CN103834728A - Method for amplification of endophytic fungi ITS gene in plant tissue and used primers - Google Patents

Abstract

The invention discloses primers for amplification of an endophytic fungi ITS gene in plant tissue. The primers include a nested PCR first round amplification primer pair (a forward primer NSA3, and a reverse primer NLC2) and a nested PCR second round amplification primer pair. The invention also provides a method for amplification of the endophytic fungi ITS gene in plant tissue by the primers. The method consists of: extraction of a plant tissue genome and PCR amplification on the extracted template DNA, with the amplification including nested PCR first round amplification and nested PCR second round amplification. The method can acquire ITS gene fragments of fungi in plant tissue with high quality, and lays the foundation for accurate analysis of the biological diversity of plant endophytic fungi.

Description

Method and the primer of endogenetic fungus ITS gene in amplification plant tissue

Technical field

The technological method that the present invention relates to endogenetic fungus ITS gene in a kind of efficient amplification plant tissue, belongs to biological technical field.

Background technology

Plant endogenesis epiphyte (Endophytic fungi) is a kind of special mycosymbiosis body, can field planting at various histoorgans such as root, stem, leaf, flower, seeds, but do not cause the obvious illness of host.Large quantity research has confirmed containing abundant endogenetic fungus monoid in plant tissue, is the important component part of global fungal diversity.Have scholarly forecast, global plant endogenesis epiphyte species quantity has 100-130 ten thousand (Dreyfuss and Chapela, 1994 at least; Ganley et al., 2004).Endogenetic fungus has the functional diversity of height equally, maintaining structure of plant community stability, promote Decomposition of leaf litter, collaborative plant-growth and the aspect such as degeneration-resistant brought into play very important effect.Meanwhile, the natural resource treasure-house of endogenetic fungus or active result.

One of important proposition of microbial ecology is species diversity and the environment responding mechanism thereof of microbe groups in research specific sample.Microorganism separates, culture & identification is the multifarious traditional method of microorganisms, also uses till today always.But, can culturing micro-organisms account for the also less than 1% of ratio of total microorganism, be therefore difficult to the overall picture of biological community structure in reaction environment sample.Along with the fast development of molecular biology and DNA sequencing technology, utilizing high-sensitive Molecular tools to detect microbial diversity (environment PCR method) has become a strong instrument in microbial ecology research field.As PCR-DGGE(denaturing gradient gel electrophoresis), Clone library(clone bank builds) and the technological method such as high-flux sequence (pyrosequencing, as Roche 454GS FLX and the Illumina MiSeq platform etc.) environmental sample such as enteron aisle, ight soil and water body that is just progressively applied to forest and agricultural land soil, deep-sea bed mud, human body and animal carry out Analysis of Microbial Diversity.

The various histoorgans of plant are also the desirable habitats of microorganism growth procreation, but the understanding of the breadth and depth to endophyte of plant species diversity is still very limited at present.Major cause is that above-mentioned Molecular Detection means all depend on the extraction to particular studies sample total genomic dna, the DNA that contains a large amount of plant origins in the DNA sample of preparation.And in total gene DNA of the samples such as soil, bed mud and enteron aisle movement, non-microorganism source genome accounts for less ratio.That is, in the time extracting plant tissue total genomic dna, the endogenetic fungus DNA wherein containing only accounts for few ratio, and in total gene DNA sample of the samples such as soil, bed mud and enteron aisle movement, the ratio that fungi accounts for other microbial DNAs is just relative high.Therefore use fungi universal primer from total genomic dna, increase fungic origin gene product (normally for the conservative gene SSU of Phylogenetic Analysis LSU ITS etc.) success ratio very low, the gene fragment that can amplify on the contrary plant (host) source, has produced non-specific amplification.This is by quality and the analytical results of remarkably influenced late gene sequence.The transcribed spacer (Internal Transcribed Spacer, ITS) of at present generally acknowledged fungi can effectively be distinguished fungi monoid in the level of planting, and is therefore the desirable barcode of research fungal diversity.

Summary of the invention

The technical problem to be solved in the present invention is to provide method and the primer of endogenetic fungus ITS gene in a kind of plant tissue that can increase; The invention belongs to a kind of efficiently increase method of two fragments of fungi ITS in plant tissue of two group-specific primerses of utilizing.

In order to solve the problems of the technologies described above, the invention provides the primer of endogenetic fungus ITS gene in a kind of plant tissue that can increase,

Described primer comprises that nest-type PRC first round amplimer is to taking turns amplimer pair with nest-type PRC second;

Described nest-type PRC first round amplimer is to being:

Forward primer NSA3:5 '-AAACTCTGTCGTGCTGGGGATA-3 ',

Reverse primer NLC2:5 '-GAGCTGCATTCCCAAACAACTC-3 ';

Described nest-type PRC second is taken turns amplimer to being following arbitrary primer pair:

①、

Forward primer ITS1-F:5 '-CTTGGTCATTTAGAGGAAGTAA-3 ',

Reverse primer ITS2:5 '-GCTGCGTTCTTCATCGATGC-3 ';

②、

Forward primer ITS1-F_KYO2:5 '-TAGAGGAAGTAAAAGTCGTAA-3 ',

Reverse primer ITS2_KYO2:5 '-TTYRCTRCGTTCTTCATC-3 ';

③、

Forward primer fITS7:5 '-GTGARTCATCGAATCTTTG-3 ',

Reverse primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ';

④、

Forward primer ITS3_KYO2:5 '-GATGAAGAACGYAGYRAA-3 ',

Reverse primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 '.

The present invention also provides the method for utilizing endogenetic fungus ITS gene in the amplification plant tissue that above-mentioned primer carries out simultaneously, comprises the genomic extraction of plant tissue, and the template DNA that extracts gained is carried out to following steps successively:

Pcr amplification:

Comprise that the nest-type PRC first round increases and nest-type PRC second is taken turns amplification;

1), the reaction system of described nest-type PRC first round amplification is:

Described forward primer is NSA3, and described reverse primer is NLC2;

Pcr amplification reaction condition is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 58 DEG C of annealing 40s, 72 DEG C are extended 50s, 30 circulations; Last 72 DEG C are extended 10min;

2), described nest-type PRC second is taken turns the reaction system of amplification and is:

Described forward primer is any one in ITS1-F, ITS1-F_KYO2, fITS7, ITS3_KYO2, and the reverse primer of described correspondence is ITS2, ITS2_KYO2, ITS4, ITS4; All the other are with the reaction system of nest-type PRC first round amplification;

Pcr amplification reaction condition is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 53 DEG C of annealing 40s, 72 DEG C are extended 50s, 30 circulations; Last 72 DEG C are extended 10min.

, above-mentioned 2 pcr amplification reaction conditions are: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 53 DEG C of annealing (wherein the annealing temperature of NSA3-NLC2 primer is 58 DEG C) 40s, 72 DEG C are extended 50s, 30 circulations; Last 72 DEG C are extended 10min.

The present invention is directed to the bottleneck of prior art, obtain the combination of primers of two ITS gene fragments (ITS1 and ITS2) the plant tissue that can efficiently increase from existing fungi ITS conserved regions universal primer kind screening, and provided corresponding test parameter and PCR reaction conditions.

Purposes and the innovation of maximum of the present invention are: utilize two-wheeled nest-type PRC can specific amplification plant tissue in the ITS gene of fungi, thereby fungal species is identified and analyte species diversity; If only take turns PCR with one, the fragment overwhelming majority who amplifies is the ITS sequence of plant origin, and reason is that the DNA of plant-sourced in genome DNA sample has disturbed the amplification efficiency of fungi ITS gene.

The invention has the advantages that: the present invention carries out the amplification of two-wheeled nest-type PRC with two groups of fungi specificity ITS transcribed spacer primers, final ITS1 and the ITS2 fragment that efficiently amplifies endogenetic fungus in root system of plant and leaf tissue, having solved the significant non-specific amplification (plant-sourced ITS) this technical bottleneck and the difficult problem that in current plant endogenesis epiphyte (environmental sample) high-flux sequence, run into, is an important innovations of utilizing in the research of high throughput sequencing technologies research environment sample fungal diversity.Adopt the method can high quality to obtain the ITS gene fragment of fungi in plant tissue, lay the first stone for accurately resolving Biological Diversity of Endophytic Fungi.

Brief description of the drawings

Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.

Fig. 1 is the conventional primer of amplification fungi rrna transcribed spacer (ITS) and site; For according to Ihrmark et al., 2012; Toju et al., 2012 amendment and obtain.

Fig. 2 utilizes ITS1-F-ITS2 and two groups of primers of fITS7-ITS4 directly increase root system (R), blade (L), rhizosphere soil (RS) and non-rhizosphere soil DNA sample (BS) acquired results figure.M:DNA Marker(labeled nucleic acid molecule).

Fig. 3 utilizes NSA3-NLC2 primer to carry out the first round amplification acquired results figure of nest-type PRC.M:DNA Marker(labeled nucleic acid molecule).

Fig. 4 be PCR product taking first round amplification as template, utilize four groups of primers such as ITS1-F-ITS2, ITS1-F_KYO2-ITS2_KYO2, ITS3_KYO2-ITS4 and fITS7-ITS4 to carry out second of nest-type PRC and take turns amplification acquired results figure.

L3-2 represents the repeat amplification protcol for the second time of leaves genomic DNA;

L3-1 represents the amplification for the first time of leaves genomic DNA sample;

R3-2 represents the repeat amplification protcol for the second time of root system genomic dna;

R3-1 represents the amplification for the first time of root system genome DNA sample;

M:DNA Marker(labeled nucleic acid molecule).

Remarks explanation:

Right side represents ITS1 fragment, and left side represents ITS2 fragment.Due to ITS1-F-ITS2; The binding site (position) of two groups of primers of ITS1-F_KYO2-ITS2_KYO2 is very approaching, and the clip size therefore amplifying is also similar; And the primer binding site of fITS7 and ITS3_KYO2 position differ 50 bp left and right, therefore fragment length is slightly variant.

Fig. 5 utilizes directly the increase BLAST result of PCR product cloning sequence of root system, leaf DNA of ITS1-F-ITS2 and two groups of primers of fITS7-ITS4.

Fig. 6 is after two-wheeled nest-type PRC, the BLAST result of extension amplification outcome sequence.

Embodiment

For realizing the object of the invention, take following technical scheme:

Embodiment 1,

One, the method for the genomic extraction of plant tissue:

Use the DNeasy Plant Mini Kit test kit of QIAGEN company and do some fine settings.Concrete steps are as follows:

1, the blade of halophytes-Suaeda salsa (Suaeda salsa) (sample number into spectrum: L3) 20mg or root system (sample number into spectrum: R3) 20mg being put into mortar carries out cooled with liquid nitrogen and is ground to Powdered.Proceed as follows respectively:

2, add 400 μ l BufferAP1 and 4 μ l RNaseA.After mixing, put into 65 DEG C of water-baths 40 minutes, slightly put upside down and mix 3 times during this time.(before use, BufferAP1 and RNaseA not being mixed).

3, add 130 μ l BufferP3, after mixing, place on ice 5 minutes; Obtain lysate.

4, by lysate at 20000g(14000rpm) lower centrifugal 5 minutes.

5, draw supernatant liquor to QIAshredder centrifugal column, insert in new 2ml centrifuge tube, 20000g filters after centrifugal 2 minutes.

6, filtrate is transferred in new centrifuge tube, adds the Buffer AW1 of 1.5 times, piping and druming immediately mixes.

7, draw 650 μ l mixed solutions to the micro-centrifugal column of DNeasy, insert in new 2ml centrifuge tube >=6000g(>=8000rpm) centrifugal 1 minute, abandon filtrate.

8, micro-DNeasy centrifugal column is put in a new 2ml centrifuge tube, adds 500ul Buffer AW2, centrifugal 1 minute of >=6000g, abandons filtrate.

9, add again 500 μ l BufferAW2, centrifugal 2 minutes of 20000g.

10, carefully from centrifuge tube, taking out centrifugal adsorbing column (do not allow the filtrate of centrifugal adsorbing column bottom below touching) is transferred in the centrifuge tube of a new 1.5ml or 2ml.

11, add 100ulBufferAE, room temperature (15-25 DEG C) is lower places 5 minutes, centrifugal 1 minute of >=6000g.

12, the solution obtaining (being the filtrate of the centrifugal gained of step 11) is rejoined in the micro-centrifugal column of DNeasy, room temperature is placed 2 minutes, centrifugal 1 minute of >=6000g.

13, DNA sample (, the filtrate of the centrifugal gained of step 12) concentration determination: use Quawell Q5000 ultramicron nucleic acid-protein determinator.The concentration recording is: R3=10.5ng/ μ l; L3=12.6ng/ μ l.

14, DNA sample retention is for subsequent use at-20 DEG C.

Remarks explanation:

Below in experiment, with rhizosphere soil DNA(RS, concentration be 30.2ng/ μ l) and non-rhizosphere soil DNA(SS, concentration is l) positive contrast of 7.8ng/ μ.

Two, PCR system and reaction conditions:

By following component preparation PCR reaction solution (50 μ l system);

Remarks explanation: in above-mentioned system, except forward primer, reverse primer, template DNA, all the other all can be purchased from precious biotechnology (Dalian) company limited.

Nest-type PRC is taking first round pcr amplification product as template, that is, second takes turns amplified production taking the first round as template.

Pcr amplification reaction condition is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 53 DEG C of annealing (wherein the annealing temperature of NSA3-NLC2 primer is 58 DEG C) 40s, 72 DEG C are extended 50s, 30 circulations; Last 72 DEG C are extended 10min.On MyCyclerPCR amplification instrument (Bio-Rad company), carry out.

Remarks explanation: except NSA3-NLC2 primer, no matter all the other are in the method one (takes turns PCR directly increases) or method two (amplification of two-wheeled nest-type PRC) of the following stated, are 53 DEG C.

The primer title relating in the present invention and sequence following (concrete site information is shown in Fig. 1):

ITS1-F（5′-CTTGGTCATTTAGAGGAAGTAA-3′）

ITS2（5′-GCTGCGTTCTTCATCGATGC-3′）

NSA3（5′-AAACTCTGTCGTGCTGGGGATA-3′）

NLC2（5′-GAGCTGCATTCCCAAACAACTC-3′）

fITS7（5′-GTGARTCATCGAATCTTTG-3′）

ITS4（5′-TCCTCCGCTTATTGATATGC-3′）

NSI1（5′-GATTGAATGGCTTAGTGAGG-3′）

NLB4（5′-GGATTCTCACCCTCTATGAC-3′）

ITS1-F_KYO2（5′-TAGAGGAAGTAAAAGTCGTAA-3′）

ITS2_KYO2（5′-TTYRCTRCGTTCTTCATC-3′）

ITS3_KYO2（5′-GATGAAGAACGYAGYRAA-3′）

Specific as follows:

Wherein NSA3-NLC2 and NSI1-NLB4 amplified production have comprised the large subunit of 18S small subunit (SSU Partial Fragment)-ITS1-5.8S-ITS2-28S (LSU Partial Fragment); ITS1F-ITS2 and ITS1-F_KYO2-ITS2_KYO2 amplified production are ITS1 region; FITS7-ITS4 and ITS3_KYO2-ITS4 amplified production are ITS2 region (referring to Fig. 1).

Test design is carried out according to following two methods:

(1), one take turns PCR and directly increase

ITS1 fragment amplification combination of primers (totally 2 groups): ITS1-F-ITS2; ITS1-F_KYO2-ITS2_KYO2.ITS2 fragment amplification combination of primers (totally 2 groups): fITS7-ITS4; ITS3_KYO2-ITS4.

(2), two-wheeled nest-type PRC amplification

First round nest-type PRC combination of primers (totally 2 groups): NSA3-NLC2; NSI1-NLB4.Second to take turns nest-type PRC combination of primers the same; That is:

ITS1 fragment amplification combination of primers (totally 2 groups): ITS1-F-ITS2; ITS1-F_KYO2-ITS2_KYO2.ITS2 fragment amplification combination of primers (totally 2 groups): fITS7-ITS4; ITS3_KYO2-ITS4.

Three, the purifying of PCR product (carrying out purifying with AxyPrep DNA Gel Extraction Kit test kit):

1, under purple lamp, cut the sepharose that contains target DNA, exhaust gel surface liquid with paper handkerchief and also shred.Calculated for gel weight (recording in advance 1.5ml centrifuge tube weight), this weight is as a gel volume (as 100mg=100ul volume).

2, add 3 (in the present invention, to use the l) BufferDE-A of gel volume of 300 μ, after mixing, in 75 DEG C of heating, be interrupted and mix (every 2-3min), until gel piece melts (about 6-3min) completely.

Note: BufferDE-A is red liquid.In melting agarose gel process, can help to observe whether completely fusing of gel.

3, the BufferDE-B(that adds 0.5 BufferDE-A volume, adds the BufferDE-B of 150 μ l), mix.In the time that the DNA fragmentation separating is less than 400bp, need add again the Virahol of 1 gel volume.Note: after adding BufferDE-B, mixture color becomes yellow, fully mixes to ensure to form the yellow solution of homogeneous.

Remarks explanation: according to design of primers site, NSA3-NLC2 amplified production clip size is 950bp, the about 800bp of amplified production fragment of NSI1-NLB4, the amplified production size of other primer is 200-250bp.

4, draw the mixed solution in step 3, transfer to DNA preparation pipe (when virgin state, this DNA prepare pipe be placed in 2ml(test kit provide) centrifuge tube) in, the centrifugal 1min of 12000g.Abandon filtrate.

5, put back 2ml centrifuge tube by preparing pipe, add the Buffer W1 of 500 μ l, the centrifugal 30s of 12000g, abandons filtrate.

6, put back 2ml centrifuge tube by preparing pipe, add the Buffer W2 of 700 μ l, the centrifugal 30s of 12000g, abandons filtrate.The Buffer W2 that adds again 700 μ l with same method washs once, the centrifugal 1min of 12000g.

Note: (1) is confirmed to add dehydrated alcohol by the designated volume on reagent bottle in Buffer W2 concentrate.

(2) use for twice Buffer W2 to rinse can guarantee that salinity is completely removed, and eliminates the impact on subsequent experimental.

7, put back in 2ml centrifuge tube the centrifugal 1min of 12000g by preparing pipe.

8, prepared by DNA to pipe clean 1.5ml centrifuge tube (providing in test kit) is provided, the filtering membrane central authorities of preparing pipe at DNA add Eluent or the deionized water of 25-30ul, and room temperature leaves standstill 1min.The centrifugal 1min(of 12000g collects filtered liquid, and what in filtrate, contain is exactly the amplified production of PCR).Eluted dna.

PCR product good purifying is connected to pGEM-T Easy carrier (Promega company), then connection product is transformed into competent cell Escherichia coli JM109(Promega company), concrete steps are carried out according to operational manual.Competent cell is coated on (ampicillin) LB flat board that contains 50 μ g/ml penbritins, (drip in advance 40 μ l3%X-gal and 7 μ l20%IPTG) and carry out blue hickie screening and obtains positive colony.Bacterium colony presents the positive colony that is of white.

On above-mentioned (ampicillin) LB flat board that contains 50 μ g/ml penbritins, the preparation method of (dripping in advance 40 μ l3%X-gal and 7 μ l20%IPTG) is:

LB solid medium, through 121 DEG C of autoclavings 15 minutes, in the time that it is cooled to 60 DEG C of left and right, adds penbritin, until the final concentration of penbritin is 50 μ g/ml, and preparation LB flat board.On flat board, add subsequently the IPTG solution that X-gal solution that 40 μ l mass concentrations are 3% and 7 μ l mass concentrations are 20%.

X-gal, the chloro-3-indoles-β-D-of the bromo-4-of 5-galactoside; IPTG, isopropylthiogalactoside.

Positive colony is identified (colony PCR) through " bacterium liquid PCR " again: picking white clone, LB liquid culture is spent the night, and (culture temperature is 37 DEG C, incubation time is 12h), drawing 1 μ l bacterium liquid verifies as DNA profiling, PCR system is that 25 μ l(are specific as follows), reaction conditions is the same, and 25 circulations are set.6-16 positive colony of last each sample picking (bacterium liquid) served Hai Shenggong order-checking, and sequencing primer is M13F(M13F:TGT AAA ACG ACG GCC AGT).LB liquid culture based formulas (1L): Tryptones 10g+ yeast powder 5g+NaCl10g.

PCR system

Forward primer is: ITS1-F, ITS1-F_KYO2, fITS7, ITS3_KYO2; Reverse primer is: ITS2, ITS2_KYO2, ITS4.

Pcr amplification reaction condition is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 53 DEG C of annealing 40s, 72 DEG C are extended 50s, 30 circulations; Last 72 DEG C are extended 10min.On MyCycler pcr amplification instrument (Bio-Rad company), carry out.

Remarks explanation: this PCR reaction is in order to verify on the pGEM-T Easy carrier in competent cell Escherichia coli JM109 whether be connected with the PCR product that purifying is good.Because to present white may be also false positive to bacterium colony sometimes.

Original series VecScreen(http: //www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html) program removes the part T carrier sequence in cloned sequence, utilize BLASTN2.2.28+ program ( http:// blast.ncbi.nlm.nih.gov/Blast.cgi) record that mates with each cloned sequence of observation analysis.

Four, interpretation of result

For method one: while directly increasing the environmental sample genomic dnas such as R3, L3, BS and SS with four groups of fungi ITS gene primer ITS1-F-ITS2, ITS1-F_KYO2-ITS2_KYO2, fITS7-ITS4, ITS3_KYO2-ITS4, two groups of primers of ITS1-F-ITS2 and fITS7-ITS4 can amplify compared with bright wisp band (Fig. 2), and two groups of primers of ITS1-F_KYO2-ITS2_KYO2 and ITS3_KYO2-ITS4 can not effectively amplify object band.Therefore, only the product of ITS1-F-ITS2 and fITS7-ITS4 amplification is tapped rubber purifying and recovery, connect product after pGEM-T Easy carrier transformed competence colibacillus cell, each amplified production selects 6-16 the positive colony through PCR checking to send company's order-checking, and sequence is verified.All sequences is carried out to blast search, find that the amplified production of ITS1-F-ITS2 and fITS7-ITS4 is the ITS gene (Fig. 5) of plant origin, Suaeda iranshahrii as fluffy in alkali etc., are fungi ITS sequence without any the matched record of sequence.

Result shows, with above two groups of primers plant genome DNA that directly increases, obvious non-specific amplification has occurred, and has also illustrated that reaction has produced and has a strong impact on plant-sourced genomic dna to PCR.

Therefore, for addressing the above problem (, in order to solve the non-specific amplification problem that in the detection of plant endogenesis epiphyte ITS gene molecule, Direct PCR brings), the present invention utilizes NSA3-NLC2 and two groups of primers of NSI1-NLB4 to carry out the first round amplification of nest-type PRC, and gene fragment can cover whole fungi ITS transcribed spacer; Carry out subsequently second taking this gene fragment as template and take turns pcr amplification, primer still uses ITS1-F-ITS2, ITS1-F_KYO2-ITS2_KYO2, fITS7-ITS4, ITS3_KYO2-ITS4.Result demonstration, NSA3-NLC2 can effectively amplify object product (the about 950bp of fragment length), and the effect of NSI1-NLB4 primer amplification is poor, without obvious band (see figure 3).By the NSA3-NLC2 purifying after gained band rubber tapping that increases, as the second template DNA of taking turns PCR.Result shows to use 4 groups of combination of primers such as ITS1-F-ITS2, ITS1-F_KYO2-ITS2_KYO2, fITS7-ITS4, ITS3_KYO2-ITS4 can amplify single bright wisp band (Fig. 4).The same, object product is verified.The result of BALST search shows that the record that totally 28 cloned sequences (at least 6 clones of every kind of amplified production) mate in GenBank database of random picking is all fungi ITS1 or ITS2 fragment (Fig. 6), as pipe is released monospore capsule bacterium (Monosporascus cannonballus) and do not cultivated root system fungi clone (Uncultured root associated fungus clone) etc.

Above result can be found out, process two-wheeled nest-type PRC, and fungi ITS gene specifically can efficiently increase; NSA3-NLC2 combination of primers can effectively be distinguished the ITS gene of fungic origin and plant-sourced, illustrates that this primer has high fungi specificity.Result of study in the present invention absolutely proves utilizes Nested PCR Technique and above combination of primers, the fungi ITS gene in plant tissue that can efficiently increase specifically, for high throughput sequencing technologies is from now on successfully applied to Biological Diversity of Endophytic Fungi and ecological study lays the foundation.

Embodiment 2, the tissue to be measured in embodiment 1 is modified to the kind benevolence tissue of tung oil tree (Vernicia fordii) by " blade (sample number into spectrum: L3) of halophytes-Suaeda salsa (Suaeda salsa) and root system (sample number into spectrum: R3) ", and only select the two-wheeled nest-type PRC amplification described in embodiment 1, all the other are equal to embodiment 1.

The result of final gained is: NSA3-NLC2 primer can amplify the product of size for 950bp left and right; Take turns nest-type PRC (4 groups of primers such as ITS1-F-ITS2, ITS1-F_KYO2-ITS2_KYO2, fITS7-ITS4, ITS3_KYO2-ITS4) through second, the object product size of acquisition is 200-250bp, carries out subsequently cloning and sequencing.Result shows that the record that totally 12 cloned sequences of picking mate in GenBank database is all fungi ITS1 or ITS2 fragment.As sickle-like bacteria (Fusarium sp.) and short stalk mould (Aureobasidium sp.) etc.

Embodiment 3,

Tissue to be measured in embodiment 1 is modified to Pinus massoniana Lamb (Pinus massoniana) needle tissue by " blade (sample number into spectrum: L3) of halophytes-Suaeda salsa (Suaeda salsa) and root system (sample number into spectrum: R3) ", and only select the two-wheeled nest-type PRC amplification described in embodiment 1, all the other are equal to embodiment 1.

The result of final gained is: NSA3-NLC2 primer can amplify the product of size for 950bp left and right; Take turns nest-type PRC (4 groups of primers such as ITS1-F-ITS2, ITS1-F_KYO2-ITS2_KYO2, fITS7-ITS4, ITS3_KYO2-ITS4) through second, the object product size of acquisition is 200-250bp, carries out subsequently cloning and sequencing.Result shows that the record that totally 8 cloned sequences of picking mate in GenBank database is all fungi ITS1 or ITS2 fragment.As speckle shell bacterium (Lophodermiumn conigenum) etc.

Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.As used the method in the present invention for the detection of (root, stem, leaf, flower, fruit etc.) fungal species in any plant tissue.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.

<110> Inst. of Subtropical Forestry, Chinese Academy of Forestry Sciences

Method and the primer of endogenetic fungus ITS gene in <120> amplification plant tissue

<160> 10

<210> 1

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> forward primer NSA3

<400> 1

aaactctgtc gtgctgggga ta 22

<210> 2

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer NLC2

<400> 2

gagctgcatt cccaaacaac tc 22

<210> 3

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> forward primer ITS1-F

<400> 3

cttggtcatt tagaggaagt aa 22

<210> 4

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer ITS2

<400> 4

gctgcgttct tcatcgatgc 20

<210> 5

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> forward primer ITS1-F_KYO2

<400> 5

tagaggaagt aaaagtcgta a 21

<210> 6

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer ITS2_KYO2

<400> 6

ttyrctrcgt tcttcatc 18

<210> 7

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> forward primer fITS7

<400> 7

gtgartcatc gaatctttg 19

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer ITS4

<400> 8

tcctccgctt attgatatgc 20

<210> 9

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> forward primer ITS3_KYO2

<400> 9

gatgaagaac gyagyraa 18

<210> 10

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer ITS4

<400> 10

tcctccgctt attgatatgc 20

Claims (2)

1. for the primer of the plant tissue endogenetic fungus ITS gene that increases, it is characterized in that:

Described primer comprises that nest-type PRC first round amplimer is to taking turns amplimer pair with nest-type PRC second;

Described nest-type PRC first round amplimer is to being:

Forward primer NSA3:5 '-AAACTCTGTCGTGCTGGGGATA-3 ',

Reverse primer NLC2:5 '-GAGCTGCATTCCCAAACAACTC-3 ';

Described nest-type PRC second is taken turns amplimer to being following arbitrary primer pair:

1., forward primer ITS1-F:5 '-CTTGGTCATTTAGAGGAAGTAA-3 ',

Reverse primer ITS2:5 '-GCTGCGTTCTTCATCGATGC-3 ';

2., forward primer ITS1-F_KYO2:5 '-TAGAGGAAGTAAAAGTCGTAA-3 ',

Reverse primer ITS2_KYO2:5 '-TTYRCTRCGTTCTTCATC-3 ';

3., forward primer fITS7:5 '-GTGARTCATCGAATCTTTG-3 ',

Reverse primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ';

4., forward primer ITS3_KYO2:5 '-GATGAAGAACGYAGYRAA-3 ',

Reverse primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 '.

2. the method for utilizing endogenetic fungus ITS gene in the amplification plant tissue that the primer described in claim 1 carries out, comprises the genomic extraction of plant tissue, it is characterized in that: the template DNA that extracts gained is carried out to following steps successively:

Pcr amplification:

Comprise that the nest-type PRC first round increases and nest-type PRC second is taken turns amplification;

1), the reaction system of described nest-type PRC first round amplification is:

Described forward primer is NSA3, and described reverse primer is NLC2;

Pcr amplification reaction condition is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 58 DEG C of annealing 40s, 72 DEG C are extended 50s, 30 circulations; Last 72 DEG C are extended 10min;

2), described nest-type PRC second is taken turns the reaction system of amplification and is:

Described forward primer is any one in ITS1-F, ITS1-F_KYO2, fITS7, ITS3_KYO2, and the reverse primer of described correspondence is ITS2, ITS2_KYO2, ITS4, ITS4; All the other are with the reaction system of nest-type PRC first round amplification;

Pcr amplification reaction condition is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 53 DEG C of annealing 40s, 72 DEG C are extended 50s, 30 circulations; Last 72 DEG C are extended 10min.

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