CN105349534A - Primer for molecular identification of panax japonicus and method for sequence-characterized amplified region (SCAR) - Google Patents
Primer for molecular identification of panax japonicus and method for sequence-characterized amplified region (SCAR) Download PDFInfo
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Abstract
The invention provides a primer for molecular identification of panax japonicus, wherein the primer is Z1, and sequences of the primer are shown as F5'-AGACGTCCACATGGGCTA-3' and R5'-TAGACGTCCACGTTAAGTTAG-3'. Meanwhile, the invention provides an identification method for labeled molecules in a sequence-characterized amplified region of the panax japonicus by virtue of the specific primer Z1, wherein specifically, the method comprises genome DNA extraction and SCAR analysis. When the specific SCAR primer Z1 is used for conducting PCR amplification on the panax japonicus and other panax plants, an amplification process comprises the following steps: pre-modifying at 94 DEG C for 5min, modifying at 94 DEG C for 30s, annealing at 55 DEG C for 30s and extending at 72 DEG C for 30s, and such cycling for 34 times; supplementing extending at 72 DEG C for 5min, conducting agarose gel electrophoresis to a PCR reaction product under voltage of 100V for 30min, and detecting radiography by virtue of a gel analysis system, so that the identification is completed. The application disclosed by the invention, on the basis of the specific primer and the PCR experimental technology, can achieve rapid identification on the panax japonicus and other panax plants. The SCAR labeling operation is simple and rapid, low in cost and is easy to implement in various laboratories.
Description
Technical field
The present invention relates to a kind of molecular assay method, be specially the sequence specific amplification region tagged molecule authentication method to rhizome of Japanese Ginseng.
Background technology
Rhizome of Japanese Ginseng is the dry rhizome of araliaceae ginseng plant rhizome of Japanese Ginseng PanaxjaponicusC.A.Mey., there is strengthening by means of tonics, blood stasis removing analgesic, the effect of stopping blooding, eliminating the phlegm, for weak after being ill, labor is coughed spitting of blood, coughing with a lot of sputum, injury from falling down, therefore has the effect of the strengthening by means of tonics of China's north medicine ginseng and the promoting blood circulation to remove blood stasis of southern medicine pseudo-ginseng concurrently, is that herbal medicine is commonly used in compact community such as Southwestern China area Tujia, Miao ethnic group etc.Panax species ginseng PanaxginsengC.A.Mey., Radix Panacis Quinquefolii PanaxquinquefoliumL., pseudo-ginseng Panaxnotoginseng (Burk.) F.H.Chen etc. are all used as medicine with rhizome, prepared slices of Chinese crude drugs outward appearance is similar to rhizome of Japanese Ginseng, easily causes mixed.Therefore, find effective ways and differentiate these medicinal materials, significant for rational use of drug.
Compared with traditional Chinese medicine authenticate technology, DNA molecular marker technology directly utilizes the difference in plant dna molecule level to differentiate, its result is not by the impact of the plant-growth time limit, physiological status and growing environment, more objective, reliable.Existing multiple DNA molecular marker technology is applied in the discriminating of plant at present, as RAPD DNA marker (RandomamplifiedpolymorphicDNA, RAPD), restriction fragment length polymorphism (Restrictionfragmentlengthpolymorphism, RFLP), single nucleotide polymorphism (Singlenucleotidepolymorphism, SNP), the Internal Transcribed Spacer (Internaltranscribedspacer, ITS) etc.Sequence specific amplification region (Sequencecharacteredamplifiedregion, SCAR) labeling technique is the molecule marker that a kind of stability is higher, specificity is stronger grown up on RAPD basis, has been applied to the discriminating of some nearly edge species.
Summary of the invention
Be intended to herein set up a kind of SCAR molecular marking technique, for quick and precisely differentiating rhizome of Japanese Ginseng and kindred plant ginseng, Radix Panacis Quinquefolii and pseudo-ginseng.Specifically comprise following content:
Plant sample comprises as follows:
The fresh blade of rhizome of Japanese Ginseng is purchased in enshi Chinese toon wood battalion rhizome of Japanese Ginseng plant development base, and the fresh blade of pseudo-ginseng is purchased in Zhe La township, mountain of papers Yanshan County, Yunnan, and the fresh blade of ginseng, Radix Panacis Quinquefolii is purchased from Jilin Province's Baishan Jingyu County.By the rapid drying for standby of silica gel after collection.
Reagent comprises as follows:
Pcr amplification reagent (ThermoScientific company), CTAB (AMRESCO company), PVP40 (Sigma company), agarose (BIOWEST company), GelRed dyestuff (BIOTIUM company), RNaseA (Sigma company), DNA sample-loading buffer (Wuhan Ke Rui Bioisystech Co., Ltd), 2 × TaqPCRMastermix[TIANGEN Biotech (Beijing) Co., Ltd.], 1kbplusDNALadder[TIANGEN Biotech (Beijing) Co., Ltd.].RAPD random primer is synthesized by Shanghai Sheng Gong company.All the other reagent are analytical pure.
Instrument comprises as follows:
BS110S electronic balance (Beijing Sai Duolisi instrument system company limited), HH-4 digital display thermostat water bath (Changzhou Guohua Electric Appliance Co., Ltd.), CT15RT high speed freezing centrifuge (Shanghai Tianmei Biochemistry Instrument Engineering Co., Ltd.), grads PCR instrument (AppliedBiosystems), DYY-6C type electrophoresis apparatus (Beijing Liuyi Instrument Factory), GeneGenius gel analysis system (SYNGNE company of Britain), LS-B50L vertical pressure steam sterilizer (Shanghai Huaxian Medical Nuclear Instruments Co., Ltd.).
By transforming RAPD analytical results thus obtaining SCAR mark, step is as follows:
The extraction of genomic dna: with liquid nitrogen by mortar, size pestle, spoon precooling, takes 200mg blade and 10%PVP (W/W) is placed in liquid nitrogen, grinds to form fine powder rapidly; Be transferred to by ground fine powder in 1.5mL centrifuge tube, the 700 μ L adding rapidly 4 DEG C of precoolings remove polysaccharide buffer, mixing, are leaving standstill 30min, 4 DEG C of 3000rmin on ice
-1centrifugal 5min, abandons supernatant, and this step repeats once, to abandon supernatant, adds 2 × CTAB extracting solution of 700 μ L65 DEG C preheatings, 65 DEG C of water-bath 40min, turns upside down mixing gently once, take out 1.5mL centrifuge tube, be cooled to room temperature, 12000rmin every 10min
-1centrifugal 10min; Get supernatant, add isopyknic chloroform: the mixed solution of primary isoamyl alcohol, mixing 10min, 12000rmin
-1centrifugal 10min, gets supernatant, repeats this step once, gets supernatant to new 1.5mL centrifuge tube, adds the Virahol of-20 DEG C of precoolings of 0.6 times of volume, mixing, in-20 DEG C of standing 1h, and 4 DEG C of 12000rmin
-1centrifugal 10min, precipitate by 70% washing with alcohol 2 times, absolute ethanol washing 1 time, dries up liquid with blower cold wind, after drying, adds 30 μ L sterilizing ddH
2o and 0.5 μ LRNaseA, 37 DEG C of water-bath 1h, standby in-20 DEG C of preservations;
RAPD-PCR reacts: use random primer H03 (sequence is 5 '-AGACGTCCAC-3 ') to carry out pcr amplification to genomic dna.PCR reaction solution cumulative volume is 25 μ L, wherein containing MgCl23 μ L, 10 × TaqbufferwithKCl2.5 μ L, Taq enzyme 1.5 μ L, dNTPMixture0.5 μ L, primer 2 μ L, template DNA 1 μ L, adds sterilizing ddH2O polishing to 25 μ L; MgCl2 concentration is 25mM, Taq enzyme concentration is that 1U/ μ L, dNTPMixture concentration is 10mM, primer concentration is 10mM.The RAPD-PCR reaction solution above-mentioned DNA program that increases is 94 DEG C of denaturation 5min; 94 DEG C of sex change 45s, 36 DEG C of annealing 60s, 72 DEG C extend 90s, circulate 39 times; 72 DEG C are supplemented extension 7min, and each PCR reaction is all established not containing the blank of template DNA, in triplicate, to determine the stability of amplified production;
Purifying, the Clone and sequence of RAPD product specific fragment: under ultraviolet lamp, rhizome of Japanese Ginseng specific DNA band (about 300bp) is cut, recovery and the purifying that test kit carries out specific DNA band is reclaimed with sepharose, the DNA fragmentation reclaimed connects with pMD18-Tvector carrier, transform and import in E.coli competent cell, select positive colony after microbial culture and extract plasmid, by plasmid enzyme restriction electrophoresis determination positive colony, check order, sequencing result is as follows:
1GAATTCGATTAGACGTCCACATGGGCTAAAAAAATTAAATTATTTTATTACTCCTTTATC
61TCTAGATTTTTCATATAATGCAACCCTTAGGATTTACACAATTAATTTTCTAAAATTTCT
121AATTTAGGCTTTAAAAATAAGAATTTAATCTTTCTAACTTATAATTAGAATTTTCTAATT
181ATATCAAAATCTCTTTATCATAATTTAAATATTACTAATTATGATTAACATTTCTTAATT
241AAAATCGCTAATTTAGCAATTCGGGTCAAAACGGTCATTAGGGTTTATAAACCAATAATT
301AGTATTTTCTAACTTAACGTGGACGTCTAATCACTAGTGAATTC
SCAR design of primers: according to sequencing result and design of primers principle, application Primer5.0 software design Auele Specific Primer a pair, primer Z1, its sequence is: F5 '-AGACGTCCACATGGGCTA-3 '; R5 '-TAGACGTCCACGTTAAGTTAG-3 '.
Application SCAR mark technology differentiates rhizome of Japanese Ginseng, and step is as follows:
Application specific SCAR primer Z1 carries out pcr amplification to rhizome of Japanese Ginseng and other panax species genomic dna, and SCAR-PCR reaction system is: template DNA 1 μ L, 2 × TaqPCRMastermix12.5 μ L, and each 1 μ L of upstream and downstream primer, adds sterilizing ddH
2o polishing to 25 μ L, wherein, upstream and downstream primer concentration is 10mmol/L.SCAR-PCR amplification program is 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, circulate 34 times; 72 DEG C are supplemented extension 5min, get PCR reaction product 20 μ L, under 100V voltage, through 1.5% sepharose (containing GelRed) electrophoresis 30min, GeneGenius gel analysis systems axiol-ogy is taken a picture, and can complete the qualification of sequence specific amplification region (SCAR) tagged molecule of rhizome of Japanese Ginseng.
Beneficial effect:
SCAR molecule marker carries out design of primers for RAPD specific fragment sequence, then from genomic dna, amplify the new technology of this specific fragment.On RAPD basis, successfully set up the SCAR molecular marking technique differentiating rhizome of Japanese Ginseng and other panax ginseng plant herein, only need depend on SCAR Auele Specific Primer and PCR experiment technology, just can realize the quick discriminating to rhizome of Japanese Ginseng and other panax ginseng plant.SCAR mark is fast simple to operate, with low cost, all easily realizes in different experiments room.
Accompanying drawing explanation
Fig. 1 be ginseng, Radix Panacis Quinquefolii, pseudo-ginseng DNA through the electrophoresis result of 1.0% sepharose, wherein, 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng.
Fig. 2 is the electrophoresis result of rhizome of Japanese Ginseng DNA through 1.0% sepharose, wherein, and 1. rhizome of Japanese Ginseng.
Fig. 3 is H03 primer extension product electrophorogram, wherein, and 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. rhizome of Japanese Ginseng.
Fig. 4 is SCAR primer extension product electrophorogram, wherein, and 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. rhizome of Japanese Ginseng.
Embodiment
Embodiment 1
CTAB method extracts genomic dna
With liquid nitrogen by mortar, size pestle, spoon precooling.Take 200mg blade and 10%PVP (W/W) is placed in liquid nitrogen, grind to form fine powder rapidly.Be transferred to by ground fine powder in 1.5mL centrifuge tube, the 700 μ L adding rapidly 4 DEG C of precoolings remove polysaccharide buffer (containing 2% beta-mercaptoethanol), mixing, are leaving standstill 30min, 4 DEG C of 3000rmin on ice
-1centrifugal 5min.Abandon supernatant, this step repeats once.Abandon supernatant, add 2 × CTAB extracting solution (containing 2% beta-mercaptoethanol) of 700 μ L65 DEG C preheatings, 65 DEG C of water-bath 40min, turn upside down mixing gently once every 10min.Take out 1.5mL centrifuge tube, be cooled to room temperature.12000rmin
-1centrifugal 10min.Get supernatant, add isopyknic chloroform: primary isoamyl alcohol (24:1), mixing 10min, 12000rmin
-1centrifugal 10min.Get supernatant, repeat this step once.Get supernatant to new 1.5mL centrifuge tube, add the Virahol of 0.6 times of volume-20 DEG C of precoolings, mixing, in-20 DEG C of standing 1h, 4 DEG C of 12000rmin
-1centrifugal 10min.Precipitate by 70% washing with alcohol 2 times, absolute ethanol washing 1 time.Liquid is dried up with blower cold wind.After drying, add 30 μ L sterilizing ddH
2o and 0.5 μ LRNaseA, 37 DEG C of water-bath 1h.Standby in-20 DEG C of preservations.
The quality examination of DNA
Measure DNA concentration with trace dna protein assay, and detect DNA purity with the ratio of A260/A280.Separately get STb gene solution 3 μ L, mix 0.6 μ L6 × DNA sample-loading buffer, under 100V voltage, through 1.0% agar gel (containing GelRed) electrophoresis 40min ~ 1h, GeneGenius gel analysis systems axiol-ogy is taken a picture, identification of dna integrity.
SCAR analyzes
(sequence is application specific SCAR primer Z1: F5 '-AGACGTCCACATGGGCTA-3 '; R5 '-TAGACGTCCACGTTAAGTTAG-3 ') pcr amplification is carried out to rhizome of Japanese Ginseng and other panax species genomic dna.SCAR-PCR reaction system is: template DNA 1 μ L, 2 × TaqPCRMastermix12.5 μ L, and each 1 μ L of upstream and downstream primer (10mmol/L), adds sterilizing ddH
2o polishing to 25 μ L.SCAR-PCR amplification condition is 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, circulate 34 times; 72 DEG C are supplemented extension 5min.Get PCR reaction product 20 μ L, under 100V voltage, through 1.5% sepharose (containing GelRed) electrophoresis 30min, GeneGenius gel analysis systems axiol-ogy is taken a picture.
Result
Total DNA extraction and quality examination
By the STb gene sterilizing ddH extracted
2after O dilution, trace dna protein assay measures OD
260and OD
280value, according to the corresponding STb gene concentration of formulae discovery (ng μ L
-1)=OD
260× extension rate × 50ng μ L
-1; Purity=OD
260/ OD
280, its ratio >1.8 is excellent, the results are shown in Table 1.STb gene electrophoresis result is shown in that in Fig. 1,2, figure, result shows that STb gene is complete, can be used for RAPD and analyzes.
Table 1 spectrophotometer method measures DNA concentration and purity
SCAR identifies
SCAR-PCR result shows, Z1 can amplify the fragment of about 300bp in rhizome of Japanese Ginseng, and this band (see Fig. 4) does not all appear in ginseng, Radix Panacis Quinquefolii, pseudo-ginseng, namely rhizome of Japanese Ginseng and ginseng, Radix Panacis Quinquefolii, pseudo-ginseng can be distinguished and differentiate by this SCAR primer rapidly.
Rhizome of Japanese Ginseng is one of China's rare Chinese traditional herbs among the people, has anti-inflammatory, resists myocardial ischemia, the multiple pharmacologically active such as antifatigue, is a natural resources of Chinese medicinal materials plant having Development volue.But because panax species is of a great variety, active various, the stem block plesiomorphism of its medicinal part, the section of especially dry stem block is difficult to differentiate especially, and the Molecular Identification of therefore carrying out DNA level by molecular biology method is expected to effectively identify these medicinal plants.
SCAR molecule marker carries out design of primers for RAPD specific fragment sequence, then from genomic dna, amplify the new technology of this specific fragment.SCAR mark is fast simple to operate, with low cost, all easily realizes in different experiments room.The maximum difficult point of SCAR mark finds RAPD specific fragment, needs to screen a large amount of random primer to obtain specific fragment.But once SCAR mark is successfully established, the plant in later stage is differentiated just to be easy to realize.We successfully establish the SCAR mark technology of rhizome of Japanese Ginseng herein, designed Z1 primer can amplify the band of about 300bp in rhizome of Japanese Ginseng, and in ginseng, Radix Panacis Quinquefolii, pseudo-ginseng equal not this bands, thus from molecular level, rhizome of Japanese Ginseng and its Closely related variety ginseng, Radix Panacis Quinquefolii, pseudo-ginseng to be distinguished.Present method is simple fast, and the preliminary evaluation for Panax Closely related variety provides one method accurately and effectively.
Claims (4)
1. for a primer for rhizome of Japanese Ginseng Molecular Identification, it is characterized in that, described primer is Z1, and its sequence is: F5 '-AGACGTCCACATGGGCTA-3 '; R5 '-TAGACGTCCACGTTAAGTTAG-3 '.
2. adopt Auele Specific Primer Z1 according to claim 1 to carry out the authentication method of rhizome of Japanese Ginseng sequence specific amplification region (SCAR) tagged molecule, it is characterized in that, comprise the steps:
The extraction of genomic dna: with liquid nitrogen by mortar, size pestle, spoon precooling, takes 200mg blade and 10%PVP (W/W) is placed in liquid nitrogen, grinds to form fine powder rapidly; Be transferred to by ground fine powder in 1.5mL centrifuge tube, the 700 μ L adding rapidly 4 DEG C of precoolings remove polysaccharide buffer, mixing, are leaving standstill 30min, 4 DEG C of 3000rmin on ice
-1centrifugal 5min, abandons supernatant, and this step repeats once, to abandon supernatant, adds 2 × CTAB extracting solution of 700 μ L65 DEG C preheatings, 65 DEG C of water-bath 40min, turns upside down mixing gently once, take out 1.5mL centrifuge tube, be cooled to room temperature, 12000rmin every 10min
-1centrifugal 10min; Get supernatant, add isopyknic chloroform: the mixed solution of primary isoamyl alcohol, mixing 10min, 12000rmin
-1centrifugal 10min, gets supernatant, repeats this step once, gets supernatant to new 1.5mL centrifuge tube, adds the Virahol of-20 DEG C of precoolings of 0.6 times of volume, mixing, in-20 DEG C of standing 1h, and 4 DEG C of 12000rmin
-1centrifugal 10min, precipitate by 70% washing with alcohol 2 times, absolute ethanol washing 1 time, dries up liquid with blower cold wind, after drying, adds 30 μ L sterilizing ddH
2o and 0.5 μ LRNaseA, 37 DEG C of water-bath 1h, standby in-20 DEG C of preservations;
SCAR analyzes: application specific SCAR primer Z1 carries out pcr amplification to rhizome of Japanese Ginseng and other panax species genomic dna, and wherein, specificity SCAR primers Z1 sequence is: F5 '-AGACGTCCACATGGGCTA-3 '; R5 '-TAGACGTCCACGTTAAGTTAG-3 '; SCAR-PCR reaction system is: template DNA 1 μ L, 2 × TaqPCRMastermix12.5 μ L, each 1 μ L of upstream and downstream primer, and add sterilizing ddH2O polishing to 25 μ L, wherein, upstream and downstream primer concentration is 10mmol/L; SCAR-PCR amplification program is 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, circulate 34 times; 72 DEG C are supplemented extension 5min, get PCR reaction product 20 μ L, under 100V voltage, through 1.5% sepharose (containing GelRed) electrophoresis 30min, GeneGenius gel analysis systems axiol-ogy is taken a picture, and can complete the qualification of sequence specific amplification region (SCAR) tagged molecule of rhizome of Japanese Ginseng.
3. authentication method according to claim 2, is characterized in that, SCAR-PCR reaction system is: template DNA 1 μ L, 2 × TaqPCRMastermix12.5 μ L, and each 1 μ L of upstream and downstream primer, adds sterilizing ddH
2o polishing to 25 μ L, wherein, upstream and downstream primer concentration is 10mmol/L.
4. authentication method according to claim 2, is characterized in that, SCAR-PCR amplification program is 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, circulate 34 times; 72 DEG C are supplemented extension 5min.
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| CN112080574B (en) * | 2020-08-18 | 2022-07-26 | 宁波城市职业技术学院 | Development of panax japonicus EST-SSR primer group and application of panax japonicus EST-SSR primer group in aspects of genetic diversity and the like |
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Application publication date: 20160224 |