture of DNA of plants and endophytic bacterium, and the sample DNA is from plant
Root, stem, leaf, flower, fruit, at least one position in seed obtain.
Compared with prior art, the beneficial effects of the present invention are：The present invention, which provides one group, is used for drawing for high-flux sequence
Thing, the primer is by the primer pair 799F/1193R for endophytic bacterium 16S rDNA V5~V7 variable region fragments and company
The Barcode sequences composition at the 5' ends of the primer pair 799F/1193R is connected on, it is various that the primer is used for endophytic bacterium
The preparation of property 16S rDNA amplicons, can substantially reduce the accounting of host in sequencing data；
On the other hand, using the primer and primer pair 799F/1392R prepare the invention provides one kind and be applicable
In the method for the endophytic bacterium diversity 16S rDNA amplicons of two generation microarray datasets, the nest-type PRC that this method passes through two-wheeled
The 16S rRNA genes of bacterium in plant tissue can be specifically expanded, host data in high-flux sequence data is greatly reduced
Accounting, improves the amplification efficiency of 16S rRNA genes, can so that turning into endophytic bacterium species progress Study on Diversity
Energy.If taking turns the amplification efficiency of the plant source DNA meeting severe jamming 16S rRNA genes in PCR, genomic samples using only one.
Brief description of the drawings
Fig. 1 is that 16S rDNA amplicons of the present invention prepare schematic diagram；
Fig. 2 is first round PCR amplification detected through gel electrophoresis figure of the invention.Swimming lane DL2000 and swimming lane mark I are
DNA molecular amount is marked, and swimming lane n01~n15 is specimen in use of the present invention, and swimming lane 1CK is negative control；B show first round PCR
The band of amplified production generation, a show the band of primer 799F/1392R amplification plant mitochondiral-DNA generations；
Fig. 3 takes turns PCR amplification detected through gel electrophoresis figures for the present invention second.Swimming lane marks I to be marked for DNA molecular amount,
Swimming lane n01~n15 is specimen in use of the present invention, and swimming lane 1CK is the negative control that first round PCR is expanded, swimming lane CKH2O is second
Take turns the negative control of PCR amplifications；B show the band of the second wheel pcr amplification product generation；
Fig. 4 takes turns pcr amplification product fragment the selection result detected through gel electrophoresis figure for the present invention second.Swimming lane mark I is DNA
Molecular weight marker, road n01~n15 is specimen in use of the present invention；Generated after second wheel pcr amplification product fragment screening shown in b
Band, a show the band of primer 799F/1193R amplification plant mitochondiral-DNA generations；
Fig. 5 is present invention nest-type PRC used and host's accounting in regular-PCR sequencing data and detection OTU richness
Line chart.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical scheme be clearly and completely described, it is clear that described embodiment is a part of embodiment of the invention, rather than
Whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creative work premise
Lower obtained every other embodiment, belongs to the scope of protection of the invention.
Embodiment
A set of primer for high-flux sequence is present embodiments provided, the primer is by for endophytic bacterium 16S
The primer pair 799F/1193R of rDNA V5~V7 variable region fragments and the 5' ends for being connected to the primer pair 799F/1193R
Barcode sequences are constituted, wherein, the sequence of the 799F is SEQ ID NO：1, the 1193R sequence are SEQ ID NO：
4, the Barcode sequences being connected with the 5' ends of the 799F are selected from SEQ ID NO：Sequence in 5-28, the 5' with the 1193R
The Barcode sequences of end connection are selected from SEQ ID NO：Sequence in 29-52.
Specifically, primer sequence involved in the present invention and title are summarized as follows：
Further, above-mentioned primer and primer pair 799F/1392R are utilized the embodiments of the invention provide a kind of, carried out
The method for preparing the endophytic bacterium diversity 16S rDNA amplicons suitable for two generation microarray datasets, including：
Step one：Sample DNA is obtained, endophytic bacterium 16Ss of the primer pair 799F/1392R to the sample DNA is used
RDNA V5~V8 variable region fragments carry out the amplification of first round PCR, obtain first round pcr amplification product；The sequence of the 799F is
SEQ ID NO：1, the 1392R sequence are SEQ ID NO：2；
Step 2：Screening described first round PCR primer acquisition endophytic bacterium 16S rDNA V5~V8 using electrophoresis can
Become area's fragment；
Step 3：Using the primer, and to screen endophytic bacterium 16S rDNA V5~V8 of acquisition in step 2
Variable region fragment is template, and second is carried out to endophytic bacterium 16S rDNA V5~V7 and takes turns PCR amplifications, second is obtained and takes turns PCR
Amplified production；The sequence of the 1193R is SEQ ID NO：4, the Barcode sequences being connected with the 5' ends of the 799F are selected from
SEQ ID NO：Sequence in 5-28, the Barcode sequences being connected with the 5' ends of the 1193R are selected from SEQ ID NO：29-52
In sequence；
Step 4：The second wheel PCR primer, which is screened, using electrophoresis obtains the plant endogenesis that 5' ends carry Barcode sequences
Bacterial 16 S rDNA V5~V7 variable region fragments.
Specifically, the acquisition methods of sample DNA include in the present embodiment, and sample DNA extraction is carried out from plant tissue, is made
Use FastDNATMSPIN Kit for Soil (Cat.No.116560200, MP Bio, USA) kit (hereinafter referred to as MP
Soil kits) extracted, operated according to kit operation instructions, step has part change, comprises the following steps that：
1) take 0.5g plant tissues to be placed in 2.0ml sterile centrifugation tubes, add the sterile water washings of 1ml 30 seconds, in triplicate；
2) by from step 1) plant tissue that obtains is transferred to the 2.0ml sterile centrifugation tubes containing the sterile alcohols of 1ml 70% and washes
Wash 2 minutes；
3) will be from step 2) obtain plant tissue and be transferred to the 2.0ml containing 1ml 2.5%NaClO (contain 0.1% Tween 80)
Sterile centrifugation tube soaks 5 minutes；
4) by from step 3) plant tissue that obtains is transferred to the 2.0ml sterile centrifugation tubes containing the sterile alcohol of 1ml 70% and washes
Wash 30 seconds；
5) by from step 4) plant tissue that obtains is transferred to the 2.0ml sterile centrifugation tubes containing 1ml sterilized waters and washs 30 seconds,
In triplicate；
6) by step 5) plant tissue after processing is transferred to cracking medium (Lysing in MP soil kits
Matrix E tube) in, carry out subsequent extracted step operation according to the specification of MP soil kits.
Further, the process of first round PCR amplifications of the present invention includes：
1) by the PCR reaction solutions expanded for first round PCR in 90-100 DEG C of pre-degeneration 2-5 minutes；
2) will be from the step 1) obtain product 90-100 DEG C be denatured 20-40 seconds；
3) will be from the step 2) obtain product 50-60 DEG C anneal 20-40 seconds；
4) will be from the step 3) obtain product 70-75 DEG C extend 1-3 minutes；
5) by the step 2) arrive step 4) process repeat 20-35 times；
6) will be from the step 5) obtain product 70-75 DEG C extend 7-15 minute, complete first round PCR expand.
Further, the component of the present invention for the first round PCR PCR reaction solutions expanded includes：
Volume parts are 3-10 buffer solution；
Concentration is 2.5mM, and volume parts are 2-5 deoxynucleotide (dNTPs)；
Concentration is 3-8 μM, and volume parts are 0.5-3 799F；
Concentration is 3-8 μM, and volume parts are 0.5-3 1392R；
Volume parts are 0.5-2 archaeal dna polymerase；
Concentration is 1-400ng/ul, and volume parts are 5-15 sample DNA；
The sterile deionized water of 10-20 volume parts.
Specifically, the amplification procedure of first round PCR described in the present embodiment includes：
1) using the DNA of said extracted as template, reaction solution (the 25ul bodies of first round PCR reaction are prepared according to following component
System)：
The forward primer is 799F, and the reverse primer is 1392R；The buffer solution is biological using the full formula gold in Beijing
The product of technology (TransGen Biotech) Co., Ltd sale, name of product is：5×FastPfu
Buffer, article No. is：GK211-01, specification is 1.2ml；The archaeal dna polymerase uses the golden biotechnology of the full formula in Beijing
The product of (TransGen Biotech) Co., Ltd sale：Name of product is：FastPfu
DNAPolymerase, article No. is：AP221-01, specification is 250units.
2) the present embodiment first round pcr amplification reaction process is：The reaction solution that the first round PCR is reacted is pre- at 95 DEG C
After denaturation 3 minutes, it is denatured 30 seconds, is then annealed 30 seconds at 58 DEG C at 95 DEG C, extended 1 minute at 72 DEG C, above amplification procedure enters
27 circulations of row；It is last to extend 10 minutes at 72 DEG C, complete the amplification of first round PCR.
3) 3ul first round pcr amplification products are taken, using 2% Ago-Gel containing 1ug/ml EB 12V/cm electricity
Depress electrophoresis 30 minutes, then observed, taken pictures using Labworks image acquisition and analysis software.B show first round PCR expansions in Fig. 2
Increase production the band of thing generation, a show the band of primer 799F/1392R amplification plant mitochondiral-DNA generations.
Further, the electrophoresis screening process in the step 2 includes：2% fine jade of first round pcr amplification product
Sepharose electrophoresis is detected, using DNA fragmentation of the glue reclaim kit gel extraction size between 600-750bp, is made
Endophytic bacterium 16S rDNA V5~V8 variable region fragments are obtained after being reclaimed with sterile deionized water elution.
Specifically, the electrophoresis screening process of the fragment of first round pcr amplification product described in the present embodiment includes：
Purpose fragment is screened using 2% agarose gel electrophoresis, with reference to shown in b in Fig. 2, AxyPrep DNA gels are used
Kit (AxyPrepTMDNA Gel Extraction Kit, AP-GX-250G, AXYGEN, USA) reclaim purpose fragment.Tool
Body step is as follows：
1) 5ul is added into 25ul first round pcr amplification products and contains the 6 of 5 × fluorescent dye (5 × SYBRGreen I)
× DNA sample-loading buffers (6 × Lodding Buffer) are mixed, and are stored at room temperature 5 minutes；
2) whole 30ul samples are added in the 2% Ago-Gel loading wells for preparing, uses 30 points of 12V/cm electrophoresis
Clock；
3) take the target stripe of 650bp areas adjacents on blue light transilluminator, the blob of viscose taken is placed in cleaning
In 2.0ml centrifuge tubes, DNA recovery is then carried out by its operation instructions using AxyPrep DNA gels QIAquick Gel Extraction Kit；
Further, the process of the second wheel PCR amplifications of the present invention includes：
1) by for the PCR reaction solutions of the second wheel PCR amplifications in 90-100 DEG C of pre-degeneration 2-5 minutes；
2) will be from the step 1) obtain product 90-100 DEG C be denatured 20-40 seconds；
3) will be from the step 2) obtain product 50-60 DEG C anneal 20-40 seconds；
4) will be from the step 3) obtain product 70-75 DEG C extend 1-3 minutes；
5) by the step 2) arrive step 4) process repeat 10-20 times；
6) will be from the step 5) obtain product 70-75 DEG C extend 7-15 minute, complete second take turns PCR expand.
The reaction solution that second wheel PCR amplifications of the present invention are used includes：
Volume parts are 5-15 buffer solution；
Concentration is 2.5mM, and volume parts are 3-6 deoxynucleotide；
Concentration is 3-8 μM, and volume parts are 0.5-3 799F；
Concentration is 3-8 μM, and volume parts are 0.5-3 1193R；
Volume parts are 0.5-2 archaeal dna polymerase；
16S rDNA V5~V8 variable region fragments of acquisition are screened in the step 2；
The sterile deionized water of 10-25 volume parts.
Specifically, the second wheel PCR amplification procedures include described in the present embodiment：
1) using first round PCR recovery product as template, prepare second according to following component and take turns PCR reaction solution (50ul bodies
System)：
The forward primer is the 799F with 5'Barcode sequences, and the reverse primer is with 5'Barcode sequences
1193R；Different samples use the primer with different 5'Barcode sequences；The buffer solution is biological using the full formula gold in Beijing
The product of technology (TransGen Biotech) Co., Ltd sale, name of product is：5×FastPfu
Buffer, article No. is：GK211-01, specification is 1.2ml；The archaeal dna polymerase uses the golden biotechnology of the full formula in Beijing
The product of (TransGen Biotech) Co., Ltd sale：Name of product is：FastPfu DNA
Polymerase, article No. is：AP221-01, specification is 250units.
2) wheel of the present embodiment second pcr amplification reaction process is：The reaction solution that the first round PCR is reacted is pre- at 95 DEG C
After denaturation 3 minutes, it is denatured 30 seconds, is then annealed 30 seconds at 58 DEG C at 95 DEG C, extended 1 minute at 72 DEG C, above amplification procedure enters
13 circulations of row；It is last to extend 10 minutes at 72 DEG C, complete second and take turns PCR amplifications；
3) take 3ul second take turns pcr amplification product, using 2% Ago-Gel containing 1ug/ml EB 12V/cm electricity
Depress electrophoresis 30 minutes, then observed, taken pictures using Labworks image acquisition and analysis software.B show the second wheel PCR expansions in Fig. 3
Increase production the band that thing is produced.
Further, the electrophoresis screening process in step 4 of the present invention includes：The second wheel pcr amplification product is used
2% agarose gel electrophoresis is detected, uses DNA piece of the glue reclaim kit gel extraction size between 400-500bp
Section, is eluted using sterile deionized water and the endophytic bacterium 16S rDNA that 5' ends are connected with Barcode sequences is obtained after reclaiming
V5~V7 variable region fragments.
Specifically, the second wheel pcr amplification product fragment electrophoretic screening process includes described in the present embodiment：
The purpose fragment (in Fig. 3 shown in b) that size is about 450bp, the method same first round are screened using 2% Ago-Gel
Pcr amplification product fragment is screened.The selection result is detected using 2% agarose gel electrophoresis, b show the second wheel PCR
The band of gained after the screening of amplified production fragment electrophoretic, a show primer 799F/1392R amplification plant mitochondiral-DNA generations
Band, with reference to Fig. 4.Screening product is quantified using fluorescent dye determination, clip size correct concentration is more than or equal to
1ng/ul sample quality is judged as A, and sample quality of the concentration between 0.5 and 1ng/ul is judged as B；For clip size not
The sample quality that correct or concentration is less than 0.5ng/ul is judged as C.Quality can be carried out directly subsequently for A or B sample
Machine sequencing experiment on storehouse is built, quality need to re-start amplification for C sample and prepare, reference table 2.
Library construction and high-flux sequence
To it is above-mentioned meet build the sample of storehouse quality requirement, use kit NEXTflexTM Rapid DNA-Seq kit
(Catalog#:5144-08, Bioo Scientific, USA) sequencing library structure (described in method by specification) is carried out, then
It is sequenced using 2 × 300 sequencing patterns of MiSeq microarray datasets, each sample provides at 30,000 sequencing data
(Reads)。
Check experiment
As shown in table 1, the 5 type samples for having carried out the sequencing of endophytic bacterium diversity, every kind of sample 3 are chosen
Individual, totally 15 samples are tested.This sequencing obtains 989,495 raw sequencing datas (Raw Reads) altogether, removes
The data (Clean Reads) after 515,938 filterings are obtained in invalid sequence, for further OTU clusters and taxology
Analysis, reference table 3.Sequencing data Chloroplast and mitochondrial data accounting are counted, is found by using 799F/1392R
The method for carrying out nested PCR amplification with 799F/1193R primer pairs significantly reduces the ratio that host pollutes in sequencing data,
The richness of detection species, reference table 4 and Fig. 5 are also improved simultaneously.
The selected DNA sample information table of the present invention of table 1.
Sample is carries out specimen in use during endophyte diversity 16S rDNA sequencings in the table 1, and sample concentration is to use
The quantitative results of NanoDrop 2000.
The quantitative table of the wheel PCR primer fragment the selection result of table 2. second
The sequencing data statistical form of table 3.
The nest-type PRC of table 4. is counted with regular-PCR sequencing host accounting
Term OUT is the abbreviation of operational taxonomic unit (operational taxonomic unit), refers to system
An external node in analysis, is the taxon of a hypothesis；It is common in animals and plants kinds System developmental analysis.For example
6clone/2OTUs refers to the individual of genealogical classification analysis to be carried out, i.e., every 2 individuals to be analyzed include 6 clones.
The description to the various embodiments of the present invention with the purpose described is supplied to those skilled in the art above.It is not
Be intended to exhaustion or it is not intended to and limits the invention to single disclosed embodiment.As described above, the present invention's is various
Substitute and change will be apparent for above-mentioned technology one of ordinary skill in the art.Therefore, although specifically beg for
Some alternative embodiments have been discussed, but other embodiment will be apparent, or those skilled in the art are relative
Easily draw.It is contemplated that including all replacements of the invention discussed herein, modification and change, and fall
Other embodiment in the spirit and scope of above-mentioned application.
Although depicting the present invention by embodiment, it will be appreciated by the skilled addressee that the present invention has many deformations
With spirit of the change without departing from the present invention, it is desirable to which appended claim includes these deformations and changed without departing from the present invention
Spirit.
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SEQUENCELISTING
<110>Shanghai Major Biological Medical Technology Co., Ltd.
<120>Endophytic bacterium diversity 16S rDNA amplicon preparation methods
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<170> PatentInversion3.5
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aacmggattagataccckg19
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acgtcatccccaccttcc18
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tggcta6
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Claims (9)
1. the primer for high-flux sequence, it is characterised in that the primer is by for endophytic bacterium 16S rDNA V5
The primer pair 799F/1193R of~V7 variable region fragments and the Barcode sequences at the 5' ends for being connected to the primer pair 799F/1193R
Row composition, wherein, the sequence of the 799F is SEQ ID NO：1, the 1193R sequence are SEQ ID NO：4, it is and described
The Barcode sequences of 799F 5' ends connection are selected from SEQ ID NO：Sequence in 5-28, is connected with the 5' ends of the 1193R
Barcode sequences are selected from SEQ ID NO：Sequence in 29-52.
2. using primer as claimed in claim 1 and primer pair 799F/1392R prepare suitable for two generation microarray datasets
The method of endophytic bacterium diversity 16S rDNA amplicons, it is characterised in that including：
Step one：Sample DNA is obtained, endophytic bacterium 16Ss of the primer pair 799F/1392R to the sample DNA is used
RDNA V5~V8 variable region fragments carry out the amplification of first round PCR, obtain first round pcr amplification product；The sequence of the 799F is
SEQ ID NO：1, the 1392R sequence are SEQ ID NO：2；
Step 2：The first round PCR primer, which is screened, using electrophoresis obtains endophytic bacterium 16S rDNAV5~V8 variable regions
Fragment；
Step 3：Using the primer, and it is variable with endophytic bacterium 16S rDNAV5~V8 that acquisition is screened in step 2
Area's fragment is template, and second is carried out to endophytic bacterium 16S rDNA V5~V7 variable regions and takes turns PCR amplifications, second is obtained and takes turns
Pcr amplification product；Wherein, the sequence of the 1193R is SEQ ID NO：4, the Barcode sequences being connected with the 5' ends of the 799F
Column selection is from SEQ ID NO：Sequence in 5-28, the Barcode sequences being connected with the 5' ends of the 1193R are selected from SEQ ID NO：
Sequence in 29-52；
Step 4：The second wheel PCR primer, which is screened, using electrophoresis obtains the endophytic bacterium that 5' ends carry Barcode sequences
16S rDNA V5~V7 variable region fragments.
3. the endophytic bacterium diversity 16S rDNA amplifications as claimed in claim 2 prepared suitable for two generation microarray datasets
The method of son, it is characterised in that the process of the first round PCR amplifications includes：
1) by the PCR reaction solutions expanded for first round PCR in 90-100 DEG C of pre-degeneration 2-5 minutes；
2) will be from the step 1) obtain product 90-100 DEG C be denatured 20-40 seconds；
3) will be from the step 2) obtain product 50-60 DEG C anneal 20-40 seconds；
4) will be from the step 3) obtain product 70-75 DEG C extend 1-3 minutes；
5) by the step 2) arrive step 4) process repeat 20-35 times；
6) will be from the step 5) obtain product 70-75 DEG C extend 7-15 minute, complete first round PCR expand.
4. the endophytic bacterium diversity 16S rDNA amplifications as claimed in claim 3 prepared suitable for two generation microarray datasets
The method of son, it is characterised in that the component for the first round PCR PCR reaction solutions expanded includes：
Volume parts are 3-10 buffer solution；
Concentration is 2.5mM, and volume parts are 2-5 deoxynucleotide；
Concentration is 3-8 μM, and volume parts are 0.5-3 799F；
Concentration is 3-8 μM, and volume parts are 0.5-3 1392R；
Volume parts are 0.5-2 archaeal dna polymerase；
Concentration is 1-400ng/ul, and volume parts are 5-15 sample DNA；
The sterile deionized water of 10-20 volume parts.
5. the endophytic bacterium diversity 16S rDNA amplifications as claimed in claim 2 prepared suitable for two generation microarray datasets
The method of son, it is characterised in that the electrophoresis screening process in the step 2 includes：The first round pcr amplification product uses 2%
Agarose gel electrophoresis is detected, using DNA fragmentation of the glue reclaim kit gel extraction size between 600-750bp,
Endophytic bacterium 16S rDNA V5~V8 variable region fragments are obtained after being reclaimed using sterile deionized water elution.
6. the endophytic bacterium diversity 16S rDNA amplifications as claimed in claim 2 prepared suitable for two generation microarray datasets
The method of son, it is characterised in that the process of the second wheel PCR amplifications includes：
1) by for the PCR reaction solutions of the second wheel PCR amplifications in 90-100 DEG C of pre-degeneration 2-5 minutes；
2) will be from the step 1) obtain product 90-100 DEG C be denatured 20-40 seconds；
3) will be from the step 2) obtain product 50-60 DEG C anneal 20-40 seconds；
4) will be from the step 3) obtain product 70-75 DEG C extend 1-3 minutes；
5) by the step 2) arrive step 4) process repeat 10-20 times；
6) will be from the step 5) obtain product 70-75 DEG C extend 7-15 minute, complete second take turns PCR expand.
7. the endophytic bacterium diversity 16S rDNA amplifications as claimed in claim 2 prepared suitable for two generation microarray datasets
The method of son, it is characterised in that the reaction solution that the second wheel PCR amplifications are used includes：
Volume parts are 5-15 buffer solution；
Concentration is 2.5mM, and volume parts are 3-6 deoxynucleotide；
Concentration is 3-8 μM, and volume parts are 0.5-3 799F；
Concentration is 3-8 μM, and volume parts are 0.5-3 1193R；
Volume parts are 0.5-2 archaeal dna polymerase；
16S rDNA V5~V8 variable region fragments of acquisition are screened in the step 2；
The sterile deionized water of 10-25 volume parts.
8. the endophytic bacterium diversity 16S rDNA amplifications as claimed in claim 2 prepared suitable for two generation microarray datasets
The method of son, it is characterised in that the electrophoresis screening process in the step 4 includes：The second wheel pcr amplification product uses 2%
Agarose gel electrophoresis is detected, using DNA fragmentation of the glue reclaim kit gel extraction size between 400-500bp,
Eluted using sterile deionized water and the endophytic bacterium 16S rDNA V5 that 5' ends are connected with Barcode sequences are obtained after reclaiming
~V7 variable region fragments.
9. the endophytic bacterium diversity 16S rDNA amplifications as claimed in claim 2 prepared suitable for two generation microarray datasets
The method of son, it is characterised in that the sample DNA is the mixture of DNA of plants and endophytic bacterium, the sample DNA from
At least one position in the root of plant, stem, leaf, flower, fruit, seed is obtained.
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| CN109706149A (en) * | 2018-11-29 | 2019-05-03 | 华中农业大学 | A kind of improved 16S-seq method and its application |
| CN111518729A (en) * | 2020-05-13 | 2020-08-11 | 中国科学院遗传与发育生物学研究所 | High-flux separation culture method for crop root system microbiome |
| CN111518730A (en) * | 2020-05-13 | 2020-08-11 | 中国科学院遗传与发育生物学研究所 | Method for separating and culturing tomato root system microbiome and obtained tomato root system microbiome |
| CN112126694A (en) * | 2019-06-24 | 2020-12-25 | 中国科学院微生物研究所 | Method for analyzing endophytic bacteria flora of plants by using primers in V5V6 region |
| WO2020258084A1 (en) * | 2019-06-26 | 2020-12-30 | 深圳华大智造科技有限公司 | Method for preparing nested multiplex pcr high-throughput sequencing library and kit |
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