CN104293955B - A kind of method utilizing ginseng telomere length to differentiate the ginseng time limit - Google Patents
A kind of method utilizing ginseng telomere length to differentiate the ginseng time limit Download PDFInfo
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- CN104293955B CN104293955B CN201410538757.6A CN201410538757A CN104293955B CN 104293955 B CN104293955 B CN 104293955B CN 201410538757 A CN201410538757 A CN 201410538757A CN 104293955 B CN104293955 B CN 104293955B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
Abstract
The invention discloses a kind of method utilizing ginseng telomere length to differentiate the ginseng time limit.The method comprises the steps: 1) get the sample that ginseng to be measured is positioned at below reed head 1 ~ 2cm place, extract STb gene; 2) STb gene is got, with can to identify and the restriction endonuclease cutting double-stranded DNA shown in 5 '-TCGA-3 ' carries out enzyme cuts; 3) with the single stranded DNA shown in 5 '-CCCTAAA-3 ' for probe, the hybridization of the Southern marking or dot blot are carried out to digestion products; 4) according to results of hybridization, the Restriction Fragment Length of hybrid product is measured, obtain the telomeric restriction fragment of described ginseng to be measured, be designated as TRF
to be measured; 5) by TRF
to be measuredsubstitute into formula y=0.827x+8.231 as y value, gained x value is the time limit of described ginseng to be measured.Experiment proves, the ginseng time limit and the physical life goodness of fit of the method mensuration are high, and sample size needed for the method is few, minimum to ginseng infringement, are a kind of Nondestructive Identification method.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method utilizing ginseng telomere length to differentiate the ginseng time limit, particularly one utilizes ginseng telomeric restriction fragment (TRF value) to differentiate the method for the ginseng time limit.
Background technology
Medicinal material ginseng, is the root of Araliaceae ginseng, in Chinese traditional medicine, employs nearly one thousand years as strengthening by means of tonics medicine.Ginseng is bloomed after plantation on the 4th year, and its root is ripe gradually and can survive centuries between 4-6.Research shows, the ginseng root time limit is longer, content of ginsenoside is higher, makes it have higher pharmaceutical use and commercial value.
Traditional discrimination method of the ginseng time limit is macroscopical identification, namely the quantity by observing Rhizoma Ginseng's bowl judges the time limit of ginseng, but part reed bowl merges more difficult observation, and mechanical damage also can cause the ginseng time limit to conclude, What is more increases reed bowl quantity by the mode of transplanting and adulterates.Chemical analysis is also usually as one of ginseng time limit mirror method for distinguishing, but the method often needs several grams of Ginseng Root Powder samples, is difficult to the integrity ensureing ginseng profile, has greatly damaged the value of ginseng.Therefore, in order to Protection of consumer rights and interests, ensure that ginseng is worth, the time limit authenticate technology that a kind of error is little, can operate, can quantize, substantially realize Nondestructive Identification must be explored.
In recent years, along with molecular biological development, the time limit being used for detecting ginseng by Molecular tools has become a breach.Telomere is made up of the reiterated DNA sequences (TTAGGG in all vertebrates) of one group of series connection, is in the requisite ribonucleoprotein complex in karyomit(e) two ends, plays a part on chromosome to safeguard Genome stability.Nobel's physiology prize scientist in 2009 discloses telomere length and shortens this characteristic gradually along with somatic division, each cell fission taken turns all can cause the shortening of telomere, which dictates that it as " annual ring " of DNA and molecular clock, can reflect the age level of biont to a certain extent.
Southern blot hybridization (Southern blot) is the universal method of carrying out genomic dna particular sequence location.Its ultimate principle is: two single nucleic acid strands with certain homology under certain conditions, can hybridize formation double-strand by the principle of base complementrity specifically.Utilize agarose gel electrophoresis separation through the DNA fragmentation of digestion with restriction enzyme, Single-stranded DNA fragments is also transferred on nylon membrane or other solid supports by DNA sex change on glue in position, fix through dry roasting or uviolizing, hybridize with the label probe of corresponding structure again, with radioautograph or enzyme reaction colour developing, thus detect the content of specific DNA molecular.
Summary of the invention
The object of this invention is to provide one utilizes ginseng telomeric restriction fragment (TRF value) to differentiate the method for the ginseng time limit.
The method of the discriminating ginseng time limit provided by the present invention, specifically can comprise the steps:
(1) get the sample that ginseng to be measured is positioned at below reed head 1 ~ 2cm place, extract genomic dna;
(2) genomic dna that step (1) is extracted is got, with can to identify and the restriction endonuclease cutting double-stranded DNA shown in 5 '-TCGA-3 ' carries out enzyme cuts;
(3) with the single stranded DNA shown in 5 '-CCCTAAA-3 ' for probe, the hybridization of the Southern marking or dot blot are carried out to step (2) gained digestion products;
(4) according to the results of hybridization of step (3), the Restriction Fragment Length (TRF) of hybrid product is measured, obtains the telomeric restriction fragment of described ginseng to be measured, be designated as TRF
to be measured;
(5) by TRF that step (4) obtains
to be measuredsubstitute into formula I as y value, gained x value is the time limit of described ginseng to be measured;
Y=0.827x+8.231 formula I
In formula, x represents the ginseng time limit, and y represents telomeric restriction fragment.
In the step (1) of described method, the amount of the described sample got can be dry weight 0.1 ~ 1g (as 0.1g).
In one embodiment of the invention, the extracting method of described genomic dna is specially CTAB method.
In the step (2) of described method, the amount of the described genomic dna got can be 1 ~ 20 μ g (as 10 μ g).
In the step (2) of described method, describedly can to identify and the restriction endonuclease cutting double-stranded DNA shown in 5 '-TCGA-3 ' can be restriction enzyme Taq
ai or described restriction enzyme Taq
athe isoschizomers of I.Describedly in one embodiment of the invention can to identify and the restriction endonuclease cutting double-stranded DNA shown in 5 '-TCGA-3 ' is specially the restriction enzyme Taq that NEB company produces
ai.
In the step (3) of described method, described probe need with fluorescent mark or nonradioactive labeling;
Described fluorescent mark can be fluorescein, texas Red, Cy5 or Cy3; Described nonradioactive labeling can be digoxin or vitamin H.In one embodiment of the invention, the mark that described probe carries is specially digoxin.
In one embodiment of the invention, in described step (3), to the Southern marking hybridization that step (2) gained digestion products specifically carries out.
In the step (4) of described method, described " according to the results of hybridization of step (3); measure the Restriction Fragment Length (TRF) of hybrid product, obtain the telomeric restriction fragment of described ginseng to be measured " is specially: the developing result Telomere length assay software (as Telomeric.2 software) of step (3) hybridization gained is calculated corresponding Restriction Fragment Length TRF according to formula II;
TRF=∑ Ai/ ∑ (Ai/Li) formula II
In formula, Ai is the absorbance of i point, and Li is the Marker length of i point.
For method provided by the present invention, described ginseng to be measured is required to be the ginseng of the time limit more than 3 years.
Experiment proves, the present invention utilizes ginseng telomere 5 '-TTTAGGG-3 ' tumor-necrosis factor glycoproteins, uses Taq
ai restriction endonuclease is cut Ginseng DNA enzyme, then with 5 '-CCCTAAA-3 ' for probe, carry out Southern blot hybridization, obtain telomere specific hybrid band, draw ginseng time limit calculation formula in conjunction with TRF value founding mathematical models, the ginseng time limit is assessed.Result show the ginseng time limit that the method measures and the physical life goodness of fit high, and sample size needed for the method is few, minimum to ginseng infringement, is a kind of Nondestructive Identification method.
Accompanying drawing explanation
Fig. 1 is the mathematical model of ginseng Southern hybridization figure and the ginseng time limit and TRF value.Wherein, A is ginseng Southern hybridization figure (time limit of the corresponding samples of Ginseng of 2,3,4,5,6,8 expression, M is DNA Molecular WeightMarker III); B is the mathematical model of the ginseng time limit and TRF value.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Table model high speed centrifuge (Eppendorf company), Olympms fluorescent microscope, WFJ Unic 7200spectrophotometer, BS2202S type electronic balance (Startorius company), SIM-F124 type ice-making machine (SANYO company), 4KBTXL-75 type vacuum freeze drier (Virtis company); 680X Labworks image acquisition and analysis software (gene company limited), PAGE gel-electrophoretic apparatus, CS101-2A electrically heated drying cabinet, SHZ-D circulating water type vacuum pump (Yu Hua Instrument Ltd. of Gongyi City); Retsch MM400 sample grinding machine, 5810R type cryogenic freezing whizzer.
UNIQ-10 pillar DNA purification kit (the raw work in Shanghai), NEB restriction enzyme Taq
ai, DNAMolecular Weight Marker III, Digoxigenin labeled, Blocking Reagent, DigiTAb Anti-Digoxigenin-AP (Roche), CDP-Star (Roche); Digoxin hybridization probe mark 5 '-CCCTAAA-3 ' is synthesized by the raw work in Shanghai.
Embodiment 1, ginseng telomere length is utilized to differentiate the foundation of the method for the ginseng time limit
For examination ginseng: from the samples of Ginseng of two time limits from different places, refer to table 1.
The samples of Ginseng of two, table 1 time limit from different places
One, the extraction of ginseng genomic dna
CTAB method is adopted to extract each genomic dna for examination ginseng.Specific as follows:
Getting the sample 0.1g dry weight (without going mouldy) that ginseng to be measured is positioned at below reed head 1 ~ 2cm place, being placed in pulverizer and grinding, crossing 40 mesh sieves.By powder transfer in the Eppendorf pipe of 2.0mL, add the sterilized CTAB extracting solution of 900 μ L (formula: 2% (2g/100ml) CTAB, 100mmol/L Tris-HCl pH=8.0,20mmol/LEDTA, 1.4mol/L NaCl), 0.02g PVP 40000,10 μ L beta-mercaptoethanol fully vibrates mixing, 65 DEG C of water-bath 1.5h-2h, period jog 2-3 time.Take out after terminating and be cooled to room temperature, add 900 μ L chloroform-isoamyl alcohol (volume ratio 24:1), mixing of fully vibrating, the centrifugal 10min of 12000g.Get supernatant, add equal-volume chloroform-isoamyl alcohol (volume ratio 24:1), mixing of fully vibrating, the centrifugal 10min of 12000g.Get supernatant, add the aqueous isopropanol of 2/3 volume precooling, place more than 2h for-20 DEG C.Take out, the centrifugal 10min of 12000g, abandons supernatant, and precipitate by 70% (volume fraction) washing with alcohol twice, 37 DEG C volatilize ethanol, add 100 μ L ddH
21% Proteinase K (5mg/ml) (namely adding the Proteinase K Solution that 1% volumetric concentration is 5mg/ml) is added, 37 DEG C of incubation 1h after O, 1%RNaseA (10mg/ml) (namely adding the RNAaseA solution that 1% volumetric concentration is 10mg/ml) 37 DEG C of incubation 30min; Again use chloroform-isoamyl alcohol as required again, 70% (volume fraction) ethanol, with reference to above step extracting, purify DNA, uses appropriate ddH
2o dissolves ,-20 DEG C of preservations.
Two, the enzyme of ginseng genomic dna is cut
Enzyme is cut system and is carried out proportioning according to such as following table 2.
The enzyme of table 2 ginseng genomic dna cuts system
65 DEG C of enzymes cut through night (12 hours), detected result display disperse shape, enzyme cut complete after the 600 μ l that add water add equal-volume chloroform again: primary isoamyl alcohol (volume ratio 24:1) purifying, the centrifugal 15min of 1200rmp, water intaking is added to the 3M NaAc of 1/10 volume, 2.5 times of volume dehydrated alcohols,-20 DEG C of precipitation more than 3h, the centrifugal 25min of 12500rpm, 70% ethanol washes 3 times, dries.
Three, Southern blots
1, transferring film
40 μ l ddH
2the genomic dna that O dissolving step two obtains, 65 DEG C of incubation 10min, are positioned on ice immediately, add loading buffer, mixing.Through 1% sepharose, constant voltage 80V electrophoresis 6 ~ 8 hours, tetrabromophenol sulfonphthalein is run and is stopped electrophoresis to gel 2/3 place.After rinsed with deionized water, put into 0.25M HCl depurination treatment 10min; Take out gel, ddH
2o rinsing, pours distortion liquid (formula: 0.5M NaOH, 1.5M NaCl) into, shaking table concussion (shaking 2 times, each 15min); Take out gel, ddH
2o rinsing, pours neutralizer (formula: 0.5mol/l Tris-Cl, 1.5mol/lNaCl) into, shaking table concussion (shaking 2 times, each 15min); Use 20 × SSC capillary transfer transferring film to spend the night (12 hours) to nylon membrane, take out in 100 DEG C of baking ovens and place 2 hours, fixed dna.
2, hybridize
Nylon membrane step 1 taken a turn for the better puts into the prehybridization solution (formula: 5 × SSC of 10ml, 0.02% (0.02g/100ml) SDS, 0.1% (0.1g/100ml) sarcosyl, 1% (1g/100ml)) prehybridization 2h in Blocking Reagent, after DNA probe (digoxin hybridization probe mark 5 '-CCCTAAA-3 ') 100 DEG C of sex change, with hybridization solution (formula: 5 × SSC, 1% (1g/100ml) Blocking Reagent, 0.1% (0.1g/100ml) sarcosyl, 0.02% (0.02g/100ml) SDS, 50% (50ml/100ml) methane amide) hybridization bottle in mixing after, 42 DEG C of hybridized overnight (14h ~ 16h) reclaim hybridization solution afterwards.
3, detect
Room temperature (25 DEG C) washing 2 times in film washing liquid 1 (formula: 2 × SSC, 0.1% (0.1g/100ml) SDS), each 10min.After washing, be placed on to wash in film bottle and wash film 2 times in 42 DEG C, each 15min with film washing liquid 2 (formula: 20 × SSC, 0.1% (0.1g/100ml) SDS).Wash ddH after film
2o rinse twice, soaks film 1min with buffer1 (formula: 100mM toxilic acid, 150mM NaCl, pH=7.5).Buffer2 (formula: 100mM toxilic acid, 150mMNaCl, 1% (1g/100ml) Blocking Reagent, pH=7.5) close after more than 2h in, with bufffer2 (filling a prescription the same) dilutedly digoxin antibody Anti-Digoxigenin-AP (dilution volume ratio 1:5000) on horizontal shaker, wash film 28min.Film is proceeded to capable square box, buffer3 (formula: 100mM Tris-Cl, 100mM NaCl, 50mMMgCl
2, pH=9.5) and balance 2min.CDP-Star on film drips equably, is positioned over after hatching 5min in X-ray magazine and develops.Data acquisition Telomere length assay software (Telomeric.2 software) calculates the mean length (i.e. the corresponding telomeric restriction fragment for examination ginseng) of restriction fragment (TRF), TRF=∑ Ai/ ∑ (Ai/Li), Ai is the absorbance of i point, and Li is the Marker length of i point.
4, result
Ginseng Southern results of hybridization figure is as shown in A in Fig. 1.Each telomeric restriction fragment for examination samples of Ginseng is obtained after Telomeric.2 software carries out data gathering computing to developing result, find that the telomeric restriction fragment of samples of Ginseng increases along with the increase of the time limit on the whole, and then with each time limit numerical value for examination samples of Ginseng for X-coordinate (x), with the telomeric restriction fragment of correspondence (TRF value) for ordinate zou (y), production standard curve, gained typical curve is as shown in B in Fig. 1.
TRF length and life in 3 years and the later ginseng time limit are remarkable positive correlation, set up Related Mathematical Models (namely as shown in the formula the equation of typical curve shown in I), and then telomeric restriction fragment (TRF value) can be adopted to predict the time limit of ginseng:
Y=0.827x+8.231 formula I
In formula, x represents the ginseng time limit, and y represents telomeric restriction fragment.
Embodiment 2, ginseng telomere length is utilized to differentiate the application of the method for the ginseng time limit
In order to further checking, what embodiment 1 was set up utilizes ginseng telomere length to differentiate the accuracy of the method for the ginseng time limit, the present inventor acquires the cultivated ginseng sample of Different years, utilizes the judgement schematics of above-mentioned foundation to carry out ginseng time limit prediction double blind experiment.
With reference to step one in embodiment 1 to three mensuration of each samples of Ginseng of the known time limit being carried out to telomeric restriction fragment.Measurement result is substituted into y=0.827x+8.231, and gained x value is the prediction time limit.
Result is as shown in table 3, shows to utilize this formula predictions ginseng time limit mutually attached with physical life.
The table 3 ginseng time limit predicts the outcome
Claims (7)
1. differentiate a method for the ginseng time limit, comprise the steps:
(1) get the sample that ginseng to be measured is positioned at below reed head 1 ~ 2cm place, extract STb gene;
(2) STb gene that step (1) is extracted is got, with can to identify and the restriction endonuclease cutting double-stranded DNA shown in 5 '-TCGA-3 ' carries out enzyme cuts;
(3) with the single stranded DNA shown in 5 '-CCCTAAA-3 ' for probe, the hybridization of the Southern marking or dot blot are carried out to step (2) gained digestion products;
(4) according to the results of hybridization of step (3), the Restriction Fragment Length of hybrid product is measured, obtain the telomeric restriction fragment of described ginseng to be measured, be designated as TRF
to be measured;
(5) by TRF that step (4) obtains
to be measuredsubstitute into formula I as y value, gained x value is the time limit of described ginseng to be measured;
Y=0.827x+8.231 formula I
In formula, x represents the ginseng time limit, and y represents telomeric restriction fragment.
2. method according to claim 1, is characterized in that: in step (1), and the amount of the described sample got is dry weight 0.1 ~ 1g.
3. method according to claim 1, is characterized in that: in step (2), and the amount of the described STb gene got is 1 ~ 20 μ g.
4. method according to claim 1, is characterized in that: in step (2), describedly can to identify and the restriction endonuclease cutting double-stranded DNA shown in 5 '-TCGA-3 ' is restriction enzyme Taq
ai or described restriction enzyme Taq
athe isoschizomers of I.
5. method according to claim 1, is characterized in that: in step (3), described probe is with fluorescent mark or nonradioactive labeling;
Described fluorescent mark is fluorescein, texas Red, Cy5 or Cy3; Described nonradioactive labeling is digoxin or vitamin H.
6. method according to claim 1, it is characterized in that: in step (4), the described results of hybridization according to step (3), the Restriction Fragment Length of hybrid product is measured, obtain the telomeric restriction fragment of described ginseng to be measured, for: the developing result Telomere length assay software of step (3) hybridization gained is calculated corresponding Restriction Fragment Length TRF according to formula II;
TRF=∑ Ai/ ∑ (Ai/Li) formula II
In formula, Ai is the absorbance of i point, and Li is the Marker length of i point.
7., according to described method arbitrary in claim 1-6, it is characterized in that: described ginseng to be measured is the ginseng of the time limit more than 3 years.
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| CN110656197B (en) * | 2019-10-17 | 2022-09-09 | 江西省科学院生物资源研究所 | Method for measuring tree age based on telomere length of camphor leaf blades |
| CN113418903A (en) * | 2021-05-12 | 2021-09-21 | 江西省中国科学院庐山植物园 | Method for identifying growth years of ginseng |
| CN113862336A (en) * | 2021-11-03 | 2021-12-31 | 天津医科大学 | High-throughput dot blot telomere length detection method based on single gene correction |
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| 基于端粒研究的人参年限鉴定及其数学模型的构建;程春松;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20140731(第07期);参见全文，尤其是摘要，第20页第19、22行，第21页第12行，第27页第3行-第31页第4行 * |
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