CN105002272A - Method for identifying varieties of RAPD (random amplified polymorphic DNA) marked panax japonicus and kindred plants thereof - Google Patents
Method for identifying varieties of RAPD (random amplified polymorphic DNA) marked panax japonicus and kindred plants thereof Download PDFInfo
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Abstract
The invention realizes the rapid identification of the panax japonicus and the related panax plants (ginseng, American ginseng, pseudo-ginseng, panax japonicus variety panax japonicus, panax lupulos and panax japonicus) thereof by combining the RAPD labeling technology with a manual drawn variety identification chart (MCID) method. Extracting total DNA of panax japonicus and kindred plants thereof from leaves by adopting an improved CTAB method, screening 4 primers which can be stably amplified and have specific bands from 16 random primers to construct a DNA fingerprint, and amplifying 46 bands in total, wherein 42 polymorphic bands are obtained, and the polymorphism ratio is 91.30%. According to the existence of the specific bands, the panax japonicus and the kindred plants thereof are distinguished one by one, and a map relation analysis map is drawn to form an artificially drawn variety identification map. The method is rapid and simple, has intuitive and reliable results, and provides an effective and feasible method for the initial identification of the panax japonicus and kindred varieties.
Description
Technical field
The present invention relates to a kind of method of kindred plant cultivar identification, be specially the variety discriminating method adopting RAPD to mark rhizome of Japanese Ginseng and kindred plant thereof.
Background technology
Rhizome of Japanese Ginseng is the dry rhizome of araliaceae ginseng plant rhizome of Japanese Ginseng Panax japonicus C.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.Specify with the Pharmacopoeia of the People's Republic of China inconsistent be, among the people also often three of rhizome of Japanese Ginseng kinds of mutation narrow leaf rhizome of Japanese Ginseng Panax japonicus C.A.Mey.var.angustifius (Burk) Cheng et Chu, Rhizoma Panacis bipinnatifidi Panax japonicus C.A.Mey.var.bipinnatidus (Seem.) C.Y.Wuet K.M.Feng, the dry rhizome of Rhizome of Bipinnatifid Ginseng Panax japonicus C.A.Mey.var.major (Burk.) C.Y.Wu et K.M.Feng is mixed does rhizome of Japanese Ginseng, but research shows, their chemical composition and pharmacologically active are not quite similar, therefore, find effective ways and differentiate these medicinal materials, significant for rational use of drug.
Summary of the invention
Differentiate to distinguish to rhizome of Japanese Ginseng and its easily be obscured Panax kindred plant (the narrow leaf rhizome of Japanese Ginseng of ginseng, Radix Panacis Quinquefolii, pseudo-ginseng and rhizome of Japanese Ginseng mutation, Rhizoma Panacis bipinnatifidi, Rhizome of Bipinnatifid Ginseng), this experiment is by RAPD molecular marking technique, build artificial drafting cultivar identification figure (MCID), set up the gene identification system of rhizome of Japanese Ginseng and kindred plant thereof.
Material
The new fresh goods of blade of rhizome of Japanese Ginseng, narrow leaf rhizome of Japanese Ginseng, Rhizoma Panacis bipinnatifidi and Rhizome of Bipinnatifid Ginseng is all purchased in enshi Chinese toon wood battalion rhizome of Japanese Ginseng plant development base, the fresh product of pseudo-ginseng blade are purchased in Zhe La township, mountain of papers Yanshan County, Yunnan, and the new fresh goods of 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
Pcr amplification reagent (Thermo Scientific company, lot number 00140623), CTAB (AMRESCO company, lot number 3186B058), PVP40 (Sigma company, lot number BCBD7485), agarose (BIOWEST company, lot number 111860), GelRed dyestuff (BIOTIUM company, lot number 13G0307), RNase A enzyme (Sigma company, lot number 036M1298G), DNA sample-loading buffer (Wuhan Ke Rui Bioisystech Co., Ltd, lot number 20150116), 1kb plus DNA Ladder [TIANGEN Biotech (Beijing) Co., Ltd., lot number M2206].RAPD random primer is synthesized by Shanghai Sheng Gong company.All the other reagent are analytical pure.
Instrument
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 (Applied Biosystems), DYY-6C type electrophoresis apparatus (Beijing Liuyi Instrument Factory), Gene Genius gel analysis system (SYNGNE company of Britain), LS-B50L vertical pressure steam sterilizer (Shanghai Huaxian Medical Nuclear Instruments Co., Ltd.).
Method
RAPD marks a method for the Variety identification of rhizome of Japanese Ginseng and kindred plant thereof, comprises the steps:
The extraction liquid nitrogen of DNA is by mortar, size pestle, spoon precooling, take rhizome of Japanese Ginseng blade, kindred plant blade respectively, be placed in liquid nitrogen with polyvinylpyrrolidone respectively, grind to form fine powder, ground fine powder is transferred in centrifuge tube, add be chilled in advance 4 DEG C remove polysaccharide damping fluid, in leaving standstill 30min on ice after mixing, at 4 DEG C, centrifugally abandon supernatant; Add 2 × cetyl trimethylammonium bromide extracting solution of 65 DEG C of preheatings toward centrifuge tube, 65 DEG C of water-bath 40min, turn upside down mixing once every 10min; Take out 1.5mL centrifuge tube, be cooled to room temperature, centrifugal, get supernatant, add the mixed solution (wherein, the volume ratio of chloroform and primary isoamyl alcohol is 24:1) of isopyknic chloroform and primary isoamyl alcohol, after mixing 10min, centrifugal, get supernatant, repeated centrifugation gets supernatant liquor 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, at 4 DEG C, centrifugal throw out; Throw out 70% washing with alcohol 2 times, 95% absolute ethanol washing 1 time, blower cold wind dries up liquid, after drying, adds sterilizing distilled water and rnase (RNase A), 37 DEG C of water-bath 1h, standby in-20 DEG C of preservations, can obtain the DNA extracted;
DNA obtained above is carried out amplified reaction by PCR reaction system under PCR reaction solution, its amplification program is denaturation, is then sex change, annealing, extension, carry out 39 circulations altogether, finally extend and determine to obtain stable amplified production, be PCR reaction product, pcr amplification reaction is all established not containing the blank of template DNA simultaneously;
RAPD primer screening collects RAPD primer, random primer is screened after application software comparison, random primer is filtered out in the PCR of RAPD the primer that in amplification rhizome of Japanese Ginseng and kindred plant thereof, DNA band is clear, reproducible again, carry out electrophoresis, gel analysis systems axiol-ogy is taken a picture;
Data analysis to be increased electrophoretogram according to arbitrarily primed PCR, the presence or absence that identical molecular weight fragment place produces specific band is added up, utilize the presence or absence of specific band rhizome of Japanese Ginseng and kindred plant thereof can be divided into groups, use the combination of the group result of multiple random primer, rhizome of Japanese Ginseng and kindred plant thereof can be made a distinction one by one, can Variety identification be completed.
The kindred plant of described rhizome of Japanese Ginseng comprises ginseng, Radix Panacis Quinquefolii, pseudo-ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi, one or more in narrow leaf rhizome of Japanese Ginseng.
In DNA extraction step, first time is centrifugal is at 4 DEG C, with 3000rmin
-1lower centrifugal 5min, other are centrifugal is 12000rmin at the corresponding temperature
-1lower centrifugal 10min.
The described polysaccharide damping fluid that goes is not containing the low salt wash liquid of CTAB, can effectively remove the impurity such as polyphenol, polysaccharide, pigment; To go in polysaccharide damping fluid, 2 × cetyl trimethylammonium bromide Extraction buffer to be all the beta-mercaptoethanol of 2% containing massfraction; Add 700 μ L in every 200mg blade and remove polysaccharide damping fluid, 700 μ L2 × cetyl trimethylammonium bromide Extraction buffers.
PCR reaction solution comprises the MgCl of 25mM
2, 10 × Taq buffer with KCl, the Taq enzyme of 1U/ μ L, dNTP Mixture, 10mM primer (the RAPD random primers of 10 bases) of 10mM be the mixing raw material of 6:5:3:1:4 by volume, other are sterilizing distilled water.
Described 10 × Taq buffer with KCl is 100mM Tris-HCl (Ph8.8), 500mM KCl, the mixture of 0.8% (v/v) Nonidet P40.
The amplification program of DNA is 90-98 DEG C of denaturation 3-10min; 92-98 DEG C of sex change 35-65s, 30-40 DEG C of annealing 33-96s, 65-80 DEG C of extension 60-120s, circulation 35-42 time; 70-75 DEG C is supplemented extension 5-10min.
More preferably, the amplification program of DNA 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.
The RAPD primer of screening comprises F02 (GAGGATCCCT), I13 (CTCTCCGCCA), H03 (AGACGTCCAC), F01 (ACGGATCCTG).
The banding pattern of discriminating ginseng is 1100bp (+) 700bp (-) of F02 primer amplification and the 800bp (+) of I13 primer amplification; The banding pattern of discriminating Radix Panacis Quinquefolii is 1100bp (+) 700bp (-) of F02 primer amplification and the 800bp (-) of I13 primer amplification; Differentiate that the banding pattern of rhizome of Japanese Ginseng is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (+) of H03 primer amplification and the 450bp (+) of I13 primer amplification; Differentiate that the banding pattern of narrow leaf rhizome of Japanese Ginseng is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (-) of H03 primer amplification and the 500bp (+) of F01 primer amplification; Differentiate that the banding pattern of Rhizoma Panacis bipinnatifidi is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (-) of H03 primer amplification and the 500bp (-) of F01 primer amplification; Differentiate that the banding pattern of Rhizome of Bipinnatifid Ginseng is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (+) of H03 primer amplification and the 450bp (-) of I13 primer amplification; Differentiate that the banding pattern of pseudo-ginseng is 1100bp (-) 700bp (-) of F02 primer amplification.Wherein there is specific band to represent with (+), represent with (-) without specific band.
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.
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 subject to the impact of environment and geographic factor, more accurately, reliably.The DNA molecular marker technology be most widely used at present is RAPD technology.RAPD technology is a kind of molecular marking technique using random primer to reflect DNA polymorphism fast, is widely used in the research of genetic diversity, Variety identification, real and fake discrimination, famous-region drug etc.The informative of RAPD result, not directly perceived comparatively speaking in the application process of identification and analysis, advise a kind of method of the artificial drafting Variety identification sketch based on DNA fingerprinting, according to specific DNA bands of a spectrum, draw collection of illustrative plates graph of a relation, clearly give expression to Variety identification result.
In the application, adopt RAPD to analyze and the artificial method of drawing Variety identification figure and combining, rhizome of Japanese Ginseng and kindred plant ginseng thereof, Radix Panacis Quinquefolii, pseudo-ginseng, narrow leaf rhizome of Japanese Ginseng, Rhizoma Panacis bipinnatifidi, Rhizome of Bipinnatifid Ginseng are differentiated to distinguish one by one DNA molecular level.The method is simple fast, and the preliminary evaluation for Panax Closely related variety provides a kind of effective and feasible method.
Adopt improved method of CTAB from blade, extract rhizome of Japanese Ginseng and kindred plant STb gene thereof, filter out from 16 random primers and can stablize amplification and 4 primer constructed dna finger printings with specific band, coamplification goes out 46 band, wherein polymorphic bands 42, polymorphism ratio is 91.30%.According to the presence or absence of specific band, rhizome of Japanese Ginseng and kindred plant thereof are differentiated to distinguish one by one, draw collection of illustrative plates relationship analysis figure, form artificial drafting cultivar identification figure.The method is simple fast, and visual result is reliable, for the preliminary discriminating of rhizome of Japanese Ginseng and Closely related variety provides a kind of effective and feasible method.
Accompanying drawing explanation
Fig. 1 be ginseng, Radix Panacis Quinquefolii, pseudo-ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi, narrow leaf rhizome of Japanese Ginseng, rhizome of Japanese Ginseng STb gene through the electrophorogram of 1.0% sepharose, wherein, 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. Rhizome of Bipinnatifid Ginseng, 5. Rhizoma Panacis bipinnatifidi, 6. narrow leaf rhizome of Japanese Ginseng, 7. rhizome of Japanese Ginseng.
Fig. 2 is the F02 primer extension product electrophorogram of blank marks, ginseng, Radix Panacis Quinquefolii, pseudo-ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi, narrow leaf rhizome of Japanese Ginseng, rhizome of Japanese Ginseng, wherein, M.Marker, 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. rhizome of Japanese Ginseng, 5. narrow leaf rhizome of Japanese Ginseng, 6. Rhizoma Panacis bipinnatifidi, 7. Rhizome of Bipinnatifid Ginseng.
Fig. 3 is the I13 primer extension product electrophorogram of blank marks, ginseng, Radix Panacis Quinquefolii, pseudo-ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi, narrow leaf rhizome of Japanese Ginseng, rhizome of Japanese Ginseng, wherein, M.Marker, 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. rhizome of Japanese Ginseng, 5. narrow leaf rhizome of Japanese Ginseng, 6. Rhizoma Panacis bipinnatifidi, 7. Rhizome of Bipinnatifid Ginseng.
Fig. 4 is the H03 primer extension product electrophorogram of blank marks, ginseng, Radix Panacis Quinquefolii, pseudo-ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi, narrow leaf rhizome of Japanese Ginseng, rhizome of Japanese Ginseng, wherein, M.Marker, 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. rhizome of Japanese Ginseng, 5. narrow leaf rhizome of Japanese Ginseng, 6. Rhizoma Panacis bipinnatifidi, 7. Rhizome of Bipinnatifid Ginseng.
Fig. 5 is the F01 primer extension product electrophorogram of blank marks, ginseng, Radix Panacis Quinquefolii, pseudo-ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi, narrow leaf rhizome of Japanese Ginseng, rhizome of Japanese Ginseng, wherein, M.Marker, 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. rhizome of Japanese Ginseng, 5. narrow leaf rhizome of Japanese Ginseng, 6. Rhizoma Panacis bipinnatifidi, 7. Rhizome of Bipinnatifid Ginseng.
Fig. 6 is the artificial drafting plant variety discriminating figure that 4 random RAPD primers differentiate to distinguish rhizome of Japanese Ginseng and kindred plant thereof, wherein, and 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. rhizome of Japanese Ginseng, 5. narrow leaf rhizome of Japanese Ginseng, 6. Rhizoma Panacis bipinnatifidi, 7. Rhizome of Bipinnatifid Ginseng.
Embodiment
Embodiment 1
Total DNA extraction
Modified 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 damping fluid (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 distilled waters and 0.5 μ L RNase A, 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 L 6 × sample-loading buffer, under 100V voltage, through 1.0% agar gel (containing GelRed) electrophoresis 40min ~ 1h, Gene Genius gel analysis systems axiol-ogy is taken a picture, identification of dna integrity.
RAPD analyzes
RAPD-PCR reaction system
PCR reaction solution cumulative volume is 25 μ L, wherein containing MgCl
2(25mM) 3 μ L, 10 × Taq buffer withKCl 2.5 μ L, Taq enzyme (1U/ μ L) 1.5 μ L, dNTP Mixture (10mM) 0.5 μ L, primer (10mM) 2 μ L, template DNA 1 μ L, add sterilizing ddH
2o polishing to 25 μ L.Pcr amplification program 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.Each PCR reaction is all established not containing the blank of template DNA, in triplicate, determines the stability of amplified production.
PCR result detects
Get PCR reaction product 20 μ L, under 100V voltage, through 1.5% sepharose (containing GelRed) electrophoresis, 1.5 ~ 2h, Gene Genius gel analysis systems axiol-ogy is taken a picture.
RAPD primer screening
From 16 random primers, filter out amplification rhizome of Japanese Ginseng and the kindred plant primer that totally 7 kind of plant STb gene bands are clear, reproducible thereof, selected primer is used for follow-up discriminatory analysis.
Data analysis
According to arbitrarily primed PCR amplification electrophoretogram, identical molecular weight fragment (electrophoretic mobility) place is produced with presence or absence of specific band and add up, utilize the presence or absence of specific band rhizome of Japanese Ginseng and kindred plant thereof can be divided into groups.Use the combination of the group result of multiple random primer, rhizome of Japanese Ginseng and kindred plant thereof can be made a distinction one by one.Draw collection of illustrative plates relationship analysis figure according to analytical results, form the artificial drafting cultivar identification figure of rhizome of Japanese Ginseng and kindred plant thereof.
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)=OD
260× extension rate × 50ng/ μ L; 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 Fig. 1, and in figure, result shows that STb gene is complete, can be used for RAPD and analyzes.
Table 1 spectrophotometer method measures DNA concentration and purity
Fig. 1 is the STb gene that extracts from the blade of ginseng, Radix Panacis Quinquefolii, pseudo-ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi, narrow leaf rhizome of Japanese Ginseng, the rhizome of Japanese Ginseng respectively according to the method described above result figure through 1.0% agarose gel electrophoresis.Wherein, 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. Rhizome of Bipinnatifid Ginseng, 5. Rhizoma Panacis bipinnatifidi, 6. narrow leaf rhizome of Japanese Ginseng, 7. rhizome of Japanese Ginseng.
In figure, result shows that the STb gene extracted is complete.
RAPD analyzes
RAPD primer screening result
By literature reading, collect RAPD primer, after application software comparison, from more than 100 primers, screen 16 random primers, screen through the PCR of RAPD again, rhizome of Japanese Ginseng and kindred plant thereof can be differentiated, and the primer that band is clear, reproducible, the selection result is in table 2.
The random primer base sequence that table 2 is analyzed for RAPD
The banding pattern of discriminating ginseng is 1100bp (+) 700bp (-) of F02 primer amplification and the 800bp (+) of I13 primer amplification; The banding pattern of discriminating Radix Panacis Quinquefolii is 1100bp (+) 700bp (-) of F02 primer amplification and the 800bp (-) of I13 primer amplification; Differentiate that the banding pattern of rhizome of Japanese Ginseng is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (+) of H03 primer amplification and the 450bp (+) of I13 primer amplification; Differentiate that the banding pattern of narrow leaf rhizome of Japanese Ginseng is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (-) of H03 primer amplification and the 500bp (+) of F01 primer amplification; Differentiate that the banding pattern of Rhizoma Panacis bipinnatifidi is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (-) of H03 primer amplification and the 500bp (-) of F01 primer amplification; Differentiate that the banding pattern of Rhizome of Bipinnatifid Ginseng is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (+) of H03 primer amplification and the 450bp (-) of I13 primer amplification; Differentiate that the banding pattern of pseudo-ginseng is 1100bp (-) 700bp (-) of F02 primer amplification.Wherein there is specific band to represent with (+), represent with (-) without specific band.
The pcr amplification of RAPD
In screened 4 random primers, rhizome of Japanese Ginseng and kindred plant STb gene coamplification thereof go out 46 bands, wherein polymorphic bands 42, polymorphism ratio is 91.30%, the band number that each primer amplification goes out is between 10 ~ 13, average each primer produces 11.5 band (table 3), and several bands large absolutely concentrate on (Fig. 2 ~ 5) between 200 ~ 2000bp.
The primer that table 3 is analyzed for rhizome of Japanese Ginseng and kindred plant RAPD thereof and amplified band polymorphism
Fig. 2 ~ 5 are the electrophorograms utilizing random primer F02, I13, H03, F01 extracted STb gene to be carried out to the product of pcr amplification according to above-mentioned PCR condition respectively.Wherein, M.Marker, 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. rhizome of Japanese Ginseng, 5. narrow leaf rhizome of Japanese Ginseng, 6. Rhizoma Panacis bipinnatifidi, 7. Rhizome of Bipinnatifid Ginseng.In figure, every kind of plant STb gene all amplifies many band, and same molecular amount place, some band is that this 7 kind of plant is common, and some band is by wherein one or more plants are peculiar.
Artificial drafting plant variety differentiates figure
Artificial drafting plant variety differentiates that figure (MICD) reflects that 4 random RAPD primers differentiate to distinguish the figure genealogical relationship of rhizome of Japanese Ginseng and kindred plant thereof totally 7 kinds fully, intuitively, as shown in Figure 6.7 kinds are divided into 3 groups by 1100bp and 700bp amplified by primers F 02 two specific band, wherein have representing with (+) of specific band, representing with (-) without specific band.One group is 1100 (+), 700 (-) 2 kinds, i.e. ginseng, Radix Panacis Quinquefolii; Two groups is 1100 (-), 700 (+) 4 kinds, i.e. rhizome of Japanese Ginseng, narrow leaf rhizome of Japanese Ginseng, leatherleaf rhizome of Japanese Ginseng, Rhizome of Bipinnatifid Ginseng; Three groups is 1100 (-), 700 (-) a kind, i.e. pseudo-ginseng, and this kind just makes a distinction.The presence or absence of the specific band 800bp that one group of kind goes out according to I13 primer amplification again can make a distinction; Two groups of kinds can be distinguished one by one according to the presence or absence of specific band 300bp, 450bp, 500bp of primer H03, I13, F01 amplification respectively.
Fig. 6 is the artificial drafting plant variety discriminating figure differentiating to distinguish rhizome of Japanese Ginseng and kindred plant thereof according to above-mentioned 4 RAPD random primers, wherein, and 1. ginseng, 2. Radix Panacis Quinquefolii, 3. pseudo-ginseng, 4. rhizome of Japanese Ginseng, 5. narrow leaf rhizome of Japanese Ginseng, 6. Rhizoma Panacis bipinnatifidi, 7. Rhizome of Bipinnatifid Ginseng.This figure reflects the analytical procedure utilizing specific band this 7 kind of plant to be differentiated one by one intuitively.
Claims (8)
1. RAPD marks a method for the Variety identification of rhizome of Japanese Ginseng and kindred plant thereof, it is characterized in that, comprises the steps:
The extraction traditional extraction rhizome of Japanese Ginseng of DNA and the leaf DNA of kindred plant thereof, qualification concentration and purity;
DNA obtained above is carried out amplified reaction by PCR reaction system under PCR reaction solution, its amplification program be denaturation, then carry out sex change, annealing, extension recirculation, last extend further after, determine to obtain stable amplified production, be PCR reaction product, pcr amplification reaction is all established not containing the blank of template DNA simultaneously;
RAPD primer screening collects RAPD primer, screen random primer after application software comparison, then the PCR of random primer through RAPD is reacted, carry out electrophoresis, gel analysis systems axiol-ogy is taken a picture, and filters out the primer that in amplification rhizome of Japanese Ginseng and kindred plant thereof, DNA band is clear, reproducible;
RAPD-PCR detects DNA cloning product in rhizome of Japanese Ginseng and kindred plant thereof and applies the primer screened, and by the PCR reaction conditions optimized, detects the RAPD-PCR product of DNA in rhizome of Japanese Ginseng and kindred plant thereof, and carries out band analysis;
Data analysis to be increased electrophoretogram according to arbitrarily primed PCR, the presence or absence that identical molecular weight fragment place produces specific band is carried out statistical study, utilize the presence or absence of specific band rhizome of Japanese Ginseng and kindred plant thereof can be divided into groups, use the combination of the group result of multiple random primer, rhizome of Japanese Ginseng and kindred plant thereof can be made a distinction one by one, can Variety identification be completed.
2. RAPD according to claim 1 marks the method for the Variety identification of rhizome of Japanese Ginseng and kindred plant thereof, and it is characterized in that, the kindred plant of described rhizome of Japanese Ginseng comprises ginseng, Radix Panacis Quinquefolii, pseudo-ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi, one or more in narrow leaf rhizome of Japanese Ginseng.
3. RAPD according to claim 1 marks the method for the Variety identification of rhizome of Japanese Ginseng and kindred plant thereof, and it is characterized in that, PCR reaction solution comprises the MgCl of 25mM
2, 10 × Taq buffer with KCl, the Taq enzyme of 1U/ μ L, dNTP Mixture, 10mM primer of 10mM, be the mixing raw material of 6:5:3:1:4 by volume, other are sterilizing distilled water, and described primer is the RAPD random primer of 10 bases.
4. RAPD according to claim 3 marks the method for the Variety identification of rhizome of Japanese Ginseng and kindred plant thereof, it is characterized in that, described 10 × Taq buffer with KCl is the mixture of the 100mM Tris-HCl of pH8.8,500mM KCl, 0.8% (v/v) Nonidet P40.
5. RAPD according to claim 1 marks the method for the Variety identification of rhizome of Japanese Ginseng and kindred plant thereof, and it is characterized in that, the amplification program of DNA is 90-98 DEG C of denaturation 3-10min; 92-98 DEG C of sex change 35-65s, 30-40 DEG C of annealing 33-96s, 65-80 DEG C of extension 60-120s, circulation 35-42 time; 70-75 DEG C is supplemented extension 5-10min.
6. RAPD according to claim 1 marks the method for the Variety identification of rhizome of Japanese Ginseng and kindred plant thereof, and it is characterized in that, the amplification program of DNA 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.
7. RAPD according to claim 1 marks the method for the Variety identification of rhizome of Japanese Ginseng and kindred plant thereof, it is characterized in that, the RAPD primer of screening comprises F02 (GAGGATCCCT), I13 (CTCTCCGCCA), H03(AGACGTCCAC), F01(ACGGATCCTG).
8. RAPD according to claim 7 marks the method for the Variety identification of rhizome of Japanese Ginseng and kindred plant thereof, it is characterized in that, differentiate that the banding pattern of ginseng is 1100bp (+) 700bp (-) of F02 primer amplification and the 800bp (+) of I13 primer amplification; The banding pattern of discriminating Radix Panacis Quinquefolii is 1100bp (+) 700bp (-) of F02 primer amplification and the 800bp (-) of I13 primer amplification; Differentiate that the banding pattern of rhizome of Japanese Ginseng is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (+) of H03 primer amplification and the 450bp (+) of I13 primer amplification; Differentiate that the banding pattern of narrow leaf rhizome of Japanese Ginseng is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (-) of H03 primer amplification and the 500bp (+) of F01 primer amplification; Differentiate that the banding pattern of Rhizoma Panacis bipinnatifidi is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (-) of H03 primer amplification and the 500bp (-) of F01 primer amplification; Differentiate that the banding pattern of Rhizome of Bipinnatifid Ginseng is 1100bp (-) 700bp (+) of F02 primer amplification, the 300bp (+) of H03 primer amplification and the 450bp (-) of I13 primer amplification; Differentiate that the banding pattern of pseudo-ginseng is 1100bp (-) 700bp (-) of F02 primer amplification.
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