CN102051409A - Method for identifying molecules of original plants of traditional Chinese medicine radix astragali - Google Patents
Method for identifying molecules of original plants of traditional Chinese medicine radix astragali Download PDFInfo
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- CN102051409A CN102051409A CN2009102364764A CN200910236476A CN102051409A CN 102051409 A CN102051409 A CN 102051409A CN 2009102364764 A CN2009102364764 A CN 2009102364764A CN 200910236476 A CN200910236476 A CN 200910236476A CN 102051409 A CN102051409 A CN 102051409A
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Abstract
The invention provides a method for identifying molecules of original plants of traditional Chinese medicine radix astragali. The method utilizes trnH-psbA intergenic sequences of cpDNA of chloroplasts to identify the original plants of the traditional Chinese medicine radix astragali, that is, Astragalus membranaceus(Fisch.)Bge., A.membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao. and A.membranaceusf.pallidipurpureus Hsiao., for the first time. Different haplotypes are obtained through DNA extraction, PCR specific amplification, sequencing and data analysis, wherein 40 individuals of 2 populations of the A.membranaceusf.pallidipurpureus Hsiao. have a haplotype of Hap1; all the populations of the A.membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao. have haplotypes of Hap2, Hap3, Hap4, Hap5 and Hap10; and the Astragalus membranaceus(Fisch.)Bge. has four haplotypes, i.e. Hap6, Hap7, Hap8 and Hap9. The three original plants of the traditional Chinese medicine radix astragali do not have crossed haplotypes, so the original plants of the traditional Chinese medicine radix astragali can be successfully identified through haplotype and cladogram analysis. The method for identifying the molecules of the original plants of the traditional Chinese medicine radix astragali makes a beneficial attempt in the application of cpDNA trnH-psbA fragments in the identification of traditional Chinese medicine.
Description
Technical field
The invention provides a kind of method of differentiating the former plant of Chinese medicinal materials, clear and definite saying so differentiated a kind of method of Radix Astragali, Radix Astagali and pale purple brightly yellowish stilbene, the molecular labeling method of design dna order-checking and haplotype.
Background technology
The Radix Astragali is one of China's conventional Chinese medicine material, and medication is with a long history, and the beginning is stated from Shennong's Herbal, tool invigorating QI to consolidate the body surface resistance, diuresis, expelling pus and toxin by strengthening QI, expelling pus and promoting granulation effect.2005 editions " dry root that records Radix Astagali Astragalusmembranaceus (Fisch.) Bge.var.mongholicus (Bge.) Hsiao. or Radix Astragali A.membranaceus (Fisch.) Bge in the Chinese pharmacopoeia is as certified products.Pale purple brightly yellowish stilbene A.membranaceus f.pallidipurpureus Hsiao. is Chinese Astragalus endemic plant, the pale purple brightly yellowish stilbene of " Chinese Plants will " record is distributed in Gansu, Ningxia, Qinghai and Sichuan (northwestward), has same pharmaceutical use to cook Radix Astragali with Radix Astragali and uses.
The Radix Astragali is a cosmopolitan species, has a plurality of variation monoids in the former plant of the Radix Astragali, and is quite similar on their forms, is difficult to difference, brought very large difficulty and confusion for evaluation work.Require discriminating person to possess abundant identification of experience, and be difficult to differentiate exactly according to morphological specificity.Along with the continuous progress of Protocols in Molecular Biology, differentiate fast that in a few hours Radix Astragali, Radix Astagali and pale purple brightly yellowish stilbene have become possibility, more and more receive publicity.Before the present invention comes forth, any disclose or reported PCR primer and method mentioned in the present patent application are not arranged as yet.
Summary of the invention
The invention provides the method that a kind of molecule marking method that adopts the segmental haplotype of chloroplast(id) trnH-psbA is differentiated the former plant of Chinese medicine astragalus.
The present invention adopts chloroplast(id) trnH-psbA fragment to differentiate the former plant of Chinese medicine astragalus, and this method comprises the following steps:
The extraction of total DNA of the former plant of the Radix Astragali;
The optimization of the screening of chloroplast(id) obligation and order-checking condition;
Pcr amplification;
The order-checking of PCR product;
Sequencing result is analyzed and is judged.
At first adopt and improve total DNA that the CTAB method is extracted the former plant of Chinese medicine astragalus (Radix Astragali, Radix Astagali and pale purple brightly yellowish stilbene), the former vegetable material of the Radix Astragali can be the dry fresh blade of the Radix Astragali, also can be the root of the Radix Astragali, all is ground into fine powder.The CTAB method can be the high salt method of CTAB, and CTAB hangs down the PH method; Next screens suitable chloroplast(id) primer.Design of primers of the present invention is with reference to primer (the Sang T that adopted of Sang T, Crawford DJ, Stuessy TF (1997) Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae) .American Journal of Botany, 84:1120-1136), what the present invention adopted is 20 μ L reaction systems, 10 μ molL
-1Upstream primer 0.5 μ L, 10 μ molL
-1Downstream primer 0.5 μ L.Reaction system enlarges or dwindles, and primer can increase thereupon or reduce.And then, the DNA that is extracted being carried out pcr amplification (" molecular cloning experiment guide ") according to known technology, the pcr amplification product electrophoresis adopts sepharose method (" molecular cloning experiment guide ".That usually, dyeing process is selected for use is ethidium bromide (EB).The PCR product checks order with dna sequencing BDT V3.1 test kit behind the PCR, directly order-checking on ABI3130 genetic analysis instrument.
At last, the sequencing result of order-checking carries out the check and correction ordering of sequence with Contig Express software, and suitably proofreaies and correct by hand.Arrange with software ClustalX version 1.81 comparisons then, all insertions (indels) or disappearance (gaps) are treated as identical sudden change.After all sequence check and correction are correct, statistics cpDNA haplotype.According to Pons and the described method of Petit, based on identical haplotype frequency, genetic diversity h in the population of cpDNA
SWith total genetic diversity h
T, genetic differentiation coefficient G between population
ST, HAPLONST calculates with program.Set with the reticulate evolution of TCS version1.13 software building haplotype.
According to method provided by the invention, all reagent comprise CTAB method agents useful for same of the present invention; The primer that is screened, the enzyme or the reagent of required usefulness in other PCR reactions, realize other enzymes essential to the invention or reagent or material, can pack into liquid form or lyophilisate is prepared into test kit in one or more acceptable containers, is used for the discriminating of the former plant of the Radix Astragali.
Description of drawings
Fig. 1 is a trnH-psbA fragment PCR identification result
M.DNA molecular weight standard: be followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.1-20 is the trnH-psbA fragment PCR amplification of 20 individualities of AMGSSCW population.
Fig. 2 is the haplotype reticulate evolution tree with TCS version1.13 software building
Fig. 3 is the sequence alignment figure of 10 haplotypes
Embodiment
1 material
Three kinds of Radix Astragali materials pick up from Gansu, the Inner Mongol, Shanxi, Heilungkiang, Jilin and Shandong Liu Sheng (seeing Table 1).Be the quick-drying fresh blade of open-air silica gel, voucher specimen is stored in Institute Of Chinese Materia Medica Of China Academy of Chinese Medical Sciences sample shop (CMMI), identifies through professor Zhu Xiangyun of Institute of Botany, Chinese Academy of Sciences.
Table 1 sample source table
2 improve total DNA that the CTAB method is extracted the former plant of the Radix Astragali (Radix Astragali, Radix Astagali and pale purple brightly yellowish stilbene)
(1) draw materials in right amount, add liquid nitrogen after the removal surface contamination and grind (to avoid the degraded of DNA) one-tenth fine powder fast in mortar, what material ground is thin more good more.
(2) take a morsel and place 1.5mL EP pipe, the CTAB that adds 65 ℃ of preheatings extracts damping fluid 700 μ L, and mixing fully vibrates.
(3) centrifuge tube is put in 65 ℃ of water-baths insulation 2 hours, during vibration for several times.
(4) take out, sample adds chloroform-primary isoamyl alcohol (24: 1) 600 μ L after being cooled to room temperature, the light and slow mixing of putting upside down, and the centrifugal 10min of 7500rpm shifts in the new EP pipe of supernatant liquor to.
(5) Virahol of adding equal-volume-20 ℃ precooling, prep dna, the light and slow mixing of putting upside down, visible white flocks is placed 30min for-20 ℃.The centrifugal 15min of 10000rpm abandons supernatant.
(6) precipitation with 70% ethanol, each washing of dehydrated alcohol once.Room temperature volatilizes ethanol.With the aseptic high purity water dissolving DNA of 20～50 μ L.
(7) detect DNA quality and content with agarose gel electrophoresis and nucleic acid quantification.
(8) 4 ℃ of preservations are standby.
The 3PCR amplification
The segmental amplimer of trnH-psbA is trnH (5 '-CGCGCATGGTGGATTCACAATCC-3 ') and psbA (5 '-GTTATGCATGAACG-TAATGCTC-3 ').
Pcr amplification is with 20 μ L amplification system: DNA50ng, 10 * PCR buffer, 2 μ L, dNTP2 μ L, 10 μ molL
-1Upstream primer 0.5 μ L, 10 μ molL
-1Downstream primer 0.5 μ L, ExTaqDNA enzyme 1U, ddH
2O supplies cumulative volume 20 μ L.
The program of amplification is: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 45s, 58 ℃ of renaturation 30s, 72 ℃ are extended 90s totally 30 circulations, and 72 ℃ are extended 7min, 4 ℃ of preservations.The product of amplification, is observed, is taken pictures under the SYNGENE type gel imaging system, electrophoresis such as Fig. 1 of 20 individualities of AMGSSCW population, 4 ℃ of preservations at 1 * TAE damping fluid electrophoresis with 1.0% sepharose (containing EB).
The order-checking of 4PCR product
On the reaction system in dna sequencing BDT V3.1 test kit Chinese operational manual and the basis of thermal cycle conditions,, after being optimized, annealing temperature obtains suitable reaction system and thermal cycling to the consumption of BigDye.
10 μ L reaction systems:
Order-checking PCR thermal cycle conditions:
→ 4 ℃ of insulations of x25 circulation of 96 ℃ of 1min → (96 ℃ of 10sec → 56 ℃ 5sec → 60 ℃ of 4min)
The purifying (10 μ L reaction systems, alcohol/EDTA/NaAc method) of 5 order-checking PCR products
(1) every pipe adds 1 μ L 125mM EDTA, and 1 μ L 3M NaAc is to the pipe end.
(2) every pipe adds 25 μ L, 100% alcohol, and aluminium foil is obturaged close, concussion mixing 4 times, and room temperature is placed 15min.
(3) the centrifugal 30min of 12000rpm is inverted, and abandons supernatant.
(4) every pipe adds 35 μ L, 70% alcohol, 4 ℃ of centrifugal 15min of 12000rpm; Be inverted at once, abandon supernatant.
(5) repeating the 4th goes on foot 1 time.
(6) the room temperature clean alcohol that volatilizees adds 10 μ L Hi-Di Formamide dissolving DNAs; Perhaps after the foil sealing in 4 ℃ of preservations.
(7) sample after the dissolving need be at 95 ℃ of sex change 4min, put in the ice behind the cooling 4min last sample electrophoresis rapidly.
6 data processing
Sequencing result carries out the check and correction ordering of sequence with Contig Express software, and suitably proofreaies and correct by hand.Carry out the comparison of sequence with clustalx software.After all sequences are proofreaied and correct comparison correctly, statistics cpDNA haplotype.Calculate the haplotype frequency with the individual number of the different haplotypes of each population and total number of individuals.Calculate the various degree of haplotype of each population then; According to Pons and the described method of Petit, based on identical haplotype frequency, average genetic diversity (h in the population of cpDNA
S) and total genetic diversity h
T, genetic differentiation coefficient G between population
STAnd N
STValue, HAPLONST calculates with program, and this program is in the website
Http:// www.pierroton.inra.fr/genetics/labo/softwareObtain.Set with the reticulate evolution of TCS version1.13 software building haplotype
7 analytical resultss
Length after the trnH-psbA intergenic sequence of 407 individual cpDNA of 21 population of the Radix Astragali is arranged is 331bp, does not have the length diversity.A/T content is abundant, reaches 74.29-74.45%, and this conforms to most of cpDNA intergenic region based composition compositions.The variation of Nucleotide has taken place in 12 sites, has formed 10 haplotypes (Hap1-Hap10).These 10 kinds of haplotype sequences are registered in the GenBank database, number of registration is respectively GQ139474～GQ139483, after comparing, the haplotype sequence finds 8 variant sites altogether, insert or lack and be respectively: (7bp), 240bp between 15bp, 31bp, 89bp, 108bp, 109bp, 131-132bp (2bp), the 133-139bp, statistics sees Table 2, sequence alignment such as Fig. 3.Each population haplotype quantity of the former plant of the Radix Astragali, haplotype composition see Table 3.Use the reticulate evolution tree (see figure 3) of 10 haplotypes of TCS program construction.This mode is used coalescence theory and is inferred sibship between haplotype, and confidence level is more than 95%.
40 individualities of 2 population of pale purple brightly yellowish stilbene have peculiar haplotype Hap1.All population of Radix Astagali have haplotype Hap2, Hap3, Hap4, Hap5 and Hap10.Most Radix Astagali (73.6%=190/258) has haplotype Hap2; Radix Astragali has haplotype Hap6, Hap7, Hap8 and four kinds of haplotypes of Hap9.Most Radix Astragali (67.9%=74/109) has haplotype Hap7.
Pale purple brightly yellowish stilbene has unique haplotype Hap1, can significantly distinguish with Radix Astagali and Radix Astragali.Radix Astragali and Radix Astagali have some exclusive haplotypes, and 3 kinds of former plants of the Radix Astragali do not have the haplotype of intersection each other.Do not intersect each other, utilize haplotype, in the employed material of test, can reach 100% discriminating.
Sequence variations site between 10 kinds of haplotypes of the former plant cpDNA of table 2 Radix Astragali trnH-psbA fragment
The cpDNA haplotype frequency of 21 population of table 3
Claims (7)
1. the discrimination method of the former plant of the Radix Astragali is characterized in that this discrimination method adopts the method for order-checking and haplotype, and this method comprises the following steps:
The extraction of total DNA of the former plant of the Radix Astragali;
The optimization of the screening of chloroplast(id) primer and order-checking condition;
Pcr amplification;
The order-checking of PCR product;
Sequencing result is analyzed and is judged.
2. the discrimination method of the former plant of a kind of Radix Astragali according to claim 1, it is characterized in that the former plant of the Radix Astragali total DNA can from do or the former vegetable material of the fresh Radix Astragali extract.
3. the discrimination method of the former plant of a kind of Radix Astragali according to claim 2 is characterized in that Radix Astragali material can be the arbitrary part or the Milkvetch Root of the Radix Astragali.
4. according to the discrimination method of the former plant of right 1 described a kind of Radix Astragali, it is characterized in that the primer of the pcr amplification that screens, screening is in the chloroplast(id) trnH-psbA of the former plant of the Radix Astragali sequence, and its sequence is:
trnH：5’-CGCGCATGGTGGATTCACAATCC-3’
psbA：5’-GTTATGCATGAACGTAATGCTC-3’
5. carry out the PCR reaction with the described primer of claim 4, the PCR reaction parameter is 94 ℃ of pre-sex change 4min; Carry out 30 circulations (94 ℃ of 45s, 58 ℃ of 30s, 72 ℃ of 90s), 72 ℃ of 7min are extended in the back, obtain amplified production.
6. after the method amplification according to claim 5, pcr amplification product directly checks order.
7. the discrimination method of the former plant of a kind of Radix Astragali according to claim 1 is characterized in that the haplotype analysis of chloroplast(id) trnH-psbA sequence and judges and can adopt software analysis, also can complicate statistics.
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| CN104878114A (en) * | 2015-06-16 | 2015-09-02 | 中国医学科学院药用植物研究所 | Caulis kadsurae medicinal material molecule identification |
| CN105505928A (en) * | 2016-02-04 | 2016-04-20 | 杭州科兴生物化工有限公司 | Nucleotide sequence, specific primer and method for identifying physalis angulata |
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| CN105648105A (en) * | 2016-04-13 | 2016-06-08 | 国家林业局泡桐研究开发中心 | Method for identifying persimmon germplasm by using cpDNA (chloroplast deoxyribonucleic acid) molecular marker |
| CN105648105B (en) * | 2016-04-13 | 2019-04-23 | 国家林业局泡桐研究开发中心 | The method for carrying out persimmon Germplasm Identification using cpDNA molecular labeling |
| CN106701998A (en) * | 2017-02-23 | 2017-05-24 | 浙江省检验检疫科学技术研究院 | Real-time fluorescent PCR (Polymerase Chain Reaction) identification method for amaranthus spinosus |
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| CN113308567A (en) * | 2021-07-21 | 2021-08-27 | 北京中医药大学 | Bar code prepared based on polymorphism region of rhubarb chloroplast genome and application thereof |
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Application publication date: 20110511 |