CN105039328A - Molecular specificity marker primer and method for detecting pinus massoniana infected by pisolithus tinctorius - Google Patents
Molecular specificity marker primer and method for detecting pinus massoniana infected by pisolithus tinctorius Download PDFInfo
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- CN105039328A CN105039328A CN201510432024.9A CN201510432024A CN105039328A CN 105039328 A CN105039328 A CN 105039328A CN 201510432024 A CN201510432024 A CN 201510432024A CN 105039328 A CN105039328 A CN 105039328A
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- molecular specificity
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Abstract
The invention relates to a molecular specificity marker primer for detecting pisolithus tinctorius and a method for quickly identifying pinus massoniana infected by the pisolithus tinctorius. The molecular specificity marker primer is characterized in that primer sequences include upstream primers 5'-CAGGAGGATGGACTCGCAGGGTTTC-3 and downstream primers 5'-CCATGACGTGGGCATTGCTTGAACT-3'. The molecular specificity marker primer and the method have the advantages that the pisolithus tinctorius in mycorrhizae can be quickly preliminarily identified by the molecular specificity marker primer, and the molecular specificity marker primer and the method are short in detection time and high in accuracy as compared with conventional morphologic detection and microscopic observation; the pinus massoniana infected by the pisolithus tinctorius can be detected by the aid of the method in only one day which is shorter than the time required by conventional morphologic observation and microscopic observation tests by two weeks at least.
Description
(1) technical field
The present invention relates to the molecular specificity labeled primers that sense colors beans Lasiosphaera fenzlii infects Pinus massoniana Lamb in early days, and the method utilizing this primer pair Pisolithus tinctorius to infect Pinus massoniana Lamb in early days to carry out differentiating and detecting.
(2) background technology
Mycorhiza (Mycorrhizas) symbiote with specific modality structure and function that to be fungi (fungal component) is formed with host plant root, can increase host plant to the absorption of nutrition, promote host plant growth, produce antibiotics and form the disease resistance etc. of mechanical barrier enhancing host plant.The ectomycorrhiza (Ectomycorrhiza) that fungi based on basidiomycetes and forest symbiosis are formed be forest-tree naturally support system, there is extremely important importance and functions in Forest ecosystems, in Forest Productivity, there is special function (Hua Xiaomei, 1997) in long-term maintenance.Utilize ectomycorrhiza to promote the development of forestry to the beneficial effect of trees and Forest ecosystems, improve the road that production of forestry power is a Sustainable development of forestry to greatest extent.
Pisolithus tinctorius (Pisolithustinctorius (Pers.) Coker & Couch) has another name called Pisolithus tinctorius (Pers) coker et Couch, it is a kind of Applying Ectomycorrhizal Fungi, can survive on the root of sapling, be the promising ectomycorrhiza Symbiont of one of Song Miao.The sapling with this fungi can grow under severe edaphic condition, and grow soon than the sapling under excellent growing conditions with other fungi ectomycorrhizas, therefore after afforestation is often first by this bacterium of seedling inoculation, then mountain area is transplanted to, to increase the survival rate of sapling.Owing to infecting the early stage of Pinus massoniana root at Ectomycorrhizal Fungus, Pisolithus tinctorius, be only the generation that cannot judge to infect by visual inspection root and infect degree, therefore identify the feature needing sample size small and highly sensitive according to molecular dna, therefore for development easy, fast and accurately authenticate technology provide effective means.And Molecular Identification means are more stable comparatively speaking, there is feature that is rapid, easy, low cost.
(3) summary of the invention
The object of the invention is to provide a kind of molecular specificity labeled primers of sense colors beans Lasiosphaera fenzlii, and can infect to Pisolithus tinctorius the method that Pinus massoniana Lamb carries out Rapid identification.
The technical solution used in the present invention is:
The molecular specificity labeled primers of Pisolithus tinctorius in mycorhiza, described primer sequence is:
Upstream primer: 5'-CAGGAGGATGGACTCGCAGGGTTTC-3';
Downstream primer: 5'-CCATGACGTGGGCATTGCTTGAACT-3'.
This primer pair is on the basis of library construction, adopt round pcr, through a large amount of shaker test, the DNA fragment specific of the Pisolithus tinctorius obtained, by this fragment cloning and sequencing, based on the DNA sequence dna obtained, designed Auele Specific Primer, carry out pcr amplification with this primer pair Pisolithus tinctorius and Pinus massoniana Lamb, the specific fragment of 349 sizes can be obtained.
The invention still further relates to and a kind ofly utilize described molecular specificity labeled primers whether to infect to the tip of a root that Pinus massoniana Lamb is formed the method that Pisolithus tinctorius carries out Identification and detection, described method is: extract Pinus massoniana Lamb tip of a root DNA to be measured as template, using described molecular specificity labeled primers as amplimer, carry out pcr amplification, electrophoresis detection is carried out to amplified production, if the DNA band of 349bp appears in electrophoresis result, then infect Pisolithus tinctorius in the Pinus massoniana Lamb tip of a root to be measured; Described molecular specificity labeled primers sequence is:
Upstream primer: 5'-CAGGAGGATGGACTCGCAGGGTTTC-3';
Upstream primer: 5'-CCATGACGTGGGCATTGCTTGAACT-3'.
The inventive method key is the selection of amplimer, and DNA extraction, PCR reaction system and reaction conditions are determined, and electrophoresis detection, all can carry out according to this area ordinary method.
Preferably, PCR reaction system of the present invention is composed as follows:
Concrete, PCR reaction conditions is as follows:
94 DEG C of denaturation 6min; 92 DEG C of sex change 40s, 54.5 DEG C of annealing 40s, 72 DEG C of extensions 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C.
PCRBuffer final concentration is 1 ×, refer to that in PCRBuffer, the concentration of each component in reaction system is identical with 1 × PCRBuffer, usually select volume to be 10 × PCRBuffer of reaction system volume 1/10.10 × PCRBuffer composition is: 100mMTris-HCl (pH8.5), 500mMKCl, 25mMMgCl2 and 1.0%Triton-X-100, and solvent is ddH
2o.
Concrete, the method for the invention is as follows:
(1) get the Pinus massoniana Lamb tip of a root to be measured, extract Pinus massoniana Lamb tip of a root genomic dna to be measured with SDS-CTAB method;
(2) genomic dna extracted with step (1), for template, using described molecular specificity labeled primers as amplimer, carries out pcr amplification:
Every 20 μ L are composed as follows for PCR reaction system:
PCR reaction conditions is as follows:
94 DEG C of denaturation 6min; 92 DEG C of sex change 40s, 54.5 DEG C of annealing 40s, 72 DEG C of extensions 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C;
(3) step (2) amplified production 5 μ L is got, mix with 1 μ L0.25% bromjophenol blue damping fluid, point sample is on the sepharose of 1.5%, electrophoresis in 1 × TAE damping fluid, under 5V/cm voltage, electrophoresis terminates rear EB and dyes, and EB dyeing is dyeed 30 minutes usually in containing the aqueous solution of 0.5 μ g/m1EB.Take a picture on automatic gel image analysis instrument, if the DNA band of 349bp appears in electrophoresis result, then infected Pisolithus tinctorius in the tip of a root to be measured, on the contrary then no.
Beneficial effect of the present invention is mainly reflected in: molecular specificity labeled primers of the present invention can carry out Early Identification fast to Pisolithus tinctorius in mycorhiza, detects, compared with microscopic examination, has detection time short, the advantage that accuracy is high with routine morphological; As long as the inventive method detects required time 1 day, and the morphological observation of routine, time at least two time-of-week required for microscopic examination test.
(4) accompanying drawing explanation
Fig. 1 is the result of the Pinus massoniana Lamb tip of a root being carried out to pcr amplification; M is DNA molecular amount standard; Arrow indication is from being numbered the special DNA band that molecular weight that the Pinus massoniana Lamb tip of a root that infected Pisolithus tinctorius amplifies is 349bp; All the other are numbered the amplification of the Pinus massoniana Lamb tip of a root not infecting Pisolithus tinctorius.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1:
(1) extraction of genomic dna: the extraction of genomic dna adopts extracts with SDS-CTAB method.The genomic dna of purifying first uses the agarose gel electrophoresis qualitative detection of 1.5%, then uses DNA/RNA ultraviolet spectrophotometer detection by quantitative.DNA extraction thing is for subsequent use in-20 DEG C of refrigerator storages.
(2) specific PCR amplification primer is designed, the sequence of primer pair is upstream primer 5'-CAGGAGGATGGACTCGCAGGGTTTC-3' and downstream primer 5'-CCATGACGTGGGCATTGCTTGAACT-3', is synthesized by Shanghai Sheng Gong biotechnology company limited.
(3) pcr amplification of mycorhiza qualification:
PCR reaction system forms:
10 × PCRBuffer2 μ l, 10mmol/LdNTPs2 μ l, 25mmol/LMgCl
22 μ l, 5U/ μ lTaqDNA enzyme 0.5 μ l, 25mM special primer is to each 1 μ l, 20ng/ μ l template DNA 3 μ l, ddH
2o complements to 20 μ l.
Amplified reaction carries out on TC-XP type amplification instrument.
PCR reaction conditions is as follows:
94 DEG C of denaturation 6min;
92 DEG C of sex change 40s, 68 DEG C of annealing 40s, 72 DEG C of extensions 2min, totally 30 circulations;
Finally fill 7min in 72 DEG C, final temperature is 4 DEG C.
(4) electrophoresis detection: get step (3) pcr amplification product 5ul, mix with 1ul0.25% bromjophenol blue damping fluid, point sample is on the sepharose of 1.5%, in 1 × TAE damping fluid, electrophoresis under 5V/cm voltage, after electrophoresis terminates, dye 30 minutes in containing the aqueous solution of 0.5 μ g/m1EB, then take a picture on the automatic gel image analysis instrument of the clear JS-380A of training.
According to the method described above, respectively detection electrophoresis result is carried out to multiple Pinus massoniana Lamb tip of a root (numbering 1 ~ 3) infecting Pisolithus tinctorius and the Pinus massoniana Lamb tip of a root (numbering 4 ~ 6) not infecting Pisolithus tinctorius and see Fig. 1.Wherein numbering 1-3 sample, all amplify the special DNA band that molecular weight is about 349bp, all the other numberings produce there are no the special DNA band of about 349bp, other non-object band is not had to produce yet, visible molecular specificity marker of the present invention is used for the qualification of Pisolithus tinctorius in mycorhiza, and its good stability, specificity are high.
Claims (4)
1. the molecular specificity labeled primers of Pisolithus tinctorius in mycorhiza, described primer sequence is as follows:
Upstream primer: 5 '-CAGGAGGATGGACTCGCAGGGTTTC-3 ',
Downstream primer: 5 '-CCATGACGTGGGCATTGCTTGAACT-3 '.
2. one kind utilizes the molecular specificity labeled primers described in claim 1 to identify the method whether infecting Pisolithus tinctorius in the Pinus massoniana Lamb tip of a root, described method is: extract Pinus massoniana Lamb tip of a root DNA to be measured as template, using described molecular specificity labeled primers as amplimer, carry out pcr amplification, electrophoresis detection is carried out to amplified production, if the DNA band of 349bp appears in electrophoresis result, then infect Pisolithus tinctorius in the Pinus massoniana Lamb tip of a root to be measured, on the contrary then no; Described molecular specificity labeled primers sequence is:
Upstream primer: 5 '-CAGGAGGATGGACTCGCAGGGTTTC-3 ',
Downstream primer: 5 '-CCATGACGTGGGCATTGCTTGAACT-3 '.
3. method as claimed in claim 2, is characterized in that described pcr amplification condition is as follows: 94 DEG C of denaturation 6min; 92 DEG C of sex change 40s, 54.5 DEG C of annealing 40s, 72 DEG C of extensions 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C.
4. method as claimed in claim 2, is characterized in that described method is as follows:
(1) get the Pinus massoniana Lamb tip of a root to be measured, add liquid nitrogen and grind, extract the genomic dna of the Pinus massoniana Lamb tip of a root to be measured with SDS-CTAB method;
(2) genomic dna extracted with step (1), for template, using described molecular specificity labeled primers as amplimer, carries out pcr amplification:
Every 20 μ L are composed as follows for PCR amplification system:
Pcr amplification condition is as follows:
94 DEG C of denaturation 6min; 92 DEG C of sex change 40s, 54.5 DEG C of annealing 40s, 72 DEG C of extensions 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C;
(3) step (2) amplified production 5 μ L is got, mix with 1 μ L0.25% bromjophenol blue damping fluid, point sample is on the sepharose of 1.5%, electrophoresis in 1 × TAE damping fluid, under 5V/cm voltage, electrophoresis terminates rear EB and dyes, take a picture on automatic gel image analysis instrument, if the DNA band of 349bp appears in electrophoresis result, then infected Pisolithus tinctorius in the tip of a root to be measured; Otherwise it is then no.
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|---|---|---|---|
| CN201510432024.9A CN105039328B (en) | 2015-07-21 | 2015-07-21 | Sense colors beans Lasiosphaera fenzlii infects the molecular specificity labeled primers and method of masson pine |
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| CN201510432024.9A CN105039328B (en) | 2015-07-21 | 2015-07-21 | Sense colors beans Lasiosphaera fenzlii infects the molecular specificity labeled primers and method of masson pine |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181544B (en) * | 2011-04-11 | 2012-11-14 | 南京林业大学 | Molecular detection method for pisolithus tinctorius of forest ectomycorrhizal fungi |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181544B (en) * | 2011-04-11 | 2012-11-14 | 南京林业大学 | Molecular detection method for pisolithus tinctorius of forest ectomycorrhizal fungi |
Non-Patent Citations (3)
| Title |
|---|
| A. AMICUCCI 等: "Identification of ectomycorrhizal fungi of the genus Tuber by species-specific ITS primers", 《MOLECULAR ECOLOGY》 * |
| 周爱东 等: "林木外生菌根真菌彩色豆马勃巢式PCR检测", 《应用与环境生物学报》 * |
| 林晓民 等: "真菌rDNA的特点及在外生菌根菌鉴定中的应用", 《西北农业学报》 * |
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