CN103834728B - Expand method and the primer of endogenetic fungus ITS gene in plant tissue - Google Patents

Abstract

The invention discloses for expanding the primer of endogenetic fungus ITS gene in plant tissue, this primer includes nest-type PRC first round amplimer and (forward primer NSA3, reverse primer NLC2) and nest-type PRC second is taken turns amplimer pair. The present invention also provides the method for endogenetic fungus ITS gene in the amplification plant tissue utilizing above-mentioned primer to carry out simultaneously, including the genomic extraction of plant tissue, the template DNA extracting gained is carried out pcr amplification, takes turns amplification including the amplification of the nest-type PRC first round and nest-type PRC second. Adopt the method can obtain the ITS genetic fragment of fungus in plant tissue by high-quality, lay the first stone for accurately resolving Biological Diversity of Endophytic Fungi.

Description

Expand method and the primer of endogenetic fungus ITS gene in plant tissue

Technical field

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

Background technology

Plant endogenesis epiphyte (Endophyticfungi) 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 disease of host. Numerous studies have demonstrated that and plant tissue contains abundant endogenetic fungus monoid, be the important component part of whole world fungal diversity. Have a scholarly forecast, whole world plant endogenesis epiphyte species quantity have at least 100-130 ten thousand (DreyfussandChapela, 1994; Ganleyetal., 2004). Endogenetic fungus has the functional diversity of height equally, maintaining structure of plant community stability, promote Decomposition of leaf litter, collaborative plant growing and degeneration-resistant etc. in played considerable effect. Meanwhile, the natural resources treasure-house of endogenetic fungus or activated product.

One of important proposition of microbial ecology is species diversity and the environment responding mechanism thereof of microbe groups in research specific sample. Microorganism separation, cultivation and qualification are the multifarious traditional methods of microorganisms, also use till today always. But, Bacterial diversity accounts for the ratio of total microorganism and is also likely to less than 1%, therefore the overall picture of biological community structure in very difficult reaction environment sample. Along with the fast development of molecular biology and DNA sequencing technology, utilize the strong instrument that high-sensitive Molecular tools detection microbial diversity (environment PCR method) has become as in microbial ecology research field. Such as PCR-DGGE(denaturing gradient gel electrophoresis), Clonelibrary(clone bank builds) and the technical method such as high-flux sequence (pyrosequencing, such as Roche 454GSFLX and IlluminaMiSeq platform etc.) be just progressively applied to the environmental sample such as forest and agricultural land soil, deep-sea bed mud, human body and the intestinal of animal, feces and water body and carry out Analysis of Microbial Diversity.

The various histoorgan of plant is also the desirable habitat of growth of microorganism procreation, but at present that the understanding of the breadth and depth of endophyte of plant bio-diversity is still extremely limited. Main reason is that above-mentioned Molecular Detection means all rely on the extraction to particular studies sample total genomic dna, the DNA sample of preparation contains the DNA of substantial amounts of plant origin. And in total gene DNA of the samples such as soil, bed mud and intestinal Excreta, non-microorganism source genome accounts for less ratio. That is, when extracting plant tissue total genomic dna, the endogenetic fungus DNA wherein contained only accounts for few ratio, and in total gene DNA sample of the samples such as soil, bed mud and intestinal Excreta, the ratio that fungus and other microbial DNAs account for is just relatively high. Therefore use fungus universal primer expand from total genomic dna fungal source gene outcome (be typically used for Phylogenetic Analysis conservative gene SSU LSU ITS etc.) success rate very low, the genetic fragment in plant (host) source can be amplified on the contrary, namely create non-specific amplification. This is by the quality of appreciable impact late gene sequence and analyzes result. Generally acknowledge that the transcribed spacer (InternalTranscribedSpacer, ITS) of fungus effectively can distinguish fungus monoid in the level planted at present, be therefore the ideal barcode of research fungal diversity.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of method that can expand endogenetic fungus ITS gene in plant tissue and the primer; The invention belongs to and a kind of utilize the method for two fragments of fungus ITS in two group-specific primers efficient amplification plant tissues.

In order to solve above-mentioned technical problem, the present invention provides a kind of can expand the primer of endogenetic fungus ITS gene in plant tissue,

Described primer includes nest-type PRC first round amplimer and takes turns amplimer pair to nest-type PRC second;

Described nest-type PRC first round amplimer to for:

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

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

Described nest-type PRC second takes turns amplimer to for 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 endogenetic fungus ITS gene in the amplification plant tissue utilizing above-mentioned primer to carry out simultaneously, including the genomic extraction of plant tissue, the template DNA extracting gained is sequentially carried out following steps:

Pcr amplification:

Amplification is taken turns including the amplification of the nest-type PRC first round and nest-type PRC second;

1), the reaction system of amplification of the described nest-type PRC first round 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 degeneration 30s, 58 DEG C of annealing 40s, 72 DEG C extend 50s, 30 circulations; Last 72 DEG C extend 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 degeneration 30s, 53 DEG C of annealing 40s, 72 DEG C extend 50s, 30 circulations; Last 72 DEG C extend 10min.

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

The present invention is directed to the bottleneck of prior art, obtain the combination of primers of two ITS genetic fragment (ITS1 and ITS2) energy efficient amplification plant tissue from the screening of existing fungus ITS conserved region universal primer kind, and give corresponding test parameters and PCR reaction condition.

Purposes and innovation that the present invention is maximum are in that: utilize two-wheeled nest-type PRC can the ITS gene of fungus in specific amplification plant tissue, thus carrying out fungal species identifying and analyte species diversity; If only taking turns PCR with one, then the fragment overwhelming majority amplified is the ITS sequence of plant origin, and the DNA that reason is in that in genome DNA sample plant source disturbs the amplification efficiency of fungus ITS gene.

The invention has the advantages that: the present invention carries out two-wheeled nested PCR amplification with two groups of fungus specificity ITS transcribed spacer primers, final efficient amplification goes out ITS1 and the ITS2 fragment of endogenetic fungus in root system of plant and leaf tissue, solve significant non-specific amplification (plant source ITS) this technical bottleneck and a difficult problem of running in current plant endogenesis epiphyte (environmental sample) high-flux sequence, be utilize an important innovations in high throughput sequencing technologies research environment sample fungal diversity research. Adopt the method can obtain the ITS genetic fragment of fungus in plant tissue by high-quality, lay the first stone for accurately resolving Biological Diversity of Endophytic Fungi.

Accompanying drawing explanation

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 fungus ribosome transcribed spacer (ITS) and site; For according to Ihrmarketal., 2012; Tojuetal., 2012 revise and obtain.

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

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

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

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

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

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

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

M:DNAMarker(labeled nucleic acid molecule).

Remarks illustrate:

Right side represents ITS1 fragment, and left side represents ITS2 fragment. Due to ITS1-F-ITS2; Closely, the clip size therefore amplified is also similar for the binding site (position) of two groups of primers of ITS1-F_KYO2-ITS2_KYO2; And the primer binding site of fITS7 differs 50 about bp with ITS3_KYO2 position, therefore fragment length slightly difference.

Fig. 5 is the BLAST result utilizing PCR primer cloned sequence that two groups of primers of ITS1-F-ITS2 and fITS7-ITS4 directly expand root system, leaf DNA.

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

Detailed description of the invention

For realizing the object of the invention, take techniques below scheme:

Embodiment 1,

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

Use the DNeasyPlantMiniKit test kit of QIAGEN company and do some fine settings. Specifically comprise the following steps that

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

2,400 μ lBufferAP1 and 4 μ lRNaseA are added. Putting into 65 DEG C of water-baths 40 minutes after mix homogeneously, period is reverse mixing 3 times slightly. (with BufferAP1 and RNaseA not being mixed before).

3, add 130 μ lBufferP3, place on ice after mixing 5 minutes; Obtain lysate.

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

5, Aspirate supernatant is to QIAshredder centrifugal column, inserts in new 2ml centrifuge tube, and 20000g filters after centrifugal 2 minutes.

6, filtrate is transferred in new centrifuge tube, adds the BufferAW1 of 1.5 times, blow and beat mixing immediately.

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

8, micro-for DNeasy centrifugal column is put in a new 2ml centrifuge tube, adds 500ulBufferAW2, centrifugal 1 minute of >=6000g, abandons filtrate.

9,500 μ lBufferAW2,20000g are added again centrifugal 2 minutes.

10, the careful centrifugal adsorbing column (not allowing centrifugal adsorbing column bottom touch following filtrate) that takes out from centrifuge tube is transferred in the centrifuge tube of new 1.5ml or 2ml.

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

12, rejoining in the micro-centrifugal column of DNeasy by the solution (namely step 11 is centrifuged the filtrate of gained) obtained, room temperature is placed 2 minutes, centrifugal 1 minute of >=6000g.

13, DNA sample (that is, step 12 is centrifuged the filtrate of gained) concentration measures: use QuawellQ5000 ultramicron nucleic acid-protein analyzer. The concentration recorded is: R3=10.5ng/ μ l; L3=12.6ng/ μ l.

14, DNA sample be saved in-20 DEG C standby.

Remarks illustrate:

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

Two, PCR system and reaction condition:

PCR reactant liquor (50 μ l system) is prepared by following component;

Remarks illustrate: in above-mentioned system, except forward primer, reverse primer, template DNA, and all the other are all purchased from precious biological engineering (Dalian) company limited.

Nest-type PRC is with first round pcr amplification product for template, i.e. second takes turns with the amplified production of the first round for template.

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

Remarks illustrate: except NSA3-NLC2 primer, no matter all the other are in method described below one (takes turns PCR directly expands) or method two (two-wheeled nested PCR amplification), are 53 DEG C.

The Primer related 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 includes 18S small subunit (SSU Partial Fragment) the big subunit of-ITS1-5.8S-ITS2-28S (LSU Partial Fragment); ITS1F-ITS2 and ITS1-F_KYO2-ITS2_KYO2 amplified production is ITS1 region; FITS7-ITS4 and ITS3_KYO2-ITS4 amplified production is ITS2 region (referring to Fig. 1).

EXPERIMENTAL DESIGN carries out according to following two method:

(1), one take turns PCR and directly expand

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 nested PCR amplification

First round nest-type PRC combination of primers (totally 2 groups): NSA3-NLC2; NSI1-NLB4. Second takes turns nest-type PRC combination of primers ibid; 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 purification (being purified with AxyPrepDNAGelExtractionKit test kit) of PCR primer:

1, under purple lamp, cut the agarose gel containing target DNA, exhaust gel surface liquid with napkin and shred. Calculated for gel weight (in advance record 1.5ml centrifuge tube weight), this weight is as a gel volume (such as 100mg=100ul volume).

2, add the BufferDE-A of 3 (using 300 μ l in the present invention) gel volumes, in 75 DEG C of heating after mix homogeneously, be interrupted mixing (every 2-3min), until gel piece is completely melt (about 6-3min).

Note: BufferDE-A is red liquid. In melting agarose gel process, it is possible to help to observe whether gel is completely melt.

3, add the BufferDE-B(of 0.5 BufferDE-A volume namely, add the BufferDE-B of 150 μ l), mix homogeneously. When the DNA fragmentation separated is less than 400bp, the isopropanol of 1 gel volume need to be added. Note: after adding BufferDE-B, mixture color becomes yellow, fully mixing is to ensure to form homogeneous yellow solution.

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

4, the mixed liquor in aspiration step 3, transfers to DNA preparation pipe (during initial condition, this DNA prepares pipe and is placed in the interior offer of 2ml(test kit) centrifuge tube) in, 12000g is centrifuged 1min. Abandon filtrate.

5, put back into 2ml centrifuge tube by preparing pipe, add the centrifugal 30s of BufferW1,12000g of 500 μ l, abandon filtrate.

6, put back into 2ml centrifuge tube by preparing pipe, add the centrifugal 30s of BufferW2,12000g of 700 μ l, abandon filtrate. The BufferW2 adding 700 μ l in the same way again washs once, and 12000g is centrifuged 1min.

Note: (1) confirms that the designated volume having pressed on reagent bottle in BufferW2concentrate adds dehydrated alcohol.

(2) use for twice BufferW2 to rinse can ensure that salinity is completely removed, and eliminates the impact on subsequent experimental.

7, putting back in 2ml centrifuge tube by preparing pipe, 12000g is centrifuged 1min.

8, DNA being prepared pipe and is placed in the 1.5ml centrifuge tube (test kit in provide) of cleaning, prepare, at DNA, Eluent or the deionized water that the filter membrane central authorities of pipe add 25-30ul, room temperature stands 1min. The centrifugal 1min(of 12000g collects filtrate, in filtrate containing be exactly the amplified production of PCR). Eluted dna.

The PCR primer that purification is good is connected to pGEM-TEasy carrier (Promega company), then connection product is transformed into competent cell EscherichiacoliJM109(Promega company), concrete steps carry out according to workbook. Competent cell is coated on (ampicillin) the LB flat board containing 50 μ g/ml ampicillin (dripping 40 μ l3%X-gal and 7 μ l20%IPTG in advance) carry out the screening of blue white macula and obtain positive colony. Bacterium colony present white be positive colony.

The preparation method (dripping 40 μ l3%X-gal and 7 μ l20%IPTG in advance) on above-mentioned (ampicillin) LB flat board containing 50 μ g/ml ampicillin is:

LB solid medium was through 121 DEG C of autoclavings 15 minutes, when it is cooled to about 60 DEG C, added ampicillin, until the final concentration of 50 μ g/ml of ampicillin, prepared LB flat board. The X-gal solution that 40 μ l mass concentrations are 3% and the IPTG solution that 7 μ l mass concentrations are 20% is added subsequently on flat board.

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

Positive colony carries out identifying (colonyPCR) then through " bacterium solution PCR ": picking white colonies, overnight (cultivation temperature is 37 DEG C to LB liquid culture, incubation time is 12h), draw 1 μ l bacterium solution to be verified as DNA profiling, PCR system is that 25 μ l(are specific as follows), reaction condition ibid, arranges 25 circulations. 6-16 positive colony of last each sample picking (bacterium solution) serves the raw work order-checking in sea, and sequencing primer is M13F(M13F:TGTAAAACGACGGCCAGT). LB liquid culture based formulas (1L): tryptone 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 degeneration 30s, 53 DEG C of annealing 40s, 72 DEG C extend 50s, 30 circulations; Last 72 DEG C extend 10min. MyCyclerPCR amplification instrument (Bio-Rad company) carries out.

Remarks illustrate: this PCR reaction is whether to be connected to the PCR primer that purification is good on the pGEM-TEasy carrier in order to verify in competent cell EscherichiacoliJM109. Because bacterium colony presents white and is also likely to be false positive sometimes.

Program removes the part carrier T sequence in cloned sequence original series VecScreen(http: //www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html), 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: when directly expanding the environmental sample genomic DNA such as R3, L3, BS and SS with four groups of funguses 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 relatively bright wisp band (Fig. 2), and two groups of primers of ITS1-F_KYO2-ITS2_KYO2 and ITS3_KYO2-ITS4 can not effectively amplify purpose band. Therefore, only the product that ITS1-F-ITS2 and fITS7-ITS4 expands is carried out rubber tapping purification and recovery, after product is connected pGEM-TEasy carrier, transformed competence colibacillus cell, each amplified production selects 6-16 the positive colony through PCR checking to send company to check order, and sequence is verified. All sequences is carried out blast search, it has been found that the amplified production of ITS1-F-ITS2 and fITS7-ITS4 is the ITS gene (Fig. 5) of plant origin, such as Herba suadeae glaucae Suaedairanshahrii etc., it does not have the coupling of any sequence is recorded as fungus ITS sequence.

Result shows that directly expanding plant genome DNA with above two groups of primers there occurs obvious non-specific amplification, also illustrate that PCR reaction is created by plant source genomic DNA and has a strong impact on.

Therefore, for solving the problems referred to above (namely, 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 two groups of primers of NSA3-NLC2 and NSI1-NLB4 to carry out the first round amplification of nest-type PRC, and genetic fragment can cover whole fungus ITS transcribed spacer; Carrying out second with this genetic fragment for template subsequently and take turns pcr amplification, primer still uses ITS1-F-ITS2, ITS1-F_KYO2-ITS2_KYO2, fITS7-ITS4, ITS3_KYO2-ITS4. Result shows, NSA3-NLC2 can effectively amplify purpose product (fragment length is about 950bp), and the effect of NSI1-NLB4 primer amplification is poor, without obvious band (see figure 3). NSA3-NLC2 is expanded purification after gained band is tapped rubber, as the second template DNA 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). Ibid, purpose product is verified. The result of BALST search show that the record that totally 28 cloned sequences (at least 6 clones of every kind of amplified production) of random picking are mated in GenBank data base is entirely fungus ITS1 or ITS2 fragment (Fig. 6), as monospore capsule bacterium (Monosporascuscannonballus) released by pipe and does not cultivate root system fungus and clones (Unculturedrootassociatedfungusclone) etc.

Result above, can efficient amplification fungus ITS gene specifically it can be seen that through two-wheeled nest-type PRC; NSA3-NLC2 combination of primers can effectively distinguish the ITS gene of fungal source and plant source, illustrates that this primer has high fungus specificity. Result of study in the present invention absolutely proves and utilizes Nested PCR Technique and above combination of primers, can fungus ITS gene in efficient amplification plant tissue specifically, be successfully applied to Biological Diversity of Endophytic Fungi for high throughput sequencing technologies from now on and ecological study lay the foundation.

Embodiment 2, the tissue to be measured in embodiment 1 is modified to by " blade (sample number into spectrum: L3) of halophytes-Suaeda salsa (Suaedasalsa) and root system (sample number into spectrum: R3) " the kernel tissue of Aleurites fordii Hemsl. (Verniciafordii), and only select the two-wheeled nested PCR 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 being sized to about 950bp; 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, it is thus achieved that purpose product be sized to 200-250bp, carry out cloning and sequencing subsequently. Result shows that the record that totally 12 cloned sequences of picking mate in GenBank data base is entirely fungus ITS1 or ITS2 fragment. Such as Fusarium spp. (Fusariumsp.) and short stalk mould (Aureobasidiumsp.) etc.

Embodiment 3,

Tissue to be measured in embodiment 1 is modified to Pinus massoniana Lamb (Pinusmassoniana) needle tissue by " blade (sample number into spectrum: L3) of halophytes-Suaeda salsa (Suaedasalsa) and root system (sample number into spectrum: R3) ", and only select the two-wheeled nested PCR 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 being sized to about 950bp; 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, it is thus achieved that purpose product be sized to 200-250bp, carry out cloning and sequencing subsequently. Result shows that the record that totally 8 cloned sequences of picking mate in GenBank data base is entirely fungus ITS1 or ITS2 fragment. Such as speckle shell bacterium (Lophodermiumnconigenum) etc.

Finally, in addition it is also necessary to be only several specific embodiments of the present invention it is noted that listed above. It is clear that the invention is not restricted to above example, it is also possible to there are many deformation. As the method in the present invention can be used for the detection of (root, stem, leaf, flower, fruit etc.) fungal species in any plant tissue. All deformation that those of ordinary skill in the art can directly derive from present disclosure or associate, are all considered as protection scope of the present invention.

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

<120>method and the primer of endogenetic fungus ITS gene in plant tissue are expanded

<160>10

<210>1

<211>22

<212>DNA

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aaactctgtcgtgctggggata22

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Claims (1)

1. expand a method for endogenetic fungus ITS gene in plant tissue, including the genomic extraction of plant tissue, it is characterized in that: the template DNA extracting gained is sequentially carried out following steps:

Pcr amplification:

Amplification is taken turns including the amplification of the nest-type PRC first round and nest-type PRC second;

1), the reaction system of amplification of the described nest-type PRC first round 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 degeneration 30s, 58 DEG C of annealing 40s, 72 DEG C extend 50s, 30 circulations; Last 72 DEG C extend 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 degeneration 30s, 53 DEG C of annealing 40s, 72 DEG C extend 50s, 30 circulations; Last 72 DEG C extend 10min;

Described nest-type PRC first round amplimer to for:

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

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

Described nest-type PRC second takes turns amplimer to for 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 '.